Phytophthora pini effector protein PpinAvh23 and coding gene and application thereof

Abstract

The invention discloses a phytophthora pini effector protein PpinAvh23 as well as a coding gene and application thereof, and belongs to the technical field of molecular biology. The amino acid sequence of the protein is as shown in SEQ ID NO. 1. Experiments show that the protein can specifically inhibit programmed death of plant cells induced by pathogenic bacteria elicitors, and it is proved that the protein can promote pathogen infection as a toxic effector in plants. The invention also provides a gene (SEQ ID NO.2) for coding the protein. The protein and the coding gene thereof can be used as a key molecular tool and a screening target for researching a plant-pathogen interaction mechanism, and a basis is provided for developing a new strategy for preventing and controlling plant diseases.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, and more specifically, relates to the effector protein PpiniAvh23 of Phytophthora indica and its encoding gene and applications. Background Technology

[0002] *Phytophthora pini* is one of the most destructive plant pathogenic oomycetes known, belonging to the genus *Phytophthora* in the family Pythaceae, phylum Oomycetes, class Oomycetes, order Peronomales. It has a wide host range and is widely distributed. It was isolated from the roots of the red pine (*Pinus resinosa* Aiton) in Minnesota, USA, by RG Pierce and named by Leonian in 1925. However, this species was largely neglected until 1956. To date, it has been found to cause root rot and wilt diseases in at least 18 plant species belonging to 12 genera in North America. Domestically and internationally, pesticide spraying is the main method for controlling the prevalence and damage of *Phytophthora pini*, but this has failed to achieve ideal control results, not only increasing the burden on farmers but also causing environmental pollution. The large distribution area of ​​*Phytophthora pini* and its ability to survive in soil or asymptomatic plants for several years make the control of *Phytophthora pini* diseases a significant challenge and difficulty.

[0003] Effector proteins are a class of exocrine proteins secreted by plant pathogens during their interaction with plants. These proteins alter the structure and metabolic pathways of host plant cells, thereby inhibiting successful infection or triggering a host defense response. They mainly include apoplast effectors (elicitors, the necrosis and ethylene inhibitory protein family, PcF / SCR proteins, etc.) and cytoplasmic effectors (RxLR and CRN protein families). During pathogen-host interaction, when the plant contains the corresponding resistance protein (R), the effector can trigger an immune response (ETI), known as an avirulence protein (AVR). When the plant lacks the corresponding resistance protein (R), the effector exerts its toxic function, inhibiting the immune response and inducing susceptibility (ETS). Most AVRs found in oomycetes today belong to the RxLR effector family, indicating that RxLR effectors play a crucial role in oomycete-host interactions.

[0004] The oomycete genome contains hundreds of highly differentiated RxLR effector genes, exhibiting diversity. These genes possess functions such as inhibiting cell death induced by the kinin protein INF1 secreted by pathogenic *Phytophthora indica*, or inhibiting PTI and ETI functions, as well as inducing cell death, and synergistically regulating plant defense responses. Examples include *Phytophthora camphorata* effector proteins Avh29, Avh49, and Avh57. However, research on the pathogenic mechanism of *Phytophthora pineensis* effectors is limited, and the structures of effector proteins vary significantly across different species.

[0005] In summary, research on the pathogenic mechanism of *Phytophthora pineensis* effectors remains insufficient. Therefore, elucidating the function of its key effectors is of great scientific significance for revealing the pathogenic molecular mechanism of *Phytophthora pineensis*, discovering new regulatory nodes of plant immunity, and providing indispensable molecular targets and tools for developing new disease control strategies based on this interaction mechanism (such as designing gene editing targets, screening novel fungicides or plant immune inducers). Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, the primary technical problem to be solved by this application is to provide the *Phytophthora indica* effector protein PpiniAvh23, which has the function of effectively inhibiting plant cell death. The secondary technical problem to be solved by this application is to provide the encoding gene of the aforementioned protein and its recombinant vector. The final technical problem to be solved by this application is to provide specific applications of the aforementioned protein and vector for use in transgenic plants or products that inhibit plant cell death, or for inhibiting plant cell death caused by various factors.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0008] An isolated Pine Pesticide effector protein, PiniAvh23, has the amino acid sequence shown in SEQ ID NO.1; or it is a derivative protein with the function of inhibiting plant cell death, which is based on the sequence shown in SEQ ID NO.1 by substitution, deletion and / or addition of one or more amino acid residues.

