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Interaction assays and IP assays suggest that MrATG4 may be regulated by MrKAT1 and MrSIR2–3, potentially through acetylation and deacetylation at lysine residues K69 and K77 (Figs 3, 5, 7 and 8). These findings enhance our understanding of post-translational modifications in autophagy regulation. These specific acetylation sites are critical for MrATG4's proteolytic activity, directly impacting ATG8 processing and autophagosome formation. In contrast, the K149R mutant showed no significant effects on MrATG4 function, indicating that not all predicted acetylation sites contribute equally to ATG4 function, highlighting the specificity of acetylation-mediated regulation.

While our findings establish MrSIR2–3 and MrKAT1 as key regulators of autophagy-mediated conidiation through dynamic acetylation of MrATG4, the upstream signals that activate these opposing enzymatic activities remain unresolved. We hypothesize that environmental cues (e.g., nutrient depletion, oxidative stress) or developmental checkpoints may trigger NAD+-dependent activation of MrSIR2–3 deacetylase activity, while acetyl-CoA availability could regulate MrKAT1 acetyltransferase function. The temporal coordination between these activities likely establishes a reversible acetylation switch that fine-tunes autophagy flux during the vegetative-to-reproductive transition.

Autophagy is essential for conidiation, and deficiencies in ATG1, ATG4, and ATG15 significantly reduce autophagy homeostasis and conidiation in filamentous fungi [4,6,35,36]. In eukaryotes, autophagy is enhanced by stresses or starvation, facilitating the degradation of increasing toxic and damaged components. The recycling of cell material is then utilized for nutrient remobilization [37]. The typical life cycle of filamentous fungi involves asexual conidiation when environmental factors limit the vegetative phase [38]. While our results do not directly demonstrate that autophagy is specifically involved in nutrient acquisition during the early stages of conidiation, they clearly indicate that autophagy is essential for maintaining cellular homeostasis, likely supporting the energy and biosynthetic demands of conidiation. Additionally, we reveal how fungi maintain autophagy homeostasis during the conidiation process through the regulation of ATG4 acetylation dynamics.

The study highlights the potential of targeting acetylation pathways to manipulate fungal conidiation and, by extension, its biocontrol efficacy. Modulating the activity of MrKAT1 or MrSIR2–3 could provide novel strategies to enhance or suppress autophagy, thereby influencing fungal development. This has significant implications for the development of more effective fungi-based biopesticides, offering avenues to optimize spore production. In a broader context, the conservation of acetylation-dependent regulation of ATG4 across eukaryotes suggests that similar mechanisms may be at play in other organisms, including plants and animals. Future studies could explore the evolutionary conservation of this regulatory system and its implications in various biological processes beyond fungal conidiation.

Overall, our study provides comprehensive insights into the molecular mechanisms governing autophagy-mediated conidiation in M. robertsii. Dynamic acetylation of MrATG4, modulated during fungal asexual reproduction, regulates autophagy to facilitate conidiation. Moreover, by elucidating the roles of MrKAT1 and MrSIR2–3 in modulating MrATG4 acetylation, we advance the understanding of post-translational regulation in fungal development and open new avenues for enhancing the application of entomopathogenic fungi in biological control.

Materials and methods

Fungal strains and culture conditions

The M. robertsii strain ARSEF 23 (ATCC no. MYA-3075) was cultured on Potato Dextrose Agar (PDA, BD Difco, BD 213300) for two weeks in the dark. The resulting conidial suspension from this culture served as the starting material for Agrobacterium-mediated transformation (ATMT). Mycelia and conidia were harvested from PDA plates at 1.5 and 2.5 days, respectively, to evaluate autophagic effects at different developmental stages of the fungus. These conditions were employed for subsequent experiments, including Laser Scanning Confocal Microscopy (LSCM, Zeiss LSM 880, Oberkochen, Germany) and immunoblotting.

