CN110734918A

CN110734918A - Phytophthora capsici effector RxLR19781 gene and application thereof

Info

Publication number: CN110734918A
Application number: CN201911068047.0A
Authority: CN
Inventors: 梁涛; 朱春原; 李壮; 张修国
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2020-01-31

Abstract

The invention discloses the biological functions of phytophthora capsici effector RxLR19781(SEQ ID NO:2) and interacting protein GME (SEQ ID NO:4) by means of various genetic engineering technologies, wherein the GME is key factors in the process of regulating and controlling the function of controlling zoospore infection by the RxLR19781, the RxLR19781 can inhibit Bax-induced necrosis, and the RxLR19781 is silenced in phytophthora capsici in steps to find that the RxLR19781 can weaken the pathogenicity of the phytophthora capsici.

Description

Phytophthora capsici effector RxLR19781 gene and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a phytophthora capsici effector RxLR19781 gene and application thereof.

Background

Phytophthora capsici (Phytophthora capsicii) is pathogenic bacteria which have strong pathogenic ability and a parasitic range of and can generate a large amount of RxLR effector molecules to cooperatively infect hosts.

Disclosure of Invention

The invention aims to provide a phytophthora capsici effector RxLR19781 gene and application thereof.

To achieve the object of the present invention, in th aspect, the present invention provides a Phytophthora capsici effector RxLR19781 gene, which is a gene encoding the following protein (a) or (b):

(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2; or

(b) And (b) the protein which is derived from the protein (a) and has the same function and is obtained by replacing, deleting or adding or more amino acids in the sequence shown in SEQ ID NO. 2.

The phytophthora capsici effector RxLR19781 gene (SEQ ID NO:1) is derived from phytophthora capsici strain SD33, the total length of the sequence is 366bp, the open reading frame ORF is 366bp, 121 amino acids are coded together, the protein molecular weight is about 4.4kD, the sequence is analyzed by using a bioinformatics analysis website, and the result shows that the signal peptide region of the RxLR19781 is 1-60 bp and has NO transmembrane region (http:// www.cbs.dtu.dk/services/TMHMM /).

In a second aspect, the invention provides biomaterials containing the genes, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors, engineered bacteria or non-regenerable plant parts.

In a third aspect, the invention provides the use of the gene or a biomaterial containing the gene for inhibiting Bax (membrane permeability regulatory protein) -mediated plant cell death.

In the invention, Bax refers to Bcl-2 related protein, and the amino acid sequence of the protein is shown as SEQ ID NO. 3.

In a fourth aspect, the invention provides the use of the gene or the biological material containing the gene in inhibiting the phytophthora capsici infection of host plants.

In a fifth aspect, the invention provides the use of the gene or a biological material comprising the gene in plant breeding. It is understood that breeding aims at improving plant disease resistance.

The aforementioned applications include: the gene is introduced into the plant by a plasmid or integrated into the plant chromosome by genetic engineering means.

In the present invention, the plant includes pepper and tobacco (Nicotiana benthamiana).

The invention takes phytophthora capsici strain SD33 as an experimental material, separates and identifies 1 effector molecule, and combines the relevant research foundation to carry out the following research on the effector molecule RxLR19781 in the phytophthora capsici strain SD 33: through qRT-PCR technology, the expression of an effector molecule RxLR19781 is found to be up-regulated in the early infection stage, which indicates that the effector molecule plays a role in the early infection stage of phytophthora capsici; the function of the RxLR19781 gene is verified in Nicotiana benthamiana by virtue of agrobacterium-mediated genetic transformation, and as a result, the RxLR19781 can not cause the necrosis of tobacco cells, but can inhibit death elicitor Bax-induced PCD and inhibit the infection and colonization of phytophthora capsici on a host; subcellular localization experiments showed that RxLR19781 has localization in both cytoplasm and nucleus.

The interaction of GDP mannose 3 '5' epimerase (GME) with RxLR19781 was demonstrated by preliminary yeast double-crossing and co-IP experiments. The gene function of GME was verified in nicotiana benthamiana by means of agrobacterium-mediated genetic transformation. As a result, GME neither causes tobacco cell necrosis nor inhibits Bax-induced cell death, and does not affect the infection of phytophthora capsici zoospores.

In order to further verify the interaction mechanism between the phytophthora capsici effector molecule RxLR19781 function and the interacting protein thereof, a VIGS-mediated gene silencing technology is utilized to obtain NbGME Bunsen silent plants, after the silencing efficiency is verified by a qRT-PCR technology, the RxLR19781 function is further verified in silenced Bunsen by steps, and the result shows that in silenced plants, compared with non-silenced plants, the RxLR19781 still cannot cause tobacco cell death, can inhibit Bax-induced cell death, but can not inhibit zoospore infection any more.

The invention verifies the biological functions of phytophthora capsici effector RxLR19781 and interaction protein GME by means of various genetic engineering technologies, and shows that the GME is key factors in the process of regulating and controlling the function of controlling zoospore infection by the RxLR19781, thereby laying a biological theoretical basis for further researching the pathogenic mechanism of phytophthora capsici.

Drawings

FIG. 1 is a plasmid map of vector pBIN-GFP.

FIG. 2 shows the results of 1% agarose gel electrophoresis of the response factor RxLR19781 gene in example 1 of the present invention.

FIG. 3 shows the results of PCR verification after Agrobacterium GV3101 was transformed with the pBIN-GFP recombinant vector of example 2 of the present invention.

FIG. 4 shows the results of the expression pattern analysis of the response factor RxLR19781 in example 3 of the present invention.

FIG. 5 shows the results of pathogenicity analysis of the response factor RxLR19781 in example 4 of the present invention; wherein, a: RxLR19781 at A, INF1 at B, pBIN-GFP idle at C, and MgCl 10mM at D₂A buffer solution; b: statistical histogram of necrosis area after inoculation; c: western Blot to verify the gene expression.

FIG. 6 shows the results of the cell death inhibition assay of the response factor RxLR19781 in example 4 of the present invention; wherein, a: RxLR19781 at A, pBIN-GFP empty at B, and MgCl 10mM at C₂A buffer solution; after 24h, Bax is inoculated at ABC 3; b: statistical histogram of necrosis area after inoculation; c: western Blot to verify the gene expression.

FIG. 7 shows the result of subcellular localization of the effector RxLR19781 in example 4 of the present invention; wherein, a: RxLR19781 subcellular localization; b: subcellular localization Western Blot experimental results.

FIG. 8 shows the effect of the effector RxLR19781 on the pathogenicity of Phytophthora capsici Leonian in example 4 of the invention.

