Identification Of Phosphoproteins And Targets Of Protein Phosphatase PPG1 In Pyricularia Oryzae

The rice blast fungus Pyricularia oryzae is a model filamentous ascomycete for the study of fungus-plant interaction,which causes the most devastating disease of worldwide cultivated rice.Protein phosphorylation is known to regulate pathogenesis,mycelial growth,conidiation and stress response in P.oryzae.However,phosphorylation mediated regulatory networks in the fungal pathogen remain largely to be uncovered.In this study,we identified 1621 phosphorylation sites of 799 proteins in mycelia of P.oryzae,including 899 new p-sites of 536 proteins and 47 new p-sites of 31 pathogenicity-related proteins that were previously reported.From the sequences flanking the phosphorylation sites,19 conserved phosphorylation motifs were identified,including 3 motifs that have not been assigned to specific protein kinases.Notably,phosphorylation was detected in 7 proteins that function upstream of Pmk1 but not in Pmkl and its downstream Mst12 and Sfll.Interestingly,phosphorylation was detected at the site oer240 of Pmp1,which is a putative protein phosphatase highly conserved in filamentous fungi but not characterized.We thus generated ?pmp1 deletion mutants and dominant allele Pmp1S240D mutants.Phenotyping analyses indicated that Pmp1 is required for virulence,conidiation and mycelial growth.Further,we found that phosphorylation level of Pmk1 in mycelia was significantly increased in ?pmp1 mutant,but decreased in Pmp1S240D mutant in comparison with the wild type,demonstrating that Pmp1 phosphorylated at Ser240 is important for regulating phosphorylation of Pmk1 in mycelia.To our surprise,phosphorylation of Mps1,another MAP kinase required for cell wall integrity and appressorial formation of P.oryzae,was also significantly enhanced in the ?pmpl mutant,but decreased in the Pmp1S240D mutant.In addition,we found that Pmp1 directly interacts with Mpsl and the region AA180-230 of Pmpl is required for the interaction.In summary,this study shed new lights on the protein phosphorylation mediated regulatory networks in P.oryzae.The author also identified targets of PPG 1,which is a protein phosphatase essential to pathogenicity of P.oryzae.With comparative and quantitative phosphoproteomic analyses,the author identified 216 up-regulated p-sites from 159 proteins in mycelia of ?moppgl.From the 216 up-regulated p-sites,5 different phosphorylation motifs were extracted that are specifically recognized by typical kinases.A KEGG pathway analysis showed that the proteins with up-regulated p-sites in mycelia of ?moppg1 are likely involved in 34 distinct pathways,indicating that PPG1 widely regulates its targets in P.oryzae.The author further chose MGG 02757 and MGG₀6712 for in detail functional characterization.MGG₀2575 in P.oryzae encodes an otholog to Dbf2 protein kinase in Saccharomyces cerevisiae and to NDR?nuclear Dbf2-related?kinase family in Homo sapiens,was thus named as MoNDRl.MoNDR1 was highly phosphorylated at Ser466 in the ?moppg1 mutant but not in the wild type strain,in the three comparative and quantitative phosphoproteomic analyses.Western blot with antibody specific to phosphorylated MoNDR at Ser466 orthologous site proteins detected three protein bands in the wild type strains but an additional band corresponding to MoNDR1 in the ?moppgl mutant,confirming that MoNDR1 was phosphorylated in the ?moppgl mutant.Yeast two-hybrid assay and Co-immunoprecipitation assay showed that PPG1 directly interact with MoNDR1 in vivo.It was also observed that PPG1::mCherry and MoNDRl::eGFP were co-localized during infection.These results indicate that PPG1 directly regulates the dephosphorylation of MoNDRl.To explore biological function of MoNDRl and impact of phosphorylation at the Ser466,?mondrl,MoNDR1S466D and MoNDR1S466A strains were generated.Phenotype assays showed that ?mondrl mutants were defective in mycelial growth,conidiation and virulence as compared with the wild type.MoNDR1S466D and MoNDR1S466A strains both also displayed defects in mycelial growth and conidiation,indicating that phosphorylation and dephosphorylation are both required for regulation of functions of MoNDR1.As compared with the?moppgl mutants,the ?mondr1 mutants had a similar mycelial growth rate,and but had more conidiation and normal conidia morphology although they were significantly reduced in conidiation and had more abnormal conidia compared to the wild type.Infection assay showed that?moppgl lost pathogenicity to infect hosts,while the ?mondrl mutants generated a few lesions although they are dramatically reduced in virulence.However,the MoNDR1S466D and MoNDR1S466A were similar to the wild type in virulence.Thus,these results suggest that for mycelial growth and conidiation in P.oryzae,PPG1 directly regulates the dephosphorylation of MoNDR1 at Ser466,which has not been characterized in any other plant pathogenic fungi.Thus,the present study provided the first comprehensive insight into regulation of MoNDR1 by PPG1 and important information for further investigating how MoNDRl is involved in growth,development and pathogenicity in pathogenic fungi.The author further investigated regulation of Met6 by PPG1.Met6 is orthologous to methionine synthase that play important roles in amino acid metabolic process.Comparative and quantitative phosphoproteomic analysis showed that Met6 was highly phosphorylated at Ser705 in ?moppgl mutants.To explore impact of phosphorylation at the Ser705 site on functions of Met6,allele mutants Met6S705D and Met6S705A were generated by using a ?met6 mutant,which was previously generated in our lab,Met6S705A restored the defects of ?met6 on growth,conidiation and pathogenicity,while Met6S705D displayed similar defects as ?met6,indicating dephosphorylation is required for function of Met6.However,yeast-two hybrid assay showed that PPG1 could not interact with Met6,indicating that the dephosporylation of Met6 regulated by PPG1 is indirectly,or may require additional mediator.It has been known that PP2A regulates metabolic process in eukaryotic cell.But the relationship between PP2A and Met6 has never been reported.Thus,our study provides cues to uncover the function of PP2A phosphatase family in metabolic pathways.Taken together,this study identified many new phosphoproteins and phosphorylation sites in mycelia of P.oryzae,and phosphorylation p-sites that are regulated by Pmpl and PPG1.In particular,the author determined that Pmp1 phosphorylated at Ser240 regulates dephosphorylation of Mps1 and Pnkl and that PPG1 regulates the dephosphorylation of MoNDRl on Ser466,and Met6 on Ser705.These new findings provided novel insights into phosphorylation-mediated regulation of asexual development and pathogenesis and will be invaluable to construction of phosphorylation-mediated regulatory networks in the fungal pathogen.