WO2020208017A1

WO2020208017A1 - Diagnostic kit and method for sweet-based rice blight resistance and resistant breeding lines

Info

Publication number: WO2020208017A1
Application number: PCT/EP2020/059893
Authority: WO
Inventors: Wolf Frommer; Joon-Seob EOM; Frank White; Bing Yang; Ricardo OLIVA
Original assignee: Wolf Frommer; Eom Joon Seob; Frank White; Bing Yang; Oliva Ricardo
Priority date: 2019-04-11
Filing date: 2020-04-07
Publication date: 2020-10-15

Abstract

The invention relates to a kit for detecting and implementing rice blight resistance based on variation in SWEET promoters, comprising (1) (i) PCR primers for amplifying SWEET11a, SWEET13, and SWEET14 cDNA; and/or (ii) rice promoter reporter lines for SWEET11 a, SWEET13, and SWEET14 accumulation; (2) rice knock out lines for SWEET11 a, SWEET13, and SWEET14 genes; and (3) tester rice lines genome-edited in the SWEET11 a, SWEET13, and/or SWEET14 promoter region for evaluating the efficacy of the respective mutation for resistance. Furthermore, the invention relates to a method for detecting and implementing rice blight resistance and to specific rice lines, which comprise at least one genome-edited SWEET promoter sequence.

Description

DIAGNOSTIC KIT AND METHOD FOR SWEET-BASED RICE BLIGHT RESISTANCE AND

RESISTANT BREEDING LINES

FIELD OF THE INVENTION

The invention relates to a kit for detecting and implementing rice blight resistance based on variation in SWEET promoters, comprising (1) (i) PCR primers for amplifying SWEETH a, SWEET13, and SWEET 14 cDNA; and/or (ii) rice promoter reporter lines for SWEET1 1 a, SWEET13, and SWEET 14 accumulation; (2) rice knock out lines for SWEET1 1 a, SWEET13, and SWEET14 genes; and (3) tester rice lines genome-edited in the SWEETH a, SWEET13, and/or SWEET14 promoter region for evaluating the efficacy of the respective mutation for resistance. Furthermore, the invention relates to a method for detecting and implementing rice blight resistance and to specific rice plants, which comprise at least one genome-edited SWEET promoter sequence.

BACKGROUND OF THE INVENTION

Rice is an important food crop in the world, especially in Africa and Asia. The rice disease“Bacterial Blight (BB)” significantly reduces crop yields and thus threatens the subsistence of farmers and the local population.

BB is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). In Xoo, key virulence factors are modular transcription-activator-like effectors (TALe), which induce SWEET sucrose transporter gene expression in the rice host, enabling the disease. TALes of Xoo bind to specific TAL effector binding elements (EBE) in the promoter region of the SWEET genes. In total, six EBEs in three of the five clade III SWEET genes are currently known to be targeted by naturally occurring TALes [(PthXo1/SWEET1 1), (PthXo2 and variants/SWEET13), (PthXo3, AvrXa7, TalC and TalF/SWEE774)]. The other two clade III SWEET genes (SWEET12 and 15) can function as susceptibility (S) genes when artificially induced, although no Xoo strains targeting these genes have been identified. Recently, a new SWEET gene belonging to clade III has been identified (named SWEETU b, since it is a close paralog of SWEET1 1). For that reason, previously named SWEET1 1 has been renamed to SWEETH a in this application. SWEETs of other clades apparently cannot function as S genes, although rice has over 20 SWEET genes. It also does not seem to matter which of the three SWEETs (1 1 a, 13, 14) is induced by a particular Xoo strain to cause the disease.

DNA polymorphisms in these EBEs can impair TALe binding to the promoter. Thereby, resistance ( R ) genes can be developed in plant hosts, providing an efficient mechanism for controlling major diseases and reducing the need for pesticides.

The first identified SWEET resistance variant was xa13, a naturally occurring promoter variant in the SWEETH a promoter. A homozygous recessive state of xa13 is needed for resistance. Today, xa13 is widely used in rice breeding programs, since the promoter variants do not appear to negatively impact plant performance in the field. Further naturally occurring recessive resistance loci have subsequently been identified in SWEET 13 (xa25) and SWEET 14 (xa41) (Hutin, M., Sabot, F., Ghesquiere, A., Koebnik, R. & Szurek, B. A knowledge-based molecular screen uncovers a broad-spectrum OsSWEET14 resistance allele to bacterial blight from wild rice. Plant J. 84, 694-703 (2015).

The number of naturally occurring EBE variants is limited, and extensive breeding efforts are required to identify and introduce desired variants into elite varieties. New approaches, such as TALEN or CRISPR-based genome editing, have been used to create targeted EBE variants for SWEETH a and SWEET14 from the japonica variety Kitaake (Zhou, J. et al. Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant J. 82, 632-643 (2015); Hutin, M., Sabot, F., Ghesquiere, A., Koebnik, R. & Szurek, B. A knowledge-based molecular screen uncovers a broad- spectrum OsSWEET14 resistance allele to bacterial blight from wild rice. Plant J. 84, 694-703 (2015); Li, T., Liu, B., Spalding, M. H., Weeks, D. P. & Yang, B. High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat. Biotechnol. 30, 390-2 (2012)).

For resistance of rice plants against BB, R gene-containing rice lines can be developed. However, Xoo will likely adapt by modifying or creating new TALe variants that target novel promoter sequences. Therefore, it will be advantageous to deploy and distribute novel BB resistant R genes specifically blocking such new strains when they emerge. The development of novel rice breeding material with durable and broad-spectrum resistance against rice blight is therefore a promising and economical way to control this disease.

Surprisingly, it was found that engineering rice breeding lines to carry multiple mutations in three SWEET gene promoters (SWEETH a, SWEET13, SWEET14) makes broad bacterial blight resistance achievable, since TALes of Xanthomonas oryzae pv. oryzae cannot bind to the corresponding binding sites in the SWEET gene promoters to activate SWEET sucrose transporter gene expression. Based on this discovery, the inventors created a kit and a method for detecting and implementing rice blight resistance based on variation in SWEET promotors. Additionally, they developed (elite) rice lines, comprising at least one genome-edited promoter sequence, preferably having a broad resistance against Bacterial Blight.

SUMMARY OF THE INVENTION

In a first aspect, the invention therefore relates to a kit for detecting and implementing rice blight resistance based on variation in SWEET promoters, comprising

(1) (i) PCR primers for amplifying SWEET1 1 a, SWEET13, and SWEET 14 cDNA and, optionally, any one or more of SWEET 1 1 b, SWEET12, and SWEET 15 cDNA; and/or

(ii) rice promoter reporter lines for SWEETH a, SWEET13, and SWEET14 accumulation and, optionally, any one or more of SWEETU b, SWEET12, and SWEET15 accumulation;

(2) rice knock out lines for SWEET1 1 a, SWEET13, and SWEET14 genes and, optionally, any one or more of SWEET 1 1 b, SWEET12, and SWEET 15 genes; and (3) tester rice lines genome-edited in the SWEETH a, SWEET13, and/or SWEET14 promoter region, and, optionally, any one or more of SWEETU b, SWEET12, and SWEET15 promoter region, for evaluating the efficacy of the respective mutation for resistance.

In various embodiments, the kit further comprises

(4) breeding lines genome-edited in the SWEETH a, SWEET13, and/or SWEET14 promoter region, and, optionally, any one or more of SWEETU b, SWEET12, and SWEET15 promoter region.

Preferably, the PCR primers comprise

(1) the primer pairs having (i) the nucleotide sequences set forth in SEQ ID Nos. 1/2 and/or 3/4 for SWEET1 1 a (ii) the nucleotide sequences set forth in SEQ ID Nos. 9/10, 1 1/12 and/or 13/14 for SWEET13, and (iii) the nucleotide sequences set forth in SEQ ID Nos. 15/16 and/or 17/18 for SWEET14, and, optionally, any one or more of the PCR primer pairs set forth in SEQ ID Nos: 5/6 (SWEETU b), SEQ ID Nos. 7/8 (SWEET12), and SEQ ID Nos. 19/20 (SWEET 15);

(2) the complements of (1); or

(3) homologues of (1) or (2) that share at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length.

Preferably, the promoter reporter lines are translational reporter lines, preferably full gene reporter lines.

The promoter reporter lines may comprise individual reporter lines for each of SWEET 1 1 a, SWEET 13, and SWEET14, and, optionally, further comprise individual reporter lines for SWEETU b and/or SWEET 12 and/or SWEET15.

Preferably, the promoter reporter lines comprise one of the promoters or functional fragments thereof selected from the group consisting of the SWEET1 1 a promoter, the SWEET13 promoter, the SWEET14 promoter, and, optionally, the SWEETU b promoter, the SWEET12 promoter, and the SWEET15 promoter, and a suitable reporter gene, preferably a b-glucuronidase (GUS) reporter gene.

In various embodiments, the rice knock out lines comprise single knock out lines for SWEETH a, SWEET13, and SWEET14, and, optionally, any one or more further single knock out lines for SWEETU b, SWEET12, and SWEET15.

The rice knock out lines may further comprise any one or more of the double knock out lines for SWEET 1 1 a/13, SWEET 13/14, SWEET 1 1 a/14 genes and SWEET 1 1 a/13/14.

In various embodiments, the rice knock out lines contain frameshift mutations that lead to early termination, preferably in the sequence corresponding to transmembrane domain I of the respective SWEET protein. Preferably, the tester rice lines or breeding lines are genome-edited in any one of the following six EBEs or homologs thereof that have at least 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length: PthXol (SEQ ID NO:21), PthXo2 (SEQ ID NO:22), PthXo2A (SEQ ID NO:23), PthXo2B (SEQ ID NO:24), PthXo2C (SEQ ID NO:25), PthXo3 (SEQ ID NO:27), TalC (SEQ ID NO:26), AvrXa7 (SEQ ID NO:28) and TalF (SEQ ID NO:29). PthXo2, 2A, 2B and 2C are homologs in the sense of the present invention and thus are different variants of the same EBE, but have been listed separately due to their respective sequences having been identified.“Genome-edited”, as used herein, refers to the artificial modification of a gene or gene sequence, typically by modification of one or more nucleotides, such as by substitution, deletion or insertion. It is generally intended, if not disclosed otherwise, that said genome-editing leads to a change in binding, i.e. reduction or abolishment, of the affected sequence region by at least one Xoo TALe. This may mean that binding affinity of a given TALe for said target sequence is reduced by at least 50%, preferably at least 90 or 100%, more preferably at least two orders of magnitude, three orders of magnitude or more, as determined by suitable methods.

In various embodiments, the rice knock-out line, the tester rice line and the rice promoter reporter line is based on the Kitaake rice line, and, wherein the breeding line is based on the indica rice line.

The kit may further comprise reagents for (q)PCR, preferably for (q)RT-PCR, of SWEET 1 1 a, 1 1 b, 12, 13, 14, and/or 15 mRNA.

In various embodiments, the kit may further comprise a set of antisera against SWEET 1 1 a, 1 1 b, 12, 13, 14, and/or 15 to detect protein accumulation, preferably by protein gel blot analyses or ELISA.

In a second aspect, the invention relates to a method for detecting and implementing rice blight resistance based on variation in SWEET promoters, comprising the steps of

(a) providing a rice blight causing bacterial pathogen;

(b) inoculating a rice line with the rice blight causing bacterial pathogen;

(c) identifying the induced SWEET glucose transporter by determining the mRNA accumulation for the SWEET1 1 a, SWEET 1 1 b, SWEET12, SWEET13, SWEET14, and/or SWEET 15 genes, preferably for at least the SWEET 1 1 a, SWEET 13, and SWEET 14 genes, in the inoculated rice line by RT-PCR and/or qPCR, and/or by determining the expression of a reporter gene in rice promoter reporter lines for SWEET 1 1 a, SWEET 1 1 b, SWEET12, SWEET 13, SWEET14, and/or SWEET15, preferably for at least SWEET1 1 a, SWEET 13 and SWEET14, inoculated with the rice blight causing bacterial pathogen;

(e) verifying dependence of the pathogen on the targeted SWEET gene(s) by inoculating rice knock out lines for the respective SWEET gene(s), wherein resistance of the respective knock out line confirms the dependence on the targeted SWEET gene(s);

(f) validating the target by inoculating tester rice lines genome-edited in the targeted and verified SWEET gene(s) promoter region(s) with the rice blight causing bacterial pathogen, wherein resistance of the respective genome-edited rice line confirms the suitability of the edited promoter gene for implementing rice blight resistance to the pathogen.

Preferably, the method uses the kit according to the invention.

In various embodiments, the method further includes the step of identifying the effectors produced by the rice blight causing bacterial pathogen and predicting the targeted SWEET promoter regions by comparison with known effectors.

In a third aspect, the invention relates to a genome-edited rice line, comprising at least one mutation in at least one effector binding element (EBE) of at least one promoter of the SWEET genes 1 1 a, 13, and 14.

Preferably, the genome-edited rice plant is genome-edited in at least one of the following EBEs or homologs thereof that have at least 95% sequence identity over the entire length: PthXol (SEQ ID NO:21), PthXo2 (SEQ ID NO:22), PthXo2A (SEQ ID NO:23), PthXo2B (SEQ ID NO:24), PthXo2C (SEQ ID NO:25), PthXo3 (SEQ ID NO:27), TalC (SEQ ID NO:26), AvrXa7 (SEQ ID NO:28) and TalF (SEQ ID NO:29).

In a preferred embodiment, the genome-edited rice plant comprises at least one mutation or is genome- edited in at least one or two different EBEs, more preferably at least three different EBEs, more preferably at least four different EBEs, even more preferably at least 5 or all 6 EBEs.

In particular, the genome-edited promoter sequence(s) comprise(s) at least one genome-edited EBE or EBE promoter region selected from the group of sequences as set forth in SEQ ID Nos. 41 -53, 55-69, 71 -96, 98-109, or 1 10-157 or close homologs thereof that share at least 95%, 96%, 97%, 98% or 99% sequence identity and retain the functionality of imparting partial or full resistance to a Xoo strain by being less receptive to TALe binding.

In a fourth aspect, the invention also relates to a method of producing a broad spectrum rice blight resistant rice plant, the method comprising down-regulating the pathogen-induced expression of at least two of the SWEET 1 1 a, 13 and 14 genes, preferably by genome-editing the promoter regions of said SWEET genes. In various embodiments, additionally the pathogen-induced expression of any one or more of the SWEETU b, SWEET12 and SWEET15 genes is also down-regulated.

For said step of genome-editing the promoter regions of said SWEET genes, all the embodiments described above in relation to the genome-edited plants as such and the kit and method of the invention, are similarly applicable.

BRIEF DESCRIPTION OF DRAWINGS Figure 1. Phenotype of sweet13 knockout mutants: (a) Phenotypes of sweet13-1 and sweet13-2 knockout mutants relative to rice cultivar (cv) Kitaake controls at the mature stage with no apparent phenotypic differences. Bar: 10 cm. (b) Relative mRNA levels (quantitative RT-PCR) of SWEET13 in flag leaf blade. Samples were harvested at 12 pm (mean ± standard error of the mean (s.e.m.), n=3 biological replicates with mRNA levels normalized to rice Ubiquitinl levels; repeated independently three times with comparable results) (c) 1 ,000 grain weight of greenhouse-grown cv Kitaake, sweet13-1 and sweet13-2. No significant differences were observed.

Figure 2. SWEET mRNA levels in uninfected rice leaves. Relative mRNA levels (quantitative RT-PCR) of SWEET1 1 a, SWEET13, SWEET 14 and SWEET 15 in different regions of rice flag leaves. Samples were harvested at 12:00 pm (mean ± s.e.m., n=3 biological replicates with expression normalized to rice Ubiquitinl levels; repeated independently three times with comparable results).

Figure 3. Example of SWEET induction as detected by RT-PCR using the PCR-Primer set (SWEET^UP) RT-PCR products for SWEETH a, 13 and 14 genes in cv Kitaake infected by the Xoo strains ME2, lacking SWEET -targeting TALes, and ME2 transformed with plasmids containing PthXol (SWEET1 1 a), PthXo2, PthXo2B (both SWEET13), PthXo3, TalC, TalF or AvrXa7 (all SWEET14), respectively. Actin served as control. Leaves were infected using leaf clipping assays, while scissors dipped in water served as additional negative control.

Figure 4. Transcriptional fusion reporter lines for SWEETH a, 13 and 14. GUS staining patterns of SWEETH a, 13 and 14 transcriptional GUS fusion lines. Transcriptional GUS fusion lines show nonspecific expression pattern in leaf tissues (a) SWEETH a transcriptional GUS fusion lines (b) SWEET13 transcriptional GUS fusion lines (c) SWEET14 transcriptional GUS fusion lines. Scale Bar: 1 mm.

Figure 5. SWEET protein accumulation in uninfected and infected transgenic rice leaves (a) GUS activity in the flag leaf blade of transgenic rice carrying a pSWEETI 1 a:SWEET1 1 a-GUS construct. No detectable GUS activity. Bar: 20 pm. (b) GUS activity in the flag leaf blade of transgenic rice carrying a pSWEETI 3:SWEET13-GUS construct. Expression is limited to minor and major veins. Bar: 20 pm. (c) GUS activity in the flag leaf blade of transgenic rice carrying a pSWEETI 4:SWEET14-GUS construct. Expression is limited to minor and major veins. Bar: 20 pm. (d) Cross-section of a rice leaf blade from pSWEETI 3:SWEET13-GUS shown in panel (b). Leaf was stained for GUS activity, fixed, embedded in paraffin and then sectioned. GUS activity is detectable within the phloem. Bar: 20 pm. (e) Induction of specific SWEET protein accumulation upon infection with Xoo strains. ME2 lacks known TAL effectors for SWEET induction. pSWEETI 1 a:SWEET1 1 a-GUS #10, pSWEETI 3:SWEET13-GUS #15 and pSWEETI 4:SWEET14-GUS #3 were used, respectively. Bar: 1 mm.

