Function Of Clnox1 And Clnox2 Oxidase Genes In The Pathogenicity Of Curvularia Lunata

Corn Curvularia leaf spot is one of the important leaf diseases in China's main corn producing areas.It was caused by Curvularia lunutaand seriously threatened corn production.In recent years,researchers had studied C.lunuta virulence factors such as toxins,melanin,and cell wall degrading enzymes,but there was no report about the components of NADPH oxidase and the relationship between pathogenicity and NADPH oxidase gene in C.lunuta.In this paper,components of NADPH oxidase was predicted by bioinformatics methods,ClNox1and ClNox2 were the main components of NADPH oxidase in C.lunuta.Transformants were obtained by knockout of target genes.NADPH oxidase gene on the growth,development and pathogenicity of C.lunata was identified through biological properties,stresses,and pathogenicity.The obtained results were as follows:1.Bioinformatics analysis of NADPH oxidase gene of Curvularia lunataIn this paper,based on the genome sequence information of C.lunata CX-3,four NADPH oxidase subunits were found in C.lunata using biological software,namely ClNox1,ClNox2,ClNoxR and ClRac.ClNox1 and ClNox2 are the main components of NADPH oxidase,and ClNoxR and ClRac are the regulatory subunits in this enzyme.There is no Nox C subunit in C.lunata.The physicochemical properties of the protein showed that ClNox1 and ClRac were unstable proteins,but ClNox2 and ClNox R were stable proteins,but the amino acid residues,molecular weight and isoelectric point of ClNox1 and ClNox2 had no difference;the protein hydrophobicity/hydrophilicity was analyzed,all of the above components were hydrophilic proteins;the location of ClNox1 was located in mitochondria,ClNox2 was located in membrane,Cl Nox R and ClRac were located in cytoplasm;the phosphorylation site analysis showed all components could be phosphorylated;functional domain analysis showed that ClNox1 and ClNox2 have similar conserved structure.The ClNoxR protein contains a TPR site that interacts with ClNox1.The ClRac protein sequence has a small GTP protein;protein signal peptide analysis indicated that ClNox are non-secretory protein.2.Cloning and deletion of ClNox1 and ClNox2 of Curvularia lunata by ATMTTo further analyze the roles of the major components of NADPH oxidase ClNox1 and ClNox2 on the growth,development and pathogenicity of C.lunata,single knockout,double knockout and complementary transformant vectors were constructed.The obtained knockout vector was introduced into Agrobacterium tumefaciens AGL-1 by freeze-thaw method,Agrobacterium containing the knockout vector were co-cultured with the conidia of C.lunata by using an improved target gene knockout method.The transformants was screened by antibiotic,transformant DNA was extracted and verified by PCR.ClNox1 and ClNox2 single and double knockouts and complementary transformants were obtained.3.Biological characteristics analysis of Clnox1 and Clnox2 transformants in Curvularia lunataSporulation of?Clnox1,?Clnox2,and?Clnox1nox2 were 3.19×10⁸,3.53×10⁸,and3.24×10⁸,respectively,which were significantly higher than wild-type strains?2.27×10⁸?.When the ClNox2 gene was knockouted,the melanin synthesis of the transformants was reduced,but there was no difference in the Clnox1 transformant compared with the wild type strain.At 4 h,the germination rates of?Clnox2 and?Clnox1nox2 were 83.3%and 82.3%,respectively,compared with the wild type?97%?,the germination rate significantly decreased.At 6 h,the germination rate of?Clnox2 conidia was not significant difference compared with the wild type strain.The results showed that ClNox2 had the role of delaying the germination of conidia,while ClNox1 did not affect the germination.The formation rates of appressoria of?Clnox1,?Clnox2,and?Clnox1nox2 were 85%,77%,and 50%,respectively,the formation rate of appressoria was significantly decreased compared to the wild-type strain?98%?.4.Function analysis of Clnox1 and Clnox2 genes in Curvularia lunata to stress and pathogenicityThe ClNox1 and ClNox2 genes showed negative regulation of mycelial growth in oxidative stress?10 mM H₂O₂?and cell wall integrity experiments?50 mg/L Congo Red and0.05%?w/v?SDS?,The growth rate of?Clnox1,?Clnox2 and?Clnox1nox2 was faster than the wild-type.The growth rates of?Clnox1,?Clnox2,and?Clnox1nox2 in the osmotic stress experiments?1 M NaCl?had no difference from wild-type and complementary transformants,suggested that the ClNox1 and ClNox2 genes are not involved in regulation osmotic stress.The results of NBT staining showed that the ROS in?Clnox1,?Clnox2 and?Clnox1nox2 was significantly lower than wild-type strains,indicated that ClNox1 and ClNox2 are the major sources of ROS in C.lunata.Wild type strains and transformants were used to inoculate self-bred corn Huangzao-4 and observed that the disease index of?Clnox1,?Clnox2,and?Clnox1nox2 decreased compared with the wild type,but typical lesions were formed.