CN108410879B - Application of rice light-harvesting pigment chlorophyll a/b binding protein Lhcb5 in rice blast germ resistance - Google Patents

Abstract

The invention discloses application of rice light-harvesting pigment chlorophyll a/b binding protein Lhcb5 in rice blast resistance. The invention discovers that Lhcb5 induces the burst of host active oxygen and induces the expression of disease-resistant related genes through phosphorylation for the first time, thereby regulating and controlling the resistance of rice to rice blast germs. The invention also relates to knockout of the LHCB5 gene in rice and acquisition of an over-expression plant, and the over-expression plant is found to have broad-spectrum resistance to different field rice blast germs.

Description

Application of rice light-harvesting pigment chlorophyll a/b binding protein Lhcb5 in rice blast germ resistance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a rice light-harvesting chlorophyll a/b binding protein Lhcb5 in rice blast resistance.

Background

Rice, which is the most widely and most important food crop planted worldwide, supplies more than half of the world population, and the demand and safety of rice production become more and more important with the rapid increase of the global population. The rice blast caused by the rice blast fungus (Magnaporthe oryzae) is the most important destructive fungal disease widely occurring in China and even in the rice field all over the world, and seriously threatens the global grain production safety. The rice blast causes 30 hundred million kilograms of grain loss in China every year, and is one of the most important diseases in each main rice production area in China. At present, the prevention and treatment of the disease mainly comprises breeding of disease-resistant varieties and chemical prevention and treatment, the chemical prevention and treatment usually has high cost, the effect is poor due to continuous generation of drug resistance of pathogenic bacteria, and the environment is polluted. The genetic diversity and toxicity of the rice blast germs formed in the evolution process are easy to change, so that the disease resistance of the disease-resistant variety is lost after the disease-resistant variety is popularized for 3 to 5 years, and the variety loses the utilization value. Therefore, the molecular mechanism of interaction between rice and rice blast germs is deeply known, which is not only beneficial to developing and applying broad-spectrum effective rice blast germ-resistant control strategies, but also provides theoretical basis for excavating new gene resources for breeding rice blast germs, and has important application value for the sustainable production of rice in the future.

In the long-term interaction selection and co-evolution process of the plant and the pathogenic microorganism, a complex and accurate military competition of attack, defense, re-attack and re-defense is continuously developed between the plant and the pathogenic microorganism. Recent studies have shown that there is an Innate immune system (animal immunity system) in plants similar to animals, which is composed of Pathogen-associated Molecular Patterns (PAMPs) and Effector molecules that induce immune responses at two levels, namely, PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity), respectively. The basic disease resistance (PTI) of plants to pathogenic bacteria is generated by recognizing conserved mode molecules (PAMPs) of the pathogenic bacteria through receptors on cell membranes, has the characteristics of stability, persistence and broad spectrum, and has important theoretical and practical values for explaining the mechanism of the PAMPs to improve the persistent disease resistance of crops. The effector is a key weapon for pathogenic bacteria to attack plants, a large number of effectors can be secreted into plant cells to interfere the disease-resistant reaction of the plants when rice blast germs infect rice, the disease-sensitive genes of the plants can be found by explaining the action targets of the effectors in the plant cells, the effector has important significance for understanding the pathogenic mechanism of the pathogenic bacteria, and the effector also has important value for constructing the disease-resistant plants by modifying the disease-sensitive genes by methods such as gene editing and the like.

