Abstract
Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal plant pathogen. Membrane proteins are important targets of fungicides and hotspots in the research and development of fungicide products. Wuyiencin affects the permeability and pathogenicity of B. cinerea, parallel reaction monitoring revealed the association of membrane protein Bcsdr2, and the bacteriostatic mechanism of wuyiencin was elucidated. In the present work, we generated and characterised ΔBcsdr2 deletion and complemented mutant B. cinerea strains. The ΔBcsdr2 deletion mutants exhibited biofilm loss and dissolution, and their functional activity was illustrated by reduced necrotic colonisation on strawberry and grape fruits. Targeted deletion of Bcsdr2 also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted gene complementation. The roles of Bcsdr2 in biofilms and pathogenicity were also supported by quantitative real-time RT-PCR results showing that phosphatidylserine decarboxylase synthesis gene Bcpsd and chitin synthase gene BcCHSV II were downregulated in the early stages of infection for the ΔBcsdr2 strain. The results suggest that Bcsdr2 plays important roles in regulating various cellular processes in B. cinerea.
Key points
• The mechanism of wuyiencin inhibits B. cinerea is closely associated with membrane proteins.
• Wuyiencin can downregulate the expression of the membrane protein Bcsdr2 in B. cinerea.
• Bcsdr2 is involved in regulating B. cinerea virulence, growth and development.
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Introduction
Botrytis cinerea Pers.: Fr. (teleomorph: Botryotinia fuckeliana Whetzel) is a typical necrotrophic ascomycete and worldwide plant pathogen that infects crop hosts during both pre- and post-harvesting phases (Bu et al. 2021; Fan et al. 2023). Moreover, B. cinerea infects > 1400 plant species including many economically important crops, leading to tremendous economic losses (Elad 2016). Due to the lack of resistant varieties, chemical control remains the most effective strategy for managing grey mould caused by B. cinerea (Weidensdorfer et al. 2019). However, through genetic plasticity, B. cinerea has developed resistance to many types of fungicides (Liu et al. 2021). Researchers use B. cinerea as a model fungus in molecular studies, therefore, exploring the molecular mechanisms underlying the development and virulence of B. cinerea will contribute to establishing more effective disease control strategies.
Membrane proteins play key roles in the physiological processes of microorganisms including transportation and intercellular communication, and they serve as important drug targets. Membrane proteins are recognised and inserted into the lipid bilayer by exquisite cellular machineries such as GlpG rhomboid protease, which is thought to allow the docking of a transmembrane substrate (Engberg et al. 2022). Transporters are integral membrane proteins with central roles in the efficient movement of molecules across biological membranes. The nucleobase ascorbate transporter UapA from Aspergillus nidulans must dimerise for correct trafficking to the membrane (Alguel et al. 2016). Many membrane proteins are primary drug targets, especially those involved in converting extracellular signals into intracellular processes. Among them, G protein-coupled receptors (GPCRs) are crucial for cellular responses to a range of bioactive molecules, and they play a key role in signalling, such as increasing the basal activity of the cannabinoid 2 receptor (Yeliseev et al. 2021). Interestingly, cell membrane proteins are important targets of fungicides in the prevention and control of fungal diseases (Huang et al. 2022). For example, natamycin inhibits the growth of yeasts and other fungi by inhibiting plasma membrane transporters that regulate amino acid and glucose transport (te Welscher et al. 2012).
