The Study Of Interaction Between Catalytic Subunits Of Protein Kinase A And CgCap20 Protein In Colletotrichum Siamense From Rubber Tree

Rubber tree(Hevea brasiliensis Musll-Arg.)is an important economic crop in the tropical region of China.One of the major causes of rubber yield loss is Colletotrichum leaf diseae(CLD).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 our China.As we known,appressorium is the specialized infection structure of many plant pathogenic fungi,and whether the appressoria were mature as normal and its turgor could penetrate the cuticle of the aerial plant surface,which is important to fungi pathogenicity.Previous studies of the CgCap20 gene in C.gloeosporioides of rubber tree in our research group showed that CgCap20 is a perilipin homologue protein and is involved in glycerol production,functional appressoria development and fungal virulence by loss of function methods.It has been reported that perilipin is phosphorylated by protein kinase A(PKA)and serves important functions in the regulation of lipolysis in the adipose tissues of mammals and insects.We opined that CgCap20 was regulated by PKA to affect the glycerol production and appressorium turgor pressure generation in plant pathogenic fungi.To verify this suggestion,we cloned the PKA catalytic gene of C.siamense strain HN08,analyzed the expression relationship between PKA subunits gene and CgCap20 using real-time fluorescence quantitative PCR technology,and detected the interaction between catalytic subunits of PKA and CgCap20 protein through yeast two hybrid technology and GST-pull down technology.The results of this study are as follows:1.For the molecular identification of the test strain HN08,the internal transcribed spacer(ITS)region of ribosomal DNA,and partion sequences of the chitin synthase(CHS-1),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),actin(ACT),partial mating type gene(Matl-2)(ApMAT),and glutamine synthetase(GS)were amplified with conserved primers.All six gene sequences of strain HN08 were deposited in GenBank(Accession No.KT924457?KT924475?KT924488?KT924504?MF036144?MF036157).A multilocus phylogenetic analysis performed with the reference sequences revealed that the HN08 isolate clustered with Colletotrichum siamense.2.PKA two catalytic subunit genes PKACl and PKAC2 were amplified by PCR and qRT-PCR methods using primers designed by homologous sequence from GenBank database.The results of the sequences analysis showed that the catalytic subunit gene of PKAC1 contained 3 introns and encoded 498 amino acids,PKAC2 gene contained 2 introns and encoded 381 amino acids,and both them have serine/threonine protein kinase catalytic domains of S_TKc and S_TK_X.Phylogenetic clustering suggested that two gene showed highest similarity to PKA catalytic genes of Colletotrichum gloeosporioides.3.To analyze the expression relationship between PKA subunits gene and CgCap20 in vivo,strain HN08 and GFP labbed strain CgCap20-GFP were tested.Both two strains were cultured in complete medium supplemented with or without the PKA activator Forskolin(concentration of 30 ?moL/L)or inhibitor IBMX(concentration of 30 mmoL/L).Then catalytic subunit genes PKAC1 and PKAC2,the regulatory subunit gene PKAR,and CgCap20 gene expression were analyzed by real-time fluorescence quantitative PCR method.The results showed that the expression of the PKA subunit genes and the CgCap20 gene on the two stains in medium supplemented with PKA activator were higer than control.On the contrary,the expression of them decreased after the treatment of the inhibitor.When the expression of PKA subunit gene increased,the expression of CgCap20 gene also increased,and vice versa.It was preliminarily confirmed that the expression of CgCap20 gene was positively affected by the expression of PKA subunit gene.4.The interaction between CgCap20 and PKAC1 or PKAC2 were verified by yeast two hybrid system.The results showed that CgCap20 protein interacted both with PKA catalytic subunit PKAC1 and PKAC2 subunits.5.The prokaryotic expression vector of two PKA catalytic subunit gene and CgCap20 were constructed.After the inducing conditions were optimized,when PKAC1 or PKAC2 was induced for 6h by lmmol/L IPTG at 28?,The PKAC1 or PKAC2 was high expressed in the supematant;when CgCap20 was induced for 20h by 0.5mmol/L IPTG at 16?,The CgCap20 was high expressed in the supematant.The precombinant protein was purified.The binding of PKAC1 and CgCap20 was confirmed by GST Pull-down assay.However,the result showd PKAC2 protein is not binding with CgCap20,which is not consistent with the result of yeast two hybrid.