It has long been found in studies of pathogen-host plant interaction that effector molecule-induced immune responses (ETI) are more rapid and intense than PTI, while the ETI process is also accompanied by the accumulation of large amounts of Reactive Oxygen Species (ROS) and allergic reactions (HR) in plant cells. There are two different excretion mechanisms for effector proteins secreted when rice blast fungi infect rice to enter plants: one is cytoplasm effector molecule which enters into rice cell by BIC (Biotrophic interface complex) special structure secretion, and the other is EIHM (extra-innovative-hyphenal membrane) structure secretion between infected hyphal cell wall and host envelope and retention in host cell exoplasmic effector molecule [1,2 ]. Either cytoplasmic or apoplastic effector molecules interact with the corresponding target genes and interfere with the rice immune response. Early studies found that the currently well-studied non-toxic effector molecule is Avr-Piz-t, which targets the host ubiquitin protease system and thus inhibits PAMP-triggered defense responses [3-5 ]. The effector molecule Slp1 participates in the pathogenic process of rice blast fungus and inhibits the host chitin-triggered defense reaction [6 ]. In addition, the avirulent effector molecule, Avr-Pi-ta, is recognized by the rice disease resistance (R) gene, Pi-ta, triggering a strong defense response [7 ]. Other nontoxic effector molecule proteins (ACE1, Avr1-CO39, AvrPiz-t, Avr-Pia, Avr-Pik/km/kp, Avr-Pii, Avr-Pib and Avr-Pi9) are also specifically recognized by corresponding disease-resistant genes in rice [8-12], thereby regulating the disease resistance of rice. However, the molecular mechanism of action of these effector molecules in rice interaction with pathogenic bacteria is not known. Therefore, the identification and cloning of the target gene of the effector molecule have important guiding value for deeply analyzing the rice disease-resistant mechanism and excavating rice disease-resistant gene resources.

During plant-pathogen interaction, plants are more dependent on photosynthetic responses, since photosynthesis is a place of biosynthesis of many defense molecule precursors [13,14 ]. Precursor for the synthesis of salicylic acid chorismate and isochorismate are both synthesized and catalyzed in chloroplasts [15], and salicylic acid plays an important role in plant defense reactions. Cmu1 interact with chorismate mutase ZmCM2 of maize species to promote the efflux of chorismate from stroma to cytoplasm, decrease chorismate in chloroplast, and result in decreased SA synthesis and reduced maize resistance [16,17 ]. Jasmonic Acid (JA) is a lipid-derived hormone whose synthesis starts in chloroplasts but is completed with peroxisomes. The biosynthesis of JA starts with the release of alpha-linolenic acid on the plastid membrane, followed by the catalysis of successive matrix-localized Lipoxygenases (LOX), Allene Oxidases (AOS) and allene cyclases (AOC), forming the precursor molecule cis-OPDA (cis 12-oxo-plant diene). cis-OPDA is subsequently transported to the peroxisome for reduction and beta-oxidation to form the final product, (+) -iso-JA. After (+) -iso-JA is released into the cytoplasm, it binds isoleucine to form the biologically active hormone JA-Ile, which in turn controls host resistance [18 ].

The related protein PsbQ in photosystem II is also induced to trigger cell necrosis, so that the infection of P.syringae is inhibited; p. syringae secreted HopN1 has cysteine protease activity and can regulate host resistance by accounting for PsbQ [19 ]. The chlorophyll a/b binding protein Lhcb5 belongs to the member of LHC protein family, and LHCII protein needs to be phosphorylated to exert the electron transfer requirement and PSII and PSI state conversion, which plays an important role in the electron transfer process [20-22 ]. However, no report is made on how the protein of the family regulates resistance of rice to rice blast fungi. Therefore, the discovery that the disease resistance of the rice is regulated by the Lhcb5 protein provides an important theoretical basis for building the disease resistance relation between photosystem proteins and hosts of the rice, is expected to provide high-quality gene resources for rice disease resistance breeding, and simultaneously screens and cultures rice plants with high disease resistance and high yield by using a molecular cloning means.

Disclosure of Invention

An object of the present invention is to provide a method for improving the rice blast resistance of rice.

The invention provides a method for improving rice blast resistance, which is to improve the phosphorylation level of Lhcb5 in a background plant.

Another objective of the invention is to improve the disease resistance of rice by improving the expression of LHCB5 gene in a background strain.

It is another object of the present invention to improve disease resistance in rice by continuously phosphorylating Lhcb5 protein.

It is a further object of the invention to provide a phosphorylation site for Lhcb 5.

Another object of the present invention is to provide an expression vector of the above-mentioned LHCB5 gene.

Another object of the present invention is to provide a transgenic plant transformed with the above vector.

The invention further aims to provide the application of Lhcb5 in continuously phosphorylating transgenic rice plants and having broad-spectrum disease resistance to different rice blast germs in the field.