Figure 6. SWEET protein accumulation in rice leaves infected with Xoo strains expressing a specific TALe. SWEET protein accumulation upon infection with specific TAL effector. Translational GUS fusion lines were infected with ME2 strain with specific effector. SWEET1 1 a was induced upon inoculation with ME2 expressing the PthXol effector. SWEET13 was induced by ME2 with PthXo2B effector. SWEET14 was induced by ME2 with PthXo3, AvrXa7, TalC or TalF.

Figure 7. Alignment of clade III SWEET to identify specific peptide sequence for immunization. Transmembrane domains are underlined and specific peptides in C-terminal region for each SWEET is marked with box.

Figure 8. Specific recognition of each antisera for its cognate antigen. Each antisera specifically recognize its cognate antigen without cross-reactivity within clade III members.

Figure 9. CRISPR-Cas9 editing of SWEET13 and SWEET14 for knockout lines and predicted truncated form of transporters. Mutagenesis of SWEET13 and SWEET14 using CRISPR/Cas9 genome editing. The guide RNA-targeting site is marked with an underline and the protospacer adjacent motif (PAM) is in bold and italic (a) Mutagenesis scheme of SWEET13 and SWEET14. Dashed line (-) denotes a deleted nucleotide in sweet13-1 (10 nt), sweet13-2 (4 nt) and sweet14-1 (1 nt), respectively. 1 nt insertion in sweet14-2 was marked by a box. Both deletion and frame shift of amino acids occurred from the 1 ^st exon and causes early termination (b) Predicted amino acid sequence of sweet13-1 , sweet13-2, sweet14-1 and sweet14-2, respectively. In sweet13-1 and sweet13-2, frameshifts occur at the position of codons 8 and 7 of the original open reading frame, respectively, leading to polypeptides with altered sequence and length due to premature stop codons (c) If it is assumed that the second ATG (codon 58 in wild type SWEET13) were used for protein production, only truncated proteins could be formed. In both mutants, the mutations will lead to loss of the first two transmembrane spanning domains, most likely leading to non-functional transporters (d) Predicted topology of the truncated SWEET14 protein in the sweet14-1 and sweet14-2 mutants in the case codon 23 would serve a start codon. In both mutants, the mutations will lead to loss of the first transmembrane spanning domain, most likely leading to non-functional transporters. Typically, premature stop codons affect RNA stability. Moreover, typically only the first ATG is used, thus is likely that all four lines have completely lost the transport functions for the respective SWEETs.

Figure 10. Molecular and phenotypic characterization of alleles of sweet13 mutant (a) Number of seeds per panicle of greenhouse-grown wild type, sweet13-1 and -2 lines grown side-by-side. No significant differences were observed (b) Total soluble sugars in wild type and sweet13-1 and -2 flag leaves. Both mutants showed similar sugar concentrations compared to wild type. Samples were harvested at dusk (8:00 pm; mean ± s.e.m, n=4 biological replicates, repeated independently at least three times with comparable results).

Figure 11. mRNA levels of clade III SWEETs in the sweet13 mutant (a-c) Relative mRNA levels of SWEET1 1 a, SWEET 14 and SWEET 15 in the sweet13-1 mutant background. SWEET 14 is the only SWEET Clade III that shows significant up-regulation in the mutant (mean ± s.e.m., n=3 biological replicates with expression normalized to rice Ubiquitinl levels, repeated independently three times with comparable results).

Figure 12. Rice knockout mutants (SWEET^k0) as diagnostic tools (a) Phenotypes of wild-type and the sweet13;14 double knock-out grown in greenhouses. No significant differences were observed. Bar: 10 cm. (b) Lesion length caused by ME2 (negative control), PX099 (positive control) and the African strain AX01947 on single, double and triple (sweetH a, sweet13 and sweet14) knockout mutants relative to cv Kitaake wild type. Lesion length measured at 14 DAI.

Figure 13. Resistance of genome-edited rice lines to different Xoo strains. Reactions of the generated cv IR64 SWEET promoter mutant lines to three representative Xoo strains (a) Lesion lengths (cm) were measured 14 DAI with Xoo strains PX099A, PX0339 and PX086. Infections were done at maximum tillering stage by inoculating 3-6 leaf samples via leaf-clip method. Four replications with two plants per replicate were performed per strain.

Figure 14. Upstream sequences (400 bp) of SWEET promoters for selected lines/varieties. Putative TATA boxes are highlighted in grey. PthXol EBEs for SWEET1 1 are marked with underline. PthXo2 EBEs for SWEET13 are marked with underline. For SWEET14, TalC EBEs are highlighted with italic. PthXo3 EBEs are highlighted with bold. AvrXa7 EBEs are underlined. TalF EBEs are highlighted with box.

Figure 15. SWEET1 1 a-13-14 triangle. Arrows indicate which TALe can overcome a particular resistance by activating any of the other SWEETs or by activating same SWEET via targeting another EBE in the same promoter, e.g. xa13-based resistance (a variant in the SWEET1 1 a promoterthat is not recognized by PthXol can be overcome by TAL effectors (e.g. PthXo2, PthXo3 etc.) that target any of the Effector- Binding Elements (EBEs) in any other SWEET promoter, or in the case of SWEET14, by targeting a different EBE in the same SWEET promoter.

Figure 16. Customized deployment of SWEET^R lines with the help of the SWEETR kit 1 .0. Farmers with Xoo-infected rice fields send samples to local breeders/pathologists, who will isolate the respective Xoo strain. After Xoo re-inoculation, the pathologist identifies both the induced and critically important SWEET using SWEET^UP for mRNA accumulation and SWEET knockout mutants (SWEET^KO). Following validation with SWEET EBE-edited Kitaake lines (SWEETp^R), the pathologist identifies the optimally resistant SWEET^R line, which would be supplied and perhaps introgressed by a local breeder. At the same time, a certified lab will isolate the Xoo DNA from infected leaves, identify the TAL effectors (TALeome), and predict targeted SWEET using the SWEETpDB. Feeding this information back into PathoTracer provides additional recommendations for Xoo-resistant rice lines to breeders developing region-specific rice lines and to farmers choosing seed for the next season. Figure 17. Alignment of the SWEET1 1 a promoter sequences from selected rice varieties. Rice varieties having nucleotide variations in PthXol EBE were identified using RiceVarMap v.2 (http://ricevarmap.ncpgr.cn/v2/). Two varieties were selected for each variation types as representative. Sequences of the first 400 bp of SWEETH a promoters of the selected varieties were extracted from the 3K database (http://snp-seek.irri.orq/). Alignment was done using ClustalW (v 2.1) in Geneious 1 1.1 .5 (https://www.qeneious.com). One A/G variation was found in the PthXol EBE. This variation occurs with a frequency of 0.002% in 4726 rice varieties.

Figure 18. Alignment of the SWEET13 promoter sequences from selected rice varieties. Rice varieties having nucleotide variations in the PthXo2 EBE were identified using RiceVarMap v.2 (http://ricevarmap.ncpqr.cn/v2/). Two varieties were selected for each variation types as representative. Sequences of the first 400 bp of SWEET13 promoters of the selected varieties were extracted from the 3K database (http://snp-seek.irri. orq/). Alignment was done using ClustalW in Geneious 1 1 .1 .5 (https://www.qeneious.com). Nine variations were found in the PthXo2 EBE with frequencies ranging from 1 .3% to 20.8%.

Figure 19. Alignment of the SWEET14 promoter sequences from selected rice varieties. Rice varieties having nucleotide variations in the PthXo3, TalC, AvrXa7, TalF EBEs were identified using RiceVarMap v.2 (http://ricevarmap.ncpqr.cn/v2/). Two varieties were selected for each variation types as representative. Sequences of the first 400 bp of SWEET14 promoters of the selected varieties were extracted from the 3K database (http://snp-seek.irri. orq/). Alignment was done using ClustalW in Geneious 1 1 .1 .5 (https://www.qeneious.com). In the PthXo3/AvrXa7 EBEs, there is one A insertion with a frequency of 7.7%. CX371 and CX372 have one G/T variation in the TalC EBE and a 18bp-deletion in the PthXo3/AvrXa7 and TalF EBEs.

Figure 20. Independent origin of SWEET-inducing TAL effectors. Neighbor-joining tree based on DisTAL distances (based on alignments of TALe repeats) between all TALes from fully sequenced Xoo genomes. Each tip represents a single TALe. Color of the tips indicates country of isolation of the corresponding strain. Groups were defined by cutting the tree at a DisTAL distance of 4. Nodes corresponding to groups containing previously described SWEET-inducing TALes are highlighted with dashed squares. Two main Xoo lineages: Xoo^s and Xoo^F are indicated with bold lines. Bar indicates scale according to DisTAL distance.

Figure 21. TALEN-induced SWEET mutants to assess prevalence of major TALes in 105 Xoo strains (a) Schematic gene structures of SWEETH a and SWEET14 with promoter sequences targeted by respective TALes (shaded) and with TALEN-bound sites (underlined). SWEET13 is shown in two rice varieties with polymorphisms of one A deletion and G/A. b, Genotypes of three Kitaake derived lines for disease assay. Figure 22. Lengths of lesions caused by 10 Xoo strains in cv IR24 (black) and cv Kitaake (grey). Each measurement was derived from 10 young fully-expanded leaves of five rice plants.

Figure 23. Alignment of PthXo2-like TALes and models for EBE interaction (a) Alignment of the Repeat variable di-residues (RVDs) of PthXo2 homologs from different Xoo strains. RVDs are shown for each homolog, with amino acids that differ from PthXo2 in italic. RVDs localized in aberrant repeats (36 aa) are shaded (b) Proposed mechanism of PthXo2B binding SWEET 13 alleles. RVD sequences of PthXo2 (row 1) and PthXo2B (row 5) aligned with the EBE of SWEET13IR₂₄ and SWEET13NM (rows 2 and 3). Low binding affinity between an RVD and a nucleotide is marked with underline. PthXo2B shows one RVD-nucleotide incompatibility with SWEET13IR₂₄ and multiple incompatibilities with SWEET13N_I . SWEET13N_I can be accommodated by looping out the 9^th or 12^th RVD of the TALe (rows 6 and 7).

Figure 24. Functional analysis of PthXo2 homologs. The genes pthXo2 and pthXo2B were cloned on the wide host range plasmid pHM1 and introduced into strain ME2 (non-virulent derivative of PX099^A, lacking pthXol). (a) EBE sequences in the selected rice varieties (b) SWEET13 induction of by Xoo strains with TALes. (c) Virulence assay on four varieties of rice with ME2 expressing pthXo2, pthXo2B and empty vector.

Figure 25. Guide RNA design. Six guide RNA genes were designed and constructed to mutate five known TALe EBEs in three SWEET promoters. Bold letters beneath shaded TALes are their target EBEs in SWEET promoters. Arrows indicate Cas9/gRNA cleavages sites at their respective binding sites.

Figure 26. Sequence information of tRNA-gRNA constructs. Six gBIock fragments synthesized by IDT (Integrated DNA Technologies, Inc., Iowa, USA) were inserted into the vector pTLN by Xbal and Xhol (in box). The dots (...) are sequences in pTLN not shown. The orientation of individual components is in order of rice glycine tRNA (in italics), gRNA scaffold (in bold) and MS2 stem-loop (in lowercase). Overhangs (shaded in dark-grey) generated by digestion of BsmBI (underlined) are identical in six plasmids. However, overhands (shaded in light gray) generated by digestion of Bsal (double underlined) are designed for assembly of the tRNA-gRNA units through Golden Gate reaction.

Figure 27. Sequence information of tRNA-gRNA recipient vector. The intermediate vector pENTR4- U6.1 P-ccdB/chl constructed as the recipient vector for tRNA-gRNA contains two Gateway recombination sequences (in italics), rice U6 promoter (in bold), two Bsal (double underlined) sites. The cassettes of ccdB (in lowercase) and chi (chloramphenicol resistant) gene (underlined were constructed to facilitate the Golden Gate assembly of multiple tRNA-gRNA units.

Figure 28. Map of the CRISPR/Cas9 construct IRS1 132 for simultaneous editing of 4 EBEs in three SWEET gene promoters in rice.

Figure 29. Resistance of SWEET edited IR64 and Ciherang-Sub1 lines. Reactions of selected mutant lines of T3 IR64-IRS1 132 and T2 Ciherang-Sub1 -IRS1 132 lines compared to parental Chiherang-Sub1 controls to infections against pthXol-, pthXo2- and avrXa7-dependent Xoo strains (PX099, PX0339 and PXO86, respectively). Different types of sequence alterations in the target SWEET promoter genes in the mutant lines resulted in varying levels of resistance to corresponding Xoo pathogens. Lesion lengths (cm) were measured at 14 days after infection (dai) of specified plants. The type of mutations for each line is indicated in brackets. Phenotyping experiments were conducted with four replications per strain, two plants each replicate and scored 3-6 inoculated leaf samples per plant.

Figure 30. Agronomic in selected genome edited mega variety lines compared to the parental controls. Location of the data points for the mutant lines as determined by MDS analysis suggest similarities of some T3 IR64-IRS1132 lines to the parental controls (IR64) control in terms of plant height, panicle length, percent reproductive tiller and percent fertility. Micro-field experiments for agronomic trait assessments were conducted using RCBD with three replications. Trials were done only in a single season.

Figure 31. Resistance of three genome edited Ciherang-Sub1 lines to three representative Xoo strains. Appearance of lesions resulting from leaf clipping with scissors for three Xoo strains (PX0399, PX099 and PX086) for three genome-edited Ciherang-Sub1 mutant lines CS-1 a, CS-4a and CS-6c (T2 generation) compared to the parental control (Ciherang-Sub1).

Figure 32. Genome-edited Komboka, Kitaake and MTU1010 rice varieties. Guide RNA design for 2.0 constructs, which include Cas9 and Cpfl in a single construct (as reported in Safari et al. (2019), Cell & Bioscience, Vol. 9, Article number: 36; doi: 10.1186/s13578-019-0298-7). Five guide RNA genes were designed and constructed to mutate five known TALe EBEs in three SWEET promoters, including five allelic EBEs targeted by two different guide RNAs for the SWEET 13 EBE. Bold letters beneath shaded TALes are their target EBEs in SWEET promoters. Arrows indicate Cas9/gRNA or Cpfl/gRNA cleavage sites at their respective binding sites: SWEET11 (PthXol EBE by Cpfl ; SEQ ID NO:160), SWEET13 (five allelic EBEs for PthXo2 and PthXo2B, edited by Cpfl : SEQ ID Nos. 161-162), and SWEET14 (TalC EBE by Cas9, AvrXa7/PthXo3 EBE and TalF EBE both by Cpfl ; SEQ Nos. 163-164).

Figure 33. Map of the CRISPR/Cas9/Cpf1 construct pMUGW5 (construct 2.0) for simultaneous editing of 5 EBEs in three SWEET gene promoters in rice.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized, and structural and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms“a”,“an” and“the” include plural referents unless context clearly indicates otherwise. Similarly, the word“or” is intended to include “and” unless the context clearly indicates otherwise. The term“comprises” means“includes”. In case of conflict, the present specification, including explanations of terms, will control.

The terms“one or more” or“at least one”, as interchangeably used herein, relate to at least 1 , or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25 or a plurality of species. In this connection, the term “plurality” means more than one, preferably 2-1000.

Numeric values specified without decimal places here refer to the full value specified with one decimal place, i.e. for example, 99 % means 99.0 %, unless otherwise defined.

The terms“about” or“approximately” or“approx.”, in connection with a numerical value, refer to a variance of ±10 %, preferably of ±5 %, more preferably of ±2 %, more preferably of ±1 %, and, most preferably less than ±1 %, with respect to the given numerical value.

When an amount, a concentration or other values or parameters is/are expressed in form of a range, a preferable range, or a preferable upper limit value and a preferable lower limit value, it should be understood as that any ranges obtained by combining any upper limit or preferable value with any lower limit or preferable value are specifically disclosed, without considering whether the obtained ranges are clearly mentioned in the context.

All items, embodiments, and examples described for the kit according to the invention also apply to the method according to the invention and to the genome-edited rice plant according to the invention, and vice versa.

In a first aspect, the invention relates to a kit for detecting and implementing rice blight resistance based on variation in SWEET promoters, comprising

(2) rice knock out lines for SWEET1 1 a, SWEET13, and SWEET14 genes and, optionally, any one or more of SWEET 1 1 b, SWEET12, and SWEET 15 genes; and

(3) tester rice lines genome-edited in the SWEETH a, SWEET13, and/or SWEET14 promoter region and, optionally, any one or more of SWEETU b, SWEET12, and SWEET15b promoter region, for evaluating the efficacy of the respective mutation for resistance.

In one embodiment, the kit according to the invention comprises (1) (i) PCR primers for amplifying SWEET1 1 a, SWEET13, and SWEET 14 cDNA and, optionally, any one or more of SWEET 1 1 b, SWEET12, and SWEET 15 cDNA;

(3) tester rice lines genome-edited in the SWEETH a, SWEET13, and/or SWEET14 promoter region, and, optionally, any one or more of SWEETU b, SWEET12, and SWEET15 promoter region, for evaluating the efficacy of the respective mutation for resistance.

