Screening Of Lipid Droplet Coating Protein CsCap20 Interaction Protein And Functional Analysis Of Hydrophobin CsHydr Of Colletotrichum Siamense

Natural rubber is an important strategic material,and its main source is Hevea brasiliensis.Colletotrichum leaf fall disease is an important leaf disease of H.brasiliensis.When it is severely damaged,it will cause leave fall of rubber trees and affect natural rubber production.There are various types of Colletotrichum species in rubber trees,of which C.siamense is the main specie of rubber tree in fields in China.Previous studies have shown that the lipid droplet coating protein CsCap20 of C.siamense affects the formation of lipid droplets in fungus and affects the appressorium turgor and pathogenicity.In this study,the yeast two-hybrid system was used to screen the CsCap20 interaction protein from the C.siamense cDNA library,and verified by Pull down and Co-IP(Co-Immunoprecipitation)technology.At the same time,lost of gene method was used to functional analysis of the hydrophobin CsHydr.The functions of the lipid droplet coating protein CsCap20 interacting protein member and the hydrophobin CsHydr were preliminary understood.It will lay the foundation for further studying the regulation of lipid droplet coating protein and understanding the pathogenic mechanism of Colletotrichum.The main findings are as follows:1.Screening and identification of the interaction proteins of CsCap20.(1)Using CsCap20 as the bait protein,16 positive clones were screened from yeast cDNA library of C.siamense by yeast two-hybrid.The results shows that they were c AMP-dependent protein kinase catalytic subunit,protein kinase,acetate kinase,ferric reductase like transmembrane component,histidine acid phosphatase,hydrophobin,translation initiation factor,glycoproteins,phosphate:h+ symporter,bys1domain-containing protein and 6 putative proteins.(2)The full length of acetic acid kinase coding gene were cloned.The results showed that the acetate kinase CsAck gene contains an intron,the DNA is 1313 bp,the cDNA is1248 bp,and it encodes 415 amino acids,contains an acetate kinase domain,and encodes a gene named CsAck.(3)It was confirmed by Pull down technology that CsCap20 can interact with CsAck in vitro.And the interaction between CsCap20 and the catalytic subunit of protein kinase A(CsPKA-C1),and the interaction between CsCap20 and acetate kinase(CsAck)in vivo was confirmed by Co-IP technology.2.Functional identification of CsHydr of C.siamense(1)The coding gene of hydrophobin was cloned and named CsHydr(accession number: MN166623).The gene contains two introns,the total length of the DNA is 520 bp,the cDNA is 417 bp,and encodes 138 amino acids.The N-terminus of the protein contains a signal peptide consisting of 19 amino acids with a hydrophobic domain HYDRO.Construction of two prokaryotic expression plasmid,PET-32a-CsHydr(including signal peptide)and PET-32a-CsHydr-18aa(removal of signal peptide).Experimentally verified the expression plasmid without the signal peptide can successfully express the hydrophobin,while the prokaryotic expression plasmid that retains the signal peptide cannot express the protein.It is shown that the presence of the hydrophobin signal peptide will affect its prokaryotic expression in E.coli.(2)Obtained a knockout mutant ΔCsHydr-66 by homologous recombination.Observed the phenotype between mutant and wild type,The results showed that the mutantΔCsHydr-66 had a slightly larger colony diameter on the PDA medium;The conidia size of mutant were reduced with 18% shorten in length and 12% in width;The hydrophobicity of hypha surface was decreased significantly;The sensitivity of the mutant to Congo red was significantly higher than that of the wild type;Pathogenicity assay showed that null mutants have significantly reduced pathogenicity.The results show that the hydrophobin CsHydr is involved in the formation of conidia,the hydrophobicity of the hyphae surface,the integrity of the cell wall and the pathogenic function.However,the interaction between hydrophobin and lipid droplet-coated protein needs to be further verified,and the biological function of the interaction between the two proteins still needs further research.