Screening And Identification Of The Proteins Interacting With Hog1 And Pbs2 In Colletotrichum Siamense From Rubber Tree

Natural rubber is strategic materials in China and is important to China’s economy and national defense construction.One of the major causes of rubber yield loss is Colletotrichum leaf diseae(CLD).Colletotrichum leaf diseae(CLD)can cause server rubber yield loss.The Colletotrichum gloeosporioides species complex and Colletotrichum acutatum species complex were the main pathogens of the CLD,and C.siamense has been reported recently as the main specie among the Colletotrichum gloeosporioides species complex in China.In previous study,it was confimed that the(HOG,high osmolarity glycerol response)response pathway consisting of the Ssk2/Ssk22-Pbs2-Hogl MAP kinase(Mitogen-Activated Protein Kinase)cascade is important for survival under hyperosmotic conditions in filamentous fungus,and it is involved in Colletotrichum resistance to the fungicide fludioxoni,but it is unclear how Colletotrichum regulate fludioxonil resistance though HOG MAPK pathway.In this study,Screening and identification of the proteins interacting with Hogl and Pbs2 in Colletotrichum siamense from Rubber tree through yeast two hybrid technology,His Pull-down technique and real-time fluorescence quantitative PCR technology.This study provides a basis for the subsequent analysis of these proteins involved in those functions,especially pathogenicity and drug resistance.On the other hand,it also provides a basis for further analysis of HOG MAPK signal path network members of C.siamense.The results of this study are as follows:1.A cDNA library of C.siamense was constructed in Colletotrichum siamense from Rubber tree.The recombination rate of this library was 83.3%,mainly distributed in 400-2000 bp,the average titer of library were 6.52x105 cfu/mL.The subsequent yeast two hybrids can be carried out.2.CsPbs2 and CsHogl gene of C.siamense strain HN08 were cloned.The results of the sequences analysis showed that the gene of CsHogl DNA sequence is 1383 bp(Accession No:MG887856),cDNA sequence is 1080 bp(Accession No:MG887857),contained 5 introns and encoded 459 amino acids,CsPbs2 gene DNA sequence is 2051 bp(Accession No:AMA 19670.1),cDNA sequence is 2004 bp(Accession No:AMA19670.1)contained 1 introns and encoded 667 amino acids,and both them have serine/threonine protein kinase catalytic domains of S_TKc.3.Cspbs2 and CsHogl bait vector was constructed to screening the cDNA library of C.siamense.There are 31 proteins interacting with CsPbs2.They mainly are cytochrome P450,cytochrome b5,histone H3,ubiqui tin-activating enzyme E1,glyceraldehyde-3-phosphate,SSK1 protein and so on.There are 33 proteins interacting with CsHogl.They mainly are cell wall protein PhiA,beta-glucosidase,glyoxal oxidase precursor,GP5 envelope protein and so on.There are 6 proteins both interacting with CsHogl and Cspbs2.They are bysl domain-containing protein,Threonine dehydrogenase,beta-glucosidase,methyltransferase,hypothetical protein and unknown protein.4.The interaction between cytochrome P450(CsP450),cytochrome b5(Cscytb5)and CsPbs2 protein was further confirmed by His Pull down.5.The effect of fludioxonil on the expression of CsP450、Cscytb5、Cspbs2 and CsHogl were analyzed using real-time fluorescence quantitative PCR.Compared with the control group,the expression of Cspbs2 and CsHogl(2 h,4 h and 8 h after treatment with fludioxonil)were significantly up-regulated.The expression of Cscytb5(4 h and 8 h after treatment with fludioxonil)and CsP450(4 h and 8 h after treatment with fludioxonil)gene were significantly down-regulated.The results showed that the expression of CsPbs2、CsHog1、Cscytb5 and CsP450 were affected by fludioxonil.