Study On Gene Function And Invasion Mechanism Of Sclerotinia Sclerotiorum DNA Virus SsHADV-1

Sclerotinia sclerotiorum,an important phytopathogenic fungus,has a wide host range.This fungus seriously affects the yield of canola in China.The diverse mycoviruses have been discovered in hypovirulent isolates of S.sclerotiorum.Those hypovirulence-associated mycoviruses are important resources for the biological control on Sclerotinia disease.In our previous study,a DNA mycovirus,SsHADV-1?Sclerotinia sclerotiorum hypovirulence associated DNA virus 1?has been characterizated in molecular level and has great potential to development a virocontrol agent.SsHADV-1can break the restriction of host vegetative incompatibility for horizontal transmission,and also can be transmitted in vitro.In this research,the function of two genes from SsHADV-1,the mechanism of virus directly infecting hyphae,and the mechanism of anti-virus of S.sclerotiorum were studied in the interaction system of S.sclerotiorum and SsHADV-1.The biological function of Capsid protein?Cp?and Replication protein?Rep?from SsHADV-1 was investigated.The overexpression transformants of Cp,Rep or both two genes,C,R and C-R,were derived from a strain Ep-1PNA367 of S.sclerotiorum,respectively.The transcripts of Cp,Rep or both two genes were detected in C,R and C-R,respectively,while correspondence encoded proteins of them failed to be detected.There was no significant difference between overexpression transformants,C,R and C-R,and strain Ep-1PNA367 in the morphological characteristics of mycelium tip and colony,the production of oxalic acid and the pathogenicity.In addition,SsHADV-1 still could be horizontally transmitted to those overexpression transformants via hyphal contact,suggesting that the overexpression of neither Cp nor Rep had no effect on the resistance to SsHADV-1.The monoclonal antibody of Cp protein was used for immunoprecipitation reaction,and results showed that 61 candidate proteins interacting with Cp protein were obtained in S.sclerotiorum.To investigate the early response mechanism of S.sclerotiorum to SsHADV-1infection,S.sclerotiorum hyphae were inoculated with purified SsHADV-1 virions.The pre-and post-infected hyphae were collected at 0,1 and 3 hours after inoculation for transcriptome analysis,respectively.The results showed that there were 187 genes dramaticly differentially expressed in the early stage of infection with SsHADV-1,of which 114 genes were up-regulated and 73 genes were down-regulated,respectively.These genes were mainly involved in metabolic processes,biosynthesis of antibiotics,and secondary metabolite.Cell structure analysis indicated that 26%of the total differentially expressed genes?DEGs?were related to membrane tissues,and 10 of them were predicted to be located in the cell membrane.Furthermore,30%of DEGs with functional annotation were predicted to be involved in the Ras-small G protein signal transduction pathway.These results revealed that SsHADV-1 virions may be able to bind host membrane proteins and influence signal transduction through Ras-small G protein-coupled receptors during early infection.The gene SS1G₀6180 was significantly down-regulated at 3 h after inoculation with SsHADV-1 virions.The length of SS1G₀6180 was 1498 bp,but its biological function was still unknown.SS1G₀6180 was predicted to encode a protein kinase with multiple phos-PKC phosphorylation sites.There was a Macoilin family transmembrane structure at the 5'-terminal of the hypothetical protein.Since this protein contains a conserved domain Pal1,it was predicted to locate in the nucleus.The results of viral horizontal transmission test indicated that the gene silencing mutant,SI,has the ablity to anti DNA,+ssRNA and dsRNA mycoviruses infection,which was different from the reported typical RNAi and methylation-induced antiviral mechanisms.The analysis on the transcriptome of SS1G₀6180 silencing mutant indicated that tyrosine and phenylalanine metabolism,secondary metabolite synthesis and glyceride metabolism were enrichment.The expression level of SS1G₀6180 was higher on the 5th day?sclerotium formation?and 9th day?sclerotium maturation?after inoculation on PDA in strain Ep-1PNA367.The colony morphology of SS1G₀6180 knockout mutant strains,KO6-1,11,12,produces less pigment and shows white color in cology morphology.The numbers of sclerotia formed by KO6-1,11,12 were significant decreased,while the size of them significant increased.The pathogenicity of mutants KO6-1,11,12 was reduced.Melanin is related to the formation of sclerotia.The polyketone synthase gene PKS13,involved in the melanin synthesis signaling pathway,was significantly down-regulated expression,while the Scytalone dehydratase-1 gene SCD1 and reductase gene T4HN were significantly up-regulated expression in SS1G₀6180 knockout mutants.Those results revealed that SS1G₀6180 is involved in the synthesis of melanin in sclerotia,and then affects the development of sclerotia formation.Previous studies suggested that SsHADV-1 could not infect Botrytis cinerea.However,the results of the present research indicated that SsHADV-1 virions could infect the protoplasts of three different strains of B.cinerea,and the virus could replicate in B.cinerea to produce virions.The average growth rate of B.cinerea strains infected with SsHADV-1?B05.10-HADV?was 11 mm/d.It was significantly lower than the average growth rate of virus-free strain B05.10?14 mm/d?.The colony morphology of B05.10-HADV was abnormal,and the frequency of conidia infected with SsHADV-1 is lower?5%?,and the virions extracted from B05.10-HADV cannot directly infect the healthy hyphae of B.cinerea.A single nucleotide of the viral nucleic acid extracted from B05.10-HADV has difference from original SsHADV-1.At the 2086^th base of the virus,the C?cytosine?base is mutated to T?thymine?base,corresponding to amino acids was mutated from threonine to serine.Single-base mutation may cause the host range change of SsHADV-1.An infection cloning vector of one and 1.6 SsHADV-1 virus per unit length was constructed and transformed into Arabidopsis thaliana Col-1.The genome of the virus can be integrated into genome of A.thaliana with a transgenic efficiency of over 68.5%.However,free viral nucleic acids could not be formed in transgenic plant,and the expression of the SsHADV-1 capsid protein was not detected by western-blot.The virus-free strain of S.sclerotiorum can infect transgenic A.thaliana and kill the entire plant.This study is the first time to explore the interactions between SsHADV-1 and S.sclerotiorum.Preliminarily establish the interaction system between DNA virus and its host,which provides abundant resources for finding the hypovirulence mechanism of the mycovirus and use it to production and application.