In another embodiment, the kit according to the invention comprises

(1) (ii) rice promoter reporter lines for SWEETH a, SWEET13, and SWEET14 accumulation and, optionally, any one or more of SWEETU b, SWEET12, and SWEET15 accumulation;

In a further embodiment, the kit according to the invention comprises

(1) (i) PCR primers for amplifying SWEET1 1 a, SWEET13, and SWEET 14 cDNA and, optionally, any one or more of SWEET 1 1 b, SWEET12, and SWEET 15 cDNA; and

The rice blight resistance imparted is durable in that it persists during multiple generations of a genome- edited rice plant. Furthermore, the resistance is preferably a broad spectrum resistance, i.e. resistance against more than one pathogenic Xoo strain, these strains optionally targeting different SWEET genes.

The kit according to the invention, comprising the knock out, promoter reporter and tester lines and primers based on the SWEET genes 1 1 a, 13 and 14, represents the basic version of the kit. Preferably, the kit is extendable to further comprise rice lines and primers based on the SWEET genes SWEET 1 1 b, SWEET 12 and/or SWEET15.

Additionally, the kit may further comprise (4) breeding lines genome-edited in the SWEETH a, SWEET13, and/or SWEET14 promoter region, and, optionally, any one or more of SWEETU b, SWEET12 and SWEET15 promoter region. The PCR primers are typically DNA oligonucleotides, usually between 15 and 50 nucleotides in length. It is preferred that they have full complementarity to the target region, i.e. are able to form Watson-Crick base pairing over their whole length with the target. The target is any one or more of the SWEET genes disclosed herein, preferably the cDNA created by reverse transcription of the mRNA population in a given rice plant (cell).

The SWEET genes referred to herein, namely SWEETH a, 1 1 b, 12, 13, 14, and 15, are known in the field and, for example, accessible in the gene databank under accession numbers NM_001068889 for SWEET1 1 a, XM_015755897 for SWEET 1 1 b, NM_001056634 for SWEET12, NM_001073287 for SWEET13, NM_001074487 for SWEET 14 and NM_001053479 for SWEET15. Also covered are homologues thereof, in particular those that occur in nature.

In various embodiments, the PCR primers of the kit according to the invention comprise

(1) at least one primer pair for SWEETH a, the at least one primer pair preferably having the nucleotide sequences set forth in (i) SEQ ID Nos. 1 and 2, and/or (ii) SEQ ID Nos. 3 and 4;

(2) at least one primer pair for SWEET13, the at least one primer pair preferably having the nucleotide sequences set forth in (i) SEQ ID Nos. 9 and 10, and/or (ii) SEQ ID Nos. 1 1 and 12, and/or (iii) SEQ ID Nos. 13 and 14; and

(3) at least one primer pair for SWEET14, the at least one primer pair preferably having the nucleotide sequences set forth in (i) SEQ ID Nos. 15 and 16 and/or (ii) SEQ ID Nos. 17 and 18; and

optionally, any one or more of

(4) at least one primer pair for SWEETU b, the at least one primer pair preferably having the nucleotide sequences set forth in SEQ ID Nos. 5 and 6;

(5) at least one primer pair for SWEET12, the at least one primer pair preferably having the nucleotide sequences set forth in SEQ ID Nos. 7 and 8; and

(6) at least one primer pair for SWEET15, the at least one primer pair preferably having the nucleotide sequences set forth in SEQ ID Nos. 19 and 20.

Also useful are the complements of any of the primer sequences disclosed above, i.e. in SEQ ID Nos. 1-20. Further contemplated are homologs of the sequences disclosed in SEQ ID Nos. 1 -20 or the complements thereof that share at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length.

In another preferred embodiment, the kit according to the invention comprises primers having the nucleotide sequences set forth in SEQ ID Nos. 1 , 2, 9, 10, 15 and 16 and, optionally, any one or more of the PCR primer pairs having the sequences set forth in SEQ ID Nos: 5 and 6, SEQ ID Nos. 7 and 8, and SEQ ID Nos. 19 and 20. Alternatively or additionally to the sequences of SEQ ID Nos. 1 and 2, the sequence of SEQ ID Nos. 3 and 4 can be used. Alternatively or additionally to the sequences of SEQ ID Nos. 9 and 10, the sequence of SEQ ID Nos. 1 1 and 12 or 13 and 14 can be used. Alternatively or additionally to the sequences of SEQ ID Nos. 15 and 16, the sequence of SEQ ID Nos. 17 and 18 can be used

The PCR primers comprised in the kit according to the invention are referred to herein using the term “SWEET^up”. Typically, the term “SWEET^up” comprises PCR primers for amplifying SWEETH a, SWEET13, and SWEET14 cDNA. In other preferred embodiments, the term comprises PCR primers for amplifying SWEET1 1 a, SWEET 1 1 b, SWEET12, SWEET13, SWEET14, and/or SWEET 15 cDNA. The preferred primer sequences care disclosed in Table 1 . In various embodiments, the “SWEET^up” component comprises all primers listed in Table 1 for amplifying SWEET1 1 a, SWEET13, and SWEET14 cDNA, more preferably all primers for all targets as listed in Table 1 .

In various embodiments, the kit may comprise additional primer sets for any one or more of the SWEET genes, such that the kit comprises two or more primer sets for (each) one of the SWEET genes. In addition to or alternatively to the primer sequences described herein, alternative sequences that target alternative target sequence stretches, such as neighboring sequences or sequences that overlap with the target sequences disclosed herein, may be used.

Although the PCR primers referred to herein are typically DNA-based primers, it is of course also possible to use modified primers that have altered backbones, alternative bases or the like.

In various embodiments, the kit according to the invention comprises a protocol for (quantitative) polymerase chain reaction ((q)PCR), preferably for (quantitative) reverse transcriptase (RT)-PCR, of SWEET 1 1 a, 1 1 b, 12, 13, 14, and/or 15 mRNA, more preferably of SWEET 1 1 a, 13, and 14 mRNA.

The kit according to the invention may further comprise reagents for PCR, preferably for RT-PCR, more preferably for qPCR or qRT-PCR, of SWEET 1 1 a, 1 1 b, 12, 13, 14, and/or 15 mRNA. In one embodiment, the kit comprises reagents for (q)PCR of SWEETH a, 13, and 14 mRNA. In another embodiment, the kit comprises reagents for (q)PCR of SWEETH a, 13, and 14, and additionally, for (q)PCR of SWEET 1 1 b, SWEET 12 and/or SWEET 15.

The reagents for PCR or RT-PCR, preferably for quantitative variants thereof, may comprise a DNA polymerase, a reverse transcriptase (RT), nucleotides, suitable buffers, and optionally specific primers, and/or specific DNA or RNA templates. Further components may comprise usual auxiliaries and excipients, such as, but not limited to, salts, such as magnesium salts, DMSO, glycerol and/or betaine. Preferably, the reagents are premixed with the exception of primers and, if present, template.

Suitable (q)PCR techniques are known to the person skilled in the art. Suitable kits designed for PCR, RT-PCR or the quantitative variants thereof are commercially available from various manufacturers, such as Qiagen (Hilden, DE). Preferably, the PCR primers and/or the (q)PCR reagents are suitable to determine SWEET mRNA accumulation in rice plants, more preferably in Xoo strain-infected leaves of rice plants. In various embodiments, the kit also allows to compare the determined mRNA levels to those of non-infected plants or plant cells, for example by including standards or a reference table. In preferred embodiments, the PCR primers are suitable for testing the effect of Xoo isolates on SWEET gene induction, preferably by measuring single mRNA levels of the SWEET genes 1 1 a, 1 1 b, 12, 13, 14, and/or 15, preferably of the SWEET genes 1 1 a, 13, and 14. These primers are typically used to determine the levels of the different SWEET genes individually, i.e. in separate aliquots of the same sample, although multiplexing techniques may also be possible.

In various embodiments, the kit may also comprise standards for SWEET gene mRNA levels in healthy, i.e. non-infected rice plants.

In various embodiments, the kit according to the invention further comprises a set of antisera and/or antibodies, for example polyclonal or monoclonal antibodies in isolated form, against SWEET 1 1 a, 1 1 b, 12, 13, 14, and/or 15 to detect protein accumulation, preferably by protein gel blot analyses or ELISA. In a preferred embodiment, the kit comprises a set of antisera/antibodies against SWEETH a, 13, and 14. Alternatively, the kit may comprise peptide antigens used for immunization to produce the antisera/antibodies. Suitable peptide sequences for immunization are disclosed in Table 7 and comprise the amino acid sequences set forth in SEQ ID Nos. 30-39.

In various embodiments, the promoter reporter lines of the kit according to the invention are transcriptional or translational reporter lines. Preferably, the promoter reporter lines are translational reporter lines, more preferably full gene reporter lines.

Typically, transcriptional reporter lines are based on a fusion of a promoter sequence of interest and a suitable reporter gene sequence to measure the transcriptional activity of the specific promoter. Translational reporter lines are, in general, based on fusion of a reporter gene, to a specific gene sequence, encoding for a protein of interest. Thereby, after translation, a combined product of the protein of interest and the reporter protein is obtained and the amount and the localization of the protein can be observed in the cell or in the organism. According to the present invention, preferably translational report lines are used.

In a preferred embodiment, the promoter reporter lines of the kit according to the invention comprise individual reporter lines for each of SWEETH a, SWEET13, and SWEET14. Additionally, the kit may additionally comprise individual promoter reporter lines for any one or more of SWEET1 1 b, SWEET12, and SWEET15.“Individual” means that the respective reporter line is designed such that it can be used to monitor the activity of only one of the SWEET genes, i.e. is a specific report for a single SWEET gene. In various embodiments, the kit comprises at least three promoter reporter lines, each comprising a promoter sequence-(SWEET gene sequence)-reporter gene sequence construct for one promoter sequence of SWEETH a, SWEET13 and SWEET14, and, optionally, a fourth, fifth or sixth promoter reporter line for any one or more of SWEET 1 1 b, SWEET 12, and SWEET 15.

Preferably, the individual promoter reporter lines comprise one of the SWEETH a, SWEETU b, SWEET12, SWEET13, SWEET14, or SWEET15 promoters or functional fragments thereof, and a suitable reporter gene, preferably a b-glucuronidase (GUS) reporter gene.

In a preferred embodiment, the promoter reporter lines comprise one of the promoters or functional fragments thereof selected form the group consisting of the SWEET1 1 a promoter, SWEET13 promoter, and SWEET14 promoter, and, optionally the SWEETU b promoter, the SWEET 12 promoter, and the SWEET15 promoter, and a suitable reporter gene, preferably a b-glucuronidase (GUS) reporter gene.

Full gene reporter lines are reporter lines with the full length sequence (e.g., an 1 to 5 kb long nucleotide sequence stretch upstream from the coding sequence) of one of the SWEETH a, SWEETU b, SWEET12, SWEET13, SWEET14, or SWEET15 promoters or fragments thereof, preferably promoter sequences of about 1 to 2 kb in length or functional fragments thereof, and the whole coding region of one of the SWEET1 1 a, SWEETU b, SWEET12, SWEET13, SWEET14, or SWEET15 genes, preferably including all introns, fused to the reporter gene, preferably to the b-glucuronidase (GUS) reporter gene, more preferably to the GUSPIus reporter gene. b-Glucuronidase is an enzyme that is capable to convert a colorless substrate (5-bromo-4-chloro-3- indole-beta-glucuronide (X-Gluc)) to a colored product, which is measurable with a suitable (microscopy) technique or can be seen with the bare eye.

In general, the promoter or the fragment thereof is combined with its corresponding coding region. That means for example that the SWEETH a promoter or the functional fragment thereof is combined with the coding region of the SWEETH a gene fused to a reporter gene.

“Functional fragment”, as used herein in relation to the promoter sequences, relates to part of the full- length promoter sequence that retains the ability to function as a promoter. Preferably, such a fragment has at least 50% of the transcriptional activity of the full-length promoter.

The reporter lines are preferably rice plant lines that have been genome-edited as described above, i.e. typically by fusing a reporter gene to any one of the SWEET genes. Thus, they allow detection of the induction of the respective SWEET gene by determination of the reporter gene product. Generally, the genome-editing as described herein can be achieved using any suitable methods, all of which are known to those skilled in the art. These methods include besides the CRISPR-Cas9 or CRISPR-Cpfl technology, described herein, also other methods such as homology driven repair, for example via transformation or transient exposition. In preferred embodiments, the kit according to the invention comprises (translational) promoter reporter lines, which comprise or consist of individual (translational) reporter lines for SWEETH a, SWEET13, and SWEET14.

In another preferred embodiment, the kit according to the invention comprises (translational) promoter reporter lines, which comprise individual (translational) reporter lines for at least three, four, five or all six of SWEET 1 1 a, SWEET 1 1 b, SWEET12, SWEET 13, SWEET 14 and/or SWEET 15, with SWEET 1 1 a, SWEET 13 and SWEET14 being preferably always included.

The promoter reporter lines are suitable for determining/monitoring protein accumulation. This technique (or kit component) can be performed/used as an alternative to the PCR primer analysis or in addition thereto.

In a preferred embodiment, various Xoo strain isolates can be tested with the reporter lines according to the invention to measure protein accumulation. In the translational reporter caused by induction of the SWEET gene leads to accumulation of the reporter gene product, i.e. a polypeptide, such as b- glucuronidase. The activity of this polypeptide/enzyme can be determined, for example as described above, for example by examination of the rice plant, preferably the leaves of the rice plant, either with the naked eye or a microscope.

The rice promoter reporter lines of the kit according to the invention are also referred to herein by use of the term“SWEET^acc“ reporter lines. Typically, the term“SWEET^acc” at least refers to rice promoter reporter lines for SWEET1 1 a, SWEET13, and SWEET14 accumulation. In some embodiments, the term covers rice promoter reporter lines for more than the three SWEET genes listed above and includes any one or more of SWEET 1 1 b, SWEET12, and SWEET 15.

The rice promoter reporter lines are typically transgenic.“Transgenic” has the meaning as a person skilled in the art would understand it. Typically, it means that a foreign gene or DNA/RNA construct has been introduced into an organism, by what the organism becomes a genetically modified organism. This transgene is, in various embodiments, the reporter gene fused to the SWEET gene, i.e. in the above described embodiments the beta-glucuronidase.

In various embodiments, the rice knockout lines of the kit according to the invention comprise single knock-out lines for SWEET 1 1 a, SWEET 13, and SWEET 14. This means that the kit comprises at least three single knock-out lines. In another embodiment, the kit further, i.e. additionally to the three aforementioned knock-out lines, comprises single knock out lines for any one or more of SWEETU b, SWEET12, and SWEET15. In some embodiments, the rice knock-out lines of the kit according to the invention comprise single knock-out lines for SWEET1 1 a, SWEET13, SWEET14, and SWEET15. Additionally, single knock-out lines for SWEET1 1 b and/or SWEET12 may be included.

“Single knock-out lines”, as used herein, relates to rice lines in which a single one of the above-listed SWEET genes has been inactivated (knocked out) by an appropriate mutation or another form of geneediting. Similarly,“double knock-out lines”, refers to rice lines in which two of the SWEET genes have been knocked out.

In various embodiments, the rice knock-out lines of the kit according to the invention may further comprise any one or more of the double knock out lines for SWEET1 1 a/13, SWEET13/14 and SWEET 1 1 a/14 genes.

Additionally, the rice knock-out lines may further comprise any one or more of the double knock-out lines for SWEET1 1 a/15, SWEET13/15, and SWEET14/15.

Other double knock-out lines that may be used include SWEET 1 1 b/13, SWEET 1 1 b/14, SWEET 1 1 b/15, SWEET1 1 a/12, SWEET12/13, SWEET12/14, SWEET12/15, and SWEET1 1 b/12. SWEET1 1 a/1 1 b double knock-out lines were found to be sterile. As a consequence, double knock-outs of these two SWEET genes can, e.g., be tested in a rice plant line is provided wherein either of the two genes is expressed under a gametophytic active promoter.

In further embodiments, the rice knock-out lines of the kit according to the invention may further comprise any one or more of the triple knock-out lines for SWEET 1 1 a/13/14 genes, SWEET 1 1 a/13/15, SWEET 1 1 a/14/15 or SWEET 13/14/15.

Other triple knock-out lines that may be used include SWEET1 1 a/12/13, SWEET1 1 a/12/14, SWEET1 1 a/12/15, SWEET1 1 b/12/13, SWEET1 1 b/12/15, SWEET 1 1 b/13/14, SWEET1 1 b/13/15, SWEET12/13/14, SWEET12/13/15, and SWEET12/14/15.

In even further embodiments, the rice knock-out lines are quadruple, quintuple or sextuple knock-out lines, wherein the SWEET genes knocked out are selected from those 6 genes disclosed herein. In various embodiments, these quadruple, quintuple or sextuple knock-out lines comprise knock-outs of SWEET 1 1 a/13/14 and any one or more of SWEET 1 1 b, SWEET 12 and SWEET 15.

Preferably, the rice knock out lines contain frameshift mutations that lead to early termination, preferably in the sequence corresponding to transmembrane domain I (TM I) of the respective SWEET protein.

The single knock out lines are, for example, obtainable by using CRISPR-Cas9, CRISPR-Cpfl , techniques or TALEN-mediated techniques. These techniques are known to the person skilled in the art and instructions for implementation are available.

In general, frameshift mutations refer to insertion or deletion of at least one base pair into/from the sequence of interest. By deletion or insertion of a number of base pairs different from 3 or multiples of 3, the open reading frame of the respective gene is changed downstream from the change such that the nucleotide sequence encodes different amino acids or a new stop codon is created. In most cases, this leads to early termination of the translation process at the newly introduced stop codon, and thereby to truncated, non-functional proteins.

