CN111718403A

CN111718403A - Related protein for inhibiting plant leaf necrosis caused by Bax and application thereof

Info

Publication number: CN111718403A
Application number: CN201910903165.2A
Authority: CN
Inventors: 张修国; 李京; 朱春原; 艾聪聪
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2019-09-24
Filing date: 2019-09-24
Publication date: 2020-09-29

Abstract

The invention relates to the technical field of biology, in particular to phytophthora capsici infected plant related protein and application thereof. The protein is the protein of A1), A2) or A3) as follows: A1) the protein with the amino acid sequence of the sequence 1 in the sequence table or the protein with the amino acid sequence of the sequence 1 in the sequence table; A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 1 in the sequence table, has identity with the protein shown in A1), and is related to leaf cell necrosis and/or phytophthora capsici infection; A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2). The protein and the application thereof can provide theoretical basis for designing a new strategy for preventing and treating the pepper phytophthora blight by effectively utilizing the disease resistance mechanism of plants.

Description

Related protein for inhibiting plant leaf necrosis caused by Bax and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to phytophthora capsici infected plant related protein and application thereof.

Background

Phytophthora capsici belongs to the genus oomycete, and is a destructive filamentous plant pathogen with relatively strong pathogenicity in plants, particularly dicotyledonous plants (Margulis and Schwartz, 2000).

Phytophthora capsici belongs to the order of Oomycetes of Oomycota of the order of Peronosporales of the family Phytophthora,

phytophthora capsici usually overwinter with oospores in soil or disease residues, and diseases are transmitted by wind, water and other agricultural activities (zheng et al, 2007). After the disease occurs, new sporangia can be generated, and zoospores are formed for re-infection. The temperature range of germ growth is 10-37 deg.C, and the optimum temperature is 20-30 deg.C. Under the conditions of continuous cropping, low-lying land, poor drainage and the like, the regions with overlarge density, weak plants and the like are all beneficial to the occurrence and spread of the disease.

The survival time of pathogenic oospores of the pepper phytophthora blight can reach about 3 years at most, and pathogenic bacteria mainly live through the winter in soil and on diseased residues in the form of the oospores so as to pass seasons with low temperature and unsuitable environment. After spring comes, the oospores will begin to germinate under appropriate temperature and humidity conditions, and zoospores can be rapidly produced to invade the roots, stem bases, leaves, etc. of the peppers (Lamour and Hausbeck, 2001). During the growth of pepper plants, pathogenic bacteria will subsequently produce sporangia and zoospores, which are spread by wind, rain, soil, etc., with multiple reinfestations (Ristaino et al, 1991; Ristain et al, 1992; Ristain0 et al, 1993; Schlub et al, 1983; Springer et al, 1982).

The current prevention and treatment work of the pepper phytophthora blight is mainly based on the traditional chemical agent prevention and treatment. The control method has the advantages that the series of problems of heavy metal exceeding and pesticide residue caused by phytophthora capsici brings great threat to human health and environmental safety.

Disclosure of Invention

The invention aims to solve the technical problem of how to inhibit plant leaf necrosis caused by Bax and/or how to inhibit phytophthora capsici from infecting plants.

The protein is the following protein A1), A2) or A3):

A1) the protein with the amino acid sequence of the sequence 1 in the sequence table or the protein with the amino acid sequence of the sequence 1 in the sequence table;

A2) protein which comprises an amino acid sequence shown in a sequence 1 in a sequence table and is related to leaf cell necrosis and/or phytophthora capsici infection;

A3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of A2), has the same property with the protein shown in A1), and is related to leaf cell necrosis and/or phytophthora capsici infection;

A4) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

Wherein the A2 is A21 or A22 as follows:

a21, protein with amino acid sequence shown as sequence 3;

a22, the amino acid sequence of which is shown as 16 th to 329 th positions in the sequence 3;

the protein in A3 can be homologous proteins RxLR101pi and RxLR101pp of a21, and the results of homology alignment with a21(RxLR101) are shown in fig. 1.

Wherein the fusion protein in A4 is A41, A42, A43 or A44, wherein:

a41, protein with amino acid sequence shown as sequence 5;

a42, protein with amino acid sequence shown as sequence 9;

a43, protein with amino acid sequence shown as sequence 7;

a44, protein with amino acid sequence shown as 16 th-340 th position in sequence 7;

a45, protein with amino acid sequence shown as sequence 11;

a46, the amino acid sequence of which is shown as 16 th-343 th position in the sequence 11.

The invention also provides a biological material related to the protein, which is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding the protein of any one of claims 1-3;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing B2) the expression cassette.

Wherein, B1) is the nucleic acid molecule with the nucleotide of R as follows B101, B102, B103, B104, B105, B106, B107, B108, B109, B110, B111 and B112:

b101, cDNA molecules or DNA molecules of a sequence 2 in a sequence table;

b102, cDNA molecules or DNA molecules of a sequence 10 in a sequence table;

b103, cDNA molecules or DNA molecules of a sequence 4 in a sequence table;

b104, is a cDNA molecule or a DNA molecule at the 46 th to the 987 th site in the sequence 4 in the sequence table;

b105, cDNA molecules or DNA molecules of a sequence 6 in a sequence table;

b106, cDNA molecules or DNA molecules of a sequence 8 in the sequence table;

b107, cDNA molecules or DNA molecules of 46 th to 1020 th sites in a sequence 8 in a sequence table;

b108, cDNA molecules or DNA molecules of a sequence 12 in a sequence table;

b109, and a cDNA molecule or a DNA molecule at the 46 th to the 1032 th position in the sequence 12 in the sequence table.

A method for inhibiting phytophthora capsici from infecting a plant, comprising introducing into the plant a recombinant vector containing said B101, B105, B106 or B107 or introducing into the plant a recombinant microorganism containing said B101, B105, B106 or B107.

The method for inhibiting phytophthora capsici from infecting plants is applied to regulation and control of phytophthora capsici infection of plants, preparation of products for resisting phytophthora capsici infection of plants, cultivation of plants resisting phytophthora capsici infection or plant breeding.

Any one of the following P1-P9 uses of the protein, or the biomaterial:

the use of P1, the protein, or the biomaterial for modulating plant leaf cell necrosis;

use of P2, the protein, or the biomaterial in the manufacture of a product for reducing necrosis of plant leaves;

use of P3, said protein, or said biological material for growing plants that reduce necrosis of plant leaves;

the use of P4, the protein, or the biological material for the manufacture of a product for reducing plant leaf necrosis;

use of P5, said protein, or said biomaterial for modulating phytophthora capsici infestation in a plant;

the use of P6, the protein, or the biomaterial in the manufacture of a product for increasing the resistance of a plant to phytophthora capsici infestation;

the use of P7, said protein, or said biological material for growing plants resistant to phytophthora capsici infestation;

the application of P8, the protein or the biological material in preparing a product for resisting phytophthora capsici infection of plants;

use of P9, the protein, or the biological material in plant breeding.

Wherein the plant is a monocotyledon or a dicotyledon.