FIG. 9 shows the functional verification of RxLR19781-M1 in embodiment 4 of the present invention; wherein, RxLR19781-M1 is inoculated at A; inoculation at B with RxLR 19781; INF1 was inoculated at C; inoculating pBIN-GFP at the position D and no-load; seeding with 10mM MgCl at E₂And (4) a buffer solution.

FIG. 10 shows the results of the inhibition of cell death by RxLR19781-M1 in example 4 of the present invention; wherein, RxLR19781-M1 is inoculated at A; inoculation at B with RxLR 19781; c, inoculating pBIN-GFP no-load; seeding with 10mM MgCl at D₂A buffer solution; after 24h Bax was inoculated at A, B, C, D four places.

FIG. 11 shows the results of comparison of the amino acid sequences of RxLR19781 of different Phytophthora capsici subspecies in example 4 of the present invention.

FIG. 12 shows the results of the inhibition of cell death by RxLR19781-M1-M in example 4 of the present invention; wherein, RxLR19781-M1-M is inoculated at A; inoculation at B with RxLR 19781; c, inoculating pBIN-GFP no-load; inoculation with 10mM MgCl at D₂A buffer solution; after 24h Bax was inoculated at A, B, C, D four places.

FIG. 13 shows the result of functional verification of RxLR19781-M2 in example 4 of the present invention; wherein, RxLR19781-M2 is inoculated at A; inoculation at B with RxLR 19781; INF1 was inoculated at C; inoculating pBIN-GFP at the position D and no-load; seeding with 10mM MgCl at E₂And (4) a buffer solution.

FIG. 14 shows the results of the inhibition of cell death by RxLR19781-M2 in example 4 of the present invention; wherein, RxLR19781-M2 is inoculated at A; inoculation at B with RxLR 19781; c, inoculating pBIN-GFP no-load; seeding with 10mM MgCl at D₂A buffer solution; after 24h Bax was inoculated at A, B, C, D four places.

FIG. 15 shows the results of the subcellular localization of NRFP-GME and RFP in example 4 of the present invention.

FIG. 16 shows the result of co-localization of NRFP-GME and GFP-RxLR19781 in example 4 of the present invention; wherein, a: GFP-RxLR19781 colocalizes with NRFP-GME; b: GFP-RxLR19781 colocalizes with RFP; c: GFP and NRFP-GME are co-localized; d: GFP co-localizes with RFP. The fluorescence intensity curves are shown by the arrows in the Merge graph on the right.

Fig. 17 shows the GME function verification result in embodiment 4 of the present invention; wherein, the part A is inoculated with GME; INF1 was inoculated at B; c, inoculating pBIN-GFP no-load; inoculation with 10mM MgCl at D₂And (4) a buffer solution.

FIG. 18 shows the results of the GME-inhibited cell death assay of example 4 of the present invention; wherein, the part A is inoculated with GME; b, inoculating pBIN-GFP; inoculation with 10mM MgCl at C₂And (4) a buffer solution. After 24h Bax was inoculated at A, B, C, D four places.

FIG. 19 is a graph showing the effect of GME on the migratory pathogenicity of Phytophthora capsici in example 4 of the present invention.

FIG. 20 shows the results of functional analysis of RxLR19781 after GME silencing in example 4 of the present invention; wherein, a: after GME is silenced, the function of RxLR19781 is verified; RxLR19781 at A, INF1 at B, pBIN-GFP at C, and 10mM MgCl at D₂A buffer solution; b: graph of GME silencing efficiency versus lysis for qRT-PCR.

FIG. 21 shows the results of the RxLR19781 inhibition of cell death assay after GME silencing in example 4 of the present invention; wherein, a: validation that RxLR19781 inhibits Bax-induced cell death following GME silencing; RxLR19781 at A, pBIN-GFP empty at B, and 10mM MgCl at C₂Buffer, 24h later Bax was inoculated at A, B, C; b: graph of GME silencing efficiency versus lysis for qRT-PCR.

FIG. 22 shows the results of analysis of inhibition of Phytophthora capsici zoospore infestation by RxLR19781 after GME silencing in example 4 of the present invention; wherein, a: after GME is silenced, RxLR19781 inhibits zoospore infection and verifies; pBIN-GFP is inoculated at A with no load, and RxLR19781 is inoculated at B; b: graph of GME silencing efficiency versus lysis for qRT-PCR.

FIG. 23 shows the result of Western Blot analysis in example 4 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

The test plants and strains referred to in The following examples were Nicotiana benthamiana, Bax and INF1 Agrobacterium strains both given by The Proc. Sinkiang university Hodgkin-David, Nanjing, at 22 ℃ and 70% humidity, 16h light, 8h dark culture (see Wang Q, Han C, Ferreira AO, et. Transcriptional Programming and Functional Interactions with The same Phytohthora sojae RXLR Effector Repentore [ J ] The Plant Cell,2011,23(6): 2064. 2086.) The vectors used include The Beijing Plant transient expression vector pBIN-GFP (i.e.pBinGFP shown in FIG. 1), The Mitsugaku et al Hodgkin-David, The P-TRV 6356, P-TRV-3683, also available from The university of Taiwan scientific and scientific university, Taiwan corporation, Taiwan).

Primers referred to in the following examples: the software SnapGene is used for designing primers, and according to the principle of primer design, a full-length primer for cloning phytophthora capsici RxLR19781 effector gene, a primer with an enzyme cutting site required for constructing a pBIN-GFP vector and an RxLR19781 mutant primer are designed. The designed primer information is shown in Table 1, and the primers were synthesized by Celui Boxing Corp.

TABLE 1 primer information

Example 1 Phytophthora capsici RxLR19781 Gene cloning and sequence analysis

Taking phytophthora capsici SD33 whole genome cDNA as a template, and amplifying an RxLR19781 gene sequence according to a corresponding PCR reaction system, wherein the reaction system is as follows: 25 μ L of 2 XPPhanta Max Master Mix, 4 μ L DNA, 17 μ L ddH2O, 2 μ L Forward Primer (10 μ M), 2 μ L Reverse Primer (10 μ M), for a 50 μ L system. The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55-65 ℃ for 30s, extension at 72 ℃ for several minutes (about 1kb/min, determined by the length of the cloned gene), 32 cycles; extension at 72 ℃ for 10 min.

The PCR product was electrophoresed in 1% agarose gel (FIG. 2), the band of interest was excised and recovered after 30min, the recovered product was ligated to pEASY-T3 vector, and then sent to the company for sequencing to obtain RxLR19781 gene sequence shown as SEQ ID NO:1 and amino acid sequence of the encoded protein shown as SEQ ID NO: 2.

