Mechanisms Of The Velvet Family Protein And Transcription Factor PacC In Pathogenesis And Pectinase Regulation In Valsa Mali

Apple is one of the pillar industries in northern China.As a destructive disease,Valsa mali restricts production of apple seriously.To control this major disease,we need explore the pathogenesis of V.mali led to speed up the theory and practice of this disease.Pectinase is virulence factors has been certified by several evidences.However,the function of these cell wall degrading enzymes is redundancy due to the numerous members of this family.Therefore,analyzing the regulation of pectinase production will facilitate the efficient reveal the pathogenesis of V.mali and might provide a new tool to develop novel,sustainable disease management strategies against Apple Valsa Canker.In this study,we conducted a functional analysis on development,virulence and pectinase expression of Velvet protein family and the pH regulatory factor VmPacC via a gene replacement strategy.We have demonstrated the pectinases were regulated in different model by Velvet protein family and VmPacC during infection.The results might provide a direction for the search of potential molecular targets of new pesticides.Furthermore,it is a new tool to develop novel,sustainable disease management strategies against Apple Valsa Canker.The main findings are as follows:?1?Two members of the Velvet family,VmVeA and VmVelB affect melanin production and abiotic stressesVelvet protein family members are important fungal-specific regulators which are involved in conidial development,secondary metabolism,and virulence.The V.mali genome contains only a single copy of all four Velvet genes designated VmVeA,VmVelB,VmVelC and VmVosA.All four genes share the common Velvet factor domain.Analysis of the amino acid sequences revealed they are the most similarities to Velvet proteins of Magnaporthe oryzae.To investigate the roles of Velvet genes in V.mali,we constructed deletion cassettes by the double-joint PCR method.The deletion cassettes were transformed into protoplasts of the wild-type strain 03-8using the polyethylene glycol?PEG?method.The Velvet genes deletion mutants were confirmed via PCR and southern blotting detection.The deletion of these genes did not affect the growth rate significantly.However,the colour of the mycelium was significantly darker in VmVeA and VmVelB deletion mutants compared with the wild-type.Quantitative real-time polymerase chain reaction showed that the transcript levels of predicted melanin biosynthesis-related genes were up-regulated in VmVeA?2.2–5.7-fold?and VmVelB?3.8–8.4-fold?deletion mutant compared with the wild-type.Similarly,the melanin content in the hyphae of VmVe A and VmVelB deletion mutants was also higher?6.62 and 5.65?g/g,respectively?compared with the wild-type?2.81?g/g?.These results showed that VmVeA and VmVelB are negative regulators of melanin synthesis.To test whether VmVeA and VmVelB are involved in abiotic stress responses,we found deletion mutants of VmVeA and VmVelB showed increased sensitivity to K⁺,H₂O₂,Congo red,and SDS.These studies suggest that VmVeA and VmVelB are involved in the regulation of the sensibility to osmotic pressure and oxidative stress,and the maintenance of cell wall integrity,in V.mali.?2?VmVeA and VmVelB could be involved in virulence via effect on pectinase gene expression in V.maliThe virulence assays showed that VmVeA and VmVelB are required for full virulence.To test whether VmVe A and VmVelB play a role in the regulation of expression of CWDEs during infection,transcript levels of pectinase genes were shown to be decreased in deletion mutants compared to those of the wild type during infection.However,the expression of other cell wall-degrading enzymes including cellulase,hemi-cellulase,or ligninase genes was not affected in the deletion mutants.Furthermore,the determination of pectinase activity and immunogold labeling of pectin demonstrated that the capacity of pectin degradation was attenuated due to deletions of VmVeA and VmVelB.Finally,the interaction of VmVeA with VmVelB was identified through co-immunoprecipitation assays.In our preliminary studies,deletion of the other two members,VmVelC and VmVosA,did not show any changes in phenotype compared to the wild type in all tests.Therefore,we did not conduct further test on these two deletion mutants.?3?VmVeA and VmVelB affect conidiation by regulating melanin synthesis