Functional Characterization Of The CDC14 Gene In Fusarium Graminearum And Magnaporthe Oryzae

Members of the Cdc14 dual specificity phosphatase family are highly selective for serine-proline phosphorylation sites generated by cyclin-dependent kinases and are common in animals and fungi but absent in plants.Although its orthologs are well conserved in plant pathogenic fungi,their functions during plant infection are not clear.Fusarium graminearum is the causal agent of head blight(FHB)or scab of wheat and barley,the pathogen not only causes severe yield losses but also contaminates infested grains with mycotoxins.Magnaporthe oryzae is the causal agent of rice blast,one of the most important diseases threatening rice production worldwide,and a model system for the study of fungal–plant interactions.In this study,we showed that CDC14 is important for plant infection and development in these two phytopathogenic fungi.In Fusarium graminearum,FgCDC14 is required for normal cell division and septum formation.Deletion of FgCDC14 is not lethal but the Fgcdc14 deletion mutant was significantly reduced in vegetative growth,conidiation.Whereas the wild type mainly produced conidia on clusters of phialides,the Fgcdc14 mutant formed much fewer,sparsely distributed phialides.The mutant produced conidia with fewer septa and multiple-nuclei per compartment compared to those of the wild-type conidia,indicating that the Fgcdc14 mutant was defective in the coordination between nuclear division and cytokinesis.Interestingly,foot cells and occasionally tip compartments of some conidia were able to exit G1-arrest and further grow and differentiate into conidiogenous cells in the Fgcdc14 mutant,resulting in the production of inter-connected conidia.Septation in Fgcdc14 hyphae also was reduced and irregular.Nuclei were distributed unevenly in the Fgcdc14 hyphae.In self-cross assays,the Fgcdc14 mutant is defective in ascosporogenesis.The Fgcdc14 deletion mutants produced morphologically normal perithecia that contained aborted ascogenous tissues.The Fgcdc14 mutant exhibited significantly reduced ascospore formation and failed to produce ascospore cirrhi.Ascospores formed by Fgcdc14 mutant were morphologically abnormal,had fewer than four compartments,and often contained more than one nucleus per compartment.Therefore,deletion of FgCDC14 affected meiotic and post-meiotic nuclear divisions and septation in developing ascospores.In infection assays with flowering wheat heads and corn silks,the Fgcdc14 mutant was almost non-pathogenic.The Fgcdc14 mutant was significantly reduced in DON production in the infested wheat kernels and rice grain cultures,suggesting that deletion of Fg CDC14 affects DON biosynthesis in F.graminearum.In vitro enzyme activity assay,the FgCdc14 protein possesses phosphatase activity with specificity for a subset of Cdk-type phosphorylation sites nearly identical to that of S.cerevisiae Cdc14.As a typical foliar pathogen,appressorium formation and penetration are critical steps in the infection cycle of M.oryzae.Because appressorium formation and penetration are closely co-regulated with cell cycle and Cdc14 phosphatases have antagonistic relationship with CDKs on proteins related to mitotic exits and cytokinesis,in this study we also functionally characterized the Mo CDC14 gene in M.oryzae.The Mocdc14 mutant was significantly reduced in growth rate and conidiation.It also was defective in septum formation and nuclear distribution.Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi-nucleate.Mutant conidia often had incomplete septa or lacked any septum.During appressorium formation,the septum delimiting appressoria from the rest of germ tubes was often formed far away from the neck of appressoria or not formed at all.Some mutant appressoria had more than one nuclei at 24 h.Besides appressoria,melanization occurred to parts of germ tubes and conidia,depending on the irregular position of the appressorium-delimiting septum.The Mocdc14 mutant also was defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells.Similar defects in septum formation,melanization,and penetration were observed with appressorium-like structures formed at hyphal tips in the Mocdc14 mutant.Often a long fragment of mutant hyphae was melanized together with the apical appressorium-like structures.These results indicate that Mo CDC14 plays a critical role in septation,nuclear distribution,and pathogenesis in M.oryzae,and proper septum formation during conidiogenesis and appressorium formation requires the Mo Cdc14 phosphatase.Taken together,our results indicate that Cdc14 phosphatase functions in cell division and septum formation in F.graminearum and M.oryzae,likely by counteracting Cdk phosphorylation,and is required for plant infection.