The Autophagy-Related Gene Aolatg4 Regulates Hyphal Growth, Sporulation, Autophagosome Formation, and Pathogenicity in Arthrobotrys oligospora

Abstract

Autophagy plays an important role in cell growth and development. The autophagy-related gene atg4 encodes a cysteine protease, which can cleave the carboxyl terminus of Atg8, thus plays a role in autophagosome formation in yeast and filamentous fungi. Arthrobotrys oligospora is well known for producing special trapping-devices (traps) and capturing nematodes. In this study, two Δ Aolatg4 mutants were generated using targeted gene replacement and were used to investigate the biological functions of autophagy in A. oligospora . Autophagic process was observed using the AoAtg8-GFP fusion protein. The mutants showed a defective in hyphal growth and sporulation and were sensitive to chemical stressors, including menadione and Congo red. The spore yield of the ΔAolatg4 mutants was decreased by 88.5% compared to the wild type (WT), and the transcript levels of six sporulation-related genes, such as abaA , fluG , brlA , and wetA , were significantly downregulated during the conidiation stage. Deletion of Aolatg4 also affected the cell nuclei and mycelial septal development in A. oligospora . Importantly, autophagosome formation and the autophagic process were impaired in the Δ Aolatg4 mutant. Moreover, the Δ Aolatg4 mutant lost its ability to form mature traps. Our results provide novel insights into the roles of autophagy in A. oligospora .

Transcriptomic Analysis Reveals That Rho GTPases Regulate Trap Development and Lifestyle Transition of the Nematode-Trapping Fungus Arthrobotrys oligospora

“…Of these, 482 genes were annotated as loss or reduction of pathogenicity and lethal. These genes, including signal transduction-related genes (e.g., ste50 , hog1 , and slt2 ), autophagy or mitophagy-related genes (e.g., atg4 , atg15 , and mtor ), small GTPases (e.g., rab7 , rac , and rho2 ), and several other genes (e.g., hog1 , slt2 , atg4 , and rab7 ), were confirmed to contribute to A. oligospora development and pathogenicity ( 13 , 14 , 45 , 46 ). Rho2 and Rac, the small GTPase of the Rho family, regulate cell wall integrity in filamentous fungi ( 44 ).…”

Aolatg1 and Aolatg13 Regulate Autophagy and Play Different Roles in Conidiation, Trap Formation, and Pathogenicity in the Nematode-Trapping Fungus Arthrobotrys oligospora

“…The fungus A. oligospora (ATCC24927) and corresponding mutants were stored in the Microbial Library of the Germplasm Bank of wild species from Southwest China (Kunming, China). Potato dextrose agar (PDA), tryptone glucose (TG), and corn-maizena yeast extract (CMY) were prepared as described previously ( Zhou et al., 2020 ; Zhou et al., 2021 ) and used to analyze fungal phenotypic traits. The complete medium (CM) and MM-N (0.5 g L −1 KCl, 0.5 g L −1 MgSO 4 , 1.5 g L −1 KH 2 PO 4 , 0.1% trace element, and 10 g L −1 glucose; pH 6.5) were used to compare colony growth and induce autophagy under nitrogen starvation ( Talbot et al., 1993 ).…”

“…The WT and mutant strains were incubated on PDA plates at 28°C for 5 days, then transferred onto PDA, CMY, TG, CM, and MM-N plates to evaluate their growth rate under different nutritional conditions, and the diameters of colonies were determined at 24 h intervals ( Zhou et al., 2020 ; Zhou et al., 2021 ). To determine the levels of stress resistance, the fungal strains were incubated on TG plates supplemented with or without (control) different concentrations of chemical stressors, including oxidative agents (H 2 O 2 and menadione) and osmotic agents (NaCl and sorbitol) at 28°C for 7 days.…”

“…To induce trap formation, the conidia of fungal strains were collected from 7-day-old cultures on CMY plates, and 50 μL suspensions (10 6 spores per mL) were incubated on water agar plates at 28°C for 3 days. Then, ~300 nematodes were added to each plate to induce trap formation, followed by microscopic observation of trap formation and nematode predation at 12 h intervals ( Zhou et al., 2020 ).…”

“…Since the sequencing of A. oligospora genome, an increasing number of studies has focused on the mechanism underlying trap formation, and several signaling proteins, such as regulators of G-protein, G-protein subunits and small GTPases, have been shown to regulate trap morphogenesis and lifestyle switch ( Yang et al., 2018 ; Yang et al., 2020 ; Bai et al., 2021 ; Ma et al., 2021 ; Yang et al., 2021 ). Moreover, three orthologous Atg proteins have been identified in A. oligospora : deletion of Aolatg8 blocked autophagy and abolished conidiation and trap formation ( Chen et al., 2013 ), whereas deletion of Aolatg4 and Aolatg5 impaired autophagy and resulted in a reduction in conidia yields, cell nucleus number, and trap production ( Zhou et al., 2020 ; Zhou et al., 2021 ). Despite their conserved features, autophagy proteins have different functions in fungi that are highly divergent morphology and lifestyle ( Pollack et al., 2009 ; Ying and Feng, 2019 ).…”