The technical scheme provided by the invention is as follows: a rice light-harvesting chlorophyll a/b binding protein Lhcb5 gene mutant: the 24 th amino acid of the protein coded by the wild gene is mutated into aspartic acid.

The coding gene of the wild type gene LHCB5 is shown as SEQ ID No.1, and the sequence of the encoded Lhcb5 protein is shown as SEQ ID No. 2.

The invention also provides application of the rice light harvesting chlorophyll a/b binding protein Lhcb5 gene or the mutant in the disease-resistant process of plants, wherein the gene is over-expressed in the plants by a transgenic method.

The use as described above, wherein the plant is a monocotyledon, preferably the plant is rice.

In the above application, the disease resistance refers to resistance to a plant disease caused by rice blast or phytophthora.

The invention also provides application of the rice light harvesting chlorophyll a/b binding protein Lhcb5 gene or the mutant in preparation of disease-resistant transgenic plants, wherein the rice light harvesting chlorophyll a/b binding protein Lhcb5 gene is overexpressed in transgenic plants through a transgenic method.

The above use, the plant is a monocotyledon, preferably the plant is rice.

The disease resistance refers to the resistance to plant diseases caused by rice blast or phytophthora.

The invention relates to application of chlorophyll a/b binding protein Lhcb5 in rice in a disease-resistant process, wherein LHCB5 gene silencing and knockout do not influence normal infection of rice blast germs; however, the overexpression transgenic plant of LHCB5 has obvious resistance to rice blast germs and can inhibit the rice blast germs from expanding in host cells. And in the process of interacting with rice blast germs, a large amount of active oxygen is induced to be generated, and cell necrosis is caused. And simultaneously induces the expression of disease-resistant related genes in host cells and the expression of NADPH oxidase genes, thereby obviously improving the resistance to rice blast germs. The transgenic plants involved in the invention do not affect normal growth and fruiting.

The invention discovers that Lhcb5 in rice is phosphorylated in the interaction process of the rice and rice blast germs, and the phosphorylation of the protein can obviously improve the basic disease resistance of the rice to the rice blast germs. The rice blast germs in different fields inoculated by the over-expressed plants are shown as resistance, which shows that the Lhcb5 has broad regulation and control spectrum and can be popularized and applied practically.

The invention also discovers that in 3000 germplasm resource scans, the expression quantity of the gene in different rice plants has obvious difference and rich polymorphism, and 26 polymorphic sites are distributed in an intron, an exon and a promoter region. The present invention also demonstrates that the LHCB5 gene encodes 283 amino acids, with threonine at position 24 being a phosphorylation site that is conserved in monocotyledonous plants but not present in dicotyledonous plants. The continuous activation of the site can induce the accumulation of active oxygen of tobacco cells and rice protoplasts and induce cell necrosis. When different rice varieties interact with rice blast germs, the phosphorylated rice varieties are shown to be disease-resistant, the non-phosphorylated rice varieties are shown to be disease-susceptible, and the phosphorylation of the Lhcb5 is important for the basic disease resistance of rice.

The invention is beneficial to the cultivation of rice disease-resistant varieties and provides a basis for screening high-water-resistance rice varieties in the later period. For example, the invention can provide an LHCB5 overexpression plant, and uses EMS mutagenesis to obtain a silencing inhibitor of LCHB5 gene and obtain a corresponding transgenic plant; in addition, the invention can further provide or apply the transgenic plant or the seed to obtain a transgenic rice plant with higher resistance by hybridization.

Drawings

FIG. 1 shows the response of LHCB5 gene to rice blast germ infection, and FIG. 1A shows the change of transcription level of LHCB5 gene at different time intervals after rice TP309 is inoculated with rice blast germ; FIG. 1B shows the change in translation level of LHCB5 gene at different time intervals after inoculation of rice TP309 with Pyricularia oryzae.

FIG. 2 shows acquisition and pathogenicity determination of an LHCB5 gene knockout mutant, and FIG. 2A shows construction of an LHCB5 gene knockout mutant target sequence obtained by a Crispr/Cas9 technology and a sequencing result of target gene knockout; FIG. 2B shows the results of pathogenic inoculation of LCHB5 knockout mutants; FIG. 2C is a statistical result of the size of the lesion of FIG. 2B.

