Genome-wide transcriptomic analysis of the response of Botrytis cinerea to wuyiencin

Liming Shi; Binghua Liu; Qiuhe Wei; Beibei Ge; Kecheng Zhang

doi:10.1371/journal.pone.0224643

. 2020 Apr 29;15(4):e0224643. doi: 10.1371/journal.pone.0224643

Genome-wide transcriptomic analysis of the response of Botrytis cinerea to wuyiencin

Liming Shi ¹, Binghua Liu ¹, Qiuhe Wei ¹, Beibei Ge ^1,^*, Kecheng Zhang ^1,^*

Editor: Kandasamy Ulaganathan²

PMCID: PMC7190121 PMID: 32348310

Abstract

Grey mould is caused by the ascomycetes Botrytis cinerea in a range of crop hosts. As a biological control agent, the nucleoside antibiotic wuyiencin has been industrially produced and widely used as an effective fungicide. To elucidate the effects of wuyiencin on the transcriptional regulation in B. cinerea, we, for the first time, report a genome-wide transcriptomic analysis of B. cinerea treated with wuyiencin. 2067 genes were differentially expressed, of them, 886 and 1181 genes were significantly upregulated and downregulated, respectively. Functional categorization indicated that transcript levels of genes involved in amino acid metabolism and those encoding putative secreted proteins were altered in response to wuyiencin treatment. Moreover, the expression of genes involved in protein synthesis and energy metabolism (oxidative phosphorylation) and of those encoding ATP-binding cassette transporters was markedly upregulated, whereas that of genes participating in DNA replication, cell cycle, and stress response was downregulated. Furthermore, wuyiencin resulted in mycelial malformation and negatively influenced cell growth rate and conidial yield in B. cinerea. Our results suggest that this nucleoside antibiotic regulates all aspects of cell growth and differentiation in B. cinerea. To summarize, some new candidate pathways and target genes that may related to the protective and antagonistic mechanisms in B. cinerea were identified underlying the action of biological control agents.

Introduction

Grey mould is a type of disease that can be severe and economically damaging to many agricultural and horticultural crops [1]. Botrytis cinerea (teleomorph: Botryotinia fuckeliana) is an airborne plant pathogen with a necrotrophic lifestyle (class: Leotiomycetes, order: Helotiales, family: Sclerotiniaceae) [2]; it has been reported to cause serious losses in over 200 crop species worldwide. After winter dormancy, B. cinerea reportedly produces a large number of conidia under relatively suitable conditions in spring (the increase of relative humidity and temperature), which disperse via air and water droplets [3].

Airborne conidia usually cause new infections. Briefly, extracellular enzymes secreted by B. cinerea, such as pectin methylesterases (PMEs), polygalacturonases (PGs), laccases, and proteases, promote cell wall degradation and consequently soften host tissues. These changes consequently facilitate the penetration and colonization of host tissues, resulting in their decay [4, 5]. Some of the aforementioned enzymes express themselves differentially according to a variety of hosts and environmental factors, which explains how B. cinerea infects a broad range of hosts [6–8]. At times, B. cinerea is present on host plants in the latent state; this implies that while B. cinerea conidia do not adversely affect the host, when post-harvest fruits are stored and transported, they can germinate under conditions of relatively high humidity and suitable temperature, eventually causing serious damage [9].

Several chemicals have been widely used to tackle the problem of grey mould, but their prolonged usage has resulted in resistance development in B. cinerea and also given rise to strains that show rapid reproduction and genetic variations. More importantly, fungicide usage usually creates a problem of resistance, resurgence, and residue. Thus, to reduce environmental pollution, researchers have begun to screen and use beneficial microorganisms and their metabolites against B. cinerea. The nucleoside antibiotic wuyiencin is one such secondary metabolite; it is produced by Streptomyces ahygroscopicus var. wuyiensis, which was first isolated from the natural soil habitat of Wuyi Mountain in China [10]. After being industrially produced (COFCC-R-0903-0070), wuyiencin has been extensively used to control various fungal diseases in vegetables and crops and to enhance their resistance to diverse pathogens. It can be regarded as an organic, pollution-free pesticide, considering its characteristics of high efficiency, broad spectrum, and low toxicity [10]. Wuyiencin can alter cytomembrane permeability and inhibit protein synthesis in the mycelium of B. cinerea, which consequently causes cytoplasm leakage and mycelial malformation, thus reducing the pathogenicity of B. cinerea. It also reportedly induces host plant resistance to pathogenic bacteria [11]. However, little is known about the molecular mechanisms underlying the action of wuyiencin, particularly in B. cinerea.

In the past few years, many researchers have focused on studying and controlling B. cinerea; it has in fact become one of the important model systems in molecular phytopathology. The first genome assemblies of two B. cinerea strains, B05.10 and T4, were sequenced using Sanger technology at low coverage [12, 13]. And the gapless, near-finished genome sequence of B. cinerea was reported in 2017 [14]. Genome sequences of B. cinerea have played a major role in facilitating genetic manipulations and analyzing the genetic basis of pathogenicity [15]. Moreover, high-coverage de novo assemblies of genome sequences have promoted the development of genome-wide transcriptomic and proteomic techniques in B. cinerea [5]. RNA sequencing (RNA-seq) and transcriptomic analyses are commonly used methods as they are very sensitive, quantitative, accurate, and affordable [16].

Here we performed a genome-wide transcriptomic analysis to study the response of B. cinerea to wuyiencin. According to our results, wuyiencin had a prominent effect on the expression of genes involved in, for example, amino acid metabolism, protein synthesis, DNA replication, and cell cycle. Moreover, it caused mycelial malformation and negatively influenced cell growth rate and conidial yield in B. cinerea.

Results

Influence of wuyiencin on the growth and morphology of B. cinerea

In response to varying concentrations of wuyiencin, B. cinerea growth was gradually inhibited; aerial mycelia and pigment production were reduced as well (Fig 1A). Notably, with an increase in wuyiencin concentration, the antibiotic resulted in tortuous, malformed mycelia, the branching decreased, and the hyphal tip expanded to form spherical vesicles (Fig 1B). In response to 50 μg/mL, 100 μg/mL, and 200 μg/mL wuyiencin, the cell growth rate of B. cinerea decreased by 25.58%, 43.95%, and 100.00%, respectively, and conidial yield declined by 96.43%, 99.90%, and 100.00%, respectively (Fig 1E). Mycelial morphology and subcellular structure of B. cinerea were observed using SEM and TEM (Fig 1C and 1D). In comparison with the control treatment, when B. cinerea was cultivated with wuyiencin, we noted not only mycelial abnormality and severe hyphal swelling but also vesicular fusion; moreover, the number of organelles in mycelium decreased and autophagic bubbles with double membrane appeared (Fig 1C and 1D). These results indicated that wuyiencin could significantly inhibit the cell growth rate and conidial yield in B. cinerea, causing mycelial malformation. As cell growth and conidiogenesis in B. cinerea are related to its pathogenicity, our data suggest that wuyiencin can considerably weaken the pathogenicity of B. cinerea.

Fig 1 — **(A)** B. *cinerea* colonies morphology; **(B)** mycelial morphology, as observed under a light microscope, scale bars: 50 μm; **(C)** mycelial morphology, as observed using transmission electron microscopy (TEM), scale bars: 25 μm; **(D)** subcellular structure, as observed using TEM, scale bars: 2 μm; and **(E)** conidial production in *Botrytis cinerea* in response to varying concentrations of wuyiencin.

Genome-wide transcriptomic analysis

We performed a genome-wide transcriptomic analysis to elucidate the molecular mechanisms underlying the action of wuyiencin. The final concentration of wuyiencin is approximately 50–100 μg/mL in agricultural production; earlier studies [11] have reported that 200 μg/mL wuyiencin is lethal to B. cinerea, but there is little effect on cell growth and conidiogenesis at 50 μg/mL wuyiencin. Thus, for RNA-seq analyses, we cultivated B05.10 in the dark at 20°C with 100 μg/mL and without wuyiencin. Three biological replicates were assessed for each sample.

Of the 12814 genes in B. cinerea, 2067 genes were differentially expressed in response to treatment with 100 μg/mL wuyiencin (Fig 2); of them, 886 and 1181 were significantly upregulated and downregulated, respectively. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were performed to classify all DEGs into the following functional categories: amino acid metabolism, protein synthesis, carbon and energy metabolism, putative secreted metabolites and proteins, DNA replication and cell cycle, and other metabolism.

Transcriptional analysis of genes involved in amino acid metabolism and protein synthesis

Amino acid metabolism and protein synthesis are fundamental cellular activities. The expression of most genes involved in the metabolism of aromatic amino acids, alanine, glycine, serine, threonine, cysteine, and methionine was upregulated when B. cinerea was cultivated with wuyiencin (S1 Table).

A major change in the metabolism of tyrosine, phenylalanine, and tryptophan was observed. These aromatic amino acids play a significant role in protein synthesis, and they also participate in the synthesis of various secondary metabolites. The structural and catalytic properties of these amino acids give specific functions to certain proteins [17]. In fungi, the terminal reactions in the biosynthesis of phenylalanine and tyrosine are achieved by converting chorismite to phenylpyruvate or 4-hydroxyphenylpyruvate, and tyrosine aminotransferase (dependent on pyridoxal-5-phosphate) catalyzes the final step in both pathways with glutamate as an amino donor [18]. Owing to the promiscuous substrate specificity of aminotransferases in the anabolism of aromatic amino acids, branched-chain and aspartate aminotransferases have activities that overlap with those of tyrosine aminotransferase [19–21]. Noticeably, having different aminotransferases with overlapping substrates would be a strategy to achieve nutritional flexibility in evolution under various growth conditions [22]. In B. cinerea, the transcriptional levels of the following key enzymes were significantly upregulated upon wuyiencin treatment: tyrosine aminotransferase (Bcin07g04780), branched-chain aminotransferase (Bcin04g01520), and L-ornithine aminotransferase (Bcin07g00730). This indicates that B. cinerea responds to wuyiencin by increasing tyrosine, phenylalanine, and tryptophan biosynthesis.

However, the expression of some key enzymes was significantly downregulated. Bcin15g05090 and Bcin16g01460 expression was downregulated (Log₂FC values, −2.0 and −4.4, respectively); both are involved in the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), a reaction catalyzed by tyrosine hydroxylase, according to KEGG enrichment analysis. The ubiquitous enzyme aromatic L-amino acid decarboxylase (Bcin06g01710) is involved in the decarboxylation of L-DOPA to dopamine, of 5-hydroxytryptophan to serotonin, and of tryptophan to tryptamine [23]. Furthermore, amine oxidases (Bcin01g08900, Bcin05g07030) convert dopamine to 3,4-dihydroxyphenylacetaldehyde and also phenylethylamine to phenylacetaldehyde. As per our results, Bcin06g01710, Bcin01g08900, and Bcin05g07030 expression was significantly downregulated (Log₂FC values, −2.9, −3.2, and −5.8, respectively). Although the expression of Bcin15g03370, which encodes 4-hydroxyphenylpyruvate dioxygenase that transforms 4-hydroxyphenylpyruvate to homogentisate, was slightly upregulated, that of two genes (Bcin11g01020 Bcin11g01050) involved in the conversion of homogentisate to fumarate and acetoacetate in tyrosine metabolism and another one (Bcin11g01060) encoding phenylacetate 2-hydroxylase, which hydroxylates phenylacetate to 2-hydroxy phenylacetate for styrene degradation, was markedly downregulated. The downregulation of these genes was in stark contrast to the upregulation of those involved in aromatic amino acid synthesis. These findings suggest that wuyiencin has an effect on both aromatic amino acid anabolism as well as catabolism.

