Functional Research Of GATA Transcription Factors SsAREA And SsSRE In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum is a typical broad host range necrotrophic pathogenic fungus,which has caused great losses to agricultural production.S.sclerotiorum has complex infection cycle and various morphological differentiation,such as apothecium,sclerotia,hyphae,infection cushion,etc.The normal formation of these morphological structures is important for S.sclerotiorum to complete the infection process and resist the harsh environment.Sclerotia is a specialized dormant structure of hyphae after suffering from adversity stress,which can survive in soil for many years and bring great difficulties to agricultural control.Infection cushion is an infection structure used to penetrate the cuticle of host,and it is a powerful weapon for S.sclerotiorum to cause diseases.There have been some reports on the formation and development mechanism of sclerotia and infection cushion,but it is far from clear.GATA transcription factor specifically binds to [AT]GATA[AG] DNA motif by using zinc finger domain,and is reported to be involved in morphogenesis,secondary metabolism,nutritional stress and virulence in filamentous fungi.Nine GATA transcription factors have been identified in S.sclerotiorum,among which many reports are involved in the growth and development,morphological transformation and pathogenicity.We focus on the effects of two classical GATA transcription factors,SsAREA and SsSRE,on nutritional stress,growth and development,sclerotia formation,infection cushion level and pathogenicity of S.sclerotiorum.SsAREA and its homologous protein are typical regulators of nitrogen metabolism,which positively regulate the expression of nitrogen utilization genes in response to glutamine concentration.In the early stage of our laboratory,the expression of SsareA was down-regulated by RNAi,and the growth of the strain was extremely slow and the utilization rate of nitrogen source decreased.To further clarify the function of SsareA,this research overexpressed SsareA in S.sclerotiorum,and analyzed the phenotypic changes of overexpressed strains.The results showed that compared with wild type,the growth rate of over-expressed strain decreased when nutrition was sufficient,but increased when nutrition was deficient,especially nitrogen deficiency.The dry weight of sclerotia decreased,melanin content decreased,and endogenous ROS production decreased.With the decrease of infection cushion level,the pathogenicity to susceptible hosts decreased,while the pathogenicity to resistant hosts was equal to that of wild type.SsSRE and its homologous are siderophore biosynthesis repressor.After Sssre expression was down-regulated by RNAi,the strain could not form sclerotia and infection cushion at all,and the endogenous ROS production increased,which made it more sensitive to oxidative stress and completely lost its pathogenicity.In order to further clarify the function of Sssre,we obtained a strain that overexpressed SsSRE.The results showed that the growth rate of the overexpressed strain decreased,the endogenous ROS decreased,the expression of Sscat was significantly up-regulated and the antioxidant capacity increased.The level of infection cushion increased significantly,and the pathogenicity to susceptible hosts decreased,while the pathogenicity to resistant hosts increased significantly,especially at the early stage of infection and to young leaves.Previous research have found that SsSRE interacts with SsMCM1,a transcription factor downstream of MAPK pathway.This research further clarified that the zinc finger domain of SsSRE is necessary for interaction through site-directed mutation and truncation.In addition,a strain overexpressing the fusion protein of SsMCM1 and GFP was obtained,the results showed that the infection cushion level of the overexpressed strain decreased significantly and the pathogenicity decreased.While SsSTE12 is the common interactive protein of SsAREA and SsSRE.In this research,Ssste12 was overexpressed in S.sclerotiorum,and it was found that the production of sclerotia was slightly decreased,and the infection cushion level and early pathogenicity were up-regulated.In this study,GATA transcription factors SsAREA and SsSRE and two interacting proteins SsMCM1 and SsSTE12 were overexpressed in S.sclerotiorum,respectively,which clarified the function of these transcription factors in regulating the growth,morphogenesis and pathogenicity of S.sclerotiorum,and preliminarily revealed the relationship between GATA transcription factors and MAPK pathway.