| Beauveria bassiana is a filamentous entomopathogen that has been widely applied for biological control of agricultural and forest insect pests. The fungal biocontrol potential is restrained in stressful environments, making it necessary to understand mechanisms involved in fungal anti-stress responses, which are either theoretical or technical basis for genetic maintenance and improvement of fungal traits against environmental stresses. This dissertation sought to characterize the functions of18heat shock proteins (Hsp) and3heat shock factors (Hsf), a P-type Ca2+-ATPase (Pmrl), a phosphotase (Cdcl4) and an adenylate cyclase (AC) in B. bassiana and to probe possible mechanisms in their regulating the fungal growth, conidiation, nutritional utilization, multi-stress responses and infection. The main results are summaried below.Functional characterization of Hsp and Hsf members in B. bassiana. The Hsp family consist of many members classified to six subfamilies in terms of their molecular sizes, including sHsp (small Hsp), Hsp40, Hsp60, Hsp70, Hsp90and Hsp100. All Hsp and Hsf homologues in budding yeast were used as queries to blast the genome of B. bassiana, resulting in30Hsp members plus Hsfl-3. The fungal Hsp family incudes three sHsp, six Hsp40, two Hsp60, fifteen Hsp70, one Hsp90and two Hsp100members plus Hsp82. Single Hsp or Hsf disruption mutants showed different degrees of changes in growth, conidiation, nutritional utilization, multi-stress responses, infectivity, cell wal composition, and intracellular solute accumulation. The sHsp deletion mutants grew slower and became more sensitive to the stresses of oxidation, cell wll perturbion, carbendazim, high temperature and UV-B irradiation during colony growth or conidial germination, followed by a slight virulence decrease. The Hsp40gene disruptions resulted in even more drastic changes in the phenotypes mentioned above. Of those, colony growth was suppressed by more than40%in Ahsp40a and Ahsp40c, which was most defective in conidiation, while osmosensitivity increased by70%in Ahsp40a, which lost pathogenicity to a susceptible host. The Hsp60genes were positive regulators of conidiation, multi-stress responses and virulence with a drastic decrease of conidial yield in Ahsp60a. The disruptions of six Hsp70genes and Hsp82caused mild defects in growth, conidiation, multi-stress tolerance and virulence while nine other Hsp70genes were indispensible for the fungal life because their disruptions were lethal. The Hsp90disruption was also lethal. Severe defects of conidiation, conidial thermotolerance and virulence were observed in Ahsp104and Ahsp78. The disruptions of three Hsf genes caused different phenotypic changes. Conidial yield decreased greatly in△hsfl, which was25-78%less tolerant to antioxidation, cell wall perturbation, high temperature and carbendazim. Severe defect occurred in the growth, conidiaiton and virulence of A.hsf2, which was more sensitive to oxidation, high osmolarity and cell wall perturbation but43%more resistant to carbendazim. The A.hs/3mutant was featured with drastic reductions in conidial yield and tolerance to cell wall stress and mild changes in other examined phenotypes. Most of the disruption mutants were altered in the contents of intracellular glecerol, mannitol and trehalose, which are impotant for cellular multi-sress responses, and some of them showed unbalanced pH change in growth substrate. Interestingly, the deletion of a Hsp or Hsf gene caused drastic transcriptional changes of many other Hsp/Hsf members, indicating complementary and interactive relationships from one to another. Among the characterized Hsp and Hsf members of B. bassiana, overall, Hsfl, Hsp78and Hsp104are key regulators of thermotolerance and Hsfl also acts as a mediator of cell wall integrity; Hsp40a is a key regulator of osmotolerance and virulence while Hsp40c, Hsp60a, Hsp78, Hsf2and Hsf3, are crucial or important regulators of conidiaiton.P-type calcium ATPase functions as a core regulator of B. bassiana growth, conidiation and responses to multiple stressful stimuli via crosstalk with signaling networks. P-type Ca2+-ATPases (Pmrl members) are core elements of calcium-calcineurin pathway in eukaryotes and experimental evidence for the regulation of physiological processes through their crosstalk with other signaling systems is of scarcity. The functions of a Ca2+-ATPase (Bbpmrl) merely existing in the Golgi complex of B. bassiana was characterized by multi-phenotypic and transcriptional analyses of wild-type and deletion mutant strains under normal and stressful conditions. Bbpmrl inactivation resulted in severe defects in nutritional uptake, growth, conidiation and germination under normal conditions, drastic reductions in cell