Moving on, the expression of three key enzymes involved in valine, leucine, and isoleucine degradation was significantly upregulated (Fig 3A). After the transamination of branched-chain aminotransferase (Bcin04g01520), valine, leucine, and isoleucine are degraded from 2-oxobutanoate or 2-oxopentanoate to butyryl-CoA or propionyl-CoA by 3-methyl-2-oxobutanoate dehydrogenase (Bcin13g01430 and Bcin13g03020) and dihydrolipoamide acetyltransferase (Bcin11g04250). The Log₂FC value of Bcin13g01430, Bcin13g03020 and Bcin11g04250 were 2.6, 2.0, and 3.4, respectively (Fig 3B, S1 Table). Concomitantly, the transcriptional levels of enoyl-CoA hydratase (Bcin03g05840) and acetyl-CoA acyltransferase (Bcin01g04960), which convert butanoyl-CoA to propanoyl-CoA, were also significantly upregulated. Similarly, the expression of genes involved in the transamination of cysteine metabolism (Bcin04g01520, Bcin14g01940) and glycine, serine, and threonine metabolism (Bcin04g03090) was upregulated, leading to an increase in 2-oxobutanoate and pyruvate production. To supportour RNA-seq data, we randomly chose Bcin07g04780, Bcin01g08900, Bcin05g07030, Bcin15g03370, and Bcin11g04250 for RT-qPCR analyses, and RT-qPCR findings supported our RNA-seq data (S2 Table). These data indicate that the substrate levels for carbon metabolism (examples being propanoate metabolism and citrate cycle) may increase and that wuyiencin promotes the catabolism of some amino acids.

Among the genes associated with protein synthesis in B. cinerea, there were 28 genes enriched in term “ribosome” by KEGG, and their expression was found to be significantly upregulated (S1 Table). Most of them encoded ribosomal proteins, including 40S ribosomal proteins S5 (Bcin03g00590), S14 (Bcin03g06970), S23 (Bcin04g05440), and S25 (Bcin03g00790), 60S ribosomal proteins L10ae (Bcin03g01820), L15 (Bcin14g04790), L24 (Bcin09g06480), L26 (Bcin13g03600), L27ae (Bcin02g05140), L29 (Bcin10g03580), L30 (Bcin09g05600), L37 (Bcin01g00770), L38 (Bcin05g06070), L43 (Bcin13g00830), and L44 (Bcin09g02490), and 60S ribosome biogenesis protein SQT1 (Bcin08g05590). A previous gene-profiling study on B. cinerea germination reported that transcripts encoding ribosomal proteins were markedly affected upon resveratrol treatment [24]. The expression of the 40S ribosomal protein S17 (Bcin12g01300), 60S ribosomal proteins L18ae (Bcin05g01600) and L9 (BC1G_16427), and 60S ribosome biogenesis protein SQT1 (Bcin08g05590) was significantly downregulated, but that of 40S ribosomal proteins S8 (Bcin04g05190) and S5 (Bcin03g00590) and 60S ribosomal proteins L14 (Bcin03g06970), L44 (Bcin09g02490), L9 (Bcin11g05920), and L35 (Bcin11g03280) was significantly upregulated [24]. However, only the trend shown by the 60S ribosomal protein L44 (Bcin09g02490) was the same when B. cinerea was treated with wuyiencin or resveratrol; all other genes were either not differentially expressed or showed contradictory expression trends upon wuyiencin treatment. These findings suggest that B. cinerea uses different protein synthesis metabolism when exposed to phytoalexin from plants or antibiotic from microorganisms. In addition, we identified only one gene encoding a chaperone (Bcin01g09530) that was involved in protein folding, but the expression of this heat-shock protein (HSP), belonging to the HSP 20 family, with the highest Log₂FC value (4.7) among the genes in protein synthesis metabolism.

Transcriptional analysis of genes involved in carbon and energy metabolism

With regard to propanoate metabolism, the expression of genes encoding glucan 1,4-alpha-glucosidase (Bcgs1, Bcin15g05660), 2-methylcitrate dehydratase (Bcin07g03110), and propionate-CoA ligase (Bcin04g03150) was markedly downregulated (Fig 3A, S1 Table). Remarkably, the necrosis-inducing glycoprotein BcGs1 (Bcin15g05660) produced by B. cinerea has elicitor activity and triggers defense response in host plants [25]. With regard to pyruvate metabolism, the expression of genes encoding L-lactate dehydrogenase (Bccyb2, Bcin01g00400), acetyl-CoA carboxylase (Bcin07g06960), pyruvate carboxykinase (Bcpck1, Bcin16g00630), and malate synthase (Bcin09g06110) was also significantly downregulated (Fig 3A). Though this downregulation might lead to a decrease in the production of pyruvate, malate, and malonyl-CoA, the genes involved in glycolysis, gluconeogenesis, and citrate cycle were not differentially expressed in response to wuyiencin treatment.

With respect to energy metabolism, six genes involved in oxidative phosphorylation were differentially expressed. Two of them, Bcin01g01100 encoding NADH dehydrogenase in complex 1 and Bcin04g00750 encoding plasma membrane ATPase in complex 5 (ATP synthase), were considerably downregulated, whereas the other four, encoding NADH oxidoreductase (Bcin10g04670 and Bcin14g00100), ATP synthase protein (Bcin10g01500), and cytochrome C oxidase (Bcin02g03350), were significantly upregulated. Moreover, the expression of Bcfdh1 (Bcin16g04640), encoding formate dehydrogenase which is potentially implicated in nitrate respiration in B. cinerea and supplies electrons, was upregulated (Log₂FC value, 2.6). This finding was, however, contrary to that reported by Zheng et al. [24]; as per their results, Bcfdh1 expression was downregulated on cultivating B. cinerea with resveratrol. Further, the expression of Bcin03g01010, which encodes a ADP/ATP mitochondrial carrier protein, was downregulated at the transcriptional level. This carrier protein is a mitochondrial translocase and promotes the transport of solutes across the mitochondrial membrane; this process between the mitochondrion and cytosol is dependent on ATP [26]. This finding was also contrary to the results reported by Zheng et al. [24]. It is noteworthy that RT-qPCR results for Bcin01g00400, Bcin07g06960, Bcin02g03350, and Bcin16g04640 were all basically consistent with our RNA-seq data (S2 Table).

Transcriptional analysis of genes encoding putative secreted metabolites and proteins

As a natural barrier, the plant cell wall provides mechanical strength and rigidity in order to prevent invasion by pathogens. To destroy the plant cell wall and successfully colonize host plants, B. cinerea secretes a diverse array of metabolites and proteins [25, 27, 28]. These proteins belong to cell wall-degrading enzyme (CWDE) families and include pectinases, xylanases, and endo-PGs; they are usually considered to be important virulence factors and act via host tissue impregnation and macromolecule degradation [29]. In response to cultivating B. cinerea with wuyiencin, the expression of 23 CWDE family-related genes was considerably influenced (S1 Table).

To degrade xylan, the major hemicellulosic component of the plant cell wall, B. cinerea needs the synergistic action of several hydrolytic enzymes. One such enzyme is endo-β-1,4-xylanase, which is encoded by the gene Bcxyn11A (Bcin03g00480). This enzyme belongs to the glycosyl hydrolase family 11 and carries out the initial breakdown of the xylan backbone [30]. For pectin degradation, B. cinerea employs a series of depolymerizing enzymes. PME is involved in one such pathway, which starts with pectin de-esterification into methanol and polygalacturonic acid (PGA). There are two genes, Bcpme1 (Bcin08g02970) and Bcpme2 (Bcin03g03830) (Fig 4A and 4B). PME activity is also important for the subsequent action of depolymerizing enzymes, which, depending on their mode of action, are classified into two groups: those of endo cleavage mode, which is random, and those of endo cleavage mode, which act on the penultimate polymer bonds [31]. Then, glycosidic bonds are interrupted by PG hydrolysis (encoded by Bcpg1, Bcin14g00850 and endo-PG; Bcpg2, Bcin14g00610; and Bcpg5, Bcin01g07330) and PGA is broken down into oligogalacturonides by the β-elimination of pectate lyases (endo-PL, Bcin03g05820). In plant–pathogen interactions, CWDEs act not only as triggers of pathogen-associated molecular pattern-triggered immunity responses in plants but also as virulence factors. In particular, in B. cinerea, xylanase (encoded by Bcxyn11A, Bcin03g00480) and endo-PG 1 (encoded by Bcpg1, Bcin14g00850) trigger immune responses and play a vital role in virulence [32]. The expression of these seven genes (Bcin03g00480, Bcin08g02970, Bcin03g03830, Bcin14g00850, Bcin14g00610, Bcin01g07330 and Bcin03g05820) was noted to be significantly upregulated (Fig 4B); the RNA-seq data generated for Bcin08g02970 and Bcin03g00480 were supported by RT-qPCR, as standard (S2 Table).

In contrast, several genes encoding putative secreted carbohydrate-active enzymes (CAZymes) were differentially expressed (Fig 4C). CAZymes are proteins with certain catalytic and carbohydrate-binding domains that degrade, modify, or create glycosidic bonds [33]. The expression of genes encoding xyloglucan (XyG) backbone-degrading enzymes was significantly upregulated. The product (glycoside hydrolase family 16 protein, GH16) of Bcin01g06010 belongs to XyG hydrolases, and the XyG backbone is hydrolyzed by endo-acting β-1,4-glucanases (encoded by Bcin03g03630) [34]. The Log₂FC values for Bcin01g06010 and Bcin03g03630 were 4.6 and 6.3, respectively, which were among the highest recorded in this study. In the primary cell wall of dicots and non-graminaceous monocots, the major pectic polysaccharides are homogalacturonan (HG) and rhamnogalacturonan (RG) [35]. We noted that the expression of almost all CAZymes involved in cleaving HG pectin backbones was upregulated at the transcriptional level, including pectinesterase (Bcin14g00860) and pectin lyase (Bcin14g03430 and Bcin03g00280). In fact, PG (Bcpg1, Bcpg2, and Bcpg5) and PME (Bcpme1 and Bcpme2) also belong to this class. However, glycoside hydrolase family 28 proteins (GH28) to the RG-I backbone (Bcin06g02140) and HG backbone (Bcin15g05030) showed low expression levels. Notably, among the genes encoding CAZymes targeting cellulose, side-chains/adducts, and xylan backbone, the expression of nearly all genes was downregulated, except the expression of Bcin12g06590 and Bcin14g05510, which was slightly upregulated. Our data thus indicate that the expression of these genes (Bcin15g05030, Bcin06g02140, Bcin13g02100, Bcin16g03020, Bcin01g05680, Bcin01g07210, Bcin05g08130, Bcin09g04150 and Bcin12g00300) is suppressed by wuyiencin.