[0009] The isolated nucleic acid molecule encoding the Pine Pesticide effector protein PopiniAvh23.

[0010] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.2.

[0011] In some embodiments, the nucleic acid molecule has a nucleotide sequence that has at least 90%, 95%, or 98% sequence identity with the sequence shown in SEQ ID NO: 2, and encodes the Pine Pesticide effector protein PopiniAvh23 as described in claim 1.

[0012] A recombinant vector comprising any of the nucleic acid molecules described above.

[0013] In some embodiments, the recombinant vector is operatively linked to an expression regulatory element that allows it to be expressed in a host cell.

[0014] A transgenic host cell comprising the recombinant vector or any of the nucleic acid molecules described herein integrated into its genome.

[0015] The use of the described protein, the described nucleic acid molecule, or the recombinant vector or host cell containing them in any of the following:

[0016] (1) As a molecular tool for studying plant cell death or plant-pathogen interaction mechanisms;

[0017] (2) As a target or tool for screening or identifying compounds that can inhibit or block the function of the PopiniAvh23 protein.

[0018] A method for screening plant immunomodulators includes the following steps: adding a test compound to a system expressing or containing the PpiniAvh23 protein of claim 1, and detecting the effect of the compound on the function of the PpiniAvh23 protein; wherein the function includes inhibiting plant cell death or promoting pathogen infection.

[0019] A method for creating a susceptible plant model for plant-pathogen interaction research, comprising introducing and expressing the said nucleic acid molecule into the plant.

[0020] Compared to existing technologies, the beneficial effects of this application are as follows:

[0021] This application fills a gap in the study of key effector functions of Phytophthora pineense. The discovered PpiniAvh23 protein, with its clearly defined functions of simultaneously inhibiting cell death and enhancing pathogenicity, provides an irreplaceable ideal model and key target for a complete molecular-level analysis of the pathogenic mechanism of oomycetes and precise localization of key inhibitory nodes in plant immunity. The molecular tools and screening system established based on this discovery can be directly used for high-throughput screening of novel compounds that specifically block the function of this effector, providing a new research and development path and solid foundation for the creation of next-generation green targeted fungicides. Attached Figure Description

[0022] Figure 1 This is a plasmid map of the recombinant expression vector pGR107 / PpiniAvh23;

[0023] Figure 2This is a diagram showing the results of transient expression of the effector PpiniAvh23 in tobacco leaves and the resulting leaf tissue necrosis. The diagram shows the results of injecting recombinant Agrobacterium containing PpiniAvh23 into tobacco leaves after injection of cell death-inducing proteins at 0, 12, and 24 h. PpiniAvh23 protein did not have an inhibitory effect at 0 and 12 h, but completely inhibited INF1 protein-induced cell death after 24 h. Furthermore, the Gus negative control did not inhibit PCD, while the INF1 positive control triggered PCD.

[0024] Figure 3 This is a diagram showing the necrosis of leaf tissue after the effector PpiniAvh23 was injected into tobacco leaves and then inoculated with Phytophthora capsici. In the diagram, the left and right leaves were injected with the Gus negative control and the effector PpiniAvh23, respectively. 36 h later, a Phytophthora capsici fungal cake of the same size was inoculated on each of the left and right leaves. After 48 h, it was observed that the lesion on the right was significantly larger than that on the left, indicating that PpiniAvh23 promoted the infection of Phytophthora capsici.