Preferably, the rice knock out lines comprised in the kit according to the invention can be used as a diagnostic tool for testing which SWEET is targeted by new Xoo strains and to predict possible resistance mechanisms and yield impacts.

In addition, the single knock out lines can be used together with the double knock out lines (or triple knock out lines) to test if up-regulation of one SWEET gene may compensate the loss of another SWEET gene or to measure phenotypic differences relative to the corresponding wildtype line. It has been found that single and double knock out variants as used herein show no significant defects with respect to plant growth or yield relative to the wildtype line. Specifically, it has been found that single and double knock outs of the genes SWEETH a, SWEET13, SWEET14, and SWEET15, do not have a negative impact on the yield.

The rice knock out lines of the kit according to the invention are referred to herein using the term “SWEET^k0“ knock out lines. Typically, as used herein, the term“SWEET^k0” relates to rice knock out lines for SWEETH a, SWEET13, and SWEET14 genes. In other embodiments, the term covers rice knock out lines for SWEET1 1 a, SWEET 1 1 b, SWEET12, SWEET13, SWEET14, and/or SWEET 15 genes, preferably for SWEET1 1 a, SWEET13, SWEET14, and/or SWEET15 genes.

Additionally, the kit comprises specific tester rice lines genome-edited in the SWEETH a, SWEET13, and/or SWEET14 promoter region and, optionally, also any one or more of SWEETU b, SWEET12 and SWEET15. These modifications in the promoter regions provide for resistance against one or more Xoo strains that target the respective SWEET promoter. These modifications are preferably base substitutions, such as single base substitutions, deletions or insertions. Typically, these modifications change one or more of the effector binding elements (EBE) within the promoter regions.

The tester rice lines can be used for evaluating the efficacy of the respective mutation for resistance.

Preferably, the tester rice lines of the kit according to the invention are genome-edited in at least one of the following six EBEs or homologs thereof that have at least 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length: PthXol (SEQ ID NO:21), PthXo2 (SEQ ID NO:22), PthXo2A (SEQ ID NO:23), PthXo2B (SEQ ID NO:24), PthXo2C (SEQ ID NO:25), PthXo3 (SEQ ID NO:27), TalC (SEQ ID NO:26), AvrXa7 (SEQ ID NO:28) and TalF (SEQ ID NO:29). The sequences of the six EBEs are summarized in Table 9 and Figure 14. It is possible that only one EBE of one promoter region of the genes SWEETH a, SWEET13, and/or SWEET14 is genome-edited. In various embodiments, the number of substituted, deleted and/or inserted nucleotides of the EBE sequence is at least one, but can also be 2, 3, 4, 5, 6, 7, 8 or more. However, it is also contemplated that the majority of the EBE region, i.e. more than 50% or more than 75% of the sequence, or even the complete sequence is altered, for example by substitution, insertion and/or deletion, provided that the resulting sequence is neither the wildtype sequence of the starting EBE nor the known sequence of any other EBE as disclosed herein. In another embodiment, not only one but two EBEs of the SWEET genes 1 1 a, 13 and 14 of a tester rice line can be modified, independently of each other. In another embodiment, three, four, five or six EBEs are altered, independently of each other. In various embodiments, the tester rice line thus comprises genome-edited version of any two or all three of the promoter regions, in particular the EBEs, of the SWEET genes 1 1 a, 13 and 14. In case the promoter region of SWEET14 is affected, the altered EBE may be any one of the four alternative ones or may comprise two, three or four of the EBEs of the promoter region of SWEET14. In various further embodiments, all the afore-mentioned embodiments directed to SWEET1 1 a, SWEET13 and SWEET14 may further comprise genome-edited versions of the promoter regions of SWEET 1 1 b, SWEET 12 and/or SWEET 15.

Different tester rice line varieties have been designed and are disclosed herein, comprising different numbers and types of resistance genes. Different sequences of genome-edited promoter regions are listed in Table 10 and 12. Tables 10 and 12 summarize genome-edited EBEs that were found to be suitable and are intended to be encompassed by the present invention in that rice plants or rice plant cells that include any of these modified sequences are covered by the scope of the present invention.

Specifically, tester rice lines may have the PthXol (SEQ ID NO:21) EBE replaced by any one of the nucleotides sequences set forth in SEQ ID Nos. 1 10-1 19. Alternatively or additionally, tester rice lines may have the PthXo2 (SEQ ID NO:22) EBE or its homologs or variants disclosed above replaced by any one of the nucleotides sequences set forth in SEQ ID Nos. 120-131 . Alternatively or additionally, tester rice lines may have the PthXo3 (SEQ ID NO:27) EBE replaced by any one of the nucleotide sequences set forth in SEQ ID Nos. 132-142. Alternatively or additionally, tester rice lines may have the TalC (SEQ ID NO:26) EBE replaced by any one of the nucleotide sequences set forth in SEQ ID Nos. 143-154. Alternatively or additionally, tester rice lines may have the TalF (SEQ ID NO:29) EBE replaced by any one of the nucleotide sequences set forth in SEQ ID Nos. 155-157. As the AvrXa7 (SEQ ID NO:28) EBE is encompassed by the PthXo3 EBE, the modifications to PthXo3 similarly apply to AvrXa7 and vice versa.

In various other embodiments, in the tester rice lines the complete promoter region of SWEETH a, SWEET 13 and/or SWEET 14 as set forth in SEQ ID NQ:40 (SWEET1 1 a), SEQ ID Nos 54 and 97 (SWEET13) and SEQ ID NO:70 (SWEET14) may be replaced by any one of the nucleotide sequences set forth in

(i) SEQ ID Nos. 41 -53, 94 and 95 (SWEET1 1 a);

(ii) SEQ ID Nos. 55-69 and 98-102 (SWEET 13); or

(iii) SEQ ID Nos. 71 -93 and 104-109 (SWEET14).

It can be preferred that such modifications for different EBEs, in particular those disclosed above, are combined in one rice plant line, in particular in the combinations disclosed in the tables included herein, such as Tables 10 and 12.

Preferably, the tester rice lines are resistant against one or more Xoo strains, which include a TAL effector that cannot bind to the corresponding genome-edited EBE. For example, rice lines that are genome-edited in the PthXo1 -EBE are intended to be resistant to Xoo strains containing the TAL effector PthXol .

In a preferred embodiment, the rice knock out line(s), the tester rice line(s), and/or the rice promoter reporter line(s) included in the kit are based on the same rice cultivar, preferably on cv Kitaake [Oryza sativa L. ssp. japonica cultivar Kitaake) or, alternatively, indica or japonica rice lines. In various embodiments, they are based on cv Kitaake or on an indica rice line, preferably on Oryza sativa L. ssp. japonica cv Kitaake or on the Oryza sativa indica cv IR64 (Mackill, D. J. & Khush, G. S. 2018. IR64: a high-quality and high-yielding mega variety. Rice 11 , 18) or cv Ciherang-Sub1 (Toledo, A. M. U. et at. 2015. Development of improved Ciherang-Sub1 having tolerance to anaerobic germination conditions. Plant Breed. Biotech. 3, 77-87) rice lines.

Cv Kitaake is a particularly good standard host for testing Xoo compatibility with rice, since the only currently known R gene for Bacterial Blight (BB) in cv Kitaake is a (recessive) R gene allele of xa25, which is, in general, dependent on strains with the TAL effector PthXo2.

Preferred genome-edited EBE sites or promoter regions in the Kitaake rice line are shown in Table 10.

The tester rice lines of the kit according to the invention are also referred to herein using the term “SWEETp^R“ genome-edited tester rice lines. Typically, the term“SWEETp^R” refers to tester rice lines genome-edited in the SWEET1 1 a, SWEET13, and SWEET14 promoter region. In various embodiments, the term relates to tester rice lines genome-edited in the SWEETH a, SWEETU b, SWEET12, SWEET13, SWEET14, and/or SWEET 15 promoter regions, preferably in the SWEET1 1 a, SWEET13, SWEET14, and/or SWEET15 promoter region. Preferably, the tester rice lines are based on cv Kitaake rice lines, preferably on Oryza sativa L. ssp. japonica cv Kitaake. Alternatively, the tester rice lines may also be indica or japonica lines, such as Oryza sativa indica cv IR64 or cv Ciherang-Sub1 . In various embodiments, the tester rice lines are usable for genotyping Xoo isolates for race characterization.

Preferably, the tester rice lines will allow the determination of which SWEET is targeted and whether use of a variant in one of the known EBEs in the respective SWEET promoter(s) is sufficient to block the infection caused by (specific) Xoo.

In a preferred embodiment, the kit according to the invention may further comprise SWEET breeding lines (also referred to herein as SWEET^R), preferably based on the (elite) rice lines IR64 or Ciherang- Sub1 . The SWEET breeding lines (SWEET^R) are mega varieties, preferably based on Oryza sativa indica IR64 and Ciherang-Sub1 .

Mega rice varieties are defined as varieties that are planted on large parts of the arable land, preferably on one million hectares or more.

Preferably, the SWEET breeding lines comprise alterations, preferably at least one mutation, in either one or more EBEs.

Preferably, the breeding lines of the kit according to the invention are genome-edited in at least one of the following six EBEs or homologs thereof that have at least 80%, 85%, 90% 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length: PthXol (SEQ ID NO:21), PthXo2 (SEQ ID NO:22), PthXo3 (SEQ ID NO:27), TalC (SEQ ID NO:26), AvrXa7 (SEQ ID NO:28) and TalF (SEQ ID NO:29) or any one of the PthXo2 homologs: PthXo2A (SEQ ID NO:23), PthXo2B (SEQ ID NO:24) or PthXo2C (SEQ ID NO:25). The sequences of the six EBEs and known homologs thereof are summarized in Table 9 and Figure 14. It is possible that only one EBE of the promoter region of the genes SWEET1 1 a, SWEET13, and/or SWEET14 is genome-edited. In various embodiments, the number of substituted, deleted and/or inserted nucleotides of the EBE sequence is at least one, but can also be 2, 3, 4, 5, 6, 7, 8 or more. Preferred are alterations by insertion or substitution of at least 1 , 2, 3, 4 or 5 nucleotides, more preferably 1 or 2 nucleotides, most preferably 1 nucleotide. Also preferred are deletions of 1 or more nucleotides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides, such as 1 to 5 nucleotides, e.g. 1 , 2 or 3 nucleotides. However, it is also contemplated that the majority of the EBE region, i.e. more than 50% or more than 75% of the sequence, or even the complete sequence is altered, for example by substitution, insertion and/or deletion, provided that the resulting sequence is neither the wildtype sequence of the starting EBE nor the known sequence of any other EBE as disclosed herein.

In various embodiments, not only one but two EBEs of the SWEET genes 1 1 a, 13 and 14 of a tester rice line can be modified, independently of each other. In another embodiment, three, four, five or six EBEs are altered, independently of each other. In various embodiments, the tester rice line thus comprises genome-edited version of any two or all three of the promoter regions, in particular the EBEs, of the SWEET genes 1 1 a, 13 and 14. In case the promoter region of SWEET14 is affected, the altered EBE may be any one of the four alternative ones or may comprise two, three or four of the EBEs of the promoter region of SWEET14. Specifically, the breeding lines may comprise genome-edited versions of the SWEETH a promoter and the SWEET13 promoter, the SWEETH a promoter and the SWEET14 promoter, the SWEET 13 and the SWEET 14 promoter, or the SWEET1 1 a, SWEET 13 and SWEET 14 promoter. If, in the afore-mentioned embodiments, the promoter of SWEETH a is genome-edited, the genome-edited region is preferably the sequence of PthXol . If, in the afore-mentioned embodiments, the promoter of SWEET13 is genome-edited, the genome-edited region is preferably the sequence of PthXo2, 2a, 2b or 2c. If, in the afore-mentioned embodiments, the promoter of SWEET14 is genome- edited, the genome-edited region is preferably the sequence of PthXo3 (SEQ ID NO:27), TalC (SEQ ID NO:26), AvrXa7 (SEQ ID NO:28) and/or TalF (SEQ ID NO:29), e.g. PthXo3/AvrXa7 and TalC, PthXol /AvrXa7 and TalF, TalC and TalF, or PthXo3/AvrXa7, TalC and TalF.

In various further embodiments, all the afore-mentioned embodiments directed to SWEETH a, SWEET13 and SWEET14 may further comprise genome-edited versions of the promoter regions of SWEET 1 1 b, SWEET 12 and/or SWEET 15.

In another embodiment, not only one but two EBEs of the SWEET genes 1 1 a, 13 and 14 of a tester rice line can be modified, independently of each other. In another embodiment, three, four, five or six EBEs of these three SWEET gene promoter regions, as disclosed herein, are altered, independently of each other.

PthXol (SEQ ID NO:21), is an EBE in the promoter sequence of SWEET1 1 a, PthXo2 (SEQ ID NO:22) and its homologs PthXo2A (SEQ ID NO:23), PthXo2B (SEQ ID NO:24) and PthXo2C (SEQ ID NO:25) are EBEs in the promoter sequence of SWEET13, and PthXo3 (SEQ ID NO:27), TalC (SEQ ID NO:26), AvrXa7 (SEQ ID NO:28) and TalF (SEQ ID NO:29) are each EBE sequences in the promoter sequence of SWEET14.

Specifically, breeding lines may have the PthXol (SEQ ID NO:21) EBE replaced by any one of the nucleotides sequences set forth in SEQ ID Nos. 1 10-1 19. Alternatively or additionally, breeding lines may have the PthXo2 (SEQ ID NO:22) EBE or its homologs or variants disclosed above replaced by any one of the nucleotides sequences set forth in SEQ ID Nos. 120-131 . Alternatively or additionally, breeding lines may have the PthXo3 (SEQ ID NO:27) EBE replaced by any one of the nucleotide sequences set forth in SEQ ID Nos. 132-142. Alternatively or additionally, breeding lines may have the TalC (SEQ ID NO:26) EBE replaced by any one of the nucleotide sequences set forth in SEQ ID Nos. 143-154. Alternatively or additionally, breeding lines may have the TalF (SEQ ID NO:29) EBE replaced by any one of the nucleotide sequences set forth in SEQ ID Nos. 155-157. As the AvrXa7 (SEQ ID NO:28) EBE is encompassed by the PthXo3 EBE, the modifications to PthXo3 similarly apply to AvrXa7 and vice versa. In various other embodiments, in the breeding lines the complete promoter region of SWEETH a, SWEET 13 and/or SWEET 14 as set forth in SEQ ID NO:40 (SWEET1 1 a), SEQ ID Nos 54 and 97 (SWEET13) and SEQ ID NO:70 (SWEET14) may be replaced by any one of the nucleotide sequences set forth in

(i) SEQ ID Nos. 41 -53, 94 and 95 (SWEET1 1 a);

(ii) SEQ ID Nos. 55-69 and 98-102 (SWEET 13); or

(iii) SEQ ID Nos. 71 -93 and 104-109 (SWEET14).

In all the above embodiments, the replacement is not limited to the actual nucleic acid sequence of the recited SEQ ID Nos. but also extends to close homologs thereof that share at least 95, 96, 97, 98 or 99% sequence identity with the recited sequence while retaining the desired impairment of TALe binding.

It can be preferred that such modifications for different EBEs, in particular those disclosed above, are combined in one rice plant line, in particular in the combinations disclosed in the tables included herein, such as Tables 10 and 12. In these breeding lines, two, three, four or five modified EBEs can thus be combined. Preferred combinations of those modified EBEs disclosed above are disclosed in Tables 10 and 12.

It is preferred that a modified, i.e. genome-edited, PthXol EBE is combined with a modified PthXo2, PthXo3/AvrXa7, TalC and/or TalF EBE. It is similarly preferred that a modified PthXo2 EBE is combined with a modified PthXol , PthXo3/AvrXa7, TalC and/or TalF EBE. It is even more preferred that a modified PthXol EBE and a modified PthXo2 EBE are combined with a modified PthXo3/AvrXa7, TalC and/or TalF EBE. If reference is made herein to PthXo2 EBE, it is understood that this similarly relates to the known variants thereof, disclosed herein as PthXo2a, 2b and 2c.

The genome-edited SWEET breeding lines of the kit according to the invention are also referred to herein using the term“SWEET^R“. Preferably, the term“SWEET^R” relates to rice lines genome-edited in the SWEET 1 1 a, SWEET 13, and SWEET 14 promoter region or may refer to rice lines genome-edited in the SWEET1 1 a, SWEET 1 1 b, SWEET12, SWEET13, SWEET14, and/or SWEET 15 promoter regions, preferably in the SWEET1 1 a, SWEET13, SWEET14, and/or SWEET15 promoter region. Preferably, the rice lines are based on Oryza sativa indica IR64 and Ciherang-Sub1 rice lines.

In a preferred embodiment, at least 3, 4 or 5 of six EBE sites in the three SWEET promoters of SWEETH a, 13 and 14 are genome-edited, preferably in the Oryza sativa indica cv IR64 and cv Ciherang-Sub1 rice lines, preferably by a CRISPR-Cas9- or CRISPR-Cpfl -mediated strategy or TALEN-mediated strategy. The thus obtained rice line is also called “elite line”. In a preferred embodiment, the line also carries a gene for flooding tolerance. In this study, a plurality of Cas9 or Cpfl -free (breeding) lines were developed encompassing 35 single mutations in the three SWEET genes 1 1 a, 13, and 14. These lines are, preferably, resistant against single or multiple Xoo strains.

Preferably, the breeding lines are transgene-free.

