Functional Analysis Of A GPI-anchored Protein SsGSSP1 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum is a widely distributed necrotrophic filamentous pathogenic fungus,which seriously affects the yield and quality of rapeseed and other crops.At present,the lack of resistance resources severely limits the resistance breeding in rapeseed.GPI-anchored proteins play an important role in plant pathogenic fungal pathogenicity,affecting the mycelium development,cell wall integrity and virulence.Therefore,identifying and studying the function of GPI-anchored proteins in S.sclerotiorum may help analyze the pathogenic mechanism of S.sclerotiorum and provide a theoretical basis for the safety prevention and control of S.sclerotiorum.In the previous study,seven differentially expressed genes encoding GPI-anchored proteins of S.sclerotiorum were identified by transcriptome analysis.Among them,SS1G＿03230（SsGSSP1）was highly expressed during inoculation,suggesting that it may be involved in the pathogenic process of S.sclerotiorum.Thereby,in this study,SsGSSP1 was selected as the candidate gene for functional analysis,and its potential in crop breeding was also explored.The main findings are as follows:（1）Bioinformatic analysis:SsGSSP1（S.sclerotiorum GPI-anchored small secreted protein 1）gene has a full length of 780 bp and a coding region of 639 bp,encoding a protein with 212 amino acids,of which the 17 amino acids in N-terminal form a signal peptide.SsGSSP1 without transmembrane domain belongs to the member of the family of GPI-anchored serine/threonine-rich membrane proteins.The GPI binding site G-ωis predicted at C-31.Moreover,subcellular localization prediction found that SsGSSP1protein was localized extracellularly,including cell wall.Consequently,it is speculated that SsGSSP1 may encode a secreted GPI-anchored protein.（2）Gene expression pattern analysis:The expression of SsGSSP1 at different stages of plant infection was analyzed by q RT-PCR.The results showed that the expression level of SsGSSP1 was increased significantly at 48 h after inoculation of rapeseed leaves,which was nine times that of on PDA medium.These results indicated that SsGSSP1 may be involved in the pathogenic process of S.sclerotiorum.（3）Functional validation of the predicted signal peptide of SsGSSP1:In order to verify the function of the predicted signal peptide of SsGSSP1,a sucrase-deficient yeast secretion system was used.The signal peptide fusion expression vector p SUC2-GSSP1SP was constructed and transferred into YTK12 yeast strain.The results showed that the strains carrying p SUC2-GSSP1SP or the positive control p SUC2-Avr1b could grow on the YPRAA medium containing raffinose,suggesting that the signal peptide of SsGSSP1is functional and the SsGSSP1 is probably a secretory protein.（4）Screening and phenotypic analysis of SsGSSP1 gene knockout strains:The gene knockout vector of SsGSSP1 was constructed and transformed into wild-type S.sclerotiorum strain 1980 via PEG-mediated protoplasts transformation.Five positive transformants were successfully screened on PDA plate containing hygromycin,and it was found that SsGSSP1 was almost not expressed in these transformants by semi-quantitative PCR and q RT-PCR.There were no significant differences in the mycelial growth rate and infection cushion formation between SsGSSP1 gene knockout strains and wild-type strain 1980,but SsGSSP1 gene knockout strains were more sensitive to hypertonicity,high salt and hyperoxia environment and cell wall inhibitors.The lesion size of SsGSSP1 gene knockout strains was significantly reduced on Arabidopsis thaliana,Nicotiana benthamiana and rapeseed,indicating that SsGSSP1 is involved in the pathogenic process of S.sclerotiorum.（5）Resistance analysis of transgenic A.thaliana lines:The SsGSSP1 host-induced gene silencing（HIGS）and over-expression vectors were constructed and transformed into wild-type A.thaliana.The stable expression lines of transgenic A.thaliana were selected from T₃ generation.Sclerotinia resistance screening showed that the lesion size of HIGS transgenic line HIGS-SsGSSP1 was significantly reduced,and the expression of SsGSSP1 was reduced at 12 h after inoculation compared with wild-type A.thaliana.These results indicate that expressing the ds RNA targeting S.sclerotiorum SsGSSP1 in A.thaliana may significantly enhance the resistance of transgenic plants.Meanwhile,the Sclerotinia resistance was enhanced in the SsGSSP1-overexpression A.thaliana lines,suggesting that SsGSSP1 may affect the defense response of host.（6）Transcriptome analysis:The S.sclerotiorum differentially expressed genes（DEGs）between wild-type strain 1980（W）and SsGSSP1 gene knockout strain（KO）were analyzed via RNA-seq.Enrichment analysis revealed that the down-regulated S.sclerotiorum DEGs were significantly enriched in oxidative stress,heat shock response and chemical resistance,suggesting that SsGSSP1 may be involved in the stress tolerance of S.sclerotiorum.While,the DEGs of A.thaliana after inoculated with W and KO were analyzed.Enrichment analysis revealed that the down-regulated A.thaliana DEGs were significantly enriched in the lignin biosynthesis and plant-pathogen interaction pathways,suggesting that SsGSSP1 may affect the plant immune defense response and therefore involved in the interaction of host and S.sclerotiorum.