Functional Analysis Of Transcription Factor CsATF1 And Screening Of Its Interacting Proteins In Colletotrichum Siamense

Hevea brasiliensis is a tropical crop and the main source of natural rubber.Anthracnose is one of the important leaf diseases of rubber tree.Colletotrichum siamense is the main pathogen of anthracnose on rubber trees in China.Basic region-leucine zipper（b ZIP）is one of the eukaryotic transcription factor family.It modulates gene expression to regulate physiological processes,such as biotic and abiotic stress response,development and other physiological processes.The HOG MAPK（High Osmolarity Glycerol Mitogen-Activated Protein Kinase）signal pathway is necessary for fungi to respond to various stress responses such as osmotic pressure.This pathway is closely related to the sensitivity of Colletotrichum species to pyrrole fungicides such as fludioxonil,but its regulatory mechanism is unclear.Previously.We screened a number of genes potentially regulated by Cs PBS2 of HOG MAPK.The expression of transcription factor Cs ATF1 was significantly down-regulated.ATF1functions downstream in HOG MAPK and play an important role in response to HOG MAPK signaling pathway.Therefore,our study investigated whether Cs ATF1 participates in regulating the sensitivity to external stressand pyrrole fungicides such as fludioxonil.The main findings are as follows:1、Cs ATF1 was cloned（accession number:MT084770）with 1758 bp DNA length,2introns and 1611 bp c DNA length,encoding a protein that consists of 536 amino acids,3 Aft1domains and a BRLZ（basic leucine zipper）domain,.The cluster analysis showed that Cs ATF1and Cfb ZIP genes of Colletotrichum fructicola are clustered into one class.2、The interaction between Cs ATF1 and Cs Hog1 was verified by yeast two hybrid system.And the expression change between Cs ATF1 and the key genes of the HOG MAPK pathway,Cs PBS2 and Cs HOG1 after treatment with fludioxonil were tested by q RT-PCR.The results showed that,compared with the control,the expression of Cs PBS2 and Cs HOG1 was significantly up-regulated and Cs ATF1 was significantly down-regulated after treatment with fludioxonil（50 mg/m L）;The results show that the expression of Cs ATF1,Cs PBS2,and Cs HOG1 is affected by fludioxonil.3、The Cs ATF1 gene deletion mutantΔCs ATF1-27 was obtained by homologous recombination technology.Phenotypic analysis showed that,There was no significant difference in the morphology of the mutantΔCs ATF1-27 colonies,and the colony diameter became extremely small compared to the wild type;The length of the conidia is slightly shortened（92.74%of the wild type）.The Cs ATF1 gene has a significant effect on the response to salt stress,and the mutant is significantly more sensitive to Congo red than the wild.Cs ATF1 affects the sensitivity of rubber tree anthracnose fludioxonil,with the increase of fludioxonil concentration,the lower the sensitivity of mutants to fludioxonil.The pathogenicity assay showed that the gene deletion mutant could form lesions,but the lesion area was reduced 62%compared to that of the wild type.These results suggest that Cs ATF1 is involved in the formation of Colletotrichum conidia morphology,cell wall integrity,stress response to fludioxonil and other pathogenic functions.Subcellular localization results showed that the Cs ATF1 gene was localized in the nucleus in both mycelia and conidia.4、Using Cs ATF1 as the bait,15 positive clones were screened by performing yeast two-hybrid,they are mucoidy inhibitor-like protein,hydrophobin 2,methyltransferase,cell wall protein Phi A,alcohol dehydrogenase,zinc-containing,putative,developmentally Regulated MAPK Interacting protein,autophagy-related protein 2,mitochondrial hypoxia responsive domain containing protein,sun domain-containing protein,c-4 methylsterol oxidase c-4 methylsterol and 5 putative proteins.Co-IP technology was used to further confirm the interaction between Cs HIG and Cs ATF1 in vivo.5、The Change in expression between Cs ATF1 and the interaction protein Cs HIG encoding gene Cs HIG was analyzed using q RT-PCR technology after treatment with fludioxonil,H₂O₂,Na Cl and sorbitol to cause anthracnose stress.However,the expression level of Cs HIG was only affected by Na Cl and sorbitol.Na Cl significantly increased the expression of Cs HIG gene,and sorbitol significantly reduced it.The results of the study suggest that the protein Cs HIG interacts with the transcription factor Cs ATF1 and participates in osmotic stress responses elicited by Na Cl and sorbitol,and does not affect the sensitivity to fludioxonil and oxidative stress.