Functional Characterization Of Histidine Kinase Family,Nuclear Kinase Weel And Phosphotase Cdc25in Beauveria Bassiana

Beauveria bassiana is a filamentous entomopathogen that has been widely applied in microbial control of anthropod pests. As active ingredients of mycoinsecticides, fungal cells are exposed to field conditions upon application, and hence their tolerances to high temperature, solar UV irradiation and agrochemicals are as important as virulence for the field persistence and efficacy of a fungal formulation. Fungal traits affecting growth and conidiation are also important for the mass production of a formulation. Thus, it is necessary to explore underlying mechanisms involved in fungal responses to developmental, environmental and host cues. This study sought to characterize the structures and functions of histidine kinase (HK) family and of cell cycle-required nuclear kinase Weel and phosphatase Cdc25in B. bassiana and eclucidate their contributions to the fungal growth, asexual development, multi-stress tolerance and virulence. The results are summarized below.Functional characterization of eigh HK orthologues in B. bassiana. Filamentous fungi may have up to11groups of hybrid-type HK homologues with each group including one or more members. Most of the HK homologues are functionally unknown and even considered as evolutionary remnants. Here we show that all eight groups of paralog-free HKs (named with their grouping codes) in B. bassiana are functionally vital and play differential roles in the fungal growth, conidiation, infection and responses to chemical and/or environmental cues. Among single HK disruption mutants, AHK3lost94%conidiation capacity, gained high iprodione resistance, and became significantly more sensitive to oxidative, cell wall perturbing, thermal and UV-B irradiative stresses but was unchanged in osmosensitivity, accompanied with severe defect of virulence through cuticle or cuticle-bypassing infection. As sole osmosensitive mutant, AHK8showed uniquely faster growth on minimal media, lost partial thermotolerance and UV-B resistance, and acquired some antioxidant capability. Less virulent ΔHK6was more tolerant to oxidation and UV-B irradiation but less tolerant to wet-heat stress at45℃while ΔHK10responded to oxidation and UV-B like ΔHK6and displayed higher sensitivity to cell wall disturbance of SDS and faster kill action through cuticle infection. Being more sensitive to oxidation and cell wall perturbation, ΔHK9was less tolerant to heat and UV-B and less virulent to host insect in cuticle-bypassing infection whilst ΔHK2, ΔHK2and AHK11had1-3phenotypes altered in response to SDS, UV-B and/or host defense. All phenotypic changes in the deletion mutants were well restored by the complementation of each target gene. Strikingly, phosphorylation levels of Hog1and Slt2hallmarking the high-osmolarity glycerol (HOG) and cell wall integrity (CWI) pathways of AHK3and AHK8were significantly altered by osmotic, oxidative and/or iprodione stresses. Our findings confirmed for the first time the multiple functions of most members in the HK family, highlighting their significances for the fungal adaptation to diverse insect hosts and environments.B. bassiana phytochrome (Bbphy) controls conidiation in response to red/far-red light and daylight length. Phytochrome (Phy) proteins in filamentous fungi are Group VIII HK homologues which share a unique N-terminal photosensory core for light sensing. Aside from the significance of B. bassiana HK8(Bbphy herein) in multi-stress responses, this protein is also essential for the fungal conidiation in reponse to red/far-red ligh and daylight length. ABbphy grew significantly slower on a nutrition-rich medium (SDAY) or faster on several minimal media with altered carbon or nitrogen source and availability, but the growth rates on different media were not affected in response to the light/dark cycles of3:21to24:0h. Strikingly, conidial yield of ABbphy on the rich medium increased at the fitted rate of3.4×107conidia per hour of white light in the light/dark cycles of0:24to16:8h, decreased greatly in the short-, long-and full-day cycles of red (660nm) or far-red (760nm) light, but was unaffected under full-day blue (450nm) light. Accompanied with the conidiaiton defects under short daylight lengths and red/far-red light were transcriptional alterations of seven conidiation-related effector genes in ABbphy versus WT under the different light conditions. All the changes in ABbphy were restored to the WT levels by the target gene complementation. The results indicate that Bbphy controls the condiation of B. bassiana by mediating the transcriptional expression of some, if not all, genes required for fungal conidiaiton in response to red/far-red light and daylight length.Weel and Cdc25controls morphogenesis, virulence and multi-stress tolerance of B. bassiana by balancing cell cycle-required Cdkl activity. Modification of cell cycle in entomopathogenic fungi is likely crucial for host infection and environmental adaptation. Here, the Cdkl activity essential for cell cycle was proven to be balanced by Weel and Cdc25in B. bassiana. Cdkl phosporylation signal was very weak in Aweel, stronger in Acdc25and absent in Aweel Acdc25. Consequently, reversed changes occurred in their cell cycles, septation patterns, and transcript profiles of some septation dependent genes. Hyphal cells were short in Aweel, slender in Acdc25, and short and swollen in Awee1Δcdc25. Conidiaiton was most defective in Δwee1, followed by Δcdc25. Mutant conidia and yeast-like blastospores also showed antagonistic changes in both size and complexity, accompanied with abnormally branched germlings in Δwee1and Δwee1Δcdc25. Conidial thermotorance and UV-B resistance decreased much more in Δwee1Δcdc25than in Δwee1but significantly increased in Δcdc25. The double deletion and the mutation CdklT14A/P15F for inhibitory phosphorylation caused most defective virulence, followed by weel deletion. All the changes were restored by targeted gene complementation. Strikingly, virulence changes in all the mutants and control strains were significantly correlated to those in blastospore size and complexity. Taken together, Weel and Cdc25control the cell cycle and biocontrol potential of B. bassiana by balancing the Cdk1activity.