Wherein the plant is Nicotiana benthamiana or Capsicum annuum.

The invention has the beneficial effects that RxLRnudix (protein A1)) and RxLR101 (protein A2)) in the invention can inhibit PCD induced by Bax, and RxLR101-P.p and RxLR101-P.i (protein A3)) can also inhibit PCD induced by Bax. RxLRnudix (protein A1)) and RxLR101-NLS fused with RxLRnudix-NLS (protein A4)) can more strongly inhibit the infection of phytophthora capsici zoospores. Thereby providing a theoretical basis for designing a new strategy for preventing and treating the pepper phytophthora blight by effectively utilizing the disease resistance mechanism of the plant.

Drawings

FIG. 1 is a diagram showing an alignment of homologous gene sequences;

FIG. 2.RxLR101 truncated mode diagram

FIG. 3. pathogenicity analysis of RxLR effector molecules on Nicotiana benthamiana, FIG. 3a is a functional verification diagram of RxLRnudix, wherein A represents RxLR101, B represents RxLRnudix, and C represents buffer; d represents the empty vector pBIN-GFP2, and E represents INF 1. FIG. 3b is a functional verification diagram of RxLR101-P.i and RxLR101-P.p, wherein A represents unloaded pBIN-GFP 2; b respectively represents RxLRnudix, RxLR101-P.i and RxLR 101-P.p; c represents buffer; d represents INF 1.

FIG. 4 shows inhibition analysis of RXRR, in 4a, A represents RxLR101, B represents RxLR101-P.p, RxLR101-P.i, C represents buffer, and D represents empty vector pBIN-GFP 2. The right panel shows trypan blue staining results, and FIG. 4B shows inhibition analysis of RxLRnudix, wherein A represents RxLR101, B represents RxLRnudix, and C represents buffer; d represents the empty vector pBIN-GFP2, and E represents INF 1. The right panel shows trypan blue staining results.

FIG. 5 Phytophthora capsici zoospore infestation of RxLR101 and-RxLRnudix in example 3

FIG. 6 qPCR detection of RxLR101 and-RxLRnudix in example 3

FIG. 7 inhibition assay of RxLR101 fusion proteins, wherein A represents RxLR101-NES, RxLRnudix-NES, B represents RxLR101-NLS, RxLRnudix-NLS, CRxLR101, D-unloaded pBIN-GFP2, E represents RxLRnudix, and F represents buffer, respectively. The right panel shows trypan blue staining results.

FIG. 8 shows the results of infection of Phytophthora capsici zoospores with RxLR101-NES/NLS in example 5

FIG. 9 results of RxLR101-NES/NLSqPCR detection in example 5

FIG. 10. results of example 5 infection of RxLRnudix-NES/NLS with Phytophthora capsici zoospores

FIG. 11 shows the results of RxLRnudix-NES/NLSqPCR assay in example 5.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Vectors and strains

The cloning Vector used for the experiments, pEASY-T3 clone Vector, was purchased from Beijing Quanyujin Biotechnology Ltd, the plant expression Vector pBIN-GFP2 (disclosed in "A Virus Essential CRN efficiency of Phytophtora capsaici supress feedbacks Defence and indecs Cell Death in plantarnulus", publicly available from the teaching laboratory of the sinus tract dragon), pYF2-PsNLS-hSpCas9, pYF2.3G-Ribo-sgRNA (disclosed in "A Phytophta capsaici efficiency Targets ACD11 binding clones of primers which regulated ROS-Mediated feedback in Argpsis", available from the teaching laboratory of the sinus tract), the technology pBIIKS + platinum.

Escherichia coli (Escherichia coli) DH 5. alpha. competent cells were purchased from Kyoto Kogyo gold Biotechnology Ltd, and Agrobacterium (Agrobacterium tumefaciens) strain GV3101 was purchased from TransGen Biotechnocroration.

Phytophthora capsici strain SD33(Phytophthora capsici) (Yong Jian Jia, Bao Zhen Feng, Wen Xiu Sun and Xiu Guo Zhuang, J. phytophathol, 157:585-591,2009) is publicly available from Shandong university of agriculture, and is only used for repeating the relevant experiments of the present invention and is not used for other purposes.

Bax Agrobacterium strains (publicly available from professor sinus professor laboratories, Nanjing university of agriculture, sinus tract), disclosed in the literature "functional analysis of 6 RxLR of Phytophthora capsici and 1 CRN effector of Phytophthora sojae").

The biological material can be obtained from Shandong agricultural university by the general university of Phytophthora capsici Jul0201 and Phytophthora capsici Aug0202 (2008), and is only used for repeating the relevant experiments of the invention and can not be used for other purposes. Primers involved in specific examples of the invention are shown in table 1:

table 1 introduction table:

example 1, RxLR101 short-cut RxLRnudix acquisition:

cloning of the RxLR101 gene: the PCR product of the reaction was electrophoresed on 1.2% agarose gel using total DNA of Phytophthora capsici SD33 strain stored in this laboratory as a template, specific primers (primer RxLR101-F and primer RxLR101-R) were designed, RxLR101 gene was amplified using primer RxLR101-F and primer RxLR 101-RPCR.

Cloning of the RxLR101-P.p gene: the total DNA of Phytophthora capsici Jul0201 stored in the laboratory is used as a template, specific primers (a primer RxLR101-P.p-F and a primer RxLR101-P.p-R) are designed, the RxLR101-P.p-F and the primer RxLR101-P.p-RPCR are used for amplifying the RxLR101-P.p gene, and a PCR product of the reaction is subjected to electrophoresis by using 1.2% agarose gel.

Cloning of the RxLR101-P.i gene: the total DNA of Phytophthora capsici Aug0202 stored in the laboratory is used as a template, specific primers (a primer RxLR101-P.i-F and a primer RxLR101-P.i-R) are designed, the RxLR101-P.i-F and the primer RxLR101-P.i-RPCR are used for amplifying the RxLR101-P.i gene, and a PCR product of the reaction is subjected to electrophoresis by using 1.2% agarose gel.

Cutting a target band, putting the cut target band into a 1.5mL centrifuge tube for gel recovery, connecting a recovered product to a pEASY-T3 vector to obtain a recombinant vector, respectively converting the recombinant vector into escherichia coli DH5 alpha, growing monoclonal bacterial plaque after 12-16h, selecting the single bacterial plaque for bacterial liquid PCR verification, and sending the bacterial liquid with the correct size of the target band to sequencing. And (5) after sequencing is carried out, absorbing bacterial liquid for amplification culture after the sequence result is correctly compared. Then, the bacterial liquid is preserved and the plasmid is extracted to obtain a cDNA plasmid connected with a pEASY-T3 recombinant vector, the pEASY-T3 recombinant plasmid containing RxLR101 is named as pEASY-T3-RxLR101, the pEASY-T3 recombinant plasmid containing RxLR101-P.i is named as pEASY-T3-RxLR101-P.i, and the pEASY-T3 recombinant plasmid containing RxLR101-P.p is named as pEASY-T3-RxLR 101-P.p.