Transcriptional analysis of genes involved in DNA replication and cell cycle

The expression of 12 genes related to DNA replication and cell cycle was downregulated when B. cinerea was treated with wuyiencin. With regard to DNA replication, the expression of Bcpol1 (Bcin16g02310), Bcpol12 (Bcin08g00800), and Bcpri2 (Bcin08g00460), encoding the components of DNA polymerase α-primase complex, was downregulated, with values of −2.6, −2.0, and −1.9 Log₂FC, respectively (S1 Table). Bcpol1 encodes Bcin16g02310, which also responds to DNA damage stimulus and cell differentiation. Besides, transcripts for Bcin08g05510 and Bcin02g07590, which encode the components of DNA polymerase δ- and ε-complex, respectively, were downregulated (Log₂FC values, −1.9 and −1.8, respectively). The expression of genes encoding DNA helicase (Bcin15g02990 and Bcin12g06740), proliferating cell nuclear antigen (Bcin01g06320), and replication protein A (Bcin12g01760) were also significantly downregulated. The expression of three cell cycle-related genes was also downregulated: Bccdc6 (Bcin01g06090, encoding cell division control protein), Bcmet30 (Bcin07g06910, encoding E3 ubiquitin ligase complex SCF subunit), and Bcmrc1 (Bcin09g01360, participating in cell cycle arrest). The downregulation of the expression of these genes may be related to cell growth impairment in B. cinerea upon wuyiencin treatment. To assess if RNA-seq data was supportable by a different method of analysis, several genes (Bcin16g02310, Bcin08g00460, Bcin01g06320 and Bcin07g06910) were randomly selected for RT-qPCR, and the results were basically consistent with RNA-seq data (S2 Table).

Transcriptional analysis of genes involved in other metabolism

With regard to vacuole membrane metabolism, some genes that probably participate in fungal physiology and morphogenesis were differentially expressed in response to wuyiencin (S1 Table). Their differential expression might be related to changes in cell morphogenesis, characterized by vesicular fusion and severe hyphal swelling (Fig 1). Three genes were enriched in fungal-type vacuole membrane and lytic vacuole membrane metabolism, as elucidated via GO analysis. The expression of Bcin08g03620 (encoding an uncharacterized protein), related to amide and peptide transporter activity and ATPase activity coupled to the movement of substances, was upregulated, whereas that of Bcin03g05090 (also encoding an uncharacterized protein), related to inorganic cation transmembrane transporter activity, and Bcin12g01770, encoding acetyltransferase, was significantly downregulated (S1 Table). Moreover, with regard to fungal cell wall metabolism, the expression of Bccrh1 (Bcin01g06010), encoding GPI-glucanosyltransferase from the glycosyl hydrolase family 16 (GH16), was considerably upregulated (S1 Table). Reportedly, this GPI-glucanosyltransferase is a homolog of the CRH1 protein that is necessary for the cross-linking of chitin to β1,6-glucan in Saccharomyces cerevisiae; Bccrh1 expression was significantly upregulated when B. cinerea was treated with resveratrol too [24, 36]. Thus, it seems like the product of Bccrh1 directly participates in the formation of cross-links between cell wall components [11].

As to fungal transporters in B. cinerea, the expression of four genes encoding ATP-binding cassette (ABC) or major facilitator (MFS) transporters was significantly upregulated (S1 Table). It is known that fungi employ active efflux by ABC and MFS transporters to resist endogenous and exogenous toxic compounds, such as antibiotics and fungicides [37–39]. In B. cinerea, the expression of BcatrB (Bcin13g00710, encoding an ABC transporter) and Bchex1 (Bcin09g00150, encoding an MFS sugar transporter) was markedly upregulated, with Log₂FC values reaching as high as 6.2 and 1.8, respectively. These results suggest that the active efflux of wuyiencin occurs in B. cinerea. This observation is also consistent with the finding that the expression of ABC and MFS transporters is significantly upregulated on cultivating B. cinerea with resveratrol [24]. Besides, the expression of MFS transporter genes Bcmfs1 (Bcin14g02870) and BcmfsM2 (Bcin15g00270) was markedly upregulated too; they are also involved in multidrug resistance, such as in transporting azole fungicides [40]. Moreover, ABC transporters contribute to B. cinerea virulence, and BcATRB is a known virulence factor [39]. These data indicate that the upregulation of the expression of these genes leads to an increase in virulence. As standard, the RNA-seq data generated for Bcin08g03620 and Bcin13g00710 were supported by RT-qPCR (S2 Table).

Discussion

Although wuyiencin has been industrially produced and widely used in China as an effective fungicide, the biological mechanisms by which this antibiotic inhibits pathogenic fungi still remain unclear [41]. We, for the first time, performed a genome-wide transcriptomic analysis to study the response of B. cinerea to wuyiencin treatment. In an earlier study to evaluate the Botrytis–wuyiencin relationship, including morphological characterization, it was reported that wuyiencin significantly reduces the production of mycelial proteins and almost completely inhibits the germination of conidiospores [11]. In this study, mycelial morphology and cell growth rate were intensely influenced in a concentration-dependent manner and conidial germination was completely inhibited upon exposure to 100 μg/mL wuyiencin; it is noteworthy that Sun et al. [11] also used a similar concentration of wuyiencin.

At present, with the development and utilization of fungicides for agricultural use, the mechanisms by which they act at a morphological, physiological, and molecular level to inhibit the growth of pathogenic microorganisms are gradually been elucidated. For example, nucleoside antibiotics are a diverse group of microbial secondary metabolites derived from nucleosides and nucleotides, which play several roles in basic cellular metabolic pathways [42]. Polyoxin and nikkomycin are typical antifungal nucleoside antibiotics that target cell wall biosynthesis and serve as competitive inhibitors of fungal chitin synthases [43]. In the fungal cell wall, the rigid carbohydrate polymer chitin is a significant structural component that provides strength and rigidity. Thus, the inhibition to chitin biosynthesis by antibiotics can cause severe hyphal swelling, consequently affecting fungal growth [44]. Moreover, the aminoglycoside antibiotic kasugamycin is known to inhibit protein biosynthesis and is widely used for managing plant diseases [45]. Kasugamycin inhibits the binding of ribosomes and aminoacyl-tRNA-message complex, but it does not affect nucleic acid synthesis [46]. Natamycin and other polyene antibiotics, which are widely used as antifungal agents, act by destroying the barrier function of cell membrane [47]. Polyene antibiotics usually consist of a macrolide core with three to eight conjugated double bonds, an exocyclic carboxyl group, and an unusual mycosamine sugar [48]. Natamycin acts by binding to principal sterols in fungal membranes, specifically to ergosterol [47]. With regard to the nucleoside antibiotic wuyiencin, not only little is known about its underlying molecular mechanism of action in B. cinerea but also the transcriptomic changes in Botrytis species upon wuyiencin treatment have not been previously studied.

In the present study, 2067 genes were differentially expressed in B05.10 after wuyiencin treatment; of these, 886 were upregulated and 1181 were downregulated. This suggests that wuyiencin activates the expression of some genes and stimulates a resistance mechanism in B. cinerea, while inhibiting the expression of some other genes. Further, wuyiencin had a remarkable effect on primary metabolism in B. cinerea, and many genes involved in amino acid metabolism were enriched. Pertaining to the metabolism of three aromatic amino acids, the expression of genes involved in anabolism was upregulated, while that of genes participating in catabolism was downregulated. After transamination and hydrolysis, aromatic amino acids are transformed to fumarate and acetoacetate, thereby linking the metabolism of amino acids and fumarate, potentially both inside and outside of mitochondria [17]. We noted that the expression of some genes encoding key enzymes involved in valine, leucine, and isoleucine degradation was upregulated; with regard to carbon metabolism, some genes encoding key enzymes showed low expression levels. This could decrease the substrate levels for pyruvate metabolism, citrate cycle, and fatty acid biosynthesis. In addition, genes related to protein synthesis and most genes involved in energy metabolism (oxidative phosphorylation) showed upregulated expression levels, which is contradictory to the response of B. cinerea to resveratrol treatment [24]. In particular, the expression of genes (Bcin01g06320, Bcin07g06910, Bcin08g03620, Bcin16g02310) involved in stress response was significantly downregulated; the products of these genes are involved in responding to DNA damage stimulus, metal ions, and toxic substances. Also, the expression of all genes involved in DNA replication and cell cycle and which were directly related to cell growth was suppressed by wuyiencin. The expression of genes encoding CAZymes was also significantly impacted upon wuyiencin treatment, indicating that gene expression might either be influenced by wuyiencin or B. cinerea adjusted the virulence and host tissue targets proactively.

Our results are in agreement with those reported by a previous study and suggest that wuyiencin plays an important role in regulating of all aspects of cell growth and differentiation in B. cinerea, in view of the broad spectrum of biological activities of nucleoside antibiotics [43]. From the experimental data, the changes caused by wuyiencin were partly similar with general stress response and induced ROS reaction [49, 50]. The relationship between biotic and abiotic stress of B. cinerea to wuyiencin need more investigation to clarification. We hypothesized that the transcriptomic response of B. cinerea to wuyiencin involves striking a balance between antagonism and competition. More importantly, in this study, some new candidate pathways and target genes that may related to the protective and antagonistic mechanisms in B. cinerea were identified underlying the action of biological control agents.

In conclusion, to further validate our results, transcriptomic and proteomic analyses in actual field cultivation situations should be performed. The signal transduction pathway and regulatory mechanisms of wuyiencin that affect the growth and development of B. cinerea need to be elucidated in future studies.

Materials and methods

Treatment of B. cinerea with wuyiencin

The B. cinerea standard strain B05.10 was separately inoculated into potato dextrose agar (PDA) medium containing no wuyiencin (control) and that containing wuyiencin at a final concentration of 50 μg/mL, 100 μg/mL and 200 μg/mL. Cultures were incubated at 20°C, and mycelial morphology was observed under a light microscope after 3 days. Conidia were eluted with 0.2% Tween 20 after 7 days and quantified using a hemocytometer under a microscope.

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

B05.10 was inoculated into PDA medium with and without wuyiencin, as mentioned above. Cultures were incubated at 20°C for 7 days, and mycelial morphology and subcellular structure were observed using SEM and TEM.

Mycelial growth was collected, washed three times with sterilized distilled water, transferred to MM–N liquid medium containing 4 mM phenylmethylsulfonyl fluoride, and incubated at 25°C for 4 h on a 200-rpm shaker. Fungal mass was then collected and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C overnight. The samples were then rinsed three times with phosphate buffer and fixed overnight at 4°C in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.0). After rinsing three times with phosphate buffer, the samples were dehydrated in an ethanol series, infiltrated with a graded series of epoxy resin in epoxy propane, and embedded in Epon 812 resin. The ultrathin sections were then stained in 2% uranium acetate followed by lead citrate, and subsequently visualized under a transmission electron microscope (Hitachi, H-7650) operating at 80 kV.

