Reduced fungal protein acetylation mediates the antimicrobial activity of a rhizosphere bacterium against a phytopathogenic fungus

Zhang, Ying-Chao; Zhan, Xin; Chen, Jun-Yu; Yu, Ding-Tian; Zhang, Tao; Zhang, Huiming; Duan, Cheng-Guo

doi:10.1038/s41467-025-60870-7

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Published: 01 July 2025

Reduced fungal protein acetylation mediates the antimicrobial activity of a rhizosphere bacterium against a phytopathogenic fungus

Nature Communications volume 16, Article number: 5644 (2025) Cite this article

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An Author Correction to this article was published on 19 August 2025

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Abstract

Rhizosphere microbes can protect plants from phytopathogens, but the molecular mechanisms are often poorly understood. Here, we report that a rhizosphere bacterium, Bacillus amyloliquefaciens strain TG1-2 displays antimicrobial activity against various phytopathogenic fungi and oomycetes, in a process that is mediated by the NatA acetyltransferase complex in the phytopathogenic fungus Verticillium dahliae. We show that acetylation of the molecular chaperone Hsp83 by NatA facilitates the formation of a co-chaperone complex Hsp83-Sti1-Hsp70 involved in protein quality control. Dysfunction of NatA or disruption of Hsp83 acetylation results in dissociation of the co-chaperon complex, increasing protein degradation and fungal apoptosis. Notably, TG1-2 and its major antimicrobial compound surfactin induce a reduction in Hsp83 acetylation, enhancing protein degradation and fungal apoptosis. Thus, our study provides insights into the mechanisms underlying the antimicrobial action of a rhizosphere strain against phytopathogenic fungi.

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Introduction

In recent years, substantial research has been conducted on plant host-associated microbiomes, with diverse microbiomes inhabiting the rhizosphere soil and playing crucial roles in plant growth and health^1,2,3,4. Beneficial microorganisms in the rhizosphere are widely utilized as biocontrol agents (BCAs) for disease prevention, highlighting their significant importance in agriculture. However, most studies have concentrated on how plants recruit beneficial microorganisms or how these microorganisms colonize plants⁵, while the molecular mechanisms by which pathogens resist BCAs remain poorly understood. Gaining insight into these mechanisms is essential for optimizing the use of BCAs in agricultural practices.

To coordinate their actions and regulate growth and antimicrobial activity, rhizosphere microorganisms produce a diversity of signaling molecules, primarily chemical compounds such as N-acyl homoserine lactones (AHLs), diffusible signal factors (DSFs), diketopiperazines, volatile organic compounds, and antibiotics^6,7,8. In addition to these chemical signals, small RNAs have emerged as key signaling molecules that shuttle between phytopathogens and plant hosts to regulate their interaction^9,10,11. Antibiotics produced by biocontrol bacteria, including Pseudomonas, Bacillus, and Trichoderma, have demonstrated anti-phytopathogen effects. These antibiotics include pseudoiodinine, lipopeptides, and chitinase^12,13,14. The inhibitory mechanisms of antibiotics on phytopathogens are usually achieved by affecting the permeability of the cell membrane¹⁵. However, their impact on signaling pathways within phytopathogens remains largely unexplored.

Protein acetylation plays a significant role in the complex interactions between plants and pathogens. This post-translational modification influences key proteins involved in various cellular processes, including protein quality control, signaling cascades, and stress responses. In the context of plant defense, protein acetylation acts as a molecular switch that can shape the plant’s response to phytopathogens, ultimately affecting disease susceptibility and the overall health of the plant. For example, a histone deacetylase inhibitor produced by the fungal pathogen Cochliobolus carbonum race 1, known as HC-toxin, enhances virulence in maize by modulating host protein acetylation¹⁶. Understanding the role of protein acetylation in phytopathogens is essential for unraveling the molecular mechanisms underlying pathogen virulence, adaptation to host environments, and evasion of plant immune responses. FolSir2, a cytoplasmic deacetylase encoded by the fungal pathogen Fusarium oxsysporum f. sp. lycopersici, promotes the deacetylation of FolGsk3, a serine/tyrosine kinase involved in various cellular functions, thereby facilitating Fol pathogenicity¹⁷. Acetylation of bacterial and fungal effectors has been reported to enhance their virulence toward the host^18,19. Recent reports indicate that the effector Fol-Secreted Virulence-related Protein 1 (FolSvp1) can translocate the tomato pathogenesis-related protein 1 (SlPR1) from the apoplast to the host nucleus, leading to the inhibition of SlPR1-meidated defense. The lysine acetyltransferase FolArd1 is required for the action of FolSvp1²⁰. In interactions between bacteria and fungi, antagonistic bacteria can inhibit the growth and virulence of Fusarium graminearum by manipulating fungal histone modification²¹. Therefore, targeting acetylation processes in phytopathogens could provide novel methods for developing innovative disease control strategies that disrupt pathogen virulence factors or enhance their susceptibility to biocontrol agents.

Verticillium dahliae Kleb., a major soil-borne pathogenic fungus responsible for vascular wilts in over 200 plant species, poses a significant threat to many economically important crops, such as cotton^22,23. V. dahliae produces microsclerotia in senescent plants, surviving in the soil and infecting hosts when stimulated by root exudates^24,25,26. The fungus’s ability to form long-lasting microsclerotia complicates control efforts, particularly as soil fumigation with methyl bromide raises environmental concerns²². Utilizing BCAs presents an eco-friendly solution for managing V. dahliae-induced wilt disease.