The sequencing result shows that the nucleotide sequence of the RxLR101 gene is shown as a sequence 4, the amino acid sequence of the RxLR101 with the coding amino acid sequence shown as a sequence 3 is shown as a sequence 3, the DNA sequence of the corresponding coding gene is shown as a sequence 4, the full length of the nucleotide sequence of the phytophthora capsici effector RxLR101 is 990bp, the coding region is provided with a protein of 329 amino acids, and the signal peptide of the effector RxLR101 is 1-15 amino acids of the sequence 3. The 16 th to 329 th positions of the sequence 3 are the amino acid sequences of mature proteins.

The homology analysis of the above-mentioned RxLR101 gene, RxLR101-P.p gene and RxLR101-P.i gene is shown in FIG. 1.

The RxLR101 is truncated according to the secondary structure (Baietal, 2012) thereof as shown in FIG. 2, and a key functional domain is selected through a vaccination experiment and is recombined to obtain a truncated RxLRnudix of the RxLR 101. The phytophthora capsici recombinant gene RxLRnudix has the full length of 231bp and encodes 77 amino acids.

4. Analysis of RxLR101 expression pattern: the cDNA of the phytophthora capsici standard strain LT1534 at each infection period in the pepper is respectively extracted as a template, the cDNA of the non-infected phytophthora capsici standard strain LT1534 mycelium period is used as a control, Pcayn is used as a reference, and the expression levels of RxLR101 at 1.5h, 3h, 6h, 12h, 24h, 48h and 72h are detected, and the results are shown in FIG. 2, and show that the expression level of RxLR101 is up-regulated at the early infection period and is highest at 3 h.

5. Construction of expression vectors

Using pEASY-T3-RxLR101 as a template, pBIN-RxLR101-F-Kpnl and pBIN-RxLR101-R-XbaI as primers, amplifying RxLR101 mature gene, performing electrophoresis on the amplified product, recovering to obtain a gel recovered product, performing enzyme digestion on the gel recovered product and a pBIN-GFP2 vector by using KpnI and XbaI endonucleases, and then performing connection by using Solution I. The recombinant pBIN-GFP2 vector containing the mature gene of RxLR101 was obtained and named pBIN-GFP2-RxLR101 recombinant vector. pBIN-GFP2-RxLR101 is a recombinant expression vector obtained by replacing a fragment between KpnI and XbaI recognition sites of pBIN-GFP2 with the RxLR101 mature gene (a mature protein consisting of 16 th to 329 th amino acids in the sequence 3 in the sequence table) and keeping other sequences of pBIN-GFP2 unchanged.

The RxLR101-P.i mature gene was amplified using pEASY-T3-RxLR101-P.i as a template and pBIN-RxLR101-P.i-F-Kpnl and pBIN-RxLR101-P.i-R-XbaI as primers, the amplified product was electrophoresed and recovered to obtain a gel recovery product, and the gel recovery product and pBIN-GFP2 vector were digested with KpnI and XbaI endonucleases and ligated with Solutioni. The pBIN-GFP2 recombinant vector containing the mature gene of RxLR101-P.i was obtained and named pBIN-GFP2-RxLR101-P.i recombinant vector. pBIN-GFP2-RxLR101-P.i is a recombinant expression vector obtained by replacing the fragment between the KpnI and XbaI recognition sites of pBIN-GFP2 with the RxLR101-P.i mature gene (encoding the mature protein consisting of amino acids 18-271 of the sequence of RxLR101-P.i in FIG. 1) while keeping the other sequences of pBIN-GFP2 unchanged.

The mature gene of-RxLR 101-P.p was amplified using pEASY-T3-RxLR101-P.p as a template and pBIN-RxLR101-P.p-F-Kpnl and pBIN-RxLR101-P.p-R-XbaI as primers, the amplified product was electrophoresed and recovered to obtain a gel recovery product, and the gel recovery product and the pBIN-GFP2 vector were digested with KpnI and XbaI endonucleases and then ligated with Solutioni. The pBIN-GFP2 recombinant vector containing the mature gene of RxLR101-P.p was obtained and named pBIN-GFP2-RxLR101-P.p recombinant vector. pBIN-GFP2-RxLR101-P.p is a recombinant expression vector obtained by replacing the fragment between KpnI and XbaI recognition sites of pBIN-GFP2 with the mature RxLR101-P.p gene (mature protein consisting of amino acids 18-359 of the sequence of RxLR101-P.p in FIG. 1) while keeping the other sequences of pBIN-GFP2 unchanged.

Using pEASY-T3-RxLR101 as a template, using pBIN-RxLR101-NLS-F-Kpnl and pBIN-RxLR101-NLS-R-Kpnl as primers, amplifying RxLR101 mature gene, carrying out electrophoresis on the amplified product, recovering the product to obtain a gel recovered product, carrying out enzyme digestion on the gel recovered product and a pBIN-GFP2 vector by using KpnI and BamHI endonuclease, and then connecting by using Solutioni. The recombinant vector pBIN-GFP2 containing the RxLR101NLS fusion protein coding gene was obtained and named pBIN-GFP2-RxLR101-NLS recombinant vector. pBIN-GFP2-RxLR101-NLS is a recombinant expression vector obtained by replacing a fragment between KpnI and BamHI recognition sites of pBIN-GFP2 with a mature gene (a fusion protein consisting of amino acids 16 to 340 in the sequence 7 of the sequence list) of RxLR101-NLS, and keeping the other sequences of pBIN-GFP2 unchanged.

Using pEASY-T3-RxLR101 as a template, pBIN-RxLR101NES-F-Kpnl and pBIN-RxLR101NES-R-Kpnl as primers, amplifying RxLR101 mature gene, performing electrophoresis on the amplified product, recovering to obtain a gel recovered product, performing enzyme digestion on the gel recovered product and pBIN-GFP2 vector by using KpnI and BamHI endoenzyme, and then performing ligation by using Solution I. The recombinant vector pBIN-GFP2 containing the RxLR101NES fusion protein-encoding gene was obtained and named pBIN-GFP2-RxLR101 NES. pBIN-GFP2-RxLR101NES is a recombinant expression vector obtained by replacing the fragment between KpnI and BamHI recognition sites of pBIN-GFP2 with the mature gene of RxLR101NES (a fusion protein consisting of amino acids 16-343 in sequence 11 of the sequence listing) and keeping the other sequences of pBIN-GFP2 unchanged.

The RxLRnudix DNA is used as a template, pBIN-RxLRnudix-F-Kpnl and pBIN-RxLRnudix-R-Kpnl are used as primers, RxLRnudix mature genes are amplified, the amplified products are subjected to electrophoresis and recovery to obtain gel recovery products, the gel recovery products and a pBIN-GFP2 vector are subjected to enzyme digestion by using KpnI and BamHI endonucleases, and then are connected by using Solution I. The recombinant vector pBIN-GFP2 containing the RxLRnudix mature protein coding gene was obtained and named pBIN-GFP2-RxLRnudix recombinant vector. pBIN-GFP2-RxLRnudix is a recombinant expression vector obtained by replacing a fragment between KpnI and BamHI recognition sites of pBIN-GFP2 with a mature gene (a fusion protein consisting of amino acids of sequence 1 in the sequence listing) carrying RxLRnudix, and keeping other sequences of pBIN-GFP2 unchanged.