FIG. 3 shows the verification of the silencing of LHCB5 gene and the expression of overexpressed transgenic plants, and FIG. 3A shows the verification of the silencing of LHCB5 gene and the expression of overexpressed plants; FIG. 3B shows western validation of Lhcb5 protein over-expressed plants.

FIG. 4 shows the determination of disease resistance of plants with LHCB5 gene silencing and overexpression, and FIG. 4A shows the determination of pathogenicity of plants with LHCB5 gene silencing and overexpression; FIG. 4B is a statistics of the incidence area of FIG. 4A; FIG. 4C shows the sporulation of the leaf lesion moisturizing culture of FIG. 4A; FIG. 4D is a microscopic observation of hypha infection of Magnaporthe grisea in LHCB5 gene-silenced and over-expressed plants; FIG. 4E shows the grade of the hyphae of FIG. 4D (grade I, no hyphae of the attached cells; grade II, only one primary hyphae of the attached cells; grade III, multiple branched hyphae of the attached cells but not extending to neighboring cells; grade IV, hyphae of the attached cells.

FIG. 5 shows that LHCB5 transgenic plants induce host active oxygen accumulation and cell necrosis, and FIG. 5A shows DAB and Trypan blue staining results of leaves of LHCB5 gene silencing and over-expression plants; FIG. 5B shows the results of determining the active oxygen in rice protoplasts of LHCB5 gene-silenced and over-expressed plants; FIG. 5C shows the DAB and Trypan blue staining results of LHCB5 gene silencing and overexpression plants after different reducing agent treatments; FIG. 5D is the DAB staining statistics of FIG. 5C; FIG. 5E is the Trypan blue stain statistics of FIG. 5C; FIG. 5F shows the results of LHCB5 gene silencing and expression of disease process-related genes in over-expressed plants; FIG. 5G is the results of the expression of two NADPH oxidases in LHCB5 gene silencing and overexpressing plants.

FIG. 6 shows the pathotypes of different rice blast fungus races.

FIG. 7 shows that LHCB5 overexpression plants regulate the broad-spectrum resistance of rice to rice blast fungus.

FIG. 8 shows that phosphorylation of Lhcb5 protein regulates disease resistance of rice against Pyricularia oryzae, and FIG. 8A shows that phosphorylation of LHCB5 overexpression plant occurs under pathogenic bacteria induction condition; FIG. 8B is a correlation analysis of expression level and disease resistance of LHCB5 gene in different rice varieties; FIG. 8C shows that phosphorylation of Lhcb5 increases resistance of rice to Magnaporthe grisea; fig. 8D is a graph of disease resistance between the disease resistance gene and the avirulence gene was not modulated by phosphorylation of Lhcb 5.

FIG. 9 shows the identification and application of phosphorylation sites of Lhcb5, and FIG. 9A shows homology alignment of Lhcb5 protein and prediction of phosphorylation sites in dicotyledonous and monocotyledonous plants; FIG. 9B shows that persistent phosphorylation of amino acid 24 of Lhcb5 induces tobacco cell necrosis; FIG. 9C shows that amino acid 24 of Lhcb5 is phosphorylated and is induced to be expressed by Phytophthora LT 263; FIG. 9D shows that continuous phosphorylation of amino acid 24 of Lhcb5 induces the accumulation of active oxygen in rice protoplasts.

Detailed Description

Example 1: infection of LHCB5 gene responding to rice blast germ

After rice TP309 is infected by a wild type strain Guy11 of rice blast fungi, RNA of rice leaves infected at different stages is extracted by using an RNA extraction kit of Tiangen company; the extracted RNA is reversely transcribed to synthesize cDNA which is used for detecting the expression quantity of LHCB5 gene, and the reverse transcription is completed by utilizing a reverse transcription kit of TaKaRa; the invention discovers that the expression quantity of LHCB5 gene is up-regulated and expressed after rice blast fungus infection (figure 1A). Further extracting rice leaf protein in different stages, and performing western detection, the invention finds that the Lhcb5 protein amount is gradually accumulated in the late stage of infection (figure 1B), and proves that the LHCB5 gene responds to the infection of rice blast germs.

