Identification And Functional Analysis Of Key Domain In Phytophthora Sojae RxLR Effector PSR1

Soybean root rot caused by Phytophthora sojae(P.sojae)is one of the devastating diseases that threaten soybean production around the world,causing direct economic losses of billions of dollars worldwide each year.Recent researches have shown that effectors secreted by Phytophthora sojae play a very important role in the process of soybeans infection,and can inhibit the host's immune response through different signal pathways and regulatory mechanisms,and promote the infection of pathogenic bacteria.We previously identified an RxLR-like effector PSR1 as an RNA silencing suppressor in P.sojae.Further research revealed that PSR1 can inhibit plant small RNA accumulation and host plants' disease-resistance reaction by binding to the Arabidopsis PINP1(PSR1-Interacting Protein 1)protein.However,the molecular properties of the PSR1 protein have not been studied.In this research,we tooke the effector PSR1 as the research object and identified a key domain of PSR1 and analyzed its function through bioinformatics analysis.We obtained the following main results:1.Through BLAST search of amino acid sequences,three PSR1-like effectors with low sequence similarity were identified on the NCBI website,there are PiPSR1L of Phytophthora infestans(P.infestans),PpPSR1L of Phytophthora parasitica(P.parasitica)and Ps Avh73 of P.sojae.All four effectors contain a predicted signal peptide(SP),an Rx LR motif and a short acidic d EER motif.Except for PSR1 at the N-terminus.With the exception of PSR1,the other three effectors are predicted to contain a single copy of the WY motif.2.It was found that PiPSR1L and PpPSR1L all have local RNA silencing suppression activity through transient expression experiments.Alignment of these 4 effectors' protein sequences shows that they have only 9 identical amino acid residues.At the same time,the results of the RNA silencing suppression test showed that changes of tryptophan at position 72 and tyrosine at position 100 in the PSR1 protein sequence will eliminate local and systemic RNA silencing suppression activity of PSR1,while mutations of the other seven conserved residues did not affect their activity.Tryptophan and tyrosine residues are important amino acids of the WY motif in Oomycetes.Therefore,we call these two amino acids the WY motif or WY domain.3.Phytophthora and virus inoculation tests found that in the WY motif,whether a single or two amino acids were mutated significantly reduced the ability of Phytophthora capsici(P.capsici)and P.infestans and the virus infection in Nicotiana benthamiana(N.benthamiana);Yeast two-hybrid and Co-Immunoprecipitation further indicate that the WY domain is required for the interaction of PSR1 and PINP1 proteins.4.Subcellular localization analysis found that the mutant of the amino acid in the WY motif of the effector PSR1 are located in the same position as the unmutated PSR1,and both are located in the cytoplasm and the nucleus,and the transcription level of the mutant does not change,however,the protein levels of the three mutants PSR1 W,PSR1Y,and PSR1 WY were significantly lower than PSR1 or PSR1 L,indicating that the WY domain of effector PSR1 is required for protein expression at the PSR1 translation level,and can maintain the stability of PSR1 protein in plants.5.Analysis of protein sequence alignment revealed that in Phytophthora,effectors that are similar to the PSR1 sequence are Rx LR effectors,and they all have very conserved WY domains.We have also demonstrated that the WY domains of Pc PSR1L and PpPSR1L,that are PSR1-like effectors in P.capsici and P.parasitica,are necessary for Phytophthora infection in the host,which indicates that the WY domain is a conserved module in the evolution and function of Phytophthora effectors.