The RxLRnudix DNA is used as a template, pBIN-RxLRnudix-NLS-F-Kpnl and pBIN-RxLRnudix-NLS-R-Kpnl are used as primers, RxLRnudix mature genes are amplified, the amplified products are subjected to electrophoresis and recovery to obtain gel recovery products, the gel recovery products and the pBIN-GFP2 vector are subjected to enzyme digestion by using Kpn I and BamH I endonuclease, and then are connected by using Solutioni. The recombinant vector pBIN-GFP2 containing the RxLRnudiNLS fusion protein coding gene is obtained and named as the recombinant vector pBIN-GFP 2-RxLRnudix-NLS. pBIN-GFP2-RxLRnudix-NLS is a recombinant expression vector obtained by replacing a fragment between Kpn I and BamH I recognition sites of pBIN-GFP2 with a mature gene (a fusion protein consisting of amino acids 1 to 88 in the sequence 5 in the sequence table) carrying RxLRnudix-NLS, and keeping other sequences of pBIN-GFP2 unchanged.

Using RxLRnudix DNA as a template, using pBIN-RxLRnudixNES-F-Kpnl and pBIN-RxLRnudixNES-R-Kpnl as primers, amplifying RxLRnudix mature genes, carrying out electrophoresis on amplified products, recovering to obtain gel recovered products, carrying out enzyme digestion on the gel recovered products and a pBIN-GFP2 carrier by using Kpn I and BamH I endonucleases, and then connecting by using Solutioni I. The recombinant vector pBIN-GFP2 containing the RxLRnudiNES fusion protein coding gene is obtained, and the recombinant vector is named as pBIN-GFP 2-RxLRnudiNES. pBIN-GFP2-RxLRnudixNES is a recombinant expression vector obtained by replacing a fragment between Kpn I and BamHI recognition site recognition sites of pBIN-GFP2 with a mature gene of RneuxNES (a fusion protein consisting of amino acids 1 to 91 in the sequence 9 of the sequence list) and keeping other sequences of pBIN-GFP2 unchanged.

6. Agrobacterium mediated phytophthora capsici RxLR effector molecule transient expression

Construction of transient expression vectors

The prepared recombinant vector pBIN-GFP2-RxLR101 is introduced into the competence of Agrobacterium GV3101 to obtain recombinant Agrobacterium, which is named as Agrobacterium-RxLR 101.

The prepared recombinant vector pBIN-GFP2-RxLRnudix is introduced into the competence of Agrobacterium GV3101 to obtain recombinant Agrobacterium, which is named as Agrobacterium-RxLRnudix.

The prepared recombinant vector pBIN-GFP2-RxLR101-P.i is introduced into the competence of Agrobacterium GV3101 to obtain recombinant Agrobacterium, which is named as Agrobacterium-RxLR 101-P.i.

The prepared recombinant vector pBIN-GFP2-RxLR101-P.p is introduced into the competence of Agrobacterium GV3101 to obtain recombinant Agrobacterium, which is named as Agrobacterium-RxLR 101-P.p.

The prepared recombinant vector pBIN-GFP2-RxLR101NLS is introduced into the competence of Agrobacterium GV3101 to obtain recombinant Agrobacterium, which is named as Agrobacterium-RxLR 101 NLS.

The prepared recombinant vector pBIN-GFP2-RxLR8-2 is introduced into the competence of Agrobacterium GV3101 to obtain recombinant Agrobacterium, which is named Agrobacterium-RxLR 101 NES.

The prepared recombinant vector pBIN-GFP2-RxLRnudix-NLS is introduced into the competence of Agrobacterium GV3101 to obtain recombinant Agrobacterium, which is named as Agrobacterium-RxLRnudix-NLS.

The prepared recombinant vector pBIN-GFP 2-RxLRnudiNES is introduced into the competence of Agrobacterium GV3101 to obtain recombinant Agrobacterium, which is named as Agrobacterium-RxLRnudiNES.

7. WesternBlot detection of effector genes: agrobacterium containing the empty vector pBIN-GFP2, Agrobacterium prepared as described above (Agrobacterium-RxLR 101, Agrobacterium-RxLRnudix, Agrobacterium-RxLR 101-P.i, Agrobacterium-RxLR 101-P.p, Agrobacterium-RxLR 101NLS, Agrobacterium-RxLR 101NES, Agrobacterium-RxLRnudix-NLS, and Agrobacterium-RxLRnudixNES) were injected into Benyan-and Capsici fructus leaves, respectively, to transiently express the effector gene and the fusion protein with pBIN-GFP2, to obtain Agrobacterium-inoculated Benyan-and Capsici fructus leaves. Extracting protein of the leaf, carrying out Western Blot detection experiment, and using primary antibody: pBIN-GFP2, secondary antibody: goat anti-mouse-HRP is detected, and the internal reference adopts an Action gene. Western blot results show that target bands with sizes similar to predicted proteins of RxLR101, RxLRnudix, RxLR101-P.i, RxLR101-P.p, RxLR101NLS, RxLR101NES, RxLRnudix-NLS and RxLRnudixES can be detected on PVDF membrane, and that the effector factors can be correctly expressed in Nicotiana capsici.

Example 2

1. Influence on the necrosis of Bunshi tobacco leaf cells

With no loading of GFP, INF1, MgCl₂For comparison, the expression of RxLRnudix, RxLR101-P.i and RxLR101-P.p transiently on Nicotiana benthamiana was analyzed for observation. Agrobacterium containing the empty vector pBIN-GFP2 and Agrobacterium-RxLRnudix, Agrobacterium-RxLR 101-P.i and Agrobacterium-RxLR 101-P.p of example 1 were inoculated onto Bentonium tabacum leaves, which were then photographed and recorded for eligibility, stained with trypan blue staining solution and decolorized with chloral hydrate and alcohol. The results are shown in FIG. 3, in which FIG. 3a shows the symptoms of leaves after transient expression of the response factor RxLRnudix, A represents RxLR101, B represents RxLRnudix, and C represents buffer; d represents the empty vector pBIN-GFP2, and E represents INF 1. FIG. 3b is a photograph of leaf blades after transient expression of the response factor RxLR101-P.i, wherein A represents unloaded pBIN-GFP 2; b represents RxLR101-P.i, RxLR 101-P.p; c represents buffer; d represents INF 1; FIG. 3c is a photograph of leaf blades after transient expression of the response factor RxLR101-P.p, wherein A represents unloaded pBIN-GFP 2; b represents RxLR 101-P.p; c represents buffer; d represents INF 1. Compared with a control, the inoculation result shows that the RxLR101-P.p generates the phenomena of shrinkage and greening of the inoculation site after inoculation for 48 hours, and the inoculation site generates obvious cell death after 7 days; RxLR101-P.i was not characterized significantly after 7d inoculation and did not cause cell death in Nicotiana benthamiana.

