Characterization Of Mannosyltransferase,Histone Acetyltransferase,Forkhead Transcript Factor And Trehalose-6-phosphate Svnthase And Their Links To Biological Control Potential Of Beauvieria Bassiana

Beauveria bassiana is a filamentous entomopathogen that has been widely ap-plied in biological control of agricultural and forest insect pests. As active ingredients of fungal insecticides, fungal cells, such as conidia, are largely influenced by high temperature, solar UV irradiation and applied agrochemicals in an environment where they are exposed upon application. Therefore, fungal biocontrol potential is largely dependent not only on conidial virulence but cellular reponses to various stresses. It is necessary to explore mechanisms involved in fungal anti-stress responses when effort is made to improve fungal genetic traits and to apply fungal formulation in a rational manner. This dissertation sought to characterize functions of several enzymes or ptoteins, including mannosyltransferses in PMT and Ktr families, histone acetyltransferase Mst2, forkhead transcription factor Fkh2 and two paralog of treha-lose-6-phosphate synthase (Tpsl). The results are summarized below.The connection of protein O-mannosyltransferase family to the biocontrol potential of B. bassiana.O-mannosylation dependent on protein O-mannosyltrans-ferase (Pmt) family is an essential posttranslational modification process in eukary-otes but their connection to the biocontrol potential of a filamentous entomopathogen against arthropod pests has not been understood. Here, we characterized the functions of three Pmt orthologs (Pmtl, Pmt2 and Pmt4) in the Pmt family of B. bassiana and found that they were positive, but differential, regulators of the fungal growth, conidiation, multi-stress tolerance and virulence. Three Pmt2 knockdown mutants (△Pmt2 was lethal), △Pmt1 and APmt4 grew 20-79% slower on nutrition-rich and limited media. Their conidial yields on a standard medium were reduced by 17-62%, accompanied with delayed germination. All the mutants became significantly less tol-erant to most stresses of cell wall perturbation, high osmolarity, oxidation, wet heat and UV-B irradiation during colony growth and conidial germination and lost viru-lence by 53-62% via cuticle infection although their virulence via hemocoel injection was not affected. Strikingly, these phenotypic defects were accompanied with re-markable cell wall damages, including thinner cell wall, lower conidial hydrophobi-city and altered cell wall composition. All the changes were well restored to wild-type levels by targeted Pmt1 or Pmt4 complementation. Our results indicate for the first time that Pmtl, Pmt2 and Pmt4 are all required for the full biocontrol potential of B. bassiana despite differential contributions.Three α-1,2-mannosyltransferases contribute differentially to conidiation, cell wall integrity, multistress tolerance and virulence of B bassiana. Members of β-1,2-mannosyltransferase (Ktr) family are required for protein O-mannosylation for the elongation of Ser/Thr mannose residues in yeasts but functionally unknown in most filamentous fungi. Here we characterized the functions of the Ktr orthologues Ktr1, Ktr4 and Kre2/Mntl in B. bassiana, and found that they were positive, but dif-ferential, mediators of many biological traits. Inactivation of Ktr4 and Kre2 resulted in 92% reduction of conidial yield on a standard medium and growth defects on sub-strates with altered carbon or nitrogen sources and availability, accompanied with re-duced conidial size and complexity. This contrasts to the dispensability of Ktrl for fungal growth and conidiation. More cell wall damage occurred in △ktr4 and △kre2 than in Aktrl, including altered contents of the cell wall components mannonproteins, a-glucans and chitin, more carbohydrate epitopes changed on conidial surfaces, much lower conidial hydrophobicity, and thinner cell walls. Consequently, Aktr4 and Akre2 became more sensitive to oxidation and cell wall perturbation than Aktrl during col-ony growth or conidial germination despite less difference in their sensitivities to two osmotic agents. Conidial thermotolerance, UV-B resistance and virulence were all lowered greatly in Aktr4 and Akre2 but only the thermotolerance decreased in Aktrl. All the phenotypical changes were well restored to wild-type levels by the comple-mentation of each target gene. Our results indicate that Ktr4 and Kre2 contribute more to the biocontrol potential of B. bassiana than Ktrl although all of them are signifi-cant