Molecular Mechanism Of Secreted Protein SsPep1 Regulating Pathogenicity Of Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum（Lib.）de Bary is a worldwide phytopathogenic fungus.Sclerotinia stem rot caused by it is one of the main diseases endangering rapeseed production,resulting in huge economic losses.However,lacking of resistant resources in rapeseed severely limits the disease-resistant breeding process of rapeseed.The complex pathogenic mechanism of S.sclerotiorum makes the prevention and control of S.sclerotiorum more difficult.Analyzing the pathogenic mechanism of S.sclerotiorum may help find the way to block the pathogenicity of S.sclerotiorum and provide new strategies to safely prevent and control of S.sclerotiorum.In the previous study,93 candidate S.sclerotiorum secretory proteins were identified differentially expressed during infecting resistant and susceptible host.Among them,a highly expressed gene SsPep1was selected.In this study,this gene was used as a candidate gene for functional analysis,and its molecular mechanism during the host-S.sclerotiorum interaction was explored.This study will provide fundamental research for the development of safety prevention and control strategies of S.sclerotiorum.The main results are as follows:（1）Bioinformatics analysis:The total length of the SsPep1 gene（SS1G＿03181）of S.sclerotiorum is 1467 bp,the length of CDS sequence is 1209 bp.This gene encodes a protein with 402 amino acids,the isoelectric point is 5.06,the fat index is 73.86,and the fat solubility index is 36.12,the average hydrophilic coefficient is 0.021,and the instability coefficient is39.16.SsPep1 contains an aspartic proteinase（Asp）domain with 19 aa as signal peptide in the N-terminal and has no transmembrane domain,suggesting that this protein may be a secreted aspartic proteinase.（2）Gene expression analysis:The expression of SsPep1 gene at different stages of hyphal growth and infection was analyzed by q RT-PCR.The results showed that the gene expression increased in the hyphal growth period and increased significantly at 6-48 hours after inoculation.These results indicated that SsPep1 may be involved in hyphal growth and infection.（3）Functional validation of the predicted signal peptide of SsPep1:The signal peptide fusion vector p SUC2-SsPep1 of SsPep1 protein was constructed and transferred into the sucrase exocytosis-deficient yeast strain YTK12,resulting in the yeast transformants being able to grow on YPRAA medium containing raffinose,which was consistent with the positive control p SUC2-Avr1b.The negative control p SUC2-Mg87 could not grow on YPRAA medium.Therefore,it is suggesting that the signal peptide of SsPep1 is functional and that SsPep1 is probably a secretory protein.（4）Screening and phenotypic analysis of SsPep1 gene silent strains:The SsPep1 gene silent vector was constructed and transformed into S.sclerotiorum wild-type atrain1980 via PEG-mediated method.The transformants were screened on PDA plate containing hygromycin,and two knockout transformants were successfully selected.q RT-PCR analysis showed that the expression levels of SsPep1 in these two knockout transformants was significantly lower than that of wild-type strain 1980.Phenotypic analysis showed that the hyphal growth rate of knockout transformants was significantly slower and no sclerotia was produced.SsPep1 gene knockout transformants produced significantly smaller lesions than that of wild-type strain 1980,suggesting that the pathogenicity of SsPep1 gene knockout transformants was significantly decreased.（5）Analysis of the interacted proteins of SsPep1:Yeast two hybrid（Y2H）techniques was performed to screen the proteins interacted with SsPep1 from the S.sclerotiorum-rapeseed interaction c DNA library.Lectin protein BnA01.LLP in rapeseed was identified interacted with SsPep1.Furthermore,SsPep1 was found function in reducing the protein expression of BnA01.LLP.（6）Effect of exogenous lectin on S.sclerotiorum resistance:Lectin and dd H₂O were sprayed on the rapeseed leaves,separately,prior to the inoculation of S.sclerotiorum 1980.The results showed that exogenous lectin could significantly reduce the pathogenicity of S.sclerotiorum.（7）Sclerotinia resistance analysis of BnA01.LLP transgenic Arabidopsis lines:Sclerotinia resistance analysis was performed on the Arabidopsis lectin mutant atllp,the results showed that the lesion area of atllp was significantly larger than that of wild type（WT）.However,the Sclerotinia resistance was significantly recovered in the BnA01.LLP complementary lines（atllp/BnA01.LLP）and enhanced in the BnA01.LLP overecpression lines（OE-BnA01.LLP）.Furthermore,the host disease response marker gene At PR1 was significantly up-regulated in WT and lightly up-regulated in atllp after inoculated with S.sclerotiorum.In the S.sclerotiorum-inoculated atllp/BnA01.LLP and OE-BnA01.LLP,the expression of At PR1 was significantly recovered and enchanced,which is coincidence with the Sclerotinia resistance.