CN116463317B - Application of StGLIP gene in regulation and control of phytophthora resistance - Google Patents

Application of StGLIP gene in regulation and control of phytophthora resistance Download PDF

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CN116463317B
CN116463317B CN202310698884.1A CN202310698884A CN116463317B CN 116463317 B CN116463317 B CN 116463317B CN 202310698884 A CN202310698884 A CN 202310698884A CN 116463317 B CN116463317 B CN 116463317B
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gene
stglip
resistance
phytophthora
amino acid
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CN116463317A (en
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杜羽
陈小康
冯雨露
冯佳淑
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Shenzhen Research Institute Of Northwest University Of Agriculture And Forestry Science And Technology
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Abstract

The invention belongs to the field of biotechnology, and relates toStGLIPApplication of gene in regulating phytophthora resistance, in particular to application of gene in regulating phytophthora resistanceStGLIPThe application of the gene and the homologous gene thereof in regulating and controlling phytophthora resistance.StGLIPThe nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The saidStGDSLThe gene and the homologous gene thereof can inhibit the infection of late blight bacteria to plants, further,StGLIPthe gene can positively regulate the basic defense PTI response of plants. The invention proves thatStGLIPThe gene can be used as a positive control factor for improving phytophthora resistance, and can be used for cultivating plant disease-resistant varieties and improving the characters.

Description

StGLIPApplication of gene in regulation and control of phytophthora resistance
Technical Field
The invention belongs to the field of biotechnology, and relates toStGLIPApplication of gene in regulating phytophthora resistance, in particular to application of gene in regulating phytophthora resistanceStGLIPThe application of the gene and the homologous gene thereof in regulating and controlling phytophthora resistance.
Background
Oomycetes (oomycetes) are a unique class of filamentous eukaryotic microorganisms that exhibit a high degree of host specialization when infesting different species of plants, including many destructive plant pathogens, severely jeopardizing the production of food crops. Late blight is an important disease affecting potato production, severely affecting sustainable potato production. The oospore can survive in soil and plant residues for many years, is a main form of overwintering of pathogenic bacteria, can be used as a primary infection source to continuously germinate and infect potatoes in the next year sowing season, and can accelerate the group variation of late blight due to oospore germination, so that the prevention and treatment difficulty of the late blight is greatly increased. At present, the prevention and control of late blight is highly dependent on chemical pesticides, but the problems of the generation of pathogen resistance, the co-evolution of plants and pathogenic microorganisms, limited effective disease-resistant resources, high disease-resistant variety cultivation difficulty and the like are accompanied, and the problem of disease resistance loss of the late blight caused by frequent virus variation are highlighted, so that the method has important significance in excavating disease-resistant genes of potatoes from a molecular level and analyzing the regulation and control mechanisms of the disease-resistant genes, and further cultivating broad-spectrum disease-resistant varieties.
The excavation and identification of disease-resistant genes are the core of breeding disease-resistant varieties, and in the interaction process of plants and late blight bacteria, plant hosts adopt a multi-layer defense system to resist pathogen attack, such as physical barriers of cell walls, epidermis horny layers and the like. More importantly, plants have evolved two innate immune systems, pathogen-associated molecular patterns (PAMP) triggered immunity (PAMPs triggered immunity, PTI) and Effector-triggered immunity (ETI) respectively. The different types of PAMPs are perceived by plant pattern recognition receptors (Pattern recognition receptor, PRR) triggering PTI, which constitutes the basic immunity of plant defense. Furthermore, when effector proteins secreted by pathogens are recognized by host resistance (Resistance protein, R) proteins, more defensive ETIs are specifically induced, often resulting in plant priming allergic reactions (Hypersensitive response, HR).
