Identification And Functional Analysis Of The Protein Kinase A Regulatory Subunit Pkr In Fusarium Graminearum

Fusarium graminearum is a causal agent of the wheat scab disease and a producer of deoxynivalenol(DON)mycotoxins.Treatment with exogenous cAMP or deletion of PDE2 cAMP phosphodiesterase increased its DON production.To better understand the role of the cAMP-PKA pathway in F.graminearum,in this study we functionally characterized the PKR gene encoding the regulatory subunit of PKA,the only known intracellular target of cAMP in fungi.Mutants deleted of PKR were viable but had severe defects in growth,conidiation,and plant infection.The pkr deletion mutant produced compact colonies with shorter aerial hyphae that were increased in the number of nuclei in hyphal compartments.Mutant conidia were morphologically abnormal,lacked typical foot cells and were shorter and less curved in comparison with those of the wild-type.Furthermore,many conidium compartments appeared to be empty in pkr conidia harvested from 5-day-old CMC cultures.When stained with propidium iodide(PI),most of these empty or highly vacuolated conidium compartments showed strong fluorescent signals,indicating that they were dead or dying.On self-mating carrot agar plates,the pkr mutant was sterile.No perithecia were observed in the mutant cultures at 14 days postfertilization(dpf)or longer.It had a disease index less than 1 and failed to spread from the inoculated kernels to neighboring spikelets,but the pkr mutant showed increased DON production(approximately three-fold)in infested wheat kernels in comparison with the wildtype.The pkr mutant was unstable and spontaneous suppressors with faster growth rate were often produced on older cultures.A total of 67 suppressor strains that grew faster than the original mutant was isolated.Three of them had similar growth rate and colony morphology with the wild type but were still defective in conidiation.In suppressor H2,conidiation was increased to approximately 30% of the wild-type level,but suppressors H1 and H3 produced fewer conidia than the original pkr mutant.Sequencing analysis with 18 candidate PKA-related genes in three representative suppressor strains(H3,H7,H14)identified mutations only in the CPK1 catalytic subunit gene.No mutation sites were detected in any of these candidate genes sequenced in suppressor H14.In suppressor strain H3,the non-synonymous mutation resulting in the E406 A change in amino acid sequences was identified in the CPK1 gene.Suppressor strain H7 also showed a mutation in CPK1 that resulted in the H531 R amino acid change.Further characterization showed that 10 of the rest 64 suppressor strains also had mutations in CPK1.Whole genome sequence of four suppressors(H22,H30,H35,H57)showed four mutations in different genes(CYC8,MIG1,NHP6A/B,SGF73).Overall,these results showed that PKR1 is important for regulating hyphal growth,reproduction,pathogenesis,and DON production,and mutations in CPK1 were partially suppressive to deletion of PKR in F.graminearum.The identification of spontaneous mutations may be helpful to further elucidate the regulation mechanism of cAMP signaling network.