CN111808179A - Four NCR proteins of Phytophthora sojae and their encoding genes and their applications - Google Patents

Abstract

The present invention discloses four NCR (Niemann Pick type C-related) proteins from Phytophthora sojae, their encoding genes and their applications. The NCR protein provided by the present invention is the protein shown in SEQ ID NO.5-8; the encoding gene thereof is shown in SEQ ID NO.1-4. It has been proved by experiments that the protein provided by the present invention plays an important role in the growth and development of Phytophthora sojae itself, which is specifically manifested in that after the protein is deleted, the growth of Phytophthora sojae is slowed down, the number of zoospores is reduced, and the Morphological changes, reduced pathogenicity, and deformity of oospore morphology. The above conclusions provide a technical basis for exploring the development and pathogenic molecular mechanism of Phytophthora sojae, and provide molecular targets for the research and development of new fungicides in the future.

Description

Four NCR proteins of Phytophthora sojae and their encoding genes and their applications

technical field

The invention belongs to the field of biotechnology, and in particular relates to four NCR (Niemann Pick type C-related) proteins PsNCR1-4 from Phytophthora sojae and their encoding genes and applications.

Background technique

Phytophthora sojae is a very important phytopathogenic oomycete that mainly causes root rot of Phytophthora sojae. The disease was first discovered and reported in North America in the 1950s; currently, it occurs in major soybean producing areas of more than 20 countries in Asia, Africa, Europe, North and South America and Oceania. In the 1980s, the disease was first discovered in northeastern China, and now it occurs in Heilongjiang Province, Anhui Province, Inner Mongolia Autonomous Region, and Shandong Province, the main soybean producing areas in China. Phytophthora sojae can infect soybeans during the entire growth period, and can cause devastating disasters to soybeans, resulting in more than 1 billion US dollars of economic losses every year. At present, it is classified as one of the ten important pathogenic oomycetes, and has been widely concerned and studied by scholars at home and abroad.

The life history of Phytophthora sojae is divided into asexual stage and sexual stage. During the asexual reproduction stage, Phytophthora sojae grows vegetatively with septate polykaryotic hyphae. Phytophthora sojae can also produce sporangia and zoospores, which can be spread over long distances by wind, rain or irrigation water. The sporangia are mostly pear-shaped and usually formed on the surface of the diseased part of the plant; they can germinate directly to form hyphae, or they can differentiate to form mononuclear, cell-free, bi-flagellated zoospores, and indirectly carry out the infection cycle of the disease. Zoospores are easily stimulated by the external environment to transform into resting spores, and then germinate to form hyphae, which directly invade or invade plant roots through wounds or natural orifices. The swimming of zoospores is autonomous and directional, which can increase the probability of successful host infection. As the main source of reinfection in the disease cycle, it plays an important role in the large-scale epidemic of diseases. During the sexual reproduction stage, Phytophthora sojae can form oospores through homologous cooperation. The oospores have thick walls and rich contents, which can withstand extreme environments and can survive in the soil for several years. Under suitable conditions, they can directly germinate to produce hyphae to infect host plants.

In conclusion, the growth rate of Phytophthora sojae hyphae and the formation of zoospores and oospores are important factors affecting the occurrence and development of the disease. If the growth rate of Phytophthora sojae hyphae can be slowed down, the formation of zoospores and oospores of Phytophthora sojae can be blocked, and the ability of pathogenic bacteria to infect host plants can be controlled, the damage of Phytophthora sojae root rot can be controlled.

SUMMARY OF THE INVENTION

The inventor's research found that the NCR protein in Phytophthora sojae is closely related to the growth rate of Phytophthora sojae hyphae, the yield and morphology of zoospores, and the normal morphological structure of oospores, while the normal infection cycle of plant diseases is closely related to the fungus Silk growth rate, zoospore and oospore production, and survival time were positively correlated. Therefore, it is possible to slow down the growth of mycelium, block the normal production of zoospores and destroy the structure of oospores by regulating the NCR protein, so as to weaken the ability of Phytophthora sojae to infect the host, thereby controlling the occurrence of Phytophthora sojae root rot. develop.

Therefore, one of the objects of the present invention is to provide a kind of Phytophthora sojae NCR protein, which comprises 4 homologous proteins, which are named as PsNCR1, PsNCR2, PsNCR3, and PsNCR4, which are derived from Phytophthora sojae strain P6497, and are as follows A1) or A2) or A3) or A4):

A1) the amino acid sequence is a protein as shown in any one of SEQ ID NO.5-8;

A2) a fusion protein obtained by linking a tag to the N-terminus and/or C-terminus of the protein shown in any one of SEQ ID NO.5-8;

A3) The amino acid sequence as shown in any one of SEQ ID NO.5-8 is obtained by substitution and/or deletion and/or addition of one or several amino acid residues and has the same function as shown in SEQ ID NO.5- A protein derived from the protein shown in any one of 8;

A4) The similarity with the amino acid sequence shown in any one of SEQ ID NO. 5-8 is more than 75%, preferably more than 85%, more preferably more than 95%, and has the similarity with the amino acid sequence as shown in SEQ ID NO. 5-8 The amino acid sequences shown are functionally identical amino acid sequences.

In order to facilitate the purification of the protein in A1), Poly-Arg(RRRRR), Poly-Arg(RRRRR), Poly-His (HHHHHH), FLAG (DYKDDDDK), Strep-tag II (WSHPQFEK), c-myc (EQKLISEEDL) and other tags.

The proteins in the above A1)-A4) can be artificially synthesized, or the encoding genes thereof can be synthesized first, and then biologically expressed. The coding gene of protein in the above-mentioned A2)-A4) can be by deleting the codon of one or several amino acid residues in the DNA sequence shown in any one of SEQ ID NO.1-4 in the sequence listing, and/or carry out A missense mutation of one or several nucleotide pairs, and/or the coding sequence for the above-mentioned tag is attached to its 5' and/or 3' end.

Wherein, in A1), SEQ ID NO.5 (PsNCR1) in the sequence listing consists of 1597 amino acid residues; SEQ ID NO.6 (PsNCR2) in the sequence listing consists of 1478 amino acid residues, and SEQ ID NO. .7 (PsNCR3) consists of 1045 amino acids, and SEQ ID NO.8 (PsNCR4) in the sequence listing consists of 1486 amino acid residues.

The second object of the present invention is to provide a nucleic acid molecule encoding the NCR protein. The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA, hnRNA or tRNA.

Wherein, the encoding gene of above-mentioned NCR protein is following B1) or B2) or B3):

B1) the DNA molecule shown in the nucleotide sequence of any one of SEQ ID NO.1-4 in the sequence listing;

B2) a cDNA molecule or DNA molecule that is identical to the nucleotide sequence shown in B1) by more than 75%, or more than 85%, or more than 95%, and encodes the above-mentioned NCR protein;

B3) a cDNA molecule or a DNA molecule that hybridizes to the nucleotide sequence defined in B1) or B2) under stringent conditions and encodes the above-mentioned NCR protein.

Above-mentioned coding gene, SEQ ID NO.1 in the sequence listing consists of 4917 nucleotides; from the 5' end of SEQ ID NO.1 1-186, 265-432, 478-4917 nucleotides It is a coding sequence, which encodes the protein (PsNCR1) shown in SEQ ID NO.5 in the sequence listing; SEQ ID NO.2 in the sequence listing consists of 4477 nucleotides; Nucleotide at position 4437 is the coding sequence, which encodes the protein (PsNCR2) shown in SEQ ID NO.6 in the sequence listing; SEQ ID NO.3 in the sequence listing consists of 3138 nucleotides; from 5 of SEQ ID NO.3 The 1-3138 nucleotides at the 'end are the coding sequence, encoding the protein (PsNCR3) shown in SEQ ID NO.7 in the sequence listing; SEQ ID NO.4 in the sequence listing consists of 4461 nucleotides; from SEQ ID NO.4 The 1-4461 nucleotides at the 5' end of NO.4 are the coding sequences, which encode the protein (PsNCR4) shown in SEQ ID NO.8 in the sequence listing.

The RNA molecule is the RNA molecule obtained by the transcription of the coding gene;

Preferably, the sequence of the RNA molecule is the following C1) or C2):

C1) The similarity of the RNA sequences transcribed from the DNA sequences shown in SEQ ID NO. 1-4 is more than 75%, more preferably more than 85%, more preferably more than 95% The RNA sequence with the same function as the RNA sequence transcribed from the DNA sequence shown in 4;

C2) RNA sequences transcribed from the DNA sequences shown in SEQ ID NO. 1-4.

The DNA sequence according to the present invention is capable of molecular hybridization with the DNA sequence shown in SEQ ID NO. 1-4 under stringent conditions and encodes the DNA sequence of any one of the above NCR proteins. The above stringent conditions can be hybridized at 65°C with a solution of 6×SSC, 0.5% SDS, and then washed once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS each.

The third object of the present invention is to provide biological materials related to the above nucleic acid molecules, including recombinant vectors, expression cassettes, recombinant microorganisms or transgenic plant cell lines. The recombinant vector can be a recombinant expression vector or a recombinant cloning vector. In the above biological materials, the vector can be plasmid, cosmid, phage or viral vector; the microorganism can be yeast, bacteria, algae or fungi, such as Agrobacterium; the transgenic plant cell line does not include propagation material. Specifically, it can be any one of the following D1) to D10) as follows:

D1) an expression cassette containing the encoding gene of claim 2;

D2) a recombinant vector containing the encoding gene of claim 2, or a recombinant vector containing the expression cassette of D1);

D3) a recombinant microorganism containing the encoded gene of claim 2, or a recombinant microorganism containing the expression cassette of D1), or a recombinant microorganism containing the recombinant vector of D2);

D4) a transgenic plant cell line containing the encoding gene of claim 2, or a transgenic plant cell line containing the expression cassette of D1);

D5) a transgenic plant tissue containing the encoding gene of claim 2, or a transgenic plant tissue containing the expression cassette of D2);

D6) a transgenic plant organ containing the encoding gene of claim 2, or a transgenic plant organ containing the expression cassette of D2);

D7) inhibits the nucleic acid molecule of the described coding gene expression of claim 2;

D8) an expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in D7);

D9) nucleic acid molecules that inhibit the translation of the above RNA molecules;

D10) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line producing the nucleic acid molecule of D9).

The fourth purpose of the present invention is to provide a set of Phytophthora sojae NCR protein combinations or DNA combinations used in sets, which are the following E1) or E2):

E1) The protein shown in SEQ ID NO.5 in the sequence listing, the protein shown in SEQ ID NO.6 in the sequence listing, the protein shown in SEQ ID NO.7 in the sequence listing, and the protein shown in SEQ ID NO.8 in the sequence listing Any two or a combination of two or more of the proteins shown;

E2) consists of the DNA molecule shown in SEQ ID NO.1 in the sequence listing, the DNA molecule shown in SEQ ID NO.2 in the sequence listing, the DNA molecule shown in SEQ ID NO.3 in the sequence listing, and the DNA molecule shown in SEQ ID NO.3 in the sequence listing Any two or a combination of two or more of the DNA molecules shown in NO.4.

The fifth object of the present invention is to provide Phytophthora sojae NCR protein and nucleic acid molecule encoding NCR protein or biological material containing nucleic acid molecule encoding NCR protein or application of the NCR protein combination or DNA combination.

The application is any one or more of the following 1)-6):

1) application in regulating the production of Phytophthora sojae zoospores and/or maintaining its form;

2) application in regulating the growth rate of Phytophthora sojae;

3) Application in regulating the ability of Phytophthora sojae to infect a host;

4) Application in maintaining the normal morphological structure of Phytophthora sojae oospores;

5) application in regulating the pathogenicity of Phytophthora sojae to a host;

6) Application in inhibiting and/or killing Phytophthora sojae;

Preferably, wherein, the application includes inhibiting one or both or both of the encoding genes described in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 Transcription or inactivation of any combination of the above, or inhibition of translation of one or two or more of the RNA molecules in any combination, or inhibition and/or inactivation of SEQ ID NO.5 and SEQ ID in the sequence listing The application of 1)-6) can be achieved by the activity of one or two or any combination of the NCR proteins described in NO.6, SEQ ID NO.7 and SEQ ID NO.8.

In the described application, by inhibiting the transcription of the above-mentioned coding gene, or inhibiting the translation of the above-mentioned RNA sequence, or inhibiting and/or inactivating the activity of the above-mentioned NCR protein, Phytophthora sojae zoospore yield and morphology, hyphae are regulated. Growth rate, ability to infect the host and normal morphological structure of oospores to inhibit and/or kill the growth of Phytophthora sojae.

The sixth purpose of the present invention is to provide the NCR protein shown in SEQ ID NO.6 in the sequence listing, SEQ ID NO.7 in the sequence listing or SEQ ID NO.8 in the sequence listing, and SEQ ID NO.2 in the sequence listing , the coded gene shown in SEQ ID NO.3 in the sequence listing or SEQ ID NO.4 in the sequence listing or the above-mentioned RNA molecule or the above-mentioned biological material or the protein combination or DNA combination as claimed in claim 5 is used as a bacteriostatic or bactericidal combination. Application of agent target screening for bacteriostatic and/or fungicides of Phytophthora sojae.

The seventh object of the present invention is to provide a method for screening or assisting the screening of Phytophthora sojae bacteriostatic and/or bactericide, the method comprising applying a substance to be detected to the Phytophthora sojae, when the substance to be detected can Inhibit the transcription of any one of the above DNA sequences or any combination of two or more DNA sequences, or inhibit the translation of any one of the above RNA sequences or any combination of two or more RNA sequences, or inhibit and/or inactivate If the activity of any one NCR protein or any combination of two or more proteins shown above, the substance to be tested is a candidate Phytophthora sojae bacteriostatic and/or fungicide.

The eighth object of the present invention is to provide a method for reducing the activity of Phytophthora sojae, comprising the steps of: inhibiting the transcription of one or two or more of the above-mentioned encoding genes in any combination, or inhibiting the RNA molecule Translation of one or two or more of any combination, or inhibition and/or inactivation of the activity of one or more of the above-mentioned NCR proteins in any combination;

Wherein, the activity of reducing Phytophthora sojae is to reduce the infectivity of Phytophthora sojae to the host and/or the pathogenicity to the host, and/or reduce the growth rate of Phytophthora sojae, and/or reducing the number and/or morphological changes of the zoospores of Phytophthora sojae, and/or deforming the oospore structure of Phytophthora sojae;

Preferably, the method is any one of the following F1)-F7):

F1) Inhibit the activity of the protein shown in SEQ ID NO.5 in the sequence listing of Phytophthora sojae and the protein shown in SEQ ID NO.6 in the sequence listing or inactivate it;

F2) inhibiting the expression or inactivating the protein shown in SEQ ID NO.6 in the sequence listing of Phytophthora sojae and/or the protein shown in SEQ ID NO.8 in the sequence listing;

F3) Inhibit the activity of the protein shown in SEQ ID NO. 6 in the Sequence Listing, the protein shown in SEQ ID NO. 7 in the Sequence Listing, and the protein shown in SEQ ID NO. 8 in the Sequence Listing in Phytophthora sojae or make it inactivated;

F4) Inhibition of the protein shown in SEQ ID NO.5 in the Sequence Listing, the protein shown in SEQ ID NO.6 in the Sequence Listing, the protein shown in SEQ ID NO.7 in the Sequence Listing and the protein shown in SEQ ID NO.7 in the Sequence Listing in Phytophthora sojae The activity or inactivation of the protein shown in ID NO.8;

F5) Inhibition of the protein shown in SEQ ID NO.5 in the Sequence Listing of Phytophthora sojae, the protein shown in SEQ ID NO.6 in the Sequence Listing, the protein described in SEQ ID NO.7 in the Sequence Listing and the SEQ ID NO.7 in the Sequence Listing One or two or more of the proteins shown in ID NO.8 are active or inactivated;

F6) Inhibit the activity of the protein shown in SEQ ID NO.6 in the sequence listing of Phytophthora sojae and the protein shown in SEQ ID NO.7 in the sequence listing or inactivate it;

F7) Inhibits the activity of or inactivates the protein shown in SEQ ID NO. 7 in the Sequence Listing and the protein shown in SEQ ID NO. 8 in the Sequence Listing in Phytophthora sojae.

In the above, F1)-F7), inactivation of the protein is achieved by inhibiting or reducing the expression of the encoding gene of the protein to be inhibited or the protein to be inactivated. Specifically, it can be achieved by gene knockout or gene silencing.

The gene knockout refers to a phenomenon in which a specific target gene is inactivated by homologous recombination. Gene knockout is the inactivation of specific target genes through changes in DNA sequence.

The gene silencing refers to the phenomenon that the gene is not expressed or expressed at a low level without damaging the original DNA. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level due to DNA methylation, heterochromatinization, and position effects, and the other is post-transcriptional gene silencing, that is, after gene transcription. Gene inactivation by specific inhibition of target RNA at the level, including antisense RNA, co-suppression, quelling, RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibit etc.

Preferably, in step F1), the gene shown in SEQ ID NO.1 in the sequence listing of Phytophthora sojae and the gene shown in SEQ ID NO.2 in the sequence listing are knocked out, so that the The protein shown in SEQ ID NO.5 and the protein shown in SEQ ID NO.6 in the sequence listing are inactivated;

In step F2), the gene shown in SEQ ID NO.2 in the sequence table in Phytophthora sojae and/or the gene shown in SEQ ID NO.4 in the sequence table is knocked out, so that the SEQ ID NO. Inactivation of the protein shown in .6 and/or the protein shown in SEQ ID NO.8 in the Sequence Listing;

In step F3), gene knocking is performed by making the gene shown in SEQ ID NO.2 in the sequence listing in Phytophthora sojae, the gene shown in SEQ ID NO.3 in the sequence listing and the gene shown in SEQ ID NO.4 in the sequence listing Divide to inactivate the protein shown in SEQ ID NO.6 in the sequence listing, the protein shown in SEQ ID NO.7 in the sequence listing and the protein shown in SEQ ID NO.8 in the sequence listing;

In step F4), the gene shown in SEQ ID NO.1 in the sequence table in Phytophthora sojae, the gene shown in SEQ ID NO.2 in the sequence table, the gene shown in SEQ ID NO.3 in the sequence table and the sequence table The gene shown in SEQ ID NO.4 in the sequence table is knocked out, so that the protein shown in SEQ ID NO.5 in the sequence table, the protein shown in SEQ ID NO.6 in the sequence table, the SEQ ID NO. Inactivation of the protein shown in 7 and the protein shown in SEQ ID NO.8 in the sequence listing;

In step F5), the gene shown in SEQ ID NO.1 in the sequence table in Phytophthora sojae, the gene shown in SEQ ID NO.2 in the sequence table, the gene shown in SEQ ID NO.3 in the sequence table and One or two or more of the genes shown in SEQ ID NO.4 in the sequence listing are knocked out, so that the protein shown in SEQ ID NO.5 in the sequence listing and the SEQ ID NO. Inactivation of one or more of the protein shown in .6, the protein shown in SEQ ID NO.7 in the sequence listing, and the protein shown in SEQ ID NO.8 in the sequence listing;

In step F6), the gene shown in SEQ ID NO.2 in the sequence table in Phytophthora sojae and the gene shown in SEQ ID NO.3 in the sequence table are knocked out, so that the gene shown in SEQ ID NO.6 in the sequence table is knocked out. The protein shown and the protein shown in SEQ ID NO.7 in the sequence listing are inactivated;

In step F7), the gene shown in SEQ ID NO.3 in the sequence table in Phytophthora sojae and the gene shown in SEQ ID NO.4 in the sequence table are knocked out, so that the gene shown in SEQ ID NO.7 in the sequence table is knocked out. The protein shown and the protein shown in SEQ ID NO. 8 in the sequence listing are inactivated.

In one embodiment of the present invention, the method for knocking out the above-mentioned gene is a CRISPR/Cas9-based knockout method.

Specifically, the CRISPR/Cas9-based gene knockout method is to transfect the Donor vector of the target gene and the sgRNA and Cas9 co-expression plasmids into Phytophthora sojae to select the recombinant bacteria inactivated by the target knockout protein.

The Donor vector is a recombination that contains the sequence of 800-1500bp upstream of the target gene to be knocked out, DodorDNA sequence (can be a gene sequence such as NPTII or GFP or RFP) and the sequence of 800-1500bp downstream of the target gene to be knocked out sequentially connected vector.

The sgRNA and Cas9 co-expression plasmid is a vector for co-expressing the coding DNA fragment of the sgRNA targeting the target gene to be knocked out and the coding DNA sequence expressing Cas9, wherein the sgRNA sequence targeting the PsNCR1 gene is GAAGCAAGAAGAGTTGAAGA, targeting the PsNCR2 gene The sgRNA sequence of GTACGTCCCGCAGATATCCT, the sgRNA sequence targeting the PsNCR3 gene is TCACGAACATGGACTGGTTG, and the sgRNA sequence targeting the PsNCR4 gene is ACGTACACAGAGTCCTGCTA.