Preferred genome-edited EBE sites and promoter regions in the indica rice lines are shown in Table 12 (Oryza sativa indica cv IR64 (Mackill, D. J. & Khush, G. S. 2018. IR64: a high-quality and high-yielding mega variety. Rice 11 , 18) or cv Ciherang-Sub1 (Toledo, A. M. U. et al. 2015. Development of improved Ciherang-Sub1 having tolerance to anaerobic germination conditions. Plant Breed. Biotech. 3, 77-87) rice lines).

In another embodiment, the kit may further comprise a SWEET promoter database access. This database is also referred to as“SWEETpDB” promoter database. This database can contain information on SWEET gene promoter region variants of existing rice lines and records of the rice genome database to collect the knowledge and enable access thereto. Typically, for each strain, the first 400 bp of the three SWEET promoters of SWEETH a, SWEET13 and SWEET14 were sequenced to generate a representative promoter sequence database. In the future, further promoter sequence variants can be added to such a promoter database.

In various embodiments, the kit according to the invention may further comprise means for providing access to a geographic information system (GlS)-based platform. This platform is also referred to as “PathoTracer”. This platform incorporates pathogen monitoring and resistance profiles of rice varieties. This may be useful for customized deployment and management of local disease outbreaks and to display the predicted involvement of SWEETH a, SWEET13 and SWEET14 in each region. In addition, the platform may suggest most effective promoter variants for breeding or deployment to control the local Xoo population.

Preferably, the kit according to the invention can be used for customized deployment of novel R gene rice variants. It may help to identify the rice lines best suited to defeat the pathogen, preferably one or more Xoo variant in a certain area, and to increase the robustness of resistance of rice lines by reducing monocultures and favoring the evolution of novel strains. Furthermore, it may allow improved monitoring of variants strain emergence.

(a) providing a rice blight causing bacterial pathogen;

(b) inoculating a rice line with the rice blight causing bacterial pathogen;

In a preferred embodiment, the method uses the kit according to the invention.

The step of providing a rice blight causing bacterial pathogen can be carried out by the user such that a sample containing a suitable pathogen is provided. Such a sample may, for example, originate from an infected rice plant. The method then allows to detect and implement rice blight resistance based on variation in SWEET promoters to this particular pathogen of interest, as provided by the user.

In a third aspect, the invention relates to a genome-edited rice plant, comprising at least one mutation in at least one EBE of at least one promoter of the SWEET genes 1 1 a, 13, and/or 14. Alternatively or additionally, such a genome-edited rice plant can also comprise at least one mutation in at least one EBE of at least one promoter of the SWEET genes 1 1 b, 12, and/or 15. In various embodiments, the genome-edited rice plant comprises at least one mutation in at least 1 , 2, 3, 4, 5 or 6 EBE(s) of at least one promoter of the SWEET genes 1 1 a, 13, and/or 14. Additionally, such rice plant may comprise at least one mutation in at least one EBE of at least one promoter of the SWEET genes 1 1 b, 12, and/or 15.

The terms“rice line”,“rice plant”, "rice variety", and "rice cultivar" are mostly used herein synonymously.

Preferably, the rice plant is genome-edited in at least one of the following six EBEs or homologs thereof that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length: PthXol (SEQ ID NO:21), PthXo2 (SEQ ID NO:22), PthXo2A (SEQ ID NO:23), PthXo2B (SEQ ID NO:24), PthXo2C (SEQ ID NO:25), PthXo3 (SEQ ID NO:27), TalC (SEQ ID NO:26), AvrXa7 (SEQ ID NO:28) and TalF (SEQ ID NO:29). Table 10 and 12 summarize the most important/suitable genome-edited promoter sequences for Kitaake/elite rice lines.

In a preferred embodiment, the single EBEs (PthXol (SEQ ID NO:21), PthXo2 (SEQ ID NO:22), PthXo2A (SEQ ID NO:23), PthXo2B (SEQ ID NO:24), PthXo2C (SEQ ID NO:25), PthXo3 (SEQ ID NO:27), TalC (SEQ ID NO:26), AvrXa7 (SEQ ID NO:28) and TalF (SEQ ID NO:29)) of the promoter regions of the SWEET genes 1 1 a, 13, and/or 14, or homologs thereof that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length can be genome-edited independently of each other. The number of altered nucleotides of the EBE sequence(s), or the number of inserted or deleted nucleotides is at least one, but can also be 2, 3, 4, 5, 6, 7, 8 or more. Preferred are alterations by insertion or substitution of at least 1 , 2, 3, 4 or 5 nucleotides, more preferably 1 or 2 nucleotides, most preferably 1 nucleotide. Also preferred are deletions of 1 or more nucleotides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides, preferably 1 to 5 nucleotides, more preferably 1 , 2 or 3 nucleotides. However, it is also contemplated that the majority of the EBE region, i.e. more than 50% or more than 75% of the sequence, or even the complete sequence is altered, for example by substitution, insertion and/or deletion, provided that the resulting sequence is neither the wildtype sequence of the starting EBE nor the known sequence of any other EBE as disclosed herein.

In various embodiments, not only one but two EBEs of the SWEET genes 1 1 a, 13 and 14 of a rice plant of the invention can be modified, independently of each other. In another embodiment, three, four, five or six EBEs are altered, independently of each other. In various embodiments, the tester rice line thus comprises genome-edited version of any two or all three of the promoter regions, in particular the EBEs, of the SWEET genes 1 1 a, 13 and 14. In case the promoter region of SWEET14 is affected, the altered EBE may be any one of the four alternative ones or may comprise two, three or four of the EBEs of the promoter region of SWEET 14. Specifically, the breeding lines may comprise genome-edited versions of the SWEETH a promoter and the SWEET13 promoter, the SWEETH a promoter and the SWEET14 promoter, the SWEET 13 and the SWEET 14 promoter, or the SWEET1 1 a, SWEET 13 and SWEET 14 promoter. If, in the afore-mentioned embodiments, the promoter of SWEETH a is genome-edited, the genome-edited region is preferably the sequence of PthXol . If, in the afore-mentioned embodiments, the promoter of SWEET13 is genome-edited, the genome-edited region is preferably the sequence of PthXo2, 2a, 2b or 2c. If, in the afore-mentioned embodiments, the promoter of SWEET14 is genome- edited, the genome-edited region is preferably the sequence of PthXo3 (SEQ ID NO:27), TalC (SEQ ID NO:26), AvrXa7 (SEQ ID NO:28) and/or TalF (SEQ ID NO:29), e.g. PthXo3/AvrXa7 and TalC, PthXol /AvrXa7 and TalF, TalC and TalF, or PthXo3/AvrXa7, TalC and TalF.

In various further embodiments, all the afore-mentioned embodiments directed to SWEETH a, SWEET13 and SWEET14 may further comprise genome-edited versions of the promoter regions of SWEET 1 1 b, SWEET 12 and/or SWEET 15. In another embodiment, not only one but two EBEs of the SWEET genes 1 1 a, 13 and 14 of a rice plant of the invention can be modified, independently of each other. In another embodiment, three, four, five or six EBEs of these three SWEET gene promoter regions, as disclosed herein, are altered, independently of each other.

Preferably, the rice plants according to the invention comprise one of these genome-edited EBEs of the promoter region of one SWEET gene 1 1 a, 13 and 14. In another preferred embodiment, two, three, four, five or six genome-edited EBEs of the promoter region of one, two or three SWEET genes 1 1 a, 13 and 14 are combined in one rice line. Preferably, the rice plant exhibits a broad resistance against Bacterial Blight, preferably against one or more, preferably against a plurality of Xoo strains.

Preferably, the genome-edited rice plant comprises at least one mutation or is genome-edited in at least one or two different EBEs, preferably at least three different EBEs, more preferably at least four different EBEs, even more preferably at least 5 or all 6 EBEs of at least one or more promoter regions of the SWEET genes 1 1 a, 13 and 14. Preferred are combinations as already recited above.

Specifically, genome-edited rice plants of the invention may have the PthXol (SEQ ID NO:21) EBE replaced by any one of the nucleotides sequences set forth in SEQ ID Nos. 1 10-1 19. Alternatively or additionally, genome-edited rice plants of the invention may have the PthXo2 (SEQ ID NO:22) EBE or its homologs or variants disclosed above replaced by any one of the nucleotides sequences set forth in SEQ ID Nos. 120-131 . Alternatively or additionally, genome-edited rice plants of the invention may have the PthXo3 (SEQ ID NO:27) EBE replaced by any one of the nucleotide sequences set forth in SEQ ID Nos. 132-142. Alternatively or additionally, genome-edited rice plants of the invention may have the TalC (SEQ ID NO:26) EBE replaced by any one of the nucleotide sequences set forth in SEQ ID Nos. 143-154. Alternatively or additionally, genome-edited rice plants of the invention may have the TalF (SEQ ID NO:29) EBE replaced by any one of the nucleotide sequences set forth in SEQ ID Nos. 155- 157. As the AvrXa7 (SEQ ID NO:28) EBE is encompassed by the PthXo3 EBE, the modifications to PthXo3 similarly apply to AvrXa7 and vice versa.

In various other embodiments, in the genome-edited rice plants of the invention the complete promoter region of SWEET1 1 a, SWEET 13 and/or SWEET 14 as set forth in SEQ ID NO:40 (SWEET1 1 a), SEQ ID Nos 54 and 97 (SWEET13) and SEQ ID NO:70 (SWEET14) may be replaced by any one of the nucleotide sequences set forth in

(i) SEQ ID Nos. 41 -53, 94 and 95 (SWEET1 1 a);

(ii) SEQ ID Nos. 55-69 and 98-102 (SWEET 13); and/or

(iii) SEQ ID Nos. 71 -93 and 104-109 (SWEET14).

It can be preferred that such modifications for different EBEs, in particular those disclosed above, are combined in one rice plant line, in particular in the combinations disclosed in the tables included herein, such as Tables 10 and 12. In these genome-edited rice plants of the invention, two, three, four or five modified EBEs can thus be combined. Preferred combinations of those modified EBEs disclosed above are disclosed in Tables 10 and 12. Preferred genome-edited rice plants of the invention are thus those that comprise a combination of modifications of those listed above that corresponds to any one of the combinations disclosed in Tables 10 and 12.

It is preferred that a modified PthXol EBE is combined with a modified PthXo2, PthXo3/AvrXa7, TalC and/or TalF EBE. It is similarly preferred that a modified PthXo2 EBE is combined with a modified PthXol , PthXo3/AvrXa7, TalC and/or TalF EBE. It is even more preferred that a modified PthXol EBE and a modified PthXo2 EBE are combined with a modified PthXo3/AvrXa7, TalC and/or TalF EBE.

In a preferred embodiment, the genome-edited promoter sequence(s) of the genome-edited rice plant comprise(s) at least one genome-edited EBE(s) or EBE promoter region(s) selected from the group of sequences as set forth in SEQ ID Nos. 41 -53, 55-69, 71 -96, 98-109, or 1 10-157, preferably more than one of these modified EBEs or EBE promoter regions in combination.

In a preferred embodiment, the genome-edited rice plant according to the invention is based on cv Kitaake or on the indica rice line, preferably on Oryza sativa L. ssp. japonica cv Kitaake or on the Oryza sativa indica cv IR64 (Mackill, D. J. & Khush, G. S. 2018. IR64: a high-quality and high-yielding mega variety. Rice 11 , 18) or cv Ciherang-Sub1 (Toledo, A. M. U. et at. 2015. Development of improved Ciherang-Sub1 having tolerance to anaerobic germination conditions. Plant Breed. Biotech. 3, 77-87) rice lines, more preferably on Oryza sativa indica cv IR64 or cv Ciherang-Sub1 , which are also called mega-varieties.

This rice plant is preferably transgene-free.

Preferred genome-edited EBE sites in the indica rice lines are shown in Table 12.

In particular, the tester rice lines and the breeding lines of the kit according to the invention are genome- edited rice plants according to the invention.

The invention also relates to a method of generating the genome-edited rice plants of the invention that exhibit resistance to the Xoo pathogen-induced expression of any one or more of the SWEET genes. In the respective method, the genome of a rice plant is edited, as described herein, to impart resistance to one or more Xoo strains by rendering the targeted SWEET gene promoters less susceptible or even resistant to pathogen-induced expression. This is achieved, as described herein, by modifying the EBEs in the SWEET genes such that the Xoo TALes can no longer bind efficiently. This loss of efficiency may be expressed in a loss of binding affinity of the modified EBE for the TALe by at least 50%, more preferably at least 90%, more preferably at least two orders of magnitude. In still another aspect, the invention also relates to a processed product comprising the DNA of a modified rice plant of the invention.

EXAMPLES

Material and methods

Promoter variation analysis

Rice varieties having nucleotide variations in six EBEs (for the TALes PthXol , PthXo2 (including 2A, 2B and 2C), PthXo3, TalC, AvrXa7, TalF) were found using the“Search for Variations in a Region" and “Search for Genotype With Variation ID“ functions in RiceVarMap v.2 (http://ricevarmap.ncpgr.cn/v2/). Two varieties were selected for each variation type as representative. Sequences of the first 400 bp of SWEETH a, 13 and 14 promoters of the selected varieties were subtracted from the 3K database (http://snp-seek.irri.org/). Alignment was done using ClustalW2.1 in Geneious 1 1 .1 .5 (https://www.geneious.com).

Sequences of the known EBEs in Oryza sativa L. ssp. japonica Kitaake are shown in Table 9 and Fig. 14.

Genotyping of rice plants. Rice genomic DNA was extracted using Cetyl trimethylammonium bromide (CTAB) (http://gsl.irri.org/services/dna-extraction-king-fisher/met). Polymerase chain reaction (PCR) was performed using ExTaq DNA polymerase (Clontech, Mountain View, CA, USA) with a melting temperature of 56 °C for SWEET 1 1 a, SWEET 13 and SWEET 14, respectively. The PCR-amplicons from the mutant alleles were validated by Sanger sequencing. Chromatograms were analyzed and aligned using Sequencher (https://www.genecodes.com/).

RNA isolation and transcript analyses. Total RNA was isolated using SpectrumTM Plant Total RNA kits (Sigma, St. Louis, MO, USA) or Trizol (Invitrogen, Carlsbad, CA, USA), and first strand cDNA was synthesized using Quantitect reverse transcription Kit (Qiagen, Hilden, Germany). qRT-PCR was performed using a LightCycler 480 (Roche, Penzberg, Germany), with the 2^~ACt method for relative quantification (Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-DD CT method. Methods 25, 402-8 (2001)).

DNA constructs and plant transformation

Generation of GUS reporter constructs. The method for constructing pSWEETI 1 a:gSWEET1 1 a- GUSplus was described (Yang, J., Luo, D., Yang, B., Frommer, W. B. & Eom, J.-S. SWEET1 1 and 15 as key players in seed filling in rice. New Phytol. 218, 604-615 (2018)). For tissue specificity analysis, a 4,354-bp genomic clone of SWEET13 containing 1 ,919 bp of the 5’ upstream region of translational start codon (ATG) and 2,435 bp of the entire coding region without stop codon, and a 4,365-bp genomic clone of SWEET14 containing 2,176 bp of the 5’-upstream region and 2,189 bp of the entire coding region without stop codon were amplified by PCR using Kitaake genomic DNA as template. The PCR amplicons were subcloned into pJET2.1/blunt (Thermo Fisher) and resulting inserts were confirmed by DNA sequencing. The cloned fragments digested with Xba\ and Kpn\ for SWEET13 or Avrll and Xmal for SWEET14 were subsequently inserted in front of GUSplus coding sequence of a promoterless GUSplus coding vector (Yang, J., Luo, D., Yang, B., Frommer, W. B. & Eom, J.-S. SWEET1 1 and 15 as key players in seed filling in rice. New Phytol. 218, 604-615 (2018)) restricted with Xba\/Kpn\ for SWEET13 and Xba\/Xma\ for SWEET14. The resulting pSWEETI 3\gSWEET13-GUSp\us and pSWEETI 4\g SWEET 14-GUSplus constructs were used to transform Oryza sativa L. ssp. japonica Kitaake. Nine independent events were obtained for pSWEETI 3:gSI/l/EET73-GUSplus and pSWEETI 4:gSI/l/EET74-GUSplus, respectively. While GUS activity levels were different in the independent lines, the GUS patterns were comparable.

Kitaake was also used for CRISPR-Cas9-mediated and TALEN-mediated genome editing of SWEETH a, SWEET13 and SWEET14 genes. The methods for the CRISPR-Cas9-induced mutant (sweet1 1 a-1) and the TALEN-induced mutant (sweet1 1 a-2) have been described previously (Yang, J., Luo, D., Yang, B., Frommer, W. B. & Eom, J.-S. SWEETH a and 15 as key players in seed filling in rice. New Phytol. 218, 604-615 (2018)). The knockout mutants sweet13-1, sweet13-2, sweet14-1, and sweet14-2 were obtained with a CRISPR-Cas9 construct targeting the coding sequence 5’- GCCTGTCCCTGCAGCATCCCTGG-3’ (SEQ ID NO. 158) of SWEET 13 and 5’- GCAT GT CT CTT CAGCATCCCTGG-3’ (SEQ ID NO. 159) of SWEET 14 (underline: PAM) common to the first exon as described (Zhou, J. et al. Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant J. 82, 632-643 (2015)). Double mutants (sweet13-2; 14-1) were created by crossing. SWEET13 RNA levels were analyzed in the sweet13 mutant and shown to be reduced (Figure 1 b). To test whether editing of the respective sites is also possible with other CRISPR enzymes, a combination strategy that comprised a construct containing both Cas9 and Cpfl (Fig. 32, 33) was successfully deployed. Cpfl has the advantage that it produces staggered cuts as opposed to Cas9, which produces blunt cuts. Cas9 therefore preferentially produces single nucleotide polymorphisms, which on the one hand may not be sufficient to block TAL effector binding due to the low selectivity of certain repeats in the TAL effector repeats and single nucleotide polymorphisms that prevent binding can likely be rapidly overcome by small changes in the TAL effector. By contrast, Cpfl produces more substantial changes, as exemplified for the African elite variety Komboka in which sites edited with Cpfl typically altered 5-10 nucleotides in the TAL effector binding site (Table 15). The combined Cas9-Cpf1 strategy allowed to generate edits in all known EBEs simultaneously in Kitaake, Komboka and MTU1010 (Fig. 32, 33, Tables 15-17).