Lipids, as important regulators of plant defense, and their derivatives are considered as signaling molecules that can regulate plant immunity. Lipids have an important role in inducing systemic acquired resistance, lipid metabolism being mainly catalyzed by lipases. GDSL lipases are a subclass of lipolytic enzymes characterized by GDSL motifs, with a relatively broad substrate range, further classified as SGNH hydrolases due to their highly conserved Ser-Gly-Asn-His residues. Although extensive research has been conducted in microorganisms, little is known about plant GDSL lipases, which have been identified in Arabidopsis, rice, canola, etc., but have not been clearly reported in Solanaceae crops such as potato. Meanwhile, the immune positive regulatory factor of the plant is identified at present, but the number of disease-resistant genes aiming at phytophthora infestans is not large, and the specific regulatory mechanism is not clear, so that the application of the immune positive regulatory factor in disease-resistant breeding is still very few.
Disclosure of Invention
At present, the immune positive regulatory factors of plants are identified, but the number of disease-resistant genes aiming at phytophthora infestans is not large, and the specific regulatory mechanism is not clear, so that the immune positive regulatory factors are rarely applied to disease-resistant breeding. The invention aims to provide a disease-resistant gene which can be applied to phytophthora infestans, is used for breeding phytophthora infestans varieties and provides a new idea for breeding disease-resistant varieties.
To solve the problems, the invention providesStGLIPThe use of genes to regulate phytophthora resistance meets this need in the art.
In one aspect, the invention relates toStGLIPUse of a gene for the regulation of phytophthora resistance, said gene comprising a gene sequence that is selected from the group consisting of seq id noStGLIPThe gene positively regulates phytophthora resistance, and the nucleotide sequence of the coding region is shown in SEQ ID NO:1 (Sol genomics network: sotub03g035790.1.1).
Further, the invention providesStGLIPUse of a gene for modulating phytophthora resistance, said gene comprisingStGLIPThe amino acid sequence of the gene coding protein is shown as SEQ ID NO:2 (Sol genomics network: sotub03g035790.1.1).
Further, the invention providesStGLIPGene overexpression in application of regulating phytophthora resistanceStGLIPThe gene improves phytophthora resistance.
Further, the invention providesStGLIPUse of a gene for modulating phytophthora resistance, over-expressing said gene in Nicotiana benthamianaStGLIPThe gene positively regulates the resistance of the Benshi tobacco to late blight bacteria.
In another aspect, the invention relates to a method of positively controlling phytophthora resistanceStGLIPGenes of the order ofStGLIPThe nucleotide sequence of the coding region of the gene is shown as SEQ ID NO:1, the amino acid sequence of the coded protein is shown as SEQ ID NO: 2.
In another aspect, the present invention relates toStGLIPThe application of the homologous gene of the gene in positive regulation of phytophthora resistance.
Further, the invention providesStGLIPHomologous genes of the genes are used for positively regulating and controlling phytophthora resistanceStGLIPThe gene has 2 homologous genes in Nicotiana benthamiana, and the amino acid sequences of the encoded proteins are respectively shown in SEQ ID NO:3 (Niben101Scf00067g01010.1) And SEQ ID NO:4 (Niben101Scf05124g00003.1) As shown.
Further, the invention providesStGLIPHomologous genes of the genes silencing Phytophthora plantarum resistance in useStGLIPHomologous genes of the genes in the Nicotiana benthamiana, and negative regulation of the resistance of the Nicotiana benthamiana to the late blight bacteria.
Further, the invention providesStGLIPHomologous genes of the genes are used for positively regulating and controlling phytophthora resistanceStGLIPHomologous genes of the genes in potatoes, and amino acid sequences of the encoded proteins are shown in SEQ ID NO:5 (Sol genomics network:Sotub03g035800.1.1) As shown.
Further, the invention providesStGLIPHomologous genes of the genes are used for positively regulating and controlling phytophthora resistanceStGLIPThe gene has 2 homologous genes in tomato, and the amino acid sequences of the coded proteins are respectively shown in SEQ ID NO:6 (Sol genomics network: solyc03g121180.4.1) and SEQ ID NO:7 (Sol genomics network: solyc03g121170.3.1).