In this study, we isolated the Bacillus amyloliquefaciens strain TG1-2 from plant rhizosphere samples, demonstrating broad-spectrum antimicrobial activity against various phytopathogens, including fungi and oomycetes. Through genetic screening for mutants hypersensitive to TG1-2, we established that the NatA acetyltransferase complex promotes tolerance in the phytopathogenic fungus V. dahliae against TG1-2-induced apoptosis. The acetylation of the molecular chaperone VdHsp83 by the NatA complex is crucial for the formation of the Hsp83-Sti1-Hsp70 co-chaperone complex, which is involved in protein quality control. The loss of the NatA complex subunits, N-Acetyltransferase 1 (Nat1) and Arrest-defect-1 (Ard1), resulted in increased susceptibility to TG1-2, while overexpression of these subunits exhibited the opposite effect. Importantly, TG1-2 inhibits VdHsp83 acetylation and disrupts protein homeostasis by negatively regulating the expression of Nat1 and Ard1, leading to dysregulation of the co-chaperone complex, enhanced protein degradation, and fungal apoptosis. Our findings reveal an antagonistic mechanism employed by phytopathogenic fungi in response to the antimicrobial activity of the biocontrol bacterium.

Results

B. amyloliquefaciens TG1-2 exhibits strong broad-spectrum inhibitory activity against a range of phytopathogens

Through phylogenetic analysis of 16S rRNA sequences, TG1-2 was identified as belonging to the genus Bacillus amyloliquefaciens, closely related to strain NBRC 15539 (Supplementary Fig. 1). To evaluate its inhibitory potential, TG1-2 was tested against a variety of phytopathogens, including the pathogenic fungi V. dahliae (isolates V592 and VdLs-17), Magnaporthe oryzae (isolates Fj-86 and Guy-11), Fusarium graminearum, Botrytis cinerea, Sclerotinia sclerotiorum, and the pathogenic oomycete Phytophthora sojae. GB03, another member of the Bacillus amyloliquefaciens genus known for its antibacterial and antifungal effects on phytopathogens^27,28,29, was also subjected to an inhibition assay as a positive control. The growth of colonies was monitored at various time points post-culture. Co-culture assays on potato dextrose agar (PDA) medium demonstrated that TG1-2 exhibited significant inhibition of all tested pathogens (Fig. 1a, b, and Supplementary Fig. 2). TG1-2 displayed a similar inhibitory effect to GB03 against most of the tested pathogens but exhibited stronger inhibitory activity than GB03 for certain pathogens, including V. dahliae-V592 and B. cinerea (Fig. 1a, b, and Supplementary Fig. 2). We next analyzed whether TG1-2 could reduce the virulence of phytopathogens during plant infection. To achieve this, cotton and Arabidopsis plants were pre-treated with TG1-2 and subsequently inoculated with V. dahliae-V592 (Fig. 1c). Observations of disease symptoms indicated that the V592 strain pre-treated with TG1-2 exhibited significantly reduced pathogenicity compared to the untreated strain (Fig. 1d, e, and Supplementary Figs. 3a, b), highlighting the biocontrol potential of TG1-2 against phytopathogenic fungi and oomycetes.

**Fig. 1: TG1-2 has broad-spectrum inhibitory activity against phytopathogenic fungi and oomycetes.**

It has been reported that certain bacterial volatile compounds can inhibit fungal development^30,31,32. To eliminate the possibility that the antimicrobial activity is primarily due to the inhibitory effects of volatile substances secreted by TG1-2, a bipartite plate co-culture experiment was conducted. The results indicated that there was no significant difference in the colony size of V. dahliae-V592 between the plates cultured with GB03 or TG1-2 compared to those cultured with DH5α or without bacteria (Supplementary Fig. 4), indicating that the antimicrobial activity of TG1-2 is not attributable to volatile substances.

Surfactin is the major antimicrobial compound produced by TG1-2

Next, we evaluated the antimicrobial activity of the culture filtrate. Dual-culture analysis revealed that the TG1-2 culture filtrate (TG1-2cf) exhibited strong inhibitory effects against V592 (Fig. 2a). Additionally, when applied to PDA plates, TG1-2 culture filtrate significantly suppressed the growth of V592 compared to the control bacterium DH5α, as illustrated in Fig. 2b, c. The diameter of the inhibition zone increased from 1.5 cm on the fifth day to 3.1 cm by the eleventh day (Fig. 2c). Notably, significant growth inhibition was also observed for seven other phytopathogens (Supplementary Fig. 5). These findings suggest that the antimicrobial substances in TG1-2 are derived from the culture filtrate and that TG1-2cf possesses broad-spectrum antimicrobial activity against most phytopathogenic fungi.

To identify the antimicrobial compound produced by TG1-2, high-performance liquid chromatography-time of flight mass spectrometry (HPLC-TOF MS) was conducted. The analysis revealed that all detected metabolites belonged to 15 chemical taxonomies (Fig. 2d). Among these,surfactin b and surfactin c, classified as organic acids and derivatives, were significantly upregulated in TG1-2, exhibiting a fold change exceeding 2500 compared to the control group, E. coli (Fig. 2e and Supplementary Fig. 7). By comparing the mass spectrum with a standard sample, we confirmed that these compounds were indeed surfactin (Fig. 2f). To validate the antimicrobial activity of surfactin, phytopathogens were cultured on plates containing 200 μM surfactin. As anticipated, the growth of all tested pathogens was significantly inhibited by surfactin (Fig. 2g, h, and Supplementary Fig. 8). Thus, our findings establish surfactin as the primary antimicrobial compound produced by TG1-2.

Under field conditions, crops frequently experience high-temperature stress. To investigate whether the antimicrobial activity mediated by surfactin can be sustained at elevated temperatures, we first pretreated the culture filtrate and surfactin at 65 °C before adding them to PDA medium, respectively. The results indicated that both the TG1-2 culture filtrate and surfactin exhibited strong inhibitory activity against all tested pathogens (Supplementary Fig. 6), suggesting that the antimicrobial activity of TG1-2 can withstand temperature increases within a certain range.