Plant materials and growth conditions. Wild-type and mutant plants were grown either in field conditions (paddy field in summer, 2016, Carnegie, Stanford, CA, USA) or in greenhouses under long- day conditions of 14-h day/10-h night, 28-30°C, 50% relative humidity, and 500-1000 pmol/m²s light intensity.

Histochemical GUS analyses. Samples were collected in 90% cold acetone for fixation, vacuum- infiltrated for 10 min and incubated for 30 min at room temperature. Leaf samples were vacuum- infiltrated in GUS washing buffer (Staining solution without 5-bromo-4-chloro-3-indole-beta-glucuronide (X-Gluc)) on ice for 10 min. The solution was changed with GUS staining solution (50 mM sodium phosphate pH 7.0, 10 mM EDTA, 20% (v/v) methanol, 0.1 % (v/v) Triton X-100, 1 mM potassium ferrocyanide, 1 mM potassium ferricyanide, 2 mM X-Gluc dissolved in dimethyl sulfoxide). Samples were incubated at 37°C. After 2 hours of incubation, samples were cleared in an ethanol series (20%, 35%, 50%) at room temperature for 30 min. Samples from Xoo inoculated leaves were incubated in 70% ethanol to remove the chlorophyll. Specimens were observed with a SteREO Discovery. V12 stereoscope (Zeiss). For paraffin sections, samples were fixed using FAA for 30 min (50% (v/v) ethanol, 3.7% (v/v) formaldehyde, 5% (v/v) acetic acid). Dehydration was performed with an ethanol series (70%, 80%, 90%, 100%, 30 min each) and 100% tert.-butanol. Samples were transferred and embedded in Histosec pastilles (Millipore, Billerica, MA, USA). Sections (10 pm) were obtained with a rotary microtome (Jung RM 2025, Wetzlar, Germany). Specimens were observed with an Eclipse e600 microscope (Nikon).

Xoo strains and infection protocols. The Xoo strains were collected from different geographic regions. Plasmid-containing Xoo strains were obtained through electroporation of competent cells with respective pHM1 -derived plasmids (e.g., pHM1/ZWpthXo1 for PthXol gene). For infection, bacterial inocula were prepared by growing bacterial cells on TS (tryptone sucrose) plates with appropriate antibiotics. Cells were scrapped from the plates and resuspended in sterile dest. water at Oϋboo ~0.5. (i) Leaf clipping (Kauffman, H. E., Reddy, A. P. K., Hsieh, S. P. Y. & Merca, S. D. An improved technique for evaluating resistance of rice varieties to Xanthomonas oryzae. Plant Dis Rep 57, 537-41 (1973)): The two youngest, fully expanded leaves of 4-5 week-old rice plants were clipped about 1 -2 cm from the tip with scissor blades immersed in bacteria immediately prior to clipping. Five plants were used for inoculation of each strain. Lesion length (distance from cut to leading edge of (gray) symptoms) was measured for each inoculated leaf 12-14 days after inoculation. The mean lesion length of ten leaves was used for each treatment. The Tukey test for analysis after ANOVA was used for statistical analyses. Leaf tissues were mounted in laminating film and photographed under white light (ii) Syringe infiltration: Bacterial suspensions were infiltrated into leaves from the bottom by pressing the opening of a needle-less syringe to the leaf. Leaf fragments with inoculated spots were cut off 48 hrs post inoculation for RNA extraction and GUS staining analysis.

Results

SWEETpDB promoter database

To predict resistance against or susceptibility to particular Xoo strains with TALes that target SWEET promoters, it would be helpful to know the sequences of any EBEs in each SWEET promoter. Variations in the promoter regions of SWEET 1 1 a, 13 and 14 from the 4726 accessions available in the rice genome database (The 3,000 rice genomes project. Gigascience 3, 7 (2014); Zhao, H. et al. RiceVarMap: a comprehensive database of rice genomic variations. Nucleic Acids Res 43, D1018-D1022 (2015)) and from an additional five rice lines relevant for India, South East Asia and Africa were thus analyzed and fifteen sequence variants found. One A/G variant in the EBE for PthXol occurred at a frequency of 0.2% (Figure 17). Seven variations were found in the PthXo2 EBE at frequencies ranging from 1 .3% to 20.8% (Figure 18). Interestingly, five variations were found at the TATA box in the promoter region of SWEET13 (TATATAAA, TATTTAAA, TATATATA, TATATAA, and TATATAAAA), which overlap with the TALe EBE. The TATATAAA variant is known to occur in japonica varieties resistant against Xoo containing PthXo2 TALe (Zhou, J. et al. Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant J. 82, 632-643 (2015)). In the PthXo3/AvrXa7 EBE, an insertion of one A nucleotide was found at a frequency of 7.7% (Figure 19). Lines CX371 and CX372 (or NERICA1 and NERICA2) have one G/T variation in the TalC EBE and an 18 bp-deletion spanning the PthXo3/AvrXa7 and TalF EBEs. These variations could lead to broad-spectrum resistance against Xoo harboring TALes TalC, PthXo3/AvrXa7 and TalF. To confirm the information mined from high-throughput genomic sequences, two varieties from the 4726 accessions were selected for each variation type and five lines relevant to India, South East Asia and Africa (MTU1010, Samba Mahsuri, Komboka, BRRI Dhan28, BRRI Dhan 29) were selected (data not shown). The first 400 bp of the three SWEET promoters were sequenced to generate a representative promoter sequence database (data not shown).

PCR primers (SWEET^up) and protocols for diagnosing SWEET gene induction

Specific primer pairs for SWEET1 1 a, 13 and 14 (SWEET 11 F1 :GGGATTTCTGGCTAGTTTCT (SEQ ID NO:1); SWEET 11 R2 : CGAGGTAG AGGACGAT GTAG (SEQ ID NO:2); SWEET 13 F1 :AGAGTTTTCAGCCAACACAT (SEQ ID NO:9); SWEET 13 R1 GTAGATCCGGTAGAACGTC (SEQ ID NO:10); SWEET 14 F2:TATTGCCTGATCATCCTCTT (SEQ ID NO:15); SWEET 14 R2:GTGAACATCTTGGCCTTCT (SEQ ID NO:16)) and protocols for both RT-PCR and qRT-PCR were generated to detect SWEET mRNA levels in uninfected leaves (Figure 2) as well as Xoo strain-specific SWEET mRNA accumulation in infected leaves (Figure 3). Analysis by qRT-PCR indicated that SWEET13 mRNA levels were the highest among the five clade III SWEET genes in uninfected leaves, followed by SWEET14. SWEET13 may play a role in phloem loading. SWEETH a mRNA levels were very low in leaves, which is consistent with its function in seed filling (Yang, J., Luo, D., Yang, B., Frommer, W. B. & Eom, J.-S. SWEET1 1 and 15 as key players in seed filling in rice. New Phytol. 218, 604-615 (2018)). Validated primer pairs as show in Table 1 below have been designed to allow efficient testing of the effect of Xoo isolates on SWEET gene induction.

Table 1 : Specific primer pairs to detect SWEET mRNA levels

The rice promoter reporter lines (SWEET^acc) for analyzing Xoo-triggered SWEET protein accumulation

In addition to the SWEET^up primers, transcriptional reporter lines for SWEET1 1 a, 13 and 14 as a second set of tools for testing RNA accumulation were generated. However, lines for the three promoters showed non-specific reporter activity in all leaf cell types (Figure 4). A similar observation had previously been made for AtSWEETH and 12 genes in Arabidopsis; only translational SWEET reporter fusions showed cell specific reporter accumulation.

To monitor protein accumulation, translational promoter reporter lines were generated using 2-kb promoter fragments and the whole coding region, including all introns, fused to the GUSPIus reporter gene. Consistent with the absolute levels of mRNA as measured by qRT-PCR, SWEET13 and SWEET14 reporter activities were detected in uninfected leaves, while SWEET1 1 a fusion lines showed no detectable GUS activity (Figure 5a-d). Both SWEET13 and SWEET14 translational fusion lines showed vein-specific expression patterns, again consistent with roles in phloem loading (Figure 5b-d). The reporter lines for SWEETH a, 13 and 14 were infected with five Xoo strains, PX061 , PX071 , PX086, PX099 and PX01 12, known to induce specific SWEETs. Xoo strain ME2, lacking TALes for SWEET induction, did not trigger induction in these SWEET^acc reporter lines. Induction of SWEETH a was detected when infected with Xoo strains PX071 and PX099, which carry PthXol , but not the other three strains. SWEET13 was induced only upon infection with PX061 , harboring PthXo2B, while SWEET 14 was induced upon infection with PXQ61 (PthXo3), PXQ86 (AvrXa7) and PXQ1 12 (PthXo3) (Figure 5e). Infection with the ME2 strain expressing either PthXol (SWEET 1 1 a), PthXo2B (SWEET 13), or PthXo3, TalC, AvrXa7 and TalF (SWEET14), further confirmed specific SWEET isoform induction (Figure 6).

Rice knockout lines (SWEET^ko) as diagnostic tools

Tools that can identify the SWEET that is targeted by a particular Xoo strain without prior knowledge of the respective EBE would be useful for engineering resistance, since variant Xoo strains could target either different promoter elements or other clade III SWEET paralogs. Moreover, it is conceivable that promoter-edited lines or variants could cause yield or performance penalties. Mutational analysis of SWEET genes would provide insight into which SWEETs have critical functions with regards to both resistance and yield. To diagnose which SWEET is targeted by new Xoo strains and to predict yield impacts, knockout mutant lines were created for four of the five described clade III SWEET genes (SWEETH a, 13, 14 and 15) using CRISPR-Cas9 (Figure 9). In all cases, lines containing frameshift mutations in the sequence corresponding to transmembrane domain I (TM I) were identified. Early termination is expected to lead to non-functional transporters (for example, premature termination in the last transmembrane domain, TM VII, led to defective transporters). While mRNA levels were expected to be near wild type levels, the sweet13 mutant lines had mRNA levels that were dramatically reduced (Fig. 1 b). The truncations in SWEETH a and SWEET15 caused seed filling defects, strongly indicative of complete loss of function of both genes. Although SWEET1 1 and SWEET15 play key roles in seed filling in rice (Yang, J., Luo, D., Yang, B., Frommer, W. B. & Eom, J.-S. SWEET1 1 and 15 as key players in seed filling in rice. New Phytol. 218, 604-615 (2018)), promoter variants of the genes, such as xa13 (SWEETH a), do not have a negative impact on yield (Laha, G. S. et at. Changes in Rice Disease Scenario in India: An Analysis from Production Oriented Survey. 1-95 (ICAR-lndian Institute of Rice Research, Rajendranagar, Hyderabad, (2016); Sakthivel, K. et al. The host background of rice influences the resistance expression of a three genes pyramid (xa5 + xa13 + Xa21) to bacterial blight ( Xanthomonas oryzae pv. oryzae) pathotypes of Indian mainland and Bay islands. Plant Breed. 136, 357-364 (2017)).

Since maize ZmSWEET13 paralogs play key roles in phloem loading, the role of rice SWEET13 in phloem transport was investigated. SWEET 13 is the most highly expressed SWEET in rice leaves, and, similar to SWEET14, is capable of transporting sucrose and localizes to the plasma membrane (data not shown) (Chen, L. Q. et al. Sucrose efflux mediated by SWEET proteins as a key step for phloem transport. Science 335, 207-1 1 (2012); Zhou, J. etal. Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant J. 82, 632-643 (2015)). SWEET13 accumulated in the phloem as judged by the analysis of GUS reporter fusions (Figure 5b, d). While SWEET14 had substantially lower mRNA levels in leaves compared to SWEET13, the SWEET14 protein still accumulated in the phloem (Figure 5c). Despite evidence for a function in phloem loading, independent CRISPR-Cas9 sweet13 and sweet14 knock-out mutant lines did not show detectable growth or yield defects under greenhouse conditions (Figure 5a, c; Figure 10), nor were obvious differences in performance observed in a single-season field experiment (data not shown). To exclude that the lack of phenotypic differences was due to potential compensatory activity from other SWEETs, expression levels of the other sucrose-transporting SWEETs were analyzed in the mutants. Only the lowly expressed SWEET14, but none of the other Clade III SWEETs, showed substantial increases in mRNA accumulation in the leaf blade of sweet13 knockout lines (Figure 1 1 a-c). To test if up-regulation of SWEET14 could compensate for the loss of SWEET13 and thereby restore apoplasmic phloem loading, additional single sweet14 and sweet13;14 double knockout lines were generated. Double mutants also did not show obvious phenotypic differences relative to the parental Kitaake (Figure 12a). Because the mutant lines showed no significant defects regarding plant growth or yield, EBE-edited lines that impact normal promoter function of SWEET13 and 14 are thus not expected to show a yield penalty (Figures 1 , 12). Further, the data indicate that phloem loading in rice, in contrast to maize and Arabidopsis, is either not critical to plant development or does not entirely depend on SWEET function (Chen, L. Q. et at. Sucrose efflux mediated by SWEET proteins as a key step for phloem transport. Science 335, 207- 11 (2012); Bezrutczyk, M. et at. Impaired phloem loading in zmsweet13a,b,c sucrose transporter triple knock-out mutants in Zea mays. New Phytol. 218, 594-603 (2018)).

Knockout lines can serve as diagnostic tools for testing Xoo strains for specific SWEET requirements. It was observed that an African strain, AX01947, which contains the effector TalC and induces SWEET14, but apparently not SWEET13, was still able to infect the Kitaake mutant edited in the TalC EBE in the SWEET14 promoter. A systematic screen for resistance using the sweet13 and sweet14 single knockout mutants as well as the sweet13;14 double mutant showed that AX01947 lost some infectivity in the sweet14 single knockout lines, but was unable to infect the sweet13;14 double knockout mutant (Figure 12b). These data demonstrated the value of the knockout lines for testing resistance and for identifying possible resistance mechanisms (Figure 1 b, Table 2 and 3). The co-dependence of strain AXQ1947 on both SWEET13 and SWEET14 function are under further investigation.

Table 2: Lesion length for individual, double and triple SWEET^ko lines (Kitaake).

PHL, The Philippines; BOLD indicates resistance; ITALIC indicates moderate resistance.

Table 3. Resistance of sweet13;14 double knockout mutants to Asian and African Xoo strains as determined by lesion length from clipping assays .

PHL, The Philippines; BOLD indicates resistance.

Genome-edited Kitaake tester rice lines (SWEETp^R) resistant to specific Xoo strains

Rice varieties carry differing numbers and types of resistance genes. The only known R gene for BB in the japonica rice variety Kitaake is a cryptic resistance gene similar to the recessive R gene xa25, which is dependent on strains with the major TAL effector PthXo2 (Zhou, J. et at. Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant J. 82, 632-643 (2015)). Thus, Kitaake is a good standard for testing Xoo compatibility with rice. In a parallel study, a set of EBE variants for SWEETH a, 13 and 14 was created by genome editing, and resistance/susceptibility was validated. SWEETp^R genome-edited Kitaake tester lines are available for genotyping Xoo isolates similar to R-gene line panels for race characterization (Ogawa, T., Yamamoto, T., Khush, G. S. & Mew, T.-W. Breeding of near-isogenic lines of rice with single genes for resistance to bacterial blight pathogen ( Xanthomonas campestris pv. oryzae). Jpn. J. Breed. 41 , 523-529 (1991)). For example, the Kitaake line #1 1 a.1 -45 was resistant to strains containing the TAL effectors PthXol and AvrXa7; line #12.2-12 was resistant to strains containing PthXo2B, PthXo3 and AvrXa7 from our collection (Tables 4 and 5).

Table 4: Resistance of Kitaake promoter-edited lines.

* Pathogenicity was scored as resistant reaction (R) with lesion lengths < 3 cm, susceptible reaction (S) with length 15-20 cm, and moderately susceptible reaction (MS) with length lesion >8 and <15 cm.

Table 5. Total number of genome-edited EBE variants in three SWEET genes.

SWEET Pathotracer visualization GIS (geographic information system)-based platforms that incorporate pathogen monitoring and resistance profiles of rice varieties are useful for customized deployment and management of local disease outbreaks (Dossa, G. S., Sparks, A., Cruz, C. V. & Oliva, R. Decision tools for bacterial blight resistance gene deployment in rice-based agricultural ecosystems. Front Plant Sci 6, 305 (2015)). Here, it is proposed to post the phenotypic outputs of the transcriptional reporter lines, knockout tester lines, and near-isogenic lines into the PathoTracer platform (http://webapps.irri.org/pathotracer/index.html). PathoTracer displays the predicted involvement of SWEETH a, SWEET13, and SWEET14 in each region and suggests the most effective promoter variants for breeding or deployment. For example, a phenotypic dataset on the ability of Xoo strains to infect the R-gene near-isogenic IRBB lines (Quibod, I. L. et at. Effector diversification contributes to Xanthomonas oryzae pv. oryzae phenotypic adaptation in a semi-isolated environment. Sci Rep 6, (2016)) was compared to the proportion of endemic strains from an area of the Philippines that may activate SWEET14 (data not shown). Based on this information, 47% of the Xoo population is predicted to be controlled by one or more of the SWEET14 EBE variants.