Wuyiencin is a nucleoside biological fungicide that has a significant control effect on B. cinerea. Previous analysis in our laboratory found that wuyiencin can inhibit B. cinerea by affecting mycelial morphology and intracellular structure (Sun et al. 2003). Using proteomics, we identified 27 proteins that are significantly regulated by wuyiencin in B. cinerea (Shi et al. 2019). Cell membrane proteins mediate the effects of fungicides in the prevention and control of fungal diseases by fungicides (Huang et al. 2022). Since wuyiencin can inhibit protein synthesis and increase cell membrane permeability of B. cinerea, we highlighted the downregulated membrane protein Bcsdr2 in network-based comparative genomic analysis, but the functions of Bcsdr2 remain poorly understood. Through homologous protein searches, we found that homologs of Bcsdr2 included alcohol dehydrogenase (BcADH1), short-chain dehydrogenase/reductase (YdfG and YMR226C), NADPH-dependent 1-acyldihydroxyacetone phosphate reductase (AYR1) and others. Among them, BcADH1 can mediate spore germination, hyphal growth and sclerotia formation in B. cinerea, decrease sporulation and affect pathogenicity and oxidative stress ability (DafaAlla et al. 2022). YdfG and YMR226C are NADP( +)-dependent short-chain dehydrogenases with L-serine dehydrogenase activity, and they play important roles in maintaining normal physiological metabolism in Escherichia coli and Saccharomyces cerevisiae (Fujisawa et al. 2003). Deletion of Ayr1 results in the loss of 1-acylglycerol phosphate reductase activity, which inhibited the synthesis of S. cerevisiae lipids and spore germination (Athenstaedt and Daum 2000). In the present study, we investigated the role of membrane protein Bcsdr2 in fungal growth and pathogenesis to reveal and explore the role of wuyiencin in controlling B. cinerea.
Materials and methods
Fungal strains and culture conditions
Strain B05.10 of B. cinerea Pers.: Fr. (B. fuckeliana (de Bary) Whetzel) was originally isolated from Vitis vinifera and has been widely used as a standard reference strain (Quidde et al. 1999). The strain B05.10 of B. cinerea was gifted by Associate Professor Liu Pengfei of China Agricultural University. B. cinerea was grown on potato dextrose agar (PDA; 200 g potato, 20 g dextrose, 20 g agar, 1 L water), complete medium (CM; 1 g yeast extract, 0.5 g casein acid hydrolysate, 0.5 g hydrolysed casein, 10 g glucose, 4 mM Ca(NO3)2ꞏ4H2O, 1.5 mM KH2PO4, 1 mM MgSO4·7H2O, 2.5 mM NaCl, 20 g agar, 1 L water) and minimal medium (MM; 10 mM K2HPO4, 10 mM KH2PO4, 4 mM (NH4)2SO4, 2.5 mM NaCl, 2 mM MgSO4, 0.45 mM CaCl2, 9 μM FeSO4, 10 mM glucose, 20 g agar, 1 L water, pH 6.9).
Conidia were quantified after 10 days of incubation on PDA medium, washed from plates, diluted to 5 mL with ddH2O and counted under a microscope. Growth tests under different stress conditions were performed on PDA plates supplemented with different agents including H2O2, KCl and sodium dodecyl sulphate (SDS) (Yan et al. 2010). The percentage of mycelial radial growth inhibition (RGI) was calculated using the formula RGI = ([C – N] / [C − 5]) × 100, where C and N indicate the colony diameter of control and treatments, respectively (Yang et al. 2018). Each experiment was repeated three times.
Gene deletion and complementation
To replace the Bcsdr2 gene in wild type (WT) B05.10 strain, 1000 bp upstream and 1000 bp downstream flanking sequences of Bcsdr2 were amplified by PCR from genomic DNA of B05.10. The resulting amplicons were fused with the HPH (hygromycin resistance gene) using double-joint PCR (Yu et al. 2004). Protoplast preparation and transformation were performed as previously described (Gronover et al. 2001). The resulting hygromycin-resistant transformants were preliminarily screened by PCR with primers (Supplementary Table S1) and further confirmed by Southern blotting analysis. The hygromycin B resistance fragment was used as a probe and labelled with digoxigenin (DIG) using a High Prime DNA Labelling and Detection Starter Kit I (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol. Genomic DNA was digested with EcoRI endonuclease. For complementation assays, a Bcsdr2-green fluorescent protein (GFP) cassette was generated by amplifying the entire open reading frame (ORF) of Bcsdr2 (without a stop codon) and cloning into the pNAN-OGG vector containing the GFP gene and the nourseothricin resistance gene (Ren et al. 2018). The resulting construct was confirmed by DNA sequencing and transformed into the Bcsdr2 deletion mutant.