2. Phytophthora capsici RxLR effector molecules inhibit Bax-induced cell death

Prepared from pBIN-GFP2, INF1 and MgCl₂As a control with RxLR101, etc., the expression of RxLRnudix, RxLR101-P.i, and RxLR101-P.p transiently on Nicotiana benthamiana was observed and analyzed. Agrobacterium containing the empty vector pBIN-GFP2 and Agrobacterium-RxLRnudix, Agrobacterium-RxLR 101-P.i and Agrobacterium-RxLR 101-P.p of example 1 were inoculated onto Hemsley tobacco leaves, when RxLRnudix was transiently expressed for 24 hours, necrosis gene Bax was inoculated, inhibition function of RxLRnudix was verified, and then the qualified Hemsley tobacco leaves were photographed and recorded, and stained with trypan blue staining solution,the decolorization treatment was carried out using chloral hydrate and alcohol, and the results are shown in FIG. 4. In FIG. 4a, A represents RxLR101, B represents RxLR101-P.p, RxLR101-P.i, C represents buffer, and D represents empty vector pBIN-GFP2, respectively. The right panel shows trypan blue staining results. FIG. 4B is a graph of inhibition assay of RxLRnudix, A for RxLR101, B for RxLRnudix, and C for buffer; d represents the empty vector pBIN-GFP2, and E represents INF 1. The right panel shows trypan blue staining results.

As shown, RxLRnudix was unable to induce cell necrosis on tobacco, and upon transient expression of RxLRnudix24h, inoculation with Bax found that RxLRnudix was still able to inhibit Bax-induced PCD.

Example 3 Regulation of Phytophthora capsici infection ability of plants

Infection of tobacco leaves experiment:

tobacco leaves of Benzilian inoculated with Agrobacterium (control), Agrobacterium-RxLR 101 and Agrobacterium-RxLRnudix containing the empty vector pBIN-GFP2 were prepared according to the method in step 2, inoculated with zoospores after transient expression for 24h, and observed for 3d to see whether the pathogenicity of Phytophthora was affected, the results are shown in FIG. 5. qRT-PCR was used to detect the expression levels at different stages. The qRT-PCR results are shown in FIG. 6.

The RxLRnudix is verified on tobacco by taking pBIN-GFP2 and RxLR101 as a contrast, and an experimental result shows that the RxLRnudix can inhibit the infection of phytophthora capsici on the tobacco. The qPCR detection result shows that both RxLR101 and-RxLRnudix can be normally expressed in the embodiment.

Example 4

1. Inhibition assays for fusion proteins RxLR101-NES, RxLR101-NLS, RxLRnudix-NES, RxLRnudix-NLS

Prepared from pBIN-GFP2, INF1 and MgCl₂By way of control, 4 effector factors of RxLR101-NES, RxLR101-NLS, RxLRnudix-NES and RxLRnudix-NLS are transiently expressed on pepper leaves, and after 24 hours of transient expression, Agrobacterium Bax is inoculated for observation and analysis. As shown in FIG. 7, A represents RxLR101-NES, RxLRnudix-NES, B represents RxLR101-NLS, RxLRnudix-NLS, C RxLR101, D-unloaded pBIN-GFP2, E represents RxLRnudix, and F represents buffer, respectively. The right panel shows trypan blue staining results. As can be seen from FIG. 7, the transients areAfter Bax7d expression, RxLR101-NES and RxLRnudix-NES still inhibited Bax-induced PCD, but neither RxLR101-NLS nor RxLRnudix-NLS inhibited Bax-induced PCD. It was demonstrated that RxLR101, RxLRnudix inhibited Bax-induced PCD in the cytoplasm and, if RxLR101, RxLRnudix were imported into the nucleus, Bax-induced PCD could no longer be inhibited, thus allowing Bax to function normally.

Example 5

Effect of RxLR101-NES, RxLR101-NLS, RxLRnudix-NES, RxLRnudix-NLS on Phytophthora capsici pathogenicity

Prepared from pBIN-GFP2, INF1 and MgCl₂By contrast, 4 effector factors of RxLR101-NES, RxLR101-NLS, RxLRnudix-NES and RxLRnudix-NLS are transiently expressed on pepper leaves, and after the transient expression is carried out for 24 hours (the transient expression method is the same as that of the agrobacterium in examples 1-4 and is not repeated here), the zoospore suspension of the prepared phytophthora capsici SD33 strain is dripped in the center of the same inoculated leaves (the zoospore amount of each leaf is consistent), then the leaves are placed in an incubator at 25 ℃ for dark treatment, and after 72 hours, the tobacco leaves are taken out, photographed under an ultraviolet lamp, photographed under a white light lamp and finally stained with trypan blue staining solution, and the staining result is shown in FIGS. 8 and 10.

qRT-PCR was used to detect the expression levels at different stages. The results of qRT-PCR are shown in FIG. 9 and FIG. 11.

The results are shown in the figure, using pBIN-GFP2, INF1, MgCl₂In contrast, RxLR101-NES and RxLRnudix-NES can hardly inhibit phytophthora capsici zoospore infection any more, and compared with positive controls RxLR101 and RxLRnudix, RxLR101-NLS and RxLRnudix-NLS can inhibit phytophthora capsici zoospore infection more strongly. Therefore, the function of RxLR101 and RxLRnudix for inhibiting phytophthora capsici zoospore infection is performed in the cell nucleus, and cytoplasm can not influence the pathogenicity of phytophthora capsici.

Sequence listing

<110> Shandong university of agriculture

<120> protein related to inhibition of plant leaf necrosis caused by Bax and application thereof

<160>12

<170>SIPOSequenceListing 1.0

<210>1

<211>77

<212>PRT

<213> Phytophthora capsici (Phytophthora capsicii)

<400>1

Gln Leu Ile Ala Ala Asn Ala Asp Arg Thr Gly Ile Ala Ala Val Asp

1 5 10 15

Ser Asn Thr Ala Leu Leu Pro Arg Val Leu Gly Val Glu Ser Lys Arg

20 25 30

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

35 40 45

Asp Ser Asp Glu Glu Ala Gly Gly Trp Asp His Gly Glu Thr Ile Glu

50 55 60

Lys Ala Val Leu Arg Glu Val Ile Glu Glu Gly Gly Val

65 70 75

<210>2

<211>231

<212>DNA

<213> Phytophthora capsici (Phytophthora capsicii)

<400>2

caattgatcg ccgccaacgc ggaccgaacc ggcattgcgg ctgtcgattc gaacactgcg 60

ctccttcctc gcgtgctagg agtcgaatcc aagcgtactc tgcgtcggta tgaccccagt 120

gagtttgatt cagaagaagc ggtcgattca gatgaggagg ccgggggctg ggatcatgga 180

gaaaccattg agaaggcagt tttgcgtgag gttattgaag aaggaggggt c 231

<210>3

<211>329

<212>PRT

<213> Phytophthora capsici (Phytophthora capsicii)

