The Identification And Virulence Mechanism Analysis Of Phytophthora Sojae Avr Effector

Phytophthora root and stem rot of soybean caused by Phytophthora sojae is one of the most destructive soybean diseases. The interaction between P. sojae and soybean fits the "gene-for-gene" hypothesis. The interaction between P. sojae Avr protein and soybean R protein determine the outcome of plant disease. So the research about Avr effector and plant R protein is significant to reveal the interaction mechanism and the control plant disease. Considering the RXLR motif; transcription and polymorphism in different races, we found Avr effectors candidates. Combining genetic mapping, we identified P. sojae Avr effector Avrld. This method gives highlight for Avr effectors identification from oomycetes or ever other pathogen. The results of polymorphism indicate that Avr3b escape the recognized by Rps3b by large number of amino acid mutation and translation termination. Avrld escapes recognition by Rpsld by absence in virulence isolates. Avrld can target E3 ubiquitin-protein ligase and affect the host plant immunity system. This shows the new mechanism of Avr effector virulence function. The major conclusions are showed in next.P. sojae Avrld gene encodes an RxLR-dEER effector recognized by Rpsld. Previous studies have described the following features of oomycete Avr effectors:(1) all of the Avr effectors identified so far contain the RXLR motif, (2) the transcripts of Avr effectors are detectable in avirulent strains, (3) many Avr effectors shows polymorphism. Considering all these features, we found the RXLR candidates similar with Avrld, and CAPS (Cleaved Amplified Polymorphic Sequence) markers of candidates are designed for genetic mapping. The Avrld avirulence and virulence strains made a cross to generate F2 genetic population, the mapping data demonstrated that Avh6 genotype co-segregated with Avrld phenotype in F2 populations. Transient expression Avrld in soybean leaves by co-bombardment indicated that Avh6 can be recognized by Rpsld, and the C terminal is the critical region for recognition. The transcription showed that Avrld expressed during infection, and the transcript levels were elevated in germinating cysts and during the mycelia and zoospores stages. The results suggested that Avrld is induced in preinfection structures and during infection. Combining the genetic mapping with bioinformatics analysis, we identifed Avrld which gave highlight for Avr effectors identification from oomycetes or other pathogens.Avrld shows polymorphism and can suppress plant immunity. The virulence phenotypes of 24 P. sojae isolates from different region were characterized on Rpsld soybean cultivars by using hypocotyl slit inoculation method. The results show that 12 isolates are avirulence and the other are virulence. The specific primers that contain Avrld coding region were designed to amplify. PCR amplification was not observed from the virulent isolates whereas PCR products of the expected size were obtained from the avirulent isolates. PCR amplification products generated with the primer set from the tested isolates were sequenced. There are two kinds of sequence AvrldP6497 and AvrldP7064 which from the strain P6497 or P7064 respectively. Compared with AvrldP6497, several amino acid polymorphisms were observed in the predicted mature protein Avrld 7064 sequences. The transient expression AvrldP6497and AvrldP7064 in Rpsld soybean leaves can trigger cell death shows that both of them can be recognized by Rpsld. The analysis of the positive selection shows that amino acid K (position 105) withstands the maximum pressure from host R protein. AvrldP6497and AvrldP7064 can suppress the cell death induced by BAX and ETI from interaction between Avrlb and Rpslb, and overexpress in tobacco can promote the infection by P. capsici. Transient expression of GFP::Avrld construct in N. benthamiana shows that Avrld might be localized in both cytoplasm and nucleus, however, expression of both Avrld::NLS and Avrld::NES does not affect Avrld recognized by Rpsld but lost the ability to promote the infection. We analysis the mechanism of variation that Avrld escapes recognition by Rpsld. The results virulence function and localization of Avrld also significant for further research about virulence function.The mechanism of Avrld regulate plant immunity. Large number of effectors from pathogen can enter host cell to destroy or interference the immunity system when P. sojae infect soybean which will promote infection and colonization. So to identify the effectos target protein is significant for revealing its virulence mechanism. In order to investigate the Avrld virulence mechanism, the Yeast Two Hybrid was to used to screen Avrld target protein. We found that Avrld can interact with Gm06860 from soybean library. We cloned the full length Gm06860 and changed the vector each other to confirm the interaction, and interaction between Avrld and Gm06860, this consistent with the result of GST Pull Down. The protein sequence analysis shows that Gm06860 contain an ubiquitin-protein ligase domain. The enzyme activity assays demonstrated that Gm06860 exhibit ubiquitin-protein ligase activity. Mutation of key residues in domain C263A and W290A abolished enzyme activity, and the mutation W290A also can not interact with Avrld, the result suggest that amino acid position 290W is significant for interaction. All the results that Avrld can target E3 ubiquitin-protein ligase and affect the host plant immunity system. This shows the new mechanism of Avr effector virulence function.Polymorphism analysis and region identification of Avr3b recognized by Rps3b. We analysed Avr3b about the sequence polymorphism; transcription and the recognition region. The results show that there are two different sequences among P. sojae isolates, commared with avirulence sequence, virulence sequence show number of amino acid mutation and early termination of translation. The analysis of the positive selection shows that amino acid position 76A and 92S withstand the maximum pressure from host R protein. Avr3b contain a Nudix motif and ADP-ribose and NADH pyrophosphorylase activity. Avr3b can express in avrulence and virulence strains and the transcription level shows higher in avirulence than virulence. The C terminal of Avr3b can be recognized by Rps3b and the mutation of Nudix motif does not affect the recognition by Rps3b. All the results indicate that Avr3b escape the recognized by Rps3b by large number of amino acid mutation and translation termination.