The Na⁺/H⁺ antiporter Nhx1 controls vacuolar fusion indispensible for life cycles in vitro and in vivo in a fungal insect pathogen

Abstract

The sole Na /H antiporter Nhx1 has been generally unexplored in filamentous fungi. We characterized Nhx1 in the entomopathogenic fungus Beauveria bassiana. An eGFP-tagged Nhx1 fusion accumulated in small punctuate structures, presumably endosomal and trans-Golgi network compartments, between septum and tubular vacuole of each wild-type cell stained with a vacuole-specific dye. Deletion of nhx1 resulted in significant acidification and severe fusion defect in vacuoles, which were fragmented and distinct from large or tubular wild-type vacuoles. The deletion also caused a drastic reduction in aerial conidiation or submerged blastospore production and more severe defect in vegetative growth than in conidial germination. The Δnhx1 mutant became more sensitive to high osmolarity, heat shock and several metal ions during growth but its conidia showed increased UV-B tolerance. Intriguingly, Δnhx1 was unable to infect a model insect through cuticle penetration or intrahaemocoel injection because it produced much less biomass and cuticle-degrading enzymes in a minimal broth and failed to form blastospores in the insect haemolymph. All changes were completely or largely restored by targeted nhx1 complementation. Our results provide novel insight into an indispensability of Nhx1 for not only vacuolar fusion but also life cycles in vitro and in vivo in B. bassiana.

Rtt109‐dependent histone H3 K56 acetylation and gene activity are essential for the biological control potential of Beauveria bassiana

“…To explore virulence‐related cellular events, 100 mL aliquots of a 10 4 conidia mL −1 suspension in Czapek broth (CZB, i.e., agar‐free CZA) containing 0.3% bovine serum albumin (BSA) as the sole nitrogen source for enzyme induction were shaken at 150 rpm for 3 days at 25 °C. Biomass levels in the resultant cultures and total activities of extracellular enzymes and Pr1 proteases in the supernatants of the cultures were quantified as described elsewhere . One unit of enzyme activity was defined as the amount of extracellular enzymes or Pr1 proteases required for an increase in the optical density at 410 or 440 nm (OD 410 or OD 440 ) of 0.01 after 1 h reaction of each supernatant versus a control, and total activity was expressed as U mL −1 supernatant.…”

“…Among the Pr1 genes, only CDEP1 and CDEP2 have been shown to be virulence factors although, as yet, most are functionally unknown in fungal insect pathogens. However, attenuated virulence was less likely to be associated with the development of fungal infection after entry into the insect hemocoel because both the fungal virulence through the cuticle‐bypassing infection and the dimorphic transition rate in the medium mimic of insect hemolymph were less affected by the absence of rtt109 as a gene essential for dimorphic transition in B. bassiana …”

“…Beauveria bassiana is an entomopathogenic fungus that usually undergoes an asexual cycle and has been integrated into pest management programs worldwide . The pest control potential of this insect pathogen is an output of diverse cellular processes and events, including conidial attachment to and germination on insect integument, hyphal penetration through the insect cuticle by means of the activities of secreted cuticle‐degrading enzymes, and the proliferation of fungal cells in insect hemocoel by yeast‐like budding until host death from mummification . Fungal response to the oxidative stress generated from host immunity defense is relevant to the success of fungal infection through cuticular penetration and of subsequent intrahemocoel proliferation, and hence important for fungal virulence .…”

Gcn5‐dependent histone H3 acetylation and gene activity is required for the asexual development and virulence of Beauveria bassiana

“…This is well in accordance with the loss of virulence or pathogenicity in the Δ gcn5 mutants of A. flavus (Lan et al ., ), C. neoformans (O'Meara et al ., ) and U. maydis (González‐Prieto et al ., ). Our data also unveil a vital role for Gcn5 in not only the biosynthesis and secretion of extracellular enzymes and Pr1 proteases presumably involved in the cuticle penetration crucial for the normal infection but also the dimorphic transition, which is essential for the rapid proliferation of fungal cells in insect haemocoel and hence determinant to the speed of host mummification to death (Wang et al ., 2016; Zhu et al ., ; Chu et al ., ; Zhang et al ., ). As far as known to date, only a very few members in the large family of cuticle degrading enzymes are functionally characterized, such as CDEP1/2 whose expression may augment the virulence of B. bassiana and bacterial crystal toxin (Zhang et al ., ; Fang et al ., ; Xia et al ., ).…”

The DUF1996 and WSC domain‐containing protein Wsc1I acts as a novel sensor of multiple stress cues in Beauveria bassiana

“…This involvement is clarified by the DUF1996‐dependent localization of Wsc1I and VLP1–3 in the vacuoles, where many cellular events, such as nutrition transport, cellular homeostasis, signalling, and stress responses, may take place (Veses et al, ). Previously, the Na + /H + antiporter Nhx1 was shown to control vacuolar fusion and acidification and function in cell response to high osmolarity, heat shock, and several metal cations in B. bassiana (Zhu, Ying, & Feng, ). Although DUF1996 is clearly required for a vacuolar localization of host protein, it remains opaque how the unknown domain aids the host protein to properly function in intracellular homeostasis of metal cations through the proton antiport system, warranting further studies.…”