[0025] Figure 4 The phylogenetic trees of PiniAvh23 between species and genera were generated using the Maximum-Likelihood (ML) method in MEGA (v7), with Bootstrap=1000;

[0026] Figure 5 The phylogenetic tree of PpiniAvh23 and the RxLR effector of Phytophthora pineensis was generated using the maximum-likelihood (ML) method in MEGA(v7), Bootstrap=1000. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] Phytophthora pini strain: described in “Hong C, Gallegly ME, Richardson PA, Kong P. Phytophthora pini Leonian resurrected to distinct species status. Mycologia. 2011 Mar-Apr;103(2):351-60. doi: 10.3852 / 10-058. Epub 2010 Oct 7.PMID: 21415291.”, available to the public from Nanjing Forestry University.

[0030] Potato Virus X (PVX) pGR107: described in the article "Nasser Beiczadeh. Investigation on Potato Virus X in North of Khorasan[A]. Chinese Society for Plant Protection. Plant Protection Towards the 21st Century----Proceedings of the International Plant Protection Congress[C]. Chinese Society for Plant Protection: Chinese Society for Plant Protection, 2004:1.", which is available to the public from Nanjing Forestry University.

[0031] Nicotiana Benthamiana: documented in the article "Louise Jones, Andrew J. Hamilton, Olivier Voinnet, et al. RNA-DNA Interactions and DNA Methylation in Post-Transcriptional Gene Silencing. The Plant Cell, 1999(11): 2291-2301.", which is available to the public from Nanjing Forestry University.

[0032] Agrobacterium tumefaciens GV3101: described in the article “González-Mula Almudena, Lang Julien, Grandclément Catherine, Naquin Delphine, Ahmar Mohammed, Soulère Laurent, Queneau Yves, Dessaux Yves, Faure Denis. Lifestyle of the biotroph Agrobacterium tumefaciens in the ecological niche constructed on its host plant.[J]. The New Phytologist, 2018.”, available to the public from Nanjing Forestry University.

[0033] Example 1: Amplification of the gene encoding the effector protein PpiniAvh23 of Phytophthora pineense

[0034] Pine mold strains were selected as test materials. Based on reported effector protein gene information, the pine mold genome sequence was analyzed to obtain the effector protein genes in the entire pine mold genome. Then, primers p1 and p2 were designed based on the obtained gene fragments to amplify and screen the target gene fragments.

[0035] 1. High-quality genomic DNA was extracted from *Phytophthora pinicola* using the CTAB-SDS method.

[0036] The *Phytophthora pineense* strain was cultured on solid V8 medium plates (formula: 340 mL V8 vegetable juice with 3.4 g calcium carbonate, mixed, centrifuged at 2000 rpm for 5 min, supernatant collected, diluted with pure water to 1 L, then 15 g agar powder added, autoclaved for 20 min). After culturing for 3 days at 25℃, three 4 mm diameter mycelial blocks were transplanted into Erlenmeyer flasks containing 100 mL of liquid V8 medium (formula: 70 mL V8 vegetable juice with 1.6 g calcium carbonate, mixed, centrifuged at 2000 rpm for 5 min, supernatant collected, diluted with pure water to 1 L, dispensed, autoclaved for 20 min). The flasks were then cultured at 25℃ for 5 days. The mycelia were filtered, ground into powder using liquid nitrogen in a mortar. Genomic DNA was then extracted from the tested strain following these steps:

[0037] (1) Transfer the mycelial powder into a 1.5 mL centrifuge tube, add 900 μL of 2% CTAB extraction solution and 90 μL of 10% SDS, vortex to mix, and incubate in a 55℃ water bath for 1 h, inverting the tube several times every 10 min. Centrifuge at 12000 rpm for 10 min.

[0038] (2) Take the supernatant and add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), mix by inversion, and centrifuge at 12000 rpm for 10 min.

[0039] (3) Transfer the supernatant to a new tube, add an equal volume of chloroform, gently invert and mix, and centrifuge at 12000 rpm for 5 min.