tolerances to oxidative, hyperosmotic, cell wall disturbing and fungicidal stresses and toxic cation types (such as EDTA, Zn2+, Cu2+and Fe3+) during colony growth or conidial germination, and half a loss of the fungal biocontrol potential represented by conidial virulence, thermotolerance and UV-B resistance. Accompanied with the multi-phenotypic defects, four important genes associated with asexual development were repressed by≥75%in the normal cultures of△Bbpmrl, and all or most of the genes encoding14cascaded MAPK proteins including those in high-osmolarity glycerol (HOG) pathway, two Ras GTPases, two protein kinases, Sskl-type response regulator, TOR signaling protein, and many downstream effector proteins were greatly downregulated in△Bbpmr1response to the chemical stresses. Our findings highlight that Bbpmrl regulates positively a series of fundamental physiological processes and infection in B. bassiana through crosstalk with cellular signaling networks including MAPK cascades and those upstream or independent of the HOG pathway.Cytokinesis-required Cdc14is a signaling hub of asexual development and multi-stress tolerance in B. bassiana. A dual-specificity, paralog-free Cdc14phosphatase located in the nuclei of B. bassiana was functionally characterized with the alterations of multiple phenotypes and effector gene transcripts caused by its inactivation. Disruption of cdc14caused defective cytokinesis due to the formation of three or more nuclei in13%of hyphal cells and89%decrease in blastospore production in liquid culture, followed by slower growth on the plates of nutrition-rich and minimal media and a loss of≥96%conidial yield under normal conditions. These defects coincided with the repression of25genes required for mitosis and conidiation. Moreover,△cdc14became hypersensitive to oxidative, osmotic, and cell wall and mitosis perturbing stresses during hyphal growth and conidial germination, and lost41-70%of conidial thermotolerance, UV-B resistance and virulence. The multi-stress tolerance defects, although rarely seen in other fungal cdc14deletion mutants, were attributable to drastic repression of61stress-responsive genes, which encode most of cascaded MAPK components as well as Ras1/Ras2, PKA/PKC, Snfl, and those enzymes or proteins responsible for the fungal antioxidation, osmotolerance and cell wall integrity. Strikingly, the phosphorylation levels of Hog1and Slt2hallmarking the high-osmolarity-glycerol and cell-wall-integrity pathways of△cdc14respectively were significantly reduced by the chemical stresses in ELISA experiments with available anti-phospho-antibodies. All the changes were well restored to wild-type level in cdc14complemented mutant. Conclusively, Cdc14regulates not only cytokinesis, asexual development and virulence but multi-stress responses by acting as a nexus of cellular signaling network in B. bassiana.Adenylate cyclase orthologues in two insect pathogens contribute differentially to growth, conidiation, pathogenicity and multi-stress responses. Adenylate cyclase (AC) is a core element of cyclic adenosine monophosphate (cAMP) signaling network essential for fungal physiological processes. This study sought to elucidate the functional diversity and differentiation of structurally similar AC orthologues, i.e., BbAC in B. bassiana and MrAC in Metarhizium robertsii. Disruptions of BbAC and MrAC resulted in different growth defects on rich and minimal media and reversed change of conidiation, i.e., drastic increase (48%-1.1fold) and decrease (52-68%) of conidial yield, during incubation on a standard medium. The growth defects of both disruptants were largely alleviated by adding cAMP to the standard medium. During colony growth under chemical stresses of different types,△BbAC was highly sensitive to oxidation, high osmolarity, cell wall perturbation, carbendazim fungicide and metal ions (Mn2+, Zn2+, Fe3+and EDTA) but more tolerant to Cu2+while△MrAC showed higher tolerance to NaCl and Fe3+and null response to either carbendazim or Congo red although it was similar to△BbAC in responses to oxidation and other metal ions. Conidial UV-B resistance decreased32%in△BbAC and22%in△MrAC although either disruptant showed an inconspicuous change in conidial theromotolerance. Strikingly,50%kill of susceptible host insects in standardized bioassays was delayed by10.9days in ABbAC but only1.4days in△MrAC. All the phenotypic changes were restored by targeted gene complementation in each disruptant. Taken together, BbAC and MrAC functioned differentially in the regulation of conidiation, pathogenicity and multi-stress responses, thereby making differential contributions to the biocontrol potential of B. bassiana and M. robertsii. |
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