RNA isolation

For each treatment sample, total RNA was extracted from frozen mycelium using Trizol reagent (Takara Bio, Japan) according to the manufacturer’s instructions. RNase-free DNase I (Takara Bio, Japan) was used to digest traces of genomic DNA. RNA concentration was spectrophotometrically determined using the NanoPhotometer^® spectrophotometer (IMPLEN, CA, USA), and RNA integrity and size distribution were assessed using 1% agarose gel electrophoresis.

Transcriptomic analysis

cDNA library construction and Illumina sequencing were completed in Novogene Bioinfomatics Technology Company (Beijing, China) following a default Illumina stranded RNA protocol. Briefly, A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext^® Ultra^™ RNA Library Prep Kit for Illumina^® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 150 bp paired-end reads were generated. We used three biological replicates of each sample. Differential expression analysis was performed using the DESeq R package (1.18.0) [51]. DESeq provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value <0.05 found by DESeq were assigned as differentially expressed. The differentially expressed genes (DEGs) were annotated using KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.genome.jp/kegg/). Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the GOseq R package, in which gene length bias was corrected. All of the raw reads are archived at the NCBI Sequence Read Archive (SRA) database (accession number: SRP212990).

Quantitative real-time RT-PCR (RT-qPCR)

To confirm RNA-seq results, 20 DEGs were chosen for RT-qPCR analyses. According to manufacturer’s instructions, RT-qPCR cycling conditions were as follows: initial denaturation for 3 min at 95°C, 45 cycles of 3-s denaturation at 95°C, 30-s annealing at 60°C, and 30-s elongation at 60°C. We used SG Fast qPCR Master Mix (High Rox) (ABI). With a minimum of two biological replicates, three technical replicates were subjected to RT-qPCR for each sample. Primers used in this study for RT-qPCR analyses are listed in S2 Table.

Supporting information

S1 Table. Gene expression changes in Botrytis cinerea cultivated with 100 μg/mL and without wuyiencin.

(XLSX)

Click here for additional data file.^{(16KB, xlsx)}

S2 Table. Gene expression changes in Botrytis cinerea cultivated with 100 μg/mL and without (ck) wuyiencin, and validation of results by RT-qPCR.

(XLSX)

Click here for additional data file.^{(22.6KB, xlsx)}

S3 Table. The information of metabolic pathways of the differentially expressed genes (DEGs) in Botrytis cinerea cultivated with 100 μg/mL and without wuyiencin.

(XLS)

Click here for additional data file.^{(14KB, xls)}

Acknowledgments

We thank the native English-speaking scientists at Elixigen Company (Huntington Beach, California) for editing our manuscript.

Data Availability

All relevant data are within the manuscript and its Supporting Information files. The RNA-seq dataset generated for this study can be found in the NCBI database under the accession number SRP212990.

Funding Statement

This work was supported by the National Natural Science Foundation (31601684), the Special Fund for Basic Scientific Research of the Chinese Academy of Agricultural Sciences (Y2017JC12), and the National Key Research and Development Plan (2017YFD0201100). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, et al. The Top 10 fungal pathogens in molecular plant pathology. Molecular plant pathology. 2012;13(4):414–30. 10.1111/j.1364-3703.2011.00783.x [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Staats M, van Kan JA. Genome update of Botrytis cinerea strains B05.10 and T4. Eukaryotic cell. 2012;11(11):1413–4. 10.1128/EC.00164-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Leroch M, Kleber A, Silva E, Coenen T, Koppenhofer D, Shmaryahu A, et al. Transcriptome profiling of Botrytis cinerea conidial germination reveals upregulation of infection-related genes during the prepenetration stage. Eukaryotic cell. 2013;12(4):614–26. 10.1128/EC.00295-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Staples RC, Mayer AM. Putative virulence factors of Botrytis cinerea acting as a wound pathogen. FEMS microbiology letters. 1995;134(1):1–7. [Google Scholar]
5.Li B, Wang W, Zong Y, Qin G, Tian S. Exploring pathogenic mechanisms of Botrytis cinerea secretome under different ambient pH based on comparative proteomic analysis. Journal of proteome research. 2012;11(8):4249–60. 10.1021/pr300365f [DOI] [PubMed] [Google Scholar]
6.Wubben JP, ten Have A, van Kan JA, Visser J. Regulation of endopolygalacturonase gene expression in Botrytis cinerea by galacturonic acid, ambient pH and carbon catabolite repression. Current genetics. 2000;37(2):152–7. 10.1007/s002940050022 . [DOI] [PubMed] [Google Scholar]
7.ten Have A, Breuil WO, Wubben JP, Visser J, van Kan JA. Botrytis cinerea endopolygalacturonase genes are differentially expressed in various plant tissues. Fungal genetics and biology: FG & B. 2001;33(2):97–105. [DOI] [PubMed] [Google Scholar]
8.ten Have A, Espino JJ, Dekkers E, Van Sluyter SC, Brito N, Kay J, et al. The Botrytis cinerea aspartic proteinase family. Fungal genetics and biology: FG & B. 2010;47(1):53–65. [DOI] [PubMed] [Google Scholar]
9.Wilson CL., Wisniewski ME., Biles CL., McLaughlin Randy, Chalutz Edo, Droby Samir. Biological control of post-harvest diseases of fruits and vegetables: alternatives to synthetic fungicides. Crop Protection. 1991;10(3):172–7. [Google Scholar]
10.Ge B, Liu Y, Liu B, Zhao W, Zhang K. Characterization of novel DeoR-family member from the Streptomyces ahygroscopicus strain CK-15 that acts as a repressor of morphological development. Applied microbiology and biotechnology. 2016;100(20):8819–28. 10.1007/s00253-016-7661-y [DOI] [PubMed] [Google Scholar]
11.Sun Y, Zeng H, Shi Y, Li G. Mode of action of Wuyiencin on Botrytis cinerea. ACTA PHYTOPATHOLOGICA SINICA. 2003;33(5). [Google Scholar]
12.Choquer M, Fournier E, Kunz C, Levis C, Pradier JM, Simon A, et al. Botrytis cinerea virulence factors: new insights into a necrotrophic and polyphageous pathogen. FEMS microbiology letters. 2007;277(1):1–10. 10.1111/j.1574-6968.2007.00930.x [DOI] [PubMed] [Google Scholar]
13.Amselem J, Cuomo CA, van Kan JA, Viaud M, Benito EP, Couloux A, et al. Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS genetics. 2011;7(8):e1002230 10.1371/journal.pgen.1002230 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Van Kan JA, Stassen JH, Mosbach A, Van Der Lee TA, Faino L, Farmer AD, et al. A gapless genome sequence of the fungus Botrytis cinerea. Mol Plant Pathol. 2017;18(1):75–89. Epub 2016/02/26. 10.1111/mpp.12384 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Melendez HG, Billon-Grand G, Fevre M, Mey G. Role of the Botrytis cinerea FKBP12 ortholog in pathogenic development and in sulfur regulation. Fungal genetics and biology: FG & B. 2009;46(4):308–20. [DOI] [PubMed] [Google Scholar]
16.T C, H DG, S M, G L, R JL, P L. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol. 2013;31(1):46–53. 10.1038/nbt.2450 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Parthasarathy A, Cross PJ, Dobson RCJ, Adams LE, Savka MA, Hudson AO. A Three-Ring Circus: Metabolism of the Three Proteogenic Aromatic Amino Acids and Their Role in the Health of Plants and Animals. Frontiers in molecular biosciences. 2018;5:29 10.3389/fmolb.2018.00029 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Prabhu PR, Hudson AO. Identification and Partial Characterization of an L-Tyrosine Aminotransferase (TAT) from Arabidopsis thaliana. Biochemistry research international. 2010;2010:549572 10.1155/2010/549572 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Mavrides C, Orr W. Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli. The Journal of biological chemistry. 1975;250(11):4128–33. [PubMed] [Google Scholar]
20.Gelfand DH, Steinberg RA. Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases. Journal of bacteriology. 1977;130(1):429–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Whitaker RJ, Gaines CG, Jensen RA. A multispecific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa. The Journal of biological chemistry. 1982;257(22):13550–6. [PubMed] [Google Scholar]
22.Rothman SC, Kirsch JF. How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase. Journal of molecular biology. 2003;327(3):593–608. 10.1016/s0022-2836(03)00095-0 [DOI] [PubMed] [Google Scholar]
23.Tison F, Normand E, Jaber M, Aubert I, Bloch B. Aromatic L-amino-acid decarboxylase (DOPA decarboxylase) gene expression in dopaminergic and serotoninergic cells of the rat brainstem. Neuroscience letters. 1991;127(2):203–6. 10.1016/0304-3940(91)90794-t [DOI] [PubMed] [Google Scholar]
24.Zheng C, Choquer M, Zhang B, Ge H, Hu S, Ma H, et al. LongSAGE gene-expression profiling of Botrytis cinerea germination suppressed by resveratrol, the major grapevine phytoalexin. Fungal biology. 2011;115(9):815–32. 10.1016/j.funbio.2011.06.009 [DOI] [PubMed] [Google Scholar]
25.Zhang Y, Zhang Y, Qiu D, Zeng H, Guo L, Yang X. BcGs1, a glycoprotein from Botrytis cinerea, elicits defence response and improves disease resistance in host plants. Biochemical and biophysical research communications. 2015;457(4):627–34. 10.1016/j.bbrc.2015.01.038 [DOI] [PubMed] [Google Scholar]
26.Williams BA, Haferkamp I, Keeling PJ. An ADP/ATP-specific mitochondrial carrier protein in the microsporidian Antonospora locustae. Journal of molecular biology. 2008;375(5):1249–57. 10.1016/j.jmb.2007.11.005 [DOI] [PubMed] [Google Scholar]
27.Kubicek CP, Starr TL, Glass NL. Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi. Annual review of phytopathology. 2014;52:427–51. 10.1146/annurev-phyto-102313-045831 [DOI] [PubMed] [Google Scholar]
28.Yang Y, Yang X, Dong Y, Qiu D. The Botrytis cinerea Xylanase BcXyl1 Modulates Plant Immunity. Frontiers in microbiology. 2018;9:2535 10.3389/fmicb.2018.02535 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Prins TW, Tudzynski P, Tiedemann Av, Tudzynski B, Have AT, Hansen ME, et al. Infection Strategies of Botrytis cinerea and Related Necrotrophic Pathogens. Fungal Pathology. 2000:33–64. [Google Scholar]
30.Brito N, Espino JJ, Gonzalez C. The endo-beta-1,4-xylanase xyn11A is required for virulence in Botrytis cinerea. Molecular plant-microbe interactions: MPMI. 2006;19(1):25–32. 10.1094/MPMI-19-0025 [DOI] [PubMed] [Google Scholar]
31.Reignault P, Valette-Collet O, Boccara M. The importance of fungal pectinolytic enzymes in plant invasion, host adaptability and symptom type. European Journal of Plant Pathology. 2008;120(1):1–11. [Google Scholar]
32.Brutus A, Reca IB, Herga S, Mattei B, Puigserver A, Chaix JC, et al. A family 11 xylanase from the pathogen Botrytis cinerea is inhibited by plant endoxylanase inhibitors XIP-I and TAXI-I. Biochemical and biophysical research communications. 2005;337(1):160–6. 10.1016/j.bbrc.2005.09.030 [DOI] [PubMed] [Google Scholar]
33.Blanco-Ulate B, Morales-Cruz A, Amrine KC, Labavitch JM, Powell AL, Cantu D. Genome-wide transcriptional profiling of Botrytis cinerea genes targeting plant cell walls during infections of different hosts. Frontiers in plant science. 2014;5:435 10.3389/fpls.2014.00435 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Gilbert HJ. The biochemistry and structural biology of plant cell wall deconstruction. Plant physiology. 2010;153(2):444–55. 10.1104/pp.110.156646 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Voragen AGJ, Coenen G-J, Verhoef RP, Schols HA. Pectin, a versatile polysaccharide present in plant cell walls. Structural Chemistry. 2009;20(263). [Google Scholar]
36.Cabib E, Blanco N, Grau C, Rodriguez-Pena JM, Arroyo J. Crh1p and Crh2p are required for the cross-linking of chitin to beta(1–6)glucan in the Saccharomyces cerevisiae cell wall. Molecular microbiology. 2007;63(3):921–35. 10.1111/j.1365-2958.2006.05565.x [DOI] [PubMed] [Google Scholar]
37.Del Sorbo G, Schoonbeek H, De Waard MA. Fungal transporters involved in efflux of natural toxic compounds and fungicides. Fungal genetics and biology: FG & B. 2000;30(1):1–15. [DOI] [PubMed] [Google Scholar]
38.Rogers B, Decottignies A, Kolaczkowski M, Carvajal E, Balzi E, Goffeau A. The pleitropic drug ABC transporters from Saccharomyces cerevisiae. Journal of molecular microbiology and biotechnology. 2001;3(2):207–14. [PubMed] [Google Scholar]
39.Stefanato FL, Abou-Mansour E, Buchala A, Kretschmer M, Mosbach A, Hahn M, et al. The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana. The Plant journal: for cell and molecular biology. 2009;58(3):499–510. [DOI] [PubMed] [Google Scholar]
40.Hayashi K, Schoonbeek HJ, De Waard MA. Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides. Applied and environmental microbiology. 2002;68(10):4996–5004. 10.1128/AEM.68.10.4996-5004.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Liu Y, Ryu H, Ge B, Pan G, Sun L, Park K, et al. Improvement of Wuyiencin biosynthesis in Streptomyces wuyiensis CK-15 by identification of a key regulator, WysR. Journal of microbiology and biotechnology. 2014;24(12):1644–53. 10.4014/jmb.1405.05017 [DOI] [PubMed] [Google Scholar]
42.Isono K. Nucleoside antibiotics: structure, biological activity, and biosynthesis. The Journal of antibiotics. 1988;41(12):1711–39. 10.7164/antibiotics.41.1711 [DOI] [PubMed] [Google Scholar]
43.Niu G, Tan H. Nucleoside antibiotics: biosynthesis, regulation, and biotechnology. Trends in microbiology. 2015;23(2):110–9. 10.1016/j.tim.2014.10.007 [DOI] [PubMed] [Google Scholar]
44.Chapman T, Kinsman O, Houston J. Chitin biosynthesis in Candida albicans grown in vitro and in vivo and its inhibition by nikkomycin Z. Antimicrobial agents and chemotherapy. 1992;36(9):1909–14. 10.1128/aac.36.9.1909 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Zhu C, Kang Q, Bai L, Cheng L, Deng Z. Identification and engineering of regulation-related genes toward improved kasugamycin production. Applied microbiology and biotechnology. 2016;100(4):1811–21. 10.1007/s00253-015-7082-3 [DOI] [PubMed] [Google Scholar]
46.Fan C, Guo M, Liang Y, Dong H, Ding G, Zhang W, et al. Pectin-conjugated silica microcapsules as dual-responsive carriers for increasing the stability and antimicrobial efficacy of kasugamycin. Carbohydrate polymers. 2017;172:322–31. 10.1016/j.carbpol.2017.05.050 [DOI] [PubMed] [Google Scholar]
47.Pedersen JC. Natamycin as a fungicide in agar media. Applied and environmental microbiology. 1992;58(3):1064–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Caffrey P, Aparicio JF, Malpartida F, Zotchev SB. Biosynthetic engineering of polyene macrolides towards generation of improved antifungal and antiparasitic agents. Current topics in medicinal chemistry. 2008;8(8):639–53. 10.2174/156802608784221479 [DOI] [PubMed] [Google Scholar]
49.Gessler NN, Aver'yanov AA, Belozerskaya TA. Reactive oxygen species in regulation of fungal development. Biochemistry (Mosc). 2007;72(10):1091–109. Epub 2007/11/21. [DOI] [PubMed] [Google Scholar]
50.Heller J, Tudzynski P. Reactive oxygen species in phytopathogenic fungi: signaling, development, and disease. Annu Rev Phytopathol. 2011;49:369–90. Epub 2011/05/17. 10.1146/annurev-phyto-072910-095355 [DOI] [PubMed] [Google Scholar]
51.Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol. 2010;11(10):R106 Epub 2010/10/29. 10.1186/gb-2010-11-10-r106 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0224643.r001

