SsAGM1-mediated UDP-GlcNAc Synthesis Is Essential For Development And Pathogenicity In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum(Lib.)de Bary is a necrotrophic phytopathogenic fungus with an worldwide distribution,causing Sclerotinia stem rot.It is very difficult to control Sclerotinia stem rot on account of the wide host range,strong viability and complex pathogenic mechanism of S.sclerotiorum.Thus,elucidating the molecular mechanism of growth,development,and pathogenicity of S.sclerotiorum,will contribute to discover potential drug targets and provide new strategies for the control of Sclerotinia stem rot caused by S.sclerotiorum.Cell wall is an essential structure in fungi,which is composed of chitin,mannan,and glucan.Chitin accounts for about 10%-20% of the cell wall in filamentous fungi and plays an important role in the survival,morphology change,and pathogenicity of phytopathogenic fungus.Chitin was synthesized by UDP-GlcNAc(uridine diphosphate-N-acetylglucosamine)as a direct precursor.In the biosynthesis of UDP-GlcNAc,the conversion of GlcNAc-6P to GlcNAc-1P catalyzed by AGM1(Nacetylglucosamine-phosphate mutase)is a critical step.AGM1 has been isolated and identified from many species,and AGM1 plays an important role in a variety of cellular processes,such as cell survival and division.However,the function of AGM1 in phytopathogenic fungi is poorly understood.In this study,N-acetylglucosamine phosphate mutase encoding gene SsAGM1 of S.sclerotiorum was used as the target gene.The gene-silenced transformant and overexpression transformant of SsAGM1 were obtained by RNAi and geneoverexpression technology.The function of SsAGM1 was clarified in S.sclerotiorum by analyzing the chitin content,UDP-GlcNAc content,vegetative growth,sclerotia and infection structure formation,stress tolerance,and pathogenicity of strains.The results of this study are as follows:1.We cloned the SsAGM1(SS1G?01582),and bioinformatics analysis showed that the full length of SsAGM1 was 1667 bp,and the encoded protein of SsAGM1 contained N-acetylglucosamine phosphate mutase domain.2.Calcofluor White staining of hyphae showed that fluorescence mainly concentrated at the tip and septum of hyphae in wild-type strain(WT),control strain(CK),and overexpressed transformants.In SsAGM1 gene-silenced transformants,fluorescence showed irregular spot-like aggregation in hyphae.Quantitative analysis showed that the contents of UDP-GlcNAc and chitin in SsAGM1 gene-silenced transformants were significantly decreased compared with WT and CK,while the contents of UDP-GlcNAc and chitin in overexpressed transformants were significantly increased.Moreover,the expression level of SsAGM1 affected the expression of GFA1,GNA1,and UAP1 in UDP-GlcNAc synthesis pathway,suggesting that SsAGM1 was involved in the UDP-GlcNAc and chitin synthesis and affected the expression of genes related to the chitin synthesis.3.Compared with WT,CK and overexpression transformants,the growth rate of SsAGM1 silenced transformants decreased significantly and could not form sclerotia.The hyphae of SsAGM1 silenced transformants flock together on hydrophobic surface,and could not form infection structure.SsAGM1 is involved in vegetative growth and morphological transformation.4.Compared with WT and CK,the sensitivity of SsAGM1 gene-silenced transformants to cell wall synthesis inhibitors(CR,SDS,CFW)and exogenous osmotic substances(Na Cl,KCl and D-sorbitol)was significantly increased,whereas the sensitivity of overexpressed transformants was significantly reduced,indicating that SsAGM1 is involved in the response to cell wall synthesis inhibitors and osmotic stress in S.sclerotiorum.5.Fluorescence microscopy showed that SsAGM1-GFP fusion proteins were distributed in cytoplasm,indicating that SsAGM1 is located in cytoplasm of S.sclerotiorum.6.Infection tests were carried out on leaves of Arabidopsis thaliana,tomato and soybean,WT,control strain and SsAGM1 overexpressed transformant caused disease symptoms at 48 h after inoculation,whereas SsAGM1 gene-silenced transformant could not invade leaves.When the infection time of SsAGM1 gene-silenced transformant was extended to 4 days,no disease spots were formed.The same result was obtained by infection test of wounded tomato leaves.These results suggest that SsAGM1 played an significant role in pathogenicity of S.sclerotiorum.The above results show that SsAGM1 is involved in UDP-GlcNAc and chitin synthesis,and involved in the vegetative growth,infection structure formation,sclerotinia formation,stress tolerance,and pathogenicity of S.sclerotiorum.This study clarified the function of SsAGM1 in the growth,development,and pathogenicity of S.sclerotiorum,which laid a foundation for further exploring the molecular mechanism of chitin synthesis and pathogenicity of S.sclerotiorum.