Genome-edited mega variety rice lines resistant to specific Xoo strains

Mega rice varieties are defined as varieties that are planted on over one million hectares. While the genome-edited BB-resistant SWEETp^R Kitaake lines can be used by breeders, SWEET^R mega variety lines would reduce the effort required for deployment, particularly for SWEET-related resistance, which is recessive. A CRISPR-Cas9-mediated strategy was used to edit five of the six EBE sites in the three SWEET promoters in the widely used indica rice mega variety IR64 and in Ciherang-Sub1 , a new elite line that also carries a gene for flooding tolerance (Toledo, A. M. U. et at. Development of improved Ciherang-Sub1 having tolerance to anaerobic germination conditions. Plant Breed. Biotech. 3, 77-87 (2015); Mackill, D. J. & Khush, G. S. IR64: a high-quality and high-yielding mega variety. Rice 11 , 18 (2018)). Using a combined Cas9-Cpf1 strategy, it was possible to successfully edit all EBEs in Kitaake, MTU101 and the elite line Komboka using the new vector pMUGW5 (Fig. 32, 33, Tables 15-17). A transformation protocol for Komboka for this purpose was established. Komboka (IR 05N 221) is a new elite variety generated by IRRI and released in Tanzania by the National Rice Research Program- KATRIN Research Centre and IRRI-Tanzania (‘Komboka’ =‘liberated’)(Kitilu et al., 2019). Komboka is high yielding (8.6 t ha ¹), semi-aromatic with good grain quality, tolerant to blast and well adapted to upland and lowland areas. Stable mutant lines with alterations either in single or multiple EBEs were generated. Overall, 32 Cas9-free lines were produced, encompassing 35 single mutations in the three genes. Agronomic assessments and pathogenicity trials have validated resistance against single or multiple Xoo strains from our collection (Figure 13; Table 6).

Table 6. Virulence of Xoo strains on promoter variants of mega varieties.

R: resistance, MR: moderate resistance, S: susceptible, *: no data, >: SNP, inserted nucleotides are indicated after the comma, deleted nucleotides are not indicated.

Peptide-derived antisera for detection of specific SWEET protein accumulation

Specific antisera for SWEETH a, 12, 13, 14 and 15 to detect SWEET protein accumulation in heterologous expressed system (Figure 7 and 8). Two specific peptides for each SWEET were used (Table 7).

Table 7. Specific peptide sequence for immunization.

Example 2: Genome editing for broad-spectrum resistance against rice bacterial blight

Material and methods

Plant material, bacterial strains, medium, and growth conditions

Rice varieties used here were Oryzae sativa L. ssp. japonica Kitaake (Zhou, J. et al. Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant J. 82, 632-643 (2015)), and Oryzae sativa L. ssp. indica IR24 (Zhou et al. 2015), IR64 (Mackill, D. J. & Khush, G. S. IR64: a high-quality and high-yielding mega variety. Rice 11 , 18 (2018)) and Ciherang-Sub1 (Toledo, A. M. U. et al. Development of improved Ciherang-Sub1 having tolerance to anaerobic germination conditions. Plant Breed. Biotech. 3, 77-87 (2015)). Rice plants were grown in growth chambers at 30°C for a 12-h light period and at 28°C for a 12-h dark period, with 60-75% relative humidity in the Yang laboratory, or under small-scale field environment in screenhouse (28°C ± 7°C day/23°C ± 4°C night; 80-85% relative humidity) at IRRI. Escherichia coli strains were grown in Luria-Bertani (LB) medium supplemented with appropriate antibiotics at 37°C. Agrobacterium tumefaciens strains were grown at 30°C under the dark. All Xoo strains were grown at 28°C in TSA (10 g/L tryptone, 10 g/L sucrose, 1 g/L glutamic acid). Antibiotics were used at the following concentration if required: 100 pg/mL ampicillin; 10 pg/mL cephalexin; 25 pg/mL chloramphenicol; 25 pg/mL kanamycin; 100 pg/mL spectinomycin; 10 pg/mL tetracyline.

Xoo genome sequencing and assembly

Total genomic DNA of Xoo strains was isolated using MagAttract HMW DNA Mini Kit (QIAGEN). DNA samples were sequenced using a single molecule real-time (SMRT-Pacbio) platform, P4-C6 chemistry. For each strain, at least two SMRT cells were used, generating around 180X coverage per genome. De novo assembly was conducted using the hierarchical assembly pipeline (HGAP) implemented in Canu V1 .5 software (Koren, S. et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 27, 722-736 (2017)). Raw PacBio reads were mapped against the resulting contigs using blasR aligner (https://github.com/PacificBiosciences/blasr), and corrections were conducted with the variant-caller software utilizing the arrow algorithm (https://github.com/PacificBiosciences/GenomicConsensus). Genome annotation and gene prediction were conducted using Prokka annotation pipeline (Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068-2069 (2014)) and the NCBI Prokaryotic Genome Annotation (PGAP) (Haft, D. H. et al. RefSeq: an update on prokaryotic genome annotation and curation. Nucleic Acids Res. 46, D851-D860 (2018)). Genome sequences were deposited in the GenBank under project accession number GenBank CP033170-CP033197. Additional reported and publicly available genomes in GenBank were retrieved and used for comparisons (Table 8). Table 8. List of all sequenced Xanthomonas oryzae pv oryzae (Xoo) strains for analysis. Thirty- one strains were sequenced herein, while thirty-six were previously published.

PHL, The Philippines; BF, Burkina Faso; 1C, Ivory Coast; SK, South Korea.

Phylogenetic and comparative genomic analyses of Xoo strains

Phylogenetic analysis was conducted by identifying pan-genome SNPs in oligo nucleotides of length k=31 and generating parsimony trees based on these SNPs, as implemented in the program kSNP (doi: 10.1093/bioinformatics/btv271 ).

TALe content analysis in Xoo

TALe annotation was conducted using the AnnoTALE software (Grau, J. etal. Computational predictions provide insights into the biology of TAL effector target sites. PLoS Comput Biol 9, (2013)). Additional annotation and comparisons were performed using Artemis genome browser and Artemis Comparative Tool (ACT) (Carver, T. J. et at. ACT: the Artemis Comparison Tool. Bioinformatics 21 , 3422-3423 (2005); Carver, T., Harris, S. R., Berriman, M., Parkhill, J. & McQuillan, J. A. Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics 28, 464-469 (2012)). A neighbor-joining tree based on the alignments of repeat regions of TALes was obtained using DisTAL (Perez-Quintero, A. L. et al. QueTAL: a suite of tools to classify and compare TAL effectors functionally and phylogenetically. Front Plant Sci 6, 545 (2015)). Target prediction for TALes was conducted using Talvez (Perez-Quintero, A. L. et al. An improved method for TAL effectors DNA-binding sites prediction reveals functional convergence in TAL repertoires of Xanthomonas oryzae strains. PLoS One 8, e68464 (2013)), and using as the target sequences in the promoter regions (1 kb upstream from the translation start site) of the rice japonica Nipponbare genome version deposited in Phytozome database V1 1 and of the indica variety IR64 (v. CSHL1 .0) (Schatz, M. C. et at. Whole genome de novo assemblies of three divergent strains of rice, Oryza sativa, document novel gene space of aus and indica. Genome Biol. 15, 506 (2014)).

CRISPR-Cas9 design and constructs

For gene editing in Kitaake, the polycistronic tRNA-gRNA (PTG) system was used to generate multiple sgRNAs with different target sequences by flanking the sgRNAs with a tRNA precursor sequence³⁸. Six intermediate vectors were constructed, pTLN-tgRNA-1 to T6, for 6 individual tRNA-gRNA units. A double-stranded DNA oligonucleotide (dsOligo) for each site was produced by annealing two complementary oligonucleotides (24-25 nt) (data not shown). Six dsOligos were individually inserted into the BsmBI-digested pTLN-tgRNA-1 to T6. The positive clones were confirmed by Sanger sequencing. All six tRNA-gRNA units were transferred into another intermediate vector named pENTR4- U6.1 P-ccdB using the Golden Gate ligation method. The gRNA cassettes were finally mobilized to pBY02-ZmllbiP-OsCas9 by using Gateway LR Clonase (Thermo Fisher Scientific, Waltham, MA) as described (Zhou, H., Liu, B., Weeks, D. P., Spalding, M. H. & Yang, B. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucl Acid Res 42, 10903-10914 (2014)). For gene editing in IR64 and Ciherang-Sub1 , four guide RNA genes, targeting PthXol EBE in SWEET1 1 a, PthXo2 EBE in SWEET13, or TalC EBE or AvrXa7 EBE in SWEET14, were constructed as described (Zhou, H., Liu, B., Weeks, D. P., Spalding, M. H. & Yang, B. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucl Acid Res 42, 10903-10914 (2014)).

Generation of rice mutant lines

Agrobacterium strains containing the respective CRISPR constructs were used for genome editing in Kitaake, IR64 and Ciherang-Sub1 . Individual transformants were selected, propagated and regenerated into whole plants (TO) as described (Hiei, Y. & Komari, T. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc 3, 824-834 (2008); Slamet- Loedin, I. H., Chadha-Mohanty, P. & Torrizo, L. Agrobacterium-mediated transformation: rice transformation. Methods Mol. Biol. 1099, 261-271 (2014)). Leaf tissues collected from individual samples were homogenized in liquid nitrogen, and genomic DNA was isolated following the CTAB method. Initial screening for transformants (TO) was done via PCR of the phosphotransferase transgene (hpt) with primers HptF and HptR. Nuclease surveyor assay using Cell enzyme was employed in TO plants to pre-select lines with possible sequence alterations in the target regions. 20 mono- and di-allelic events for IR64 were selected and the EBE regions sequenced using site-specific PCR amplicons obtained with primer pair 8NKpl-F5 and 8N3-R for SWEETH a, primer pair 12N3-F1 and 12N3-R1 for SWEET13, and primer pair 1 1 N3-F2 and 1 1 N3-R for SWEET14. To detect the CRISPR transgenes, paired primers OsCas9-F and OsCas9-R were used for Cas9, primers g8N3-F and g12N3-R for gRNA genes targeting SWEET1 1 and SWEET13, and primers g1 1 N3-F and g1 1 N3-R for gRNA gene targeting SWEET14. These primer pairs were also used for selection of candidate mutant lines in the advanced generations (T1 to T2). Nineteen gRNA gene-free and Cas9-free IR64 T1 plants were selected and further analyzed. Thirty plants for each of the 19 IR64 T2 lines were phenotyped for resistance to Xoo strains PX0339, PX099, and PX086 and target promoter regions of the candidate lines were sequenced to confirm mutations. Seeds of T2 plants were bulked (from 30 plants per line), phenotyped for Xoo resistance and analyzed for agronomic traits as described below. Seeds from 13 individual T3 plants were advanced to T4 based on three criteria: (1) mutation type, (2) consistent resistance to the three Xoo strains, and (3) seed count. For Ciherang-Sub1 , seven independent transformation events were obtained. Eighteen Cas9-free T1 plants were further analyzed for resistance and amplicon sequencing as described above till T3.

Evaluation for agronomic traits of the rice mutant lines

It was expected that mutant lines would show potential changes in agronomic traits due to disruptive expression of the SWEET genes, off-target mutations across the genome, or somaclonal variation. To assess the performance of the selected mutant lines, four agronomic characters in IR64-IRS1 132 and Ciherang-Sub1 -IRS1 132 lines were assessed under a paddy screenhouse condition in an RBD experimental design with three replications. At maturity, the plant height, panicle length, percent reproductive tillers (number of tillers with panicle/total number of panicles per plant) and percent fertility (number of filled grains/ total number of grains) were measured.

Disease assays

Fully expanded leaves of rice plants (6-8 weeks old) were inoculated using a leaf-tip clipping method. Xoo stocks, preserved in -80°C freezer, were streaked out on TSA plates supplemented with appropriate antibiotics and incubated at 28°C for 2-4 days. The cells were harvested from the plates and resuspended in sterilized distilled water (Oϋboo 0.5, =10⁸ cfu/ml). Scissor blades were immersed in Xoo suspension and used to clip about 2 cm from the leaf tip. The lesion lengths were measured 14 days after inoculation. Lesions were measured from each test plant. Lesion length measurements <5 cm were scored as resistant (R), 6-10 cm as moderately resistant (MR), 1 1 -14 cm as moderately susceptible (MS), and >15cm as susceptible (S). Three replications with five plants per replicate were inoculated per strain.

Results

Diversity of the major TALe genes from Xoo genome sequences

An assessment of the diversity of the major TALes in extant strains of Xoo was obtained by analyzing Xoo genome sequence data and virulence assays on rice lines with SWEET promoter polymorphisms. TALe gene content was derived from complete genome sequences of 67 strains from Asia (Xoo^s) and Africa (Xoo^F), which were either in databases or newly sequenced here (Table 9). Whole-genome, SNP- based parsimony trees clearly separated Xoo^s from Xoo^F genomes, revealing two distinct evolutionary lineages (data not shown). Trees based on alignments of repeat regions of 856 TALes revealed two major clusters (Figure 20). TALes predicted to induce SWEET genes (major TALes) were present in all strains, regardless of origin (data not shown). Indeed, all strains possessed one or more close homologs of the known major TALe gene groups (PthXol , PthXo2, and PthXo3/AvrXa7). PthXo3/AvrXa7 is treated as a single class. Asian strains had approximately equal numbers of PthXo2 (targeting SWEET13) and PthXo3/AvrXa7 (SWEET14) (data not shown). The majority of Asian strains had multiple major TALes, consisting of PthXo2 with either PthXo3 or AvrXa7 (data not shown). None of the strains had both PthXo3 and AvrXa7 (data not shown). In African lineages, the two known major TALes of Xoo are TalC and TalF, which each induce SWEET14 using non-overlapping EBEs (Streubel, J. et al. Five phylogenetically close rice SWEET genes confer TAL effector-mediated susceptibility to Xanthomonas oryzae pv. oryzae. New Phytol. 200, 808-19 (2013); Yu, Y. et al. Colonization of rice leaf blades by an African strain of Xanthomonas oryzae pv. oryzae depends on a new TAL effectorthat induces the rice nodulin-3 Os1 1 N3 gene. Mol Plant Microbe Interact 24, 1 102-13 (201 1)). Half of the analyzed African strains had both TalC and TalF, while none had TalF alone (data not shown). Overall, six EBEs in three SWEET promoters were found (Table 9). The results supported the hypothesis that activation of SWEET genes was a critical evolutionary step that occurred independently in different geographic regions during Xoo adaptation to rice.

Table 9. Known EBEs in SWEET promoters.

Diversity of the major TALes from infection trials

To estimate the prevalence and variation of major TALes in geographically diverse Xoo strains, a collection of 105 Xoo strains was screened against the japonica rice variety Kitaake and its derivative lines carrying mutations in the three SWEET genes normally targeted by TALes. Kitaake contains a naturally incompatible variant allele of SWEET13 that cannot be targeted by PthXo2 (Zhou, J. et al. Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant J. 82, 632-643 (2015)). Strains were screened against lines with alternate alleles of SWEETH a and SWEET14 created using TAL effector nucleases both singly and in combination (Figure 21). Ten strains in the collection were not virulent on Kitaake but were still virulent on IR24 (Figure 22), indicating that either these strains are solely dependent on PthXo2, since the EBEs in the other SWEET gene promoters are the same in Kitaake and IR24, or Kitaake has an R gene that is effective against these strains. These ten strains were removed from further screening. Of the remaining 95, 18 strains were not virulent on line #1 , which is defective in the EBE targeted by PthXol in SWEET1 1 a (Figure 21); 69 strains were not virulent on line #5, which is defective in the EBEs of AvrXa7 and PthXo3 within SWEET14 (Figure 21). These 87 strains were also not virulent on line #52-1 , with mutant alleles of both SWEET1 1 a and SWEET14 (data not shown). Thus, of the 95 strains, 18 have a PthXol -like TALe, and 69 have at least one AvrXa7/PthXo3-like TALe. These phenotypes are in agreement with the available genomic sequences of the TALes (67 of strains). In total, 85 strains from the screen were predicted to rely on major TALes that target known EBE regions of SWEET1 1 a, SWEET13 and SWEET14. Eight of the 105 strains had different virulence abilities and, presumably, different major TALes, since these strains were virulent against plants carrying all three modified SWEET promoters. These eight strains were collected in the Philippines (PX061 , PX0364, PXO404, PX0421 and PX0513), from Korea (JW8901 1 and KX085) and one from Africa (AX01947) (data not shown). The virulence assay could not distinguish strains which individually carry both avrXa7/pthXo3-like and pthXo2-like or pthXol and pthXo2-like TALe genes.

