Novel Regulation Modes Of Appressorium Development And Identification Of Appressorium Associated Proteins In Setosphaeria Turcica

Host,environment and pathogen are a community of destiny that interacts,depends on each other,and co-evolves.The survival of pathogenic fungi requires host,and its growth is affected by the environment and host,which is a complex regulatory process.The fungal pathogen Setosphaeria turcica is the hemibiotrophic which causes northern corn leaf blight(NCLB)disease in maize.S.turcica develops a specialized cel,called an appressorium,which generates enormous pressure to breach the leaf cuticle and gain entry into plant tissue,ultimately affecting maize yield.Although there are many studies on its infection process and form.However,how S.turcica establishes host infection is largely unknown.In this study,S.turcica and maize were used as the research object to analyze the molecular mechanism of appressorium development,infection and pathogenicity.The main results were as follows:1.We found that the DNA genotoxic reagents,DNA replication inhibitor hydroxyurea(HU),the alkylating agent methyl methane-sulfonate(MMS)and camptothecin(CPT),could significantly reduce the pathogenicity of S.turcica.It found that the pathogenicity was decreased and the appressorium formation was inhibited when the conidia and hyphae were exposures to genotoxic stress,and the length of germ tube was significantly increased,we speculated that this event may be an active regulation process of pathogens.The S-phase checkpoint inhibitors CAF and HU cooperate to restore appressorium formation,and the expression of the S-phase checkpoint marker gene RNR1 was restored.We speculated that appressorium formation is regulated by the S-phase checkpoint of the cell cycle.In order to clarity the mechanism of appressorium formation,a silent mutant of the StATR of S.turcica was created.The inhibition of appressorium formation was restored in StATR knockdown mutants compared to wild type.These results demonstrate that the central checkpoint kinase StATR is critical for blocking appressorium initiation on genotoxic stress in S.turcica.Appressorium developmental regulation is a new branch of StATR-mediated S-phase checkpoint signaling pathway.2.The content of melanin of S.turcica increased with HU concentration.we found that melanin content could not be significantly increased like wild-type pathogens with HU when StATR-RNAi mutants and S-phase checkpoint inhibitor CAF treatment to detect melanin content.It inferred that the melanin of S.turcica is also regulated by the S-phase checkpoint.The melanin synthesis inhibitor tricyclazole(TCZ)was used to treat S.turcica,the result was that the hyphae were obviously whitish.HU and ultraviolet radiation(UV)combined with TCZ showed that colony growth was significantly inhibited.The quantitative data strongly supported that melanin accumulation is a defence response to genotoxic stress for self-protection.q-PCR analysis of melanin synthase revealed that StPKS and StLAC2 were induced by HU and regulated by the StATR-mediated S-phase checkpoint.The interaction of the ATR regulatory pathway was performed,and we found that StATR downstream kinase StCdsl inactivates StNrm1,and StNrm1 interacts with the transcription factor StMbp1.Yeast one-hybrid and EMSA assays proved StMbp1 bound to the CGCG-box site in-750 bp from the melanin synthase StPKS promter.These results suggest that StATR-induced the synthesis of melanin through the StCds 1-Nrm 1-Mbp1-PKS signaling pathway in response to genotoxic stress,and melanin synthesis is another new branch of the StATR-mediated S-phase checkpoint signaling pathway.3.We next investigated whether the responses activated by S.turcica are evolutionarily conserved among phytopathogenic fungi.We selected four fungal pathogens for a simple test,including Cochliobolus heterostrophus,Cochliobolus carbonum,Alternaria solani,and Alternaria kikuchiana.Appressorium initiation was significantly inhibited in the presence of HU in all species,and HU caused dramatic accumulation of melanin in all the indicated species.Taken together,these observations suggest that the inhibition of appressorium formation and melanin accumulation in the presence of genotoxic stress are universal responses in a wide range of phytopathogenic fungi.4.By analyzing the transcriptome of S.turcica with HU,a series of autophagy genes and secondary metabolites were found to respond to genotoxic stress.Yeast two-hybrid and q-PCR assays proved that autophagy genes(StATG7,StATG16,et al.)were regulated by ATR-mediated S-phase checkpoints,and autophagy acts as another self-protection.In conclusion,S.turcica activate an ATR-dependent pathway to suppress appressorium-mediated infection and induce melanin-related and autophagy to self-protection in response to environmental stress.5.In this study,we report several novel factors contributing to S.turcica pathogenicity,identified using a genomic and transcriptional screen at different stages of S.turcica appressorium development.We identified two cytoskeleton regulators,SLM1 and SLM2,which are crucial for hypha and appressorium development.The SLM1 and SLM2 transcripts accumulated during germling stage but their levels were notably reduced at the appressorium stage.Deletion of SLM2 dramatically affected cell morphology,penetration ability,and pathogenicity.We also identified three different types of S.turcica glycosyl hydrolases(GH12,GH28 and GH74)that are critical for plant cell wall degradation.Their transcripts accumulated during the appressorium infection stage induced by cellophane and maize leaf.Most importantly,we characterized a novel and specific S.turcica effector,appressorium-coupled effector 1(StACE1),whose expression is coupled to appressorium formation in S.turcica.This protein is required for maize infection and induces cell death on expression in Nicotiana benthamiana.As well as several other genes with unknown functions,whose expression patterns indicated that they might be candidate factors essential for S.turcica.These observations suggest that the phytopathogen S.turcica is primed in advance with multiple strategies for maize infection,which are coupled to appressorium formation at the early infection stages.6.In order to further explore the mechanism between maize and StACE1 of S.turcica.StACE1 was mainly localized in the nucleus by subcellular localization.Multiple interacting proteins were obtained by yeast two-hybrid maize cDNA library,including two transcription factors ZmTIFY-1OB and ZmbHLH93.ZmbHLH93 has two conserved domains,bHLH(basic helix-loop-helix)and ACT(aspartokinase,chorismate,and TyrA)by bioinformatics analysis.And StACE1 interacted specifically with the ACT domain,but not bHLH domain.Moreover,ZmbHLH93 itself forms a dimer,and the bHLH and ACT domains was not interact,but the bHLH and ACT domains can interact with itself in ZmbHLH93 dimers.The biological effect mechanism of StACE1 in the host remains to be further clarified.In conclusion,this study demonstrates a new mode of ATR-mediated S-phase checkpoint regulation of S.turcica.We showed that in response to genotoxic stress,StATR inhibits appressorium development,promotes melanin synthesis,and regulates a series of new modes such as autophagy and secondary metabolism.And we identified a series of key proteins of appressorium development,which play a role in cytoskeleton regulation,plant cell wall degradation and the interaction of key host proteins.The results of the study deepened our understanding of the appressorium development and related regulatory mechanisms of S.turcica,provide new ideas for the subsequent study of the mechanism of interaction between phytopathogenic fungi and hosts,and also lay a theoretical foundation for the prevention and control of pathogenic fungi.