Functional Characterization Of20Signaling And Effector Proteins Essential For Cell Wall Integrity And Their Connections To Biocontrol Potential Of Entomopathogenic Fungi

Beauveria bassiana and Metarhizium robertsii are fungal entomopathogens widely applied in biological control of arthropod pests. Thir biocontrol potential is largely dependent upon their virulence and tolerance to environmental stresses, such as high temperature, solar UV irradiation, and agrochemicals. Fungal cell wall is the first barrier to adverse stimuli and thus is crucial for both multi-stress tolerance and virulence. This study sought to elucidate a relationship between cell wall integrity and fungal biocontrol potential by functional characterization of three mitogen-activated protein （MAP） kinases constituting cell wall integrity （CWI） signaling pathway and of other CWI-related proteins in B. bassiana and/or M. robertsii. The results are summarized below.Functional characterization of three MAP kinases in the CWI signaling pathway. Bckl, Mkkl and Slt2are MAPKK kinase （MAPKKK）, MAPK kinase （MAPKK） and MAP kinase （MAPK） cascaded in fungal CWI pathway that may control multi-stress responses via crosstalk with high-osmolarity glycerol （HOG） pathway in budding yeast. In this study, Bckl, Mkkl and Slt2orthologues in B. bassiana were confirmed as the three-module cascade essential for CWI because cell wall impairment occurred in the hyphae and conidia of Δbckl, Δmkkl and Δslt2examined in multiple experiments. Strikingly, all the deletion mutants became more sensitive to hyperosmotic NaCl and sorbitol with the Western blot of Hog1phosphorylation being weakened in Abckl and absent in Amkkl and Aslt2. Apart from crossing responses to cell wall perturbation and high osmolarity, three deletion mutants exhibited faster growth and conidiation on nutrition-rich medium, much less virulence to Galleria mellonella larvae, and higher sensitivity to nutritional, fungicidal, thermal and UV-B irradiative stresses, accompanied with less accumulation of intracellular mannitol and trehalose. Moreover, Δmkkl and Δslt2were equally more sensitive to all the stresses of different types except wet-heat stress than wild type, and were more or less different from Abckl in sensitivity to most of the stresses despite their null responses to two oxidants. All the changes in three deletion mutants were restored by each target gene complementation. Taken together, the CWI-required Bckl, Mkkl and Slt2are all positive, but differential, regulators of multi-stress tolerance and virulence perhaps due to interplay with the HOG pathway essential for osmoregulation, thereby contributing greatly to the biocontrol potential of the fungal entomopathogen.Functional comparison of GPI-anchored protein Ecm33between B. bassiana and M. robertsii. Ecm33is one of several glycosylphosphatidylinositol （GPI） anchored proteins. This protein is known to be involved in fungal CWI but its contribution to multi-stress tolerance is largely unknown. Here we characterized the functions of two Ecm33orthologues, i.e., Bbecm33in B. bassiana and Mrecm33in M. robertsii. Bbecm33and Mrecm33were both confirmed as GPI-anchored cell wall proteins in immunogold localization. Single-gene disruptions of Bbecm33and Mrecm33caused slight growth defects but conidial yield decreased much more in ABbecm33（76%） than in AMrecm33（42%）, accompanied with significant reductions of intracellular mannitol and trehalose contents in both mutants and weakened cell wall in ABbecm33only. Consequently, ΔBbecm33was far more sensitive to cell wall perturbation by Congo red and sodium dodecyl sulfate （SDS） than AMrecm33, which showed null response to SDS. Both deletion mutants became significantly more sensitive to two oxidants （menadione and H₂O₂）, two fungicides （carbendazim and ethirimol）, osmotic salt NaCl and Ca2+during growth despite some degrees of differences in their sensitivities to the chemical stressors. Strikingly, conidial UV-B resistance decreased by55%in ABbecm33but was unaffected in AMrecm33, unlike a similar decrease （25-28%） of conidial thermotolerance in both. All the changes were restored to wild-type levels by target gene complementation in each fungus. However, neither ABbecm33nor AMrecm33showed a significant change in virulence to a susceptible insect host. The results indicate that Bbecm33and Mrecm33are important, but differential, contributors to the conidiation and multi-stress tolerance of B. bassiana and M. robertsii.Functional comparison of cell wall biogenesis phosphatase Ssdl between B. bassiana and M. robertsii. The Saccharomyces cerevisiae Ssdl is a cytoplasmic RNA binding protein with a conserved RNA binding domain and regulates CWI, cell cycle and pathogenicity. The genomes of B. bassiana and M. robertsii harbor the Ssdl orthologues Bbssdl and Mrssdl which are distributed in cytoplasm, as confirmed in immunogold localization. Both Bbssdl and Mrssdl were functionally characherized by constructing single-gene disruption and complementation mutants, followed by analyses of various phenotypes. As a result, colony growth became defective in ABbssdl but not in AMrssdl while conidiation was more defective in AMrssdl than in ABbssdl, accompanied with slower germination of their conidia. Both deletion mutants were less tolerant to cell wall disturbation, oxidation, high osmolarity, fungicides, high temperature and UV-B irradiation, and were less virulent to susceptible insect species in standardized bioassays, accompanied with opposite changes of cell wall surface carbohydrate epitodes and similar reductions of intracellular mannitol and trehalose contents. All the changes were restored to wild-type levels by target gene complementation in each fungus. The results indicate overlapped functions of Bbssdl and Mrssdl in the two entomopathogenic fungi and suggest a differret mechanism involved in their effects on CWI.Functional comparisons of13WSC domain-containing proteins in B. bassiana. WSC （water-soluble carbohydrate） domain exists in fungal transmembrane proteins, which are characteristic with at least eight conserved cysteines called cysteine-rich domain （CRD） and function upstream of the CWI signaling pathway in budding yeast. The WSC domain also exists in (3-1,3-exoglucanases in fungi. In this study,13WSC domain-containing proteins （Wscl-13） were found existing in B. bassiana and functionally characterized in the experiments of various phenotypes in their single-gene disruption mutants versus wild-type and complemented strains. All of them were functionally vital and diverse and took parts in CWI maintainance. Compared to wild type, Awscl, Awsc2and Awsc4showed mild growth defects while severe conidiation defects occurred in Awsc3and Awsc4. All of the disruption mutants were hypersensitive to the cell wall stressors like calcofluor white, Congo red, SDS and caffeine. Most of them were less tolerant to oxidation, high osmolarity, metal ions, fungicides and alkali environment during colony growth. Conidial resistance to UV-B irradiation decreased significantly in Δwscl-Δwscl3while conidial thermotolerance was lowered in Δwsc2-Δwsc5and Awsc8. Moreover, all the deletion mutants except Awsc4, Awsc6and Δwscl2exhibited much lower virulence to G. mellonella larvae infected through cuticle. The results indicate functional diversity, complexity and differention of all the WSC domain-containing proteins in B. bassiana for the first time, and highlight their important roles in maintaining the fungal cell wall integrity and biocontrol potential.