Transcriptome analyses
Mycelia of WT B05.10 and Bcsdr2 gene deletion mutant ΔBcsdr2 strains were harvested after growth on PDA medium at 22 °C under 12-h light and 12-h dark conditions for 3 days (with three biological replicates). Total RNA was extracted using a fungal RNA kit(R6840-01; Omega Bio-Tek, Norcross, GA, USA) and tested for quality using Nanodrop. RNA sample having a RNA integrity number > 7.0, 260/280 ratio > 1.8 and 260/230 ratio > 1.9 were analysed by Allwegene Technology Co., Ltd. (Beijing, China). Briefly, Trimmomatic (v0.33) software was used to filter the sequencing data (Goulin et al. 2023). A reference genome index was built, and filtered reads were mapped to the reference genome using STAR (v2.5.2b) (Acevedo et al. 2024). Mapping statistics are shown in Supplementary Table S2. HTSeq (v0.5.4) was used to compare the read count values for each gene with the original gene expression level, and fragments per kilobase of exon model per million mapped reads (FPKM) were used to standardise expression. DESeq (v1.10.1) was used to analyse differentially expressed genes (DEGs) with absolute log2 value > 1 and p < 0.05 as cut-off criteria (Saputro et al. 2023). All DEGs are listed in Supplementary Table S3. Gene Ontology (GO) categories of up- and downregulated genes were identified using the g:Profiler toolset (Raudvere et al. 2019).
Nucleic acid manipulation and qRT-PCR
Fungal genomic DNA was extracted as described previously by McDonald and Martinez (1990). Plasmid DNA was isolated using a RapidLyse Plasmid Mini Kit (DC211; Vazyme, Nanjing, China).
Real-time quantitative reverse transcription (qRT-PCR) was used to measure the expression of disease-related genes in the Bcsdr2 disruption mutant ΔBcsdr2 and the WT B05.10 strain. The total RNA remaining from the transcriptome experiment was used. RNA was reverse-transcribed using a HiScript III 1st Strand cDNA Synthesis Kit (R312; Vazyme). qRT-PCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Q712; Vazyme). The expression level of each transcript was calculated using the ΔΔCt method (Livak and Schmittgen 2001). For normalisation of the data, the transcription level of each gene in hyphae of strain B05.10 was given a value of 1.0, and the scale was used to calibrate the transcript levels of genes in hyphae of ΔBcsdr2. qRT-PCR was repeated three times. All genes and primers used for qRT-PCR are listed in Supplementary Table S4.
Pathogenicity and infection-related morphogenesis assay
Infection tests were performed on grape fruits and leaves. Briefly, the tested plant tissues were point-inoculated with 5-mm-diameter mycelial plugs of 3-day-old cultures. Before inoculation, the cuticle of the hosts was wounded with a sterilised needle tip to facilitate penetration of the fungus into plant tissues. The inoculated samples were placed under high relative humidity conditions (~ 95%) at 25 °C with 16 h of daylight. These experiments were repeated three times, and each included ten samples. Infection-related morphogenesis was observed on the onion epidermis using a published method (Zhang et al. 2022).
Morphology and ultrastructure of fungal hyphae
To investigate the role of Bcsdr2 on hyphal morphology and ultrastructure in ∆Bcsdr2 and WT B05.10 strains, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed. Mycelial morphology and ultrastructure were observed by SEM/TEM according to a modified method (Zou et al. 2022). Hyphae on coverslips were immersed in 4 °C glutaraldehyde (4%) and incubated in darkness at 4 °C for 16 h. Mycelia were washed three times with phosphate-buffered saline (PBS), dehydrated and dried in a vacuum freeze-dryer. Samples were sprayed with gold powder and examined using an SU8000 SEM instrument (Hitachi, Tokyo, Japan). One millilitre of spore suspension (1 × 105 spores mL−1) was added to 100 mL of PDB and incubated at 25 °C with shaking a 150 rpm for 72 h. Mycelia were centrifuged, washed three times with PBS and postfixed with 1% osmium tetroxide for 2 h. Samples were washed three more times with PBS, further dehydrated in a graded ethanol series (30%, 50%, 60%, 70%, 80%, 90%, 95% and 100%) and then embedded in Spurr’s low viscosity resin. Sections were observed using an H-7500 TEM instrument (Hitachi, Tokyo, Japan).