Bioinformatics analysis

Putative Mrsir2 and Mrkat1 genes were identified from generalist sequences through online BLAST analysis on NCBI. Protein sequences of MrSIR2 were aligned from different fungi including Metarhizium robertsii, M. acridum; M. rileyi; Moelleriella libera; Beauveria bassiana; Fusarium graminearum; Magnaporthe oryzae; Aspergillus fumigatus; Saccharomyces cerevisiae. The SMART program was used for structural comparisons, and phylogenetic analysis was conducted using the neighbor-joining method in MEGA11 [39].

Genes deletion and phenotype assays

Agrobacterium-mediated homologous recombination was employed for the gene knockout of Mrsir2s, Mratg4, and Mrkat1, following our previous studies [39,40]. Specifically, pDHt-bar (conferring glufosinate resistance) was utilized for gene knockout, while pDHt-ben (conveying benomyl resistance) was used for generating the complementary strain (Comp). At least three mutants were obtained for each gene knockout, and one strain was randomly selected for testing. All primers used in this are listed in S2 Table.

Phenotype assays for different strains were conducted as follows: 1 μL aliquots of the conidial suspension (1 × 107 conidia/mL) were centrally spotted onto PDA. Additionally, a conidium suspension of 100 μL was spread on PDA, SDAY, CM plates for conidial production statistics after 7 and 14 days [39].

Deacetylase activity assays

The total protein from each strain, treated with a 100 nM HDAC inhibitor TSA, underwent incubation with a fluorescent AMC peptide (Ac-Arg-His-Lys-Lysac) that was acetylated through Nε-acetylated lysine [41]. Subsequently, the deacetylation activity of the enzyme within the samples was assessed using a fluorescence microplate reader (Synergy HTX, BioTek, America).

Transcriptomics analysis

To delineate the expression patterns of genes, samples from the WT strain were collected at various stages of fungal development. These stages included the induction of two-week-old conidia on PDA and tissues cultured on PDA for 1.5 days, 2.5 days, and 5 days, respectively. RNA-Seq analysis was conducted by the Beijing Genomics Institute (BGI, Shenzhen, China), employing the fragments per kilobase per million mapped reads (FPKM) method as previously described [42]. Additionally, the relative expression of conidiation-associated genes was evaluated after culturing on PDA for 2.5 days [39].

Protein fusions and localization assays

To investigate the subcellular localization of MrSIR2s, we employed a plasmid, pDHt-bar-gapdh-GFP, which carries the glyceraldehyde 3-phosphate dehydrogenase (gapdh) promoter. This plasmid was utilized for protein fusions, where each MrSIR2 was fused to the terminus of GFP [39]. Subsequently, a DAPI staining assay was conducted to facilitate fluorescence observation.

For the evaluation of autophagic levels and the localization of MrATG8 in different strains, we constructed the plasmid pDHt-ben-Np-GFP-MrATG8, which incorporates the native promoter of Mratg8 [4]. The localization assays were observed through LSCM under various culture conditions.

Western blotting analysis

Total protein extraction was carried out using a RIPA lysis buffer (Beyotime, P0013K), and the total protein concentration was estimated using the bicinchoninic acid protein assay kit (Beyotime, P0010). In the context of western blotting, 20 or 30 μg of total proteins were loaded and adjusted. The subsequent western blotting analysis utilized anti-GFP antibody (Abmart, M20004), anti-FLAG antibody (Sigma, F1804). To serve as loading controls, parallel-running SDS-PAGE gels were stained and imaged. After 1.5 d, 2.5 d culture on PDA or SDAY, proteins from WT, ΔMrsir2–3 or ΔMrkat1 strains were immunoprecipitated using anti-FLAG antibody and immunoblotted with pan anti-acetyl-lysine antibody (PTMBiolabs, Hangzhou, China) [21].