The NatA acetyltransferase complex enhances the tolerance of V. dahliae to TG1-2

To identify the genes responsible for TG1-2-mediated inhibitory activity, a library of T-DNA insertion mutants of V592 was screened for sensitivity to TG1-2cf (Fig. 3a). Three mutants were identified that exhibited enhanced sensitivity to TG1-2cf (Fig. 3b and Supplementary Fig. 9). Through tail-PCR, these mutations were mapped to VdNat1 (VDAG_07819) and two uncharacterized genes, VDAG_06189 and VDAG_06587. Among the three T-DNA mutants, the VdNat1-T mutant displayed the most pronounced inhibitory effect compared to the other two mutants. We also investigated their sensitivity to surfactin. The quantitative data demonstrated that only the VdNat1-T mutant exhibited enhanced inhibition of V. dahliae growth following surfactin treatment. No significant difference in inhibitory effect was observed between the wild-type V. dahliae and the mutants of VDAG_06189 and VDAG_06587, indicating that the enhanced sensitivity to surfactin is specific to VdNat1. Therefore, we focused on the VdNat1 gene in subsequent studies. To confirm the role of VdNat1 in V. dahliae tolerance to TG1-2, a knockout mutant of VdNat1, designated VdΔnat1, was generated according to a previously reported method¹¹. 11. Co-culture results confirmed its hypersensitivity to TG1-2cf compared to the wild-type V592 strain (Fig. 3c, d). Conversely, overexpression of VdNat1 in V. dahliae substantially increased its tolerance, highlighting the role of VdNat1 in promoting V. dahliae tolerance to TG1-2.

**Fig. 3: NatA acetyltransferase complex functions in *V. dahliae* tolerance to TG1-2.**

VdNat1 encodes a subunit of the NatA acetyltransferase complex, which directs the nascent polypeptide to the catalytic subunit Ard1³³. Interaction analyses using yeast two-hybrid (Y2H) and split luciferase assays confirmed the interaction between VdNat1 and the V. dahliae homolog of Ard1 (Fig. 3e, f). Dysfunction of VdNat1 and VdArd1 resulted in a significant reduction in overall acetylated protein levels (Fig. 3g), indicating the functional role of the NatA complex in protein acetylation. To validate the complex’s function in V. dahliae tolerance to TG1-2, knockout (VdΔard1) and overexpression (VdArd1OE) strains of VdArd1 were generated. Similar to the VdΔnat1 mutant, the VdΔard1 mutant exhibited increased sensitivity to TG1-2cf, while the VdArd1OE strain demonstrated reduced sensitivity (Fig. 3h, i), highlighting the role of the NatA complex in enhancing V. dahliae tolerance to TG1-2.

Next, we investigated whether the TG1-2 culture filtrate and surfactin play a role in the overall protein acetylation levels. To this end, the wild-type V592 strain was treated with either TG1-2cf or 200 μM surfactin for 48 h. Compared to the untreated control, both TG1-2cf and surfactin treatments significantly decreased the overall protein acetylation levels (Fig. 3j), suggesting that surfactin has a substantial effect on protein acetylation homeostasis. Consistent with this hypothesis, both the RNA and protein levels of VdNat1 and VdArd1 were significantly reduced following TG1-2cf treatment (Fig. 3k, l) or surfactin treatment (Supplementary Fig. 10). These data strongly support the notion that the NatA acetyltransferase complex plays a critical role in V. dahliae tolerance to TG1-2, which, in turn, counteracts the NatA complex to antagonize V. dahliae tolerance.

The NatA complex functions as an antagonist to TG1-2-induced apoptosis in V. dahliae

Protein acetylation plays a pivotal role in various cellular processes, and its dysregulation can impede cell growth and trigger apoptosis^34,35. To assess whether TG1-2 can induce apoptosis in V. dahliae and the involvement of the NatA acetyltransferase complex in this process, we examined apoptosis in V592, VdΔnat1, and VdΔard1 before and after surfactin treatment using Fluorescein (FITC)-12-dUTP labeling. Our results indicated a substantial increase in apoptotic V. dahliae cells following treatment with TG1-2cf and surfactin compared to untreated cells, underscoring the apoptotic-inducing capability of surfactin (Fig. 4a, b, and Supplementary Fig. 11). Notably, the deletion of VdNat1 and VdArd1 significantly heightened surfactin-induced apoptosis, suggesting that the NatA acetyltransferase complex counteracts TG1-2-induced fungal apoptosis, thereby augmenting V. dahliae tolerance to TG1-2.

**Fig. 4: NatA complex antagonizes TG1-2-induced *V. dahliae* apoptosis.**

The NatA complex facilitates the acetylation of the molecular chaperone Hsp83

Acetylation mediated by the NatA complex is a prevalent post-translational protein modification known to impact cellular survival³⁶. To identify acetylated substrates of the NatA complex relevant to TG1-2 tolerance, Nat1 immunoprecipitation-mass spectrometry (IP-MS) was performed (Supplementary Data 1) to investigate the interaction of VdNat1 with co-purified proteins. Notably, the Y2H assay, split luciferase assay, and co-immunoprecipitation (co-IP) assay demonstrated that the molecular chaperone protein VdHsp83 (encoded by VDAG_04645) interacts with VdNat1 in vivo. VdHsp83, which is homologous to mammalian Hsp90 (Supplementary Fig. 12), is essential for protein folding and maturation within the molecular chaperone system, a critical regulator of protein homeostasis in eukaryotes^37,38.

The intriguing interaction between VdHsp83 and VdNat1 prompted further investigation into whether VdHsp83 is a target of acetylation by the NatA complex. In an in vitro acetylation assay, the purified recombinant VdHsp83 protein was found to be acetylated by VdArd1 (Fig. 5d, left panel). To identify the target sites on VdHsp83, purified GST-VdHsp83 protein was subjected to an in vitro acetylation assay, followed by digestion into peptides and subsequent micro-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Additionally, a VdHsp83 immunoprecipitation assay followed by LC-MS/MS was performed. The data indicated that two peptides containing Lysine 381 and Lysine 410 (K381 and K410) were acetylated (Fig. 5e) and exhibited increased acetylation levels in the presence of VdArd1 protein (Supplementary Data 2). To confirm these results, both peptides were biosynthesized and subjected to an in vitro acetylation assay. In the presence of VdArd1, both K381- and K410-containing peptides were acetylated (Fig. 5d, middle and right panels). Mutagenesis of K381 and K410 to deacetylation-mimic arginine (K381R and K410R, respectively) significantly reduced the acetylation levels of VdHsp83 peptides in the in vitro acetylation assay (Fig. 5d), confirming K381 and K410 as specific target sites for VdArd1-mediated acetylation of VdHsp83.

