Pleiotropic effects of Ubi4, a polyubiquitin precursor required for ubiquitin accumulation, conidiation and pathogenicity of a fungal insect pathogen

Abstract

Summary Ubi4 is a polyubiquitin precursor well characterized in yeasts but unexplored in insect mycopathogens. Here, we report that orthologous Ubi4 plays a core role in ubiquitin‐ and asexual lifestyle‐required cellular events in Beauveria bassiana. Deletion of ubi4 led to abolished ubiquitin accumulation, blocked autophagic process, severe defects in conidiation and conidial quality, reduced cell tolerance to oxidative, osmotic, cell wall perturbing and heat‐shock stresses, decreased transcript levels of development‐activating and antioxidant genes, but light effect on radial growth under normal conditions. The deletion mutant lost insect pathogenicity via normal cuticle infection and was severely compromised in virulence via cuticle‐bypassing infection due to a block of dimorphic transition critical for acceleration of host mummification. Proteomic and ubiquitylomic analyses revealed 1081 proteins differentially expressed and 639 lysine residues significantly hyper‐ or hypo‐ubiquitylated in the deletion mutant, including dozens of ubiquitin‐activating, conjugating and ligating enzymes, core histones, and many more involved in proteasomes, autophagy‐lysosome process and protein degradation. Singular deletions of seven ubiquitin‐conjugating enzyme genes exerted differential Ubi4‐like effects on conidiation level and conidial traits. These findings uncover an essential role of Ubi4 in ubiquitin transfer cascade and its pleiotropic effects on the in vitro and in vivo asexual cycle of B. bassiana.

Dsk2 involves in conidiation, multi‐stress tolerance and thermal adaptation in Beauveria bassiana

“…To construct the Dsk2::GFP fusion protein, our previous plasmid p0380‐TEF‐GFP (Wang et al ., 2020a) were used as backbones for the insertion of the Dsk2 (tag locus: BBA_05501) ORF. Briefly, the ORF of Dsk2 was amplified from the cDNA of WT strains with paired primers (Table S1), followed by ligating with the p0380‐TEF‐GFP digested with restriction enzyme BamHI under the action of a one‐step cloning kit (Vazyme, Nanjing, China).…”

“…The conidial virulence of each strain was assessed by two infection modes as our previous protocol (Wang et al ., 2020a). Briefly, groups of ~40 G .…”

“…The conidial virulence of each strain was assessed by two infection modes as our previous protocol (Wang et al, 2020a). Briefly, groups of 40 G. mellonella larvae were immersed for 15 s in 40 ml of a 10 7 conidia ml −1 suspension for normal infection through cuticular penetration.…”

“…Ubiquitin (Ub) is a conserved protein, consisting of 76 amino acids. Among those, seven Lys residues act as a symbol for protein modification in all eukaryotic cells (Leach et al ., 2011; Wang et al ., 2020a). Ubiquitination is a fundamental post‐translation modification to determine the fate of misfolded or aged proteins (Gemayel et al ., 2017).…”

“…Ubiquitination is a fundamental post‐translation modification to determine the fate of misfolded or aged proteins (Gemayel et al ., 2017). This cellular modification relies upon the sequential cooperation between target proteins, (E1) Ub‐activating enzymes, (E2) Ub‐conjugating enzymes, (E3) Ub‐protein ligases and degradation‐machine proteasome (Jin et al ., 2007; Rape, 2018; Wang et al ., 2020a). Meanwhile, transporter proteins, like Dsk2, were recruited to help proteasome recognize and digest these ubiquitinated misfolded proteins, or to induce their autophagy.…”