[0040] (4) Transfer the supernatant to a new tube, add 2 volumes of anhydrous ethanol and 1 / 10 volume of 3 M NaAc (pH 5.2), precipitate at -20℃ (>1 h); centrifuge at 12000 rpm for 10 min, discard the supernatant, wash the precipitate twice with 70% ethanol, and air dry at room temperature.

[0041] (5) Add 20 μL of sterile ultrapure water or TE (pH 8.0) to dissolve the precipitate (containing 20 μg / mL RNase) and treat at 37℃ for 1 h.

[0042] Take 5 μL of DNA sample and perform 1% agarose gel electrophoresis to detect the DNA fragment length. Then, store the DNA in a -20℃ freezer for long-term storage.

[0043] 2. PCR amplification of the target gene fragment

[0044] The primers are p1 and p2, and their sequences are as follows:

[0045] p1: 5'-ATGATGGGGTCGAGCGTGTTC-3';

[0046] p2: 5'-CATGCACTTGTCTAAGCCGGA-3'.

[0047] The reaction system consisted of 1 μL each of upstream and downstream primers (p1 and p2), DNA (100 ng), PrimeSTAR Max Premix (25 μL), and ddH2O to a final volume of 50 μL.

[0048] The PCR reaction program was as follows: 95℃ for 5 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 30 s, for 32 cycles; 72℃ for 7 min.

[0049] Two μL of the reaction product was electrophoresed to identify single clones containing the target gene, and then sequenced. Homology sequence alignment of the obtained gene fragments was performed using the BLAST program in NCBI to identify the target gene, PiniAvh23, whose nucleotide sequence is shown in SEQ ID No. 2, and the amino acid sequence of the expressed protein is shown in SEQ ID No. 1.

[0050] Example 2: Transient expression of the effector PpiniAvh23 in tobacco.

[0051] 1. Construction of PVX recombinant expression vector

[0052] (1) The target gene fragment PpiniAvh23 was amplified by PCR digestion with SmaI enzyme, and the insert fragment was recovered, with a size of approximately 254 bp.

[0053] Reaction system: QuickCut™ Sma I (2 μL), plasmid (<1 μg), 10X QuickCut Green Buffer (5 μL), add ddH2O to bring the volume to 50 μL, 37℃, 15 min.

[0054] (2) The target fragment was ligated with the enzyme-digested pGR107 vector and transformed into Escherichia coli DH5α.

[0055] (3) After transformation, DH5α was screened for resistance by Kan, and the colonies were incubated overnight in a shaker at 37°C before plasmids were extracted.

[0056] (4) The recombinant plasmid was digested and identified using the restriction endonuclease SmaI. The recombinant plasmid that was preliminarily identified by restriction enzyme digestion (two target bands of approximately 10098 bp and 254 bp in size) was sent to Genscript Biotech Co., Ltd. for sequencing. The recombinant plasmid that was sequenced to show that the DNA fragment of SEQ ID No. 2 was inserted between the SmaI restriction sites of the pGR107 vector was named pGR107 / PpiniAvh23 (plasmid map as shown in Figure 1). Figure 2 (As shown). In the recombinant expression vector pGR107 / PpiniAvh23, the promoter for transcription of the DNA fragment shown in SEQ ID No. 2 is the 35S promoter. The primer sequences used are shown in Table 1 below:

[0057] Table 1 Primer sequences for pGR107 / PpiniAvh23

[0058]

[0059] 2. Agrobacterium-mediated transformation

[0060] 2.1. Extraction of recombinant plasmid pGR107 / PpiniAvh23

[0061] (1) Inoculate Escherichia coli containing recombinant plasmid pGR107 / PpiniAvh23 into LB medium containing appropriate amount of antibiotics and culture at 37℃ with shaking at 220-250 rpm until the logarithmic growth phase.

[0062] (2) Take 1-5 mL of overnight culture (12-16 h) and add it to a centrifuge tube (prepared by yourself). Centrifuge at 10,000 rpm (11,500 × g) for 1 min. Discard the culture medium and invert the tube onto absorbent paper to remove any remaining liquid.