**Fig. 5: The NatA complex acetylates Hsp83 at the K381 and K410 residues.**

Hsp83 acetylation enhances the tolerance of V. dahliae to TG1-2

In mammalian cells, the acetylation of molecular chaperones plays a crucial role in maintaining protein homeostasis during cellular stress³⁶. Next, we investigated whether the acetylation of Hsp83 contributes to fungal tolerance to TG1-2. To this end, we mutated the K381 and K410 residues of VdHsp83 to deacetylation-mimic arginine (K381R and K410R) and acetylation-mimic glutamine (K381Q and K410Q), respectively. These mutated Hsp83 proteins were then complemented into the knockout VdΔhsp83 strain. A growth inhibition assay was conducted on plates containing TG1-2cf to assess the sensitivity of the different strains. The results demonstrated that both the K381R and K410R mutant strains exhibited increased sensitivity to TG1-2cf compared to the V592 strain (Fig. 6a and Supplementary Fig. 13). Furthermore, the growth inhibition rates were even higher in the K381/410 R double mutant compared to the single mutants. In contrast, there was no significant change in sensitivity between the K381Q and K410Q mutants and the V592 strain (Fig. 6a and Supplementary Fig. 13). These findings strongly suggest that the acetylation of VdHsp83 enhances the tolerance of V592 to TG1-2.

**Fig. 6: Hsp83 acetylation promotes the tolerance of V592 and is essential for the inhibition of TG1-2-induced apoptosis.**

Hsp83 acetylation is crucial for inhibiting TG1-2-induced apoptosis in V. dahliae

Next, we further investigated whether the acetylation of VdHsp83 plays a role in the NatA complex-mediated inhibition of TG1-2- and surfactin-induced apoptosis in V. dahliae. As depicted in Fig. 6b, c, and Supplementary Fig. 14, the acetylation-mimic mutation strains K381Q and K410Q did not exhibit signs of apoptosis on standard culture medium. When grown on culture medium containing TG1-2cf or surfactin, these strains displayed apoptosis levels comparable to those of the V592 and VdHsp83-Flag/VdΔhsp83 strains. In contrast, the deacetylation-mimic strains K381R and K410R showed mild apoptosis even when cultured on standard medium (Fig. 6b, c, and Supplementary Fig. 14). Upon exposure to culture medium containing TG1-2cf or surfactin, the levels of apoptosis significantly increased compared to V592, with further enhancement observed in the double mutant. Based on these results, we conclude that NatA complex-mediated acetylation of VdHsp83 is crucial for preventing TG1-2-induced apoptosis in V. dahliae.

Hsp83 acetylation controls the binding complexes of co-chaperones associated with Hsp83

In mammals, acetylated Hsp70 binds to the co-chaperone Hop and Hsp90 to facilitate protein refolding. The deacetylation of Hsp70 diminishes its capacity to bind with Hop and Hsp90, resulting in protein degradation³⁶. Our investigation revealed that V. dahliae Hsp83 is the homolog of mammalian Hsp90 (Supplementary Fig. 12), leading us to speculate that the acetylation of VdHsp83 may also affect protein homeostasis by modulating its interactions with homologous proteins such as VdHop and VdHsp70. Sti1 serves as the homolog of mammalian Hop1 in V. dahliae. Y2H and split luciferase assays indicated that both VdHsp83 and VdHsp70 can interact with VdSti1 (Fig. 7a, b). However, no direct interaction was observed between VdHsp70 and VdNat1 (Fig. 7a) or between VdHsp83 and VdHsp70 (Supplementary Fig. 15). This suggests that VdHsp83 may form a co-chaperone complex with VdHsp70 and VdSti1, thereby contributing to protein degradation.

**Fig. 7: Hsp83 acetylation stabilizes the co-chaperone complex to regulate protein homeostasis.**

Subsequently, we investigated whether the acetylation of VdHsp83 is necessary for the formation of the VdHsp83 co-chaperone complex. To this end, VdSti1-GFP was expressed in the wild-type V592 strain, as well as in the VdHsp83-Flag/VdΔhsp83, K381/410R-Flag/VdΔhsp83, and K381/410Q-Flag/VdΔhsp83 strains. The binding affinity of VdHsp83 to the co-chaperone VdSti1 was assessed. Co-IP assays demonstrated that the deacetylation-mimic K381/410R mutation significantly reduced the binding affinity to Sti1 compared to wild-type VdHsp83-Flag, while the acetylation-mimic K381/410Q mutation did not visibly affect the binding affinity (Fig. 7c). To further confirm this result, competitive Co-IP assays were also performed in the VdΔard1 strain, as well as in TG1-2cf and surfactin-treated V592 strains. As shown in Fig. 7d, the loss of function of VdArd1 significantly reduced the binding affinity of Hsp83 to Sti1 and Hsp70 (left panel). Similar reductions were also observed in V592 strains treated with TG1-2cf (middle panel) or surfactin (right panel). Therefore, we conclude that the acetylation of VdHsp83 is essential for the formation of the co-chaperone complex, and that the NatA complex and surfactin play a substantial role in this process.