Through the technical scheme and the combination of the embodiments, the technical scheme provided by the invention has at least the following beneficial effects or advantages:
positive regulatory factor of the inventionStGLIPThe gene is cloned as the gene of the positive regulation plant phytophthora resistance of the solanaceae crops for the first time, and is transiently over-expressed on the Nicotiana benthamiana through an agrobacterium-mediated transient expression systemStGLIPGene, proveStGLIPGene inhibition of infection of plants by late blight bacteria, obtaining plant material with silenced tobacco homologous genes by virus-mediated gene silencing technique (Virus induced gene silencing, VIGS), andthe material proves that the resistance of plants to late blight bacteria is inhibited; burst test with active oxygen, proveStGLIPPositive regulation of the plant basic defense response PTI response demonstratedStGLIPThe application of the gene as a positive regulatory factor in improving phytophthora resistance can be used for breeding phytophthora resistant varieties.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic view ofStGLIPGel electrophoresis gel diagram of gene. StGLIP-1 and StGLIP-2 are target gene fragments amplified from potato cDNA respectively, and Marker is DNA molecular weight standard Marker III, and the size is 200bp-5000bp.
FIG. 2 is a schematic diagram showing the result of StGLIP positive regulation plant immunization. A-C, SP-GFP in FIG. 2 is a negative control; D-F, TRV-GUS in FIG. 2 is a negative control for the silenced plants. FIG. 2A is a photograph showing representative photographs taken under a white light background after 5 days of inoculation of plant leaves with phytophthora infestans after transient expression of SP-GFP and StGLIP-GFP, respectively, in left and right regions of Nicotiana benthamiana; FIG. 2B is a photograph showing representative photographs taken under a blue light background after 5 days of inoculation of plant leaves with phytophthora infestans after transient expression of SP-GFP and StGLIP-GFP, respectively, in left and right regions of Nicotiana benthamiana; c in FIG. 2 is a histogram of the lesion area statistics of A and B; d in FIG. 2 is a representative photograph taken of TRV-GUS plant leaves against a blue light background 5 days after inoculation with Phytophthora infestans; e in FIG. 2 is a representative photograph taken of TRV-NbGLIP plant leaves in a blue light background 5 days after inoculation with Phytophthora infestans; f in fig. 2 is a chart of the spot area statistics of D and E. Wherein, stGLIP-GFP is marked as an experimental group, namely, the region containing StGLIP-GFP agrobacterium is injected into Nicotiana benthamiana;Pinfestins is Phytophthora infestans strain Pi14-3-GFP; TRV-NbGLIPFor experimental group plants, i.e. in Benshi tobaccoPlants of two nicotiana benthamiana homologous genes corresponding to StGLIP were silenced in grasses.
FIG. 3 is a graphical representation of the results of comparing plant growth phenotype after homologous gene silencing of tobacco according to the present invention with wild type. FIG. 3A is a phenotype photograph showing that TRV-GUS plants and TRV-NbGLIP plants and TRV-SGT1 plants grow for about 3 weeks; b in FIG. 3 is a Benshi tobacco homologous geneNiben101Scf05124g00003.1Is a silencing efficacy detection graph of (1); c in FIG. 3 is the Orthosiphon aristatus homologous geneNiben101Scf00067g01010.1Is a silencing efficacy test of (2). TRV-GUS and TRV-SGT1 are used as controls; TRV-GUSFor silencing the beta-glucosidase gene (beta-glucuronidase) in nicotiana benthamiana is a stable negative control plant in a tobacco virus-induced gene silencing system; TRV-NbGLIPSilencing two plants of the Nicotiana benthamiana homologous genes corresponding to StGLIP in Nicotiana benthamiana; SGT1 (suppressor of the G sole of SKP 1) is an essential component of a variety of plant disease resistance gene-mediated disease resistance signaling pathways; TRV-SGT1 is used in silencing tobacco in Nicotiana benthamianaSGT1And (5) gene plants.
FIG. 4 is a schematic representation of the flg 22-induced reactive oxygen species enhancement by a transient expression StGLIP plant on Nicotiana benthamiana of the present invention. SP-GFP is a tobacco leaf in which the control, namely, the signal peptide C-terminal of StGLIP protein is fused with GFP protein and transiently expressed in Nicotiana benthamiana; stGLIP-GFP is a tobacco leaf in which Agrobacterium containing StGLIP-GFP is transiently expressed in Nicotiana benthamiana.
