| Apple tree Valsa canker,caused by Valsa mali,is one of the most destructive fungal diseases of apple.The utilization of chemical treatment is the main method of controlling apple tree Valsa canker.Since the pathogen penetrates extensively into the host phloem and xylem,chemical treatment cannot effectively cure or control Valsa canker,leading to heavy damage to apple production and economic benefits.The study for molecular mechanism of Valsa mali is crucial for developing more effective disease management strategies.In fungi,heterotrimeric guanine-nucleotide binding proteins(G-proteins)controlled cAMP/PKA(the cyclic AMP/ protein kinase A)pathway to regulate the virulence.G protein α subunits,regulators of G protein signaling(RGS)proteins,and catalytic subunit of cAMP-dependent protein kinase A(PKA)were three important parts of cAMP/PKApathway.The genome sequence of V.mali opened new opportunities and perspectives for basic research to study its mechanism of plant infection.In this study,we did research in G protein α subunits genes,Regulators of G-protein signaling(RGS)and catalytic subunit of cAMP-dependent protein kinase A genes in cellular signal transduction,to analyze their functions in development and pathogenicity of V.mali.This study is great significance to explore the molecular mechanism of V.mali pathogenesis,and also provide important theoretical basis for the development of new strategies for durably controlling the disease.The main research results are as follows:1.Identification and functional analysis of Gvm2 and Gvm3 genesIn fungi,heterotrimeric guanine-nucleotide binding proteins(G-proteins)are key elements of signal transduction pathways,which control growth,asexual and sexual development,and virulence.In this study,we have identified genes encoding two heterotrimeric G protein α subunits Gvm2 and Gvm3 from Valsa mali,the causal agent of apple Valsa canker.Characterization of gvm2 and gvm3 mutants showed that Gvm3 may be a crucial regulator of vegetative growth.Deletion of the corresponding gene resulted in a 20% reduction in growth rate.Besides,Gvm2 and Gvm3 seemed to be involved in asexual reproduction and mutants were hypersensitive to oxidative stress,and cell membrane stress.By contrast,neither gvm2 nor gvm3 mutants were affected by salt stress.Interestingly,both G protein α subunits seemed to be involved in virulence.In infection assays with Malus domestica Borkh.cv.‘Fuji’ leaves lesion size of deletion mutants gvm2 and gvm3 were reduced 40% and 35%,respectively.Similarly,infection assays on twigs showed a reduction of lesion length of 28% and 33%,respectively.Cell wall degrading enzymes is an important virulence factors in V.mali.Many hydrolytic enzyme-coding genes including pectin-degrading enzymes,cellulose enzymes and hemicellulose genes were expressed at lower levels in the mutants gvm2 and gvm3 than in the wild type.The results of this study suggested that Gvm2 and Gvm3 play an important role in virulence probably by regulation of cell wall degrading enzyme genes expression.To further analyze the response of gvm2,or gvm3 mutant on the expression of the genes in the cAMP/PKA pathway,we checked the transcript levels of different genes.The results showed that adenylate cyclase VmAC,protein kinase A(PKA)regulator subunit VmPKR,and PKA catalytic subunit VmPKA1 were down-regulated in both mutants.Further analysis showed that the intracellular cAMP level and PKA activity were down-regulated in gvm3 mutant,but didn’t have a significant change in gvm2 mutant.Overall,our findings indicated that both Gvm2 and Gvm3 played various roles in the modulation of vegetative growth,asexual development,and virulence in V.mali.2.Regulators of G protein signaling(RGS)proteins play an important role in Valsa maliRegulators of G protein signaling(RGS)proteins play an important role in controlling heterotrimeric G protein signaling.In this study,three RGS genes(VmFlbA,VmRgsA,and VmRgsB)were functionally characterized in the plant pathogenic fungus,Valsa mali.We found that VmFlbA and VmRgsA are important for vegetative growth and mycelial morphology.Growth rates and hyphae morphology of VmrgsB mutant were similar to those of wild type strain 03-8.In addition,the mutant VmflbA produced more melanin than the corresponding wild type strain 03-8 and the expression levels of melanin biosynthesis genes were higher in the VmflbA mutant than in the wild type 03-8,suggesting a negative regulatory role of VmflbA in melanin biosynthesis in V.mali.Besides,mutants of VmflbA and VmrgsA seemed to be less hypersensitive to osmotic stree,oxidative stress,and cell membrane stress.We have also shown that VmFlbA and VmRgsA are involved in the virulence.In infection assays using Malus domestica cv.‘Fuji’ leaves and twigs,the size of lesions caused by deletion mutants VmflbA,or VmrgsA are significantly reduced.However,virulence in VmrgsB mutant strain had no obvious change.RNA-seq analysis of mutants VmflbA revealed that Gvm3 was up-regulated expression,which mean VmFlbA could negatively regulate Gvm3.Besides,many hydrolytic enzyme-coding genes were expressed at lower levels in the mutants than in the wild type.Cell wall degrading enzymes is an important virulence factors in V.mali.The results of this study suggested that VmFlbA play an important role in virulence probably by regulation of cell wall degrading enzyme genes expression.Taking together,in addition to VmRgsB,VmFlbA and VmRgsA proteins are involved in a complex process governing vegetative development,hyphae morphology,abiotic stress,and pathogenicity.3.Roles of the catalytic subunit of cAMP-dependent protein kinase A in Valsa maliPrevious study showed that G protein α subunit Gvm3 involved in regulating cAMP-PKA signaling pathway.To determine the importance of the cyclic AMP(cAMP)-protein kinase A(PKA)signaling pathway in development and virulence,two PKA catalytic subunit genes VmPKA1 and VmPKA2 were cloned and characterized.VmPKA1 and VmPKA2 encode typical catalytic subunits with a highly conserved protein kinase domain.To determine the gene function of VmPKA1 and VmPKA2,the mutants of them were constructed.The results suggest that the Vmpka1 mutants of V.mali exhibited the defects of mycelial morphology,the reduction of conidiation and growth rate,loss of virulence.Growth rates,virulence and hyphae morphology of Vmpka2 mutant were similar to those of wild type strain 03-8,but only the conidia production was significantly reduced.The Vmpka1 mutants of V.mali showed loss of PKA activity.However,PKA activity in Vmpka2 mutant strains had no obvious change.In addition,VmPKA1 also negatively regulate responses to heat,osmotic,and SDS stresses in V.mali.Interestingly,Fast-growing sectors of the Vmpka1 mutant formed when cultured 40 d at 25℃.Taken together,these findings indicate that the PKA signal transduction pathway play important but distinct roles,in the growth,virulence,conidiation formation,and stress,in the fungus V.mali. |