Decision Letter 0

Kandasamy Ulaganathan

27 Nov 2019

PONE-D-19-28852

Genome-wide Transcriptomic Analysis of the Response of Botrytis cinerea to Wuyiencin

PLOS ONE

Dear ProProfessor Zhang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Jan 11 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Kandasamy Ulaganathan

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

1. Thank you for including the following funding statement within your acknowledgements section of your manuscript; "This work was supported by the National Natural Science Foundation (31601684), the Special Fund for Basic Scientific Research of the Chinese Academy of Agricultural Sciences (Y2017JC12), and the National Key Research and Development Plan (2017YFD0201100). "

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"NO"

2. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

Additional Editor Comments (if provided):

You are requested to undertake a major revision of the paper addressing all the points raised by the reviewers. Please provide your reply for each of the comments made by the reviewers

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

Reviewer #4: Partly

Reviewer #5: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a decent study of genome-wide transcriptomic analysis of B. cinerea treated with wuyiencin. The authors identified 2067 differentially expressed genes, and performed and discussed pathway analysis, which revealed the molecular mechanisms of wuyiencin in treating B. cinerea.

Here are some minor questions.

1, Table S1 and Table S2 may be mis-labeled or uploaded in the opposite order. Can you double check?

2, Figure 2 looks nice. However, for the inner circle, I can barely read their colors and match them to the legend. Is there anyway to improve? Also for the third circle, what does the length of each bar represent?

Reviewer #2: The authors describe the inhibitory effects on Botrytis cinerea of the antifungal metabolite wuyiencin from the biological control bacterium Streptomyces ahygroscopicus var. wuyiensis, and present transcriptome data showing changes in gene expression in wuyiencin-treated B. cinerea compared to untreated controls.

The morphological effects of wuyiencin treatment are quite superficially, from the pictures shown in Fig. 1, it is difficult to draw any deeper conclusion about the molecular processes that are affected. The main part of the manuscript deals with the analyis of differential gene expression based on RNA sequencing. As expected from a potent growth inhibitor, many genes encoding metabolic enzymes are up- or downregulated, however, it is difficult to draw more specific conclusions about the molecular target(s) of wuyiencin. This applies also to the differential upregulation by wuyiencin of major plant cell degrading enzymes encoding genes (Bcxyn11A, Bcpg1/2, Bcpme1/2), which are interesting observations but don’t provide clear evidences towards a certain mode of action. In general, the interpretation of differential transcriptome studies taken at only one time point is very difficult because they are unable to describe the dynamics of gene expression changes induced by inhibitor treatment, and therefore yield only limited informations.

Part of the text is not written very well and carefully. Line numbers are missing. Some recent papers (e.g. about the latest B. cinerea genome sequence, van Kan et al., Mol Plant Pathol. 2017 18:75-89) have not been cited.

Reviewer #3: In this manuscript, Shi and colleagues present the transcriptome-wide response of Botrytis cinerea to exposure to the anti-fungal agent, wuyiencin. The manuscript is well written and provides an in-depth characterization of the genes which are differentially expressed in response to the drug. I had no major criticisms with the science, although I did find some of the results/conclusions a bit perplexing (discussed below) and felt that inclusion of p-values at certain points in the text would be informative to the reader (also discussed below). Aside from these two points, which can be easily fixed by some minor additions to the text, I feel that this manuscript is appropriate for publication in PLOS One.

Comments:

1. When first using the abbreviation for control (I assume this is what ck means) please define it (first paragraph of results).

2. When using phrases such as "almost significantly" please include p-values. My interpretation of "almost" may different than another reader's, particularly since the adjusted p-value cutoff was 0.05.

3. I found the upregulation of certain genes involved in pathogenesis, such as the CWDEs and the CAZymes, in response to the antifungal agent to be counter-intuitive. Perhaps a little more discussion on why this might be occurring would be helpful.

4. It is interesting that no genes involved in cell death are upregulated. In addition, the genes associated with stress responses that were identified in this study were all down-regulated.

5. Out of the pathways shown to be differentially expressed, could the authors point to a particular one which they believe is most likely the target of wuyiencin? The authors mention in the discussion that this work highlights potential mechanisms but I think I missed the ones that they predict to be most likely.

Reviewer #4: In order to elucidate the effects of wuyiencin on the transcriptional regulation in B. cinerea, the authors used genome-wide transcriptomic analysis of B. cinerea treated with wuyiencin.

The found that putative genes involved in amino acid metabolism were influenced in response to wuyiencin treatment. Moreover, the suggest expression of genes involved in protein synthesis and energy metabolism and of those encoding ATP-binding cassette transporters was markedly upregulated, whereas that of genes participating in DNA replication, cell cycle, and stress response was downregulated. Furthermore, wuyiencin resulted in mycelial malformation and negatively influenced cell growth rate and conidial yield in B. cinerea. Even though the subject is very important and the transcriptional analysis can give hint on wuyencin mode of actions this paper is mainly descriptive and not supported by functional analysis.

Major concerns:

1. Is there any correlation between the phenotypic influence by SEM and TEM to the transcriptional changes- this need to be addresses and discussed.

2. Lack of functional analysis of the effect demonstrated by transcriptional changes (e.g. are AA levels or metabolism really changed? ; are CWDE active?; Do cell cycle/DNA replication impaired?; how can athoures conclude that wuyiencin active efflux is occurs?)

3. Not enough details on the RNA-seq experiment and analysis such as PCA , reads number etc.

4. Missing statistic on qRT-PCR analysis

5. Figure 2 is unreadable! - must be replaced with better way of demonstration the transcriptional changes by GO- annotation diagrams or pies., KEGG analysis is missing and need to be added

6. The authors fail to discuss how come some of the pathway they suggested to be involved is upregulate but other genes in the same pathway are down regulated- in AA metabolism or CWDE (Figures 3 and 4)?