Example 2: acquisition of LHCB5 gene knockout mutant and pathogenicity determination

The rice knockout mutant of the LCHB5 gene is obtained by entrusting Wuhanbo remote biology company by using a Crispr/Cas9 method. And the gene is knocked out through sequencing, and two forms of different mutations exist (figure 2A); further, the wild type strain Guy11[23] of Pyricularia oryzae is inoculated on rice leaves, the sizes of scabs are counted after 7 days, the experiment is repeated for 3 times, stable and consistent results are obtained, and the LHCB5 gene knockout mutant shows susceptibility to Pyricularia oryzae (fig. 2B and C).

Example 3: LHCB5 gene silencing and verification of over-expression plant

The invention utilizes pUCCRNAi carrier to test the silencing of LHCB5 gene, firstly, sequences of LHCB5 gene from 489bp to 696bp of initiation codon are respectively constructed on the pUCCRNAi carrier in forward and reverse directions, and then the remote Wuhan Boeher company is entrusted to transform to obtain LHCB5 gene silencing mutant; the invention also constructs the coding region of the target gene LHCB5 into a pCAM2300 vector by utilizing an enzyme digestion connection method, XbaI and PstI enzyme digestion sites are respectively added to upstream and downstream primers of LHCB5 during primer design, the LHCB5 gene is amplified, then the target gene and the vector are subjected to enzyme digestion linearization by using restriction enzymes XbaI and PstI, and the vector and the fragment are connected by T4 ligase to form the vector of the pCAM2300-LHCB 5. Then the LHCB5 gene is expressed by an actin promoter through the vector, and the vector is provided with a Flag label for later verification; the vector is constructed in the laboratory, and a Wuhan Boehfar biological company is entrusted with transformation to obtain a plant with the LHCB5 gene over-expression; the expression quantity of the LHCB5 gene of the obtained transgenic plant is quantitatively analyzed through qRT-PCR, the silencing and expression of the LCHB5 gene are proved to have no problems (figure 3A), and the western detection shows that the Lhcb5 protein quantity in the over-expressed plant is obviously increased (figure 3B), so that the obtained transgenic plant can be used for subsequent experiments.

The invention carries out heavy load on the obtained transgenic rice plant to obtain 3 rd generation stable genetic progeny plant for pathogenicity determination. The wild type strain Guy11 spore liquid of rice blast fungus is inoculated by spraying on rice leaves, and the concentration is 5x10⁴In each ml, a silent mutant of the LHCB5 gene is found to be infected, an overexpression plant of the LHCB5 gene is resistant to the disease of the Guy11, and conidia cannot be formed after the lesion spots on the resistant leaves are subjected to moisture culture, so that the lesion spots are necrotic spots (FIGS. 4A, B and C); further, by using a rice leaf sheath infection experiment, it is found that Guy11 can not normally expand infected hypha in an overexpression plant of the LHCB5 gene (fig. 4C and D), and the fact that the overexpression plant of the LHCB5 gene can inhibit the infection of rice blast germs and improve disease resistance is demonstrated.

Example 4: LHCB5 gene overexpression plant for inducing host active oxygen accumulation and cell necrosis

The method utilizes diaminobenzidine (DAB, sigma) to dye and observe the active oxygen of the host, and utilizes trypan blue (TB, Biyunnan) to dye and observe the necrotic cells of the host. Inoculating rice blast spores on rice leaves for 24 hours, collecting, dyeing the leaves for more than 8 hours under the room temperature dark condition by using DAB with the concentration of 1mg/ml, and then adding ethanol: acetic acid (94:4) is used for decoloring, the leaves with active oxygen accumulation are dyed into dark brown, and the experimental result shows that the LHCB5 gene overexpression material accumulates a large amount of active oxygen after the rice blast fungus is infected; further staining the leaves with trypan blue staining solution, and decolorizing with aqueous trichloroethanol, the necrotic cells could be stained dark blue (fig. 5A), and as a result, the phenomenon of massive cell necrosis of the LHCB5 gene overexpression material was found. In addition, the same results were obtained in rice protoplasts, when the LHCB5 gene overexpression material was treated with the purified rice blast fungus mycelia, a large amount of active oxygen was accumulated, and the burst of active oxygen was detected by a Luminol chemiluminescence detector and a kinetic curve was drawn (FIG. 5B).