Co-IP assays

The Rp27 promoter was incorporated into the pDHt-gapdh-bar plasmid and utilized to generate the plasmids pDHt-gapdh-MrSIR2–3-GFP-bar-Rp27-FLAG-MrATG4 and pDHt-gapdh-MrKAT1-GFP-bar-Rp27-FLAG-MrATG4. The plasmids pDHt-ben-Rp27-FLAG, pDHt-gapdh-GFP-bar-Rp27-FLAG-MrATG4 were constructed and subsequently transformed into the WT or mutant strains to serve as controls. These plasmids were separately introduced into the WT using ATMT. Similarly, point-mutant plasmids, including pDHt-gapdh-MrSIR2–3-GFP-bar-Rp27-FLAG-MrATG4K69R, pDHt-gapdh-MrSIR2–3-GFP-bar-Rp27-FLAG-MrATG4K77R, pDHt-gapdh-MrSIR2–3-GFP-bar-Rp27-FLAG-MrATG4K149R and pDHt-gapdh-MrSIR2–3-GFP-bar-Rp27-FLAG-MrATG4K69,77R, were constructed to examine ATG4 acetylation levels from different point mutant strains.

Total protein was extracted from the transformed strains using a RIPA lysis buffer (Beyotime, P0013K) and subjected to incubation with anti-FLAG Magnetic Beads (Bimake, Shanghai, China) at 4 °C for 10 hours. Immunoblotting was performed using FLAG, GFP or acetyl-lysine antibodies to detect the respective proteins.

Yeast two-hybrid assays

To investigate potential interactions between MrSIR2–3 and ATG8-conjugation system proteins, or between MrATG4 and various acetyltransferase proteins, yeast two-hybrid assays were performed [4]. Specifically, the plasmids pGBKT7-MrSIR2–3 and pGADT7-MrATG3/4/7/8 were constructed and co-transformed into the yeast strain Y2HGold. Additionally,pGADT7-MrATG4 was co-transformed with pGBKT7-Gcn5/Esa1/Kat1/Rtt109/Sas2/Sas3 into Y2HGold, respectively. As controls, the pair of plasmids pGADT7-T and PGBKT7–53 were used as a positive control, while the paired vectors (pGADT7 and pGBKT7-MrSIR2–3; pGADT7-MrATG4 and pGBKT7) served as negative controls. The interactions were assessed using synthetic dropout-Leu-Trp medium and Leu-Trp-His-Ade/X-α-gal/AbA plates.

BiFC assays

The plasmids pDHt-gapdh-MrSIR2–3-YVN-bar-gapdh-MrATG4-YVC and pDHt-gapdh-MrKAT1-YVN-bar-gapdh-MrATG4-YVC were constructed and then separately transformed into the WT. The plasmids pDHt-gapdh-MrSIR2–3-YVN-bar-gapdh-YVC, pDHt-gapdh-YVN-bar-gapdh-MrATG4-YVC, pDHt-gapdh-MrKAT1-YVN-bar-gapdh-YVC and pDHt-gapdh-YVN-bar-gapdh-MrATG4-YVC were constructed and then separately transformed into the WT, as the control. The YFP fluorescence was observed under a LSCM [43].

TEM analysis

Transmission electron microscopy (TEM) was employed to examine the autophagic bodies within the conidia of different strains. The initial conidium production from both the WT and mutant strains were collected, washed, and fixed in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) at 4°C for 16 hours. Following three washes with PBS buffer, the samples were dehydrated and embedded in resin. Subsequently, ultrathin sections of each sample were visualized using a TEM (HT-7700, Hitachi, Japan) operating at 80 kV [4,44].

ATG4 activity assay

ATG4 activity was measured as described previously [45] using the fluorogenic substrate AU4S. Briefly, cells or tissues were lysed in buffer containing 2 mM dithiothreitol (DTT). Lysates were incubated with 0.2 μM AU4S for 40 min at 37°C. Fluorescence intensity was quantified using a luminescence microplate reader (Synergy HTX, BioTek, America).

Statistical analysis

All data presented in this paper are derived from a minimum of three biological replicates. The two-tailed Student's t-test was employed to assess statistical differences between the WT and various mutants [46]. Significance levels are indicated in the Fig legends, with '*' representing p < 0.01.

Supporting information

S1 Fig. Schematic diagram of targeted disruption by homologous recombination approach.