Preferably, the sgRNA and Cas9 co-expression plasmid is based on the PYF515 vector as the starting vector, and the double-stranded sgRNA coding sequence obtained by annealing the sgRNA of the PsNCR1 gene or the PsNCR2 gene or the PsNCR3 gene or the PsNCR4 gene is inserted into the Nhe I and Bsa of the PYF515 vector Between the recognition sites of I enzyme, sgRNA and Cas9 co-expression plasmids of PsNCR1 gene or PsNCR2 gene or PsNCR3 gene or PsNCR4 gene were obtained.

The application of the substance inhibiting the expression and/or activity of NCR protein in the preparation of a fungicide for Phytophthora sojae also belongs to the protection scope of the present invention.

In the above application, the substance that suppresses NCR protein expression and/or activity is a substance that suppresses NCR protein expression and/or suppresses the transcription of the encoding gene of NCR protein and/or suppresses the translation of the RNA molecule obtained by the transcription of the encoding gene of NCR protein. .

Experiments have proved that the NCR protein provided by the present invention plays a role in the growth and development of Phytophthora sojae itself. Different types of knockout mutants were obtained by using CRISPR/Cas9 gene editing technology, and their growth and development were significantly changed compared with the wild-type parental strain, mainly including: slowed hyphal growth rate of PsNCR1 single-knockout mutants, zoospores The yield decreased and the ability to infect host plants became weaker; the ability of PsNCR2 single knockout mutants and PsNCR4 single knockout mutants to infect host plants became weaker; while the biological traits of PsNCR3 single knockout mutants did not change significantly; PsNCR2/PsNCR4 double-knockout mutants, PsNCR2/PsNCR4/PsNCR3 triple-knockout mutants, and PsNCR2/PsNCR4/PsNCR3/PsNCR1 quad-knockout mutants exhibited reduced hyphal growth rate, little zoospore formation, and infection The ability of the host plant was weakened; in addition, the morphological structure of the oospores of the PsNCR2/PsNCR4/PsNCR3/PsNCR1 quad-knockout mutants changed, the contents could not be filled, the vacuolation increased, and the oocysts appeared deformed. Therefore, NCR protein in Phytophthora sojae can play an important role in various processes of vegetative growth, asexual reproduction, sexual reproduction and host infection of Phytophthora sojae. The invention provides technical support for the research on the pathogenic mechanism of Phytophthora sojae, and provides a potential molecular target for the research and development of new fungicides in the future.

Description of drawings

Figure 1 shows Phytophthora sojae strain P6497 (WT), empty vector control transformants CK (ΔN1-CK, ΔN2-CK, ΔN3-CK, ΔN4-CK), PsNCR1 single knockout transformants ΔN1 series strains (A in Figure 1 ), PsNCR2 single-knockout transformants ΔN2 series strains (B in Figure 1), PsNCR3 single-knockout transformants ΔN3 series strains (Fig. 1, C), PsNCR4 single-knockout transformants ΔN4 series strains (Fig. 1, D) Colony expansion diameter histogram (cultured on V8 solid medium for 5d);

Figure 2 shows Phytophthora sojae strain P6497 (WT), empty vector control transformants CK (ΔN1-CK, ΔN2-CK, ΔN3-CK, ΔN4-CK), PsNCR1 single knockout transformants ΔN1 series strains (A), PsNCR2 Histogram of pathogenicity of single knockout transformant ΔN2 series strains (B), PsNCR3 single knockout transformant ΔN3 series strains (C), and PsNCR4 single knockout transformant ΔN4 series strains (D);

Figure 3 shows Phytophthora sojae strain P6497 (WT), empty vector control transformant CK (ΔN2-CK), PsNCR2 single knockout transformant ΔN2 series strain (ΔN2-44), ΔN2/4 series double knockout transformant (Fig. 3 A), ΔN2/4/3 series triple-knockout transformants (Fig. 3, B) and ΔN2/4/3/1 series four-knockout transformants (Fig. 3, C) Histogram of colony expansion diameter and colony Morphological photo (D in Figure 3) (cultured on V8 solid medium for 5d);

Fig. 4 shows Phytophthora sojae strain P6497 (A, B and C in Fig. 4), PsNCR2/PsNCR4 double knockout mutants (ΔN2/4 series double knockout transformants, D, E and F in Fig. 4) in ordinary optics Morphological photos of zoospores released from sporangia under microscope (40x or 20x objective);

Figure 5 shows the oospore morphology of Phytophthora sojae strain P6497 (WT) and PsNCR2/PsNCR4/PsNCR3/PsNCR1 quadruple-knockout mutant under ordinary light microscope (40x objective) (A) and transmission electron microscope (B) and strain P6497 (WT), Histogram of oospore malformation rates in PsNCR1 single knockout mutants, PsNCR2/PsNCR4 double knockout mutants, PsNCR2/PsNCR4/PsNCR3 triple knockout mutants, and PsNCR2/PsNCR4/PsNCR3/PsNCR1 quadruple knockout mutants (C);

Figure 6 shows Phytophthora sojae strain P6497 (WT), empty vector control transformant CK, PsNCR2 single knockout mutant, PsNCR2/PsNCR4 double knockout mutant, PsNCR2/PsNCR4/PsNCR3 triple knockout mutant and PsNCR2/PsNCR4/ Incidence pictures (A) and lesion length (B) of hypocotyls of soybean yellow seedlings infected by PsNCR3/PsNCR1 four-knockout mutants.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Phytophthora sojae strain P6497: a standard strain donated by Professor Brett M. Tyler of Oregon State University, USA, stored in the Laboratory of Seed Pathology and Fungicide Pharmacology, School of Plant Protection, China Agricultural University, and available to the public from China Agricultural University.

Medium or Reagent Recipe:

10% V8 solid medium: 100ml V8 vegetable juice, 1.4g CaCO ₃ , stir and mix well, dilute 10 times with deionized water, add 900ml deionized water, add 15g agar, and sterilize at 121°C for 20min.

10% V8 liquid medium: 100ml V8 vegetable juice, 1.4g CaCO ₃ , stir and mix, centrifuge at 12,000 rpm for 5 minutes, take the supernatant, dilute it with deionized water 10 times, and sterilize by high pressure moist heat at 121° C. for 20 minutes.

Nutrient pea broth (NPB): 125g of peas were added with 1l of deionized water, sterilized by high pressure moist heat at 121°C for 20min, filtered with gauze to obtain a pea nutrient solution; 2.0g of yeast extract, 5.0g of glucose, 5.0g of manna were weighed alcohol, 5.0g sorbitol, 2.0g CaCO ₃ , 0.1g CaCl ₂ , 0.5g MgSO ₄ , 3.0g KNO ₃ , 1.0g K ₂ HPO ₄ , 1.0g KH ₂ PO ₄ , stir and mix well, centrifuge at 3000rpm for 10min or stand still 30min, take the supernatant, dilute to 1l with pea nutrient solution, add 15g agar powder to solid medium (NPBA), and sterilize by moist heat for 20min. Before use, in a sterile bench, add 2ml of vitamin stock solution (Biotin 6.7× ^10-7 g/ml; Folic acid 6.7× ^10-7 g/ml; L-inositol 4.0× ^10-5 g/ml; Nicotinic acid 4.0 x 10 ^-5 g/ml; Pyrido xine-HCl 6.0 x 10 ^-4 g/ml; Riboflavin 5.0 x 10 ^{- 5} g/ml; Thiamine-HCl 1.3 x 10 ^-3 g/ml) and 2 ml of trace element stock solution (FeC ₆ H ₅ O ₇ ·3H ₂ O 5.4×10 ^-4 g/ml; ZnSO ₄ ·7H ₂ O 3.8×10 ^-4 g/ml; CuSO ₄ ·5H ₂ O 7.5×10 ^-4 g/ml; MgSO ₄ ·H ₂ O 3.8×10 ⁻⁵ g/ml; H ₃ BO ₃ 2.5×10 ⁻⁵ g/ml; Na ₂ MoO ₄ ·H ₂ O 3.0×10 ⁻⁵ g/ml).

Pea Mannitol Medium (Pea Mannitol, PM): Accurately weigh 91.1g mannitol, 1g CaCl ₂ , 2g CaCO ₃ , add about 900ml of pea nutrient solution, stir and mix for about 30min, centrifuge at 3000rpm for 10min or stand for 30min, take the Clear, dilute to 1 liter with pea nutrient solution, add 15 g of agar powder to solid medium (PMA), and sterilize by moist heat for 20 min.

Mycelial hydrolysis solution (20ml): 10ml 0.8M mannitol, 0.8ml 0.5M KCl, 0.8ml 0.5M 4-morpholineethanesulfonic acid, 0.4ml 0.5M _CaCl2 , 0.12g cellulase (Calbiochem, cat. No.219466), 0.12g lyase (Sigma, cat.No.L1412), dilute to 20ml with sterile ultrapure water, mix gently to dissolve, filter and sterilize with 0.22μm filter membrane, and prepare for use now.

MMG solution (250ml): 18.22g mannitol, 0.76g MgCl ₂ ·6H ₂ O, 2.0ml 0.5M 4-morpholineethanesulfonic acid (pH=5.7), make up to 250ml with ultrapure water, filter with 0.22μm membrane Sterilized.

W5 solution: 0.1g KCl, 4.6g CaCl ₂ ·2H ₂ O, 2.25g NaCl, 7.8g glucose, dissolve in ultrapure water to 250ml, filter and sterilize with 0.22μm filter membrane.

PEG-CaCl ₂ solution (40% w/v): 12 g of PEG 4000, 3.75 ml of 0.5 M CaCl ₂ , 3 ml of sterile ultrapure water, sterilized by filtration through a 0.22 μm filter.

Example 1. Acquisition of 4 NCR proteins of Phytophthora sojae, PsNCR1, PsNCR2, PsNCR3, PsNCR4 and their encoding genes

In this example, the four NCR proteins PsNCR1, PsNCR2, PsNCR3, PsNCR4 and their encoding genes (or cDNAs) of Phytophthora sojae can all use the DNA (or cDNA) of the standard strain of Phytophthora sojae P6497 as a template, and the primers listed in Table 1 can be used to amplify the increase gain. Wherein, the material for DNA or RNA extraction can be the mycelium of the standard strain P6497 of Phytophthora sojae. Among them, the encoding gene of PsNCR1, PsNCR1, is shown in SEQ ID NO.1 in the sequence listing, and SEQ ID NO.1 in the sequence listing consists of 4917 nucleotides; , 265-432, 478-4917 nucleotides are coding sequences, the protein PsNCR1 shown in SEQ ID NO.5 in the coding sequence table; the coding gene PsNCR2 of PsNCR2 is shown in SEQ ID NO.2 in the sequence table, SEQ ID NO.2 in the sequence listing is composed of 4477 nucleotides; the nucleotides 1-4437 from the 5' end of SEQ ID NO.2 are the coding sequence, which are shown in SEQ ID NO.6 in the coding sequence listing. The protein PsNCR2; the encoding gene of PsNCR3, PsNCR3, is shown in SEQ ID NO.3 in the sequence listing, and SEQ ID NO.3 in the sequence listing consists of 3138 nucleotides; The nucleotide is the coding sequence, which encodes the protein PsNCR3 shown in SEQ ID NO.7 in the sequence listing; the coding gene PsNCR4 of PsNCR4 is shown in SEQ ID NO.4 in the sequence listing, and SEQ ID NO.4 in the sequence listing consists of 4461 Nucleotide composition; nucleotides 1-4461 from the 5' end of SEQ ID NO. 4 are the coding sequence, which encodes the protein PsNCR4 shown in SEQ ID NO. 8 in the sequence listing. The above-mentioned proteins or genes can also be artificially synthesized.

Table 1. Amplification primers for full-length coding genes of PsNCR1, PsNCR2, PsNCR3, and PsNCR4

Example 2. Construction of Phytophthora sojae PsNCR1, PsNCR2, PsNCR3, PsNCR4 gene knockout vectors

The construction method of gene knockout vector based on CRISPR/Cas9 in this example and the sequence of related vector and NPT II gene sequence are described in the document "Fang, Y., and Tyler, B.M. (2016). Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9. Molecular plant pathology, 17(1), 127-139.” and “Fang, Y., Cui, L., Gu, B., Arredondo, F., and Tyler, B.M. (2017). Efficient genome editing in the oomycete Phytophthora sojae using CRISPR/Cas9.Curr.Protoc.Microbiol.44,21A.1.1-21A.1.26.". The pBluescript II SK+ homology arm vector plasmid (Donor vector), sgRNA and Cas9 co-expression plasmid PYF515 used in this example were donated by Professor Brett M. Tyler of Oregon State University, USA.

The gene sequence of the insert PcMuORP1 used in this example is in the literature "Wang, W., Xue, Z., Miao, J., Cai, M., Zhang, C., Li, T., Zhang, B., Tyler , B.M. and Liu, X. (2019) PcMuORP1, an Oxathiapiprolin-Resistance Gene, Functions as a Novel Selection Marker for Phytophthora Transformation and CRISPR/Cas9 Mediated GenomeEditing.Front.Microbiol.10,2402.” published in.

The Donor vectors used in this embodiment are pBS-NPTII-NCR1, pBS-NPTII-NCR2, pBS-NPTII-NCR3, pBS-NPTII-NCR4, pBS-GFP-NCR1, pBS-RFP-NCR3; sgRNA and Cas9 co-expression plasmids PYF515-NCR1, PYF515-NCR2, PYF515-NCR3, PYF515-NCR4; and the specific construction of co-expression plasmids PYF515-PcMuORP1-NCR1, PYF515-PcMuORP1-NCR3, PYF515-PcMuORP1-NCR4 with fluthiazopyridine selectable markers The method is as follows:

HD CloningKit将三个扩增片段依次融合连接入克隆载体pBluescript II SK+(EcoR V酶切)，连接产物转入大肠杆菌DH5α感受态细胞中，37℃过夜培养后，使用通用引物M13F(序列：5’-TGTAAAACGACGGCCAGT-3’)/M13R(序列：5’-CAGGAAACAGCTATGACC-3’)扩增、测序验证克隆，将验证正确的含有依次连接的PsNCR1上游1000bp序列、NPTII基因序列和PsNCR1下游1000bp序列的重组表达载体命名为pBS-NPTII-NCR1。1) Construction of pBS-NPTII-NCR1: Using the DNA of Phytophthora sojae strain P6497 as a template, the TaKaRa-In-Fusion_Tools online website (http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Cloning_Resources/On line_In -Fusion_Tools) designed primers to amplify the 1000bp upstream sequence of the target gene PsNCR1 (shown in SEQ ID NO. The sequence of NPTII gene (the NPTII gene was obtained by amplifying Pbs-NPTII-NCR1-F2 and Pbs-NPTII-NCR1-R2 using the PYF515 backbone plasmid as the template and the primer sequences shown in Table 2), 1000bp downstream of PsNCR1 Sequence (shown in SEQ ID NO. 12 in the sequence table, obtained by primer amplification of Pbs-NPTII-NCR1-F3 and Pbs-NPTII-NCR1-R3 shown in Table 2), using

The three amplified fragments were successively fused and ligated into the cloning vector pBluescript II SK+ (EcoR V digestion) with the HD Cloning Kit, and the ligated products were transferred into E. coli DH5α competent cells. After overnight incubation at 37°C, the universal primer M13F (sequence: 5 '-TGTAAAACGACGGCCAGT-3')/M13R (sequence: 5'-CAGGAAACAGCTATGACC-3') was amplified and sequenced to verify the clone, and it will be verified that the correct recombination containing the sequence of 1000bp upstream of PsNCR1, NPTII gene sequence and 1000bp downstream of PsNCR1 sequence is connected in sequence The expression vector was named pBS-NPTII-NCR1.

2) Construction of pBS-NPTII-NCR2: step as described in 1), the target gene PsNCR2 upstream 1000bp sequence (shown in SEQ ID NO. -NCR2-F1 and Pbs-NPTII-NCR2-R1 amplified), NPTII gene sequence (NPTII gene is based on PYF515 backbone plasmid as the template, the primer sequences are shown in Table 2 Pbs-NPTII-NCR2-F2 and Pbs-NPTII- The fragment obtained by the amplification of NCR2-R2), the 1000bp downstream sequence of PsNCR2 (including the 40bp untranslated region sequence behind the stop codon TAA, shown in SEQ ID NO. NPTII-NCR2-F3 and Pbs-NPTII-NCR2-R3 amplified) were sequentially fused and linked into the backbone vector pBluescript II SK+ (EcoR V digestion), and sequenced to verify the positive clones. The recombinant expression vector of 1000bp sequence, NPTII gene sequence and 1000bp downstream sequence of PsNCR2 was named pBS-NPTII-NCR2.

3) Construction of pBS-NPTII-NCR3: step as described in 1), the target gene PsNCR3 upstream 1000bp sequence (shown in SEQ ID NO. -NCR3-F1 and Pbs-NPTII-NCR3-R1 were amplified), NPTII gene sequence (NPTII gene is based on the PYF515 backbone plasmid as the template, the primer sequences are shown in Table 2 Pbs-NPTII-NCR3-F2 and Pbs-NPTII- The fragment obtained by the amplification of NCR3-R2), the downstream 1000bp sequence of PsNCR3 (shown in SEQ ID NO. Amplify and obtain) successively fused and connected into backbone vector pBluescript II SK+ (EcoR V digestion), and sequenced to verify positive clones, and the correct recombination containing 1000 bp upstream of PsNCR3 sequence, NPTII gene sequence and 1000 bp downstream of PsNCR3 sequence connected in turn will be verified. The expression vector was named pBS-NPTII-NCR3.

4) Construction of pBS-NPTII-NCR4: According to the steps described in 1), the upstream 1000bp sequence of the target gene PsNCR4 (shown in SEQ ID NO.17 in the sequence listing, through the primer Pbs- NPTII-NCR4-F1 and Pbs-NPTII-NCR4-R1 were amplified by amplification), NPTII gene sequence (NPTII gene is based on the PYF515 backbone plasmid as the template, and the primer sequences are shown in Table 2. Pbs-NPTII-NCR4-F2 and Pbs-NPTII -The fragment obtained by amplification of NCR4-R2), the 1000bp downstream sequence of PsNCR4 (shown in SEQ ID NO. Amplify and obtain) successively fused and connected into backbone vector pBluescript II SK+ (EcoR V digestion), and sequenced to verify positive clones, and the correct recombination containing the sequentially connected PsNCR4 upstream 1000bp sequence, NPTII gene sequence and PsNCR4 downstream 1000bp sequence will be verified. The expression vector was named pBS-NPTII-NCR4.

5) Construction of pBS-GFP-NCR1: According to the steps described in 1), the upstream 1000bp sequence of the target gene PsNCR1 (shown in SEQ ID NO. GFP-NCR1-F1 and Pbs-GFP-NCR1-R1 amplification), GFP gene sequence (SEQ ID NO. GFP-NCR1-R2 amplification), the downstream 1000bp sequence of PsNCR1 (shown in SEQ ID NO. Add to obtain) successively fuse into the backbone vector pBluescript II SK+ (EcoR V digestion), and sequence to verify the positive clone, will verify the correct recombinant expression containing the sequentially connected PsNCR1 upstream 1000bp sequence, GFP gene sequence and PsNCR1 downstream 1000bp sequence The vector was named pBS-GFP-NCR1.

6) Construction of pBS-RFP-NCR3: According to the steps described in 1), the upstream 1000bp sequence of the target gene PsNCR3 (shown in SEQ ID NO. - NCR3-F1 and Pbs-RFP-NCR3-R1 amplification), RFP gene sequence (SEQ ID NO. RFP-NCR3-R2 amplification), PsNCR3 downstream 1000bp sequence (SEQ ID NO. 16 in the sequence listing, amplified by primers Pbs-RFP-NCR3-F3 and Pbs-RFP-NCR3-R3 shown in Table 2 Obtained) successively fused and connected into the backbone vector pBluescript II SK+ (EcoR V digestion), and sequenced to verify the positive clone, and the correct recombinant expression vector containing the sequentially connected PsNCR3 upstream 1000bp sequence, RFP gene sequence and PsNCR3 downstream 1000bp sequence will be verified. Named pBS-RFP-NCR3.

