Fungal insect pathogens have evolved diverse mechanisms to evade host immune recognition and defense responses. However, identification of fungal factors involved in host immune evasion during cuticular penetration and subsequent hemocoel colonization remains limited. Here, we report that the entomopathogenic fungus Beauveria bassiana expresses an endo-β-1,3-glucanase (BbEng1) that functions in helping cells evade insect immune recognition/ responses. BbEng1 was specifically expressed during infection, in response to host cuticle and hemolymph, and in the presence of osmotic or oxidative stress. BbEng1 was localized to the fungal cell surface/ cell wall, where it acts to remodel the cell wall pathogen associated molecular patterns (PAMPs) that can trigger host defenses, thus facilitating fungal cell evasion of host immune defenses. BbEng1 was secreted where it could bind to fungal cells. Cell wall β-1,3-glucan levels were unchanged in ΔBbEng1 cells derived from in vitro growth media, but was elevated in hyphal bodies, whereas glucan levels were reduced in most cell types derived from the BbEng1 overexpressing strain (BbEng1^OE). The BbEng1^OE strain proliferated more rapidly in the host hemocoel and displayed higher virulence as compared to the wild type parent. Overexpression of their respective Eng1 homologs or of BbEng1 in the insect fungal pathogens, Metarhizium robertsii and M. acridum also resulted in increased virulence. Our data support a mechanism by which BbEng1 helps the fungal pathogen to evade host immune surveillance by decreasing cell wall glucan PAMPs, promoting successful fungal mycosis.

Summary: Laccases are widely present in bacteria, fungi, plants and invertebrates and involved in a variety of physiological functions. Here, we report that Beauveria bassiana , an economic important entomopathogenic fungus, secretes a laccase 2 (BbLac2) during infection that detoxifies insect immune response‐generated reactive oxygen species (ROS) and interferes with host immune phenoloxidase (PO) activation. BbLac2 is expressed in fungal cells during proliferation in the insect haemocoel and can be found to distribute on the surface of haemolymph‐derived in vivo fungal hyphal bodies or be secreted. Targeted gene‐knockout of BbLac2 increased fungal sensitivity to oxidative stress, decreased virulence to insect, and increased host PO activity. Strains overexpressing BbLac2 showed increased virulence, with reduced host PO activity and lowered ROS levels in infected insects. In vitro assays revealed that BbLac2 could eliminate ROS and oxidize PO substrates (phenols), verifying the enzymatic functioning of the protein in detoxification of cytotoxic ROS and interference with the PO cascade. Moreover, BbLac2 acted as a cell surface protein that masked pathogen associated molecular patterns (PAMPs), enabling the pathogen to evade immune recognition. Our data suggest a multifunctional role for fungal pathogen‐secreted laccase 2 in evasion of insect immune defenses.

Polymalic acid (PMA) is a novel polyester polymer that has been broadly used in the medical and food industries. Its monomer, L-malic acid, is also a potential C4 platform chemical. However, little is known about the mechanism of PMA biosynthesis in the yeast-like fungus, Aureobasidium pullulans. In this study, the effects of different nitrogen concentration on cell growth and PMA biosynthesis were investigated via comparative transcriptomics and proteomics analyses, and a related signaling pathway was also evaluated. A high final PMA titer of 44.00 ± 3.65 g/L (49.9 ± 4.14 g/L of malic acid after hydrolysis) was achieved in a 5-L fermentor under low nitrogen concentration (2 g/L of NH4NO3), which was 18.3 % higher yield than that obtained under high nitrogen concentration (10 g/L of NH4NO3). Comparative transcriptomics profiling revealed that a set of genes, related to the ribosome, ribosome biogenesis, proteasome, and nitrogen metabolism, were significantly up- or down-regulated under nitrogen sufficient conditions, which could be regulated by the TOR signaling pathway. Fourteen protein spots were identified via proteomics analysis, and were found to be associated with cell division and growth, energy metabolism, and the glycolytic pathway. qRT-PCR further confirmed that the expression levels of key genes involved in the PMA biosynthetic pathway (GLK, CS, FUM, DAT, and MCL) and the TOR signaling pathway (GS, TOR1, Tap42, and Gat1) were upregulated due to nitrogen limitation. Under rapamycin stress, PMA biosynthesis was obviously inhibited in a dose-dependent manner, and the transcription levels of TOR1, MCL, and DAT were also downregulated. The level of nitrogen could regulate cell growth and PMA biosynthesis. Low concentration of nitrogen was beneficial for PMA biosynthesis, which could upregulate the expression of key genes involved in the PMA biosynthesis pathway. Cell growth and PMA biosynthesis might be mediated by the TOR signaling pathway in response to nitrogen. This study will help us to deeply understand the molecular mechanisms of PMA biosynthesis, and to develop an effective process for the production of PMA and malic acid chemicals.