Functional Analysis Of CsSCS7 And Screening Of Its Interacting Proteins In Colletotrichum Siamense

Colletotrichum genus is an important type of plant pathogen,and it has many species and a wide range of hosts.Among them,C.siamense is the dominant species of Colletotrichum leaf fall disease in China,and it is also the main pathogen of anthracnose in many other tropical crops.HOG MAPK(High-Osmolarity Glycerol Mitogen-Activated Protein Kinase)signal pathway plays an important role in osmotic stress,growth and development,pathogenicity and fungicide sensitivity.Our previous studies have shown that the HOG MAPK pathway member Cs PBS2 is involved in the regulation of the fungicide fludioxioil sensitivity of C.siamense.In order to understand how fungi depend on this signal pathway to regulates the fungicide sensitivity,CsSCS7,one of the interaction proteins of a key components(Cs PBS2)of the HOG MAPK pathway,was screened in a c DNA library of C.siamense by yeast two-hybrid(Y2H)system.Its homologue is SCS7,a fatty acid 2-hydroxylase,the key enzyme of the synthesis of monohydroxylated inositolphosphateceramide of Saccharomyces cerevisiae,and it had been confirmed that it is related to the regulation of drug sensitivity.Therefore,we speculate that Cs PBS2 interacts with CsSCS7 and regulates drug sensitivity in C.siamense.In this study,we further analyzed the function of CsSCS7 gene and screened the candidate interaction proteins of CsSCS7,which will help us to understand the functions of CsSCS7 and whether CsSCS7 is involved in the regulation of drug sensitivity,and also lay a foundation for further analyzing how fungi depend on HOG MAPK signal pathway to regulates the fungicide sensitivity.The results of this study are as follows:1.The protein structure of CsSCS7 from C.siamense were analyzed.The results of the sequences analysis showed that the gene of CsSCS7 DNA sequence was 1271bp and c DNA sequence was 1215 bp,contained 1 intron and encoded 404 amino acids.The protein contained a Cyt b5 domain,a low complexity area,a transmembrane domain,and a hydroxylase domain,it is similar in structure to yeast SCS7 protein.Cluster analysis showed that it was 50%similar to the fatty acid hydroxylase SCS7 in S.cerevisiae,it is a homologous gene of yeast SCS7 gene.2.The transcriptional expression relationship between CsSCS7 gene and two key genes(Cs PBS2 and Cs Hog1)of HOG MAPK pathway were evaluated.The three genes expression levels were analyzed in the wild-type HN08 strain treated with activator anisomycin(2×10^-3?g/m L)and inhibitor SB203580(53?M),and fludioxonil(50?g/m L)for 2 h.The results showed that the m RNA expression levels of Cs PBS2,Cs Hog1 and CsSCS7 genes were 1.89,9.72 and 2.88 fold increased with the activator anisomycin treatment and 0.27,0.25,0.26 fold decreased with inhibitor SB203580 treatment,respectively,compared with those in the control group.And the m RNA expression levels of Cs PBS2,Cs Hog1 and CsSCS7 were 6.46,4.31 and 1.34fold increased with fludioxonil treatment,respectively.The results indicated that the expression of Cs Pbs2,Cs Hog1 and CsSCS7 was cooperative expression under three test conditions.3.The function of CsSCS7 gene were characterized.The gene deletion mutant?CsSCS7 and the complemented mutant?CsSCS7/CsSCS7 were obtained.The phenotype analysis showed that the colony growth rate of mutant?CsSCS7 was significantly reduced,and the colony growth diameter of the mutant?CsSCS7 was only 6.88-18.15%of that of the wild type strain in PDA,CM,MM,and V8 media.The conidia is smaller than that of wild type,its length and width are reduced by 19.63%and 20.83%,respectively,compared with the wild type.the mutant conidia germinate later than that of the wild type,and the germination rate is reduced.The pathogenicity assay showed that both the disease rate and lesion area of mutant were extremely significantly reduced.The sensitivity to fludioxonil and prochloraz is increased because the growth of the mutant was completely inhibited when the concentration of the two kinds of fungicide were 0.1?g/m L but wild type strain and complemented transformant can still grow.The results suggested that CsSCS7 gene is an important growth factor in C.siamense,and it is also involved in the formation of conidia size and gemination,pathogenicity,and drug sensitivity.The content of short-chain fatty acid and mid-long-chain fatty acids in the wild-type strain and the mutant?CsSCS7 were detected by GC-MS technology.The results showed that the content of 17 fatty acids in mid-long-chain fatty acids were significantly reduced in mutant than that of wide-type strain,including Methyl linoleate,Methyl oleate,Methyl palmitate,Methyl stearate,Methyl linolenate,Methyl palmitoleate,Methyl elaidate,Methyl heptadecanoate,Methyl cis-10-heptadecenoate,Methyl myristate,Methyl pentadecanoate,Methyl dodecanoate,Methyl heneicosanoate,Methyl tricosanoate,cis-11,14,17-Eicosatrienoic acid methyl ester,cis-11,14-Eicosadienoic acid methyl ester,Methyl octanoate.The content of acetic acid and isovaleric acid in the short-chain fatty acids were significantly lower,but the content of propionic acid and isobutyric acid was significantly higher than those in the wild-type.This result indicates that the CsSCS7 gene may affect the synthesis of some mid-long-chain fatty acids and short-chain fatty acids.4.The candidate interaction proteins of CsSCS7 protein were screened by the yeast two-hybrid method in C.siamense.The results showed that 35 proteins may interact with CsSCS7,including sphingolipid C4-hydroxylase SUR2,acetate kinase,glucoamylase,sterol 24-C-methyltransferase,cyanate hydratase,enolase,beta-lactamase hydrolase,c-5 sterol desaturase,22kda glycoprotein,hypersensitive response-inducing protein,hydrophobin 2,antigenic thaumatin,translocator protein,1,3-beta-glucan synthase component FKS1,Collagen alpha-5(VI)chain,ATP synthase 9(mitochondrion),Transcription factor atf21,Pyruvate kinase,transcription initiation factor IIB,Stress protein DDR48,Peroxisomal catalase,F-box-like/WD repeat-containing protein TBL1X,dna J heat shock family protein,Rodlet protein,Pyranose dehydrogenase 3,Rho1 guanine nucleotide exchange factor 3,alcohol dehydrogenase and 8 uncharacterized proteins and hypothetical proteins.