Screening Of Mycoviral Biocontrol Factors Of Colletotrichum And Exploring Of Pathogenesis-Related Genes

Colletotrichum spp.is a pathogenic fungus with important economic significance,which could cause serious damage to agricultural production and has been rated as the eighth most important plant pathogenic fungus in the world.Mycoviruses are a kind of viruses that infect fungi and widespread in all major taxonomic groups of fungi.Some mycoviruses infection can induce visible abnormal symptoms on the host fungi and have the potential for green prevention and control in plant fungal diseases.The interactions between hypovirulence-associated mycoviruses and their hosts also provide important insights for studying the pathogenic mechanism of fungi at the molecular level and searching of potential fungicide targets.In this study,the mycoviruses diversity of the C.fructicola,C.camelliaea,C.alienum,C.liriopes,C.cliviicola and C.plurivorum were analyzed,and the molecular and biological characteristics of two partitiviruses were mainly studied.Furthermore,three key functional genes were screened and explored using the mycovirus-host interaction model,and their functions were functional characterized.These results obtained were showed as follows:(1)Thirteen new mycoviruses were obtained from C.fructicola,C.camelliaea,C.alienum,C.liriopes,C.cliviicola,C.plurivorum.The whole or partial genome sequence of these viruses were obtained by dsRNA extraction combined with the RNA-Seq and Sanger sequencing.Based on sequence analysis of RNA-dependent RNA polymerase(Rd Rp),four mycoviruses were identified as dsRNA viruses,of which three belonged to Partitiviridae and one belonged to Totiviridae;eight mycoviruses were +ssRNA viruses,among which four were belonged to Mitoviridae,two were Botourmiaviridae,and two were Virgaviridae;one mycovirus whose genome has been characterized as between dsRNA and +ssRNA was belonged to a provisional family(Polymycoviridae).(2)We report a novel dsRNA virus designated Colletotrichum liriopes partitivirus 1(ClPV1)from the plant pathogenic fungus C.liriopes.ClPV1 genome has two dsRNA segments,named as dsRNA1 and dsRNA2,which in the lengths of 1807 and 1706 bp,respectively,and each contained an open reading frame(ORF).The ORF1 of ClPV1 was predicted to encode a Rd Rp comprised of 571 aa and the ORF2 was predicted to encode CP comprised of 498 aa.The Rd Rp and CP of ClPV1 had 65.21% and 50.10% aa identity with the Metarhizium brunneum partitivirus 1.The Rd Rp-based phylogenetic tree showed that ClPV1 might be affiliated to the new putative genus Epsilonpartitivirus in the family Partitiviridae.The viral particles of ClPV1 were isometric in shape,with an average diameter of 35 nm.The virus-free strain CL-B-2-P1 was obtained by eliminating the virus in Cl-B-2.Compared with Cl-B-2-P1,the conidial production and pathogenicity of Cl-B-2 were significantly reduced.In addition,we transfected the purified virus particles of ClPV1 into a virus-free,wild type C.liriopes strain SJM3-2 by a PEG-mediated protoplast transfection method.An isogenic fungal strain of SJM3-2,SJM3-2-PT5,that was infected by ClPV1 also showed a reduced virulence when compared to SJM3-2,indicating that ClPV1 could induce hypovirulence on the C.liriopes fungus.(3)We report a novel dsRNA virus designated Colletotrichum alienum partitivirus 1(CaPV1)that was isolated from the C.alienum strain YC-31.CaPV1 genome has two dsRNA segments,named as dsRNA 1 and dsRNA 2,which in the lengths of 1770 and 1651 bp,respectively,and encoded the Rd Rp and CP,respectively.The Rd Rp and CP of CaPV1 had88.31% and 70.85% aa identity with the Metarhizium Brunneum Partitivirus 2 and Colletotrichum Partitivirus 1,respectively.Phylogenetic analysis showed that CaPV1 might be affiliated to Gammapartitivirus genus in the family Partitiviridae.Virus elimination,virus transfection with purified viral particles and biological comparison revealed that CaPV1 could reduce the virulence of C.alienum.It is noteworthy that CaPV1 affected host virulence on the one hand,but on the other hand,it has the ability to enhance conidial and pigment production in the host,which is significantly different from other hypovirulence-associated mycoviruses.CaPV1 impacted the conidial germination and appressorium formation rate of strain YC-31,and some conidial were abnormal and appressorium could not form melanin pigmentation as well as that the invasive mycelia could not be produced in the epidermal invasion test of onion.It was found that CaPV1 reduced host sensitivity of the cell wall integrity,oxidative stress,endoplasmic reticulum stress and andazoxystrobin sensitivity,and increased sensitivity of osmotic stress,carbendazim and prochloraz sensitivity.These results indicated that CaPV1 affected the sensitivity of C.alienum to different kinds of fungicide and external stress.CaPV1 could also infect C.fructicola,C.spaethianum and C.gloeosporioides and induce hypovirulence by transfecting of protoplasts with viral particles,thus extending the host range of CaPV1.