Autophagy-related gene BbATG11 is indispensable for pexophagy and mitophagy, and contributes to stress response, conidiation and virulence in the insect mycopathogen Beauveria bassiana.

Autophagy is a conserved degradation system in eukaryotic cells that includes non-selective and selective processes. Selective autophagy functions as a selective degradation mechanism for specific substrates in which autophagy-related protein 11 (ATG11) acts as an essential scaffold protein. In B. bassiana, there is a unique ATG11 family protein, which is designated as BbATG11. Disruption of BbATG11 resulted in significantly reduced conidial germination under starvation stress. The mutant ΔBbATG11 displayed enhanced sensitivity to oxidative stress and impaired asexual reproduction. The conidial yield was reduced by approximately 75%, and this defective phenotype could be repressed by increasing exogenous nutrients. The virulence of the ΔBbATG11 mutant strain was significantly impaired as indicated in topical and intra-hemocoel injection bioassays, with a greater reduction in topical infection. Notably, BbATG11 was involved in pexophagy and mitophagy, but these two autophagic processes appeared in different fungal physiological aspects. Both pexophagy and mitophagy were associated with nutrient shift, starvation stress and growth in the host hemocoel, but only pexophagy appeared in both oxidation-stressed cells and aerial mycelia. This study highlights that BbATG11 mediates pexophagy and mitophagy in B. bassiana and links selective autophagy to the fungal stress response, conidiation and virulence.

Interactome analysis of transcriptional coactivator multiprotein bridging factor 1 unveils a yeast AP-1-like transcription factor involved in oxidation tolerance of mycopathogen Beauveria bassiana.

Oxidation tolerance is an important determinant to predict the virulence and biocontrol potential of Beauveria bassiana, a well-known entomopathogenic fungus. As a transcriptional coactivator, multiprotein bridging factor 1 mediates the activity of transcription factor in diverse physiological processes, and its homolog in B. bassiana (BbMBF1) contributes to fungal oxidation tolerance. In this study, the BbMBF1-interactomes under oxidative stress and normal growth condition were deciphered by mass spectrometry integrated with the immunoprecipitation. BbMBF1p factor has a broad interaction with proteins that are involved in various cellular processes, and this interaction is dynamically regulated by oxidative stress. Importantly, a B. bassiana homolog of yeast AP-1-like transcription factor (BbAP-1) was specifically associated with the BbMBF1-interactome under oxidation and significantly contributed to fungal oxidation tolerance. In addition, qPCR analysis revealed that several antioxidant genes are jointly controlled by BbAP-1 and BbMBF1. Conclusively, it is proposed that BbMBF1p protein mediates BbAP-1p factor to transcribe the downstream antioxidant genes in B. bassiana under oxidative stress. This study demonstrates for the first time a proteomic view of the MBF1-interactome in fungi, and presents an initial framework to probe the transcriptional mechanism involved in fungal response to oxidation, which will provide a new strategy to improve the biocontrol efficacy of B. bassiana.

The cellular proteome is affected by a gelsolin (BbGEL1) during morphological transitions in aerobic surface versus liquid growth in the entomopathogenic fungus Beauveria bassiana.

The gelsolin superfamily includes seven protein members: gelsolin, villin, adseverin, CapG, advillin, supervillin and flightless I. The gelsolin proteins are actin-binding proteins that contain three or six gelsolin-like domains, and they play important roles in remodelling actin dynamics and cellular processes in eukaryotes. The entomopathogenic fungus Beauveria bassiana expresses a unique CapG protein (BbGEL1) that contains three gelsolin-like domains. BbGEL1p is associated with actin during mycelial growth and plays an important role in fungal morphological transitions under both aerobic and submerged conditions. The ΔBbGEL1 mutant displays abnormal spore-producing structures that reduce the conidial and blastospore yields by approximately 70% and 90% respectively. The virulence of the ΔBbGEL1 mutant is notably reduced as indicated by topical and intrahemocoel injection assays. Two comparative proteomics analyses indicated that BbGEL1 has significantly different roles in the development of conidia and blastospores, and the results revealed the potential targets of BbGEL1 in the corresponding developmental processes. Additionally, as an overlapping downstream protein of BbGEL1, the hydrophobin-like protein gene BbHyd3 is required for conidiation but has a negative role in blastospore formation. Our findings indicate that in addition to its function as an actin-interacting protein, BbGEL1 contributes to fungal morphological transitions via broad genetic pathways.

The autophagy-related genes BbATG1 and BbATG8 have different functions in differentiation, stress resistance and virulence of mycopathogen Beauveria bassiana.