7) Construction of PYF515-NCR1: using the sgRNA design website EuPaGDT (http://gRNA.ctegd.uga.edu/) and the RNA structure online analysis tool (http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/ Predict1/Predict1.html), select the sgRNA sequence that specifically targets the PsNCR1 gene and has a weak secondary structure (sgNCR1: GAAGCAAGAAGAGTTGAAGA, targeting the 2895-2914th position of SEQ ID No.1 of the PsNCR1 gene), and send it to the company for synthesis Forward and reverse sgRNA sequence primers with NheI and BsaI restriction sites and HH ribozyme. Dissolve in sterile water to a 100 μM solution. Annealing reaction to synthesize double-stranded sgRNA sequence, reaction system: 3 μl forward strand solution, 3 μl reverse strand solution, 3 μl 10×T4 DNA Ligase Buffer (NEB), 4 μl 0.5M NaCl, 21 μl ultrapure sterile water, pipette and mix well , react at 100 °C for 2 min, let it cool at room temperature for 4 h, and then dilute the reaction solution by 500 times. Then take 2μl of 10×T4 DNA Ligase Buffer (NEB), 50ng of PYF515 vector (Nhe I/Bsa I double digestion), 4μl of diluted double-stranded sgRNA solution, 1μl of T4 DNA Ligase, and sterile ultrapure water to make up to 20μl , react at room temperature for 30 min, and use 5 μl of the ligation product to transform E. coli DH5α competent cells. After overnight incubation at 37 °C, use the primer pair RPL41_Pseq_F (sequence: 5'-CAAGCCTCACTTTCTGCTGAC TG-3')/M13F (sequence: 5'-TGTAAAACGACGGCCAGT- 3') Perform colony PCR verification, and sequence to verify positive clones, and name the recombinant vector that can express the above sgRNA correctly as PYF515-NCR1.

8) Construction of PYF515-NCR2: According to the steps described in 4), the designed sgRNA sequence (sgNCR2: GTACGTCCCGCAGATATCCT, targeting the antisense of the reverse complementation of the 3278-3297th position of SEQ ID No.2 of the PsNCR2 gene) The sequence on the chain) was ligated into the PYF515 vector (Nhe I/Bsa I double digestion), and the positive clones were verified by sequencing, and the correct recombinant vector was named PYF515-NCR2.

9) Construction of PYF515-NCR3: According to the steps described in 4), the designed sgRNA sequence (sgNCR3: TCACGAACATGGACTGGTTG, targeting the 2406-2425th reverse complement of the SEQ ID No.3 of the PsNCR3 gene The sequence on the sense strand) was ligated into the PYF515 vector (Nhe I/Bsa I double digestion), and the positive clones were verified by sequencing, and the correct recombinant vector was named PYF515-NCR3.

10) Construction of PYF515-NCR4: According to the steps described in 4), the designed sgRNA sequence (sgNCR4: ACGTACACAGAGTCCTGCTA, targeting the 1024-1043 position of SEQ ID No. 4 of the PsNCR4 gene) was linked into the PYF515 vector (Nhe I/Bsa I double-enzyme digestion), and sequenced to verify the positive clones, and the verified correct recombinant vector was named PYF515-NCR4.

HD Cloning Kit将PcMuORP1连接入如7)中构建的载体pYF515-NCR1(PacI/NotI双酶切)，连接产物转入大肠杆菌DH5α感受态细胞中，37℃过夜培养后，使用引物Resg-F(序列：5’-ACTCGCCCACGATCGGAAGG-3’)和Resg-R(序列：5’-CACAAAATCTGCAACTTCGC-3’)进行菌落PCR验证，并测序验证阳性克隆，将验证正确的重组载体命名为pYF515-PcMuORP1-NCR1。11) Construction of pYF515-PcMuORP1-NCR1: use the TaKaRa-In-Fusion_Tools online website (the same URL as 1) to design primers (PcMuORP1-F1 and PcMuORP1-R1 as shown in Table 2) to amplify the PcMuORP1 gene sequence, using

The HD Cloning Kit ligated PcMuORP1 into the vector pYF515-NCR1 (PacI/NotI double digestion) constructed as described in 7), and the ligated product was transferred into E. coli DH5α competent cells. After overnight incubation at 37°C, primers Resg-F ( Sequence: 5'-ACTCGCCCACGATCGGAAGG-3') and Resg-R (sequence: 5'-CACAAAATCTGCAACTTCGC-3') were verified by colony PCR, and the positive clones were verified by sequencing, and the correct recombinant vector was named pYF515-PcMuORP1-NCR1.

12) Construction of pYF515-PcMuORP1-NCR3: According to the steps described in 11), the PcMuORP1 gene sequence was ligated into the vector pYF515-NCR3 (PacI/NotI double digestion) constructed in 9), and the sequence was confirmed to be positive Cloning, the verified correct recombinant vector was named pYF515-PcMuORP1-NCR3.

13) Construction of pYF515-PcMuORP1-NCR4: According to the steps described in 11), the PcMuORP1 gene sequence was ligated into the vector pYF515-NCR4 (PacI/NotI double digestion) constructed in 10), and the sequence was confirmed to be positive Cloning, the verified correct recombinant vector was named pYF515-PcMuORP1-NCR4.

Table 2. Primer sequences used for vector construction

Example 3. Obtainment of Phytophthora sojae PsNCR1-4 knockout transformants

PsNCR1-4 gene knockout transformants were prepared by CaCl ₂ -PEG-mediated protoplast transformation. The method of oomycete genetic transformation is described in the document "Fang, Y., and Tyler, BM (2016). Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9. Molecular plant pathology, 17(1), 127-139.".

The single knockout transformants were obtained by using the Donor vector, sgRNA and Cas9 co-expression plasmids (pBS-NPTII-NCR1 and PYF515-NCR1, pBS-NPTII-NCR2 and PYF515-NCR2, pBS-NPTII-NCR3 and PYF515-NCR3, or pBS-NPTII-NCR4 and PYF515-NCR4) were co-transformed into the protoplasts of Phytophthora sojae P6497, and the grown transformants were subjected to G418-resistant V8 solid medium plates were cultured and screened at 25°C, the mycelia of suspected transformants were collected, DNA was extracted for PCR sequencing verification, and RNA was extracted from positive transformants for Q-PCR verification. PsNCR1 single knockout transformants ΔN1 series strains, PsNCR2 single knockout transformants ΔN2 series strains, PsNCR3 single knockout transformants ΔN3 series strains, and PsNCR4 single knockout transformants ΔN4 series strains were obtained. At the same time, the transformants transformed into the same vector plasmid and subjected to the same transformation steps but without homologous substitution were used as CK control transformants, namely ΔN1-CK, ΔN2-CK, ΔN3-CK, ΔN4-CK.

Since PsNCR2 and PsNCR4 have high protein homology (protein sequence identity>85%), it is speculated that they may have redundant complementation in function, so these two genes were subjected to double knockout experiments. The double knockout transformants were obtained by transferring the vector plasmid of the PsNCR4 gene obtained in Example 1 (Donor vector pBS-NPTII-NCR4, sgRNA and Cas9 co-expression plasmid pYF515-PcMuORP1-NCR4) into Phytophthora sojae PsNCR2 single knockout In addition to the protoplasts of the transformant ΔNCR2-44, the grown transformants were cultured and screened at 25°C on fluthiazolyl-resistant V8 solid medium plates, the mycelia of the suspected transformants were collected, and DNA was extracted for PCR sequencing verification. ; Extracted RNA for Q-PCR verification. After verification, ΔN2/4 series double-knockout transformants with inactivated PsNCR2 and PsNCR4 proteins of Phytophthora sojae were obtained.

The three-knockout transformants were obtained by transferring the PsNCR3 gene vector plasmid (Donor vector pBS-RFP-NCR3, sgRNA and Cas9 co-expression plasmid pYF515-PcMuORP1-NCR3) obtained in Example 1 into Phytophthora sojae double knockout In the protoplasts of the transformant ΔNCR2/4-26 (which has lost the resistance to fluthiazopyridone after multiple transgenerations), the grown transformants were cultured on the fluthiazopyridone-resistant V8 solid medium plate at 25°C Screening, collecting the mycelia of suspected transformants, extracting DNA for verification by PCR sequencing; extracting RNA for verification by Q-PCR. After verification, ΔN2/4/3 series triple knockout transformants inactivated by PsNCR2 protein, PsNCR3 protein and PsNCR4 protein of Phytophthora sojae were obtained.

The four-knockout transformants were obtained by transferring the PsNCR1 gene vector plasmid (Donor vector pBS-GFP-NCR1, sgRNA and Cas9 co-expression plasmid pYF515-PcMuORP1-NCR1) obtained in Example 1 into Phytophthora sojae three-knockout In the protoplast of the transformant ΔNCR2/4/3-70 (which has lost the resistance to fluthiazopyridone after multiple transmutations), the grown transformant was plated on a solid medium plate of fluthiazopyridone resistance V8 25 Cultivate and screen at ℃, collect the mycelia of suspected transformants, extract DNA for PCR sequencing verification; extract RNA for Q-PCR verification. After verification, the ΔN2/4/3/1 series of four-knockout transformants with inactivated PsNCR1, PsNCR2, PsNCR3 and PsNCR4 proteins were obtained.

Example 4. Biological trait analysis of Phytophthora sojae PsNCR1, PsNCR2, PsNCR3 and PsNCR4 gene knockout transformants

1. Detection of mycelial growth rate

The wild-type Phytophthora sojae strain P6497 (WT), CK control transformants (ΔN1-CK, ΔN2-CK, ΔN3-CK, ΔN4-CK), various types of knockout transformants obtained in Example 3: PsNCR1 single Knockout transformants ΔN1 series strains (ΔN1-69, ΔN1-106, ΔN1-30, ΔN1-15, ΔN1-49), PsNCR2 single knockout transformants ΔN2 series strains (ΔN2-12, ΔN2-14, ΔN2-23 , ΔN2-42, ΔN2-44), PsNCR3 single knockout transformants ΔN3 series strains (ΔN3-11, ΔN3-23, ΔN3-8, ΔN3-14), PsNCR4 single knockout transformants ΔN4 series strains (ΔN4-12 , ΔN4-18, ΔN4-19, ΔN4-29, ΔN4-109); PsNCR2 protein and PsNCR4 protein inactivated ΔN2/4 series double knockout transformants (ΔN2/4-2, ΔN2/4-6, ΔN2/ 4-8, ΔN2/4-16, ΔN2/4-18, ΔN2/4-26); ΔN2/4/3 series triple knockout transformants (ΔN2/4/ 3-70, ΔN2/4/3-71, ΔN2/4/3-100, ΔN2/4/3-105, ΔN2/4/3-122); PsNCR1 protein, PsNCR2 protein, PsNCR3 protein and PsNCR4 protein inactivated The ΔN2/4/3/1 series of four knockout transformants (ΔN2/4/3/1-31, ΔN2/4/3/1-107, ΔN2/4/3/1-125, ΔN2/4/3 /1-174, ΔN2/4/3/1-211), respectively inoculated in the center of a sterile petri dish (diameter 9cm) with 15ml V8 solid medium, cultured at 25°C for 5 days in the dark, using the cross method The colony diameter of each strain was measured, and each strain was replicated 3 times.

The results showed that the hyphal growth rates of all the tested PsNCR1 single-knockout transformants ΔN1 series strains were significantly lower than those of the wild-type Phytophthora sojae strain P6497 (WT) and the control transformant ΔN1-CK; The hyphal growth rate of the single knockout transformants of the NCR gene was not significantly different as a whole compared with P6497 and CK, and there was a slight difference between the transformants (Fig. 1). The mycelial growth rates of ΔN2/4 series double knockout transformants, ΔN2/4/3 series triple knockout transformants and ΔN2/4/3/1 series quad knockout transformants were significantly different from those of Phytophthora sojae strains P6497(WT) and Compared with the control transformants, CK was significantly decreased. The experimental results indicated that the NCR protein was involved in regulating the hyphal growth of Phytophthora sojae ( FIG. 3 ).

2. Quantity and morphological detection of sporangia and zoospores

10% V8 solid and liquid media were prepared, and wild-type Phytophthora sojae strain P6497 (WT), empty vector control transformant CK, and various types of knockout transformants obtained in Example 3: PsNCR1 single knockout transformant ΔN1 Series strains, PsNCR2 single knockout transformants ΔN2 series strains, PsNCR3 single knockout transformants ΔN3 series strains, PsNCR4 single knockout transformants ΔN4 series strains; PsNCR2 and PsNCR4 double knockout transformants ΔN2/4 series strains; PsNCR2, PsNCR3 and PsNCR4 triple knockout transformants ΔN2/4/3 series strains; PsNCR1, PsNCR2, PsNCR3 and PsNCR4 four knockout transformants ΔN2/4/3/1 series strains were inoculated on V8 solid medium, respectively, at 25°C Dark culture for 5-7 days, use a 5mm hole punch to punch out 10 bacterial cakes for each strain and put them into a sterile petri dish (9cm in diameter) of 20ml V8 juice liquid medium. After 3 days of dark culture at 25°C, use 20ml Rinse with sterile deionized water, once every 30min, for a total of 5 times, then add 10ml of deionized water to make up the volume, put it in the dark at 25 °C for 4-6 hours, and observe the sporangia on the bacterial cake through a microscope. Quantity and morphology; 8-10 hours later, the number and morphology of zoospores produced in sterile water were observed by microscope, repeated 3 times.

The results showed that compared with the wild-type Phytophthora sojae strain P6497 (WT) and the empty vector control transformant CK, the number of sporangia and the number of released zoospores in the PsNCR1 single-knockout transformant ΔN1 series strain obtained in Example 3 decreased significantly. (observed after standing for 10 hours), but the morphology of sporangia and zoospores was normal, which indicated that PsNCR1 protein mainly affected the number of sporangia and zoospores of Phytophthora sojae. Compared with P6497 and CK, the sporulation yield of the other three single-knockout transformants of Phytophthora sojae NCR gene was not significantly different as a whole, and there was a slight difference between the transformants (Table 3). The number of ΔN2/4 series double knockout transformants, ΔN2/4/3 series triple knockout transformants and the number of sporangia were not different within the same detection time, but normal zoospores could not be released (observed after standing for 10 hours), The mass content was released (Table 3, Figure 4). Figure 4 is a photograph of the morphology of the sporangia released from the sporangia of Phytophthora sojae strain P6497 (A, B and C in Figure 4), and ΔN2/4 series double knockout transformants (D, E and F in Figure 4). The morphology of the sporozoites released by the ΔN2/4/3 series triple-knockout transformants and the ΔN2/4/3/1 series four-knockout transformants was consistent with the ΔN2/4 series double-knockout transformants. The ΔN2/4/3/1 series of four-knockout transformants significantly decreased the number of sporangia and could not release normal zoospores within the same detection time (Table 4). This indicates that NCR protein may affect the cytokinesis of Phytophthora soja sporangia, so that normal zoospores cannot be produced.

Table 3. The sporulation yield of each single-gene knockout mutant of Phytophthora sojae strain P6497

Table 4. Sporulation of ΔN2/4 series double knockout transformants, ΔN2/4/3 series triple knockout transformants, and ΔN2/4/3/1 series quad knockout transformants of Phytophthora sojae strain P6497.

3. Number of oospores, morphological detection and statistics of deformity rate

Wild-type Phytophthora sojae strain P6497 (WT), control transformants CK (ΔN1-CK, ΔN2-CK, ΔN3-CK, ΔN4-CK), various types of knockout transformants obtained in Example 3 were inoculated into The center of a sterile petri dish (diameter 9cm) with 15ml V8 solid medium was cultured at 25°C in the dark for 7-14 days. The number and morphology of oospores produced were observed by microscope, and the deformity rate was counted, repeated 3 times.

The results showed that the number of oospores of various types of knockout transformants obtained in Example 3 did not change significantly compared with those of the wild-type strain P6497 (WT) and the control transformant CK. However, by ordinary optical microscope (40x objective) and transmission electron microscope, it was found that the oospore morphology of the ΔN2/4/3/1 series four-knockout transformants was deformed. And the fat body region could not be filled, shrinking (Fig. 5A,B). Through statistical analysis of the deformity rate, it was found that the oospore deformity rate of the ΔN2/4/3/1-31 quadruple-knockout transformants was almost 100%, while that of the PsNCR1 single-knockout transformants ΔN1-15 also had less than 5%. The deformity rate of oospores in other types of knockout transformants was not significantly different from that of wild-type strain P6497 (WT) (Fig. 5C). It indicated that NCR proteins may be involved in regulating the morphological structure of Phytophthora sojae oospores.

4. Pathogenicity testing

The tested soybean plant variety was Nippon Green, which was planted in a seedling tray (540mm × 280mm, 80 plants per hole). The culture medium was peat soil and vermiculite in a ratio of 2:1. (27±2℃; 24h dark treatment) culture for 7d for use.

For strains that can produce zoospores, prepare a zoospore suspension (2×10 ⁴ zoospores/ml) with reference to the above method; for strains that cannot produce zoospores, prepare their hyphae suspensions, and culture them for 5-7 days. Phytophthora sojae V8 solid medium was beaten with 5 mm of bacterial cake. Inoculate 10 μl zoospore suspension or a fungus cake at about 1 cm of the hypocotyl of soybean yellow flower seedlings, and inoculate 10-20 yellow flower seedlings per strain. After culturing for 3 days at 25°C in the dark and moisturizing, investigate the infection of Phytophthora soybean under the yellow flower seedlings. Lesion length (mm) of hypocotyl.

The results show that, compared with the wild-type Phytophthora sojae strain P6497 (WT) and the empty vector control transformant CK, various types of knockout transformants obtained in Example 3: PsNCR1 single-knockout transformants ΔN1 series strains, PsNCR2 single-knockout transformants Knockout transformants ΔN2 series strains, PsNCR4 single knockout transformants ΔN4 series strains; PsNCR2 and PsNCR4 double knockout transformants ΔN2/4 series strains; PsNCR2, PsNCR3 and PsNCR4 triple knockout transformants ΔN2/4/3 series strains; The pathogenicity of PsNCR1, PsNCR2, PsNCR3 and PsNCR4 quadruple-knockout transformants ΔN2/4/3/1 series strains were all significantly reduced (Fig. 2, Fig. 6). However, the pathogenicity of the ΔN3 series strains of the PsNCR3 single-knockout transformants did not decrease significantly. And there was no significant difference in pathogenicity between the ΔN2/4 series double knockout transformants and the ΔN2/4/3 series triple knockout transformants, while the ΔN2/4/3/1 series four knockout transformants almost lost the infection yellow The ability of flower seedlings to produce lesions of about 5mm only at the inoculated fungus cake. This indicates that NCR protein has the ability to participate in the regulation of Phytophthora sojae infection of host plants.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as It is the protection scope of the present invention.

sequence listing

<110> China Agricultural University; Northwest A&F University

<120> Four NCR proteins of Phytophthora sojae and their encoding genes and applications