Abiotic stress and pathogenic factor assay
Mycelia-responsive trials were carried out to determine the responses of B05.10, ΔBcsdr2 and complemented strain ΔBcsdr2-C to abiotic stresses, including osmotic pressure, H2O2, sodium dodecyl sulphate (SDS), protease, polygalacturonase and cellulase, and their ability to produce infectious agent. Specifically, mycelial agar plugs were removed from the margin area of a 2-day-old PDA culture of an isolate and inoculated in Petri dishes containing PDA with KCl (1 M), H2O2 (10 mM) and 20 mg/L SDS (w/v). Cultures were incubated at 20 °C for 2 days. Secretion of proteases, polygalacturonases and cellulases was assessed using nutrient agar (NA; 5 g NaCl, 10 g sucrose, 3 g beef extract, 3 g yeast extract, 20 g agar, 1 L water, pH 7.0), polygalacuronic acid agar (PGAA; 10 g polygalacuronic acid, 20 g sucrose, 2 g (NH4)2SO4, 20 g agar, 1 L water) and carboxymethyl cellulose sodium agar (CMC-Na; 10 g carboxymethyl cellulose sodium salt, 10 g yeast extract, 1 g tryptone, 4 g (NH4)2SO4, 2 g K2HPO4, 0.5 g MgSO4ꞏ7H2O, 20 g agar, 1 L water) medium, respectively. Cultures were incubated at 22 °C for 3 days. Experiments included one mycelial agar plug per dish and three dishes (replicates) for each treatment. The diameter of each colony was measured, and the mycelial growth inhibition rate (MGIR) was calculated using the following formula (Zhou et al. 2022):
where ADCK is the average colony diameter of an investigated isolate in the control treatment, and Ds is the diameter of that isolate in the presence of a stress generation chemical (KCl, H2O2 or SDS). Each assay was repeated three times.
Statistical analyses
All assays were conducted in triplicate unless otherwise indicated. The number of conidia, colony diameter and lesion diameter date was assessed using IBM SPSS statistics 20.0 software (IBM Corp., Armonk, NY, USA). The significance of different treatments on various indices was evaluated by analysis of variance (ANOVA) with least significant difference (LSD’s) multiple comparisons, and p ≤ 0.05 was considered statistically significant. After analysis, the average angular values were individually back-transformed to numerical values.
Results
Identification of Bcsdr2 in B. cinerea
The Bcsdr2 gene (Bcin10g01350) of B. cinerea was identified by transcriptome data analysis. Bioinformatics analysis by NCBI (https://www.ncbi.nlm.nih.gov/) showed that this 1505-bp gene comprising three exons and two introns encodes 300 amino acid polypeptide. Homologs of Bcsdr2 were identified by BLASTp and phylogenetic trees of Bcsdr2 proteins were constructed by MEGA 10.0.5 (Fig. 1) (Tribhuvan et al. 2022). The evolutionary history was inferred using the neighbour-joining method with bootstrap replications 1000 (Russo and Selvatti 2018). The Bcsdr2 protein is highly homologous to EMR85959.1 and P024480.1 from B. cinerea BcDW1 and Bofu T4 strains, respectively.
Deletion and complementation of Bcsdr2 in B. cinerea
To investigate the functions of the Bcsdr2 protein in B. cinerea, we generated single ΔBcsdr2 gene deletion mutants using homologous recombination (Fig. 2A). The left and right arms (1000 bp) of the Bcsdr2 gene and the hygromycin B resistance gene (2145 bp) of plasmid pUCHYG were amplified, and the recombinant Bcsdr2 gene containing the above fragments was obtained by fusion PCR.
We obtained independent transformants by screening on selection medium supplemented with hygromycin B and PCR verification. After single spore isolation, transformants were verified as homozygous by PCR and further confirmed to be single-copy insertions by Southern blotting analysis (Fig. 2 B and C). To confirm that the phenotypic changes of mutants were due to gene deletion, ΔBcsdr2 mutants were complemented with the full-length Bcsdr2 gene to generate ΔBcsdr2-C complemented strains.