transcription factor Vm Cmr1.To evaluate whether VmVeA and VmVelB affect conidiation in V.mali,we found both of them are negative regulators of conidiation regardless of light in V.mali.Also,the VmVeA and VmVelB deletion mutants weakened conidia the dependence on the light.To investigate the relationship between melanin biosynthesis and conidiation which seem both negatively regulated by VmVe A and VmVelB,we chose to delete VmCmr1,the homolog of the transcription factor Cmr1 that regulates melanin biosynthesis,in the wild type,?VmVeA,and?VmVelB strains.The VmCmr1 deletion mutant exhibited the same growth rates but produced a lower level of melanin than wild type.Moreover,it failed to produce pycnidia on PDA at 15dpi as compared to wild type that produced numerous pycnidia.However,pycnidia could be observed at 30 dpi.The double deletion mutants named?VmCmr1/?VmVeA and?VmCmr1/?VmVelB showed the same phenotype with respect to growth rate,melanin synthesis and conidiation as the single deletion mutant??VmCmr1?.This result indicated VmVeA and VmVelB mutants lost the regulation of melanin synthesis and conidiation when VmCmr1 was deleted.In addition,we also demonstrated that VmCmr1 was independent on virulence.?4?The pH regulatory factor VmPacC could be involved in virulence via effect on the secretion of citric acid and succinic acid in V.maliThe transcription factor PacC is the terminal component of the PAL signaling pathway for responsing pH condition.The role of PacC in pathogenic fungi has been explored in few species,and each time its partaking in virulence has been found.The genome of V.mali contains single copy genes of pH-signaling pathway genes.Analysis of the amino acid sequences revealed significant similarities to various conservation of each of the protein sequence between the four fungi pH-signaling pathway.VmPacC protein consists of 560 amino acid residues,which is smaller than the PacC/Rim101 orthologues in yeast?635 aa?and A.nidulans?678 aa?.And contains classical zinc finger domain and Zinc-finger double domain at its N-terminal region.We found that the expression of VmPacC was up-regulation 9.9-fold and 17-fold in pH 7 and pH 9 than pH 4,and 2-5.6-fold during infection.The optimum pH value of V.mali is 3-5.The radial growth of the VmPacC deletion mutant on PDA was similar to that of the wild type at pH 3 to pH 5.Deletion of VmPacC impaired the radial growth of the VmPacC deletion mutant was decreased comparing to wild type.At pH 6–pH 10,the radial growth of the VmPacC deletion mutant was decreased.So,VmPacC was involve in fungal responses to neutral and alkaline pH.We measured the pH dynamic change of wild type,?VmPacC and?VmPacC-C strains in the liquid medium and during infection.V.mali can acidizing pH condition for growth and infection.And the capacity of acidize pH condition was impaired when VmPacC have been deleted.When VmPacC deletion mutant inoculated onto detached apple leaves and twigs,an obvious decrease in lesion size were observed.Analysis of organic acids secreted of V.mali by high performance liquid chromatography?HPLC?indicated that the accumulation of citric acid and succinic acid was reduced in VmPacC deletion mutant comparing to wild type.The virulence of VmPacC deletion mutant and wild type were increase when externally treated with citric acid and succinic acid.The VmPacC involve in virulence partly due to the regulation secretion of citric acid and succinic acid.?5?VmPacC is involved in suppress production of pectinaseWe tested if VmPacC affected the demonstrated virulence factors pectinase by qRT-PCR.Unexpected,transcript levels of a majority of pectinase genes was up regulated in different degrees in deletion mutant during infection.To further confirm the negative regulation,we generated dominant activating allele mutants,C-27,which can persistent express VmPacC.When grown on solid mediums mended pectin as the carbon source,the growth rate of C-27was reduce comparing with wild type and?VmPacC strains.Also,C-27 strains were significantly reduced in virulence.The transcript levels and pectinase activity of C-27 strains were reduced when culture in pH 4 pectin medium.In p H 7 medium,the pectinase genes of VmPacC deletion were up-regulated 3-7-fold in pH 7.And protein content was increase in deletion mutant as well.But the pectinase activity was the same level all the strains.All this result showed that the pectinases were suppressed by VmPacC in V.mali.