Xoo strains carrying pthXo2-like genes with aberrant repeats and novel predicted targeting activities

Genome sequencing analysis confirmed that the 7 deviant strains with undefined EBE targets (PX061 , PX0364, PXO404, PX0421 , PX0513, JW8901 1 and KX085) encoded PthXo2 proteins with altered RVD configurations (Figure 20). All PthXo2-like effectors contained 22 RVDs. PthXo2C, in the 2 Korean strains, contains 7 different RVDs, while PthXo2B, in the 5 Philippine strains, differs in 8 of its RVDs (Figure 23). In addition to these amino acid differences in the RVDs, PthXo2B and PthXo2C have longer, 36-amino acid long, repeats at RVD 9 and 12 relative to PthXo2 (Figure 23). All 5 Philippine isolates also carry a gene for PthXo3, while the Korean strains have a gene for AvrXa7 (Figure 23). The differences between PthXo2B and PthXo2C and the addition of PthXo3 or AvrXa7 reflect the overall separation of the sub-lineages from the Philippines and Korea (data not shown), indicating co-evolution towards more options to target the SWEET13 locus. Flexible binding of SWEET13 by PthXo2B and PthXo2C, through looping of RVD9 or RVD12 (data not shown) as has been proposed for an aberrant repeat of AvrXa7 (Richter, A. et at. A TAL effector repeat architecture for frameshift binding. Nat Commun 5, 3447 (2014)), could explain the virulence of these strains on Kitaake and derived edited lines. EBE prediction programs indicated a binding site for PthXo2B-12L (with PthXo2B looped at RVD 12) and PthXo2B-9L (with PthoXo2B looped at RVD9) within SWEET 13 in Kitaake and Nipponbare (both japonica varieties). The predicted EBE sequence is TATAAAGCACCACAACTCCCTTC (Figure 24, lines 6 and 7).

Using a PX061 genomic DNA library, the candidate gene for PthXo2B was identified by sequence analysis, cloned, and introduced into strain PX099^AME2, hereafter ME2, which is not pathogenic on any rice line due to the null mutation in the sole major TALe gene pthXol of PX099^A (Yang, B., Sugio, A. & White, F. F. Os8N3 is a host disease-susceptibility gene for bacterial blight of rice. Proc. Natl. Acad. Sci. U.S.A. 103, 10503-8 (2006)). Whereas PthXo2 enables virulence on indica but not japonica varieties, ME2 carrying PthXo2B adds virulence to japonica varieties Kitaake and Nipponbare but loses virulence on indica varieties (Figure 24a, b). Expression analysis indicated that SWEET13 is induced in the susceptible lines and not induced in the incompatible lines (Figure 23b and c).

Stacking edited EBEs in SWEET gene promoters confers broad-spectrum resistance to Xoo

It was tested if stacking multiple mutations in the EBEs of three SWEET promoters known to be targeted by major TALes would provide resistance to most, if not all, Xoo strains in a single rice. Using a CRISPR multiplex editing approach (Figures 25 - 27), a series of mutant lines were generated from Kitaake (Table 10). The EBE mutant lines were first challenged with ME2 transformants, each carrying a gene for either PthXol , PthXo2B, AvrXa7, TalC orTalF. The lines with the respective promoter mutations were resistant to ME2 derivatives carrying the corresponding TALes (Table 1 1). When challenged with eleven field strains, including the seven RVD-variant Xoo strains that can induce SWEET 13, one rice line (#1 1 a.1 .3) was resistant to all (Table 1 1). The line conferred only moderate resistance to the Xoo^F lineage strain AX01947, which, of the known TALes, only contains TalC, suggesting the presence of another effector. A similar observation was obtained upon inoculation of Xoo^F strain CFBP7325, which has both TalF and TalC (data not shown).

Table 10. EBE sequences in the SWEET promoters in Kitaake and Kitaake-derived, CRISPR- induced SWEET promoter mutants. DNA sequences shaded in grey or dark-grey are SWEET EBEs for respective TALes. Letters for PAM for Cas9 and TATA box are in bold upper letters. Bold lower letters are insertion mutations, while dashed lines represent nucleotide deletions. Lines in bold font were subjected to disease assays (Table 11 ).

Table 11. Disease reactions genome edited SWEET promoter lines and the parental Kitaake line caused by different representative Xoo strains.

Disease phenotypes are classified into 4 categories: S, susceptible, lesion length 15-20 cm; MS, moderately susceptible, lesion length >8 cm; MR moderately resistant, lesion length between 4 and 8 cm; R resistant, lesion length <3 cm.

PHL: The Philippines; SK: South Korea; A: Africa.

* heterozygous line.

Genome-edited mega varieties with broad-spectrum resistance to Xoo

Rice mega varieties, or lines cultivated over one million hectares, are semi-dwarf and high-yielding lines that usually perform uniformly across multiple environments (Mackill, D. J. & Khush, G. S. IR64: a high- quality and high-yielding mega variety. Rice 11 , 18 (2018)). If combined SWEET mutations do not affect agronomic traits in a rice mega variety, the same combinations are likely to be beneficial in all derived varieties and, thus, useful for rice breeding programs. Therefore, CRISPR-Cas9 (Figure 28) was used to target SWEET genes in the rice mega varieties IR64 and Ciherang-Sub1 , and key agronomic traits were measured in confined paddy experiments. Prior to trials, the CRISPR-edited SWEET promoters of all mutant lines were sequenced, and lines were selected based on the diversity of mutations in the respective EBEs (Table 12). Overall, 13 IR64-IRS1 132 and 18 Ciherang-Sub1 -IRS1 132 Cas9-free lines, spanning 30 combinations of EBE mutations in SWEETH a, SWEET13, and SWEET14, were tested (Table 13). Agronomic assessments and multivariant analysis of plant height, panicle length, number of reproductive tillers, and fertility rate indicated that most lines performed similar to the wild-type parents (Figure 30). Only line IR64-9 showed phenotypic differences in yield, panicle length, and fertility (reduced by 10% under paddy conditions).

After agronomic tests, a range of different insertions, deletions, and substitutions in SWEETH a (n=3), SWEET13 (n=8) and SWEET14 (n=20) were selected, on the basis that more extensive mutations in the EBEs would be primed to match TALe flexibility and population variants of pathogen. These 31 SWEET variants were in 10 rice lines, which were challenged with tester Xoo strains (Figure 29). Nine of the lines were resistant to all tested Xoo strains (Figure 29). Seven variants of SWEET13, ranging from two-nucleotide insertions (+2) to a seven-nucleotide deletion (-7), all conferred resistance. The line IR64-6 carried only a single nucleotide insertion or a single substitution in SWEETH a, which was insufficient to abrogate PthXol function (Figures 29 and 31).

Table 12. DNA sequence of EBEs in IR64 and Ciherang-sub and their CRISPR-induced SWEET promoter mutants. DNA sequences shaded in gray or light-gray are SWEET EBEs for respective TALes. Bold upper letters indicate the PAM for Cas9 and TATA box. Bold lower letters are insertion mutations, while dashed lines represent nucleotide deletions. Lines in bold font were subjected to disease assays (Table 13).

Table 13. EBE-edited variants in IR64 and Ciherang-Sub1 subjected to pathogenicity tests (also Figure 29). Nucleotide changes in EBEs of promoters.

R: resistant, MR: moderately resistant, S: susceptible, *: no data, >: SNP, inserted nucleotides are indicated after the comma, deleted nucleotides are not indicated.

Genome-edited mega rice varieties have no yield penalty in Confined Field Trials for agronomic performance in the Philippines/Evaluation for agronomic traits of the rice mutant lines

For the field trial 8 advanced IR64-IRS1 132 and 4 Ciherang-Sub1 -IRS1 132 lines were selected. These edited lines were generated through successive selfing and were selected based on the distinct variants in the SWEET promoters (Table 14). The SWEET-edited IR64 and Ciherang-Sub1 lines were sown at the IRRI trial site on April 26, 2019. The site is monitored by both external and internal biosafety committees in compliance with the Philippine biosafety regulations. The trial was performed from April 22 to August 27, 2019 within the F8-F9 sites approved by the Department of Science and Technology (DOST) in the Philippines. An alpha lattice incomplete block design randomization with 3 replicates each was used. Wild type IR64 and Ciherang-Sub1 were used as controls. Preharvest and post-harvest data collected include reproductive tiller number, fertility, single plant yield, and bulk yield (Table 14). Overall, yield parameters (fertility, single plant yield, and bulk yield) of edited lines were not different from the wild type controls. Several lines showed a small increase in single plant yield, which could be due to a lower performance of the wild type control, but this did not translate into bulk yield differences. In summary, the edited lines in both genetic backgrounds IR64 and Ciherang-Sub1 behave similar as the controls and show no obvious defects in the key parameters reported here.

Table 14. Agronomic traits of genome edited IR64-IRS1132 and Ciherang-Sub1-IRS1132 lines

Plant ID Reproductive Fertility Single plant Bulk yield

_ tillers (%) (%) _ yield (gr) _ (tons/ha)

IR64-IRS1 132-005-55-1 -8-B 97.7 54.6 22.1 2.2

IR64-IRS1 132-007-18-1 -2-B 99.9 55.7 24.2* 2.8

IR64-IRS1 132-007-45-1 -3-B 100.5 57.3 24.8* 2.7

IR64-IRS1 132-009-32-1 -2-B 97.7 62.4* 24.4* 2.6

IR64-IRS1 132-134-37-1 -2-B 99.0 67.2* 29.3* 3.6*

IR64-IRS1 132-134-4-1 -2-B 98.6 60.0 22.5 2.5

IR64-IRS1 132-134-53-1 -8-B 98.8 58.6 24.4* 2.5

IR64-IRS1 132-136-3-1 -2-B 99.7 59.1 24.7* 2.4

Wildtype IR64 98.4 54.1 20.2 2.3

Ciherang-Sub1 -IRS1 132- 99.1 53.7 27.8* 3.3

001 -1 1 -7-B

Ciherang-Sub1 -IRS1 132- 99.2 56.1 23.6 3.0

003-23-1 -B Ciherang-Sub1 -IRS1 132- 99.6 57.1 24.6 2.9

004-24-1 -B

Ciherang-Sub1 -IRS1 132- 98.3 56.3 24.7 3.1

006-19-5-B

Wildtype Ciherang-Sub1 98.7 56.4 23.7 3.0

* significantly higher Least Square means compared to the control. Yellow color indicate significant lower LS means compared to the control.

Transformation/backcross protocol for genome editing with 2.0 constructs

The purpose of recurrently backcrossing of the edited rice lines to the parental lines is to a) segregate out the gene editing components, b) select for lines homozygous for the edited alleles, and c) remove unintended genomic changes derived from the gene editing process (e.g., tissue culturing, off-targeting if any). Two rounds of backcrossing with recurrent parental lines are sufficient and performed as described below. Multiple individuals are used to perform backcrossing in parallel. For the backcross, select plants in primary, TO or second generation, T1 that contain the desired edits in the genes of interest as pollen donor (male) and cross with the parental plant (female). Harvest the seeds (BC1/F1) and grow the seed into individual plants (BC1/F2 population). Genotype ~500 individual plants with (PCR-) markers for identification of edits and detect the gene editing reagents (e.g., guide RNA and Cas9 genes to identify individual plants free of gene editing reagents and homozygous for the desired edits. Select the identified individual plants with normal morphology, most vigorous growth, development and reproduction and use the selected individual plants as pollen donor (male) to cross to the parental plants (female). Harvest the seeds (BC2/F1) and grow the seeds into individual plants (BC2/F2 population). Genotype ~300 individual plants with the same markers as above to identify individuals homozygous for the desired edits. Select the homozygous edited plants with normal morphology, most vigorous growth, development and reproduction and propagate the seeds from the selected plants.

Table 15. EBE sequences in the SWEET promoters in Komboka and Komboka-derived CRISPR- induced SWEET promoter mutants. DNA sequences shaded in gray or light-gray are SWEET EBEs for respective TALes. Bold upper letters indicate the PAM for Cas9 and TATA box. Bold lower letters are insertion mutations, while dashed lines represent nucleotide deletions.

Table 16 EBE sequences in the SWEET promoters in Kitaake and Kitaake-derived CRISPR- induced SWEET promoter mutants. DNA sequences shaded in gray or light-gray are SWEET EBEs for respective TALes. Bold upper letters indicate the PAM for Cas9 and TATA box. Bold lower letters are insertion mutations, while dashed lines represent nucleotide deletions.

Table 17 EBE sequences in the SWEET promoters in MTU1010 and MTU1010-derived CRISPR- induced SWEET promoter mutants. DNA sequences shaded in gray or light-gray are SWEET EBEs for respective TALes. Bold upper letters indicate the PAM for Cas9 and TATA box. Bold lower letters are insertion mutations, while dashed lines represent nucleotide deletions and nucleotide exchanges are boxed.

Claims

1. Kit for detecting and implementing rice blight resistance based on variation in SWEET promoters, comprising

(3) tester rice lines genome-edited in the SWEETH a, SWEET13, and/or SWEET14 promoter region and, optionally, any one or more of SWEETU b, SWEET12, and SWEET15 promoter region, for evaluating the efficacy of the respective mutation for resistance.

2. The kit of claim 1 , further comprising

(4) breeding lines genome-edited in the SWEET1 1 a, SWEET13, and/or SWEET14 promoter region and, optionally, any one or more of SWEETU b, SWEET12 and SWEET15 promoter region.

3. The kit of claims 1 or 2, wherein the PCR primers comprise

(2) the complements of (1); or

4. The kit of any one of claims 1 to 3, wherein the promoter reporter lines are translational reporter lines, preferably full gene reporter lines.

5. The kit of claim 4, wherein the promoter reporter lines comprise individual reporter lines for each of SWEETH a, SWEET13, and SWEET14, and, optionally, further comprise individual reporter lines for SWEET 1 1 b, SWEET12, and/or SWEET 15.

6. The kit of claim 4 or 5, wherein the promoter reporter lines comprise one of the promoters or functional fragments thereof selected from the group consisting of the SWEETH a promoter, the SWEET13 promoter, the SWEET14 promoter and, optionally, the SWEETU b promoter, the SWEET12 promoter, and the SWEET15 promoter, and a suitable reporter gene, preferably a b- glucuronidase (GUS) reporter gene.

7. The kit of any one of claims 1 to 6, wherein the rice knock out lines comprise single knock out lines for SWEET11 a, SWEET13, and SWEET14 and, optionally, any one or more further single knock out lines for SWEET 11 b, SWEET12, and SWEET 15.

8. The kit of claim 7, wherein the rice knock out lines further comprise any one or more of the double knock out lines for SWEET 11 a/13, SWEET 13/14 and SWEET 11 a/14 genes.

9. The kit of any one of claims 1 to 8, wherein the rice knock out lines contain frameshift mutations that lead to early termination, preferably in the sequence corresponding to transmembrane domain I of the respective SWEET protein.

10. The kit of any one of claims 1 to 9, wherein the tester rice lines or breeding lines are genome- edited in at least one of the following six EBEs or homologs thereof that have at least 95% sequence identity over the entire length: PthXol (SEQ ID NO:13), PthXo2 (SEQ ID NO:14), PthXo3 (SEQ ID NO:15), TalC (SEQ ID NO:16), AvrXa7 (SEQ ID NO:17) and TalF (SEQ ID NO:18).

11. The kit of any one of claims 1 to 10, wherein the rice knock out line, the tester rice line, and/or the rice promoter reporter line is based on the Kitaake rice line or an indica rice line, and/or, wherein the breeding line is based on an indica rice lines.

12. The kit of any one of claims 1 to 11 , further comprising reagents for (q)PCR, preferably for (q)RT-PCR, of SWEET 11 a, 11 b, 12, 13, 14, and/or 15 mRNA.

13. The kit of any one of claims 1 to 12, further comprising a set of antisera against SWEET 11 a, 11 b, 12, 13, 14, and/or 15 to detect protein accumulation, preferably by protein gel blot analyses or ELISA.

14. Method for detecting and implementing rice blight resistance based on variation in SWEET promoters, comprising the steps of

(a) providing a rice blight causing bacterial pathogen;

(b) inoculating a rice line with the rice blight causing bacterial pathogen;

(c) identifying the induced SWEET glucose transporter by determining the mRNA accumulation for the SWEET11 a, SWEET 11 b, SWEET12, SWEET13, SWEET14, and/or SWEET 15 genes, preferably for at least the SWEET 11 a, SWEET 13, and SWEET 14 genes, in the inoculated rice line by RT-PCR and/or qPCR, and/or by determining the expression of a reporter gene in rice promoter reporter lines for SWEET 11 a, SWEET 11 b, SWEET12, SWEET 13, SWEET14, and/or SWEET15, preferably for at least SWEET11 a, SWEET 13 and SWEET14, inoculated with the rice blight causing bacterial pathogen; (e) verifying dependence of the pathogen on the targeted SWEET gene(s) by inoculating rice knock out lines for the respective SWEET gene(s), wherein resistance of the respective knock out line confirms the dependence on the targeted SWEET gene(s);

15. The method of claim 14, wherein the method uses the kit of any one of claims 1 to 13.

16. The method of claim 14 or 15, wherein the method further includes the step of identifying the effectors produced by the rice blight causing bacterial pathogen and predicting the targeted SWEET promoter regions by comparison with known effectors.

17. A genome-edited rice plant, comprising at least one genome-edited promoter sequence of the SWEETH a, SWEET13 or SWEET14 gene, preferably selected from the group of sequences as set forth in SEQ ID Nos. 41-53, 55-69, 71-96, 98-109, or 1 10-157.

18. The genome-edited rice plant of claim 17, wherein the plant comprises at least two genome- edited promoter sequences of the SWEETH a, SWEET13 or SWEET14 gene, preferably is genome-edited in at least two different EBEs, preferably at least three different EBEs, more preferably at least four different EBEs, even more preferably at least 5 or all 6 EBEs.

19. Method of generating a broad spectrum rice blight resistant rice plant, the method comprising down-regulating the pathogen-induced expression of at least two of the SWEET 11 a, 13 and 14 genes, preferably by genome-editing the promoter regions of said SWEET genes.

20. The method of claim 19, wherein the method further comprises down-regulating the pathogen- induced expression of any one or more of the SWEET11 b, SWEET12 and SWEET15 genes.