7. Is ROS effected by wuyiencin, may check similar effect demonstrated by Vogel et al., 2011

8. Authors fail to discuss their results and explain properly how specific the changes wuyiencin are and not part of general stress response??

9. Reference 11 is very important backbone for this paper and it is not available in pubmed

Minor concerns:

1. Figures legends are not sufficient

2. Statistic is missing Figures 3-4

3. Fig1. no size bars, arrows are not defined, pigment is not shown?

4. wuyiencin is not biological control agent??

5. Is there any correlation between RNAseq and qRT-PCR?

6. Sometime there is no correlation between genes marked on Fig 3 to the text in result section

7. In results section may parts belong to discussion

8. Conidial germination S1 figure should be in main text, also demonstrate other results on conidia

9. Why control called ck?? It is very confusing

10. Some earlier studies references are missing (e.g. in results second part line 3)

11. In discussion part paragraph 4 “ In the past…” is not connected and need to be removed

12. The manuscript is not written fluently and very hard to read

13. The MS must go through English professional editing

Reviewer #5: The manuscript titled "Genome-wide Transcriptomic Analysis of the Response of Botrytis cinerea to Wuyiencin" by Shi et al is well written and provides a solid base for the future investigation of the mode of action of Wuyiencin however, the authors did not provide access to their data nor did they provide citations for how their analyses were carried out. Just stating that a given software package was used does provide sufficient information to asses the statistical analyses carried out. A further point is that the quality of the images in the figure in the version of the manuscript I dowloaded was insufficient to illustrate what the authors indicated in the text especially for figure 1 and 2. A final major point is that there are instances in where the RT-qPCR data does not fully support the RNA-seq data and as such the athors need to moderate statments that suggest it is fully consistant with the RNA-seq data

Apart from these major issues that need to be addressed the authors also need to improve the language associated with their analyse of the data.

For example In the abstract the sentence beginning "We could identify 2067 differentially .." should be edited to improve clarity

Change the phrase "remarkably influence" to improve precision of the statement give specifics, up regulated ? down regulated? or even change the proceeding structure to something like " transcript levels of genes involved in amino acid metabolism and those encoding putative secreted proteins were altered..."

IN this regard it is more precise that the authors refer to altered transcript levels since they are not specifically measuring gene expression.

Also the last sentence of the abstract is too general the data presented does not suppot this statement so it should be edited to clarify what the data does support.

In the first paragraph of the introduction edit the phrase "relatively suitable condition" to more precisely indicate which conditions are required for conidiation.

Introduction second paragraph the evidence is not conclusive enough to make the statment that differential expression of enzymes is the only basis for B. cinerea infection of multiple hosts.

QRT_PCR should be RT-qPCR since it is the PCR that is quantitative and RT-qPCR does not validate RNA-seq data it provides data that support that obtained by RNA-seq.

IN the results, Figure 2 does not illustrate clearly the number of genes with altered transcript levels

KEGG does not simply classify genes it assess the representation of specific functional groups of genes edit the text to clarify this indicating that the DEGs were enriched in the following functional categories ...

The phrase almost significantly down regulated is meaningless indicate specifically what the data shows.

Statements like "Protein synthesis, although an exceedingly complicated process ..." are overly obvious and add nothing to the text add such statements to indicate more precisely what the authors are trying to convey

The sentence "These findings suggest that B. cinerea uses different protein synthesis metabolism when exposed to phytoalexin from plants and antibiotic in microorganisms." is not clear do the authors mean " ... and when exposed to antibiotics from microorganisms"?

The phrase "most significant" is not meaningful, edit to clarify precisely what the authors intend to convey

The statement "The downregulation of the expression of these genes is directly related to cell growth

rate impairment in B. cinerea upon wuyiencin treatmen." is poorly worded and not support. The authors need to cite data that supports the contention that altering the transcript levels of these genes alters growth rate

To further validate was not the reason RT-qPCR was carried out it was carried out to assess if RNA_seq data was supportable by a different method of analysis

The authors state "significantly downregulated" but never define this nor do they provide sufficient information on their statistical analyses such that this can be independently assessed. this must be edited for clarification.

Sun et al is referenced but no date is provided for this reference

The statement "We strongly believe" has no place in a scientific publication. This is not religion, evidence must be presented to support conclusions.

The authors state "...in this study, we identified candidate pathways and target genes that may offer insights into the protective and antagonistic mechanisms underlying the action of biological control agents." but do not eleborate on what information they can gain from the data about the potential mode of action of the antifungal used and how that relates to other antifungal of the same class

In the conclusion the phrase "actual situations" is meaningless the authors must edit this phrase and the sentence it is in to provide clarity of the point they are trying to convey

The authors must define how they denatured the RNA during agarose gel electrophoresis

The authors must provide more information on how they used DESeq R and provide a citation for this analysis

Eliminate any reference of RT-qPCR being carried out to confirm RN-seq data this suggests that you had expectations of what the data would show which would have influenced interpretation of he reults

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Matthias Hahn

Reviewer #3: Yes: Andrew D. L. Nelson

Reviewer #4: No

Reviewer #5: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Apr 29;15(4):e0224643. doi: 10.1371/journal.pone.0224643.r002

Author response to Decision Letter 0

11 Feb 2020

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

Reviewer #4: Partly

Reviewer #5: No

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: No

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: No

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

Review Comments to the Author

Here are some minor questions.

1, Table S1 and Table S2 may be mis-labeled or uploaded in the opposite order. Can you double check?

Answer: Table S1 and Table S2 were checked and revised.

Answer: Figure 2 was revised. The legend of Figure 2 has been modified. Thanks for your kind comment, we put the information of metabolic pathways of DEGs in the supplemental Table S3.

For instance:

Figure 2. Circular map of B05.10 genome and genes that were differentially expressed (DEGs) upon wuyiencin treatment. Moving inward, the outer two rings show the length (red ring) and gene (green ring) density of every chromosome, respectively. The third ring represents differentially expressed genes (DEGs): upregulated and downregulated DEGs are highlighted as red and blue bars, respectively. The length of each bar represent the fold change.

Answer: Line numbers are adding and the recent paper have been cited.

Comments:

1. When first using the abbreviation for control (I assume this is what ck means) please define it (first paragraph of results).

Answer: In the first paragraph of results (Line 104), it was revised.

Answer: Line 151, "almost" was deleted, to avoid misunderstanding.

Answer: The related content was in Discussion part.

4. It is interesting that no genes involved in cell death are upregulated. In addition, the genes associated with stress responses that were identified in this study were all down-regulated.

Answer: Yes.

Answer: The most likely target of wuyiencin is the cell growth and differentiation of Botrytis cinerea, especially in amino acid metabolism, protein synthesis and energy metabolism.

Reviewer #4: In order to elucidate the effects of wuyiencin on the transcriptional regulation in B. cinerea, the authors used genome-wide transcriptomic analysis of B. cinerea treated with wuyiencin.

Major concerns:

1. Is there any correlation between the phenotypic influence by SEM and TEM to the transcriptional changes- this need to be addresses and discussed.

Answer: In this study, we didn’t find any relationship between SEM and TEM to the transcriptional changes-. For the further study, the phenotypic change of unique gene or pathway may easily to observed by SEM and TEM.

3. Not enough details on the RNA-seq experiment and analysis such as PCA , reads number etc.

Answer: The details on RNA-seq experiment and analysis were added in Methods part.

4. Missing statistic on qRT-PCR analysis

Answer: The statistic result of qRT-PCR analysis was added in Table S2.

5. Figure 2 is unreadable! - must be replaced with better way of demonstration the transcriptional changes by GO- annotation diagrams or pies., KEGG analysis is missing and need to be added.

Answer: Figure 2 was revised and KEGG analysis is added in Method part.

Answer: This is difficult to explain the reversed gene expression just from transcriptional data. The affection of wuyiencin may trigger the host gene feedback regulation for survival. This speculate need more single-gene experimental data to support.

7. Is ROS effected by wuyiencin, may check similar effect demonstrated by Vogel et al., 2011

Answer: We didn’t find this ref, but ROS effected by wuyiencin was similar with part of general stress response, and this was further discussed in revised manuscript in the second paragraph from bottom of discussion.

8. Authors fail to discuss their results and explain properly how specific the changes wuyiencin are and not part of general stress response??

Answer: From this study, we just know that there are some similar changes that caused by the wuyiencin compared with general stress response. The further discussion was added to the revised manuscript in the second paragraph from bottom of discussion and add the related refs below.

Gessler, N. N.; Aver'yanov, A. A.; Belozerskaya, T. A. Reactive oxygen species in regulation of fungal development. Biochemistry (Mosc).2007, 72, 1091-109.

Heller, J.; Tudzynski, P. Reactive oxygen species in phytopathogenic fungi: signaling, development, and disease. Annu Rev Phytopathol.2011, 49, 369-90.

9. Reference 11 is very important backbone for this paper and it is not available in pubmed

Answer: The reference 11can be found in Baiduxueshu website (www. xueshu.baidu.com/).

Minor concerns:

1. Figures legends are not sufficient

Answer: Figures legends had been revised.

2. Statistic is missing Figures 3-4

Answer: We used the unique mapped reads to annotate the readcount of each genes. And the significant difference analysis about the expression level of each gene was carried out by DESeq. And there is just one readcount number for each gene in the result file. Statistic is contained in the calculating of P-values.

3. Fig1. no size bars, arrows are not defined, pigment is not shown?

Answer: Size bars were added. Pigment was calculated using blood counting chamber and showed in histogram of Fig 1E.

4. wuyiencin is not biological control agent??

Answer: Wuyiencin is biological control agent.

5. Is there any correlation between RNAseq and qRT-PCR?

Answer: The selected genes used in qRT-PCR are all representative ones which significant differentially expressed in RNAseq. The qRT-PCR is the verification of RNAseq result and they are consistent.

6. Sometime there is no correlation between genes marked on Fig 3 to the text in result section

Answer: The key genes in Fig 3 are mainly involved in amino acid metabolism. And some of them also participate in protein synthesis or carbon and energy metabolism. Some sentences have been adjusted in order to the correlation between genes marked on Fig 3 to the text.

7. In results section may parts belong to discussion.

Answer: Yes.

8. Conidial germination S1 figure should be in main text, also demonstrate other results on conidia

Answer: Figure S1 was added to Figure 1 as 1E.

9. Why control called ck?? It is very confusing

Answer: In the first paragraph of results (Line 104), it was revised.

10. Some earlier studies references are missing (e.g. in results second part line 3)

Answer: The reference had been added.

11. In discussion part paragraph 4 “ In the past…” is not connected and need to be removed

Answer: It was revised.

12. The manuscript is not written fluently and very hard to read.

Answer: We tried our best to improve the manuscript and made some changes for better reading.

