Genomic Sequencing Of Trichothecium Roseum And The Regulation Of TrPacC And TrNox On Its Growth,Development And Pathogenicity

Trichothecium roseum is an important postharvest pathogenic fungus that could infect apple,muskmelon,tomato and other fruits and vegetables.In addition to causing postharvest losses and quality deterioration,T.roseum also produces toxins in the host,creating a potential safety hazard.In this study,the basic biological characteristics of T.roseum were first observed.Based on the whole genome sequencing of the pathogen,the p H signaling pathway transcription factor PacC and the key enzyme NADPH oxidase for reactive oxygen production were selected for bioinformatics analysis.By constructing deletion mutants of Tr PacC,Tr Nox A and Tr Nox B(ΔPacC,ΔNox A andΔNox B),gene function verification and transcriptome analysis were carried out to clarify the regulatory mechanism of Tr PacC,Tr Nox A and Tr Nox B on the growth and development,stress and pathogenicity of T.roseum.The main results are as follows:1.The growth cycle of T.roseum mainly goes through four stages:spore germination,germ tube elongation,mycelial growth and sporulation.Spores germinate after 2.5～3 h of culture,and can sprout at any part.Spores are produced after 44 h,and the size of spores is about 15～20μm.The optimum growth temperature of T.roseum is 25～28℃,the optimum p H is 7,the optimum carbon source is glucose,the utilization rate of nitrate is high,and the dark condition is conducive to the growth of mycelium.The lethal temperature of hyphae was 5°C for10 min,and the lethal temperature of spores was 58°C for 10 min.2.The whole genome size of T.roseum is about 34.4 Mb,and the GC content is53.4%.T.roseum contains 10421 coding genes,GC content is 59.04%,the maximum length is 52224 bp,the minimum length is 120 bp,of which 92.24%are in the range of 0～3000 bp.A total of 9769 genes were annotated,among which 9679,6084,4514and 3728 genes were annotated in Nr,Swissprot,KOG and KEGG databases,respectively.In addition,2884,1774 and 1455 genes were annotated in the pathogen-host interaction database,fungal virulence factors database and carbohydrate-active enzymes database,respectively.3.The full length of Tr PacC gene is 1886 bp,encoding 595 amino acids,containing three highly conserved zinc finger domains.The promoter region contains two core promoter elements,four enhancer elements and three DNA binding sites.The protein encoded by Tr PacC is unstable,does not contain transmembrane helix,and has strong hydrophilicity,which is closely related to Fusarium graminearum.The full length of Tr Nox A and Tr Nox B genes were 1796 bp and 1784 bp,encoding 539and 571 amino acids,respectively.The protein encoded by Tr Nox A is an unstable protein,containing three transmembrane helixes,which has strong hydrophilicity and close relationship with F.oxysporum.The protein encoded by Tr Nox B is a stable protein with seven transmembrane helices,which has weak hydrophilicity and close relationship with Acremonium chrysogenum.4.Acidic environment inhibited the colony growth of T.roseum,but induced the expression of Tr PacC.At p H 5,the expression of Tr PacC was the highest.TheΔPacC weakened the unique‘ring structure’of the colony,resulting in edge depression,smaller spores and sparse hyphae.The expression of sporulation-related genes and chitinase genes was significantly down-regulated,and colony growth,biomass,sporulation and spore germination rate were inhibited to varying degrees.Tr PacC is involved in the response to ion,oxidation and cell wall integrity stress,and is most sensitive to ion stress.The expression of ammonia synthesis and transport-related genes inΔPacC was significantly down-regulated,resulting in reduced ammonia accumulation and weakened alkaline ability to environmental p H.In addition,ΔPacC also inhibited the expression of extracellular enzyme genes,reduced the level of intracellular reactive oxygen species,and significantly weakened the pathogenicity to muskmelon,tomato and apple fruits.Transcriptomics results showed that a total of 564 differentially expressed genes were screened inΔPacC,338up-regulated and 226 down-regulated,among which some genes related to maintaining acid-base balance,Na~+transport,cell wall synthesis and hydrolase were significantly down-regulated.5.The colony growth ofΔNox A andΔNox B was inhibited,the spores became smaller,the spore germination was delayed,and the sporulation was reduced.The expression of sporulation-related genes and chitinase genes was significantly down-regulated.At the same time,Tr Nox A and Tr Nox B are also involved in the response to oxidation,ion and cell wall integrity stress.ΔNox A andΔNox B inhibited the level of reactive oxygen species,reduced antioxidant capacity,down-regulated the expression of extracellular enzyme genes and Tr PacC,resulting in a significant decrease in the pathogenicity of melon,tomato and apple fruits.The transcriptome results showed that 2439 differentially expressed genes were screened inΔNox A,of which 1169 were up-regulated and 1270 were down-regulated.A total of 1653 genes were differentially expressed inΔNox B,of which 876 were up-regulated and 777 were down-regulated.Some genes related to growth and development,rhythm,signal transduction,oxidative stress and secondary metabolite synthesis inΔNox A andΔNox B were significantly down-regulated.To sum up the above,the whole genome of T.roseum is about 34.4 Mb,containing 10421 coding genes,with a maximum length of 52224 bp and a minimum length of 120 bp.Tr PacC plays an important role in the process of T.roseum alkalization environment p H.In addition,Tr PacC is also involved in growth and development,response to different stresses,especially ionic stress,and regulation of pathogenicity.Similarly,Tr Nox A and Tr Nox B are also involved in the growth,development and stress response of T.roseum,in which Tr Nox A is crucial for sporulation.In addition,Tr Nox A and Tr Nox B also participate in the pathogenesis of T.roseum by regulating redox balance,extracellular enzyme gene expression and p H signaling pathway.