After company sequencing, RxLR19781 sequence information was obtained. The sequence of the RxLR19781 has the full length of 366bp, the ORF of an open reading frame is 366bp, the total code length is 121 amino acids, the protein molecular weight is about 4.4kD, the sequence is analyzed by using a bioinformatics analysis website, and the result shows that the signal peptide region of the RxLR19781 is 1-60 bp, and no transmembrane region (http:// www.cbs.dtu.dk/services/TMHMM /).

EXAMPLE 2 construction of plant expression vectors

In order to verify the function of phytophthora capsici effector molecules RxLR19781, a gene specific primer with a corresponding enzyme cutting site and used for removing a signal peptide is designed according to the enzyme cutting site on a plant expression vector pBIN-GFP, a T3 plasmid with RxLR19781 is used as a template, a gene sequence with the enzyme cutting site and used for removing the signal peptide is cloned, a target gene is constructed on the plant expression vector by a double enzyme cutting method, a sample is sent to a company for sequencing through the verification of PCR of a bacterial liquid, agrobacterium tumefaciens GV3101 is transformed after the sequencing is correct, and a inoculation experiment can be carried out after the successful transformation is verified. The results of PCR validation after transformation of Agrobacterium GV3101 with the pBIN-GFP recombinant vector are shown in FIG. 3.

Example 3 Phytophthora capsici Effector RxLR19781 expression Pattern analysis

After phytophthora capsici SD33 bacterial strain zoospores infect pepper leaves, collecting samples after 1.5h, 3h, 6h, 12h, 24h, 48h and 72h respectively, extracting RNA and performing reverse transcription to obtain cDNA, taking the cDNA as a template, taking the cDNA in the SD33 mycelium period as a reference, taking phytophthora capsici Actin as an internal reference gene, and performing qRT-PCR to detect the expression quantity of effector molecules RxLR19781 in different periods. The results of triplicate qRT-PCR were averaged and analyzed for variance, and the results are shown in figure 4. Analysis of expression patterns of RxLR19781 shows that the effector molecule is most obviously up-regulated in 1.5h at the early infection stage, and then the expression level gradually decreases, and slightly increases after 24 h.

Example 4 Phytophthora capsici Effector RxLR19781 functional assay

() Experimental methods

1. Agrobacterium transient expression technology

(1) Taking out the preserved agrobacterium containing the constructed vector, streaking on an LB (lysogeny broth) plate containing antibiotic (Rif, Kana 50 mug/muL), carrying out inverted culture at 28 ℃ for 36h, and growing a single colony on the plate;

(2) picking single colony to 50mL liquid LB culture medium containing antibiotic (Rif, Kana 50 ug/ul), culturing at 28 deg.C and 200rpm for 20h with shaking;

(3) centrifuging at room temperature and 4000rpm for 5min to collect thalli;

(4) discard the supernatant, 10mM MgCl₂Washing thallus for 3 times;

(5) adding appropriate amount of 10mM MgCl₂The cells were suspended in a solution (containing 10mM MES, 200. mu.M As) to prepare a suspension OD₆₀₀The value reaches 0.5 to 0.6;

(6) 5-week-old lamina of Nicotiana benthamiana was selected and inoculated to the same site. Penetrating the bacterial liquid into the blades from the back of the blades by using a 1mL injector;

(7) symptoms were observed daily and recorded by photography.

2. Trypan blue staining

(1) Preparing trypan blue dye liquor mother liquor: 200mL of lactic acid, 200mL of glycerol, 200mL of phenol, 200mL of deionized water and 0.4g of trypan blue;

(2) uniformly mixing the mother liquor and absolute ethyl alcohol according to the proportion of 1:2 for later use;

(3) boiling water, placing into a beaker containing trypan blue liquid, and preheating for 1 min;

(4) transferring the inoculated leaves into a beaker, and carrying out water bath for 1-2min in boiling water (paying attention to times not to boil too many leaves to prevent uneven sample boiling);

(5) after water bath, the leaves and trypan blue stain were transferred to a large petri dish and soaked overnight (approximately 12 h);

(6) after soaking, pouring out trypan blue dye solution, pouring in chloral hydrate solution, and changing the chloral hydrate saturated solution for times every 12 hours;

(7) and after the leaves are transparent, pouring out the chloral hydrate solution, pouring into 95% alcohol for soaking for 12 hours, and taking a picture for recording.

3. Western Blot detection

(1) Taking 2-3 inoculated Nicotiana benthamiana leaves, putting the leaves into a mortar, grinding the leaves into fine powder by using liquid nitrogen, and subpackaging the fine powder into 1.5mL centrifuge tubes;

(2) adding 600mL of sample adding buffer solution, uniformly mixing, and carrying out ice bath for 10 min;

(3) boiling water bath for 5min, shaking for 30s, and boiling water bath again for 5 min;

(4) centrifuging at 7000rpm at room temperature for 10min, taking the supernatant, transferring to a new centrifugal tube of 1.5mL at room temperature of 14000rpm, centrifuging for 10min, taking the supernatant, and storing for later use;

(5) performing SDS-PAGE electrophoresis on the prepared Western Blot sample, wherein the loading amount is 30 mu L;

(6) after electrophoresis is finished, transferring the protein from the SDS-PAGE gel to a PVDF membrane by a wet transfer method, and keeping constant pressure at 220V for 1.5 h;

(7) taking down the PVDF membrane, sealing with 5% skimmed milk powder for 12 h;

(8) adding monoclonal antibody mouse Anti- Anti-GFP (3000: 1) according to the proportion, and incubating for 90 min;

(9) washing the membrane with TBST for 5min for 3 times;

(10) diluting a rabbit anti-mouse secondary antibody (5000-;

(11) repeating the step (9);

(12) adding prepared ECL color developing solution, developing and taking a picture.

4. Subcellular localization of Bunsen cells

The plant expression vector pBin-GFP used in the experiment has a GFP fluorescent protein label, the target gene is transiently expressed in the Nicotiana benthamiana for 48 hours by an agrobacterium transformation method, fluorescence is preliminarily observed by a long-wave ultraviolet lamp, the expression of the target gene is determined, and then a subcellular localization observation test of the target gene is carried out by using an ultrahigh-resolution confocal microscope.