The invention utilizes microscopic observation technology to authenticate the accumulation of host active oxygen of the LHCB5 gene overexpression material. Injecting the magnaporthe grisea spore liquid into rice leaf sheaths, dyeing with DAN and Trypan, after decolorizing as above, tearing off the inner epidermis of the leaf sheaths by using surgical forceps, and carrying out microscopic observation to find that a large amount of active oxygen is accumulated in the leaf sheath cells of the LHCB5 gene overexpression material, and the extension of magnaporthe grisea hypha is inhibited; further treatment of leaf sheath cells with reducing agent and staining also revealed that the reducing agent treatment inhibited the accumulation of part of active oxygen (FIGS. 5C, D and E), indicating that the LHCB5 gene-overexpressed plants did induce host active oxygen production. The present invention also found that the disease process related genes PR1, PBZ1, AOS2 and LOX1 were all up-regulated in plants overexpressing LHCB5 gene (fig. 5F), and that two NADPH oxidase genes were also up-regulated in the overexpressing material (fig. 5G).

Example 5: LHCB5 gene overexpression plant for regulating and controlling broad-spectrum disease resistance of rice to rice blast germs

The invention discovers that the LHCB5 gene overexpression plant has resistance to different rice blast germs in the field. We isolated 21 rice blast germs from different pathogenic types in the field (figure 6), sprayed and inoculated LHCB5 gene overexpression plants and TP309 wild type plants respectively, observed the disease occurrence of rice leaves 7 days after inoculation, found that the LHCB5 gene overexpression plants show resistance to 21 field strains, and the TP309 shows infection (figure 7). The resistance regulated by the LHCB5 gene overexpression plant has broad spectrum.

Example 6: phosphorylation of Lhcb5 protein regulates disease resistance of rice to rice blast bacteria

The invention finds that disease resistance regulated by Lhcb5 is closely related to phosphorylation of the protein. A rice TP309 and LHCB5 gene overexpression rice material is inoculated by a rice blast fungus wild type strain Guy11, after 48 hours, leaf protein is extracted, and western detection is carried out, so that the TP309 is not phosphorylated regardless of inoculation, and the LHCB5 gene overexpression material can be phosphorylated after inoculation (figure 8A), which indicates that phosphorylation occurs after response to rice blast fungus infection by Lhcb 5.

The invention also finds that the expression quantity of the LHCB5 gene has certain correlation with the disease resistance of rice. RNA of different rice varieties is extracted, expression quantity of LHCB5 gene is quantitatively analyzed through qRT-PCR, expression difference of LHCB5 gene in different rice is found, resistance of the rice to rice blast germs is also found, strain with high expression quantity of LHCB5 gene shows resistance to rice blast germs, and correlation value is-0.713 (figure 8B).

The invention also relates to the relation between the expression quantity and phosphorylation of LHCB5 gene, and the difference exists in phosphorylation of Lhcb5 protein after different rice varieties are inoculated with rice blast germs, rice varieties with Lhcb5 protein being not phosphorylated show diseases to the rice blast germs, and rice varieties with Lhcb5 protein being phosphorylated show diseases to the rice blast germs (figure 8C). However, this disease resistance phenomenon was not applicable to the recognition between the disease resistance gene and the avirulence gene (FIG. 8D).

