Molecular Mechanisms Of The High-affinity Phosphodiesterase PcPdeH In Regulation Of The Pathogenicity In Phytophthora Capsici.

Signal transduction by heterotrimeric G protein signaling utilizes cyclic adenosine monophosphate（cAMP）as a secondary messenger to respond to extracellular signals and control a range of intracellular downstream signaling components in many eukaryotes.The cAMP signaling pathway is the best studied paradigm of signal transduction and influences and regulates physiological processes such as metabolism,morphogenesis and pathogenicity.The intracellular levels of cAMP are dependent on the actions of many different proteins that affect its biosynthesis and hydrolysis.The inactivation of cAMP to 5’-AMP is carried out by cAMP phosphodiesterases（PDEs）.PDEs can target specific intracellular sites or signaling complexes and reset the strength and intensity of the cAMP/PKA cascade.Phytophthora capsici is a highly destructive pathogen of Solanaceae crops（pepper and tomato）,leguminous crops（lima bean and green sword bean）and most cucurbit plants.In the filed,zoospores,formed from sporangia on the tips of fl ooded hyphae,are the main method of dispersal and inoculum.In P.capsici,which is a severely dynamic and destructive heterothallic species,the role of the cAMP signaling pathway and cAMP phosphodiesterases that hydrolyze cAMP have not yet been identifi ed.To elucidate the function of PcPdeH,PcPdeH-knockout mutants were obtained using a type II CRISPR/Cas9 system in Phytophthora capsici.In our study,we were interested in investigating the role of phosphodiesterases in regulating the growth,intracellular cAMP level and pathogenicity of P.capsici.The knockout of PcPdeH resulted in a reduction in pathogenicity.The knockout mutants lost almost all virulence.Furthermore,our data suggest that the pathogenicity defect was not associated with reduced vegetative growth or penetration,and the typical symptoms of water-stained lesions on N.benthamiana and C.annuum did not expand after zoospores were incubated for 5-7 dpi or on the wounded leaves.In addition,the deletion of PcPdeH resulted in a defect in the establishment of a successful infection and the formation of restricted infectious hyphae.The PcPdeH-deletion mutants displayed higher sensitivity to H₂O₂ than the wild type and CK.The mycelial growth of the mutant was severely inhibited on Plich’s medium containing 1-5 m M H₂O_2.In addition,the loss of PcPdeH resulted in abnormal cyst germination,indicating that the polar growth of hyphae was affected,as indicated by inflexion,branches and partial swelling.PcPdeH-knockout mutants exhibited increased（1.5-2-fold）cAMP levels relative to the wild-type and CK strains,indicating that the high-affinity phosphodiesterase in P.capsici regulated the intracellular level of cAMP.Based on these phenotypic features,we propose that PcPdeH is crucial for vegetative growth,cyst germination and pathogenicity in P.capsici.