Three Mitogen-Activated Protein Kinases Required for Cell Wall Integrity Contribute Greatly to Biocontrol Potential of a Fungal Entomopathogen

Abstract

Bck1, Mkk1 and Slt2 are three mitogen-activated protein (MAP) kinases constituting cell wall integrity (CWI) pathway that may control multi-stress responses via crosstalk with high-osmolarity glycerol (HOG) pathway in budding yeast. In this study, Bck1, Mkk1 and Slt2 orthologues in Beauveria bassiana were confirmed as the three-module cascade essential for CWI because cell wall impairment occurred in the hyphae and conidia of Δbck1, Δmkk1 and Δslt2 examined in multiple experiments. Strikingly, all the deletion mutants became more sensitive to hyperosmotic NaCl and sorbitol with the Western blot of Hog1 phosphorylation being weakened in Δbck1 and absent in Δmkk1 and Δslt2. 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, Δmkk1 and Δslt2 were equally more sensitive to all the stresses of different types except wet-heat stress than wild type and more or less different from Δbck1 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 targeted gene complementation. Taken together, the CWI-required Bck1, Mkk1 and Slt2 are 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.

The high osmotic response and cell wall integrity pathways cooperate to regulate morphology, microsclerotia development, and virulence in Metarhizium rileyi

“…In addition, the disruption of Mrhog1 or Mrslt2 leads to restricted vegetative growth and abnormal morphological clones on SMAY plates. This result was unlike the observations in ΔBbhog1 26 and ΔBbslt2 2728. Moreover, similar phenotypic changes have been observed in other fungi26272840, in which the single-gene disruptions increased sensitivity to cell wall damage, high osmolarity, and oxidative stress, as well as showing convoluted colonies.…”

“…This result was unlike the observations in ΔBbhog1 26 and ΔBbslt2 2728. Moreover, similar phenotypic changes have been observed in other fungi26272840, in which the single-gene disruptions increased sensitivity to cell wall damage, high osmolarity, and oxidative stress, as well as showing convoluted colonies. As in other fungi, these results indicated that the CWI and HOG pathways in M. rileyi could functionally overlap in responding to cell wall damage, osmoregulation, and oxidative stress171924.…”

“…Traditionally, Hog1p, the core MAPK of the HOG pathway, is activated in sense and responds to fluctuations in environmental osmotic pressure2526. Slt2p, the central MAPK of the CWI pathway, is activated in response to cell wall stress to maintain cell wall integrity272829. However, the two MAPKs are not restricted to individual stimuli, but their responses can be elicited by various environmental stresses303132.…”

Stress response signaling and virulence: insights from entomopathogenic fungi

“…Not too surprisingly, the ΔBbslt2 mutant showed defects in virulence using both topical and intrahemocoel injection assays. All three members of the B. bassiana CwI pathway, bck1, mkk1, and slt2, were further investigated (Chen et al 2014b). Single mutants of each gene generally resulted in similar phenotypes although some differences were noted.…”

Distinct roles of two cytoplasmic thioredoxin reductases (Trr1/2) in the redox system involving cysteine synthesis and host infection of Beauveria bassiana

“…To further confirm subcellular localization of Trr11 and Trr2 revealed by their eGFP-tagged fusion proteins, mouse monoclonal anti-eGFP antibodies (Cell Signaling Technology, Boston, MA, USA) were used to probe the fusion proteins in the protein extracts from the 3-day-old hyphal cultures of the WT and the selected transgenic strains following a protocol of Western blot hybridization described previously (Chen et al 2014). Briefly, the protein extract samples were separated through SDS-PAGE and transferred to ployvinyldene difluoride (PVDF) membranes (Millipore, Germany).…”