The Structural And Functional Analysis Of StCHS6 Gene In Setosphaeria Turcica

Our previous study showed that there were 8 chitin synthase genes（StCHS1-StCHS8）in the Setosphaeria turcica.However,the expression pattern and function of each gene were not clear.Based on the previous study,RNA-Seq technology was used to analyze the expression patterns of the above chitin synthase genes in five typical developmental stages of S.turcica,and the structure and function of the highly expressed gene StCHS6 were further analyzed to define the roles the gene played in the growth,development and pathogenesis in S.turcica.The main findings were as follows:1.RNA-Seq data analysis of 5 typical developmental stages of hypha,conidium,germ tube,appressorium and penetration peg in S.turcica showed that StCHS1,StCHS2,StCHS5 and StCHS6 had higher expression levels at the above five periods,and StCHS3,StCHS4,StCHS7 and StCHS8 had lower expression levels（FPKM<1）or no expression.It was worth noting that StCHS6 had the highest expression at all the above five periods,indicating that StCHS6 might play important roles in the growth,development and infection process of S.turcica.2.StCHS6 was located at the scaffold₁:2066293-2069876（-）of the genome of S.turcica.The full length of the gene was 3584 bp and the size of the CDS sequence was2703 bp.StCHS6 contained 4 introns and 5 exons.The combination of two adjacent intron phases were（1–2）,（2–2）and（2–1）.The StCHS6 protein was localized on the cell membrane and contained the typical chitin synthase catalytic domain Chitin_synth₁（682aa-694aa）and multiple transmembrane domains.Its molecular formula was C₄₅₈₆H₆₉₈₅N₁₂₀₃O₁₃₀₉S₃₀,which encoded 900 amino acid residues.The relative molecular mass,isoelectric point,fat index,average hydrophilicity and the instability index were100877.98Da,8.36,81.86,-0.178,and 36.94,respectively.In the StCHS6 protein,random coils,alpha-helix,extended strands and beta-turns accounted for 43.56%,38.44%,13.44%,and 4.56%,respectively.3.Two StCHS6 knockout mutants were obtained.The StCHS6 knockout vector was successfully constructed by cloning and ligating the upstream,downstream fragments of StCHS6 gene and the Bar resistance gene.Based on the principle of homologous recombination,the StCHS6 gene knockout transformants were obtained by Agrobacterium-mediated transformation.The transformants were verified by Bar resistance screening,Bar sequence amplification,specific primer amplification,and RT-PCR.It was finally confirmed that two StCHS6 knockout mutants were obtained and named asΔStCHS6-1,ΔStCHS6-2,respectively.4.StCHS6 gene was involved in the growth and development of the S.turcica.Compared with the wild type strain,the colony morphology of StCHS6 knockout mutants became lighter in color,the mycelium was more dense,the growth rate of colonies were slowed down,the length of hyphal cell was shortened and no conidia were found.5.StCHS6 gene was involved in the synthesis of the cell wall in S.turcica.Compared with the wild type strain,the StCHS6 gene knockout mutants had different sensitivities to Congo red,SDS and CFW.The mutant strains enhanced their tolerance to100μg/mL Congo red;the mutant strains were more sensitive to the 20μg/mL CFW.Compared with the wt strain,the chitin content of the hypha in the two StCHS6 gene knockout mutants increased by 1.47 and 1.44 times,respectively.The chitin distribution in the hypha of the StCHS6 knockout mutants were observed by fluorescence microscopy,and obvious chitin accumulation was found at the hypha tip of the mutant strains,which suggested that the deletion of StCHS6 gene may affect the assembly of chitin with other components in the cell wall of the mycelial tip.6.StCHS6 gene was involved in the pathogenic process of the S.turcica.Although the StCHS6 gene knockout mutants had the ability to penetrate cellophane,their penetration ability were weaker than wt strain.Compared with the wt strain,the process of appressorium induced by hypha were delayed in StCHS6 gene knockout mutants.The StCHS6 gene knockout mutants could only cause limited necrosis in intact leaves,and the pathogenicity of StCHS6 knockout mutants decreased to a certain extent in the stabbed leaves.7.StCHS7 and StCHS8 have complementary effects on the function of StCHS6.In△StCHS6-1,the expression levels of StCHS7 and StCHS8 were increased by 3 times and2.23 times,respectively.In△StCHS6-2,the expression levels of StCHS7 and StCHS8increased 3.98 times and 2.64 times,respectively.