13. The MS must go through English professional editing.

Answer: Yes.

however, the authors did not provide access to their data nor did they provide citations for how their analyses were carried out. Just stating that a given software package was used does provide sufficient information to assess the statistical analyses carried out.

Answer: All of the raw reads have been uploaded to NCBI Sequence Read Archive (SRA) database. And more detailed analysis method that we carried out and the accession number has been added to Methods part and data availability statement.

A further point is that the quality of the images in the figure in the version of the manuscript I dowloaded was insufficient to illustrate what the authors indicated in the text especially for figure 1 and 2.

Answer: The quality of the images were improved.

A final major point is that there are instances in where the RT-qPCR data does not fully support the RNA-seq data and as such the athors need to moderate statments that suggest it is fully consistant with the RNA-seq data

Answer: The two results are not fully consistant, but the overall trend is the same. The sentence has been revised to express more accurately.

Apart from these major issues that need to be addressed the authors also need to improve the language associated with their analyse of the data.

For example In the abstract the sentence beginning "We could identify 2067 differentially .." should be edited to improve clarity. Change the phrase "remarkably influence" to improve precision of the statement give specifics, up regulated ? down regulated? or even change the proceeding structure to something like " transcript levels of genes involved in amino acid metabolism and those encoding putative secreted proteins were altered..."IN this regard it is more precise that the authors refer to altered transcript levels since they are not specifically measuring gene expression.

Answer: The sentences in the abstract and other part had been revised to improve clarity.

Also the last sentence of the abstract is too general the data presented does not suppot this statement so it should be edited to clarify what the data does support.

Answer: The last sentence of the abstract has been modified according to reviewer’s suggestion.

In the first paragraph of the introduction edit the phrase "relatively suitable condition" to more precisely indicate which conditions are required for conidiation.

Answer: The sentences in the first paragraph of the introduction had been revised to improve clarity.

Introduction second paragraph the evidence is not conclusive enough to make the statment that differential expression of enzymes is the only basis for B. cinerea infection of multiple hosts.

Answer: The sentences in the second paragraph of the introduction had been revised to improve clarity, and the differential expression of enzymes is just one way for B. cinerea infection of multiple hosts.

QRT_PCR should be RT-qPCR since it is the PCR that is quantitative and RT-qPCR does not validate RNA-seq data it provides data that support that obtained by RNA-seq.

Answer: The qRT-PCR statement and related sentences had been corrected.

IN the results, Figure 2 does not illustrate clearly the number of genes with altered transcript levels

Answer: Figure 2 was revised.

Answer: Yes.

The phrase almost significantly down regulated is meaningless indicate specifically what the data shows.

Answer: The sentences in line 151 had been revised to improve clarity.

Answer: The sentences in line 191 had been deleted.

Answer: The sentences in line 213 had been revised to improve clarity.

The phrase "most significant" is not meaningful, edit to clarify precisely what the authors intend to convey

Answer: The sentences in line 216 had been revised to improve clarity.

The statement "The downregulation of the expression of these genes is directly related to cell growth rate impairment in B. cinerea upon wuyiencin treatmen." is poorly worded and not support. The authors need to cite data that supports the contention that altering the transcript levels of these genes alters growth rate

Answer: The sentences in line 325 had been revised to improve clarity.

To further validate was not the reason RT-qPCR was carried out it was carried out to assess if RNA_seq data was supportable by a different method of analysis

Answer: The sentences in line 326 had been revised to improve clarity.

Answer: The sentences in line342 and line345 were added ‘(Table S1)’ which could provide information.

Sun et al is referenced but no date is provided for this reference

Answer: The reference were added here (Line 381).

The statement "We strongly believe" has no place in a scientific publication. This is not religion, evidence must be presented to support conclusions.

Answer: The sentences in line 435 had been revised to improve clarity.

Answer: According to the analysis of transcriptome, the genes relating to amino acid metabolism, protein synthesis and cell cycle/DNA replication are differentially expressed. And the expression of some genes in these metabolism are verified by RT-qPCR. The most likely target of wuyiencin is the cell growth and differentiation of Botrytis cinerea, especially in amino acid metabolism, protein synthesis and energy metabolism.

In the conclusion the phrase "actual situations" is meaningless the authors must edit this phrase and the sentence it is in to provide clarity of the point they are trying to convey

Answer: The sentences in line 441 had been revised to improve clarity and the phrase "actual situations" means the cultivation environment in actual field.

The authors must define how they denatured the RNA during agarose gel electrophoresis

Answer: We did not denature the RNA during agarose gel electrophoresis. Only RNA integrity and size distribution were assessed using 1% agarose gel electrophoresis.

The authors must provide more information on how they used DESeq R and provide a citation for this analysis

Answer: The Methods part Transcriptomic analysis were supplemented and the reference had been cited.

Eliminate any reference of RT-qPCR being carried out to confirm RNA-seq data this suggests that you had expectations of what the data would show which would have influenced interpretation of the results

Answer: To confirm RNA-seq results, 20 DEGs were chosen for RT-qPCR randomly without expectations.

Attachment

Submitted filename: Response to Reviewer Comments-1(1).docx

Click here for additional data file.^{(32.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0224643.r003

Decision Letter 1

Kandasamy Ulaganathan

27 Mar 2020

Genome-wide Transcriptomic Analysis of the Response of Botrytis cinerea to Wuyiencin

PONE-D-19-28852R1

Dear Dr. Zhang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Kandasamy Ulaganathan

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #5: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Reviewer #1: The author has addressed all my concerns. I have no further questions. I would suggest to accept this paper.

Reviewer #3: I thank the authors for addressing my concerns. I still do not think that they really address in the discussion that they observe downregulation of stress response genes (lines 420-423) and upregulation of genes that a naive reviewer would assume to help the pathogen be more virulent (lines 277-283) or better degrade the cell wall of their host. Finally, the authors mention on line 323-324 that downregulation of the expression of these genes (Bcin09g01360, etc) are directly related to cell growth impairment in B. cinerea... This sentence is speculation and as such should be moved to the discussion or changed to "may be related".

Overall though, I think the data will be useful to the community. Aside from the above points and a few areas where grammar needs to be addressed, I think this manuscript is suitable for publication.

Reviewer #5: I feel that the authors have addressed the concerns I raised adequately. The improved images make a big difference. There is still some minor issues with wording but it is acceptable.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: Yes: Andrew Nelson

Reviewer #5: No

PLoS One. doi: 10.1371/journal.pone.0224643.r004

Acceptance letter

Kandasamy Ulaganathan

20 Apr 2020

PONE-D-19-28852R1

Genome-wide Transcriptomic Analysis of the Response of Botrytis cinerea to Wuyiencin

Dear Dr. Zhang:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Kandasamy Ulaganathan

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Gene expression changes in Botrytis cinerea cultivated with 100 μg/mL and without wuyiencin.

(XLSX)

Click here for additional data file.^{(16KB, xlsx)}

S2 Table. Gene expression changes in Botrytis cinerea cultivated with 100 μg/mL and without (ck) wuyiencin, and validation of results by RT-qPCR.

(XLSX)

Click here for additional data file.^{(22.6KB, xlsx)}

S3 Table. The information of metabolic pathways of the differentially expressed genes (DEGs) in Botrytis cinerea cultivated with 100 μg/mL and without wuyiencin.

(XLS)

Click here for additional data file.^{(14KB, xls)}

Attachment

Submitted filename: Response to Reviewer Comments-1(1).docx

Click here for additional data file.^{(32.6KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files. The RNA-seq dataset generated for this study can be found in the NCBI database under the accession number SRP212990.

[pone.0224643.ref001] 1.Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, et al. The Top 10 fungal pathogens in molecular plant pathology. Molecular plant pathology. 2012;13(4):414–30. 10.1111/j.1364-3703.2011.00783.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref002] 2.Staats M, van Kan JA. Genome update of Botrytis cinerea strains B05.10 and T4. Eukaryotic cell. 2012;11(11):1413–4. 10.1128/EC.00164-12 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref003] 3.Leroch M, Kleber A, Silva E, Coenen T, Koppenhofer D, Shmaryahu A, et al. Transcriptome profiling of Botrytis cinerea conidial germination reveals upregulation of infection-related genes during the prepenetration stage. Eukaryotic cell. 2013;12(4):614–26. 10.1128/EC.00295-12 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref004] 4.Staples RC, Mayer AM. Putative virulence factors of Botrytis cinerea acting as a wound pathogen. FEMS microbiology letters. 1995;134(1):1–7. [Google Scholar]

[pone.0224643.ref005] 5.Li B, Wang W, Zong Y, Qin G, Tian S. Exploring pathogenic mechanisms of Botrytis cinerea secretome under different ambient pH based on comparative proteomic analysis. Journal of proteome research. 2012;11(8):4249–60. 10.1021/pr300365f [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref006] 6.Wubben JP, ten Have A, van Kan JA, Visser J. Regulation of endopolygalacturonase gene expression in Botrytis cinerea by galacturonic acid, ambient pH and carbon catabolite repression. Current genetics. 2000;37(2):152–7. 10.1007/s002940050022 . [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref007] 7.ten Have A, Breuil WO, Wubben JP, Visser J, van Kan JA. Botrytis cinerea endopolygalacturonase genes are differentially expressed in various plant tissues. Fungal genetics and biology: FG & B. 2001;33(2):97–105. [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref008] 8.ten Have A, Espino JJ, Dekkers E, Van Sluyter SC, Brito N, Kay J, et al. The Botrytis cinerea aspartic proteinase family. Fungal genetics and biology: FG & B. 2010;47(1):53–65. [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref009] 9.Wilson CL., Wisniewski ME., Biles CL., McLaughlin Randy, Chalutz Edo, Droby Samir. Biological control of post-harvest diseases of fruits and vegetables: alternatives to synthetic fungicides. Crop Protection. 1991;10(3):172–7. [Google Scholar]

[pone.0224643.ref010] 10.Ge B, Liu Y, Liu B, Zhao W, Zhang K. Characterization of novel DeoR-family member from the Streptomyces ahygroscopicus strain CK-15 that acts as a repressor of morphological development. Applied microbiology and biotechnology. 2016;100(20):8819–28. 10.1007/s00253-016-7661-y [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref011] 11.Sun Y, Zeng H, Shi Y, Li G. Mode of action of Wuyiencin on Botrytis cinerea. ACTA PHYTOPATHOLOGICA SINICA. 2003;33(5). [Google Scholar]