<130> MP2019630Z

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4917

<212> DNA

<213> Phytophthora sojae

<400> 1

atgtggctgt tcgcggccat cagcctcgcg atcgcgacgc cgtgcttccg cgcgtggctc 60

gaggcgctgt tcgccgagct gtccatcatc ttcttcacgt acctgcggct ggtgtcggcc 120

ggcctgagcc tcgtgctgct gctggcgctg gccttccacg gccgccgctt cctgcgcagt 180

cgtcgggtcg ccaaggcgcc gcgagcgggc ggcagcggca gccaagcggc ggatgaggga 240

tccaaggcgt cgctgtcggc caagacgggc ctggcgcgcg tccactggcg gcgcggcacc 300

aggcgccgcg cgctcatgct gcgcgtcaag agcacgacca gcgagatcgc gggcagcggc 360

ttctacccgc cgcagctctg gacctcgtcg ctcttcgcgc tggccatgct gcccattatc 420

aacagcgtcg tggtcggggg cggaggcggc tcgagtctgc gcggactcaa gagcgagacg 480

gcggccgccg cccaaacggc tttcgacgcc gacgcggcgg cactcgtgac gctgcagcag 540

cagctgacga cctgcaagta ctcgggaggc gacgactgtc ttgacgacgt gactgccatc 600

tctgtgctgg ggagctatca gcaggcgagt ggatactgcg tggcctttga cgcggcctat 660

gtgaatgtga cgacgggggc ggcactaccg gcccagtact tccccatcgg ggtggaggag 720

gcccacgccc agggctttgc caacaacttc tcggcgtggt ccgagaccaa ccaggagaag 780

ttcaagacgg actgccccct gctgttcaac gagacggtga gtggagacgg agaggggctg 840

ctgtgctgta ccgagacgca gtacgagatg ctgagcctgc aggtccggaa gctgcctgga 900

gagtgcacgt cctgcaagca gaacctgcgc aacctgtggt gccagttcac gtgccatccg 960

agcaacagcc tgttcgtgga cgtgacgcag gtgcggctta tggagggaga cgcggaccac 1020

gcggacgagg tcttccccgc catcgaggag gccacctact acgtgggcag tgacatggtg 1080

cgcgacctgc acgacttttg tgaggccgac tcgggcttca tgccgctgtt gtgcgggatg 1140

aatgccgatg gtaattgctc gacgacaggc tcggacatgt tggggtacct tggagcgtac 1200

agctttgacg gtgtgggatc gccctcgcag gtgatcttca ctacgatgga gcagctgtca 1260

gcggctgagc aggaggacaa gatctgtgcc tgtgacagca gcaacacgac tggctgcttt 1320

tcgccgatgg atacgcggct tgagtcgtgc gtggatacgt gtggctcgct ctgtgctgtg 1380

agtgatgatg acagccggca gtatcaggcg gcttgctaca gttccggcag tacgtcggca 1440

tccgacgatt tgagcactgt cactacagcc acgaccagca ccagcgctgc tgataagctt 1500

gagtctttac tgtcggattt gtcgtcccgt gcggagggtg gtagttttgc cgtgctaaac 1560

tatgtccttg ctgttctcgc attctttggc gccacagcgc ttgcgctcgg cttcgcgtac 1620

tcgacgcggt acggcaggaa gaagcgccag tctgtgctgg acgaccctgt taacggtttg 1680

ggatctggaa tgctgtcact cgtgaatttg gatcagttga agggaatcgg acgttgggat 1740

gaccggctga cgatgcactt gaaacgctgg ggagatttcg tggccatggg gaaccacccg 1800

ttgtacatta ttctactgtc gttgatggtt gtcgtttgct gctcgagtgg gttgatccgc 1860

atggaagtgg agacggactc catgaagctg tgggtttccg gtaggagctc tgtttttcag 1920

gagaggacgc gcttcggcga gatgcttggg ccagtggatc gtatggaacg actggtcctt 1980

gtcactaagg acggcggcgc agtgaccaga ccagcgtata tcaaggaggc gatccgcttg 2040

cagcaagtca ttggaagcga ggttgccgct gacagtatta cactgagtga gatctgcgtc 2100

aaagacgcct cgagctctcc atgccaggtg aattccgtaa cgcagtactt ccagaacagc 2160

atggatcact tcaacatgta cgatgcgtat gggctcgtag gcaagcactt gagcaactgc 2220

gctaatgctc cggaacgagc ggacggtaat gtgtgcagtg agctgcaagt gcaactcaat 2280

gcctcgggtg cttcgcttcc gacgagtatg agcggctgcc cttgtgcgtc gtcgtttggg 2340

gtgccgatgg ctgagctgga gaagtacctg ggcggattga gtactgacgg tggatcattg 2400

aatgccagtg cttatctgga gcaagcgaca actctctttt caactgccat ggtgactaac 2460

caccaggatg gtgctaagaa cgcagatgca atcgcttggg aacgcgcgta tatcgcgcgg 2520

atggagaaag aatcggatac caacacgatg tatgatatct attatgctgc agaggtgtct 2580

gctgacgacg agttcgtggc tgcttccaac ttggacatcg tcttcaaggc tggaattgca 2640

ggctttctct ttatgtttgt ttacgtggtc attggactga accactggaa gctggactac 2700

cgcttcttcc attcgtcaaa gatcggtgtg ggtttcatgg gcgttgcgtg catcctgatg 2760

gccgtgggcg ggaccttggg tatctttgcg tggactggag taaagcttca gatcgtgacg 2820

ctcgttgtga tgccggtggt tgtacttgcg atcggcactg gaaacatctt cctgatcctg 2880

catgccgtcg acctgaagca agaagagttg aagatggagc agcgctcact gtttgtgggt 2940

cttgaagaca acgatttcgg tattcatgag atcacgtgtg tgctgctgtg cgaggcaacg 3000

ggttatatcg gccccagcat gatcgcgacg tgcgtatgcg agtgctgtat cgttgctttt 3060

gcagcatatt cgaccatgcc agctgcacag tggctcgctg gatcgttggt gcttgggctc 3120

gcagccagtt ttgctttgca aatgaccttg ttcctcgcca tcgtagcgct ggacaagcgt 3180

cgtgagctca gcggcacgta cgatgtgatc tgctgtaagc gggcatcgtt cgctcgccgc 3240

cctcgcctct ccgaagacga gacgactgct gcgaccgaaa actcgtcgtt ccctggaagc 3300

actatttcac tgcccgactt gaacttgatg aaccgatgcg ttgctggata catccatgtg 3360

ctgctcaaga aggtgtcgaa ggtgttggtg ctcctcgtgt ttgccgcgtg tactctggcg 3420

gcgattgtct ccatcgaggc aatggatcgc ggtctttcac cgaactcgtt catcccgacg 3480

aactcgtatc tgcacgcata ctaccgtgca gtggacgaaa atgacttgtc gacgaaggag 3540

ttccctgcct atttcgttgt tgaagctggc tacggaagca accctaccgg attcaacgat 3600

ctcgccaacg atgcggaagc tcaatgcaaa ctgtgctcgt cgaaggagtt ctgcgacgac 3660

ttgtcgatcc cgaatattct cagtgcgttg gttgccgctg gggaaagcaa cgtcacgttt 3720

ttcaaggacg gcactgttgt tggatcatgg ttggacgact tctggagctt tgttgaccca 3780

gccagtgagt gctgccgcgt cgatgcggag aacgactact cgtattacgc tattctaccg 3840

gaagagagca gcgctgaata tgtgctcaag cgcgcgtcca acgcgccatc gtgtctcgct 3900

gactcaagtg cggtgctctc agtgcccgat gaatcgttca tgtcgttgtt cagcatgttc 3960

tcgactgcgg ctgctggacc cttgtgctcg tacgcagcag gcacgcgcta ccatggacag 4020

ctcagcgttg atagccaacc tattcctgcc atgagcagta gtgctgctgc gggcgtgact 4080

ctcaatggca ctggctacgg cagtgatgtg acggcgtttg tgtacaaggt tctgagcact 4140

acagtcggct cgtcaaagat ttcaggaagc caagaaggag caatcgcagc ctactcgcaa 4200

gctcagcaca ttgccaagtg gatcagcgag gaaactggca tcgatgtgtg ggcgtactcc 4260

cccgagtacg tttatctgga tcagttccac tcggttcgtc gtactgcgta cattgtggtg 4320

ggtgtcggac tcgcagtggt gttcgtgctg cagagtttgg ctctggggag ttactggtat 4380

ggatttgcgg tgacttgtgt cgctgcagcc acggttgtcc aagtcgctgg cctcatgatg 4440

ccgatggggg tcccaatcaa ctcgctttcg attgtgagcc tctcgatcgc cgtcaccttt 4500

tccgtcggat tctcggggca ttttgctcgt ctttttgcca aggctcgcac catcactgac 4560

gacttgggct actcacctgg cggcgatgcc tgcgttcgga aggtgttggc gcagcttctc 4620

gcgtcgtgga cgttgggcgt cgccgtctcc aagttcgttg ccatcgcagc gctcgcgctt 4680

gtcgccacgc ccgtcttcga acccgctggg aactgtttct tccggacgct gatggctgca 4740

gcggtgtgcg cgtggctgaa cagcgccgtg ctgcttcccg tcgggctcag catttgtgtg 4800

gatgccacgg aaggccgcgt gcgtgacgtg aagccgacga atgaagaggg cggggagtac 4860

tcgcgtgaga gcccttcgtc gtcataccac actgcgccgc cgactagcaa gtactga 4917

<210> 2

<211> 4477

<212> DNA

<213> Phytophthora sojae

<400> 2

atgcgcgtct cggccctcgc gtcgctgctg ctgccgctgg gcgtggcccg cgccgccagc 60

tcctcgagct ccacgacgtc gtcgggcgtg tcgtccgcct ccagctacgt gccgtggagc 120

ctcgccgaca acgccaccaa cctggccatc atcgagtcgc agctcaccat ctgcacgtac 180

tccaaggtgg aggagtgcat ccaggacccc aagctcgtgc aggagctcgg ctcgctctac 240

cgcgcgccgg gctactgcgt agccttcgac tcggcctacg tcaacatcac cacgaccagc 300

gctgccatcc ccaaccgcta ctacccgacg tccgtggagg acgcgtacga cgccggcttc 360

tccaacaagt tctccgagtg gtcggacacc aaccgcgagc agttcgaagt ggactgtccg 420

ctgctctaca atgagaccat cgccctgggc gacgacatgc tctgctgtac tgagtcccag 480

tacacagggc tgtccacgca ggtgcgcatg atccccggtc tgtgctcggc ctgcaaggag 540

aacctgcgca acatcttctg ccagttcacg tgcaacccga acaacagcat gttcctggac 600

atcaacgagg tgcgcatcat gtcgggcgac gacgagcacg aaggtcagat cttcccagcg 660

gtggaggagg caacgtacta cgtgggcaag gactggatcc gcgacatcta cgacttctgc 720

gaagacgaca gctccttctc gctgctgtgc aaccccaacc aggactgtac cgacggctac 780

ggcctcatgg agtacatggg caagtacgcc tacaacagca tcgggtcccc agagcagatc 840

aacgtgacaa ctatggatca gctttcggag gagctgcaaa tgaccgagtt ctgccattgc 900

gactacgcca acgagaccaa ttgcatttcg cccatgaaca acaagatgac gtcgtgtgtg 960

ggcgtctgcg gctcgctttg tgccgttagc tccgacgata cgcgcacgta cacggatgct 1020

tgctacggtg ccaaatccgc gtcctcttcc ggtagtggca gcgtcgtgga caacgatgac 1080

gacgcgactt ggatggagct caacgagtac ctcgccaaca acctgcccgc gacggattgg 1140

accccgttca actacttcct cgtgatcttt ggaggcgtgg tgggtgtgct gctgattgtg 1200

ggcttcatcg tcgccatgct ccgtgagagg aggtacagcg cagtcgagcg taactcgggc 1260

actccgcagg tagttggccc taacacacca gacttccatg gcgtggcgag cgcgatggct 1320

ggcagcgtcg gatacttgag cttcttggac gatctgatga caaacaagct gcggttgtgg 1380

gcggcgttcg tctccaaggg taaccgaccg aagaagatca ccccgatggt gctgtgcgtt 1440

gtcgtcattt gcgccgtcgg cttgtacaac attgatatcg aaacggaccc gatcaagctg 1500

tgggtttcta cgtcaagtac ttcgtacgag cagcgccaac attatggcga gatcttcaac 1560

ccgttctacc gctcggagca gatcattatg gtcccgaagg acggtggcaa catctaccgc 1620

tcgtcgatca tcaaggaggc catccgcgtg cagactgtgg cagccaacgt gacgtacact 1680

tcggacgacg gtgacgagac gattactctg gacgacattt gctggaaagc tacgggcacg 1740

ggctgcaccg tcaactcgat cacgcagtac ttccagaaca acatggagca cttcgagttc 1800

tacgagaagt atggattgga gctggagcac tttagtaact gcctttactc tccgaccacg 1860

tcggacgtgg cgctttgcac cgagttgaag aacgctttgg aggatggcga ttcgctcccg 1920

tcgagcatga gcgactgccc ctgcttgtcg gcgtttggct cgcccatgaa cctgtacaac 1980

acgtacctgg gtgggttccc tgacggcgct gagagcaact acacgctgtt cctcgaatcg 2040

gaagcgtttg tttcttcgta cctcaactac aactacgctg atgacgacaa gaacgaacct 2100

gccatcaagt gggagcgcga gtacatcaag acgatgaaga aggaggctgc gtctaacacg 2160

atcttcgacg tctatttcta cgccgaaatc tccgtcaacg atgaggtcga cgttgaatca 2220

aacaacggaa tgggccctgt ggcgctcagt tactgtttga tgctcatcta catttcgttg 2280

ggcattaatc gcatcaagtt cagtcgagag ttcttcattt cgtccaagat cctggccgga 2340

ttctgcggtg tgataagtat tgcctgcggt gtggcttcga cgatcggtat ctacatgtgg 2400

gctggagtca agctccagct tattatcatg gaagtggttc ccttcctgtc gctggccatc 2460

ggtgtcgaca acatcttctt gatcatccac gccatgacgg agaaggaaga tcagctccga 2520

cgcgaccagc caagtctctt tattggtctg gagcacaacc ccaaggcgat cgaggagatc 2580

acaacggtca tcttgtctga gagtatcgcg tacatcggcc ccagtatctt catggcctcc 2640

gctgctgagt cggtggcatt tgcgttcggt tccatctcgg cgatgcctgt cgtgctgtgg 2700

ttcgctgcga tggcatgctg tgctgtggcg atcaactttt gcttccagat gacgttcttc 2760

ctctctgttc ttactctgga caagcgtcgc gagctgagcg gcaagtacga catcattttc 2820

aagcgggcgt cagcggtggc agcacaggca cccgccgctc ctgagactgt tcagcattcc 2880

tcagagccgc ttgtgtcgct gcagcccaag actccagcag cggacgatgt ccgtccttct 2940

gtcacgcccg agaacagcac gctgacggat gtccttgact attgcgtgga tgtctacgcg 3000

tcgatattaa cccacaagct tgtgaagctc gtcgtgctgt tactgttcct cgcgtggaca 3060

ttgtggtcta tctactcgat ggaatcgctg gaccagggcc tgccgcagaa ggaggccatg 3120

ccttcagact cgtacatgat cgagtacttc aacgcactgg atgtgtacct cgccacgggt 3180

gtgccggtgt acttcgtggt cgagactggc tatggtcgga accccgatgc ttggtcactg 3240

aacgatgaga gtgtcgagac gatcttctgc aagtccaagg atatctgcgg gacgtactca 3300

attcccaaca ttatgaacgc attggcaaac gacggtgaca aaacgaatac ccacatcagc 3360

cccggcacga cctactcgtg gatggatgac ttctggggct tcgtcaaccc tgacagcgag 3420

tgttgccgtg tggattccga gggcgcctac gtcccgatcg aaactggcaa cgacacgtac 3480

acgactctgc gcgcggacga cgacacgtgc cttgctacgt cggtgatgat tccccccgtg 3540

cctgaggccc aatacatgtc gctcttcagt atgttcgcga cggcaagtgc tggtacgtcg 3600

tgctcgtacg gtggtggttc gatctaccgt ggccagttca gtatcgactc ggagcccatt 3660

ccgaccgtca acgcctcgac gcctgcggtg aagatcaaca gcagtggcta cggtgacgag 3720

atcacggctt ggtcgtacat ggtgacgggc acgtcgaacc cgacgcagca gcgctacatc 3780

gactcgtaca agcagaacct tgcggcggcc gagtggatca gcgagaagac tggtgtggac 3840

atctgggtgt actcgctgac gtacgtgtac ttcgagcagt acctgactgt ggtcgacgac 3900

gcctacaagc tcatcggtct ggcgctggcg gctatcttcg tgatcacgac gctgtacctg 3960

ggcaacgtgt tctactcgct ggtgatcgcg ttgacggcga ccaacatcgt cgtgctcgtg 4020

ctgggtctga tgcagccgct ggacatcatg ctcaacggtc tgtccattgt gaacctgatc 4080

atcgcggctg gcatctcggt tgagttctgc ggtcactacg tccggttctt cgcgaaggct 4140

cgtggtacgg gcgacgagcg agctcgcgat gctcttcgcc aggtcttgac gtcggttgtg 4200

tttggcatca ccatcacgaa ggtcatcggt ctgagtgtgc tgacactcgc ggactcgcgc 4260

gtgttcaaga agtactactt ccgcatgtac atgatggtcg tggtgtgcgg tgtgctcaac 4320

ggcatgttgc tgctgcctgt ggtgctgagc accatcatgg acgtcaagaa ctttttcctc 4380

cgcaagcgct cgaggaagag cgagctcccg ttggcccctg tcactcgcgt agagtaagtc 4440

agcctactct gtcaccaact gtgtggagct ttcaacg 4477

<210> 3

<211> 3138

<212> DNA

<213> Phytophthora sojae

<400> 3

atggcgcgcg ccttctatcg cctgggcgcg ctctgcagcg gctcccccgt gctcatggcg 60

ctggcggcgc tgctctgcgg cggcgtgctg tgcctggggc tgctcaacat gcggctgcag 120

accgacccgc agggcctgtg ggtgcctccg cgcagcgtcg cggcccggga gcaggcgcgc 180

ttcgacgagc tcttcgggcc cttcttccgc gtccagcagc tcatcttcta cgcggactca 240

gactcagatg gtctctctgc gacgtgtgac gcctcgcgcg acctggtgca gcgccgcttc 300

ctgctgcaga tggccaaggt gcaggcgcag atcgccgacg ccgccgtcac agtgcagggc 360

gacggcgccc agggcaaggt gacgctctcg ctcgaggact tctgctaccg ccccatccgc 420

ggcaagggct gcctcgtcac cagccccttc cagtactggc tcggcaacgc gtcgctgctg 480

gagggcgacc cggacatcaa gctcacgacc gcgtgccaga ccacggaccc acagctccag 540

gagcgcgcgc cctgcatgga ccagaatggc gtccccgtca tgcgcgacgt cgtgttcggc 600

ggcctgtcga gggacgactg ccaccagaac ccggacccgt gcggggaggc cacgccgcag 660

gcccaggcgc tcgtggtcac gttcctgctc aacaacaggc ccgagaacga gacgtacacg 720

aggtacgtgg agcagtggga gcagcaggcg ttcctcaaga ttgcagcaca ggcggcggag 780

gcgcttaagc cctcctcgac cgccaacaag agcgacgagt tcatctggga cagcgtgcag 840

gaccaggagc tggcggacgt gggcgtcgac ggcatgcgcc tatcctacat ggcggagcgg 900

tcggtggccg actcgctcgt ggtgcagacc aaccagaacg cgtttattgt ggtggtgagc 960

tacctcgtca tgttcttgta cgtgtcggcg tcgctcggca agttcacgga cccggtgcgc 1020

tcgcggttcg ggctggggct cacgggcatc ctgatcgtgc tgctgtcgct gggggcggct 1080

atgggggtga gctgcgccat tctgcagatg gaggtgacga tgatcacgtt ggaggtcgtg 1140

ccgttcctgg tgctggctat cggcgtcgac aacatgttta tcttgaccaa cgagttcgat 1200

cgactcgcgg ctctgcgtgg cctcgcaacg ctggacacca ggcgcaacac gcgcgaccgg 1260

gcagaggacg aactgttgat gttgaagcag gtgctgggag aaaccatggt caatgtcgga 1320

ccctcgatcg tcgtggcggc tgttgcagaa acgttggcgt tcttggtggg ggctctcaca 1380

cgtatccccg ccctaacgag cttctgcgtg gttgctgcgc tggctgttgc cgctgacttt 1440

gcgctgcaaa tgacgtggtt tgcctcggcc ctcgtgttgg acgcgcgccg cgtgcgtgcc 1500

cgacgctacg atttgttccc gtggatgaag cagaaactca ccctcacgcc gcccacaaag 1560

ggcaagcgga ggatcgagtc caagattcac taccagtatg acttgctggt tgacgaaagc 1620

gagcgaagtg acgagccagc agctcgtgtg agcagcactg gcacacttca gcgtttcgtg 1680

gagaaaacct acatcccgtt cctgttacgc cggtccacga aagtgctggt gcttgtgacg 1740

gctctttcag tggtaactct tagtgctttt ggctcctccg aactcccact cggactcgag 1800

caggagcttg cggtgcctac ggatttttac ctgcatgagt acttcaagaa acagaccgcg 1860

cttggtgagg cggggccacc agcttacgtt gttctggata gcgatgtgga ttacacagat 1920

gcacgcctcc agcaagatgt gaacgtgcta ttggatcagc tttccggtct tcgccagtat 1980

atccagctgc cggtgtgctc gtggctgcat acgttcaacc aatggcgtca aatgcgcttc 2040

ttcctacagg acaagattaa acaaggtcag tgcgattgtc cggtgcagcc aatggatccg 2100

ttcccgtacg aactcgcgaa tgttggagtc gaggacccca gtggagacgt tgatttgttc 2160

ctagcgcccg agtatgggta tggcgctctg gcaacggcgc atgtgacccc caatgcgctg 2220

ttctacccgt tggtgaagaa cttcacaaag atctcaattg actcaacgtg ctgccagcat 2280

ttcgggctgtgtggagcgcagtatgaaggagacatcatattcaacgagccaaatgcaggt2340

gatgacgact ctacgggcat gtctatcgtc ggttctcgta tacgcttcca gctgaatgca 2400

ctccgcaacc agtccatgtt cgtgaactcg tactactacc ttcatgatgt ggtggggcgt 2460

tggagcatcg accatgccgc cactgccttc ccgtacgctc ttgtcttcgt gtacgaagag 2520

caatacacgt acatccaggg agtggcgctg caaagtgtgc tgctggcgct ggccgtggtg 2580