[0063] (3) Add 250 μL of Buffer P1 to the centrifuge tube containing bacterial precipitate (please check first whether RNase A has been added to Buffer P1), and mix with a pipette or vortex.

[0064] (4) Add 250 μL of Buffer P2 to step 2 and gently invert the container 8-10 times to fully lyse the cells.

[0065] (5) Add 350 μL of Buffer P3 to step 3, and immediately gently invert the container 8-10 times to completely neutralize Buffer P2. A white flocculent precipitate should appear at this point. Centrifuge at 12,000 rpm (13,400 × g) for 10 min.

[0066] (6) Place the FastPure DNA Mini Columns adsorption column into a 2 ml collection tube. Carefully transfer the supernatant from step 4 into the adsorption column using a pipette, being careful not to aspirate any precipitate. Centrifuge at 12,000 rpm (13,400 × g) for 30-60 seconds. Discard the waste liquid in the collection tube and return the adsorption column to the collection tube.

[0067] (7) Add 600 μL of Buffer PW2 (please check if it has been diluted with anhydrous ethanol) to the adsorption column. Centrifuge at 12,000 rpm (13,400 × g) for 30-60 seconds. Discard the waste liquid and put the adsorption column back into the collection tube.

[0068] (8) Repeat step 7.

[0069] (9) Return the adsorption column to the collection tube. Centrifuge at 12,000 rpm (13,400 × g) for 1 min to dry the adsorption column, in order to completely remove the residual washing solution in the adsorption column.

[0070] (10) Place the adsorption column into a new sterile 1.5 mL centrifuge tube. Add 30-100 μL of Elution Buffer to the center of the membrane of the adsorption column. Let stand at room temperature for 2 min, then centrifuge at 12,000 rpm (13,400 × g) for 1 min to elute the DNA.

[0071] (11) Discard the adsorption column and store the DNA product at -20°C to prevent DNA degradation.

[0072] 2.2. Preparation of Agrobacterium competent cells

[0073] (1) Select single colonies of Agrobacterium GV3101 in 4 ml LB liquid medium for 2 days, and culture at 28℃ with shaking at 200 rpm for 24 h;

[0074] (2) Take 3 mL of culture medium and place it in a 200 mL Erlenmeyer flask and shake for 8 h until OD=0.8, then place on ice for 10 min;

[0075] (3) Dispense into 50 mL centrifuge tubes and centrifuge at 5000 rpm and 4℃ for 10 min;

[0076] (4) Discard the supernatant, resuspend in 10 mL of sterile ultrapure water, and centrifuge at 5000 rpm and 4℃ for 10 min;

[0077] (5) Repeat the above process 3 times;

[0078] (6) Discard the supernatant, add 2 mL of 5% glycerol, and gently aspirate with a pipette to resuspend the cells;

[0079] (7) Aliquot 100 μL of cells onto ice and resuspend them in pre-chilled 1.5 ml EP tubes;

[0080] (8) Quick-freeze with liquid nitrogen and store in a refrigerator at -80°C.

[0081] 2.3. Electroporation transformation of Agrobacterium-competent cells

[0082] (1) Place the electroshock cup and cup holder on ice to cool. Set the electroconversion device parameters: capacitor C = 25 μF, voltage V = 2 kV (for a 0.2 kV electroshock cup), and pulse control unit to 800. .

[0083] (2) Add 100 μL of competent cell suspension thawed on ice or freshly prepared and plasmid in ice to a cooled 1.5 mL EP tube, mix gently and place on ice for about 1 min.

[0084] (3) Transfer the mixture of cells and plasmids to a cold electroporation cup and gently tap it to bring the mixture to the bottom of the electroporation cup.

[0085] (4) Apply a pulse under the above conditions and the resulting time constant is 4.8~5.1 ms.

[0086] (5) Immediately add 1 mL of LB culture medium to the electroporation cup (at room temperature). Resuspend the cells and transfer them to a 17 mm × 100 mL EP tube. Incubate at 200 rpm and 30 °C for 3 h.

