The Developmental Regulatory Function Analysis Of CAMP Phosphodiesterases In Setosphaeria Turcica

Setosphaeria turcica which causing Northern Corn Leaf Blight, is one of importantphytopathogenic fungi, and always results in significant corn yield losses. cAMP signaltransduction pathway is a widespread extracellular signal transduction pathway in fungi,and playes important roles in regulating the growth, morphogenesis, development,secondary metabolism and pathogenicity of phytopathogenic fungi. In this research, twokey enzyme genes of Setosphaeria turcica, involved in cAMP signal transduction pathway,were cloned and named as StH-PDE and StL-PDE respectively. StH-PDE was encodedhigh-affinity cAMP phosphodiesterase and StL-PDE was encoded low-affinity cAMPphosphodiesterase. Functional analysis of StH-PDE and StL-PDE genes were explored bycreating the gene-knockout mutants. Main results in this paper were as follows:1. One high-affinity cAMP phosphodiesterase gene (StH-PDE) and one low-affinitycAMP phosphodiesterase gene (StL-PDE) were cloned with the candidate gene cloningstrategy. Among them, the full length DNA and cDNA of StH-PDE and StL-PDE had beenobtained. StH-PDE included3208bp DNA sequence with2898bp coding region andconsisted of6exons and5introns, and its predicted protein contained965aa with amolecular weight of107.16kDa. StL-PDE gene included5054bp and was interrupted byone intron and its ORF of3089bp was encoded1019amino acid residues and wasinterrupted by4intons. All introns were accordance with GT-AG rules.2. The low identity for the StH-PDE on nucleic acid level found for the high-affinitycAMP phosphodiesterase from Aspergillus fumigatus, Botryotinia fuckeliana, Metarhiziumacridum, Neosartorya fischeri was between33.12%and36.60%. PPDEase_Ⅰ3’5’ cyclicnucleotide phosphodiesterase, catalytic domain and metal dependent phosphodiesterase,HD/domain, and PDEase_Ⅰ3’5’ cyclic nucleotide phosphodiesterase, conserved site wasfound, whereas high-affinity cAMP phosphodiesterase was20.33%sequence similarity tolow-affinity cAMP phosphodiesterase of S. turcica. It was probable that two genesbelonged to two groups of phosphodiesterase and shared different catalyse substrate. Thelow identity for the StL-PDE on nucleic acid level found for the low-affinity cAMPphosphodiesterase from Trichoderma reesei, Beauveria bassiana, Colletotrichumhigginsianum, Talaromyced stipitatus was between21.98%and23.06%. PDEase_Ⅱ3’5’ cyclic nucleotide phosphodiesterase domain and translation elongation factor EFIB,gamma chain, conserved site was found in StL-PDE.3. StH-PDE mRNA expression level was determined in the different developmentstage of S. turcica. The result showed that StH-PDE mRNA expression level was thehighest in mycelium and lowest in conidium and infection hypha.4. The StH-PDE gene-disruption vector was constructed based on the genedouble-cross homologous combination theory and PEG-mediated gene transformationsystem. Nine transformants named as△StH-PDE1~△StH-PDE9were screened byhygromycin B and PCR with specific primers corresponding to hygromycinphosphotransferase gene and StH-PDE gene.△StH-PDE2,△StH-PDE3and△StH-PDE8were obtained by Southern blot analysis performed with the DIG-labeled HPHgene and StH-PDE gene as probes respectively. Furthermore, single spore of△StH-PDE2and△StH-PDE3were isolated and verification by RT-PCR. The function analysis wasindicated that mutants were dark yellow, showed different color with the wild type strain.The number of aerial hypha and the growth in mutants was reduced. The cell of hypha wasswollen and the compartmentation of the cell was shorter. The surface of the hypha wasdisplayed irregular lined. The cell wall integrity was destroyed. The red secretion wasadhered on the surface of the hypha. The sporulation defect was showed in mutants, whilehypha could germinated and formed appressorium associating gwith postphonement,furthermore, the penetrate ability on the cellophane surface was declined. Theaccumulation of intra-cellular glycerin content mutants was increased and showedreinforced salt-stress resistance. The hydrophobicity of mutants was impaired andpresented an wettable phenotype. The HT-toxin activity of mutants was resemble with wildtype strain. The pathogenicity test showed that mutants could penetrate on the host, butcouldn’t result in lession. The mRNA expression level of CHS、GFA、FKS、GSC、GEL、Tubulin、MPG1and FPS in mutants was lower than wild type strains in different extent.5. StL-PDE mRNA expression level was determined in the different developmentstage of S. turcica. The result showed that StH-PDE mRNA expression level was thehighest in mycelium and lower in spore germination and appressorium formation.6. The StL-PDE gene replacement vector was also constructed based on the samestrategy as StH-PDE. StL-PDE transformants named△StL-PDE1and△StL-PDE10wereisolated and verificated by PCR、southern blot and RT-PCR. The function analysis ofmutants exhibited increased aerial hypha formation and second-infect hypha branching,reduced conidiation, lower penetration ability on the cellophane surface, enhancedaccumulation of intra-cellular glycerin content, reinforced salt-stress resistance, reducedmelanin content and declined laccase activity. The PT-PCR analysis indicated that mRNAexpression level of CHS、GFA、FKS、GSC、GEL、Tubulin and MPG1was no differencein the wild type and mutant strains. Only the mRNA expression level of FPS displayed a little declined in the mutant strains.The above results can summarize the function of these genes:1) StH-PDE gene wasinvolved in cell wall integrity, sporulation, second metabolism, sense to nutrition, melaninbiosynthesis, salt-stress resistant and pathogenicity in S. turcica respectively.2) StL-PDEgene played an important role in the regulation of sporulation, penetration ability of thehypha and laccase activity in Setosphaeria turcica.3）StH-PDE gene might have apredominant role and StL-PDE gene played a secondary role in the regulation of growthand development n S. turcica.