ttcggagcgc tcttcgtgct gatggacggg agcttgcggc tgaccaccgt tgtcaccttg 2640

tgcgtgctgt cgatgacttt ctcgcagttg ggcttcctct tcgtgtggaa tatgattgct 2700

gggccgggcg cagaaacgtc gatcaatgca gtctcggtgg tgaatctgct cgcctgtgtt 2760

gggcttggtg tcgagttttg cgtgcacacg gcacaccagt ttgccttttc gcgacggcat 2820

cacctgggca caactgccaa cgaccacaca cgatacgctc tgagcagtgt gggcgcatcc 2880

atcttctccg gtattacgct caccaagttc tgcggcatcg gtgtccttgc gttcgcgccg 2940

tccatgttgt tccgcgtgta cttcttccgc atgtacctgg gcattgtcgt gttgggctgc 3000

ttccacggtc tcgtactgct gccagtgctg ctgagtctca ttggccagcc gcagaagtac 3060

ccgaacgatc tcagctcgtt tctactttct gaagagcgag acgacgagtt agaggacgaa 3120

gtgagagcgt tcaagtga 3138

<210> 4

<211> 4461

<212> DNA

<213> Phytophthora sojae

<400> 4

atgcgcgtct cggccctcgc gtcgctgctg ctgccgctgg gcgtggcccg cgccgccagc 60

tcctcgggct ccacgacgtc cacggcgacc acgacgacaa cgggcacgtc gtccgtctcc 120

agctacgtgc cctggacgct ggccgacaac gccaccaacc tggccgacat caaggcgcag 180

ctcaccatgt gcacgtactc caaggtggag gagtgcatcc aggacccggc gctggtgcac 240

gagctcggcg cgctcgtgcg cgctcctggc cactgcgtgg ccttcgactc gtcctacgtc 300

aacgtcacca cggccggtgt cgccatcccc aaccgctact acccgacgtc cgtggaggac 360

gcgtacgacg ccggcttctc caacaagttc tccgagtggt cggacaccaa ccgcgagcag 420

ttcgaggtgg actgcccgct gctctacaac gagaccatcg cccagggcga cgacatgctc 480

tgctgtaccg agaaccagta cacggggctg tccacgcagg tgcgcatgat ccccggtctg 540

tgctcggcct gcaaggagaa cctgcgcaac atcttctgtc agatgacgtg cagccccaac 600

aacagcatgt tcctggacgt caacgaggtg cgcatcatgc ccggcgacga cgagcacccg 660

gacgccgtgt tccccgccgt ggaggaggtc acgtactacg tgggcagcga ctggatccgc 720

gacatctacg acttctgcga ggccgacagc tccttctcgc tgctgtgcaa ccccaatcag 780

gactgccatg acggctacgg cctcctcgag tacatgggca agtacgcctt caacagcatc 840

gggtctccgc tccagattaa cgtgacgacc atggacaagg tccccgagat caaccaaatg 900

accgagttct gccactgcga caacgtgaat gccaccaact gcatcctgcc gcagaacagc 960

aggatgacgt cctgcgtcgg cacctgcggc tcgctgtgtg ccgtgagctc gagcgacgac 1020

cgcacgtaca cagagtcctg ctacggcgcc agcaatgcgg tcgccacgtc ctcctcggcc 1080

agcggaagcg tcggctccgg ctcggacgac tcgacgtggg ccgagctgaa cgcgtacctg 1140

gccagcaaca tcccggtgac ggactggacg ggcctcaact acttcctcgt catcttcggt 1200

ggagccgtcg ccctgctgct cattgtgggc ttcattgtcg ctggctgccg cgagcggagg 1260

gcccgcatcc ccaacccgca cacgggcacg ccccacatcg gcccgtacac gcctgaggct 1320

cacggcgtgg cgcacgcgat ggagacgagc aacacgcgtc tgagctatct ggacgagctc 1380

atgacgaaca agctgcgcac gtgggcggta ttcgtgtcga cgggcaaccg gcccaagaag 1440

atgatcccga tggtgctgtg cgttgtcgct gtctgcgttg tcggcctgta caacattgat 1500

atcgagacgg acccgatcaa gctgtgggtg tcctcgtcca gtacctcgta ccagcagcgc 1560

cagcactacg gcgagatctt caacccgttc taccgctccg agcaggtcat tatggtgccc 1620

aaggacggcg gcaacatcta ccgctcgtcg atcatcaagg aggccatccg cgtgcagact 1680

gtggcagcca acgtgacgta cacttcggac gacggtgacg agacgattac tctggacgac 1740

atttgctgga aggctacggg cacgggctgc accgtcaact cgatcacgca gtacttccag 1800

aacaacatgg agcacttcga gttctacgag aagtatgggc tggagatgga gcactttagt 1860

aactgccttt actctccgac cacgtcggac gtggcgcttt gcaccgagtt gaagaacgct 1920

ctggaggatg gcgactcgct cccgtcgagc atgagcgact gcccctgctt gtcagcgttt 1980

ggctcgccca tgaacctgta caacacctac ctgggtggct tccctgacgg cgctgaaagc 2040

aactacacgt tgttcctcga ctctgtggcg tttgtttcct cgtacctgaa ctacaactac 2100

gctgacgacg acaagaacga acctgccatc aagtgggagc gcgagtacat caagacgatg 2160

aaggaggagg ctgcgtccaa cacgatcttc gacgtctact tctatgccga aatctccgtc 2220

aacgatgaga tcgacgccga gtcgagcaac ggtatgggcc ctgtggcgct cagttactgt 2280

ttgatgatca tctacatctc gctcggtatc aaccgcatca agttcagccg cgagtttttc 2340

atctcgtcca agatcgtggc cggtttctgc ggtgtcatga gcattgtgtg cggtgtggcg 2400

tcgacgatcg gtatctacat gtgggctgga gtcaagctcc agcttattat catggaagtg 2460

gttcccttcc tgtcgctggc catcggtgtc gacaacatct tcttgatcat ccacgctatg 2520

acggagaagg aagaccagat gcgccgtgag cagccaagtc tcttcatcgg tctggagcac 2580

aaccccacgg cgatcgagga gatcaccacg acgattctgt ccgagagtct ggcgtacatc 2640

ggccccagta tcttcatggc ctccgctgcc gagtccgtgg ccttcgcgtt cggttccatt 2700

tcgccgatgc ctgtcgtgct gtggttcgcc gccatggctt gctgcgctgt ggcgatcaac 2760

ttctgcctcc agatgaccct gttcctgtct gttcttacgc tggacaagcg ccgcgagctg 2820

agcggcaagt acgacattat cttcaagcgc gcctcgtacg tcaggtcgca gccgcctgcc 2880

ggtggcccgg agactcagca gactgcccaa ccccttgtct ctctcgagcc caagaccccg 2940

gcgcccgaag atgcccgccg ctctatcacg cctgagaacc gcacgctgac ggacgtcctc 3000

gactactgcg tggatgtcta cgcgtcgatc ctgacgtaca agattgtgaa gctcgtcgtg 3060

ctgctggtct tcctcttctg gacgttgtgg tcgatctact cgatggaatc gttggaccag 3120

ggtctgccgc agaaggaggc catgccttca gactcgtaca tgattgagta cttcaacgcg 3180

ctggatgtgt acctcgccac gggtgtgcct gtgtacttca tcgttgaaac tggctacggt 3240

cgcaacccgg acacgtggtc gctgaacgac gagagcgtcg agacgctctt ctgcaagtcg 3300

aaggacatct gtggaacgta ctcaatcccc aacatcatga acgcgctggc caaccacgga 3360

gacaagaatg tgacgcacat cagccccggc acgacctact cgtggatgga tgacttctgg 3420

ggcttcgtca accctgacag cgagtgttgc cgtgtggatt ccgagggcgc ctacgtcccg 3480

atcgaaactg gcaacgacac gtacacgact ctgcgcgcgg acgacgacac gtgccttgct 3540

acgtcggtga cgattccccc cgtgcctgag gcccaataca tgtcgctctt cagtatgttc 3600

gcgacggcaa gtgctggtac gtcgtgctcg tacggtggtg gttcgatcta ccgtggccag 3660

ttcagtatcg actcggagcc cattccgacc gtcaacgcct cgacgcctgc ggtgaagatc 3720

aacagcagtg gctacggtga cgagatcacg gcttggtcgt acatggtgac gggcacgtcg 3780

aacccgacgc agcagcgcta catcgactcg tacaagcaga accttgcggc ggccgagtgg 3840

atcagcgaga agactggtgt ggacatctgg gtgtactcgc tgacgtacgt gtacttcgag 3900

cagtacctga ctgtggtcga cgacgcctac aagctcatcg gtctggcgct ggcggctatc 3960

ttcgtgatca cggcgctgta cctgggcaac gtgttctact cgctggtgat cgcgttgacg 4020

gcgaccaaca tcgtcgtgct cgtgctgggt ctgatgcagc cgctggacat catgctcaac 4080

ggtctgtcca ttgtgaacct gatcatcgcg gccggtatcg ccgtcgagtt ctgcggtcac 4140

tacgtccggt tcttcgcgaa ggctcgcggt acgggcgacg agcgcgctcg cgatgctctt 4200

cgccaggtct tgacgtcggt tgtgtttggc atcaccatca cgaaggtcat cggtctgagt 4260

gtgctgacac tcgcggactc gcgcgtgttc aagaagtact acttccgtat gtacatgatc 4320

gtcgtgctct gcggtgtgct caacggcatg ctgctgctgc ccgtgctgct cagcaccatc 4380

acagacgtca aggacttctt cttgcgtaga ggatcacgga aaaccgacct gccttcggcg 4440

cctgttactc gggtggagta a 4461

<210> 5

<211> 1597

<212> PRT

<213> Phytophthora sojae

<400> 5

Met Trp Leu Phe Ala Ala Ile Ser Leu Ala Ile Ala Thr Pro Cys Phe

1 5 10 15

Arg Ala Trp Leu Glu Ala Leu Phe Ala Glu Leu Ser Ile Ile Phe Phe

20 25 30

Thr Tyr Leu Arg Leu Val Ser Ala Gly Leu Ser Leu Val Leu Leu Leu

35 40 45

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

50 55 60

Leu Ala Arg Val His Trp Arg Arg Gly Thr Arg Arg Arg Ala Leu Met

65 70 75 80

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

85 90 95

Pro Pro Gln Leu Trp Thr Ser Ser Leu Phe Ala Leu Ala Met Leu Pro

100 105 110

Ile Ile Asn Ser Val Val Thr Ala Ala Ala Ala Gln Thr Ala Phe Asp

115 120 125

Ala Asp Ala Ala Ala Leu Val Thr Leu Gln Gln Gln Leu Thr Thr Cys

130 135 140

Lys Tyr Ser Gly Gly Asp Asp Cys Leu Asp Asp Val Thr Ala Ile Ser

145 150 155 160

Val Leu Gly Ser Tyr Gln Gln Ala Ser Gly Tyr Cys Val Ala Phe Asp

165 170 175

Ala Ala Tyr Val Asn Val Thr Thr Gly Ala Ala Leu Pro Ala Gln Tyr

180 185 190

Phe Pro Ile Gly Val Glu Glu Ala His Ala Gln Gly Phe Ala Asn Asn

195 200 205

Phe Ser Ala Trp Ser Glu Thr Asn Gln Glu Lys Phe Lys Thr Asp Cys

210 215 220

Pro Leu Leu Phe Asn Glu Thr Val Ser Gly Asp Gly Glu Gly Leu Leu

225 230 235 240

Cys Cys Thr Glu Thr Gln Tyr Glu Met Leu Ser Leu Gln Val Arg Lys

245 250 255

Leu Pro Gly Glu Cys Thr Ser Cys Lys Gln Asn Leu Arg Asn Leu Trp

260 265 270

Cys Gln Phe Thr Cys His Pro Ser Asn Ser Leu Phe Val Asp Val Thr

275 280 285

Gln Val Arg Leu Met Glu Gly Asp Ala Asp His Ala Asp Glu Val Phe

290 295 300

Pro Ala Ile Glu Glu Ala Thr Tyr Tyr Val Gly Ser Asp Met Val Arg

305 310 315 320

Asp Leu His Asp Phe Cys Glu Ala Asp Ser Gly Phe Met Pro Leu Leu

325 330 335

Cys Gly Met Asn Ala Asp Gly Asn Cys Ser Thr Thr Gly Ser Asp Met

340 345 350

Leu Gly Tyr Leu Gly Ala Tyr Ser Phe Asp Gly Val Gly Ser Pro Ser

355 360 365

Gln Val Ile Phe Thr Thr Met Glu Gln Leu Ser Ala Ala Glu Gln Glu

370 375 380

Asp Lys Ile Cys Ala Cys Asp Ser Ser Asn Thr Thr Gly Cys Phe Ser

385 390 395 400

Pro Met Asp Thr Arg Leu Glu Ser Cys Val Asp Thr Cys Gly Ser Leu

405 410 415

Cys Ala Val Ser Asp Asp Asp Ser Arg Gln Tyr Gln Ala Ala Cys Tyr

420 425 430

Ser Ser Gly Ser Thr Ser Ala Ser Asp Asp Leu Ser Thr Val Thr Thr

435 440 445

Ala Thr Thr Ser Thr Ser Ala Ala Asp Lys Leu Glu Ser Leu Leu Ser

450 455 460

Asp Leu Ser Ser Arg Ala Glu Gly Gly Ser Phe Ala Val Leu Asn Tyr

465 470 475 480

Val Leu Ala Val Leu Ala Phe Phe Gly Ala Thr Ala Leu Ala Leu Gly

485 490 495

Phe Ala Tyr Ser Thr Arg Tyr Gly Arg Lys Lys Arg Gln Ser Val Leu

500 505 510

Asp Asp Pro Val Asn Gly Leu Gly Ser Gly Met Leu Ser Leu Val Asn

515 520 525

Leu Asp Gln Leu Lys Gly Ile Gly Arg Trp Asp Asp Arg Leu Thr Met

530 535 540

His Leu Lys Arg Trp Gly Asp Phe Val Ala Met Gly Asn His Pro Leu

545 550 555 560

Tyr Ile Ile Leu Leu Ser Leu Met Val Val Val Cys Cys Ser Ser Gly

565 570 575

Leu Ile Arg Met Glu Val Glu Thr Asp Ser Met Lys Leu Trp Val Ser

580 585 590

Gly Arg Ser Ser Val Phe Gln Glu Arg Thr Arg Phe Gly Glu Met Leu

595 600 605

Gly Pro Val Asp Arg Met Glu Arg Leu Val Leu Val Thr Lys Asp Gly

610 615 620

Gly Ala Val Thr Arg Pro Ala Tyr Ile Lys Glu Ala Ile Arg Leu Gln

625 630 635 640

Gln Val Ile Gly Ser Glu Val Ala Ala Asp Ser Ile Thr Leu Ser Glu

645 650 655

Ile Cys Val Lys Asp Ala Ser Ser Ser Pro Cys Gln Val Asn Ser Val

660 665 670

Thr Gln Tyr Phe Gln Asn Ser Met Asp His Phe Asn Met Tyr Asp Ala

675 680 685

Tyr Gly Leu Val Gly Lys His Leu Ser Asn Cys Ala Asn Ala Pro Glu

690 695 700

Arg Ala Asp Gly Asn Val Cys Ser Glu Leu Gln Val Gln Leu Asn Ala

705 710 715 720

Ser Gly Ala Ser Leu Pro Thr Ser Met Ser Gly Cys Pro Cys Ala Ser

725 730 735

Ser Phe Gly Val Pro Met Ala Glu Leu Glu Lys Tyr Leu Gly Gly Leu

740 745 750

Ser Thr Asp Gly Gly Ser Leu Asn Ala Ser Ala Tyr Leu Glu Gln Ala

755 760 765

Thr Thr Leu Phe Ser Thr Ala Met Val Thr Asn His Gln Asp Gly Ala

770 775 780

Lys Asn Ala Asp Ala Ile Ala Trp Glu Arg Ala Tyr Ile Ala Arg Met

785 790 795 800

Glu Lys Glu Ser Asp Thr Asn Thr Met Tyr Asp Ile Tyr Tyr Ala Ala

805 810 815

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

820 825 830

Val Phe Lys Ala Gly Ile Ala Gly Phe Leu Phe Met Phe Val Tyr Val

835 840 845

Val Ile Gly Leu Asn His Trp Lys Leu Asp Tyr Arg Phe Phe His Ser

850 855 860

Ser Lys Ile Gly Val Gly Phe Met Gly Val Ala Cys Ile Leu Met Ala

865 870 875 880

Val Gly Gly Thr Leu Gly Ile Phe Ala Trp Thr Gly Val Lys Leu Gln

885 890 895

Ile Val Thr Leu Val Val Met Pro Val Val Val Leu Ala Ile Gly Thr

900 905 910

Gly Asn Ile Phe Leu Ile Leu His Ala Val Asp Leu Lys Gln Glu Glu

915 920 925

Leu Lys Met Glu Gln Arg Ser Leu Phe Val Gly Leu Glu Asp Asn Asp

930 935 940

Phe Gly Ile His Glu Ile Thr Cys Val Leu Leu Cys Glu Ala Thr Gly

945 950 955 960

Tyr Ile Gly Pro Ser Met Ile Ala Thr Cys Val Cys Glu Cys Cys Ile

965 970 975

Val Ala Phe Ala Ala Tyr Ser Thr Met Pro Ala Ala Gln Trp Leu Ala

980 985 990

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

995 1000 1005

Leu Phe Leu Ala Ile Val Ala Leu Asp Lys Arg Arg Glu Leu Ser Gly

1010 1015 1020

Thr Tyr Asp Val Ile Cys Cys Lys Arg Ala Ser Phe Ala Arg Arg Pro

1025 1030 1035 1040

Arg Leu Ser Glu Asp Glu Thr Thr Ala Ala Thr Glu Asn Ser Ser Phe

1045 1050 1055

Pro Gly Ser Thr Ile Ser Leu Pro Asp Leu Asn Leu Met Asn Arg Cys

1060 1065 1070

Val Ala Gly Tyr Ile His Val Leu Leu Lys Lys Val Ser Lys Val Leu

1075 1080 1085

Val Leu Leu Val Phe Ala Ala Cys Thr Leu Ala Ala Ile Val Ser Ile

1090 1095 1100

Glu Ala Met Asp Arg Gly Leu Ser Pro Asn Ser Phe Ile Pro Thr Asn

1105 1110 1115 1120

Ser Tyr Leu His Ala Tyr Tyr Arg Ala Val Asp Glu Asn Asp Leu Ser

1125 1130 1135

Thr Lys Glu Phe Pro Ala Tyr Phe Val Val Glu Ala Gly Tyr Gly Ser

1140 1145 1150

Asn Pro Thr Gly Phe Asn Asp Leu Ala Asn Asp Ala Glu Ala Gln Cys

1155 1160 1165

Lys Leu Cys Ser Ser Lys Glu Phe Cys Asp Asp Leu Ser Ile Pro Asn

1170 1175 1180

Ile Leu Ser Ala Leu Val Ala Ala Gly Glu Ser Asn Val Thr Phe Phe

1185 1190 1195 1200

Lys Asp Gly Thr Val Val Gly Ser Trp Leu Asp Asp Phe Trp Ser Phe

1205 1210 1215

Val Asp Pro Ala Ser Glu Cys Cys Arg Val Asp Ala Glu Asn Asp Tyr

1220 1225 1230

Ser Tyr Tyr Ala Ile Leu Pro Glu Glu Ser Ser Ala Glu Tyr Val Leu

1235 1240 1245

Lys Arg Ala Ser Asn Ala Pro Ser Cys Leu Ala Asp Ser Ser Ala Val

1250 1255 1260

Leu Ser Val Pro Asp Glu Ser Phe Met Ser Leu Phe Ser Met Phe Ser

1265 1270 1275 1280

Thr Ala Ala Ala Gly Pro Leu Cys Ser Tyr Ala Ala Gly Thr Arg Tyr

1285 1290 1295

His Gly Gln Leu Ser Val Asp Ser Gln Pro Ile Pro Ala Met Ser Ser

1300 1305 1310

Ser Ala Ala Ala Gly Val Thr Leu Asn Gly Thr Gly Tyr Gly Ser Asp

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

Gln His Ile Ala Lys Trp Ile Ser Glu Glu Thr Gly Ile Asp Val Trp

1365 1370 1375

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

1380 1385 1390

Arg Thr Ala Tyr Ile Val Val Gly Val Gly Leu Ala Val Val Phe Val

1395 1400 1405

Leu Gln Ser Leu Ala Leu Gly Ser Tyr Trp Tyr Gly Phe Ala Val Thr

1410 1415 1420

Cys Val Ala Ala Ala Thr Val Val Gln Val Ala Gly Leu Met Met Pro

1425 1430 1435 1440

Met Gly Val Pro Ile Asn Ser Leu Ser Ile Val Ser Leu Ser Ile Ala

1445 1450 1455

Val Thr Phe Ser Val Gly Phe Ser Gly His Phe Ala Arg Leu Phe Ala

1460 1465 1470

Lys Ala Arg Thr Ile Thr Asp Asp Leu Gly Tyr Ser Pro Gly Gly Asp

1475 1480 1485

Ala Cys Val Arg Lys Val Leu Ala Gln Leu Leu Ala Ser Trp Thr Leu

1490 1495 1500

Gly Val Ala Val Ser Lys Phe Val Ala Ile Ala Ala Leu Ala Leu Val

1505 1510 1515 1520

Ala Thr Pro Val Phe Glu Pro Ala Gly Asn Cys Phe Phe Arg Thr Leu

1525 1530 1535

Met Ala Ala Ala Val Cys Ala Trp Leu Asn Ser Ala Val Leu Leu Pro

1540 1545 1550

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

1555 1560 1565

Val Lys Pro Thr Asn Glu Glu Gly Gly Glu Tyr Ser Arg Glu Ser Pro

1570 1575 1580

Ser Ser Ser Tyr His Thr Ala Pro Pro Thr Ser Lys Tyr

1585 1590 1595

<210> 6

<211> 1478

<212> PRT

<213> Phytophthora sojae

<400> 6

Met Arg Val Ser Ala Leu Ala Ser Leu Leu Leu Pro Leu Gly Val Ala

1 5 10 15

Arg Ala Ala Ser Ser Ser Ser Ser Thr Thr Ser Ser Gly Val Ser Ser

20 25 30

Ala Ser Ser Tyr Val Pro Trp Ser Leu Ala Asp Asn Ala Thr Asn Leu

35 40 45

Ala Ile Ile Glu Ser Gln Leu Thr Ile Cys Thr Tyr Ser Lys Val Glu

50 55 60

Glu Cys Ile Gln Asp Pro Lys Leu Val Gln Glu Leu Gly Ser Leu Tyr

65 70 75 80

Arg Ala Pro Gly Tyr Cys Val Ala Phe Asp Ser Ala Tyr Val Asn Ile

85 90 95

Thr Thr Thr Ser Ala Ala Ile Pro Asn Arg Tyr Tyr Pro Thr Ser Val

100 105 110

Glu Asp Ala Tyr Asp Ala Gly Phe Ser Asn Lys Phe Ser Glu Trp Ser