5. Rapid site-directed mutagenesis

(1) Designing a forward mutation primer and a reverse mutation primer (table 1);

(2) configuring a mutation PCR system: DNA with 0.1. mu.g of T3 product, 2. mu.L of forward mutation primer, 2. mu.L of reverse mutation primer, 10. mu.L of 5 Xfast Alteration Buffer, 1.5. mu.L of Fast Alteration DNA Polymerase, and make up to 50. mu.L with sterile water; fast alternation Buffer and Fast alternation DNA Polymerase were from the Rapid site-directed mutagenesis kit (TIANGEN Co.).

(3) Mutation PCR procedure: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 20s, annealing at 55 ℃ for 10s, extension at 68 ℃ for 3min, and 18 cycles; extending for 5min at 68 ℃;

(4) adding the PCR product into a new 1.5 mu L centrifuge tube, adding 1 mu L Dpn I restriction enzyme, fully mixing, carrying out water bath at 37 ℃ for 1h, and digesting circular plasmid;

(5) transferring the treated PCR product to escherichia coli competent DH5 α, screening by an Amp resistant culture medium, picking a single colony, shaking the colony, and then sending the colony to a company for sequencing.

6. TRV virus induced gene silencing

(1) Carrying out homologous analysis and comparison on a target gene to be silenced, and finding out a gene sequence of about 200bp of the specificity of the target gene;

(2) constructing a specific sequence into a TRV2 virus silencing vector by using a double enzyme digestion method (Table 1);

(3) after the sequencing is correct, the TRV2 vector connected with the specific fragment is transferred into an agrobacterium-sensitive GV3101, and simultaneously, the TRV1 helper plasmid is transferred into another agrobacterium-sensitive GV 3101;

(4) mixing TRV1 and Agrobacterium with specific fragment TRV2 at ratio of 1:1 to obtain final concentration at OD₆₀₀Inoculating to 2-week-sized Nicotiana benthamiana leaf;

(5) after 10 days, collecting inoculated Bos-nian tobacco, extracting total RNA, performing reverse transcription to form cDNA, taking the cDNA as a template, performing qRT-PCR experiments by using a primer of a target gene, and verifying the expression quantity of the target gene so as to determine the silencing efficiency.

(II) results of the experiment

1. Phytophthora capsici effector molecule RxLR19781 pathogenicity analysis

RxLR19781 was constructed into pBIN-GFP vector, recombinant plasmid was transferred into Agrobacterium GV3101, leaf discs of Nicotiana benthamiana were inoculated by compression injection, INF1 was used as a necrosis positive control, pBIN-GFP was used as an empty control, and 10mM MgCl was used₂Buffer served as blank control. Taking off the leaves after 7d, taking a picture, recording, and carrying out trypan blue stainingAnd (3) decoloring chloral hydrate, and repeating the experiment for 3-4 times, wherein the number of leaves is more than 15. The results of the experiment are shown in FIG. 5. The inoculation results show that RxLR19781 does not cause necrosis of plant cells compared to the control after 7d inoculation.

2. Phytophthora capsici effector molecule RxLR19781 inhibited cell death assay

The Nicotiana benthamiana was inoculated with RxLR19781, pBIN-GFP as an empty control, and 10mM MgCl₂Buffer served as blank control. And inoculating Bax after 24h, picking up leaves after 7d, taking pictures, recording, performing trypan blue staining and chloral hydrate decoloring, and repeating the experiment for 3-4 times, wherein the number of the leaves is more than 15. The results of the experiment are shown in FIG. 6. The vaccination results showed that RxLR19781 inhibited Bax-induced cell death compared to controls.

3. Phytophthora capsici effector molecule RxLR19781 subcellular localization

The molecule RxLR19781 is in the area of the plant cell which performs the function, the effector molecule is inoculated to Nicotiana benthamiana for 48 hours, and the subcellular localization condition is observed by using an ultrahigh resolution confocal microscope. The RxLR19781 subcellular localization is shown in figure 7. Subcellular localization results showed that RxLR19781 was not clearly localized, and was localized in the cytoplasm and nucleus similar to GFP localization. Western Blot experiment shows RxLR19781 protein expression.

4. RxLR19781 test for inhibiting zoospore infection

To verify the effect of the effector molecule RxLR19781 on the infectivity of Phytophthora capsici, the RxLR19781 was inoculated 24h after the inoculation of Nicotiana benthamiana and then inoculated with Phytophthora capsici zoospores. Control was performed by inoculating unloaded GFP-pBIN 24 hours later with zoospores. And 3d, observing the infection condition, and respectively photographing and recording under a fluorescent lamp and a long-wave ultraviolet lamp, dyeing by trypan blue, decoloring by chloral hydrate, and photographing and recording. The results of the experiment are shown in FIG. 8. The zoospore infection condition shows that the phytophthora capsici effector molecule RxLR19781 can inhibit the zoospore infection compared with a control.

5. Phytophthora capsici effector molecule RxLR19781 structure analysis

The three-dimensional structure of the protein of the effector molecule RxLR19781 was further analyzed , and since the function of RxLR19781 is different from that of Avr3a (RxLR19781 does not cause cell necrosis but can suppress cell death caused by Bax, while Avr3a can cause cell necrosis and does not suppress cell death caused by Bax), we compared the protein structures of the two effector molecules. analysis revealed that there are two major differences between , in that th α -helix of 19781 is composed of two small helices, while th α th helix of Avr3a is longer and more complete, and in that loop-4 of 19781 (located between W3-domain and W4-domain) is closer to the interior of the structure.

6. Phytophthora capsici effector molecule RxLR19781 mutation

The th difference region (amino acids 35-40) is mutated into alanine, named as RxLR19781-M1, and the second difference region (amino acids 76-80) is mutated into alanine, named as RxLR 19781-M2.

(1) RxLR19781-M1 functional validation

RxLR19781-M1 was constructed into a plant expression vector, and the recombinant plasmid was transferred into Agrobacterium GV3101 and inoculated with Nicotiana benthamiana. RxLR19781 as a positive control, INF1 as a necrosis positive control, pBIN-GFP as an empty control, and 10mM MgCl₂Buffer served as blank control. And 7d, taking off the leaves, taking a picture, carrying out trypan blue staining and chloral hydrate decoloring, and repeating the experiment for 3-4 times, wherein the number of the leaves is more than 15. The experimental results are shown in FIG. 9, and the Western blot detection results are shown in FIG. 23.

The experimental result shows that the phenotype after RxLR19781-M1 inoculation is the same as that of RxLR19781, and the cell necrosis can not be caused, which indicates that the amino acid in the region has no influence on the self-necrosis function of effector molecules.