Autophagy-related proteins play significantly different roles in eukaryotes. In the entomopathogenic fungus Beauveria bassiana, autophagy is associated with fungal growth and development. BbATG1 (a serine/threonine protein kinase) and BbATG8 (a ubiquitin-like protein) have similar roles in autophagy, but different roles in other processes. Disruption mutants of BbATG1 and BbATG8 had impaired conidial germination under starvation stress. The mutant ΔBbATG8 exhibited enhanced sensitivity to oxidative stress, while a ΔBbATG1 mutant did not. BbATG1 and BbATG8 showed different roles in spore differentiation. The blastospore yield was reduced by 70% and 92% in ΔBbATG1 and ΔBbATG8 mutants, respectively, and the double mutant had a reduction of 95%. Conidial yield was reduced by approximately 90% and 50% in ΔBbATG1 and ΔBbATG8 mutants, respectively. A double mutant had a reduction similar to ΔBbATG1. Additionally, both BbATG1 and BbATG8 affected the levels of conidial protein BbCP15p required for conidiation. The virulence of each autophagy-deficient mutant was considerably weakened as indicated in topical and intrahemocoel injection assays, and showed a greater reduction in topical infection. However, BbATG1 and BbATG8 had different effects on fungal virulence. Our data indicate that these autophagy-related proteins have different functions in fungal stress response, asexual development and virulence.

Qualitative ubiquitome unveils the potential significances of protein lysine ubiquitination in hyphal growth of Aspergillus nidulans.

Protein ubiquitination is an evolutionarily conserved post-translational modification process in eukaryotes, and it plays an important role in many biological processes. Aspergillus nidulans, a model filamentous fungus, contributes to our understanding of cellular physiology, metabolism and genetics, but its ubiquitination is not completely revealed. In this study, the ubiquitination sites in the proteome of A. nidulans were identified using a highly sensitive mass spectrometry combined with immuno-affinity enrichment of the ubiquitinated peptides. The 4816 ubiquitination sites were identified in 1913 ubiquitinated proteins, accounting for 18.1% of total proteins in A. nidulans. Bioinformatic analysis suggested that the ubiquitinated proteins associated with a number of biological functions and displayed various sub-cellular localisations. Meanwhile, seven motifs were revealed from the ubiquitinated peptides, and significantly over-presented in the different pathways. Comparison of the enriched functional catalogues indicated that the ubiquitination functions divergently during growth of A. nidulans and Saccharomyces cerevisiae. Additionally, the proteins in A. nidulans-specific sub-category (cell growth/morphogenesis) were subjected to the protein interaction analysis which demonstrated that ubiquitination is involved in the comprehensive protein interactions. This study presents a first proteomic view of ubiquitination in the filamentous fungus, and provides an initial framework for exploring the physiological roles of ubiquitination in A. nidulans.

RNA sequencing analysis identifies the metabolic and developmental genes regulated by BbSNF1 during conidiation of the entomopathogenic fungus Beauveria bassiana.

Conidiation promotes fungal dispersal and survival in the environment, and is a determinant for the biocontrol potential of Beauveria bassiana. The SNF1/AMPK protein kinases function as an important regulator of fungal development and energy metabolism, and play a crucial role in conidiation of the filamentous fungi. In previous study, it has been established that the B. bassiana homolog (BbSNF1) controls conidial production. This study showed that the ΔBbSNF1 mutants displayed a delayed development of mycelia and conidia, but the conidiophore morphogenesis was not significantly changed in the mutants. Ablation of BbSNF1 significantly changed the metabolic homeostasis of intracellular amino acids during conidiation, and caused a notable reduction in the contents of seven amino acids (i.e., arginine, alanine, valine, phenylalanine, lysine, leucine, and glutamic acid). All above amino acids could recover conidiation of the mutants in different extents (ranging from 43.3 to 300 %). Transcriptomic analysis revealed many putative target genes regulated by BbSNF1 and associated with conidial development, and these genes were primarily involved in metabolism, cell rescue, and transport. Particularly, four categories related to the amino acid degradation were over-represented in the up-regulated genes, and three categories related to the amino acid biosynthesis were over-represented in the down-regulated genes. Moreover, the ΔBbSNF1 mutants displayed reduced expression level of the upstream and central regulators of conidiation, as well as the other regulator and cytoskeleton genes. Our data indicate that SNF1 kinase contributes to B. bassiana conidiation by regulating the metabolism and the central regulators of conidiation.