Study On The Function Of Transcription Factor SsGCN4 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum(Lib.)de Bary is one of the most devastating soilborn fungal pathogens,infecting hundreds of species of host plants worldwide.Ascospores discharged during sexual reproduction can cause the widespread prevalence of sclerotinia diseases.In this study,we found a gene that may be involved in the regulation of sexual reproduction,named Ssgcn4.The encoding protein SsGCN4 belongs to bZIP family transcription factors and is an unstable hydrophilic protein.In this study,the Ssgcn4 knockout mutant was constructed by homologous recombination strategy.The homozygous Ssgcn4 knockout mutant was successfully obtained with the methods of carpogenic germination and single ascospore purification(ΔSsgcn4-1 and ΔSsgcn4-2),and Ssgcn4 complementation transformant(Ssgcn4-C)was also successfully constructed by Agrobacterium tumefaciens transfection.Biological phenotype experiments were carried out on the obtained strains in the aspects of mycelial growth,sclerotia development,apothecia and ascospores formation,oxalic acid production capacity,pathogenicity and abiotic stress.The results showed that the deletion of Ssgcn4 would slow down the mycelial growth rate and produce more sclerotia and its dry weight was correspondingly increased,but it did not affect the shape and volume of sclerotia;besides,more aerial hyphae and pigmented compound appressoria with dark color but low density were produced on the surface of hydrophobic slide.The deletion of Ssgcn4 improved the tolerance of S.sclerotiorum to osmotic stress and increased its sensitivity to cell wall inhibitors,but it had no significant effect on ability to produce oxalic acid,oxidative stress response and pathogenicity.Furthermore,the deletion of Ssgcn4 also led to significant changes in the sexual reproduction stage of S.sclerotiorum.Ssgcn4 knockout mutants showed aberrant apothecium morphology and development retardation,and failed to enter the mature stage of apothecia.The top of ascus was abnormally enlarged and ascospores cannot be formed inside.In conclusion,this study clarified that SsGCN4 affects the mycelial growth and development,sclerotia yield and pigmented compound appressoria formation of S.sclerotiorum,revealed that SsGCN4 plays an important role in cell wall integrity and osmotic stress response,and found that SSGCN4 participates in sexual reproduction regulation and plays an vital role in the ascospores germination of S.sclerotiorum,especially the blocking of ascosporogenesis pathway is expected to become an attractive target site for disease control of S.sclerotiorum.