<400>3

Met Arg Val Leu Ser Leu Val Thr Leu Leu Ala Phe Val Ser Ala Gln

1 5 10 15

Leu Ile Ala Ala Asn Ala Asp Arg Thr Gly Ile Ala Ala Val Asp Ser

20 25 30

Asn Thr Ala Leu Leu Pro Arg Val Leu Gly Val Glu Ser Lys Arg Thr

35 40 45

Leu Arg Arg Tyr Asp Pro Ser Glu Phe Asp Ser Glu Glu Ala Val Asp

50 55 60

Ser Asp Glu Glu Ala Asp Pro Val Glu Val Ala Asp Ser Asp Glu Glu

65 70 7580

Val Val Ser Glu Gly Glu Glu Arg Val Gly Ile Pro Gly Met Glu Lys

85 90 95

Val Ala Ser Lys Ala Thr Lys Ala Asp Asp Met Val Pro Asn Ala Ala

100 105 110

Lys Gly Ala Met Lys Trp Lys Ile Leu Val Gln Lys Asn Met Gly Gln

115 120 125

Leu Ala Glu Thr Ala Lys Leu Val Lys Lys Leu Lys Val Gly Ser Phe

130 135 140

Tyr Ser Asn Val Glu Leu Glu Lys Met Ser Leu Ser Ala Leu Arg Gln

145 150 155 160

Leu Asp Asp Ile Gln Gln Leu Gln Lys Ala Asp Ile Lys Ser Asn Val

165 170 175

Phe Gly Thr Lys Ala Thr Ala Asn Gly Met Arg Arg Lys Met Thr Arg

180 185 190

Thr Glu Asn Met Lys Leu Pro Pro Glu Gln Phe Leu Val Ser His Val

195 200 205

Gly Arg Gly Ala Gln Arg Leu Gly Glu Asn Gly Gln Arg Leu Leu Ser

210 215 220

Ala Ala Val Ile Ser Lys Gly Asp Asp Ile Asn Ser Lys Val Leu Leu

225 230 235 240

Ile Ser Ser Ser Asp Thr Lys Lys Gly Asp Phe Leu Leu Pro Lys Gly

245 250 255

Gly Trp Asp His Gly Glu Thr Ile Glu Lys Ala Val Leu Arg Glu Val

260 265 270

Ile Glu Glu Gly Gly Val Asn Gly Gln Leu Leu His Lys Leu Gly Glu

275 280 285

Tyr Pro Phe Lys Lys Gly Ala Thr Ala Tyr Ala Tyr Met Met Lys Ala

290 295 300

Ser Thr Val Tyr Asp Asp Trp Ala Glu Ser Ile Arg Tyr Arg Ile Trp

305 310 315 320

Val Arg Thr Glu Met Leu Trp Lys Tyr

325

<210>4

<211>990

<212>DNA

<213> Phytophthora capsici (Phytophthora capsicii)

<400>4

atgagagttc tttcgctcgt gactttgctc gcattcgtat ctgcccaatt gatcgccgcc 60

aacgcggacc gaaccggcat tgcggctgtc gattcgaaca ctgcgctcct tcctcgcgtg 120

ctaggagtcg aatccaagcg tactctgcgt cggtatgacc ccagtgagtt tgattcagaa 180

gaagcggtcg attcagatga ggaggccgat cccgttgagg tggctgattc agatgaggaa 240

gttgtttccg aaggtgaaga gagagtcggg atccccggca tggagaaagt tgcctccaag 300

gcaaccaaag cggacgacat ggtgcccaat gcagccaagg gggcgatgaa gtggaaaatt 360

ctggtgcaga aaaacatggg tcagcttgct gagactgcta agctggtgaa aaagctcaag 420

gttggcagtt tctacagtaa cgtagagctg gagaaaatga gcttgtcggc gctgaggcag 480

ctggacgaca tccaacagct ccagaaggct gatatcaaaa gcaatgtctt tggaaccaag 540

gcaactgcta acggaatgcg taggaaaatg acgcgcactg agaacatgaa gctccctcct 600

gagcagttcc tggtgtcgca cgttggtcgc ggagcacaac gcttgggcga gaacgggcag 660

cgcctcttgt cggctgctgt catttctaag ggcgatgaca tcaatagcaa ggtgctcttg 720

atttcgagtt cagacacaaa gaagggcgac ttcctgttgc ctaagggggg ctgggatcat 780

ggagaaacca ttgagaaggc agttttgcgt gaggttattg aagaaggagg ggtcaatggt 840

cagctactcc ataagcttgg agagtacccg tttaaaaagg gtgccaccgc ttacgcttac 900

atgatgaagg cttctacggt ttatgacgat tgggccgaga gtattcgcta tcgtatctgg 960

gtacgtactg aaatgttatg gaagtattga 990

<210>5

<211>88

<212>PRT

<213> Phytophthora capsici (Phytophthora capsicii)

<400>5

Gln Leu Ile Ala Ala Asn Ala Asp Arg Thr Gly Ile Ala Ala Val Asp

1 5 10 15

Ser Asn Thr Ala Leu Leu Pro Arg Val Leu Gly Val Glu Ser Lys Arg

20 25 30

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

35 40 45

Asp Ser Asp Glu Glu Ala Gly Gly Trp Asp His Gly Glu Thr Ile Glu

50 55 60

Lys Ala Val Leu Arg Glu Val Ile Glu Glu Gly Gly Val Ser Leu Tyr

65 70 75 80

Leu Ser Phe Leu Leu Arg Ser Ser

85

<210>6

<211>264

<212>DNA

<213> Phytophthora capsici (Phytophthora capsicii)

<400>6

caattgatcg ccgccaacgc ggaccgaacc ggcattgcgg ctgtcgattc gaacactgcg 60

ctccttcctc gcgtgctagg agtcgaatcc aagcgtactc tgcgtcggta tgaccccagt 120

gagtttgatt cagaagaagc ggtcgattca gatgaggagg ccgggggctg ggatcatgga 180

gaaaccattg agaaggcagt tttgcgtgag gttattgaag aaggaggggt ctcactttac 240

ctttcgtttc ttcttcggag ctcc 264

<210>7

<211>340

<212>PRT

<213> Phytophthora capsici (Phytophthora capsicii)

