Adenylate Cyclase Regulates The Growth Of Mycelium And Formation Of Fruit Body In Flammulina Velutipes

The adenylate cyclase(AC)and protein kinase A(cAMP-depend protein kinase,PKA)are the major components of the cAMP/PKA signaling pathway.Previous studies indicate that the cAMP/PKA signaling pathway plays an important role in growth of mycelium and formation of fruiting body.In this study,we applied biological information method to construct the cAMP/PKA signaling pathway based on the genome and transcriptome datas of Flammulina velutipes.In order to study the function of cAMP/PKA signaling pathway in growth of mycelium and formation of fruiting body in F.velutipes,RNA interference(RNAi)and overexpression were applied to elucidate the function of fvac gene encoding adenylate cyclase,which is the crucial component of cAMP/PKA pathway.And the main results are showed as followed:(1)Real-Time Quantitative PCR(qRT-PCR)was used to analysis the expression patterns of this pathway,and the result revealed that expression of those genes encoding adenylate cyclase(AC)and protein kinase A(PKA)increased in primordia stage.Analysis of homology domains using InterProScan indicated that all of those proteins in signal pathway contained conserved domains belonging to the adenylate cyclase(AC)and protein kinase A(PKA),respectively.And phylogenetic analysis demonstrated adenylate cyclase(AC)and protein kinase A(PKA)were clustered with AC and PKA from other basidiomycete,respectively.These results indicated that the cAMP/PKA signaling pathway was conserved in F.velutipes.(2)To determine the function of cAMP/PKA signaling in growth of mycelium and formation of fruiting body in F.velutipes,we constructed the RNAi vector and overexpression vector using the binary expression vector pBHg-BCA1-gpd by recombinant clone method and the vectors were named pBHg-fvac-RNAi and pBHgfvac-OE respectively.The vectors named pBHg-fvac-RNAi and pBHg-fvac-OE were transformated into the homokaryon mycelium of F.velutipes strain L11 by the method of agrobacterium mediated transformation.After screening with hygromycin,genomic DNA of the putative transformants which can grow on the PDA plates containing hygromycin and cefotaxime was extreated and was applied to amplify hyg gene and our target gene containing partial sequence of vector.Finally,we obtained two overexpression transformants(named OE1 and OE2)and four interference transformants(named Ri101-1,Ri101-2,Ri918-1 and Ri918-3).Genome sequencing showed that the two overexpression transformants(named OE1 and OE2)were sensu stricto transformants.In addition,the result of genome sequencing demonstrated that the amplification of hyg gene and our target gene,containg partial sequence of vector,can be useful for identification of the real transformations.(3)The anaylisis of expression of fvac adenylate cyclase gene in the six transformants and wild type strain L11 was conducted via qR-TPCR.In comparison with the wild type strain L11,the expression levels of the fvac gene was down-regulated in the four interference transformants(Ri101-1,Ri101-2,Ri918-1 and Ri918-3).While,the expression level of fvac was up-regulated in the two overexpression transformants(OE1 and OE2).In addition,high performance liquid chromatograph(HPLC)was applied to detect the content of intracellular cAMP in transformants.The four interference transformants(Ri101-1,Ri101-2,Ri918-1 and Ri918-3)were significantly reduced in the intracellular cAMP levels.And the cAMP content in OE1 and OE2 mutants increased about 4.3-fold and 2.8-fold,respectively.Protein kinase A(PKA),consisting of two regulatory subunits(PKAr)and two catalytic subunits(PKAc),are the downstream target of cAMP.Similar to the trend in the expression level of fvac gene,the expression levels of fvpkar,fvpkacl and fvpkac2,encoding the regulatory subunits and two catalytic subunits of protein kinase A,increased in overexpression transformants but reducded in RNAi transformants.(4)Observation of morphological phenotypes indicated that both of the two overexpression mutants(OE1 and OE2)formed very thick aerial mycelium mats on the PDA plates and,the growth rate of hyphal of OE1and OE2 was increased compared to the wild type strain L11.But in contrast to OE1 and OE2,the four interference transformants exhibited reduced hyphal growth rate with low mycelial density.Furthermore,the accumulation of green pigmentation,absent in the control strain L11 and the two overexpression mutants,were observed around the colonies of the four interference transformants.When cultured on PDA plates containg inhibitors of adenylate cyclase and protein kinase A(PKA),the overexpression transformants reduced in growth rate,which were more than the reduction observed in the wild-type strain L11.Addition of exogenous cAMP and 8-Br-cAMP,an activator of protein kinase A,to agar cultures of four interference transformants mutants almost fully restore normal growth.And the growth and morphology of Ri101-1 and Ri101-2 was visibly altered in the presence of 2 ?M 8-Br-cAMP,resembling those of the OE1 and OE2 mutant grew on the PDA plates.These data demonstrated that fvac gene played an important role in the growth of mycelium,the density of hyphae and the regularity of the colony edges.Because hydrophobin genes have been reported to be involved in the formation of aerial hyphae and fruit body,we detected the expression levels of hydrophobin genes with qRT-PCR and,the result showed that the expression of six hydrophobin genes was up-regulated in OE1 and OE2 mutants.While the expression of these hydrophobin genes were downregulated in the four interference mutants.Thus,we concluded that fvac positively regulated these hydrophobin genes(5)When grown on PDA plates containing 5 mM H2O2,the four interference transformants and wild type strain L11 showed significantly reduced colony size.In contrast,vegetative growth of the OE1 and OE2 mutants were not dramatically affected by H2O2.One hour exposure to 42?,the OE1 and OE2 mutants were still able to grow after incubation at 25? for 24h,while the growth of the four interference transformants and wild type strain L11 were completely abolished under the same condition.The expression levels of fvhsp26 and fvsod2 were down-regulated in the four interference transformants,while the expression levels of those genes were up-regulated in the two overexpression mutants.These data demonstrated that fvac gene positively regulated fvhsp26 and fvsod2,and the two overexpression mutants had increased tolerance against heat shock and H2O2 in F.velutipes.(6)The fruiting trial for heterokaryotic strains consisting of these fvac gene transformants mated with compatible homokaryotic strain L22 were carried out.And the results of fruiting trial displayed that heterokaryotic strain OE1× L22 and OE2×L22 were able to form fruiting body and,there were more lateral mushroom compared to wild type strain L11×L22.However,heterokaryotic strains Ri101-1×L22,Ri101-2×L22,Ri918-1×L22 and Ri918-3×L22 showed markedly suppressed formation of fruiting body.These results indicated that cAMP/PKA signal pathway played a regulatory role in the formation and development of fruit bodies.According to experimental results mentioned above,we conclude that of fvac encoding adenylate cyclase played a definitive role in growth of mycelium and formation of aetial hyphae and mushroom in F.velutipes.Adenylate cyclase regulated protein kinase A activity through adjusting the secondary messenger cAMP level for regulating growth of mycelium.In addition,fvac gene positively regulated the formation of pigment in F.velutipes.