(TIF)

S2 Fig. Verification of gene deletions. (A) PCR verification of Mrsir2–1 gene deletion. (B). PCR verification of Mrsir2–2 gene deletion. (C) PCR verification of Mrsir2–3 gene deletion. (D) PCR verification of Mrsir2–4 gene deletion. Within panels A - D, Δ represents the knockout mutant; WT, the wild-type strain; P, the plasmid containing the gene knockout cassette; R, the randomly insert mutants; M, the DNA marker.

(TIF)

S3 Fig. Quantification of conidial yields. (A) Quantification of conidial yields from different strains after one weeks of growth on PDA. * p < 0.01. (B and C) The conidia production of different strains cultured on CM medium for 7 or 14 days. * p < 0.01.

(TIF)

S4 Fig. Relative expression levels of conidiation-related genes from different strains. Mrgpdah was used as a control.

(TIF)

S5 Fig. BiFC assay for the patterns of MrATG4-MrSIR2-3 and MrATG4-MrKAT1 in vivo. (A-D) All the hypha tips were examined by DIC and fluorescence microscopy. Strains expressing MrATG4-nYFP and empty cYFP, MrSIR2–3-cYFP and empty nYFP, MrKAT1-cYFP and empty nYFP were used as negative controls. Scale bar: 10 μm.

(TIF)

S6 Fig. Co-immunoprecipitation assays for the patterns of MrATG4 in vivo. (A and B) Total proteins, suspensions, and proteins eluted from anti-FLAG agarose from transformants co-expressing Mr-GFP and MrATG4-FLAG. The blots were probed with anti-FLAG or anti-GFP antibodies. T, total; S, suspensions; E, elution.

(TIF)

S7 Fig. The fluorescent intensity of GFP-Atg8 in the ΔMrsir2–3 mutant and WT. (A-B) The laser power of the confocal microscope was set to a range of 5% to 35% to detect the autophagic fluorescence intensity of ATG8-GFP in WT and ∆Mrsir2–3 mutant strains. Scale bar: 10 μm.

(TIF)

S8 Fig. The fluorescence of various mutants expressing GFP-ATG8.

(TIF)

S9 Fig. Bioinformatics analysis and verification of gene deletion of MrKAT1. (A) Schematic representation of KAT1 proteins. Kat1_N, histone acetyl transferase KAT1 N-terminus. (B) Phylogenetic analysis of KAT1-related proteins from several fungi. (C) PCR verification of Mrkat1 gene deletion. Δ represents the knockout mutant; P, the plasmid containing the gene knockout cassette; R, the randomly insert mutants; M, the DNA marker.

(TIF)

S10 Fig. Loading control of ATG4 acetylation assay. (A and B) The Mr-FLAG was constructed into WT, ΔMrsir2–3 and ΔMrkat1 strains. These strains were cultured on PDA or SDAY for 1.5 d, 2.5 d and proteins were immunoprecipitated with antibody to FLAG followed by immunoblotting with antibody to acetylated-lysine.

(TIF)

S11 Fig. For the analysis of GFP-MrAtg8 degradation in the WT and mutant mycelial cells cultured in different medium (A and B) The parallel-running protein gels were stained as references, respectively.

(TIF)

S12 Fig. ΔMratg4 consisted with ΔMratg4K69,77,149R. (A) Colony phenotyping of WT and mutant strains on different media after growth for 14 days at 25 °C. (B) Quantification of conidial yields for those strains. * p < 0.01.

(TIF)

S1 Table. Interacting proteins screening of MrSIR2–3.

(XLSX)

S2 Table. PCR primers used in this study.

(XLSX)

Author contributions

Conceptualization: Deshui Yu, Yulong Wang, Bo Huang.

Investigation: Deshui Yu, Rui Xie, Rong Zhou, Zhenbang Liu, Najie Shi, Jiaojiao Qu.

Methodology: Deshui Yu, Yulong Wang.

Supervision: Xiangyun Xie, Yang Yang.

Writing – original draft: Deshui Yu, Yulong Wang, Guang Yang, Bo Huang.

Writing – review & editing: Deshui Yu, Yulong Wang, Guang Yang, Bo Huang.

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