Next, we investigated whether the acetylation-mediated formation of the Hsp83 co-chaperone complex plays a role in regulating protein homeostasis. To this end, we examined ubiquitin-mediated protein degradation in Hsp83 complementation strains as well as in acetylation/deacetylation-mimic strains. The results indicated that the Hsp83 and K381/410Q strains displayed similar levels of protein degradation, while the deacetylation-mimic K381/410 R strain exhibited a significantly higher level of protein degradation (Fig. 7e). This suggests that Hsp83 acetylation is essential for maintaining protein homeostasis of V. dahilae. We also investigated protein degradation levels in the TG1-2cf/surfactin-treated V592 strains and in the VdΔard1 mutant strain. Consistent with the previous results, the protein degradation level was dramatically increased in the VdΔard1 mutant strain and in the V592 strains treated with TG1-2cf or surfactin compared to the untreated V592 strain (Fig. 7e). This suggests that VdArd1-mediated acetylation is crucial for protein degradation, and that both TG1-2 culture filtrate and surfactin can enhance protein degradation by disrupting VdArd1-mediated Hsp83 acetylation

Based on the data presented above, we propose a working model of the NatA acetyltransferase complex-mediated acetylation in V. dahliae tolerance to the beneficial bacterium TG1-2 (Fig. 7f). In this model, VdNat1 and VdArd1 function within the NatA acetyltransferase complex to acetylate the molecular chaperone VdHsp83, thereby promoting the formation of the co-chaperone complex Hsp83-Sti1-Hsp70. This Hsp83-Sti1-Hsp70 complex facilitates protein refolding, which leads to increased tolerance to TG1-2. Conversely, TG1-2 induces the downregulation of VdNat1 and VdArd1, disrupting the acetylation of Hsp83. This disruption results in the dysregulation of the Hsp83-Sti1-Hsp70 complex, ultimately promoting protein degradation and cellular apoptosis.

Discussion

A growing body of research indicates that beneficial microorganisms play a crucial role in promoting plant growth and controlling plant diseases^3,39,40. For soil-borne diseases, the development and utilization of rhizosphere biocontrol microbial agents represent effective and environmentally friendly strategies for disease prevention and control. In this study, we identified a biocontrol bacterium strain, TG1-2, from Bacillus amyloliquefaciens, which exhibits broad-spectrum and high inhibitory activity against phytopathogenic fungi and oomycetes. We provided evidence that surfactin is the primary antimicrobial compound produced by TG1-2. Notably, compared to the reported biocontrol strain GB03 from B. amyloliquefaciens, which is a member of B. subtilis, TG1-2 exhibited stronger inhibitory effects on various phytopathogens. Therefore, TG1-2 has the potential to serve as an effective biocontrol strain for managing phytopathogenic diseases.

Over the past decade, the majority of studies on plant-microbiome interactions have concentrated on how plants recruit beneficial bacteria and how these bacteria colonize plants⁵. However, the mechanisms by which pathogens counteract the antimicrobial effects of beneficial bacteria remain poorly understood. Gaining insight into this aspect is essential for developing effective biocontrol strategies against pathogens. In this study, we screened hypersensitive mutants for their response to TG1-2 and uncovered an antagonistic mechanism in the phytopathogenic fungus V. dahliae. We found that the NatA acetyltransferase complex promotes V. dahliae’s tolerance to TG1-2. Notably, a significant reduction in the inhibitory effect was observed in V. dahliae strains overexpressing the NatA complex subunits VdArd1 and VdNat1 (Fig. 3c, h). In this mechanism, the NatA complex directly acetylates the molecular chaperone Hsp83, and the acetylated Hsp83 facilitates the formation of a co-chaperone complex, thereby antagonizing protein degradation and preventing cell apoptosis.

Post-translational modification, an important molecular marker, has been shown to dynamically regulate multiple cellular functions of various proteins. Lysine acetylation is a type of post-translational modification that can occur on both histones and non-histone proteins^41,42,43. A growing body of research has confirmed that lysine acetylation plays a role in intra- and interspecific interactions. Bacterial and fungal effectors have been found to undergo acetylation, and the loss of this modification reduces the effectors’ virulence toward the host^18,19. Histone acetylation has been reported to be involved in the interactions between bacteria and fungi. For example, antagonistic bacteria can inhibit the growth and virulence of Fusarium graminearum by manipulating fungal histone modifications²¹. In this study, we revealed that the acetylation of the molecular chaperone Hsp83 at two conserved lysine sites, catalyzed by the NatA acetyltransferase complex, is essential for the formation and function of the co-chaperone complex Hsp83-Sti1-Hsp70 in regulating protein homeostasis and the tolerance of V. dahliae to the biocontrol bacterium TG1-2. Additionally, the two subunits of the NatA complex, VdArd1 and VdNat1, were downregulated by TG1-2 at both the RNA and protein levels, indicating that the transcriptional regulation of NatA complex-mediated fungal acetylation is an important strategy employed by biocontrol bacteria to inhibit phytopathogenic fungi. Furthermore, in addition to acetylation, other protein modifications also play a role in regulating the chaperone-mediated protein triage decision between refolding and degradation. For instance, in mammalian cells, it has been reported that the phosphorylation of Hop decreases its binding affinity to Hsp70⁴⁴. Therefore, it is reasonable to speculate that there may be interactions between these two modifications that jointly coordinate the regulation of protein homeostasis. This warrants further investigation in future studies. Intriguingly, we noticed that knocking out VdNat1 and VdArd1 would produce a certain degree of apoptosis, even without surfactin treatment (Fig. 6). In humans, human arrest defective 1 (hARD1) and N-acetyltransferase human (NATH) are the homologs of VdArd1 and VdNat1. In a previous study³⁵, the authors found that RNAi-based knockdown of hARD1 and/or NATH triggers apoptosis in human cell lines. They also found that knockdown of hARD1 also sensitized cells to daunorubicin-induced apoptosis. In this case, knockdown of hARD1 and/or NATH also induces an increase of apoptosis without treatment. As the NATH-hARD1 complex-mediated N-terminal acetylation is a co-translational process associated with ribosome, it is speculated that NATH-hARD1 or VdNat1-VdArd1 complex affect apoptosis through the ribosomal-linked acetylation activity. It has been documented those post-translational modifications are closely correlated with activation of apoptosis⁴⁵. Thus, we speculate that VdNat1-VdArd1 module may utilize similar mechanism to regulate protein homeostasis and apoptosis.