<400>7

Met Arg Val Leu Ser Leu Val Thr Leu Leu Ala Phe Val Ser Ala Gln

1 510 15

Leu Ile Ala Ala Asn Ala Asp Arg Thr Gly Ile Ala Ala Val Asp Ser

20 25 30

Asn Thr Ala Leu Leu Pro Arg Val Leu Gly Val Glu Ser Lys Arg Thr

35 40 45

Leu Arg Arg Tyr Asp Pro Ser Glu Phe Asp Ser Glu Glu Ala Val Asp

50 55 60

Ser Asp Glu Glu Ala Asp Pro Val Glu Val Ala Asp Ser Asp Glu Glu

65 70 75 80

Val Val Ser Glu Gly Glu Glu Arg Val Gly Ile Pro Gly Met Glu Lys

85 90 95

Val Ala Ser Lys Ala Thr Lys Ala Asp Asp Met Val Pro Asn Ala Ala

100 105 110

Lys Gly Ala Met Lys Trp Lys Ile Leu Val Gln Lys Asn Met Gly Gln

115 120 125

Leu Ala Glu Thr Ala Lys Leu Val Lys Lys Leu Lys Val Gly Ser Phe

130 135 140

Tyr Ser Asn Val Glu Leu Glu Lys Met Ser Leu Ser Ala Leu Arg Gln

145 150 155 160

Leu Asp Asp Ile Gln Gln Leu Gln Lys Ala Asp Ile Lys Ser Asn Val

165 170175

Phe Gly Thr Lys Ala Thr Ala Asn Gly Met Arg Arg Lys Met Thr Arg

180 185 190

Thr Glu Asn Met Lys Leu Pro Pro Glu Gln Phe Leu Val Ser His Val

195 200 205

Gly Arg Gly Ala Gln Arg Leu Gly Glu Asn Gly Gln Arg Leu Leu Ser

210 215 220

Ala Ala Val Ile Ser Lys Gly Asp Asp Ile Asn Ser Lys Val Leu Leu

225 230 235 240

Ile Ser Ser Ser Asp Thr Lys Lys Gly Asp Phe Leu Leu Pro Lys Gly

245 250 255

Gly Trp Asp His Gly Glu Thr Ile Glu Lys Ala Val Leu Arg Glu Val

260 265 270

Ile Glu Glu Gly Gly Val Asn Gly Gln Leu Leu His Lys Leu Gly Glu

275 280 285

Tyr Pro Phe Lys Lys Gly Ala Thr Ala Tyr Ala Tyr Met Met Lys Ala

290 295 300

Ser Thr Val Tyr Asp Asp Trp Ala Glu Ser Ile Arg Tyr Arg Ile Trp

305 310 315 320

Val Arg Thr Glu Met Leu Trp Lys Tyr Ser Leu Tyr Leu Ser Phe Leu

325 330335

Leu Arg Ser Ser

340

<210>8

<211>1020

<212>DNA

<213> Phytophthora capsici (Phytophthora capsicii)

<400>8

atgagagttc tttcgctcgt gactttgctc gcattcgtat ctgcccaatt gatcgccgcc 60

aacgcggacc gaaccggcat tgcggctgtc gattcgaaca ctgcgctcct tcctcgcgtg 120

ctaggagtcg aatccaagcg tactctgcgt cggtatgacc ccagtgagtt tgattcagaa 180

gaagcggtcg attcagatga ggaggccgat cccgttgagg tggctgattc agatgaggaa 240

gttgtttccg aaggtgaaga gagagtcggg atccccggca tggagaaagt tgcctccaag 300

gcaaccaaag cggacgacat ggtgcccaat gcagccaagg gggcgatgaa gtggaaaatt 360

ctggtgcaga aaaacatggg tcagcttgct gagactgcta agctggtgaa aaagctcaag 420

gttggcagtt tctacagtaa cgtagagctg gagaaaatga gcttgtcggc gctgaggcag 480

ctggacgaca tccaacagct ccagaaggct gatatcaaaa gcaatgtctt tggaaccaag 540

gcaactgcta acggaatgcg taggaaaatg acgcgcactg agaacatgaa gctccctcct 600

gagcagttcc tggtgtcgca cgttggtcgc ggagcacaac gcttgggcga gaacgggcag 660

cgcctcttgt cggctgctgt catttctaag ggcgatgaca tcaatagcaa ggtgctcttg 720

atttcgagtt cagacacaaa gaagggcgac ttcctgttgc ctaagggggg ctgggatcat 780

ggagaaacca ttgagaaggc agttttgcgt gaggttattg aagaaggagg ggtcaatggt 840

cagctactcc ataagcttgg agagtacccg tttaaaaagg gtgccaccgc ttacgcttac 900

atgatgaagg cttctacggt ttatgacgat tgggccgaga gtattcgcta tcgtatctgg 960

gtacgtactg aaatgttatg gaagtattca ctttaccttt cgtttcttct tcggagctcc 1020

<210>9

<211>91

<212>PRT

<213> Phytophthora capsici (Phytophthora capsicii)

<400>9

Gln Leu Ile Ala Ala Asn Ala Asp Arg Thr Gly Ile Ala Ala Val Asp

1 5 10 15

Ser Asn Thr Ala Leu Leu Pro Arg Val Leu Gly Val Glu Ser Lys Arg

20 25 30

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

35 40 45

Asp Ser Asp Glu Glu Ala Gly Gly Trp Asp His Gly Glu Thr Ile Glu

50 55 60

Lys Ala Val Leu Arg Glu Val Ile Glu Glu Gly Gly Val Asn Glu Leu

65 70 75 80

Ala Leu Lys Leu Ala Gly Leu Asp Ile Asn Lys

85 90

<210>10

<211>276

<212>DNA

<213> Phytophthora capsici (Phytophthora capsicii)

<400>10

caattgatcg ccgccaacgc ggaccgaacc ggcattgcgg ctgtcgattc gaacactgcg 60

ctccttcctc gcgtgctagg agtcgaatcc aagcgtactc tgcgtcggta tgaccccagt 120

gagtttgatt cagaagaagc ggtcgattca gatgaggagg ccgggggctg ggatcatgga 180

gaaaccattg agaaggcagt tttgcgtgag gttattgaag aaggaggggt caacgagctt 240

gctcttaagt tggctggact tgatattaac aagtag 276

<210>11

<211>343

<212>PRT

<213> Phytophthora capsici (Phytophthora capsicii)