[0087] (6) Collect cells by centrifugation at 5000 rpm for 3 min and spread them on LB selective plates containing kanamycin at an appropriate dilution. Colonies that grow after incubation at 30℃ for 2 days are positive clones.

[0088] 3. Recombinant Agrobacterium-positive transformants were injected into the leaves of Nicotiana benthamiana seedlings.

[0089] Nicotiana Benthamiana was used as the test plant. Single colonies of recombinant Agrobacterium transformants (GV3101 / pGR107 / PpiniAvh23, pGR107 / GFP (negative control), and pGR107 / INF1) screened and identified in step 2 were picked up with sterile toothpicks and inoculated into LB broth (containing 50 mg / ml kanamycin and rifampin each). The cultures were incubated at 30°C for 48 h, centrifuged at 5000 rpm for 3 min to collect the cells, resuspended in 10 mM MgCl2, and repeated three times. The final volume was adjusted to OD using 10 mM MgCl2. 600 =0.5. The experiment used *Nicotiana benthamiana* grown in a greenhouse (22-25℃, high light intensity) for 4 to 6 weeks. The third to sixth leaves from the top were used for *Agrobacterium* inoculation. A small wound was made in the lower epidermis of the tobacco using a needle, and 100 μL of *Agrobacterium* suspension carrying the effective molecule (pGR107::PpiniAvh23) was injected into the *Nicotiana benthamiana* leaf using a 1 mL needleless syringe. Figure 2 The following were used as positive controls: *Agrobacterium* transformed with Gus (pGR107::Gus) was used as a negative control; *Gus* and *Agrobacterium* suspension containing the INF1 (pGR107::INF1) gene were injected simultaneously; *PpiniAvh23* was injected alone; *Agrobacterium* suspension containing the candidate gene and *Agrobacterium* suspension containing the INF1 gene were injected simultaneously (PpiniAvh23+INF1); and *Agrobacterium* transformed with INF1 was used as a positive control. After inoculation, tobacco leaves were cultured in the dark at 22℃ with 75% humidity, then transferred to a climate chamber. Symptom changes in the inoculated portion were observed and recorded daily after inoculation.

[0090] like Figure 2As shown, when recombinant Agrobacterium containing PpiniAvh23 was injected into tobacco leaves after injection of a cell death-inducing protein, INF1 could induce programmed cell death (PCD) at 0, 12, and 24 h. PpiniAvh23 protein had no inhibitory effect at 0 and 12 h, but inhibited Bax protein-induced cell death after 24 h. Furthermore, the Gus negative control showed weak inhibition of PCD, while only the INF1 positive control triggered PCD.

[0091] Example 3: Effector PpiniAvh23 enhances the pathogenicity of Phytophthora capsici.

[0092] The test material was Nicotiana Benthamiana grown in a greenhouse (22-25℃, high light intensity) for 4 to 6 weeks.

[0093] Using a toothpick, single colonies of the recombinant Agrobacterium transformants GV3101 / pGR107 / PpiniAvh23 (screened and identified positively in step 2 of Example 2) were inoculated into LB liquid medium (containing 50 mg / ml each of kanamycin and rifampin) and cultured at 30°C for 48 h. Positive clones were then added to 3 mL of LB medium supplemented with kanamycin (50 μg / mL) and cultured at 200 rpm at 30°C for 48 h. The cells were collected by centrifugation at 5000 rpm for 3 min, resuspended in 10 mM MgCl2, and repeated three times. The final volume was adjusted to OD using 10 mM MgCl2. 600 =0.5.

[0094] Leaves of roughly the same size and in good growth were selected for effector injection. GV3101 / pGR107 was injected on the left half of the leaf as a control, and GV3101 / pGR107 / PpiniAvh23 was injected on the right half. The transiently expressed tobacco leaves were removed and placed in 90 mm petri dishes lined with moistened filter paper. 24 h after injection, fresh *Phytophthora capsici* mycelium cakes, punched with a 5 mm punch, were inoculated near the injection site. The petri dishes were placed in a 25°C incubator for growth in the dark, and at least three independent experiments were conducted. After inoculation, the plants were incubated at 25°C, and symptom changes at the inoculated sites were observed and recorded daily.