contributors.Histone acetyltransferase Mst2 is required for gene expression, cell cycle, multistress tolerance and virulence of B. bassiana. Histone lysine acetylation is as-sociated with transcriptional activity, but the connection of each acetylated lysine to cellular functions is not well understood due to limited specificity for most histone acetyltransferases and deacetylases. Confirmed in this study, B. bassiana Mst2, an ortholog of Schizosaccharomyces pombe SpMst2, is a highly specific H3 lysine 14 (H3K14) acetyltransferases. Deletion of Mst.2 in B. bassiana caused severe defects in vegetative growth, conidiation, conidial germination, cell tolerance to oxidative, cell wall disturbing and osmotic stresses, and hence partial loss of the fungal biocontrol potential represented by conidial virulence, thermotolerance and UV-B resistance. In addition, AMst2 showed altered cell cycle in yeast-like blastospores in vitro and lost a capability of DNA damage repair. Taken together, Mst2 is significant for the biolog-ical control potential of B. bassiana.Transcriptional control of fungal cell cycle and cellular events by Fkh2, a forkhead transcription factor in B. bassiana. Transcriptional control of the cell cy-cle by forkhead (Fkh) transcription factors is likely associated with fungal adaptation to host and environment. Here we show that Fkh2, an ortholog of yeast Fkh1/2, or-chestrates cell cycle and many cellular events of B. bassiana. Deletion of Fkh2 re-sulted in dramatic down-regulation of the cyclin-B gene cluster and hence altered cell cycle (longer G2/M and S, but shorter G0/G1, phases) in unicellular blastospores. Consequently, AFkh2 produced twice as many, but smaller, blastospores than wild-type under submerged conditions, and formed denser septa and shorter/broader cells in aberrantly branched hyphae. In these hyphae, clustered genes required for septation and conidiation were remarkedly up-regulated, followed by higher yield and slower germination of aerial conidia. Moreover, AFkh2 displayed attenuated virulence and decreased tolerance to chemical and environmental stresses, accompanied with altered transcripts and activities of phenotype-influencing proteins or enzymes. All the changes in AFkh2 were restored by Fkh2 complementation. All together, Fkh2-dependent transcriptional control is vital for the adaptation of B. bassiana to diverse habitats of host insects and hence contributes to its biological control potential against arthropod pests.Two Tps1 paralogs play major and minor, but additive, roles in trehalose synthesis, conidiation, multistress responses and host infection in B.bassiana. Trehalose accumulation level in fungal cells is relevant to fungal life and pathogenic-ity. Trehalose-6-phosphate synthase (Tps1) is known to control the first step of treha-lose synthesis but multiple Tps1 paralogs do not necessarily function in the process of some filamentous fungi. Here we characterized two Tps1 paralogs (TpsA and TpsB) existing in B. bassiana by multiple analyses of single/double tpsA/B deletion mutants. As a result, total TPS activity and trehalose content in the hyphal cells exposed to various stresses or not stressed were reduced by 71-75% and 72-80% in AtpsA, and 21-30% and 15-45% in AtpsB, respectively, but consistently undetectable in AtpsAAtpsB compared with wild-type. Conidiation level under normal conditions was decreased by 33%,50% and 98% in the three mutants respectively. Conidial quality indicated by trehalose content, viability, density, cell wall composition and hydropho- bicity was more impaired in △tpsA than in △tpsB and poorest in △tpsA△tpsB. Conse-quently, △tpsA△tpsB was most sensitive to nutritional, osmotic, oxidative, cell wall perturbing, thermal and UV-B irradiative stresses. The fungal virulence to Galleria mellonella larvae was most attenuated in △tpsA△tpsB, followed in △tpsA and AtpsB in order. The levels of almost all the phenotypic defects in AtpsAAtpsB approached to the sums of those observed in △tpsA and △tpsB. All the changes were restored by ei-ther tpsA or tpsB complementation. Taken together, TpsA and TpsB play major and minor, but additive, roles in co-regulating trehalose synthesis, conidiation capacity, conidial quality, multi-stress tolerance and virulence, which are determinant to the biological control potential of B. bassiana against arthropod pests. Our findings high-light an additive significance of the fungal Tpsl paralogs not only for intracellular trehalose accumulation but for environmental adaptation and host infection.