FIG. 5 is a potato of the present inventionStGLIPThe amino acid sequence of the gene and 2 homologous genes of Nicotiana benthamiana are shown in the schematic diagram. The black background is the amino acid with the same sequence, the dark gray background is the amino acid sequence with the homology of more than or equal to 75 percent but less than 100 percent, the light gray background is the amino acid sequence with the homology of more than or equal to 50 percent but less than 75 percent, and the white background is the amino acid sequence with the homology of less than 50 percent; the same amino acid sequences are marked with lowercase letters in the figures.
FIG. 6 is a schematic diagram showing the amino acid sequence alignment of StGLIP and 1 homologous gene of potato and 2 homologous genes of tomato. The black background is the amino acid with the same sequence, the dark gray background is the amino acid sequence with the homology of more than or equal to 75 percent but less than 100 percent, the light gray background is the amino acid sequence with the homology of more than or equal to 50 percent but less than 75 percent, and the white background is the amino acid sequence with the homology of less than 50 percent; the same amino acid sequences are marked with lowercase letters in the figures.
Detailed Description
The following describes the embodiments of the present invention with reference to examples, but the present invention is not limited to the examples.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products available on the market without the manufacturer's attention.
Example 1
The present embodiment providesStGLIPCloning of the Gene.
Step one: obtaining plant material, namely potato number 9 (which can be obtained through a public channel), and extracting RNA from leaves of the plant material;
step two: extracting RNA, extracting potato leaf RNA by using an RNA extraction kit (Tiangen Biochemical technology Beijing Co., ltd., product number DP 419), identifying the integrity of the RNA by agarose gel electrophoresis, and then measuring the purity and concentration of the RNA on a spectrophotometer;
step three: cloning genes, namely obtaining cDNA of a nine-leaf blade of the potato green potato by using a reverse transcription kit (Accurate Biotechnology, product AG 11728), designing an upstream primer and a downstream primer according to the full-length coding sequence of SEQ ID NO.1, and amplifying by using the cDNA as a template; PCR product was subjected toXhoI andHinthe vector StGLIP-GFP-pART27 is constructed after dIII digestion, ligation and bacterial liquid PCR verification, and the correct plasmid is used for subsequent experiments by sequencing and comparing with the sequence of SEQ ID NO. 1.
The primer sequences used were as follows:
StGLIPXhoIF:ccgctcgagATGAGTATCCACAGACA GCAAA
StGLIPHindIIIR:cccaagcttCAAGGCATCAACTTAATTGGA。
StGLIPgel electrophoresis gel diagram of the gene is shown in figure 1. The results show that a single target appears at the position of 800-1200 bp of agarose gelStrip, proof ofStGLIPGenes were successfully amplified from the potato genome.
Example 2
The present example demonstrates genes using Agrobacterium-mediated transient expression in combination with in vitro leaf inoculationStGLIPEnhancing the resistance of plants to late blight bacteria.
In this example, the expression level of StGLIP-GFP in plants was increased by transiently expressing StGLIP-GFP in tobacco using Agrobacterium-mediated transient expression. The specific implementation is as follows.
The plasmid StGLIP-GFP-pART27 is transformed into agrobacterium strain C58C1, a small amount of StGLIP-GFP glycerinum and a control SP-GFP glycerinum are dipped on a solid LB culture medium (corresponding to antibiotics and glycerinum) by a sterilizing gun head in an ultra-clean workbench, the solid LB culture medium is reversely placed at 28 ℃ for culturing 48 h, bacterial plaques are picked up in a 5 mL liquid culture medium, shake culturing is carried out at 28 ℃ for 10-16h at 200 rpm, after the bacterial plaques are taken out, the bacterial plaques are collected by centrifugation at 3000xg for 5min at room temperature, the prepared MES heavy suspension (200 mM acetosyringone is added for dilution at 1:1000) is added for gently heavy suspension bacterial colony precipitation, 1-3 h is placed at room temperature, and bacterial liquid OD is measured 600 And (3) diluting and preparing a target bacterial liquid with OD=0.3, selecting leaf blades in the middle of the Nicotiana benthamiana growing for about 5 weeks, injecting the target bacterial liquid into the back of the leaf surface by using a 1 mL injector, and performing subsequent experiments after 24 hours of expression.