To summarize, we identified the B. amyloliquefaciens strain TG1-2 as a potential biocontrol agent with broad-spectrum antimicrobial activity against various phytopathogens. Surfactins have been revealed as the primary antimicrobial compounds. Importantly, our data demonstrate that the NatA acetyltransferase complex plays a crucial role in establishing TG1-2’s tolerance to the pathogenic fungus V. dahliae. The acetylation of the molecular chaperone Hsp83 in V. dahliae by the NatA complex promotes the formation of a co-chaperone complex consisting of Hsp83, Sti1, and Hsp70, which regulates protein homeostasis and counteracts TG1-2-induced apoptosis. Disruption of Hsp83 function leads to the dissociation of the co-chaperone complex and an increase in apoptosis. Notably, the NatA complex-mediated acetylation serves as a target site for TG1-2 to enhance fungal apoptosis. These findings provide new targets for the development of effective bacterial biopesticides against phytopathogenic fungi.

Methods

Isolation, identification, and anti-microbial activity of TG1-2

Bacteria were isolated from rhizosphere samples collected from weeds thriving in high-salinity environments in Fujian, China. Soil samples were suspended in 0.85% NaCl, and the culture filtrate was plated on LB agar for cultivation at 28 °C. Once a single colony had developed, it was transferred to a new LB plate until no extraneous bacteria were present around the colony. The isolated strain was then preserved in 50% glycerol. To evaluate the inhibitory activity of TG1-2 against V592, a co-culture assay was performed on potato dextrose agar (PDA), which provides nutrients for both bacterial and fungal growth. The PDA plate had a diameter of 9 cm, with bacteria cultured in the center and fungi cultured around the perimeter. Additionally, fungi were also cultured in the center with bacteria surrounding them. The inhibition zones were measured after 5 days of co-culture at 28 °C. Furthermore, the antagonistic activities of TG1-2 against eight other plant pathogens were assessed, with GB03 serving as a positive control. All strains were tested in triplicate, and all pathogen strains were obtained from our laboratory’s strain collection. To identify TG1-2, the 16S rRNA gene was amplified using the universal primers 27 F and 1492 R and sequenced by Sangon Biotech (Shanghai, China). The resulting sequence was compared against the National Center for Biotechnology Information (NCBI) database. The analysis identified 19 Bacillus species with a homology greater than 90%. To further investigate the phylogenetic relationship between TG1-2 and the 19 Bacillus species, a phylogenetic tree was constructed using MEGA 5.0 software, employing the maximum likelihood method⁴⁶.

Inhibitory activity of TG1-2 secondary metabolites

The inhibitory effect of TG1-2 volatile compounds on phytopathogens was examined using a co-culture method on a bipartite plate. Bacteria were cultured on one side of the plate, while fungi were cultured on the other side. A horizontal partition was placed in the middle of the plate to prevent direct contact between the bacteria and fungi.

To assess the inhibitory activity of the TG1-2 culture filtrate, TG1-2 was cultivated in 300 ml of LB broth until an optical density (OD) of 3.0 was reached at 28 °C and 220 rpm. The culture filtrate was subsequently obtained through centrifugation and filtration. This filtrate was then added to a PDA plate with a diameter of 6 cm at a 20% concentration. Activated pathogens were transferred to the center of the plate and incubated at 28 °C. To evaluate whether the inhibitory activity of the TG1-2 culture filtrate and surfactin was influenced by high temperature, both the culture filtrate and surfactin were heated to 65 °C for 2 h before being added to PDA plate.

Characterization of the secondary metabolites in TG1-2

To prepare the sample for mass spectrometry analysis, the culture filtrate of TG1-2 was mixed with pre-cooled methanol, acetonitrile, and water in a volume ratio of 2:2:1. This mixture was vortexed and then subjected to low-temperature ultrasound for 30 min. After standing at −20 °C for 10 min, the mixture was centrifuged at 14,000 × g at 4 °C for 20 min. The resulting culture filtrate was vacuum-dried. For mass spectrometry analysis, 100 μL of a 50% acetonitrile aqueous solution was added to the dried culture filtrate, followed by vortexing and centrifugation at 14,000 × g at 4 °C for 15 min. High-performance liquid chromatography (HPLC) was employed to separate the components of the culture filtrate, and a Triple TOF mass spectrometer was used to collect spectra. The mass spectrometry data were analyzed using XCMS software. Additionally, the secondary metabolites of the E. coli strain DH5α were detected as a control group. After sum normalization, the processed data were analyzed using the R package (ropls, version 3.6.3). Multivariate analysis, including Pareto-scaled principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA), was conducted. A seven-fold cross-validation combined with response permutation testing was used to assess model robustness. The variable importance in projection (VIP) values from the OPLS-DA model were calculated to determine each variable’s contribution to the classification. Student’s t-test was applied to evaluate differences between two groups of independent samples, and metabolites with VIP > 1 and p < 0.05 were considered significantly altered. Pearson’s correlation analysis was performed to assess the relationships between variables. The identified secondary metabolites could be purchased metabolites (MedChemExpress, #HY-129555) and were added to PDA plate at concentrations of 50 μM, 100 μM, and 200 μM. Subsequently, the activated pathogen was transferred to the plate and cultured at 28 °C for 5 days.