[pone.0224643.ref012] 12.Choquer M, Fournier E, Kunz C, Levis C, Pradier JM, Simon A, et al. Botrytis cinerea virulence factors: new insights into a necrotrophic and polyphageous pathogen. FEMS microbiology letters. 2007;277(1):1–10. 10.1111/j.1574-6968.2007.00930.x [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref013] 13.Amselem J, Cuomo CA, van Kan JA, Viaud M, Benito EP, Couloux A, et al. Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS genetics. 2011;7(8):e1002230 10.1371/journal.pgen.1002230 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref014] 14.Van Kan JA, Stassen JH, Mosbach A, Van Der Lee TA, Faino L, Farmer AD, et al. A gapless genome sequence of the fungus Botrytis cinerea. Mol Plant Pathol. 2017;18(1):75–89. Epub 2016/02/26. 10.1111/mpp.12384 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref015] 15.Melendez HG, Billon-Grand G, Fevre M, Mey G. Role of the Botrytis cinerea FKBP12 ortholog in pathogenic development and in sulfur regulation. Fungal genetics and biology: FG & B. 2009;46(4):308–20. [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref016] 16.T C, H DG, S M, G L, R JL, P L. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol. 2013;31(1):46–53. 10.1038/nbt.2450 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref017] 17.Parthasarathy A, Cross PJ, Dobson RCJ, Adams LE, Savka MA, Hudson AO. A Three-Ring Circus: Metabolism of the Three Proteogenic Aromatic Amino Acids and Their Role in the Health of Plants and Animals. Frontiers in molecular biosciences. 2018;5:29 10.3389/fmolb.2018.00029 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref018] 18.Prabhu PR, Hudson AO. Identification and Partial Characterization of an L-Tyrosine Aminotransferase (TAT) from Arabidopsis thaliana. Biochemistry research international. 2010;2010:549572 10.1155/2010/549572 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref019] 19.Mavrides C, Orr W. Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli. The Journal of biological chemistry. 1975;250(11):4128–33. [PubMed] [Google Scholar]

[pone.0224643.ref020] 20.Gelfand DH, Steinberg RA. Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases. Journal of bacteriology. 1977;130(1):429–40. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref021] 21.Whitaker RJ, Gaines CG, Jensen RA. A multispecific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa. The Journal of biological chemistry. 1982;257(22):13550–6. [PubMed] [Google Scholar]

[pone.0224643.ref022] 22.Rothman SC, Kirsch JF. How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase. Journal of molecular biology. 2003;327(3):593–608. 10.1016/s0022-2836(03)00095-0 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref023] 23.Tison F, Normand E, Jaber M, Aubert I, Bloch B. Aromatic L-amino-acid decarboxylase (DOPA decarboxylase) gene expression in dopaminergic and serotoninergic cells of the rat brainstem. Neuroscience letters. 1991;127(2):203–6. 10.1016/0304-3940(91)90794-t [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref024] 24.Zheng C, Choquer M, Zhang B, Ge H, Hu S, Ma H, et al. LongSAGE gene-expression profiling of Botrytis cinerea germination suppressed by resveratrol, the major grapevine phytoalexin. Fungal biology. 2011;115(9):815–32. 10.1016/j.funbio.2011.06.009 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref025] 25.Zhang Y, Zhang Y, Qiu D, Zeng H, Guo L, Yang X. BcGs1, a glycoprotein from Botrytis cinerea, elicits defence response and improves disease resistance in host plants. Biochemical and biophysical research communications. 2015;457(4):627–34. 10.1016/j.bbrc.2015.01.038 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref026] 26.Williams BA, Haferkamp I, Keeling PJ. An ADP/ATP-specific mitochondrial carrier protein in the microsporidian Antonospora locustae. Journal of molecular biology. 2008;375(5):1249–57. 10.1016/j.jmb.2007.11.005 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref027] 27.Kubicek CP, Starr TL, Glass NL. Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi. Annual review of phytopathology. 2014;52:427–51. 10.1146/annurev-phyto-102313-045831 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref028] 28.Yang Y, Yang X, Dong Y, Qiu D. The Botrytis cinerea Xylanase BcXyl1 Modulates Plant Immunity. Frontiers in microbiology. 2018;9:2535 10.3389/fmicb.2018.02535 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref029] 29.Prins TW, Tudzynski P, Tiedemann Av, Tudzynski B, Have AT, Hansen ME, et al. Infection Strategies of Botrytis cinerea and Related Necrotrophic Pathogens. Fungal Pathology. 2000:33–64. [Google Scholar]

[pone.0224643.ref030] 30.Brito N, Espino JJ, Gonzalez C. The endo-beta-1,4-xylanase xyn11A is required for virulence in Botrytis cinerea. Molecular plant-microbe interactions: MPMI. 2006;19(1):25–32. 10.1094/MPMI-19-0025 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref031] 31.Reignault P, Valette-Collet O, Boccara M. The importance of fungal pectinolytic enzymes in plant invasion, host adaptability and symptom type. European Journal of Plant Pathology. 2008;120(1):1–11. [Google Scholar]

[pone.0224643.ref032] 32.Brutus A, Reca IB, Herga S, Mattei B, Puigserver A, Chaix JC, et al. A family 11 xylanase from the pathogen Botrytis cinerea is inhibited by plant endoxylanase inhibitors XIP-I and TAXI-I. Biochemical and biophysical research communications. 2005;337(1):160–6. 10.1016/j.bbrc.2005.09.030 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref033] 33.Blanco-Ulate B, Morales-Cruz A, Amrine KC, Labavitch JM, Powell AL, Cantu D. Genome-wide transcriptional profiling of Botrytis cinerea genes targeting plant cell walls during infections of different hosts. Frontiers in plant science. 2014;5:435 10.3389/fpls.2014.00435 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref034] 34.Gilbert HJ. The biochemistry and structural biology of plant cell wall deconstruction. Plant physiology. 2010;153(2):444–55. 10.1104/pp.110.156646 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref035] 35.Voragen AGJ, Coenen G-J, Verhoef RP, Schols HA. Pectin, a versatile polysaccharide present in plant cell walls. Structural Chemistry. 2009;20(263). [Google Scholar]

[pone.0224643.ref036] 36.Cabib E, Blanco N, Grau C, Rodriguez-Pena JM, Arroyo J. Crh1p and Crh2p are required for the cross-linking of chitin to beta(1–6)glucan in the Saccharomyces cerevisiae cell wall. Molecular microbiology. 2007;63(3):921–35. 10.1111/j.1365-2958.2006.05565.x [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref037] 37.Del Sorbo G, Schoonbeek H, De Waard MA. Fungal transporters involved in efflux of natural toxic compounds and fungicides. Fungal genetics and biology: FG & B. 2000;30(1):1–15. [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref038] 38.Rogers B, Decottignies A, Kolaczkowski M, Carvajal E, Balzi E, Goffeau A. The pleitropic drug ABC transporters from Saccharomyces cerevisiae. Journal of molecular microbiology and biotechnology. 2001;3(2):207–14. [PubMed] [Google Scholar]

[pone.0224643.ref039] 39.Stefanato FL, Abou-Mansour E, Buchala A, Kretschmer M, Mosbach A, Hahn M, et al. The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana. The Plant journal: for cell and molecular biology. 2009;58(3):499–510. [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref040] 40.Hayashi K, Schoonbeek HJ, De Waard MA. Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides. Applied and environmental microbiology. 2002;68(10):4996–5004. 10.1128/AEM.68.10.4996-5004.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref041] 41.Liu Y, Ryu H, Ge B, Pan G, Sun L, Park K, et al. Improvement of Wuyiencin biosynthesis in Streptomyces wuyiensis CK-15 by identification of a key regulator, WysR. Journal of microbiology and biotechnology. 2014;24(12):1644–53. 10.4014/jmb.1405.05017 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref042] 42.Isono K. Nucleoside antibiotics: structure, biological activity, and biosynthesis. The Journal of antibiotics. 1988;41(12):1711–39. 10.7164/antibiotics.41.1711 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref043] 43.Niu G, Tan H. Nucleoside antibiotics: biosynthesis, regulation, and biotechnology. Trends in microbiology. 2015;23(2):110–9. 10.1016/j.tim.2014.10.007 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref044] 44.Chapman T, Kinsman O, Houston J. Chitin biosynthesis in Candida albicans grown in vitro and in vivo and its inhibition by nikkomycin Z. Antimicrobial agents and chemotherapy. 1992;36(9):1909–14. 10.1128/aac.36.9.1909 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref045] 45.Zhu C, Kang Q, Bai L, Cheng L, Deng Z. Identification and engineering of regulation-related genes toward improved kasugamycin production. Applied microbiology and biotechnology. 2016;100(4):1811–21. 10.1007/s00253-015-7082-3 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref046] 46.Fan C, Guo M, Liang Y, Dong H, Ding G, Zhang W, et al. Pectin-conjugated silica microcapsules as dual-responsive carriers for increasing the stability and antimicrobial efficacy of kasugamycin. Carbohydrate polymers. 2017;172:322–31. 10.1016/j.carbpol.2017.05.050 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref047] 47.Pedersen JC. Natamycin as a fungicide in agar media. Applied and environmental microbiology. 1992;58(3):1064–6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0224643.ref048] 48.Caffrey P, Aparicio JF, Malpartida F, Zotchev SB. Biosynthetic engineering of polyene macrolides towards generation of improved antifungal and antiparasitic agents. Current topics in medicinal chemistry. 2008;8(8):639–53. 10.2174/156802608784221479 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref049] 49.Gessler NN, Aver'yanov AA, Belozerskaya TA. Reactive oxygen species in regulation of fungal development. Biochemistry (Mosc). 2007;72(10):1091–109. Epub 2007/11/21. [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref050] 50.Heller J, Tudzynski P. Reactive oxygen species in phytopathogenic fungi: signaling, development, and disease. Annu Rev Phytopathol. 2011;49:369–90. Epub 2011/05/17. 10.1146/annurev-phyto-072910-095355 [DOI] [PubMed] [Google Scholar]

[pone.0224643.ref051] 51.Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol. 2010;11(10):R106 Epub 2010/10/29. 10.1186/gb-2010-11-10-r106 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Genome-wide transcriptomic analysis of the response of Botrytis cinerea to wuyiencin

Liming Shi

Binghua Liu

Qiuhe Wei

Beibei Ge

Kecheng Zhang

Roles

Abstract

Introduction

Results

Influence of wuyiencin on the growth and morphology of B. cinerea

Fig 1. Phenotypes of Botrytis cinerea cultivated with various concentrations of wuyiencin (50 μg/mL, 100 μg/mL, and 200 μg/mL wuyiencin).

Genome-wide transcriptomic analysis

Fig 2. Circular map of B05.10 genome and genes that were differentially expressed (DEGs) upon wuyiencin treatment.

Transcriptional analysis of genes involved in amino acid metabolism and protein synthesis

Fig 3. Expression profiles of genes involved in amino acid metabolism.

Transcriptional analysis of genes involved in carbon and energy metabolism

Transcriptional analysis of genes encoding putative secreted metabolites and proteins

Fig 4. Expression profiles of genes encoding putative secreted metabolites and proteins.

Transcriptional analysis of genes involved in DNA replication and cell cycle

Transcriptional analysis of genes involved in other metabolism

Discussion

Materials and methods

Treatment of B. cinerea with wuyiencin

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

RNA isolation

Transcriptomic analysis

Quantitative real-time RT-PCR (RT-qPCR)

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Kandasamy Ulaganathan

Roles

Author response to Decision Letter 0

Decision Letter 1

Kandasamy Ulaganathan

Roles

Acceptance letter

Kandasamy Ulaganathan

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases