Protein acetylation is an evolutionarily conserved post-translational modification (Weinert et al., 2013), which is catalyzed by lysine acetyltrasferases and reversed by lysine deacetylases (Henriksen et al., 2012). Lysine acetylation has been found to occur in almost every compartment of a cell, and to play important roles in various cellular processes including protein-protein interactions (Hou et al., 2010), enzymatic activity (Starai and Escalante-Semerena, 2004; Nambi et al., 2013), metabolic pathways (Hou et al., 2010; Wang et al., 2010; Guan and Xiong, 2011; Nambi et al., 2013; Xie et al., 2015), cell morphology (Choudhary et al., 2009), calorie restriction (Imai and Guarente, 2010; Henriksen et al., 2012), protein-nucleic acid interactions (Arif et al., 2010; Ren et al., 2016), transcription factors, hormone receptors, signal transducers, chaperones and cytoskeleton (Singh et al., 2010; Zhang et al., 2013). Lysine acetylation is believed to be a main signaling modulator due to its widely occurrences and diverse cellular functions (Norvell and McMahon, 2010).

In recent years, with the advance of mass spectrometry (MS) and high affinity purification of acetylated peptides, acetylproteome studies have been successfully performed in some prokaryotic microorganisms, such as Escherichia coli (Kuhn et al., 2014), Salmonella enterica (Wang et al., 2010), Bacillus subtilis (Kim et al., 2013), Geobacillus kaustophilus (Lee et al., 2013), Erwinia amylovora (Wu et al., 2013), Thermus thermophilus (Okanishi et al., 2013) and Saccharopolyspora erythraea (Huang et al., 2015); the budding yeast: Saccharomyces cerevisiae (Henriksen et al., 2012), Oomycete: Phytophthora sojae (Li et al., 2016) and in filamentous fungi: Botrytis cinerea (Lv et al., 2016) and Fusarium graminearum (Zhou and Wu, 2019) et al. The knockout of some acetylated related genes caused the reduced mycelium growth, spore-production and virulence of fungi (Ding et al., 2010; Chen et al., 2018; Zhou and Wu, 2019), which indicated that lysine acetylation play important role in the growth and virulence of pathogens.

Apple valsa canker (AVC), caused by Cytospora sp., is one of the most destructive diseases of apple trees in eastern Asia (Cao et al., 2009; Ma et al., 2020). Bacillus velezensis L-1 was characterized as an effective biocontrol agent (BCA) against AVC in our previous work submitted to European Journal of Plant Pathology. Genome analysis revealed that there are a large number of acetyltransferase and deacetylase orthologs in the genomic of C. mali (Yin et al., 2015), which suggests that protein acetylation may play a critical role in the biological process, pathogenesis and the responses to BCA of C. mali. During the infection of AVC into apple barks, genes associated with plant cell wall degrading enzymes, toxic secondary metabolite biosynthesis were up-regulated, indicating their roles in the pathogenicity of C. mali (Ke et al., 2014), accordingly, BCA could inhibit the AVC by disrupting the cell wall-related genes of AVC, affecting the carbohydrate metabolic pathway of pathogens (Liu et al., 2018). On the other side, pathogens recruit numerous genes to resist the BCA. Sclerotinia sclerotiorum (the causing agent of sclerotinia stem rot) could up-regulate cell wall, cell membrane synthesis, antioxidants, glutathione S-transferase (GST), ABC transporter, and the autophagy pathway to alleviate the pressure of BCA (Yang et al., 2020). Study on the biological control mechanism of BCA and the response mechanism of AVC to biotic stress are in need for the further application of BCA in controlling AVC. In this study, a comparative acetyl-proteomics study based on Tandem Mass Tag (TMT) technology (McAlister et al., 2012) was carried out between C. mali QH2 and the C. mali QH2 suppressed by B. velezensis L-1. These data will complement current knowledge of the C. mali acetylproteome and provide an expanded platform for better understanding of the function of acetylation in the cell metabolites of pathogens and responses to the BCA stress.

In this study, we performed the different expression of lysine acetylated proteins in the C. mali QH2, during the suppresion of B. velezensis L-1. A total of 733 lysine acetylation sites in 416 proteins were identified, and 195 identified acetylation proteins were differently expressed at the level of|Fold change|>1.2 (1.2-fold over, or 0.83 less) and P < 0.05 in the TR. Among DEAPs, 101 proteins were up-regulated, and 94 proteins were down-regulated in the TR. Some DEAPs were predicted to be involved in the mycelium growth, pathogenicity of C. mali and its response to the biostress.

Two lysine acetylated proteins, located in the cytoplasm and related to the fatty acids and biofilm formation, respectively, were significantly down regulated in the TR. Fatty acids play important role in forming the plasma membrane and maintaining the fluidity of the cell membrane (Lattif et al., 2011). The formation of biofilm is closely related to the pahogenic and the drug resistance of the fungi (Wang et al., 2020). The significant down regulation of fatty acids and biofilm formation related acetylated proteins in the TR, indicating that lysine acetylated proteins are involved in the biofilm formation of C. mali QH2, and B. velezensis L-1 could inhibit the C. mali QH2 by inhibiting its synthesis of biofilms.

BCAs could affect the carbohydrate metabolic pathway, and inhibit the energy production in fungi (Liu et al., 2018). In this study, the lysine acetylated proteins were involved in multiple processes of energy production. Galactose is widely utilized by organisms through its conversion to glucose-6-phosphate, which can be then metabolized in the cytoplasm of the cell through the glycolysis pathway to produce ATP (Martchenko et al., 2007). The galactose metabolism depicts the conversion of galactose into glucose, lactose, and other sugar intermediates that may be used for a range of metabolic process. Down regulation of galactose metabolism/starch and sucrose metabolism indicating the suppression of B. velezensis L-1 on the carbohydrate metabolism. Pyruvate is the end production of glycolysis, and plays an important role as the central metabolite in the branching point of the tricarboxylic acid (TCA) cycle as well as the synthesis of amino acids (Zhang et al., 2020). Specifically, the pyruvate can be converted into Acetyl-CoA, which was used as the primary energy source for the TCA cycle, or converted into oxaloacetate to replenish TCA cycle intermediates. In addition, pyruvate can also be used to synthesize carbohydrates, fatty acids, ketone bodies, alanine, and steroids. Malic acid synthase (MS) is one of the key enzymes in the glyoxylate cycle, down regulation of the NADP-dependent malic enzyme may also reduce the use of acetyl-CoA through the glyoxylate cycle, what's more, the acetoacetyl-CoA synthetase and acetyl-CoA acetyltransferase IA were significantly down regulated. Decreased carbohydrate metabolism, and the down production of acetyl-CoA all lead to the energy shortage in C. mali.

Glutathione is an endogenous component of cellular metabolism, also referred to as GSH, which functions in detoxification and antioxidant processes by catalyzing the binding GSH⁻ to electrophilic centers on toxic substrates (Zettl et al., 1994; Marrs et al., 1995; Tian et al., 2016), and related to the virulence of fungi (Liu et al., 2018; Yang et al., 2020). Histone acetyltransferase GCN5 was proved as an important acetylated protein in a variety of fungi (Rösler et al., 2016; Chen et al., 2018). In our study, the B. velezensis L-1 suppressed C. mali showed the significantly down expression glutathione metabolism and GCN5. The down regulation of these virulence related proteins indicated that the acetylated proteins were involved in the virulence of C. mali QH2, and B. velezensis L-1 could reduce the virulence of C. mali QH2 by down expression of its virulence related genes.

C. mali has made multiple responses to the inhibition of BCAs. Autophagy pathway removes damaged organelles, and acts as an intracellular “scavengers” to maintain cellular homeostasis (Wang et al., 2019). BCAs cause oxidative damage to fungal cells, thus, antioxidant, hydrolase enzymes and other detoxification activities are regulated in the fungi to improve the tolerance (Van Acker et al., 2014). POD are enzymes that catalyze oxidation-reduction reaction by mechanism of free radical that transform several compounds into oxidized or polymerized products, they play important roles in eliminating reactive oxygen species damage, and are important enzymes involved in the biological antioxidant defense system (Horiguchi et al., 2001). In eukaryotes, CYP450 is membrane-binding protein, related to the metabolism of exogenous substance, and plays an important role in mytotoxin metabolism of necrotrophic fungus (Kelly et al., 1997; Syed et al., 2010; Ning and Wang, 2012). ABC transporters are important efflux transporters, are capable of translocating antifungal items out of cells and contribute to the development of tolerance to antifungals or biostress. Hsp 70 and other chaperones determine cell death and cell transformations; it has been implicated in the cellular stress initiation process (Pujhari et al., 2019). The upregulation of acetylated proteins in autophyge, POD, CYP450, ABC transporter and Hsp70 are the ways C. mali alleviate the suppression from biostress.

Taken together, these findings suggested that several lysine acetylated proteins related to fatty acids, biofilm formation, galactose metabolism/starch and sucrose metabolism pathway, TCA cycle, glutathione, and GCN5 were involved in the normal mycelium growth and pathogenicity of C. mali, and on the other side, the acetylated proteins involved in the autophagy, POD, CYP450, ABC transporter, and Hsp70 were expressed to release the stress from the biostress. Further verification of acetylated proteins involved in the interaction between B. velezensis L-1 and C. mali QH2 will be focused in our future work. It will be helpful to understand roles of the protein with acetylated lysine at the post-translational modification level in C. mali.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article.

PS and LZ designed the experiments, analyzed the data, and wrote the manuscript. PS and QM performed the experiments. All authors contributed to the article and approved the submitted version.

This research was funded by the National Natural Science Foundation of China (Grant Nos. 32060594 and 31860540), partially supported by the Research Program of Science and Technology at Universities of Inner Mongolia Autonomous Region (NJZY21458), and Research Start-up for High-level Researchers in Inner Mongolia Agricultural University (Grant Nos. NDYB2018-14 and NDYB2019-1).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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