Then, taking leaf blades of Benshi tobacco after 24 hours of expression of StGLIP-GFP, washing off floating and sinking impurities on the surface of the leaf, sucking excessive water by filter paper, wrapping leaf stalks by wet degreased cotton, placing the leaf stalks in a white plastic tray paved with wet filter paper, inoculating about 1000 zoospores of Phytophthora infestans on the back of the leaf blades, covering the mouth of the plastic tray by a preservative film, placing the leaf stalks in a 16 ℃ incubator for photographing in a dark place of about 5-6 d, and measuring the disease spots. Photographing the blades subjected to different treatments, measuring the diameter of the disease spots of the blades by using a vernier caliper, and analyzing the data. The results show that the area of the disease spots of the over-expressed StGLIP-GFP is significantly smaller than that of the control SP-GFP, and the over-expressed StGLIP-GFP in plants is proved to significantly enhance the resistance of the plants to late blight bacteria by (note: the disease spots are circled by a white dotted line frame)
Example 3
In this example, the expression of the target gene was reduced by using virus-induced gene silencing technique, and the gene was verifiedStGLIPThe resistance of the plant to late blight bacteria is reduced after silencing.
According to FIGS. 1, 2 and 5, the present embodiment uses virus-induced gene silencing technique to reduce the expression of target genes, which is implemented as follows.
Step one: the silencing vector TRV2-NbGLIP was transformed into C58C1 Agrobacterium for competence and the successfully transformed clones were picked up in liquid LB medium for 24-36 hours.
Step two: using a constant temperature centrifuge, collecting thallus at 3000xg for 5min, re-suspending thallus with MES solution containing acetosyringone, diluting part of thallus, and detecting OD with spectrophotometer 600 Is a concentration of (3).
Step three: and (3) regulating the bacterial liquid to a proper concentration by using an MES buffer solution, and mixing and injecting the bacterial liquid with TRV1 bacterial liquid in a ratio of 1:1.
Step four: selecting 4-leaf stage Nicotiana benthamiana seedlings, injecting 3 rd leaf leaves, and selecting leaves with the upper parts completely flattened after 2-3 weeks for inoculation experiments. TRV-GUS plants were used as controls.
In FIG. 2, (A-C) is StGLIP over-expression promoting plant resistance to late blight bacteria; (D-F) to silence the homologous genes of the Nicotiana benthamiana to promote the infection of the plants by the late blight bacteria. The results show that: against a controlTRV-GUSIn contrast to the plants which were used for the cultivation,TRV-NbGLIPthe lesions of (1) are significantly increased in area (note: lesions are circled with white dashed boxes), demonstrating silencing in plantsGLIPThe gene significantly promotes late blight bacteria to infect plants.
Example 4
The present example provides potatoesStGLIPSequence information and homology analysis of genes.
According to the embodiment shown in FIGS. 5 and 6, the potato is providedStGLIPAnalysis of sequence information and homology of genes, wherein potatoStGLIPThe full-length open reading frame sequence of the gene is 1080bp, and the detailed sequence is Sol genomics network: sotub03g035790.1.1, amino acid sequence of 359 amino acids total, signalP5.0 (https:// services. Healthcare. Dtu. Dk/services/SignalP-5.0)V.) the predicted signal peptide consists of the N-terminal 27 amino acids, see Sol genomics network for detailed sequences: sotub03g035790.1.1.