115 120 125

Asp Thr Asn Arg Glu Gln Phe Glu Val Asp Cys Pro Leu Leu Tyr Asn

130 135 140

Glu Thr Ile Ala Leu Gly Asp Asp Met Leu Cys Cys Thr Glu Ser Gln

145 150 155 160

Tyr Thr Gly Leu Ser Thr Gln Val Arg Met Ile Pro Gly Leu Cys Ser

165 170 175

Ala Cys Lys Glu Asn Leu Arg Asn Ile Phe Cys Gln Phe Thr Cys Asn

180 185 190

Pro Asn Asn Ser Met Phe Leu Asp Ile Asn Glu Val Arg Ile Met Ser

195 200 205

Gly Asp Asp Glu His Glu Gly Gln Ile Phe Pro Ala Val Glu Glu Ala

210 215 220

Thr Tyr Tyr Val Gly Lys Asp Trp Ile Arg Asp Ile Tyr Asp Phe Cys

225 230 235 240

Glu Asp Asp Ser Ser Phe Ser Leu Leu Cys Asn Pro Asn Gln Asp Cys

245 250 255

Thr Asp Gly Tyr Gly Leu Met Glu Tyr Met Gly Lys Tyr Ala Tyr Asn

260 265 270

Ser Ile Gly Ser Pro Glu Gln Ile Asn Val Thr Thr Met Asp Gln Leu

275 280 285

Ser Glu Glu Leu Gln Met Thr Glu Phe Cys His Cys Asp Tyr Ala Asn

290 295 300

Glu Thr Asn Cys Ile Ser Pro Met Asn Asn Lys Met Thr Ser Cys Val

305 310 315 320

Gly Val Cys Gly Ser Leu Cys Ala Val Ser Ser Asp Asp Thr Arg Thr

325 330 335

Tyr Thr Asp Ala Cys Tyr Gly Ala Lys Ser Ala Ser Ser Ser Gly Ser

340 345 350

Gly Ser Val Val Asp Asn Asp Asp Asp Ala Thr Trp Met Glu Leu Asn

355 360 365

Glu Tyr Leu Ala Asn Asn Leu Pro Ala Thr Asp Trp Thr Pro Phe Asn

370 375 380

Tyr Phe Leu Val Ile Phe Gly Gly Val Val Gly Val Leu Leu Ile Val

385 390 395 400

Gly Phe Ile Val Ala Met Leu Arg Glu Arg Arg Tyr Ser Ala Val Glu

405 410 415

Arg Asn Ser Gly Thr Pro Gln Val Val Gly Pro Asn Thr Pro Asp Phe

420 425 430

His Gly Val Ala Ser Ala Met Ala Gly Ser Val Gly Tyr Leu Ser Phe

435 440 445

Leu Asp Asp Leu Met Thr Asn Lys Leu Arg Leu Trp Ala Ala Phe Val

450 455 460

Ser Lys Gly Asn Arg Pro Lys Lys Ile Thr Pro Met Val Leu Cys Val

465 470 475 480

Val Val Ile Cys Ala Val Gly Leu Tyr Asn Ile Asp Ile Glu Thr Asp

485 490 495

Pro Ile Lys Leu Trp Val Ser Thr Ser Ser Thr Ser Tyr Glu Gln Arg

500 505 510

Gln His Tyr Gly Glu Ile Phe Asn Pro Phe Tyr Arg Ser Glu Gln Ile

515 520 525

Ile Met Val Pro Lys Asp Gly Gly Asn Ile Tyr Arg Ser Ser Ile Ile

530 535 540

Lys Glu Ala Ile Arg Val Gln Thr Val Ala Ala Asn Val Thr Tyr Thr

545 550 555 560

Ser Asp Asp Gly Asp Glu Thr Ile Thr Leu Asp Asp Ile Cys Trp Lys

565 570 575

Ala Thr Gly Thr Gly Cys Thr Val Asn Ser Ile Thr Gln Tyr Phe Gln

580 585 590

Asn Asn Met Glu His Phe Glu Phe Tyr Glu Lys Tyr Gly Leu Glu Leu

595 600 605

Glu His Phe Ser Asn Cys Leu Tyr Ser Pro Thr Thr Ser Asp Val Ala

610 615 620

Leu Cys Thr Glu Leu Lys Asn Ala Leu Glu Asp Gly Asp Ser Leu Pro

625 630 635 640

Ser Ser Met Ser Asp Cys Pro Cys Leu Ser Ala Phe Gly Ser Pro Met

645 650 655

Asn Leu Tyr Asn Thr Tyr Leu Gly Gly Phe Pro Asp Gly Ala Glu Ser

660 665 670

Asn Tyr Thr Leu Phe Leu Glu Ser Glu Ala Phe Val Ser Ser Tyr Leu

675 680 685

Asn Tyr Asn Tyr Ala Asp Asp Asp Lys Asn Glu Pro Ala Ile Lys Trp

690 695 700

Glu Arg Glu Tyr Ile Lys Thr Met Lys Lys Glu Ala Ala Ser Asn Thr

705 710 715 720

Ile Phe Asp Val Tyr Phe Tyr Ala Glu Ile Ser Val Asn Asp Glu Val

725 730 735

Asp Val Glu Ser Asn Asn Gly Met Gly Pro Val Ala Leu Ser Tyr Cys

740 745 750

Leu Met Leu Ile Tyr Ile Ser Leu Gly Ile Asn Arg Ile Lys Phe Ser

755 760 765

Arg Glu Phe Phe Ile Ser Ser Lys Ile Leu Ala Gly Phe Cys Gly Val

770 775 780

Ile Ser Ile Ala Cys Gly Val Ala Ser Thr Ile Gly Ile Tyr Met Trp

785 790 795 800

Ala Gly Val Lys Leu Gln Leu Ile Ile Met Glu Val Val Pro Phe Leu

805 810 815

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

820 825 830

Thr Glu Lys Glu Asp Gln Leu Arg Arg Asp Gln Pro Ser Leu Phe Ile

835 840 845

Gly Leu Glu His Asn Pro Lys Ala Ile Glu Glu Ile Thr Thr Val Ile

850 855 860

Leu Ser Glu Ser Ile Ala Tyr Ile Gly Pro Ser Ile Phe Met Ala Ser

865 870 875 880

Ala Ala Glu Ser Val Ala Phe Ala Phe Gly Ser Ile Ser Ala Met Pro

885 890 895

Val Val Leu Trp Phe Ala Ala Met Ala Cys Cys Ala Val Ala Ile Asn

900 905 910

Phe Cys Phe Gln Met Thr Phe Phe Leu Ser Val Leu Thr Leu Asp Lys

915 920 925

Arg Arg Glu Leu Ser Gly Lys Tyr Asp Ile Ile Phe Lys Arg Ala Ser

930 935 940

Ala Val Ala Ala Gln Ala Pro Ala Ala Pro Glu Thr Val Gln His Ser

945 950 955 960

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

965 970 975

Val Arg Pro Ser Val Thr Pro Glu Asn Ser Thr Leu Thr Asp Val Leu

980 985 990

Asp Tyr Cys Val Asp Val Tyr Ala Ser Ile Leu Thr His Lys Leu Val

995 1000 1005

Lys Leu Val Val Leu Leu Leu Phe Leu Ala Trp Thr Leu Trp Ser Ile

1010 1015 1020

Tyr Ser Met Glu Ser Leu Asp Gln Gly Leu Pro Gln Lys Glu Ala Met

1025 1030 1035 1040

Pro Ser Asp Ser Tyr Met Ile Glu Tyr Phe Asn Ala Leu Asp Val Tyr

1045 1050 1055

Leu Ala Thr Gly Val Pro Val Tyr Phe Val Val Glu Thr Gly Tyr Gly

1060 1065 1070

Arg Asn Pro Asp Ala Trp Ser Leu Asn Asp Glu Ser Val Glu Thr Ile

1075 1080 1085

Phe Cys Lys Ser Lys Asp Ile Cys Gly Thr Tyr Ser Ile Pro Asn Ile

1090 1095 1100

Met Asn Ala Leu Ala Asn Asp Gly Asp Lys Thr Asn Thr His Ile Ser

1105 1110 1115 1120

Pro Gly Thr Thr Tyr Ser Trp Met Asp Asp Phe Trp Gly Phe Val Asn

1125 1130 1135

Pro Asp Ser Glu Cys Cys Arg Val Asp Ser Glu Gly Ala Tyr Val Pro

1140 1145 1150

Ile Glu Thr Gly Asn Asp Thr Tyr Thr Thr Leu Arg Ala Asp Asp Asp

1155 1160 1165

Thr Cys Leu Ala Thr Ser Val Met Ile Pro Val Pro Glu Ala Gln

1170 1175 1180

Tyr Met Ser Leu Phe Ser Met Phe Ala Thr Ala Ser Ala Gly Thr Ser

1185 1190 1195 1200

Cys Ser Tyr Gly Gly Gly Ser Ile Tyr Arg Gly Gln Phe Ser Ile Asp

1205 1210 1215

Ser Glu Pro Ile Pro Thr Val Asn Ala Ser Thr Pro Ala Val Lys Ile

1220 1225 1230

Asn Ser Ser Gly Tyr Gly Asp Glu Ile Thr Ala Trp Ser Tyr Met Val

1235 1240 1245

Thr Gly Thr Ser Asn Pro Thr Gln Gln Arg Tyr Ile Asp Ser Tyr Lys

1250 1255 1260

Gln Asn Leu Ala Ala Ala Glu Trp Ile Ser Glu Lys Thr Gly Val Asp

1265 1270 1275 1280

Ile Trp Val Tyr Ser Leu Thr Tyr Val Tyr Phe Glu Gln Tyr Leu Thr

1285 1290 1295

Val Val Asp Asp Ala Tyr Lys Leu Ile Gly Leu Ala Leu Ala Ala Ile

1300 1305 1310

Phe Val Ile Thr Thr Leu Tyr Leu Gly Asn Val Phe Tyr Ser Leu Val

1315 1320 1325

Ile Ala Leu Thr Ala Thr Asn Ile Val Val Leu Val Leu Gly Leu Met

1330 1335 1340

Gln Pro Leu Asp Ile Met Leu Asn Gly Leu Ser Ile Val Asn Leu Ile

1345 1350 1355 1360

Ile Ala Ala Gly Ile Ser Val Glu Phe Cys Gly His Tyr Val Arg Phe

1365 1370 1375

Phe Ala Lys Ala Arg Gly Thr Gly Asp Glu Arg Ala Arg Asp Ala Leu

1380 1385 1390

Arg Gln Val Leu Thr Ser Val Val Phe Gly Ile Thr Ile Thr Lys Val

1395 1400 1405

Ile Gly Leu Ser Val Leu Thr Leu Ala Asp Ser Arg Val Phe Lys Lys

1410 1415 1420

Tyr Tyr Phe Arg Met Tyr Met Met Val Val Val Cys Gly Val Leu Asn

1425 1430 1435 1440

Gly Met Leu Leu Leu Pro Val Val Leu Ser Thr Ile Met Asp Val Lys

1445 1450 1455

Asn Phe Phe Leu Arg Lys Arg Ser Arg Lys Ser Glu Leu Pro Leu Ala

1460 1465 1470

Pro Val Thr Arg Val Glu

1475

<210> 7

<211> 1045

<212> PRT

<213> Phytophthora sojae

<400> 7

Met Ala Arg Ala Phe Tyr Arg Leu Gly Ala Leu Cys Ser Gly Ser Pro

1 5 10 15

Val Leu Met Ala Leu Ala Ala Leu Leu Cys Gly Gly Val Leu Cys Leu

20 25 30

Gly Leu Leu Asn Met Arg Leu Gln Thr Asp Pro Gln Gly Leu Trp Val

35 40 45

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

50 55 60

Phe Gly Pro Phe Phe Arg Val Gln Gln Leu Ile Phe Tyr Ala Asp Ser

65 70 75 80

Asp Ser Asp Gly Leu Ser Ala Thr Cys Asp Ala Ser Arg Asp Leu Val

85 90 95

Gln Arg Arg Phe Leu Leu Gln Met Ala Lys Val Gln Ala Gln Ile Ala

100 105 110

Asp Ala Ala Val Thr Val Gln Gly Asp Gly Ala Gln Gly Lys Val Thr

115 120 125

Leu Ser Leu Glu Asp Phe Cys Tyr Arg Pro Ile Arg Gly Lys Gly Cys

130 135 140

Leu Val Thr Ser Pro Phe Gln Tyr Trp Leu Gly Asn Ala Ser Leu Leu

145 150 155 160

Glu Gly Asp Pro Asp Ile Lys Leu Thr Thr Ala Cys Gln Thr Thr Asp

165 170 175

Pro Gln Leu Gln Glu Arg Ala Pro Cys Met Asp Gln Asn Gly Val Pro

180 185 190

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

195 200 205

Gln Asn Pro Asp Pro Cys Gly Glu Ala Thr Pro Gln Ala Gln Ala Leu

210 215 220

Val Val Thr Phe Leu Leu Asn Asn Arg Pro Glu Asn Glu Thr Tyr Thr

225 230 235 240

Arg Tyr Val Glu Gln Trp Glu Gln Gln Ala Phe Leu Lys Ile Ala Ala

245 250 255

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

260 265 270

Glu Phe Ile Trp Asp Ser Val Gln Asp Gln Glu Leu Ala Asp Val Gly

275 280 285

Val Asp Gly Met Arg Leu Ser Tyr Met Ala Glu Arg Ser Val Ala Asp

290 295 300

Ser Leu Val Val Gln Thr Asn Gln Asn Ala Phe Ile Val Val Val Ser

305 310 315 320

Tyr Leu Val Met Phe Leu Tyr Val Ser Ala Ser Leu Gly Lys Phe Thr

325 330 335

Asp Pro Val Arg Ser Arg Phe Gly Leu Gly Leu Thr Gly Ile Leu Ile

340 345 350

Val Leu Leu Ser Leu Gly Ala Ala Met Gly Val Ser Cys Ala Ile Leu

355 360 365

Gln Met Glu Val Thr Met Ile Thr Leu Glu Val Val Pro Phe Leu Val

370 375 380

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

385 390 395 400

Arg Leu Ala Ala Leu Arg Gly Leu Ala Thr Leu Asp Thr Arg Arg Asn

405 410 415

Thr Arg Asp Arg Ala Glu Asp Glu Leu Leu Met Leu Lys Gln Val Leu

420 425 430

Gly Glu Thr Met Val Asn Val Gly Pro Ser Ile Val Val Ala Ala Val

435 440 445

Ala Glu Thr Leu Ala Phe Leu Val Gly Ala Leu Thr Arg Ile Pro Ala

450 455 460

Leu Thr Ser Phe Cys Val Val Ala Ala Leu Ala Val Ala Ala Asp Phe

465 470 475 480

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

485 490 495

Arg Val Arg Ala Arg Arg Tyr Asp Leu Phe Pro Trp Met Lys Gln Lys

500 505 510

Leu Thr Leu Thr Pro Pro Thr Lys Gly Lys Arg Arg Ile Glu Ser Lys

515 520 525

Ile His Tyr Gln Tyr Asp Leu Leu Val Asp Glu Ser Glu Arg Ser Asp

530 535 540

Glu Pro Ala Ala Arg Val Ser Ser Thr Gly Thr Leu Gln Arg Phe Val

545 550 555 560

Glu Lys Thr Tyr Ile Pro Phe Leu Leu Arg Arg Ser Thr Lys Val Leu

565 570 575

Val Leu Val Thr Ala Leu Ser Val Val Thr Leu Ser Ala Phe Gly Ser

580 585 590

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

595 600 605

Phe Tyr Leu His Glu Tyr Phe Lys Lys Gln Thr Ala Leu Gly Glu Ala

610 615 620

Gly Pro Pro Ala Tyr Val Val Leu Asp Ser Asp Val Asp Tyr Thr Asp

625 630 635 640

Ala Arg Leu Gln Gln Asp Val Asn Val Leu Leu Asp Gln Leu Ser Gly

645 650 655

Leu Arg Gln Tyr Ile Gln Leu Pro Val Cys Ser Trp Leu His Thr Phe

660 665 670

Asn Gln Trp Arg Gln Met Arg Phe Phe Leu Gln Asp Lys Ile Lys Gln

675 680 685

Gly Gln Cys Asp Cys Pro Val Gln Pro Met Asp Pro Phe Pro Tyr Glu

690 695 700

Leu Ala Asn Val Gly Val Glu Asp Pro Ser Gly Asp Val Asp Leu Phe

705 710 715 720

Leu Ala Pro Glu Tyr Gly Tyr Gly Ala Leu Ala Thr Ala His Val Thr

725 730 735

Pro Asn Ala Leu Phe Tyr Pro Leu Val Lys Asn Phe Thr Lys Ile Ser

740 745 750

Ile Asp Ser Thr Cys Cys Gln His Phe Gly Leu Cys Gly Ala Gln Tyr

755 760 765

Glu Gly Asp Ile Ile Phe Asn Glu Pro Asn Ala Gly Asp Asp Asp Ser

770 775 780

Thr Gly Met Ser Ile Val Gly Ser Arg Ile Arg Phe Gln Leu Asn Ala

785 790 795 800

Leu Arg Asn Gln Ser Met Phe Val Asn Ser Tyr Tyr Tyr Leu His Asp

805 810 815

Val Val Gly Arg Trp Ser Ile Asp His Ala Ala Thr Ala Phe Pro Tyr

820 825 830

Ala Leu Val Phe Val Tyr Glu Glu Gln Tyr Thr Tyr Ile Gln Gly Val

835 840 845

Ala Leu Gln Ser Val Leu Leu Ala Leu Ala Val Val Phe Gly Ala Leu

850 855 860

Phe Val Leu Met Asp Gly Ser Leu Arg Leu Thr Thr Val Val Thr Leu

865 870 875 880

Cys Val Leu Ser Met Thr Phe Ser Gln Leu Gly Phe Leu Phe Val Trp

885 890 895

Asn Met Ile Ala Gly Pro Gly Ala Glu Thr Ser Ile Asn Ala Val Ser

900 905 910

Val Val Asn Leu Leu Ala Cys Val Gly Leu Gly Val Glu Phe Cys Val

915 920 925

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

930 935 940

Thr Ala Asn Asp His Thr Arg Tyr Ala Leu Ser Ser Val Gly Ala Ser

945 950 955 960

Ile Phe Ser Gly Ile Thr Leu Thr Lys Phe Cys Gly Ile Gly Val Leu

965 970 975

Ala Phe Ala Pro Ser Met Leu Phe Arg Val Tyr Phe Phe Arg Met Tyr

980 985 990

Leu Gly Ile Val Val Leu Gly Cys Phe His Gly Leu Val Leu Leu Pro

995 1000 1005

Val Leu Leu Ser Leu Ile Gly Gln Pro Gln Lys Tyr Pro Asn Asp Leu

1010 1015 1020

Ser Ser Phe Leu Leu Ser Glu Glu Arg Asp Asp Glu Leu Glu Asp Glu

1025 1030 1035 1040

Val Arg Ala Phe Lys

1045

<210> 8

<211> 1486

<212> PRT

<213> Phytophthora sojae

<400> 8

Met Arg Val Ser Ala Leu Ala Ser Leu Leu Leu Pro Leu Gly Val Ala

1 5 10 15

Arg Ala Ala Ser Ser Ser Gly Ser Thr Thr Ser Thr Ala Thr Thr Thr

20 25 30

Thr Thr Gly Thr Ser Ser Val Ser Ser Tyr Val Pro Trp Thr Leu Ala

35 40 45

Asp Asn Ala Thr Asn Leu Ala Asp Ile Lys Ala Gln Leu Thr Met Cys

50 55 60

Thr Tyr Ser Lys Val Glu Glu Cys Ile Gln Asp Pro Ala Leu Val His

65 70 75 80

Glu Leu Gly Ala Leu Val Arg Ala Pro Gly His Cys Val Ala Phe Asp

85 90 95

Ser Ser Tyr Val Asn Val Thr Thr Ala Gly Val Ala Ile Pro Asn Arg

100 105 110

Tyr Tyr Pro Thr Ser Val Glu Asp Ala Tyr Asp Ala Gly Phe Ser Asn

115 120 125

Lys Phe Ser Glu Trp Ser Asp Thr Asn Arg Glu Gln Phe Glu Val Asp

130 135 140

Cys Pro Leu Leu Tyr Asn Glu Thr Ile Ala Gln Gly Asp Asp Met Leu

145 150 155 160

Cys Cys Thr Glu Asn Gln Tyr Thr Gly Leu Ser Thr Gln Val Arg Met

165 170 175

Ile Pro Gly Leu Cys Ser Ala Cys Lys Glu Asn Leu Arg Asn Ile Phe

180 185 190

Cys Gln Met Thr Cys Ser Pro Asn Asn Ser Met Phe Leu Asp Val Asn

195 200 205

Glu Val Arg Ile Met Pro Gly Asp Asp Glu His Pro Asp Ala Val Phe

210 215 220

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

225 230 235 240

Asp Ile Tyr Asp Phe Cys Glu Ala Asp Ser Ser Phe Ser Leu Leu Cys

245 250 255

Asn Pro Asn Gln Asp Cys His Asp Gly Tyr Gly Leu Leu Glu Tyr Met

260 265 270

Gly Lys Tyr Ala Phe Asn Ser Ile Gly Ser Pro Leu Gln Ile Asn Val

275 280 285

Thr Thr Met Asp Lys Val Pro Glu Ile Asn Gln Met Thr Glu Phe Cys

290 295 300

His Cys Asp Asn Val Asn Ala Thr Asn Cys Ile Leu Pro Gln Asn Ser

305 310 315 320

Arg Met Thr Ser Cys Val Gly Thr Cys Gly Ser Leu Cys Ala Val Ser

325 330 335

Ser Ser Asp Asp Arg Thr Tyr Thr Glu Ser Cys Tyr Gly Ala Ser Asn

340 345 350

Ala Val Ala Thr Ser Ser Ser Ala Ser Gly Ser Val Gly Ser Gly Ser

355 360 365

Asp Asp Ser Thr Trp Ala Glu Leu Asn Ala Tyr Leu Ala Ser Asn Ile

370 375 380

Pro Val Thr Asp Trp Thr Gly Leu Asn Tyr Phe Leu Val Ile Phe Gly

385 390 395 400

Gly Ala Val Ala Leu Leu Leu Ile Val Gly Phe Ile Val Ala Gly Cys

405 410 415

Arg Glu Arg Arg Ala Arg Ile Pro Asn Pro His Thr Gly Thr Pro His

420 425 430

Ile Gly Pro Tyr Thr Pro Glu Ala His Gly Val Ala His Ala Met Glu

435 440 445

Thr Ser Asn Thr Arg Leu Ser Tyr Leu Asp Glu Leu Met Thr Asn Lys

450 455 460

Leu Arg Thr Trp Ala Val Phe Val Ser Thr Gly Asn Arg Pro Lys Lys

465 470 475 480

Met Ile Pro Met Val Leu Cys Val Val Ala Val Cys Val Val Gly Leu

485 490 495

Tyr Asn Ile Asp Ile Glu Thr Asp Pro Ile Lys Leu Trp Val Ser Ser

500 505 510

Ser Ser Thr Ser Tyr Gln Gln Arg Gln His Tyr Gly Glu Ile Phe Asn

515 520 525

Pro Phe Tyr Arg Ser Glu Gln Val Ile Met Val Pro Lys Asp Gly Gly