(4)The molecular mechanism of hypovirulence caused by CaPV1 were elucidated and the key pathogenic genes and signaling pathways of Colletotrichum were screened by CaPV1-host interaction.By comparative transcriptome and proteome,the changes of gene expression,signaling pathway and metabolic pathway that were affected by CaPV1 were analyzed.From the selective analysis and q RT-PCR verification,we found that the vesicular transport,endocytosis,autophagy,pentose,glucuronic acid conversion and protein-Ofucosyltransferase 1(POFUT1)pathway and gene expression were significantly changed.The host endocytosis were affected by CaPV1 which was observed by staining with lipid affinity dye FM4-64.The key gene CaRab7 that differentially expressed in endocytosis was knocked out,which resulted to impaired growth and pathogenicity,preliminarily suggesting that CaPV1 regulated host pathogenicity by affecting host endocytosis.Based on the above virus-host interaction system,the candidate pathogenesis-related proteins including Rab protein CfRab7,polygalacturonase CfEpg1 and CfPOFUT1 were further screened.(5)The function of candidate pathogenesis-related gene CfRab7 in the growth and pathogenicity of C.fructicola was analyzed.Bioinformatics analysis showed that the full length of the CfRab7 gene was 981 bp,contained 205 aa and possessed the unique domain of Ras superfamily and Rab family proteins,which was evolutionally conserved in Colletotrichum.Knockout and complementation of CfRab7 showed that CfRab7 deletion mutants were serious affected in colony growth,conidial production,appressorium formation,appressorium turgor pressure and pathogenicity.The external stress sensitivity tests showed that CfRab7 was involved in the regulation of cell wall integrity,osmotic stress,oxidative stress and endoplasmic reticulum stress.Conidial germination and onion epidermal invasion tests showed that the appressorium formation rate and turgor pressure of the CfRab7 deletion mutants were significantly reduced,and primary and secondary hyphae could not be formed in the infection process,thus they could not infect host plants,resulting in the basic loss of pathogenicity.(6)The function of candidate pathogenesis-related gene CfEpg1 in the growth and pathogenicity of C.fructicola was analyzed.Bioinformatics analysis showed that CfEpg1 gene was 1173 bp in length and encoded 363 aa,which had a Glyco＿hydro＿28 domain.The1-17 aa was the signal peptide region of CfEpg1 gene which has no transmembrane region present,the typical characteristics of secreting protein.Knockout and complementation of CfEpg1 showed that they have no significant difference in colony growth and conidial production between CfEpg1 deletion mutants and the wild type.However but some appressorium of the mutants could not melanin pigmentation and turgor pressure decreased,resulting in decrease of pathogenicity.The external stress sensitivity tests showed that CfEpg1 was involved in regulating the cell wall integrity and oxidative stress process of C.fructicola.Subcellular localization of CfEpg1 showed thst it was localized to the nucleus and membrane in Nicotiana benthamiana.(7)The function of candidate pathogenesis-related gene CfPOFUT1 in the growth,pathogenicity and fungicide sensitivity of C.fructicola was analyzed.CfPOFUT1 gene was1452 bp in length and encoded 484 aa,which had a O-Fuc T＿like domain.In order to study the function of CfPOFUT1,the silencing and overexpression mutants of CfPOFUT1 gene were successfully obtained.The conidial production and pathogenicity of overexpression mutant strains OCfOFUT1 were remarkably increased compared to the wild-type strain LY5-1,less sensitive to azoxystrobin but more sensitive to carbendazim and prochloraz.The conidial production and pathogenicity of silencing mutant strains Si CfOFUT1 were reduced compared to the wild-type strain LY5-1,more sensitive to cell wall integrity and ER stress,more sensitive to azoxystrobin but less sensitive to carbendazim and prochloraz.CfPOFUT1 was involved in conidial production,pathogenicity,cell wall integrity,ER stress and fungicide sensitivity of C.fructicola.In conclusion,this study was carried out for virus bio-control factors screening for Colletotrichum spp.Two hypovirulence-associated partitivirus were identified,among which the mechanism of hypovirulence of one virus was preliminary elucidated.Moreover,three key pathogenesis-related genes in Colletotrichum were screened,by using the mycovirus-host interaction system,and functional identified.This study provided new biocontrol resource for control of anthracnose,promoted the elucidation of pathogenic mechanism of Colletotrichum,and also provided a theoretical basis for searching for effective control agent targets of Colletotrichum.