The method comprises the following specific steps: homology search of the amino acid sequence of potato StGLIP was performed by BLAST program, and as a result, it was found to be homologous to 2 genes in Nicotiana benthamianaNiben101Scf00067g01010.1AndNiben101Scf05124g00003.1homology is greater than 80%, and similarity of amino acid sequences is greater than 70%; at the same time 1 is found in the search of genome in potatoStGLIP2 homologous genes in tomato sequencing genome, the similarity of the amino acid sequences of which is above 70%, deducing that 5 homologous genes have the same sequence as the homologous genesStGLIPThe same function;
wherein: potatoStGLIPThe amino acid sequences of the genes and 2 homologous genes in Nicotiana benthamiana are shown in FIG. 5. The figure shows that two homologous genes in StGLIP and Nicotiana benthamiana have highly conserved amino acid sequences at the C end, including sequences with consistent black background, and have the largest ratio, and about 200 amino acid sequences are consistent, so that the high homology of the two homologous genes in StGLIP and Nicotiana benthamiana is proved.
PotatoStGLIPThe results of the alignment of the amino acid sequence of the gene-encoded protein with the amino acid sequence of 1 homologous gene in potato are shown in FIG. 6. The figure shows that 1 homologous gene in StGLIP and potato has highly conserved amino acid sequences, including sequences with consistent black background, the duty ratio is maximum, and about 300 amino acid sequences are consistent, which proves that the 1 homologous gene in StGLIP and potato has high homology and similar functions.
PotatoStGLIPThe amino acid sequence of the gene-encoded protein was aligned with the amino acid sequences of 2 homologous genes in the tomato sequencing genome as shown in FIG. 6. The figure shows that the two homologous genes of StGLIP and tomato have highly conserved amino acid sequences, including sequences with consistent black background, and the ratio is maximum, so that the high homology of the 2 homologous genes in StGLIP and tomato is proved.
Example 5
The present example provides overexpressionStGLIPGene promotion of plant ROSBurst test.
By means of an agrobacterium transient expression system, after StGLIP-GFP and a control SP-GFP are transiently expressed in Nicotiana benthamiana for 48 hours, cutting round leaves of Nicotiana benthamiana with the diameter of 5mm by using a puncher, and placing the round leaves in ultrapure water in a dark place for 8-12 h to heal the wounds of the leaves; subsequently preparing a reaction substrate, namely luminol and horseradish peroxidase (the adding ratio is 1:1000), adding the reaction substrate to a 96-well ELISA plate, and picking up the same number of small discs in each treatment; rapidly adding diluted bacterial flagellin flg22 (final concentration is 10 mu M), and rapidly placing into a multifunctional enzyme-labeled instrument; under the chemiluminescent condition, reading the values at intervals of 1 min, reacting for 30-50 min, and drawing an active oxygen burst curve after the reaction is finished.
Positive regulatory factor of the inventionStGLIPThe gene is cloned as the gene of the positive regulation plant phytophthora resistance of the solanaceae crops for the first time, and is transiently over-expressed on the Nicotiana benthamiana through an agrobacterium-mediated transient expression systemStGLIPGene, proveStGLIPThe gene promotes the resistance of plants to late blight bacteria, plant materials for silencing the tobacco homologous genes are obtained through a virus-mediated gene silencing technique (VIGS), and the materials are proved to inhibit the resistance to late blight bacteria; through the active oxygen burst test, it is proved thatStGLIPPositive regulation of the plant basal defense PTI response demonstratedStGLIPThe application of the gene as a positive regulatory factor in improving phytophthora resistance can be used for breeding phytophthora resistant varieties.
The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.

Claims (4)

1.StGLIPUse of a gene for the regulation of phytophthora resistance, characterized in that the gene comprisesStGLIPThe gene positively regulates phytophthora resistance, and the nucleotide sequence of the coding region is shown in SEQ ID NO:1, shown in the figureThe phytophthora is phytophthora infestans.
2. The use according to claim 1, wherein theStGLIPThe amino acid sequence of the gene coding protein is shown as SEQ ID NO: 2.
3. The use according to claim 1, wherein the expression of the expression is over-expressedStGLIPThe gene improves the resistance of plants to phytophthora infestans.
4. Use according to claim 3, characterized in that the overexpression of the said expression in nicotiana benthamianaStGLIPThe gene positively regulates the resistance of the Benshi tobacco to late blight bacteria.
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