530 535 540

Asn Ile Tyr Arg Ser Ser Ile Ile Lys Glu Ala Ile Arg Val Gln Thr

545 550 555 560

Val Ala Ala Asn Val Thr Tyr Thr Ser Asp Asp Gly Asp Glu Thr Ile

565 570 575

Thr Leu Asp Asp Ile Cys Trp Lys Ala Thr Gly Thr Gly Cys Thr Val

580 585 590

Asn Ser Ile Thr Gln Tyr Phe Gln Asn Asn Met Glu His Phe Glu Phe

595 600 605

Tyr Glu Lys Tyr Gly Leu Glu Met Glu His Phe Ser Asn Cys Leu Tyr

610 615 620

Ser Pro Thr Thr Ser Asp Val Ala Leu Cys Thr Glu Leu Lys Asn Ala

625 630 635 640

Leu Glu Asp Gly Asp Ser Leu Pro Ser Ser Met Ser Asp Cys Pro Cys

645 650 655

Leu Ser Ala Phe Gly Ser Pro Met Asn Leu Tyr Asn Thr Tyr Leu Gly

660 665 670

Gly Phe Pro Asp Gly Ala Glu Ser Asn Tyr Thr Leu Phe Leu Asp Ser

675 680 685

Val Ala Phe Val Ser Ser Tyr Leu Asn Tyr Asn Tyr Ala Asp Asp Asp

690 695 700

Lys Asn Glu Pro Ala Ile Lys Trp Glu Arg Glu Tyr Ile Lys Thr Met

705 710 715 720

Lys Glu Glu Ala Ala Ser Asn Thr Ile Phe Asp Val Tyr Phe Tyr Ala

725 730 735

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

740 745 750

Gly Pro Val Ala Leu Ser Tyr Cys Leu Met Ile Ile Tyr Ile Ser Leu

755 760 765

Gly Ile Asn Arg Ile Lys Phe Ser Arg Glu Phe Phe Ile Ser Ser Lys

770 775 780

Ile Val Ala Gly Phe Cys Gly Val Met Ser Ile Val Cys Gly Val Ala

785 790 795 800

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

805 810 815

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

820 825 830

Ile Phe Leu Ile Ile His Ala Met Thr Glu Lys Glu Asp Gln Met Arg

835 840 845

Arg Glu Gln Pro Ser Leu Phe Ile Gly Leu Glu His Asn Pro Thr Ala

850 855 860

Ile Glu Glu Ile Thr Thr Thr Ile Leu Ser Glu Ser Leu Ala Tyr Ile

865 870 875 880

Gly Pro Ser Ile Phe Met Ala Ser Ala Ala Glu Ser Val Ala Phe Ala

885 890 895

Phe Gly Ser Ile Ser Pro Met Pro Val Val Leu Trp Phe Ala Ala Met

900 905 910

Ala Cys Cys Ala Val Ala Ile Asn Phe Cys Leu Gln Met Thr Leu Phe

915 920 925

Leu Ser Val Leu Thr Leu Asp Lys Arg Arg Glu Leu Ser Gly Lys Tyr

930 935 940

Asp Ile Ile Phe Lys Arg Ala Ser Tyr Val Arg Ser Gln Pro Pro Ala

945 950 955 960

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

965 970 975

Pro Lys Thr Pro Ala Pro Glu Asp Ala Arg Arg Ser Ile Thr Pro Glu

980 985 990

Asn Arg Thr Leu Thr Asp Val Leu Asp Tyr Cys Val Asp Val Tyr Ala

995 1000 1005

Ser Ile Leu Thr Tyr Lys Ile Val Lys Leu Val Val Leu Leu Val Phe

1010 1015 1020

Leu Phe Trp Thr Leu Trp Ser Ile Tyr Ser Met Glu Ser Leu Asp Gln

1025 1030 1035 1040

Gly Leu Pro Gln Lys Glu Ala Met Pro Ser Asp Ser Tyr Met Ile Glu

1045 1050 1055

Tyr Phe Asn Ala Leu Asp Val Tyr Leu Ala Thr Gly Val Pro Val Tyr

1060 1065 1070

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

1075 1080 1085

Asn Asp Glu Ser Val Glu Thr Leu Phe Cys Lys Ser Lys Asp Ile Cys

1090 1095 1100

Gly Thr Tyr Ser Ile Pro Asn Ile Met Asn Ala Leu Ala Asn His Gly

1105 1110 1115 1120

Asp Lys Asn Val Thr His Ile Ser Pro Gly Thr Thr Tyr Ser Trp Met

1125 1130 1135

Asp Asp Phe Trp Gly Phe Val Asn Pro Asp Ser Glu Cys Cys Arg Val

1140 1145 1150

Asp Ser Glu Gly Ala Tyr Val Pro Ile Glu Thr Gly Asn Asp Thr Tyr

1155 1160 1165

Thr Thr Leu Arg Ala Asp Asp Asp Thr Cys Leu Ala Thr Ser Val Thr

1170 1175 1180

Ile Pro Pro Val Pro Glu Ala Gln Tyr Met Ser Leu Phe Ser Met Phe

1185 1190 1195 1200

Ala Thr Ala Ser Ala Gly Thr Ser Cys Ser Tyr Gly Gly Gly Ser Ile

1205 1210 1215

Tyr Arg Gly Gln Phe Ser Ile Asp Ser Glu Pro Ile Pro Thr Val Asn

1220 1225 1230

Ala Ser Thr Pro Ala Val Lys Ile Asn Ser Ser Gly Tyr Gly Asp Glu

1235 1240 1245

Ile Thr Ala Trp Ser Tyr Met Val Thr Gly Thr Ser Asn Pro Thr Gln

1250 1255 1260

Gln Arg Tyr Ile Asp Ser Tyr Lys Gln Asn Leu Ala Ala Ala Glu Trp

1265 1270 1275 1280

Ile Ser Glu Lys Thr Gly Val Asp Ile Trp Val Tyr Ser Leu Thr Tyr

1285 1290 1295

Val Tyr Phe Glu Gln Tyr Leu Thr Val Val Asp Asp Ala Tyr Lys Leu

1300 1305 1310

Ile Gly Leu Ala Leu Ala Ala Ile Phe Val Ile Thr Ala Leu Tyr Leu

1315 1320 1325

Gly Asn Val Phe Tyr Ser Leu Val Ile Ala Leu Thr Ala Thr Asn Ile

1330 1335 1340

Val Val Leu Val Leu Gly Leu Met Gln Pro Leu Asp Ile Met Leu Asn

1345 1350 1355 1360

Gly Leu Ser Ile Val Asn Leu Ile Ile Ala Ala Gly Ile Ala Val Glu

1365 1370 1375

Phe Cys Gly His Tyr Val Arg Phe Phe Ala Lys Ala Arg Gly Thr Gly

1380 1385 1390

Asp Glu Arg Ala Arg Asp Ala Leu Arg Gln Val Leu Thr Ser Val Val

1395 1400 1405

Phe Gly Ile Thr Ile Thr Lys Val Ile Gly Leu Ser Val Leu Thr Leu

1410 1415 1420

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

1425 1430 1435 1440

Val Val Leu Cys Gly Val Leu Asn Gly Met Leu Leu Leu Pro Val Leu

1445 1450 1455

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

1460 1465 1470

Arg Lys Thr Asp Leu Pro Ser Ala Pro Val Thr Arg Val Glu

1475 1480 1485

<210> 9

<211> 717

<212> DNA

<213> Artificial sequence

<400> 9

atgggcaagg gcgaggaact gttcactggc gtggtcccaa tcctggtgga actggatggt 60

gatgtgaacg ggcacaagtt ctccgtcagc ggagagggtg aaggtgatgc cacctacgga 120

aagctcaccc tgaagttcat ctgcactacc ggaaagctcc ctgttccgtg gccaaccctc 180

gtcaccactt tcacctacgg tgttcagtgc ttctcccggt acccagatca catgaagcag 240

catgacttct tcaagagcgc catgcccgaa ggctacgtgc aagaaaggac tatcttcttc 300

aaggatgacg ggaactacaa gacacgtgcc gaagtcaagt tcgaaggtga taccctggtg 360

aaccgcatcg agctgaaagg catcgatttc aaggaagatg gaaacatcct cggacacaag 420

ctggagtaca actacaactc ccacaacgta tacatcatgg ccgacaagca gaagaacggc 480

atcaaggtga acttcaagat caggcacaac atcgaagatg gaagcgtgca actggcggac 540

cactaccagc agaacacgcc catcggcgat ggccctgtcc tgctgccgga caaccattac 600

ctgtccacgc aatctgccct ctccaaggac cccaacgaga agagggacca catggtcctg 660

ctggagttcg tgacggctgc tgggatcacg catggcatgg atgaactcta caagtga 717

<210> 10

<211> 678

<212> DNA

<213> Artificial sequence

<400> 10

atggcctcct ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc 60

tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 120

acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc 180

ctgtcccctc agttccagta cggctccaag gcctacgtga agcaccccgc cgacatcccc 240

gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 300

gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac 360

aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc 420

atgggctggg aggcctccac cgagcggatg taccccgagg acggcgccct gaagggcgag 480

atcaagatga ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc 540

tacatggcca agaagcccgt gcagctgccc ggcgcctaca agaccgacat caagctggac 600

atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc 660

cactccaccg gcgcctaa 678

<210> 11

<211> 1000

<212> DNA

<213> Phytophthora sojae

<400> 11

gtcgtcgcga ggatacatcg acgtagacgt ctctgccgat ggtggagccg ctgagctcct 60

tgagctcctt cgctcgaagc cggcaccact ggtggtgtgc tgctggccgg cccgccgtcc 120

tggcggtgca gcaacgatga ctcagcgccg ccgcgcgtca aagtgaacaa gtccagacaa 180

gttcagtcga gctgaaggcg tcgccacgtc gagcggcttc gctttcggtg tgcgacaagc 240

gatgcccctc tcttcaaggg acaatgctgt ccctacagca aggggccggc acgctgtgcc 300

cctggagcag acgccgcagt tgatcccttc gcggcaacag caacagcgac tcgaacaggc 360

agctcggcga gctctcgctg cccgcgtctg ctccggatgg gagtgccgca cccactttgc 420

aggcccacgc tcgaccaggt tgattcaagc ccgcgacagc aagcccgaga tttgaggcgg 480

gagggaacag tagaccatgc tgcagcccaa cgccatgctg ccgtccttgt cgtccaaggc 540

gacagagctc acagtcaagt tgtcgccagg cttacttgcg atgagaagac ctccagcgcg 600

cgagcgagcg gaggctccgt gcagccaaga aggcggcggc agcgagcgag cagattcact 660

cgagcacgtc gatgtgcgtc tgtcgaccga cagactcgga agaggtgtcg caacggaccg 720

catactgagc ccaggtcact ttatggaaaa aaccatatgg atacgcgcat ctggtttcag 780

aggttttttac acagcctctc attacctgaa cgccaatgtc gataccgtcg tgctatacag 840

gcaatgtgtt gtcgaaggtg atggcccctc tccgtcgccc cgtcgctgca gtgatgaagt 900

gccactctgt cactatcctc ccccgcttta cgctttcgcc ttgcggctgc tatgacatca 960

tttcgatatc ggcgcccaga ggatcacagg accaggcgcc 1000

<210> 12

<211> 1000

<212> DNA

<213> Phytophthora sojae

<400> 12

tgggctggct ggctatccga ccgctcacta gcactcggta tgcatgtata tgaaccacaa 60

gacgacaaaa cacgagaaaa aagcgacgaa gacgctggag atgatcccga atgaacaaga 120

agcccatgtg tacacttctg ctatgggcaa gcagcaaaag gacaaacaaa agccggagat 180

tgcaacagcg catgaatgta ttttatattt tcatttcctg agggcttgcc cccccgtccc 240

ctttgcgtcc actgcttcga gtcggggttt cctgccttgg tcgcgaatgg cgagtatttc 300

ggccccggaa gctacaggta agtcctctcc aaaatagttg tgccagcacc acttcaacgt 360

tgtccccatt taggctgaag gagctggcgc tgctgcggtt gagcggtcga acaacctgaa 420

ggtgtccagg cgacggcgtg cttcctctcg cgaccatgag caacgcgcct acggcggggg 480

gcgccatgtc caacatgttc agtcctcgca gtacgaatgg gaacaaccgg tatcacctgg 540

ccagcggtgg agctcgtacg ccgcgtgtgt ttccgccaga tgtgcagagt atcccgctga 600

gtccgtcgcg gctcgagagc ccgatgtttc gagagcgggt gacggcgctg atgcagcaag 660

gcgacacgta cgcgaagcgg ctggacagcg agcgtcgtcg gtcccatgac ctggatctag 720

cgctgcgcac cctgcgagtc gagcacttcc aagcgcgcaa ggcgctgtgt gacgccacga 780

acgcccagct ctgcgctgct acgacagagc tgaagccgat tcggacgctc gagaaccgcc 840

tggacaaagt gctcacgcgc tacaacgagg tctgcaacgc gaacaaggcg ctgcgcgagc 900

agatccaggc cttgcgccgt gaaaaggtgc agcaggctgc agtgctcgac aagctcgagc 960

gggagactgc gcaggcccag gcggaggctg ctcaagtcgc 1000

<210> 13

<211> 1000

<212> DNA

<213> Phytophthora sojae

<400> 13

agggggcggc aaacgctgcg gcgcgggggc tgccgcattg cttcctgggc gttggcgacg 60

agttggcagc tccgagcaga cccacgaccc gaagcaccga ctggctgcgg acggctcggt 120

cggcgtcgtc ctccgcgtgg agactgtccg cgtccacgta gcgaggaaac agcagcggcc 180

ctgccgccgg cagctcgagg cgaggcgtcg gcattggcat gggcatgggc gcgaggaagc 240

gatatgtagc caaatgtatt tttaccttca acgaccatca ctctggtgga acatatggtg 300

agagctcatc cagcatacca ccacgaccta cactgtactt ggcccagctt gcctgcgtgt 360

atccgtcgcg tcgtcttggt gatcctcgcg tcttggcgat ccctgtgcct gcccccgctg 420

aagcggctgc acgcaccctg aatcccacga ccataatcat ctcgtctctc ggtatgacgc 480

ttggccttcc ttccaacgcc tcgagcgctg actcagcccc catgcaacaa ggcggatgcc 540

gcctccttcc tcgcgcgcgc atcgactcgt gtgggccaag ccgaccttcg gctggctgtc 600

agccctccgc cgccgtctcc tttcaccgtt aggtcaggag tgcctgttac ctaaccgtgt 660

ggtgcatttt gatccgggga ccgcgcaggc gcagccgagc gcggctgaag caggcccgac 720

taacggagcc attcggcact acggggccgc cagacgcgcg atgcagacgc acagcgacac 780

cctccaccat tgacaaggcc ggcgagtcga gcgtgaccca cggcggtcaa gccgacggca 840

acgccggcga tgccgagtgc cacccctgga tacatctgat ccgtctcaaa ctcgagcgcg 900

gggatccccg tgctccccta tgcgacctgc acctgcaccc ccactcctcc cgagctcctc 960

acttcccaat cgaccggcgc caggacaagt cgacaccctc 1000

<210> 14

<211> 1000

<212> DNA

<213> Phytophthora sojae

<400> 14

gtcagcctac tctgtcacca actgtgtgga gctttcaacg cgtcgagtgc gtaggtataa 60

acgagtatat aatcaatcaa gcacatcttc aaaatgcgcc tacatgtaca tgaatgaatg 120

cttcttgggt tgctggaagc cgcgtggagg ttgcaccgca acttcaacat gtacgaccat 180

cactctgcat tagatcatgt cgccgccacg ggcttctcct cctccttggc cccgttggca 240

gcctcctcca cgacggcgcg gccgatcggt tcgggcgccg tatccgtcgc ctcggcctgc 300

tgcagcacca cgagcgtgaa cctcaagtgc tgggcgatga tcatctcgtg catcttggtg 360

cgcagccgca gccactcgat ctcgctgtcg ttcgggtccg ggaagagctt ctgcaggtgt 420

ttcttggtgc gtccgtagag gctcaggacg tggtaggcgt gcatgatggc ctccttgtac 480

tcgaggttct cgtacttgac cacgatgccg tcgttgttca tgaggatgta ggacgagaag 540

ccgggcttgc tcttgagctg cttgatcacc tcctccacct cgttcggagc cgctgccgcc 600

atcgtatccc gccttgtctt gtcttggaga gagagaggaa cgcaaagtga tcaaggatcc 660

gtgcgactcg actcgaaccg acgcaaaacg gctggggtca acaggactct tcaactgctc 720

gagcgtcaag ccaagcatgg cccagggctc cggctcgtac tacgatgact acatgcgcag 780

caacgcgatg ctcaagcact gcggcgaaaa gaacgccaag aagctgctga cggaccggaa 840

cgcctacatt tccttcctcg aggtgcagct cgagcgcgtg tcggccgcgt gcttgaccac 900

gcaaaccttc gagcggcgtc tggcggagct cgagtgcgcc cagctcgcca acgaccagaa 960

ggtggggtcg ctctccaagg tcttccgact gaaccaggag 1000

<210> 15

<211> 1000

<212> DNA

<213> Phytophthora sojae

<400> 15

gcccagtccc gtcttcttgt acaggtggcc ggccttgcgc aggaaggagg aggacgacga 60

gccgttgccc gcggagctcg agagcgcggc ggcggccgat gacgccaatg gcgcaggcgg 120

cggcgactgc ggcgagttgt gcatcgtagc ggcggcgcca gcagtagcgg cttcttggct 180

ggtgcttggg tgggcgctgg gcagcgactt gcggactgat gagaagctcg ccatctttcg 240

cttgtcacgc agttacgcgg agggatcagg ccgggcccat actgggcgct agtgaaatgg 300

caaggacggg gcaggttgaa gcccagttct gcgtactgtg ggtgacctgg cgccgccagt 360

ttcacgcgcg atgcgcgcac gtcaagctcc cgctttgctt gacggcaacg tcctcgccga 420

tctgccttgg tctgccatgc tgacttgcat ccgcggcctt cgtgcattgc ggattcgtta 480

gtgtttctgc ccaggtggat gccagcggtt caacgccagc ttcggctcgg cacattggcg 540

ttggctgact cacttggcgc tggtggtgga gtccacgcca ccagcatttt gtcgctcgag 600

cagactgcgg cagcattgtc gatcgaatcc caccagattg ctggagctca atgccgctcg 660

tttgtccgtc gggtccgtcg ttcaactgac cccaagctct tctataggag ctataggtct 720

gcatttgaaa gcgttatcag agcacgtttg actaccatgg ttggctggcc aaggagcact 780

cgaagcatgt tggctagacg gccaaagtca tcgactttgt ggtactcgaa gtgaagacgc 840

acgaactcgt ctgctgcgtt ttcaagcagc acagacagcc ctcgtatcag ctcgcacttc 900

aaccactcac ttggtctttg taatgatccc tcgtctcgac gctcattggc caatagtccc 960

cattcagctt caatctcctt cctccctccc tctccgagcc 1000

<210> 16

<211> 1000

<212> DNA

<213> Phytophthora sojae

<400> 16

gtggtgctag gtgtcaacaa tacttctctg gcccacataa gaaacaacac ttcggttttc 60

tggacataac tatttacaaa aaaatcgttt ggcgactcgc cgaacactgc gcatttgctc 120

cgtttcacgc tatgatttaa aaaatctagc agcagaaaga aaagagaaac agccttttat 180

ctgaagtccg ttttaggcaa cattaggttt tttagcgagg tgccgtcaaa aattagcgat 240

attagaaaat aaaatatcac cgtaatcgcg agtgactgct ttacccttat gttcggttgt 300

cgatgttgta gcgcctgttg gagacgagtt agaagggcta cagttttgag ggttttctac 360

gcgattatct cgacatgcgc agacccattt attttggcca atagtaagct gaaacagttg 420

attataccca atcatatgtg acatgtggtc cacatcaaat tgaggtttaa tgccgaatgt 480

tgttacttaa gtgggccaga gaagtataaa ccagactgtg ctgaagtcac gtggttttca 540

attcgaggta gactcttact gttcgcaagt ctacgaattt gcgttgtcat agaaaatgat 600

ctcgcggatg agtggatgcc agcctaccac gcattcttgc gccagaactg ccaccattcc 660

ttggccagca atcccttttt gcgtcgccag agcgcagaga actccaaata ctcatggaag 720

cgcgggtcgt ctttcagttt ttagtcccag cgtagtcaag atggtaatcg ggttttcatc 780

ctccttgagc cactggcggt actttttgtt tttcgcgatc ttctccagct tcgcggggtc 840

cttggtgaac cggttcgtta gcttgatgat caaggtgaac gccctctcct cctcctcctc 900

cgcaatgccc gtgctgtcaa ctcgaagccg tctcttcctg gccaggctgt ccccatcggc 960

gagtccgtca aagtcagacg cggttacctt cgtcaatctc 1000

<210> 17

<211> 1000

<212> DNA

<213> Phytophthora sojae

<400> 17

ttaattagga cgtagtgggt atttacatga aaacaatgta tccattcata ttgataatga 60

tcgacttgga atggctattt aatcacgaca ggtacatttg taatgcgacg cttcagaagc 120

agctgtacca ctctgagtcc gtgttgccct tgatgttgcc cccgttgcac cgtgtatcct 180

tcttgcactt caggaagcag cttctcccgc gcgtccatct tggtgaaagc agactctgca 240

gagagtgcat tcacttggca tggtgcgact ctaagctgag ctgacggcgt taattttgct 300

gcgctttaat cgcggatgct tttggtacgc ttgctgtccg aggcactcgg agcaccgacg 360

gtatcaacac cacacggcaa tgccatcggt gcggccatcc ttacgaatct aaaatttaga 420

ttctaaatgc cccgacagga gtgccgcgcg agcatattcc gagtcgatgc agcctcgaac 480