Example 7: prediction and application of Lhcb5 protein phosphorylation site

The phosphorylation site of the Lhcb5 protein, threonine at position 24, is found through website prediction and homology alignment, and the site is not existed in dicotyledonous plants and is relatively conserved in monocotyledonous plants (FIG. 9A). We performed persistent activation mutation of this site to threonine to aspartate, and expressed in tobacco cells using pBIN vector containing 35S promoter and stained with DAB, and found that persistent activation induced active oxygen accumulation (FIG. 9B). Further research shows that Lhcb5 can respond to the infection of phytophthora LT263 and generate phosphorylation; the threonine 24 was mutated to alanine, and inactivating mutation was performed, and it was found that phosphorylation of Lhcb5 could not occur after inactivating mutation (fig. 9C), indicating that the amino acid 24 of Lhcb5 was a phosphorylation site. And the same result is obtained in rice protoplasts, when the continuously activated Lhcb5 is expressed in LHCB5 gene silencing material, the rice protoplasts accumulate a large amount of active oxygen, but the inactivation mutation does not accumulate the active oxygen, and the detection of the active oxygen burst is carried out by using a Luminol chemiluminescence detector and drawing a dynamic curve (figure 9D).

Reference to the literature

1.Valent B,Khang CH(2010)Recent advances in rice blast effector research.Curr Opin Plant Biol 13:434‐441.

2.Mosquera G,Giraldo MC,Khang CH,Coughlan S,Valent B(2009)Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1‐4as biotrophy‐associated secreted proteins in rice blast disease.Plant Cell 21:1273‐1290.

3.Park CH,Chen SB,Shirsekar G,Zhou B,Khang CH,et al.(2012)The Magnaporthe oryzae Effector AvrPiz‐t Targets the RING E3Ubiquitin Ligase APIP6to Suppress Pathogen‐Associated Molecular Pattern‐Triggered Immunity in Rice.Plant Cell 24:4748‐4762.

4.Park CH,Shirsekar G,Bellizzi M,Chen SB,Songkumarn P,et al.(2016)The E3Ligase APIP10Connects the Effector AvrPiz‐t to the NLR Receptor Piz‐t in Rice.Plos Pathogens 12.

5.Wang RY,Ning YS,Shi XT,He F,Zhang CY,et al.(2016)Immunity to Rice Blast Disease by Suppression of Effector‐Triggered Necrosis.Current Biology 26:2399‐2411.

6.Mentlak TA,Kombrink A,Shinya T,Ryder LS,Otomo I,et al.(2012)Effector‐Mediated Suppression of Chitin‐Triggered Immunity by Magnaporthe oryzae Is Necessary for Rice Blast Disease.Plant Cell 24:322‐335.

7.Chuma I,Isobe C,Hotta Y,Ibaragi K,Futamata N,et al.(2011)Multiple Translocation of the AVR‐Pita Effector Gene among Chromosomes of the Rice Blast Fungus Magnaporthe oryzae and Related Species.Plos Pathogens 7.

8.Bohnert HU,Fudal I,Dioh W,Tharreau D,Notteghem JL,et al.(2004)A putative polyketide synthase peptide synthetase from Magnaporthe grisea signals pathogen attack to resistant rice.Plant Cell 16:2499‐2513.

9.Ribot C,Cesari S,Abidi I,Chalvon V,Bournaud C,et al.(2013)The Magnaporthe oryzae effector AVR1‐CO39 is translocated into rice cells independently of a fungal‐derived machinery.Plant J 74:1‐12.

10.Wu J,Kou YJ,Bao JD,Li Y,Tang MZ,et al.(2015)Comparative genomics identifies the Magnaporthe oryzae avirulence effector AvrPi9 that triggers Pi9‐mediated blast resistance in rice.New Phytologist 206:1463‐1475.

11.Zhang SL,Wang L,Wu WH,He LY,Yang XF,et al.(2015)Function and evolution of Magnaporthe oryzae avirulence gene AvrPib responding to the rice blast resistance gene Pib.Scientific Reports 5.

12.Li W,Wang BH,Wu J,Lu GD,Hu YJ,et al.(2009)The Magnaporthe oryzae Avirulence Gene AvrPiz‐t Encodes a Predicted Secreted Protein That Triggers the Immunity in Rice Mediated by the Blast Resistance Gene Piz‐t.Molecular Plant‐Microbe Interactions 22:411‐420.

13.Hammerschmidt R(1999)Induced disease resistance:how do induced plants stop pathogensPhysiological and Molecular Plant Pathology 55:77‐84.