V. dahliae infection analysis and fungal biomass measure in planta

A. thaliana (Col-0) and the cotton cultivar Xinluzao No. 16, known for its high susceptibility to V. dahliae, were selected for infection studies. The plants were inoculated using the root-dipping method, as previously described¹¹. Briefly, V592 was cultured in liquid Czapek-Dox medium for five days to achieve a conidial concentration of 1 × 10⁷ conidia per milliliter. Subsequently, 4-week-old Arabidopsis and cotton plants were dipped in the conidial suspension for 20 min and one hour, respectively. Prior to inoculation with V592, TG1-2 was introduced into the roots of Arabidopsis (for 20 min) and cotton (for two hours) at an optical density (OD) of 0.6, two weeks before the V592 inoculation. Additionally, two groups of Arabidopsis roots were soaked in 200 μM surfactin and TG1-2 culture filtrate for 20 min, respectively, two weeks prior to the V592 inoculation. For fungal biomass analysis, all aboveground tissues of infected plants were collected at 21 days post-inoculation (dpi) and ground in liquid nitrogen for DNA extraction using the DNAsecure Plant kit (Tiangen Biotech, #A1227A). Quantitative real-time PCR was employed to estimate fungal biomass in infected cotton plants by comparing fungus-specific DNA levels to those of Arabidopsis/cotton-specific DNA. SYBR Green-labeled fragments (Transgen Biotech, #Q41210) were amplified, and qPCR was performed using a CFX96 Touch Deep Well Real-time PCR Detection System (Bio-Rad). Three biological replicates were conducted. The primers used in this study are listed in Supplementary Table 1.

Generation of fungal knockout and overexpression strains

To construct the knockout plasmids pGKO-HPT-Vdnat1, pGKO-HPT-Vdard1, and pGKO-NAT-Vdhsp83, upstream and downstream sequences flanking the target genes (VdNat1, VdArd1, and VdHsp83) were amplified using gene-specific primer pairs (Nat1-ko-up-F/R, Nat1-ko-dn-F/R; Ard1-ko-up-F/R, Ard1-ko-dn-F/R; Hsp83-ko-up-F/R, Hsp83-ko-dn-F/R; see Supplementary Table 1). These sequences were subsequently cloned into the pGKO vector containing the appropriate resistance markers via homologous recombination. For the generation of overexpression strains (VdNat1-GFP, VdNat1-Flag, VdArd1-Flag, VdHsp83-Flag, and VdSti1-GFP), the coding sequences of the respective genes were amplified using primers listed in Supplementary Table 1 and ligated into the 2b-silent vector.

Fungal transformation was conducted as previously described⁴⁷. Briefly, target sequences were amplified using Phanta Flash Master Mix (Vazyme, #P510), and homologous recombination was performed with the ClonExpress MultiS One Step Cloning Kit (Vazyme, #C113). Putative transformants were selected on agar plates supplemented with the corresponding antibiotics and further validated by PCR. All primers employed in this study are provided in Supplementary Table 1.

IP-MS analysis

The IP assay was conducted as previously described with minor modifications^48,49. Fresh mycelia of the Nat1-GFP overexpression strain were harvested from cultures, flash-frozen in liquid nitrogen, and ground to a fine powder. Total protein was extracted by resuspending the powder in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM MgCl₂, 10% glycerol, 0.1% NP-40, 0.5 mM DTT) followed by incubation at 4 °C on a rotator for 30 min. The lysate was centrifuged at 12,000 × g for 16 min at 4 °C, and the resulting supernatant was incubated with GFP-conjugated magnetic beads (GFP antibody, Abmart, #M20004; magnetic beads, Invitrogen, #3104361) for 2 h at 4 °C. After binding, the beads were washed three times with lysis buffer and twice with PBS buffer. Bound proteins were eluted using glycine buffer (50 mM, pH 2.8) and subjected to tryptic digestion.

Digested peptides were analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) coupled to an Easy nLC 1200 system. Peptides were loaded onto a C18 reversed-phase column (25 cm × 75 μm inner diameter; in-house packed with 1.9 μm RP-C18 resin) and separated using a linear gradient of buffer B (80% acetonitrile, 0.1% formic acid) at 300 nL/min. Buffer A consisted of 0.1% formic acid in HPLC-grade water.

MS data were acquired in data-dependent acquisition mode, selecting the top 20 most abundant precursor ions (350–1800 m/z) for higher-energy collisional dissociation (HCD) fragmentation. Key parameters included: Survey scan resolution: 60,000 at m/z 200. HCD scan resolution: 15,000 at m/z 200. Normalized collision energy: 28 eV. Dynamic exclusion: 45 ms. Precursor mass tolerance: 10 ppm. Fragment ion mass tolerance: 0.02 Da. Raw data were processed using Thermo Proteome Discoverer (version 2.5.0.400) and searched against the TAIR10 database. Trypsin specificity was applied, allowing up to two missed cleavages. A GFP vector-expressing strain served as the negative control.

Protein interaction analysis

The Yeast Two-Hybrid (Y2H) assay was performed using the Matchmaker Gold Y2H system (Clontech) following the manufacturer’s instructions. Briefly, full-length genes of interest were cloned into the pGADT7 and pGBKT7 vectors to generate activation domain (AD) and binding domain (BD) constructs, respectively. These constructs were co-transformed into Y2HGold yeast cells and plated on selective Synthetic Defined (SD) medium lacking leucine and tryptophan (SD/-Leu/-Trp; SD/-LW). Plates were incubated at 28 °C for 48 h. Colonies were subsequently transferred to higher-stringency SD medium lacking leucine, tryptophan, and histidine (SD/-Leu/-Trp/-His; SD/-LWH) and incubated at 28 °C for 3–6 days. Protein-protein interactions were inferred from the growth of transformants on SD/-LWH medium. For the split luciferase assay, candidate proteins fused to N- or C-terminal luciferase fragments were transiently expressed in leaves of 4-week-old Nicotiana benthamiana via agroinfiltration. Luciferase activity was measured 48 h post-infiltration using a charge-coupled device (CCD) imaging system (Tanon). Fresh mycelia co-expressing Tef-Nat1-GFP and Tef-Hsp83-Flag were harvested for protein extraction. Total proteins were extracted and subjected to Co-IP using GFP-conjugated magnetic beads at 4 °C, as previously described⁵⁰. Co-precipitated Hsp83-Flag was detected by immunoblotting with an anti-Flag antibody (Sigma-Aldrich, #F1804).