(2) Validation of inhibition of cell death by RxLR19781-M1

The B.benthamiana was inoculated with RxLR19781-M1, RxLR19781 as a positive control, pBIN-GFP as an empty control, and 10mM MgCl₂Buffer served as blank control. Inoculating Bax after 24h, picking off leaves after 7d, photographing, recording, and performing trypan blue staining and hydrationAnd (3) decolorizing with chloral, and repeating the experiment for 3-4 times, wherein the number of leaves is more than 15. The experimental results are shown in FIG. 10, and the Western blot detection results are shown in FIG. 23.

The experimental results show that RxLR19781-M1 inoculated with Nicotiana benthamiana and inoculated with Bax 24h later does not inhibit Bax-induced cell death any more, therefore, the mutant region of RxLR19781-M1 is the key functional region of the effector molecule for inhibiting Bax-induced cell death.

(3) RxLR19781-M1-M mutation

Since the RxLR19781-M1 region has been verified as a key functional region for regulating the inhibition of Bax-induced cell death by the effector molecule, and in order to further verify which amino acids play the most critical role in the functional region, the present invention also aligns the amino acid sequence of RxLR19781 with the amino acid sequence of RxLR19781 in different Phytophthora capsici strains races, and the alignment result is shown in FIG. 11.

In the mutant region of RxLR19781-M1, the difference is that amino acid N is mutated to amino acid S, so we mutated this amino acid, N to S, named RxLR 19781-M1-M.

(4) Validation of inhibition of cell death by RxLR19781-M1-M

RxLR19781-M1-M was inoculated onto B.benthamiana, RxLR19781 was used as a positive control, pBIN-GFP was used as an empty control, and 10mM MgCl was used₂Buffer served as blank control. And inoculating Bax after 24h, picking up leaves after 7d, taking pictures, recording, performing trypan blue staining and chloral hydrate decoloring, and repeating the experiment for 3-4 times, wherein the number of the leaves is more than 15. The experimental results are shown in FIG. 12, and the Western blot detection results are shown in FIG. 23.

The experimental results show that RxLR19781-M1-M no longer inhibits Bax-induced cell death after mutating the amino acid N to S. Therefore, asparagine (N) in this region is a key amino acid for regulating RxLR19781 to inhibit Bax-induced cell death.

(5) RxLR19781-M2 functional validation

RxLR19781-M2 was constructed into a plant expression vector, and the recombinant plasmid was transferred into Agrobacterium GV3101 and inoculated with Nicotiana benthamiana. RxLR19781 as positive control, INF1 as necrosis positive control, and PBIN-GFP as idling controlControl, MgCl at 10mM₂Buffer served as blank control. And 7d, taking off the leaves, photographing and recording, carrying out trypan blue staining and chloral hydrate decoloring, and repeating the experiment for 3-4 times for more than 15 leaves. The experimental results are shown in FIG. 13, and the Western blot detection results are shown in FIG. 23.

The experimental result shows that the phenotype after RxLR19781-M2 inoculation is the same as that of RxLR19781, and the cell necrosis can not be caused, which indicates that the amino acid in the region has no influence on the self-necrosis function of effector molecules.

(6) Validation of inhibition of cell death by RxLR19781-M2

The B.benthamiana was inoculated with RxLR19781-M2, RxLR19781 as a positive control, pBIN-GFP as an empty control, and 10mM MgCl₂Buffer served as blank control. And inoculating Bax after 24h, picking up leaves after 7d, taking pictures, recording, performing trypan blue staining and chloral hydrate decoloring, and repeating the experiment for 3-4 times, wherein the number of the leaves is more than 15. The experimental results are shown in FIG. 14, and the Western blot detection results are shown in FIG. 23.

The results of the experiments show that RxLR19781-M2 inoculated with Byan and inoculated with Bax 24 hours later can not inhibit cell death induced by Bax any more, because the amino acids in the RxLR19781-M2 region are not different from those in other Phytophthora capsici races by sequence analysis and structural alignment analysis, and are whole Loop regions in structure, -step mutation is not suitable, therefore, the amino acid-asparagine (N) in the RxLR19781-M2 mutation region and the RxLR19781-M1-M mutation region jointly regulate the function of the effector molecule for inhibiting cell death induced by Bax.

8. Amplification of the RxLR19781 interacting protein GDP mannose 3 '5' epimerase (GME)

Experiments have now demonstrated that RxLR19781 interacts with the 3 '5' epimerase (GME) enzyme GDP mannose from capsicum, and GME function was verified in order to verify the role of GME in RxLR19781 function at step .

Extracting total RNA of hot pepper, reverse transcribing to cDNA, and amplifying GME gene with cDNA as template. GME was constructed into the T3 cloning vector and sequenced by the company. The amino acid sequence of the pepper GME gene coding protein is shown as SEQ ID NO. 4.

9. Subcellular localization of the interacting protein GME

(1) Self subcellular localization of the interacting protein GME

Constructing the interaction protein GME into a plant expression vector with an RFP label at the N end, transferring to agrobacterium GV3101, and obtaining the final concentration OD of the bacterial liquid₆₀₀Adjusting to about 0.5, inoculating Benzen tobacco with unloaded RFP as control, and performing subcellular localization observation on the inoculated portion of Benzen tobacco with ultrahigh resolution confocal microscope, the localization observation result is shown in FIG. 15. The experimental results show that the interacting protein GME localizes itself in the cytoplasm compared to the RFP empty control.

(2) Interaction protein GME and RxLR19781 subcellular co-localization

NRFP-GME and GFP-RxLR19781 are mixed and inoculated into Nicotiana benthamiana, and the final concentration OD of bacterial liquid₆₀₀Adjusted to about 0.5, inoculated with Benyan using the empty RFP and GFP as controls, and then sub-cellular localization observation was performed on the inoculated portion of Benyan using an ultra-high resolution confocal microscope, and the results of co-localization observation are shown in FIG. 16. The experimental results show that the interacting protein GME colocalizes with RxLR19781 in the cytoplasm compared to the control. Therefore, we hypothesized that RxLR19781 interacts with GME in the cytoplasm and thus performs its corresponding function.

10. Interaction protein GME functional verification

(1) Interoperable protein GME pathogenicity analysis

The interacting protein GME is constructed into a pBIN-GFP plant expression vector, transferred to agrobacterium GV3101, inoculated with Nicotiana benthamiana, INF1 as a positive control, pBIN-GFP as an idle control, and 10mM MgCl₂As a blank control, the verification results are shown in fig. 17. The results of the experiment show that GME does not cause cell death compared to the control.