14.Swarbrick PJ,Schulze‐Lefert P,Scholes JD(2006)Metabolic consequences of susceptibility and resistance(race‐specific and broad‐spectrum)in barley leaves challenged with powdery mildew.Plant Cell and Environment 29:1061‐1076.

15.Sendon PM,Seo HS,Song JT(2011)Salicylic Acid Signaling:Biosynthesis,Metabolism,and Crosstalk with Jasmonic Acid.Journal of the Korean Society for Applied Biological Chemistry 54:501‐506.

16.Djamei A,Kahmann R(2012)Ustilago maydis:Dissecting the Molecular Interface between Pathogen and Plant.Plos Pathogens 8.

17.Djamei A,Schipper K,Rabe F,Ghosh A,Vincon V,et al.(2011)Metabolic priming by a secreted fungal effector.Nature 478:395‐398.

18.Wasternack C,Hause B(2013)Jasmonates:biosynthesis,perception,signal transduction and action in plant stress response,growth and development.An update to the 2007review in Annals of Botany.Annals of Botany 111:1021‐1058.

19.Rodriguez‐Herva JJ,Gonzalez‐Melendi P,Cuartas‐Lanza R,Antunez‐Lamas M,Rio‐Alvarez I,et al.(2012)A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses.Cellular Microbiology 14:669‐681.

20.Allen KD,Staehelin LA(1992)Biochemical Characterization of Photosystem II Antenna Polypeptides in Grana and Stroma Membranes of Spinach.Plant Physiol 100:1517‐1526.

21.Fristedt R,Wasilewska W,Romanowska E,Vener AV(2012)Differential phosphorylation of thylakoid proteins in mesophyll and bundle sheath chloroplasts from maize plants grown under low or high light.Proteomics 12:2852‐2861.

22.Chen YE,Yuan S,Du JB,Xu MY,Zhang ZW,et al.(2009)Phosphorylation of Photosynthetic Antenna Protein CP29 and Photosystem II Structure Changes in Monocotyledonous Plants under Environmental Stresses.Biochemistry 48:9757‐9763.

23.Qi Z,Liu M,Dong Y,Zhu Q,Li L,et al.(2016)The syntaxin protein(MoSyn8)mediates intracellular trafficking to regulate conidiogenesis and pathogenicity of rice blast fungus.New Phytol 209:1655‐1667。

<110> Nanjing university of agriculture

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<212> amino acid

<400> 2

MAALAPSKMLGTRLNFAGSSRYATAAPTTGAQKIVSLFSKKPAPKPKPAAVTSSSPDIGDELAKWYGPDRRIFLPEGLLDRSEVPDYLNGEVPGDYGYDPFGLSKKPEDFSKYQAYELIHARWAMLGAAGFIIPEACNKFGANCGPEAVWFKTGALLLDGNTLNYFGNSIPINLIVAVAAEVVLVGGAEYYRIINGLDLEDKLHPGGPFDPLGLASDPDQAALLKVKEIKNGRLAMFSMLGFFIQAYVTGEGPVENLSKHLSDPFGNNLLTVISGAAERTPSL*

Claims (4)

1. A mutant of rice light-harvesting pigment chlorophyll a/b binding protein LHCB5 is characterized in that the 24 th amino acid of a protein coded by a wild type gene is mutated into aspartic acid, and the amino acid sequence of the protein coded by the wild type gene is shown in SEQ ID NO. 2.

2. The mutant according to claim 1, wherein the gene encoding the wild-type gene is represented by SEQ ID No. 1.

3. The application of the rice light-harvesting pigment chlorophyll a/b binding protein LHCB5 gene or the mutant as claimed in claim 1 in plant disease resistance, wherein the LHCB5 gene or the mutant is over-expressed in plants by a transgenic method; the plant is rice; the disease resistance refers to the resistance of the plant diseases caused by rice blast germs.

4. The application of the rice light-harvesting pigment chlorophyll a/b binding protein LHCB5 gene or the mutant as claimed in claim 1 in preparing disease-resistant transgenic plants, wherein the LHCB5 gene or the mutant is over-expressed in the plants by a transgenic method; the plant is rice; the disease resistance refers to the resistance of the plant diseases caused by rice blast germs.

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