Functional Study On Transcription Factor ChhapX Of Cochliobolus Heterostrophus

Southern corn leaf blight caused by Cochliobolus heterostrophus is an important maize disease.Currently,the pathogenicity research of C.heterostrophus is mainly focused on the genome of T and O races and molecular mechanism of HMT toxin synthesis.During infection process,C.heterostrophus dealed with a variety of stress responses from itself and the host,and produced toxins to maintain its pathogenicity.Multiple transcription factors were involved in this process.Transcription factor HapX,as one main regulator of iron homeostasis,plays an important role in maintaining virulence of plant pathogenic fungi.The influence of ChhapX on C.heterostrophus iron homeostasis and pathogenicity and whether it used iron regulation system to coordinate its iron balance to respond to iron ions changes are still not clear.In this study,ChappX was used as the research object to establish and optimize the Agrobacterium tumefaciens-mediated transformation system of C.heterostrophus.ChhapX knockout mutants were obatined,and iron homeostasis,development and pathogenicity of the mutants were analysed.to lay the foundation for clarifying C.heterostrophus pathogenic mechanism.The method and results are as follows:1.Gene cloning and physicochemical properties analysis of transcription factor ChhapX of C.heterostrophusThe HapX gene sequence of Aspergillus fumigatus was used as a BLAST query to obtain full sequence of ChhapX gene of C.heterostrophus Bioinformatics analysis showed that ChhapX gene of C.heterostrophus is 1440bp-long and has an open reading frame?ORF?.ChhapX protein consisting of 422 amino acids contains a b ZIP-type domain and is located in nucleus.Phylogenetic tree analysis showed that the ChhapX gene of C.heterostrophus had the closest phylogenetic relationship with HapX of C.lunata.2.Establishment and optimization of Agrobacterium tumefaciens-mediated transformation of C.heterostrophus and acquisition of ?ChhapX strain.The optimal system for Agrobacterium-mediated genetic transformation of C.heterostrophus: C.heterostrophus conidia(105conidia mL^-1)were incubated at 25 ? in the dark for 8 hours.Agrobacterium strain AGL-1 containing binary vector was cultured to f OD₆₀₀ = 0.5 and was mixed with germinating conidia at a ratio of 1: 1.Induction lasted 30 minutes before co-culture was performed for 48 hours.ChhapX knockout vector p PZP100 ChhapX was constructed using overlapping PCR and one step cloning methods and was introduced into Agrobacterium strain AGL-1 by electric shock method.Based on the above optimal system of ATMT,candidate transformants were obtained and DNA was extracted to further verify the transformants.3.Effects of ChhapX on growth,development,stress adaption and pathogenicity of C.heterostrophus under different iron concentrationUnder different iron concentrations,deletion of ChhapX had no effect on C.heterostrophus mycelium growth,but significantly reduced mycelial dry weight with thinnercolonies and increased conidia production.Under sufficient iron and excessive iron conditions,?ChhapX produced less melanin pigments.In the stress adaption assay,ChhapX deletion mutants were more sensitive to CR,SDS and NaCl,but were less sensitiveto H₂O₂ in comparision with the wild type strain under deficient iron and sufficient iron conditions.While and ChhapX deletion strains had increased sensitivity to SDS and decreased sensitivity to CR and NaCl under excessive iron condition,.Besides,maize inbred line B73 was used for pathogenicity testing.Compared with the wild type strain,ChhapX deletion mutants showed significantly reduced pathogenicity.All these data suggested that transcription factor ChhapX affects mycelial growth,conidiation,stress adaption and pathogenicity.4.Transcription regulation of ChhapX on iron homeostasis and oxidative homeostasis related genes under different iron concentrations.The real-time fluorescence quantitative PCR results showed that the transcription level of C.heterostrophus ChappX gene changes with different iron ion concentrations.Under low iron ion concentrations,transcription level of ChhapX was significantly up-regulated.Moreover,ChhapX gene regulated transcriptionallyiron homeostasis-related genes NRPS6,NRPS2,Sid A,mir B and CYCA Under different iron concentrations..Under deficient iron condition,,the expression of Sid A,NRPS6,NRPS2 and CYCA in ChhapX deletion mutants were significantly down-regulated;under sufficient iron conditions Sid A,NRPS6,NRPS2 and mir B genes were transcriptionally down-regulated;under excessive iron condition,.transcription of Sid A,NRPS6 and mir B genes were significantly up-regulated.These data indicates that ChhapX regulate iron absorption and iron consumption genes.ChhapX regulated oxidative homeostasis related genes SOD1,SOD2,CAT1,CAT3,NOX2,NOX4,NOX5,GR and GSS: under deficient iron condition,,the transcriptions of CAT3 and NOX4 in ChhapX deletion mutants were significantly up-regulated;under sufficient iron condition,SOD1 expression is up-regulated but CAT1,CAT3,NOX2,NOX5 and GR were significantly down-regulated;under excessive iron condition,the expressions of SOD1,SOD2,CAT1,NOX2,NOX4 and GSS were down-regulated.These results indicate that ChhapX regulates the oxidative homeostasis of C.heterostrophus.