(2) Validation of cell death inhibition by interacting protein GME

GME was inoculated in N.benthamiana, pBIN-GFP as an empty control, 10mM MgCl₂Buffer served as blank control. And inoculating Bax after 24h, picking up leaves after 7d, taking pictures, recording, performing trypan blue staining and chloral hydrate decoloring, and repeating the experiment for 3-4 times, wherein the number of the leaves is more than 15. The results of the experiment are shown in FIG. 18. Results of the experimentIt was shown that GME did not inhibit Bax-induced cell death as the control. Preliminary verification that phytophthora capsici effector molecule RxLR19781 inhibits Bax-induced cell death independently of its interacting protein GME.

(3) Verification of GME inhibiting phytophthora capsici zoospore infection

And (3) inoculating GME into the indigowoad tobacco, inoculating phytophthora capsici zoospores after 24 hours, inoculating the indigowoad tobacco with GFP, inoculating the phytophthora capsici zoospores after 24 hours as a control, observing the infection condition of the phytophthora capsici after 3 days, respectively taking pictures under a fluorescent lamp and a long-wave ultraviolet lamp for recording, carrying out trypan blue staining, decoloring chloral hydrate, and taking pictures for recording. The results of the experiment are shown in FIG. 19. The experimental results show that GME has no influence on the infection capacity of phytophthora capsici zoospores compared with the control.

11. Transient silencing of the GmE gene of Nicotiana benthamiana

Comparing the pepper GME gene with the genome DNA of the Nicotiana benthamiana to find out homologous GME in the Nicotiana benthamiana in order to verify the function of the GME in step , and preparing for instantly silencing the GME in the Nicotiana benthamiana, wherein the amino acid sequence of the GME gene coding protein is shown as SEQ ID NO. 5, a specific sequence GME-S of about 300bp is screened from NbGME, the sequence fragment is constructed into a pTRV2 virus silencing vector, and is transferred into agrobacterium GV3101 after the successful construction of sequencing verification, and the sequence of the GME-S is shown as SEQ ID NO. 6.

Selecting 2 weeks old Nicotiana benthamiana, transferring pTRV1 to Agrobacterium GV3101, co-inoculating with pTRV2-GME-S Agrobacterium, and inoculating with each bacterial solution with final concentration OD₆₀₀The value is about 0.5. pTRV1 was co-inoculated with pTRV2 in empty cells as a control. After 10 days, collecting leaves, extracting RNA, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR (quantitative reverse transcription-polymerase chain reaction) verification on silencing efficiency by using NbGME specific primers.

12. RxLR19781 functional verification after transient silencing of GME

(1) Self-function verification of RxLR19781 after transient silencing of GME

In Nicotiana benthamiana transiently silenced for GME, RxLR19781 was inoculated to verify self-function, INF1 as a positive control, pBIN-GFP as an empty control, 10mM MgCl₂Buffer served as blank control. The experiment was carried out on non-silent B.benthamiana after inoculation with pTRV2As a control. And 7d, observing the surface change, taking a picture and recording after the leaves are taken off, and recording the picture after trypan blue staining chloral hydrate is decolored. The results of the experiment are shown in FIG. 20. The experimental results show that compared with non-silent plants, the function of the RxLR19781 is not changed, and the cell necrosis can not be caused. Therefore, we conclude that RxLR19781 itself functions independently of its interacting protein GME interaction.

(2) Validation of RxLR19781 inhibition of cell death after transient silencing of GME

In transient GME-silenced B.benthamiana, RxLR19781 was inoculated to verify self-function, pBIN-GFP as an empty control, 10mM MgCl₂As a blank control. This experiment was performed on non-silenced B.benthamiana after inoculation with pTRV2 as a control. After 24h, Bax is inoculated, and after 7d, the surface change is observed, the leaves are taken off and photographed for recording, trypan blue is stained, chloral hydrate is decolored, and photographed for recording. The experimental results are shown in fig. 21. Experimental results show that RxLR19781 is also able to inhibit Bax-induced cell death in GME-silenced burley tobacco. It is therefore postulated that the function of RxLR19781 to inhibit Bax-induced cell death is independent of its interaction with the interacting protein GME.

(3) Experiment for inhibiting zoospore infection by RxLR19781 after transient GME silencing

In the Bunsen tobacco with transient GME silencing, RxLR19781 is inoculated into the Bunsen tobacco, phytophthora capsici zoospores are inoculated after 24h, the Bunsen tobacco is inoculated with GFP, and the phytophthora capsici zoospores are inoculated after 24h as a control. This experiment was also performed on non-silenced B.benthamiana after inoculation with pTRV2 as a control. And 3d, observing the phytophthora capsici infection condition, and respectively taking a picture under a fluorescent lamp and a long-wave ultraviolet lamp for recording, dyeing trypan blue, decoloring chloral hydrate and taking a picture for recording. The results of the experiment are shown in FIG. 22. The experimental results show that compared with GME non-silent plants, RxLR19781 can not inhibit phytophthora capsici zoospore infection any more after GME is silenced in Nicotiana benthamiana. Therefore, we conclude that the function of RxLR19781 in inhibiting zoospore infestation is related to the interacting protein GME.

The invention clones phytophthora capsici effector molecule RxLR19781 by using cDNA of phytophthora capsici virulent strain SD33 as a template, and deeply researches the effector molecule. The research shows that the effector molecule RxLR19781 is up-regulated in early stage of phytophthora capsici infection and cannot cause the death of the nicotiana benthamiana cells, but can inhibit the cell death induced by Bax and inhibit the infection of phytophthora capsici zoospores. Therefore, the effector molecule is inferred to have an immune function in the early infection stage of phytophthora capsici and can improve the self-resistance of plants by expressing in plant cells.

Through a previous yeast two-hybrid experiment and a co-IP experiment, an interacting protein GDP mannose 3 '5' epimerase (GME) of RxLR19781, which can catalyze the conversion of GDP-d-mannose to GDP-1-galactose, is a key step of a synthesis pathway of ascorbic acid of higher plants, ascorbic acid can regulate the balance of Reactive Oxygen Species (ROS) in plants so as to prevent plant cell death, and heat shock protein 70(HSP70) is reported to be a molecular chaperone of the GME, so that the GME is involved in a plant disease-resistant process.

The transient expression of GME in Nicotiana benthamiana has been found to be non-involved in the process of cellular necrosis and non-inhibition of Bax-induced cell death, and non-inhibition of Phytophthora capsici zoospore infestation, subcellular localization has been found to be that GME is expressed in the cytoplasm of Nicotiana benthamiana, transient silencing of GME homologous genes in Nicotiana benthamiana has been performed to further to verify the function of the effector molecule RxLR 19781.

Through the function research of phytophthora capsici effector molecule RxLR19781 and interacting protein GME thereof, the mechanism that RxLR19781 inhibits phytophthora capsici zoospore infection is preliminarily proved, a solid foundation is laid for further steps of researching other functional pathways of RxLR19781, and a reliable method is provided for the function research of pathogenic oomycete effector molecules.

Although the invention has been described in detail with respect to and its specific embodiments, it will be apparent to those skilled in the art that variations or modifications may be made thereto without departing from the spirit of the invention.

Sequence listing

<110> Shandong university of agriculture

<120> phytophthora capsici effector factor RxLR19781 gene and application thereof

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>366

<212>DNA

<213> Phytophthora capsici (Phytophthora capsicii)

<400>1

atgcgtttcg ctttccttct gttcgtggct gcagtttccc tcattgcttc cggcgacgcg 60

ctgtcgacac aagctgacgc cacaagccgc catttgcgct ctcaccacca gacgaacacc 120

tacgacgctg aagaagaaga acgtgctctt gacagatcca ttgtcaagaa tctccctgaa 180

caattcaaga acatgtacaa gtatcctagc aaaatggaca atgttctcga gtcatggcgc 240

acgggtcttc agtcggtgga cgacgctgtc atgtacatga agtctcttgg catggacttc 300

gacgctatct cgcactttgt ggacgcgtac cggaagcata ttaacaagaa aggacttcct 360

tactaa 366

<210>2

<211>121

<212>PRT

<213> Phytophthora capsici (Phytophthora capsicii)

<400>2

Met Arg Phe Ala Phe Leu Leu Phe Val Ala Ala Val Ser Leu Ile Ala

1 5 10 15

Ser Gly Asp Ala Leu Ser Thr Gln Ala Asp Ala Thr Ser Arg His Leu

20 25 30

Arg Ser His His Gln Thr Asn Thr Tyr Asp Ala Glu Glu Glu Glu Arg

35 40 45

Ala Leu Asp Arg Ser Ile Val Lys Asn Leu Pro Glu Gln Phe Lys Asn

50 55 60

Met Tyr Lys Tyr Pro Ser Lys Met Asp Asn Val Leu Glu Ser Trp Arg

6570 75 80

Thr Gly Leu Gln Ser Val Asp Asp Ala Val Met Tyr Met Lys Ser Leu

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Gly Met Asp Phe Asp Ala Ile Ser His Phe Val Asp Ala Tyr Arg Lys

100 105 110

His Ile Asn Lys Lys Gly Leu Pro Tyr

115 120

<210>3

<211>192

<212>PRT

<213> mouse (Mus musculus)

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Met Asp Gly Ser Gly Glu Gln Leu Gly Ser Gly Gly Pro Thr Ser Ser

1 5 10 15

Glu Gln Ile Met Lys Thr Gly Ala Phe Leu Leu Gln Gly Phe Ile Gln

20 25 30

Asp Arg Ala Gly Arg Met Ala Gly Glu Thr Pro Glu Leu Thr Leu Glu

35 40 45

Gln Pro Pro Gln Asp Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Arg

50 55 60

Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Glu Leu Gln Arg Met Ile

65 70 75 80

Ala Asp Val Asp Thr Asp Ser Pro Arg Glu Val Phe Phe Arg Val Ala

85 90 95

Ala Asp Met Phe Ala Asp Gly Asn Phe Asn Trp Gly Arg Val Val Ala

100 105 110

Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Lys Ala Leu Cys Thr Lys

115 120 125

Val Pro Glu Leu Ile Arg Thr Ile Met Gly Trp Thr Leu Asp Phe Leu

130 135 140

Arg Glu Arg Leu Leu Val Trp Ile Gln Asp Gln Gly Gly Trp Glu Gly

145 150 155 160

Leu Leu Ser Tyr Phe Gly Thr Pro Thr Trp Gln Thr Val Thr Ile Phe

165 170 175

Val Ala Gly Val Leu Thr Ala Ser Leu Thr Ile Trp Lys Lys Met Gly

180 185 190

<210>4

<211>376

<212>PRT

<213> Pepper (Capsicum annuum)

<400>4

Met Gly Thr Ser Val Glu Thr Lys Tyr Gly Glu Tyr Thr Tyr Glu Asn

1 5 10 15

Leu Glu Arg Glu Pro Tyr Trp Pro Ser Glu Lys Leu Arg Ile Ser Ile

20 25 30

Thr Gly Ala Gly Gly Phe Ile Ala Ser His Ile Ala Arg Arg Leu Lys

35 40 45

Thr Glu Gly His Tyr Ile Ile Ala Ser Asp Trp Lys Lys Asn Glu His

50 55 60

Met Ser Glu Asp Met Phe Cys His Glu Phe His Leu Val Asp Leu Arg

65 70 75 80

Val Met Asp Asn Cys Leu Lys Val Thr Lys Gly Val Asp His Val Phe

85 90 95

Asn Leu Ala Ala Asp Met Gly Gly Met Gly Phe Ile Gln Ser Asn His

100 105 110

Ser Val Ile Met Tyr Asn Asn Thr Met Ile Ser Phe Asn Met Met Glu

115 120 125

Ala Ser Arg Ile Asn Gly Ile Lys Arg Phe Phe Tyr Ala Ser Ser Ala

130 135 140

Cys Ile Tyr Pro Glu Phe Lys Gln Leu Glu Thr Asn Val Ser Leu Lys

145 150 155 160

Glu Ser Asp Ala Trp Pro Ala Glu Pro Gln Asp Ala Tyr Gly Leu Glu

165 170 175

Lys Leu Ala Thr Glu Glu Leu Cys Lys His Tyr Asn Lys Asp Phe Gly

180 185 190

Ile Glu Cys Arg Ile Gly Arg Phe His Asn Ile Tyr Gly Pro Phe Gly

195 200 205

Thr Trp Lys Gly Gly Arg Glu Lys Ala Pro Ala Ala Phe Cys Arg Lys

210 215 220

Ala Leu Thr Ser Thr Asp Lys Phe Glu Met Trp Gly Asp Gly Lys Gln

225 230 235 240

Thr Arg Ser Phe Thr Phe Ile Asp Glu Cys Val Glu Gly Val Leu Arg

245 250 255

Leu Thr Lys Ser Asp Phe Arg Glu Pro Val Asn Ile Gly Ser Asp Glu

260 265 270

Met Val Ser Met Asn Glu Met Ala Glu Ile Val Leu Gly Phe Asp Gly

275 280 285

Lys Asn Leu Pro Ile His His Ile Pro Gly Pro Glu Gly Val Arg Gly

290 295 300

Arg Asn Ser Asp Asn Thr Leu Ile Lys Glu Arg Leu Gly Trp Ala Pro

305 310 315 320

Thr Met Lys Leu Lys Asp Gly Leu Arg Ile Thr Tyr Phe Trp Ile Lys

325 330 335

Glu Gln Ile Glu Lys Glu Lys Val Gln Gly Ser Asp Val Ser Ala Tyr

340 345 350

Gly Ser Ser Lys Val Val Gly Thr Gln Ala Pro Val Glu Leu Gly Ser

355 360 365

Leu Arg Ala Ala Asp Gly Lys Glu

370 375

<210>5

<211>450

<212>PRT

<213> Ben's tobacco (Nicotiana benthamiana)

<400>5

Arg Leu Val Leu Cys Leu Ser Met Thr Tyr Arg Ser Arg Val Arg Val

1 5 10 15

Gly Ala Ala Thr Asn Val Tyr Ile Arg Asn Gly Lys Phe Trp Arg Asn

20 25 30

Gln Leu Trp Arg Tyr Ile Tyr Ile Cys Ile Ile Leu Ala Ile Asn Gln

35 40 45

Arg Ser Val Thr Gln Asn Tyr Ser Pro Leu Ser Leu Ser Phe Ala Ile

50 55 60

Ser Phe Asp Thr Gly Pro Ile Ser Ser Arg Met Gly Ser Ser Gly Gly

65 70 75 80

Thr Ser Tyr Gly Glu Tyr Thr Tyr Glu Asn Leu Glu Arg Glu Pro Tyr

85 90 95

Trp Pro Ser Glu Lys Leu Arg Ile Ser Ile Thr Gly Ala Gly Gly Phe

100 105 110

Ile Ala Ser His Ile Ala Arg Arg Leu Lys Thr Glu Gly His Tyr Ile

115 120 125

Ile Ala Ser Asp Trp Lys Lys Asn Glu His Met Ser Glu Asp Met Phe

130 135 140

Cys His Glu Phe His Leu Val Asp Leu Arg Val Met Asp Asn Cys Leu

145 150 155 160

Lys Val Thr Lys Gly Val Asp His Val Phe Asn Leu Ala Ala Asp Met

165 170 175

Gly Gly Met Gly Phe Ile Gln Ser Asn His Ser Val Ile Met Tyr Asn

180 185 190

Asn Thr Met Ile Ser Phe Asn Met Met Glu Ala Ser Arg Ile Thr Gly

195 200 205

Val Lys Arg Phe Phe Tyr Ala Ser Ser Ala Cys Ile Tyr Pro Glu Phe

210 215 220

Lys Gln Leu Glu Thr Asn Val Ser Leu Lys Glu Ser Asp Ala Trp Pro

225 230 235 240

Ala Glu Pro Gln Asp Ala Tyr Gly Leu Glu Lys Leu Ala Thr Glu Glu

245 250 255

Leu Cys Lys His Tyr Asn Lys Asp Phe Gly Ile Glu Cys Arg Ile Gly

260 265 270

Arg Phe His Asn Ile Tyr Gly Pro Phe Gly Thr Trp Lys Gly Gly Arg

275 280 285

Glu Lys Ala Pro Ala Ala Phe Cys Arg Lys Ala Gln Thr Ser Ala Asp

290 295 300

Lys Phe Glu Met Trp Gly Asp Gly Lys Gln Thr Arg Ser Phe Thr Phe

305 310 315 320

Ile Asp Glu Cys Val Glu Gly Val Leu Arg Leu Thr Lys Ser Asp Phe

325 330 335

Arg Glu Pro Val Asn Ile Gly Ser Asp Glu Met Val Ser Met Asn Glu

340 345 350

Met Ala Glu Met Val Leu Ser Phe Glu Asp Lys Lys Leu Pro Val His

355 360 365

His Ile Pro Gly Pro Glu Gly Val Arg Gly Arg Asn Ser Asp Asn Thr

370 375 380

Leu Ile Lys Glu Lys Leu Gly Trp Ala Pro Ser Met Lys Leu Lys Asp

385 390 395 400

Gly Leu Arg Ile Thr Tyr Phe Trp Ile Lys Glu Gln Ile Glu Lys Glu

405 410 415

Lys Val Gln Gly Ala Asp Val Ser Val Tyr Gly Ser Ser Lys Val Val

420 425 430

Gly Thr Gln Ala Pro Val Gln Leu Gly Ser Leu Arg Ala Ala Asp Gly

435 440 445

Lys Glu

450

<210>6

<211>336

<212>DNA

<213> Ben's tobacco (Nicotiana benthamiana)

<400>6

ggcgttaagc ggttctttta tgcgtccagt gcttgcatct accctgaatt taagcagttg 60

gaaactaacg tcagcttaaa ggagtctgat gcttggcctg cggagcctca agatgcttat 120

ggcttagaaa aactggcaac agaggagcta tgtaagcatt acaacaagga cttcggaatt 180

gaatgtcgca ttggacgttt ccataacatt tatggcccat ttggaacatg gaaaggtgga 240

cgcgagaaag cgccagcagc tttttgtaga aaagcccaaa cttccgctga taaattcgag 300

atgtggggag atggaaagca aactcgatct ttcacc 336

Claims

1. Phytophthora capsici effector RxLR19781 gene, which is characterized in that it is a gene encoding the following protein (a) or (b):

(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2; or

2. A biological material comprising the gene of claim 1, said biological material comprising recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors, engineered bacteria, or non-regenerable plant parts.

3. Use of the gene of claim 1 or the biomaterial of claim 2 to inhibit Bax-mediated plant cell death.

4. The application according to claim 3, wherein the application comprises: the gene is introduced into the plant by a plasmid or integrated into the plant chromosome by genetic engineering means.

5. Use according to claim 3 or 4, wherein the plant comprises capsicum, tobacco.

6. Use of the gene of claim 1 or the biomaterial of claim 2 for inhibiting phytophthora capsici infestation of a host plant.

7. The application according to claim 6, wherein the application comprises: the gene is introduced into the plant by a plasmid or integrated into the plant chromosome by genetic engineering means.

8. Use according to claim 6 or 7, wherein the plant comprises capsicum, tobacco.

9. Use of the gene of claim 1 or the biomaterial of claim 2 in plant breeding.