Functional Research On A Secreted Protein SsCP1 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum?Lib.?de Bary is a necrotrophic fungal pathogen,causes disease in a wide range of plants worldwide.It also causes yield losses and quality decline in crop production.Studies on pathogenesis mechanism of S.sclerotiorum can provide potential target for Sclerotinia disease management and offer a reference basis for breeding of resistant plant.Cerato-platanin?CP?proteins are phytotoxic proteins secreted by fungi.CPs are distributed among many filamentous fungi.In this study,a gene?SS1G₁0096?coding a protein named S.sclerotiorum Cerato-platanin protein 1?SsCP1?was cloned and identified from S.sclerotiorum.The full-length of Sscp1 gene is 969 bp,possess only one exon with 5?-UTR 359 bp,3?-UTR 196 bp,and coding sequences?cds?414 bp in length.The SsCP1 protein has 137 aa with 18 aa signal peptide at N-terminal,the molecular weight is 12.1 kDa and isoelectric point?pI?is 4.61 for mature SsCP1 protein.The percentage of hydrophobic amino acid residues is 39%,while the Grand Average of Hydropathy?GRAVY?is 0.029 that suggests SsCP1 protein is a soluble protein.SsCP1 protein has threes motifs in sequence which are conserved in CPs family from different fungi.The transcript level of Sscp1 increased by 12-fold at 12 hours post inoculation?hpi?and then maintained at a high level expression during late stages?24 48 hpi?,indicating that Sscp1 may be involved in infection of S.sclerotiorum.The ?Sscp1 gene mutants and complementary strains showed no significant changes on mycelial growth,colony morphology,sclerotial development and oxalic acid production compared with WT strain.Virulence of ?Sscp1 mutants was significant declined on detached leaves on Arabidopsis and tomato,suggesting that Sscp1 gene is involved in the virulence of S.sclerotiorum.To study the function of SsCP1 protein during Sclerotinia-plant interacion,a series of plasmids for heterologous proteins expression with protein tags and proteins subcellular localization tagged with multi-colour fluorescent proteins?FPs?in plants was constructed.When expressed in Nicotiana benthamiana using a TRV-based virus expression system,SsCP1 protein with or without signal peptide both could induce necrosis like cell death on leaves,and the cell death induced by SsCP1 with signal peptide was more strong.35s:Sscp1 transgenic plants were constructed via Agrobacterium-mediated transformation of A.thaliana with floral dip method.The 35s:Sscp1 transgenic plants showed no obvious changes in growth and development.The concentration of endogenous salicylic acid significant increased in 35s:Sscp1 transgenic plants compared with Col-0,and the expression level of transcripts of isochorismate synthase 1?ICS1?gene,which is essential for SA biosynthesis,was up-regulated.QRT-PCR results showed that the marker genes of SA signaling pathway PR1,PR2 and PR5 were highly induced in 35s:Sscp1 transgenic plants.The expression levels of BIK1 and RBHOD,which are key genes in basal resistance or PTI,were also up-regulated.The lesion produced by S.sclerotiorum WT strain on 35s:Sscp1 transgenic plants have no differences compared to that of Col-0,while ?Sscp1 mutants produced larger lesion on 35s:Sscp1 transgenic plants than on Col-0.Meanwhile,the 35s:Sscp1 transgenic plants showed more resistance to necrotrophic pathogens Botrytis cinerea and Alternaria brassicicol,and a biotrophic pathogen Golovinomyces orontii.At PR1 in Arabidopsis thaliana was identified to interacted with SsCP1 protein via immunoprecipitation?IP?,LC-MS/MS and Yeast two-hybrid?Y2H?techniques.The interaction between SsCP1 and At PR1 was further confirmed Co-immunoprecipitation?Co-IP?and bimolecular fluorescence complementation?BIFC?techniques.Sequence region between motif 1 and motif 2 is essential for the interaction of SsCP1 and At PR1.Protein subcellular localization showed that SsCP1 and At PR1 co-localized in apoplast of plant cells.Furthermore,35S:PR1 transgenic plants showed increased resistance to S.sclerotiorum.Our results suggested that SsCP1 is involved in the pathogenesis of S.sclerotiorum,and SsCP1 is an important virulence factor.SsCP1 could act as a PAMP to induce the host plant PTI response;on the other hand,SsCP1 protein interacts with Arabidopsis protein At PR1 to inhibit the plant resistance to S.sclerotiorum and facilitate its infection.