[0095] like Figure 3 As shown, the lesion area at the injection site of the effector PpiniAvh23 was significantly larger than that at the injection site of the blank vector. The results indicate that after PpiniAvh23 is secreted into host cells, it significantly promotes pathogen infection and inhibits the plant's immune response.

[0096] Example 4: Phylogenetic tree of effector PpiniAvh23 between species and genera.

[0097] BlastP (E≤1e-15) was used to perform homology searches on several oomycete species in the FungiDB database (http: / / fungidb.org), NCBI (https: / / www.ncbi.nlm.nih.gov / ), and genome websites. The alignment results yielded 20 species with similar homology, and sequence alignment was performed using BioEdit (v7.2.5). The phylogenetic tree was generated using Neighbor-Joining (NJ) in MEGA (v6) with default parameters. The reliability of the NJ phylogenetic tree was evaluated using the bootstrap value obtained from 1000 analyses, such as... Figure 4 As shown.

[0098] The *P. pini*Avh23 gene and four intracellular effectors (Avh29, Avh87, Avh49, Avh57) of *P. pini* were downloaded from the *P. pini* information resource website (https: / / fungidb.org / fungidb / app / ). A phylogenetic tree was constructed using the maximum likelihood (ML) method with MEGA (v7) software. Comparison of *P. pini*Avh23 with the other four *P. pini* RxLR effectors showed that *P. pini*Avh23 is on a single branch. Figure 5 As shown.

[0099] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. In all examples shown and described herein, any specific value should be interpreted as merely exemplary and not as a limitation, unless otherwise specified; therefore, other examples of exemplary embodiments may have different values.

Claims

1. An isolated Phytophthora infestans effector protein PpinAvh23, characterized in that, the amino acid sequence of which is shown as SEQ ID NO. 1; or a derivative protein which is based on the sequence shown as SEQ ID NO. 1 with one or several amino acid residues being substituted, deleted and / or added, and which has the function of inhibiting plant cell death.

2. An isolated nucleic acid molecule encoding the Phytophthora effector protein PpinlAvh23 of claim 1.

3. The nucleic acid molecule of claim 2, wherein, the nucleotide sequence of which is shown as SEQ ID NO.

2.

4. The nucleic acid molecule of claim 2, wherein, the nucleotide sequence of which has at least 90%, 95% or 98% sequence identity with the sequence shown as SEQ ID NO: 2, and which encodes the Phytophthora effector protein PpinlAvh23 as claimed in claim 1.

5. A recombinant vector, characterized in that, comprising the nucleic acid molecule of any one of claims 2 to 4.

6. The recombinant vector of claim 5, wherein, the nucleic acid molecule is operably linked to expression regulatory elements allowing its expression in a host cell.

7. A transgenic host cell, wherein, comprising the recombinant vector of claim 5 or 6, or the nucleic acid molecule of any one of claims 2 to 4 integrated into its genome.

8. Use of the protein of claim 1, the nucleic acid molecule of claims 2-4, or the recombinant vector or host cell comprising them, in any one of: (1) as a molecular tool for studying plant cell death or plant-pathogen interaction mechanisms; (2) as a target or tool for screening or identifying compounds capable of inhibiting or blocking the function of PpinlAvh23 protein.

9. A method of screening for plant immunomodulators, characterized in that, comprising the following steps: adding a test compound to a system in which the PpinlAvh23 protein of claim 1 is expressed or present, and detecting the effect of the compound on the function of the PpinlAvh23 protein; wherein the function includes inhibiting plant cell death or promoting pathogen infection.

10. A method of creating a model of a susceptible plant for plant-pathogen interaction studies, characterized in that, comprising introducing and expressing into a plant the nucleic acid molecule of claims 2-4.

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