cgaggtcctc ggtcaccgag tcggtgcggt tgctgtagga gagccgggtg tacagtatcc 540

gctcgaccag caggatggcc tcgttttcgc tggcgccgac ctgattcgtt cgtctgcggt 600

gatggaggtc tgccggccag cgtcgtgagt aacccagcgc gagaccagcc gcccgcacca 660

agacgccgct catcggggca gacgcccgag gtgatcacta tgtcggcggg ggatccatgc 720

atagccgcgc ccaggcgccg tcgcaggcgc agtatccgcg ctgcgcctct ggtcgagctc 780

ttgctgatcg acggtctctt taatccgcgc ttctggtcgc gggttcatgc agctccgcgc 840

cactctttcg ctcgctgctc ccgctgtccg aggtgtccgc ggtgcagtct cagcctcatt 900

tagaaatcta ccggcagtcg agcacgcgca gcgctagcgt ccgactgatt agggttggct 960

gcctccactg gttctcctcc cttccacaca tccccacatc 1000

<210> 18

<211> 1000

<212> DNA

<213> Phytophthora sojae

<400> 18

gccgacgtcc aagctgtgag caacttcgtg tatcctgcgt tgaagccaat ctgtacatat 60

gaggacgcca ccgatcccgt agccagcatc caagtcaagc ccggacgagt gcgatggagc 120

aatagagaag tgtgcgcgtg actacttcgt actactagta gtagtaagta gtacccagtc 180

gttaagtaaa taaaataggg accccctgtc ttccttctta ctctttgtgc tttggactcg 240

gcttggttgg cgacttcaag gcggtgtttg cctttgctgt tgaggtgagc ctgcatgacc 300

ctctcaatga gagtatcacg ccaacgttgt gagaggttga aagggctttt cacatggagt 360

ctggcagcag taagcttagc caattcaggg aactctgcag ccagggtatc aaactcccca 420

gctaggaagg cagccgacaa ctggccgatg gctcggagcg cgtggaagtg gtcgatcttg 480

ccggcggact tcagtagccg gttgagcgtt ggtgaaaaca cggggatgtc cgagaagttg 540

ttgtgccgag tgccgcggag ccacgtaagg cgcgactgct cgtgcgtaca ccccctcgca 600

tggcgctcag tgctctccgc attggggcgc cagtggagga agtgctccga catcaagtgg 660

agcgcaggaa cacgcgagcc gaggccgtcg cgcacatctg catccagggg ctccatccag 720

gcgtcgagca gtaccatctt cttgaaccgc gcgtcttggt gagcagcgct gagtgccgtc 780

gccgcaccga acgagtgtcc agcaacgctc acgttggcga ggttgcagtg gccgaagacg 840

ctgcccgatg cggcggcatc ttgcttgatg gcgtccagaa ctgcgcgcac ttcgctcaca 900

cgctgctgca gttgtccatt gcggaagcgg aagcctgctc cgtcgacgtc gtcccgaacc 960

tccggcgtga tctgctggta gtaatcgatg cggccagcct 1000

Claims (10)

1. A class of Phytophthora sojae (Phytophthora sojae) NCR protein, which is the protein of the following A1) or A2) or A3) or A4): A1) the amino acid sequence is a protein as shown in any one of SEQ ID NO.6-8; A2) A fusion protein obtained by linking a tag to the N-terminus and/or C-terminus of the protein shown in any one of SEQ ID NO.6-8; A3) The amino acid sequence shown in any one of SEQ ID NO. 6-8 is obtained by substitution and/or deletion and/or addition of one or several amino acid residues and has the same function from SEQ ID NO. 6- A protein-derived protein of any one of 8; A4) The similarity with the amino acid sequence shown in any one of SEQ ID NO. 6-8 is more than 75%, preferably more than 85%, more preferably more than 95% and has the same amino acid sequence as SEQ ID NO. 6-8 The amino acid sequences shown are functionally identical amino acid sequences. 2. the encoding gene of the NCR protein shown in encoding claim 1; Preferably, the encoding gene is as follows B1) or B2) or B3): B1) the DNA molecule shown in the nucleotide sequence of any one of SEQ ID NO.2-4 in the sequence listing; B2) has more than 75% or more than 85% or more than 95% identity with the nucleotide sequence shown in B1), and encodes a cDNA molecule or DNA molecule of the NCR protein described in claim 1; B3) a cDNA molecule or a DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in B1) or B2) and encodes the NCR protein of claim 1. 3. the RNA molecule that the coding gene described in claim 2 is transcribed; Preferably, the sequence of the RNA molecule is the following C1) or C2): C1) The similarity of the RNA sequence transcribed from the DNA sequence shown in SEQ ID NO.2-4 is more than 75%, more preferably more than 85%, more preferably more than 95% The RNA sequence with the same function as the RNA sequence transcribed from the DNA sequence shown in 4; C2) RNA sequence transcribed from the DNA sequence shown in any one of SEQ ID NO. 2-4. 4. the biological material containing the nucleic acid molecule relevant to the coding gene shown in claim 2 or the RNA molecule described in claim 3, is any one of following D1) to D10): D1) an expression cassette containing the encoding gene of claim 2; D2) a recombinant vector containing the encoding gene of claim 2, or a recombinant vector containing the expression cassette of D1); D3) a recombinant microorganism containing the encoded gene of claim 2, or a recombinant microorganism containing the expression cassette of D1), or a recombinant microorganism containing the recombinant vector of D2); D4) a transgenic plant cell line containing the encoding gene of claim 2, or a transgenic plant cell line containing the expression cassette of D1); D5) a transgenic plant tissue containing the encoding gene of claim 2, or a transgenic plant tissue containing the expression cassette of D2); D6) a transgenic plant organ containing the encoding gene of claim 2, or a transgenic plant organ containing the expression cassette of D2); D7) inhibits the nucleic acid molecule of the described coding gene expression of claim 2; D8) an expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in D7); D9) a nucleic acid molecule that inhibits the translation of the RNA molecule of claim 3; D10) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line producing the nucleic acid molecule of D9). 5. A set of Phytophthora sojae NCR protein combinations or DNA combinations used in a set is the following E1) or E2): E1) consists of the protein shown in SEQ ID NO.6 in the sequence listing, the protein shown in SEQ ID NO.7 in the sequence listing, the protein shown in SEQ ID NO.8 in the sequence listing, and the protein shown in SEQ ID NO.5 in the sequence listing Any two or a combination of two or more of the proteins shown; E2) consists of the DNA molecule shown in SEQ ID NO.2 in the sequence listing, the DNA molecule shown in SEQ ID NO.3 in the sequence listing, the DNA molecule shown in SEQ ID NO.4 in the sequence listing, and the DNA molecule shown in SEQ ID NO.4 in the sequence listing Any two or a combination of two or more of the DNA molecules shown in NO.1. 6. NCR protein according to claim 1, coding gene according to claim 2 or RNA molecule according to claim 3 or biological material according to claim 4 or protein combination according to claim 5 or DNA combination Application, it is characterized in that: described application is any one or more in following 1)-6): 1) application in regulating the production of Phytophthora sojae zoospores and/or maintaining its form; 2) application in regulating the growth rate of Phytophthora sojae; 3) Application in regulating the ability of Phytophthora sojae to infect a host; 4) Application in maintaining the normal morphological structure of Phytophthora sojae oospores; 5) application in regulating the pathogenicity of Phytophthora sojae to a host; 6) Application in inhibiting and/or killing Phytophthora sojae; Preferably, wherein, the application includes inhibiting or inactivating the transcription of one or two or more of the encoding genes according to claim 2, or inhibiting the RNA according to claim 3 The translation of one or two or more arbitrary combinations in the molecule, or inhibit the activity of one or two or more arbitrary combinations in the NCR protein as claimed in claim 1 or inactivate it, or inhibit or inactivate the claims 5. The NCR protein combination or the DNA combination, or the transcription of the DNA combination according to claim 5 is inhibited to realize the application of 1)-6). 7. The NCR protein according to claim 1, the encoding gene according to claim 2, or the RNA molecule according to claim 3, or the biological material according to claim 4, or the combination of proteins according to claim 5, or the combination of DNA. Use in screening Phytophthora sojae as a bacteriostatic or fungicide target. 8. A method for screening or assisting screening of Phytophthora sojae bacteriostatic and/or bactericide, the method comprising applying a substance to be detected to the Phytophthora sojae, when the substance to be detected can inhibit as claimed in the claim Transcription of any combination of one or more of the coding genes described in 2, or inhibit the translation of one or more of the arbitrary combinations of the RNA molecules as claimed in claim 3, or inhibit and/or inactivate According to the activity of any combination of one or two or more of the NCR proteins according to claim 1, the substance to be detected is a bacteriostatic and/or bactericide of the Phytophthora sojae. 9. A method for reducing the activity of Phytophthora sojae, comprising the steps of: inhibiting transcription or deletion of any combination of one or more of the encoding genes according to claim 2, or inhibiting as claimed in claim 2 Translation of one or two or more arbitrary combinations in the RNA molecule of claim 3, or inhibit the activity of one or more arbitrary combinations in the NCR protein as claimed in claim 1 or inactivate it, or, Inhibit or inactivate the NCR protein combination or DNA combination of claim 5; Wherein, the activity of reducing Phytophthora sojae is reducing the infectivity of Phytophthora sojae to the host and/or the pathogenicity to the host, and/or reducing the mycelial growth rate of Phytophthora sojae, and/or reducing the number and/or morphological changes of the zoospores of Phytophthora sojae, and/or deforming the oospore structure of Phytophthora sojae; Preferably, the method is any one of the following F1)-F7): F1) Inhibit the expression or inactivate the protein shown in SEQ ID NO.5 in the sequence listing and the protein shown in SEQ ID NO.6 in the sequence listing in Phytophthora sojae; F2) inhibiting the expression or inactivating the protein shown in SEQ ID NO.6 in the sequence listing of Phytophthora sojae and/or the protein shown in SEQ ID NO.8 in the sequence listing; F3) Inhibit the expression of the protein shown in SEQ ID NO. 6 in the Sequence Listing, the protein shown in SEQ ID NO. 7 in the Sequence Listing, and the protein shown in SEQ ID NO. 8 in the Sequence Listing in Phytophthora sojae or make it inactivated; F4) Inhibition of the protein shown in SEQ ID NO.5 in the Sequence Listing, the protein shown in SEQ ID NO.6 in the Sequence Listing, the protein shown in SEQ ID NO.7 in the Sequence Listing and the protein shown in SEQ ID NO.7 in the Sequence Listing in Phytophthora sojae Expression or inactivation of the protein shown in ID NO.8; F5) Inhibition of the protein shown in SEQ ID NO.5 in the sequence listing of Phytophthora sojae, the protein shown in SEQ ID NO.6 in the sequence listing, the protein described in SEQ ID NO.7 in the sequence listing, and the protein in the sequence listing Expression or inactivation of one or more of the proteins shown in SEQ ID NO.8; F6) inhibit the expression or inactivate the protein shown in SEQ ID NO.6 in the sequence listing and the protein shown in SEQ ID NO.7 in the sequence listing in Phytophthora sojae; F7) Inhibit the expression of the protein shown in SEQ ID NO.7 in the sequence listing of Phytophthora sojae and the protein shown in SEQ ID NO.8 in the sequence listing or inactivate it; More preferably, in step F1), the gene shown as SEQ ID NO.1 in the sequence listing in Phytophthora sojae and the gene shown in SEQ ID NO.2 in the sequence listing are knocked out, so that the sequence listing The protein shown in SEQ ID NO.5 and the protein shown in SEQ ID NO.6 in the sequence listing are inactivated; In step F2), the gene shown in SEQ ID NO.2 in the sequence table in Phytophthora sojae and/or the gene shown in SEQ ID NO.4 in the sequence table is knocked out, so that SEQ ID NO.4 in the sequence table is knocked out. Inactivation of the protein shown in 6 and/or the protein shown in SEQ ID NO.8 in the sequence listing; In step F3), gene knocking is performed on the gene shown in SEQ ID NO.2 in the sequence table in Phytophthora sojae, the gene shown in SEQ ID NO.3 in the sequence table and the gene shown in SEQ ID NO.4 in the sequence table Divide to inactivate the protein shown in SEQ ID NO.6 in the sequence listing, the protein shown in SEQ ID NO.7 in the sequence listing and the protein shown in SEQ ID NO.8 in the sequence listing; In step F4), by comparing the gene shown in SEQ ID NO.1 in the sequence table in Phytophthora sojae, the gene shown in SEQ ID NO.2 in the sequence table, the gene shown in SEQ ID NO.3 in the sequence table and the sequence table The gene shown in SEQ ID NO.4 in the sequence table is knocked out, so that the protein shown in SEQ ID NO.5 in the sequence table, the protein shown in SEQ ID NO.6 in the sequence table, the SEQ ID NO. Inactivation of the protein shown in 7 and the protein shown in SEQ ID NO.8 in the sequence listing; In step F5), by comparing the gene shown in SEQ ID NO.1 in the sequence listing of Phytophthora sojae, the gene shown in SEQ ID NO.2 in the sequence listing, the gene and sequence shown in SEQ ID NO.3 in the sequence listing One or two or more of the genes shown in SEQ ID NO.4 in the list are knocked out, so that the protein shown in SEQ ID NO.5 in the sequence table and the protein shown in SEQ ID NO.6 in the sequence table are knocked out Inactivation of one or more of the protein, the protein shown in SEQ ID NO.7 in the sequence listing, and the protein shown in SEQ ID NO.8 in the sequence listing; In step F6), the gene shown in SEQ ID NO.2 in the sequence table in Phytophthora sojae and the gene shown in SEQ ID NO.3 in the sequence table are knocked out, so that the gene shown in SEQ ID NO.6 in the sequence table is knocked out. The protein shown and the protein shown in SEQID NO.7 in the sequence listing are inactivated; In step F7), the gene shown in SEQ ID NO.3 in the sequence table in Phytophthora sojae and the gene shown in SEQ ID NO.4 in the sequence table are knocked out, so that the gene shown in SEQ ID NO.7 in the sequence table is knocked out. The protein shown and the protein shown in SEQ ID NO. 8 in the sequence listing are inactivated. 10. the application of the substance inhibiting the expression and/or activity of the NCR protein of claim 1 in the preparation of a plant Phytophthora sojae fungicide; preferably, the substance inhibiting the expression and/or activity of the NCR protein is to inhibit the expression of the NCR protein and/or substances that inhibit the transcription of the gene encoding the NCR protein and/or the translation of the RNA molecule obtained by the transcription of the gene encoding the NCR protein.

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