<400>11

Met Arg Val Leu Ser Leu Val Thr Leu Leu Ala Phe Val Ser Ala Gln

1 5 10 15

Leu Ile Ala Ala Asn Ala Asp Arg Thr Gly Ile Ala Ala Val Asp Ser

20 25 30

Asn Thr Ala Leu Leu Pro Arg Val Leu Gly Val Glu Ser Lys Arg Thr

35 40 45

Leu Arg Arg Tyr Asp Pro Ser Glu Phe Asp Ser Glu Glu Ala Val Asp

50 55 60

Ser Asp Glu Glu Ala Asp Pro Val Glu Val Ala Asp Ser Asp Glu Glu

65 70 75 80

Val Val Ser Glu Gly Glu Glu Arg Val Gly Ile Pro Gly Met Glu Lys

85 90 95

Val Ala Ser Lys Ala Thr Lys Ala Asp Asp Met Val Pro Asn Ala Ala

100 105 110

Lys Gly Ala Met Lys Trp Lys Ile Leu Val Gln Lys Asn Met Gly Gln

115 120 125

Leu Ala Glu Thr Ala Lys Leu Val Lys Lys Leu Lys Val Gly Ser Phe

130 135 140

Tyr Ser Asn Val Glu Leu Glu Lys Met Ser Leu Ser Ala Leu Arg Gln

145 150 155 160

Leu Asp Asp Ile Gln Gln Leu Gln Lys Ala Asp Ile Lys Ser Asn Val

165 170 175

Phe Gly Thr Lys Ala Thr Ala Asn Gly Met Arg Arg Lys Met Thr Arg

180 185 190

Thr Glu Asn Met Lys Leu Pro Pro Glu Gln Phe Leu Val Ser His Val

195 200 205

Gly Arg Gly Ala Gln Arg Leu Gly Glu Asn Gly Gln Arg Leu Leu Ser

210 215 220

Ala Ala Val Ile Ser Lys Gly Asp Asp Ile Asn Ser Lys Val Leu Leu

225 230 235 240

Ile Ser Ser Ser Asp Thr Lys Lys Gly Asp Phe Leu Leu Pro Lys Gly

245 250 255

Gly Trp Asp His Gly Glu Thr Ile Glu Lys Ala Val Leu Arg Glu Val

260 265 270

Ile Glu Glu Gly Gly Val Asn Gly Gln Leu Leu His Lys Leu Gly Glu

275 280 285

Tyr Pro Phe Lys Lys Gly Ala Thr Ala Tyr Ala Tyr Met Met Lys Ala

290 295 300

Ser Thr Val Tyr Asp Asp Trp Ala Glu Ser Ile Arg Tyr Arg Ile Trp

305 310 315 320

Val Arg Thr Glu Met Leu Trp Lys Tyr Asn Glu Leu Ala Leu Lys Leu

325 330 335

Ala Gly Leu Asp Ile Asn Lys

340

<210>12

<211>1032

<212>DNA

<213> Phytophthora capsici (Phytophthora capsicii)

<400>12

atgagagttc tttcgctcgt gactttgctc gcattcgtat ctgcccaatt gatcgccgcc 60

aacgcggacc gaaccggcat tgcggctgtc gattcgaaca ctgcgctcct tcctcgcgtg 120

ctaggagtcg aatccaagcg tactctgcgt cggtatgacc ccagtgagtt tgattcagaa 180

gaagcggtcg attcagatga ggaggccgat cccgttgagg tggctgattc agatgaggaa 240

gttgtttccg aaggtgaaga gagagtcggg atccccggca tggagaaagt tgcctccaag 300

gcaaccaaag cggacgacat ggtgcccaat gcagccaagg gggcgatgaa gtggaaaatt 360

ctggtgcaga aaaacatggg tcagcttgct gagactgcta agctggtgaa aaagctcaag 420

gttggcagtt tctacagtaa cgtagagctg gagaaaatga gcttgtcggc gctgaggcag 480

ctggacgaca tccaacagct ccagaaggct gatatcaaaa gcaatgtctt tggaaccaag 540

gcaactgcta acggaatgcg taggaaaatg acgcgcactg agaacatgaa gctccctcct 600

gagcagttcc tggtgtcgca cgttggtcgc ggagcacaac gcttgggcga gaacgggcag 660

cgcctcttgt cggctgctgt catttctaag ggcgatgaca tcaatagcaa ggtgctcttg 720

atttcgagtt cagacacaaa gaagggcgac ttcctgttgc ctaagggggg ctgggatcat 780

ggagaaacca ttgagaaggc agttttgcgt gaggttattg aagaaggagg ggtcaatggt 840

cagctactcc ataagcttgg agagtacccg tttaaaaagg gtgccaccgc ttacgcttac 900

atgatgaagg cttctacggt ttatgacgat tgggccgaga gtattcgcta tcgtatctgg 960

gtacgtactg aaatgttatg gaagtataac gagcttgctc ttaagttggc tggacttgat 1020

attaacaagt ag 1032

Claims

1. The protein is the protein of A1), A2), A3) or A4) as follows:

2. The protein of claim 1, wherein a2) is a21 or a22 wherein:

a21, protein with amino acid sequence shown as sequence 3;

a22, the amino acid sequence of which is shown as 16 th to 329 th positions in the sequence 3.

3. The protein of claim 1, wherein the fusion protein in A4) is A41, A42, A43 or A44, wherein:

a41, protein with amino acid sequence shown as sequence 5;

a42, protein with amino acid sequence shown as sequence 9;

a43, protein with amino acid sequence shown as sequence 7;

a45, protein with amino acid sequence shown as sequence 11;

a46, the amino acid sequence of which is shown as 16 th-343 th position in the sequence 11; .

4. The biomaterial related to the protein of any one of claims 1 to 3, which is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding the protein of any one of claims 1-3;

B2) an expression cassette comprising the nucleic acid molecule of B1);

5. The related biological material according to claim 4, wherein: B1) the nucleic acid molecule has the following nucleotides of B101, B102, B103, B104, B105, B106, B107, B108, B109 and B109:

b101, cDNA molecules or DNA molecules of a sequence 2 in a sequence table;

b102, cDNA molecules or DNA molecules of a sequence 10 in a sequence table;

b103, cDNA molecules or DNA molecules of a sequence 4 in a sequence table;

b105, cDNA molecules or DNA molecules of a sequence 6 in a sequence table;

b106, cDNA molecules or DNA molecules of a sequence 8 in the sequence table;

b108, cDNA molecules or DNA molecules of a sequence 12 in a sequence table;

6. A method for inhibiting phytophthora capsici infection in a plant, comprising introducing into the plant a recombinant vector comprising B101, B105, B106 or B107 as defined in claim 5 or introducing into the plant a recombinant microorganism comprising B101, B105, B106 or B107 as defined in claim 5.

7. The method of inhibiting phytophthora capsici from infecting a plant according to claim 6, for use in regulating phytophthora capsici infection in a plant, preparing a product for the plant to resist phytophthora capsici infection, growing a plant to resist phytophthora capsici infection, or breeding a plant.

8. Use of any one of the following P1-P9 of the protein of any one of claims 1-3, or the biomaterial of claim 4 or 5:

use of P1, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for modulating leaf cell necrosis in a plant;

use of P2, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 in the manufacture of a product for reducing necrosis of plant leaves;

use of P3, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for the cultivation of a plant for the reduction of leaf necrosis in a plant;

use of P4, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for the manufacture of a product for reducing leaf necrosis in a plant;

use of P5, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for modulating phytophthora capsici infestation in plants;

use of P6, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 in the manufacture of a product for increasing the resistance of a plant to phytophthora capsici infestation;

use of P7, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for growing plants resistant to phytophthora capsici infestation;

use of P8, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for the preparation of a product for combating phytophthora capsici infestation in plants;

use of P9, a protein according to any one of claims 1 to 3, or a biological material according to claim 4 or 5 in plant breeding.

9. Use according to claim 6, characterized in that: the plant of claim 6, which is a monocotyledonous plant or a dicotyledonous plant.

10. Use according to claim 6, characterized in that: the plant of claim 6, which is Nicotiana benthamiana or Capsicum annuum.