| Phosphodiesterases(PDEs)play the function of hydrolyzing intracellular second messenger cAMP(cyclic adenosine monophosphate),terminating the signal transduction pathways involved in these second messengers.Currently,the phosphodiesterase family reported in filamentous fungi contain high-affinity phosphodiesterase(pdeH/pde2)and low-affinity phosphodiesterase(pdeL/pdel),which play a very important regulatory role in the vegetative growth and the pathogenesis development in fungi.A representative strain of nematode-trapping fungus,Arthrobotrys oligospora,produces three-dimensional networks under starvation conditions as a predator to capture and digestive nematodes for growth,however,the signaling mechanisms of trap formation are still unclear.In this study,the genes coding for high-affinity phosphodiesterase(AoPdeH,AOL s00083g160)and low-affinity phosphodiesterase(AoPdeL,AOL s00097g378)were disrupted in Arthrobotrys oligospora,the functions of two phosphodiesterases were characterized in the vegetative growth and pathogenic development of A.oligospora.The main experimental results:1.Functional domain and cluster analysis of two phosphodiesterasesThe conserved domains of two phosphodiesterases in A.oligospora were predicted using InterProScan,AoPdeH has a 3'5'-cyclic nucleotide phosphodiesterase catalytic domain superfamily(IPR036971),a HD/PDEase domain(IPR003607),a 3'5'-cyclic nucleotide phosphodiesterase catalytic domain(IPR002073)and a 3'5'-cyclic nucleotide phosphodiesterase conserved site(IPR023174);while AoPdeL contains a leucine-rich repeat domain superfamily(IPR032675),a ribonuclease Z/hydroxyacyl glutathione hydrolase-like(IPR036866)and a cyclic-AMP phosphodiesterase class?(IPR000396).Cluster analysis showed that AoPdeH and AoPdeL were divided in two different evolutionary branches,respectively,which suggested that they may play different roles in fungi.2.Analyses of vegetative growth,stress resistance and pathogenicity of phosphodiesterase mutantsThe two phosphodiesterase-encoding genes were knocked out using homologous recombination method,and two mutants were successfully obtained.The hyphal morphology,growth rate,stress tolerance ability,nematicidal ability,the extracellular protease activity,and trap formation were statistically compared between the wild-type(WT)strain and mutants.The deletion of AoPdeH caused significantly change in phenotypic traits,such as the growth rate of the mutant strain was slowe down,the hyphal septum was increased and the mutants became sensitive to the environmental pressure.In addition,the ?AoPdeH mutants lost the ability to form mature three-dimensional networks,but the nematicidal ability only significantly reduced.Meanwhile,analysis of the proteolytic activity of the fermentation broth in the?AoPdeH mutants showed that no significant change in proteolytic activity compared with the WT strain.However,the growth rate and stress resistance of the ?AoPdeL mutants showed no significantly different from those of the WT strain,while the nematicidal ability and the traps number were decreased,and the hyphal morphology was swollen and deformed.Our results indicated that AoPdeH is involved in the regulation of the biological processes related to vegetative growth,stress resistance and pathogenic development in A.oligospora.3.Comparison of conidiation and sporulation-related genes between the WT and mutantsThe conidia yield of the ?AoPdeH mutants were significantly decreased to 2-6%compared with the WT strain,and the conidia yield of the ?AoPdeL mutants had no significantly different from that of the WT strain.The conidial morphology of the two phosphodiestera mutants was deformed and became narrow and long without septum.In addition,the conidiophores of ?AoPdeH mutants were significantly decreased compared with the WT strain.However,the conidia yield and conidiophores of the?AoPdeL mutants had no significantly different from the WT strain.To further investigate the regulation of phosphodiesterase on sporulation,13 sporulation-related genes were selected for real-time PCR analysis in the early period(3 days),middle period(5 days)and later period(7 days)of sporulation.The results showed that the expression levels of FlbA,Acr1,FluG,VelB,NsdD,FlbC,PkaCl,Plp1,AbaA,LreA and LreB were down-regulated in the ?AoPdeH mutants compared with the WT strain.while the transcription levels of 13 sporulation-related genes in the ?AoPdeL mutants showed no change significantly compared with the WT strain.The above experimental results indicated that AoPdeH is involved in the regulation of conidiation in A.oligospora.4.Phosphodiesterases involves in the regulation of intracellular cAMP levels in A oligosporaThe cAMP content in mycelia of the WT and mutants was determined by enzyme-linked immunosorbent assay(ELISA).The cAMP content in the mycelia of the?AoPdeH and ?AoPdeL mutants at all tested time points was higher than that of the WT strain.To further analyze the regulation mechanism of phosphodiesterase on cAMP levels,16 genes located on the upstream and downstream of phosphodiesterase or involved in the regulation of cAMP level were selected for real-time PCR analysis,in which the expression levels of cAMP receptor-like protein,cAMP reaction element binding protein and adenylate cyclase changed significantly(significantly up-regulated during mycelial growth and development,significantly downregulated during pathogenic development),indicating that phosphodiesterase participates in the regulation of cAMP level by regulating the expression of the above genes in A oligospora.Innovations in this paper:1.The coding genes of two phosphodiesterase family were successfully knocked out in the A.oligospora for the first time.It was found that AoPdeH is involved in the regulation of important biological processes,such as the vegetative growth,asexual reproduction,stress resistance,trapformation and intracellular cAMP levels.2.The transcriptional levels of several genes related to conidiation and cAMP synthesis were compared in the WT,?AoPdeH and ?AoPdeL mutants,our results suggested that two phosphodiesterase may involve in the regulation of these genes in A oligospora. |
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