The Mechanism Of SsHADV-1 Afftects Sclerotinia Sclerotiorum Infecting Brassica Napus

Sclerotinia sclerotiorum(Lib.)de Bary is a devastating fungal pathogen and leads to sclerotinia rot of hundreds of crops.There are no effective and environmentally friendly methods to control.Mycoviruses are viruses that parasitize fungi,and hypovirulence-associated mycoviruses have potential to control fungal diseases.Many hypovirulence-associated mycoviruses have been isolated from Sclerotinia sclerotiorum,and Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1(SsHADV-1)is one of them.SsHADV-1 is the first identified DNA fungal virus,it has strong infectivity,and its particles could infect the hyphae of S.sclerotiorum directly and could spread among host‘s vegetative incompatible individual efficiently.To understand the mechanism of hypovirulence of S.sclerotiorum caused by SsHADV-1,we compared the gene expression profiles of SsHADV-1 infected strain DT-8 and virus-free strain DT-8VF on living rapeseed(Brassica napus)plants.Hyphal fragments of strain DT-8 and strain DT-8VF were separately inoculated on the leaves of living plants of rapeseed and incubated at 20? cultivation room.At 6 hpi,12 hpi,18 hpi and 24 hpi,mycelial mass of S.sclerotiorum were collected from inoculated leaves and used to extract total RNA.Next generation sequencing(NGS)was applied to get different transcript data of strain DT-8 and DT-8VF.Differentially expressed genes(DEGs)of these two strains were selected to show the transcriptional difference after the infection of SsHADV-1.Compared with strain DT-8VF,816 genes of strain DT-8 were significantly up-regulated and 587 were down-regulated at 6 hpi.795 were up-regulated and 835 were down-regulated at 12 hpi,991 genes were up-regulated and 717 were down-regulated at 18 hpi,while 981 genes were up-regulated and 756 down-regulated at 24 hpi.A total of 687 genes were significantlly differentially expressed at the four sampling time points,among them,435 genes were up-regulated and 252 down-regulated.The down-regulated genes of the four time points were mainly enriched in secondary metabolism,glycan metabolism,protein binding and fatty acid metabolism,while the up-regulated genes were related to DNA repair,DNA recombination and replication.Gene ontology(GO),KEGG pathway enrichment and FunCat analysis of the DEGs indicated that SsHADV-1 mainly influenced the metabolic pathways and the DNA damage response related pathways of S.sclerotiorum.Up-regulated genes were enriched in pathways of DNA damage repair,DNA replication,oxidation-reduction and transport in the early infection,toxin transport and nitrogen metabolism in the late infection.Down-regulated genes were enriched in pathways of sugar and lipid metabolism,virulence factors and detoxification.Genes of carbohydrate metabolism and secondary metabolism were down-regulated at 12 hpi and 18 hpi.Glycosyl hydrolase genes of strain DT-8 were down regulated which were known as related to pathogenecity of S.sclerotiorum.108 significantly down-regulated carbohydrate activity enzymes and 6 significantly down-regulated small secreted proteins were obtained.Further analysis and verification experiment was needed for these enzymes and secretory proteins that may be involved in the pathogenic process of S.sclerotiorum.Infection of DNA virus could induce the DNA damage of host,KU proteins are key factors in Non-homologous end-joining(NHEJ)which firstly recognize the double-strand break(DSB)and recruit DNA-dependent protein kinase(DNA-PK)to repair.There are two KU protein encoding genes,Ssku70 and Ssku80 in S.sclerotiorum.In this study,the genes Ssku70 and Ssku80 of strain DT-8 were up-regulated 2 times to 4 times at the four inoculated time points compared with strain DT-8VF.The function of these genes was studied by gene knockout technique.The Ssku70 gene mutants showed sectorial-shaped colony morphology and denser mycelia,which exhibited hypovirulence on plants.The Ssku80 gene mutants had more aerial hyphae and the pathogenicity to tomato leaves was decreased significantly with strain DT-8VF.The ability of mutants on virus replication and accumulation needs to be investigated further.Briefly,in this study,we explored RNA_Seq analysis to investigate the difference of gene expression between strain DT-8 and DT-8VF on living rapeseed leaves.Our study provides a reliable data and theoretical basis for the further study of the hypovirulence of S.sclerotiorum induced by the virus SsHADV-1,and also references for revealing the interaction mechanism between virus and plant,it also supplies important cues for study on biological control of Sclerotinia rots with mycoviruses.