Acetylation analysis

For the in vitro acetylation assay, E. coli BL21 (DE3) cells harboring either the pGEX-4T-1-Ard1 or pGEX-4T-1-Hsp83 plasmid were cultured to mid-log phase (OD600 = 0.4–0.8). Protein expression was induced by adding 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), followed by overnight incubation at 16 °C. Cells were pelleted by centrifugation, and proteins were extracted using lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1 mM PMSF). GST-tagged Ard1 and Hsp83 were affinity-purified using Glutathione Sepharose 4B beads (GE Healthcare, #17-0756-05) and eluted with elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM reduced glutathione, 5% glycerol). Acetylation reactions were performed as described with minor modifications³⁶. Reaction products were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with an anti-acetyllysine antibody (Abmart, #M20004). To confirm acetylation sites, parallel reactions were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Control reactions lacking Ard1 acetyltransferase were included to rule out non-specific acetylation. For the identification of in vivo acetylation, an IP-MS assay was employed as described. Hsp83 protein was immunoprecipitated from Hsp83-Flag strain using Flag-conjugated beads (Flag antibody, Sigma-Aldrich, #F1804; beads, Invitrogen, #3104361). For acetylation site analysis, carbamidomethylating of cysteines was defined as fixed modification, while methionine oxidation and acetylation of lysine residues were specified as the variable modification. FDR cutoff on protein, peptide, and PSM level were 1%. Acetylation site probability cutoff was set 0.75. To assess the acetylation site mutations in small peptides, a dot blot analysis was carried out. The reaction products of peptides, both with or without Ard1 protein, were dotted onto a PVDF membrane and incubated with an anti-Lys-ac antibody for detection.

Cell apoptosis analysis

Confocal imaging (TCS SP8, Leica) was used to observe cellular apoptosis induced by surfactin, following labeling with FITC-12-dUTP. V592 conidia were cultured in liquid Czapek-Dox medium for 3 days to reach a concentration of 3×10^6 conidia per milliliter. For the surfactin treatment, 200 μM of surfactin was added to the conidia grown in Czapek-Dox medium after 1 day of incubation, followed by an additional 2 days of growth. The conidia were then collected via centrifugation and washed with PBS. The harvested conidia were fixed with 1% paraformaldehyde for 20 min at 4 °C, and subsequently permeabilized with 0.2% Triton X-100 for 5 min at room temperature. After permeabilization, the conidia were incubated with a FITC-12-dUTP (Vazyme, #A111-01)-labeled mixture at room temperature for 60 min. The reaction was terminated by adding 20 mM EDTA. The apoptotic conidia were visualized using confocal microscopy with excitation/emission settings of 500/540 nm. Apoptotic conidia exhibited green fluorescence, facilitating their identification and analysis. Cellular apoptosis induced by the TG1-2 culture filtrate was observed using 7-AAD labeling. Conidia treated with the TG1-2 culture filtrate were incubated with 7-AAD and subsequently visualized under confocal microscopy with excitation/emission settings of 546/647 nm. Apoptotic conidia displayed a distinct red fluorescence.

Trypan blue staining

Seven-day-old cotton roots were collected from the soil and washed with clean water. The cleaned roots were soaked in 200 μM surfactin for 20 min and then stained with trypan blue solution for 30 s. After staining, the roots were rinsed with clean water to remove excess dye from the surface. Roots treated with sterile water served as a negative control, while needle-damaged roots served as a positive control.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The raw data of 16S rRNA amplicon sequencing in this study have been deposited in NCBI GenBank under accession code PP939635. The metabolome data have been deposited in the National Genomics Data Center under accession code PRJCA028780. The mass spectrometry proteomics raw data have been deposited in the ProteomeXchange Consortium via the PRIDE⁵¹ partner repository with the dataset identifier PXD061243. Other data that support the findings of this study are available in the published article and its Supplementary Information file. Source data are provided with this paper.

Change history

19 August 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41467-025-63043-8

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Acknowledgements

C.G.D. was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB27040203) and National Natural Science Foundation of China (32270200). The authors thank Prof. Zonghua Wang (Fujian Agriculture and Forestry University) for providing M. oryzae, F. graminearum, B. cinerea, S. sclerotiorum, thank Prof. Yongli Qiao (Shanghai Normal University) for providing P. sojae strain, and thank Shanshan Wang from the Core Facility of Center for Excellence in Molecular Plant Sciences, CAS, for her mass-spectrometry technical support.

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State Key Laboratory of Plant Trait Design, Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
Ying-Chao Zhang, Xin Zhan, Jun-Yu Chen, Ding-Tian Yu, Huiming Zhang & Cheng-Guo Duan
University of the Chinese Academy of Sciences, Beijing, China
Xin Zhan, Jun-Yu Chen, Ding-Tian Yu & Cheng-Guo Duan
Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi, China
Tao Zhang

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Contributions

C.G.D. and Y.C.Z. designed the study. Y.C.Z., X.Z. and T.Z. performed the experiments and analyzed the data. D.T.Y. performed data deposition. X.Z. performed pathogenic experiments. Y.C.Z. and J.Y.C. purified the proteins. T.Z. performed the inhibitory experiment of TG1-2. C.G.D. wrote the manuscript. C.G.D. and H.Z. edited the manuscript. All authors discussed the results and reviewed the manuscript.

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Zhang, YC., Zhan, X., Chen, JY. et al. Reduced fungal protein acetylation mediates the antimicrobial activity of a rhizosphere bacterium against a phytopathogenic fungus. Nat Commun 16, 5644 (2025). https://doi.org/10.1038/s41467-025-60870-7

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Received: 20 September 2024
Accepted: 05 June 2025
Published: 01 July 2025
Version of record: 01 July 2025
DOI: https://doi.org/10.1038/s41467-025-60870-7

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