Functional Analysis Of Sclerotinia Sclerotiorum Transcription Factor SsFZC1 During Infection

Sclerotinia sclerotiorum（Lib.）de Bary is an important plant pathogenic fungi with worldwide distribution.Sclerotinia stem rot（SSR）which is caused by S.sclerotiorum brought great losses to rape planting industry.S.sclerotiorum has complex virulence factors such as plant cell wall degrading enzymes（PCDEs）,oxalic acid（OA）and effectors,but there are few studies on transcription factors in regulating sclerotia development and pathogenicity of S.sclerotiorum,and the mechanism of it in the growth and development and pathogenic processes of S.sclerotiorum needs to be further explored.In this study,Ssfzc1（SSIG₀7003）was cloned from S.sclerotiorum which is up-regulated during infection,indicating that Ssfzc1 might be involved in the pathogenicity of S.sclerotiorum.Through bioinformatics analysis,it was found that there was a Zn（II）2Cys6 zinc finger domain at its N-terminal,that SsFZC1 is a C6 zinc finger transcription factor.The full-length sequence of Ssfzc1 is 3125 bp and have five exons,including 455 bp for 5’ UTR and 743 bp for 3’ UTR,and the coding sequence（CDS）is 1927 bp.By using the PEGmediated protoplast transformation and agrobacterium tumefaciens mediated transformation,the Ssfzc1 gene knock-out and complement mutants were constructed respectively.The homozygous mutant strain was obtained by subculture of mycelium tip and PCR and RTPCR verification.The mycelial growth rate of Ssfzc1 deletion mutans decreased compared with the wild type strain（WT）,suggesting that SsFZC1 may be involved in the regulation of vegetative growth of S.sclerotiorum;Ssfzc1 mutants had no significant difference in the number of sclerotia formation compared with WT,but the total dry weight of sclerotia decreased by 40%-50%,suggesting that SsFZC1 might be involved in the development and maturation of sclerotia of S.sclerotiorum;It was found that SsFZC1 did not affect the hypersaline hypertonic sensitivity of S.sclerotiorum,but was closely related to the response of S.sclerotiorum to oxidative stress.The ΔSsfzc1 mutant could still successfully infect the host（Brassica napus,Nicotiana benthamiana,Lycopersicon esculentum and Capsicum annuum L）,but its pathogenicity decreased significantly,while its pathogenicity recovered on the leaves of plants with wounds.In addition,the formation time of ΔSsfzc1 mutants’ infection cushions was delayed and the number of infection cushions was reduced,indicating that Ssfzc1 may be involved in the pathogenesis of S.sclerotiorum by regulating the formation and development of infection cushions.SsFZC1 regulates the expression of secreted proteins SsCP1,SsSVP2,SsIEB1 and Ssv263.Transcriptomic sequencing further analyzed and screened 39 candidate effector proteins and genes encoding cell wall degrading enzymes that may be regulated by SsFZC1.It was speculated that SsFZC1 regulates the expression of genes related to cell wall degrading enzymes and secreted proteins,and is involved in the pathogenic process of S.sclerotiorum.Using host-induced gene silencing（HIGS）based on TRV system,targeted silencing of Ssfzc1 reduced the virulence of S.sclerotiorum,suggesting that Ssfzc1 can be used for the prevention and treatment of S.sclerotiorum in vitro.In conclusion,the transcription factor SsFZC1 of S.sclerotiorum was involved in the regulation of vegetative growth,sclerotia development,infection cushion formation and pathogenicity,laying a foundation for further clarifying the molecular mechanism of S.sclerotiorum pathogenicity,and providing a theoretical basis for the screening of the targeted fungicide and creation of SSR resistant germplasm resources.