Bcsdr2 is involved in hyphal growth and conidiation
The mycelial growth rate and conidia of ΔBcsdr2 were significantly different from the WT parent B05.10. The ΔBcsdr2 strain had a slower growth rate than the ΔBcsdr2-C complemented strains and the B05.10 WT strain on PDA, CM and MM, especially on PDA (Fig. 3 A and B). Conidia are the primary inoculum for the disease cycle of B. cinerea (Abbey et al. 2019). After incubating on PDA for 10 days, the abundance of conidia was significantly less for ΔBcsdr2 than for B05.10 and ΔBcsdr2-C (Fig. 3C). However, there were no significant changes in morphology or size (Fig. 3D). In addition, when incubated on PDA medium at 22 °C for 10 h, all spores of B05.10 germinated, whereas the average germination rate of ΔBcsdr2 was only 75.32%.
Effects of Bcsdr2 deletion on mycelial growth, sporulation and conidial germination. Bars represent standard errors from three replicates. Values on bars followed by different letters indicate significant differences at p = 0.05. A Mycelial growth of ∆Bcsdr2, B05.10 and ∆Bcsdr2-C strains on PDA plates after 3 days of cultivation. B Quantification of colony diameter of the indicated strains grown on PDA plates for 3 days. C Quantification of conidia produced by the indicated strains. D Conidia morphology of different strains. E SEM and TEM observations of hyphae of B. cinerea and ∆Bcsdr2 strains grown on PDA plates (diameter 4 cm)
SEM results showed that hyphae of control strain B05.10 grew upright, with uniform thickness, a smooth surface and no distortion or deformities, and apical hyphae branches were relatively uniform; by contrast, those of ΔBcsdr2 displayed swollen tips, reduced hyphal size and uneven apical branches. TEM results showed that compared with control strain B05.10, the ΔBcsdr2 strain exhibited dissolution and disappearance of various biological membranes, including mitochondrial membranes, endoplasmic reticulum membranes, nuclear membranes and cell membranes. The above features are indicated by the red arrows in the figure (Fig. 3E). These results also indicate that Bcsdr2 is important for vegetative growth and conidiation of B. cinerea.
Bcsdr2 participates in regulating the pathogenicity of B. cinerea
To determine whether Bcsdr2 is involved in regulating pathogenicity in B. cinerea, strawberry fruits and tobacco leaves were inoculated with ΔBcsdr2 mutants. Compared with the WT strain, ΔBcsdr2 mutants exhibited reduced virulence in different hosts (Fig. 4 A and B). At 96 h, tobacco leaves inoculated with ΔBcsdr2 mutants displayed small lesions, while WT-inoculated leaves had an average lesion size of 0.78 cm and 2.01 cm for tobacco leaves and strawberry fruits, respectively. Similarly, lesion size was considerably decreased on ΔBcsdr2 mutant-inoculated strawberry fruits compared with WT-inoculated fruits (Fig. 4C). Complemented strain ΔBcsdr2-C exhibited almost the same level of virulence as the WT strain. These results suggest that Bcsdr2 plays a crucial role in the virulence of B. cinerea.
Effects of Bcsdr2 deletion on mycelial infection and pathogenicity. Bars represent standard errors from three replicates. Values on bars followed by different letters indicate significant differences at p = 0.05. A and B Disease symptoms caused by each strain on strawberry fruits wounded and tobacco leaves. Images were captured at 96 h after inoculation. C Pathogenicity on tobacco leaves and strawberry fruits after 96 h of incubation
Effects of Bcsdr2 deletion on sensitivity to abiotic stresses and pathogenicity factors
The results of mycelial response assays showed that compared with ΔBcsdr2-C and B05.10, ΔBcsdr2 mutants exhibited suppressed mycelial growth in the presence of KCl, CR, SDS and H2O2, and the ability to produce proteases, polygalacturonase and cellulases was significantly reduced (Fig. 5A). These results suggest that disruption of Bcsdr2 may have marginal effects on mycelial growth in response to abiotic stresses, and pathogenicity factor production capacity may also be affected.
Sensitivity of ΔBcsdr2, B05.10 and ΔBcsdr2-C strains to abiotic stresses. Bars represent standard errors from three replicates. Values on bars followed by different letters indicate significant differences at p = 0.05. A All strains were grown on PDA plates amended with KCl, CR, SDS or H2O2 at the indicated concentrations at 20 °C for 2 days, and with skimmed milk powder, polygalacuronic acid or carboxymethyl cellulose at 20 °C for 3 days. B Sensitivity of ΔBcsdr2, B05.10 and ΔBcsdr2-C strains to KCl, CR, SDS and H2O2. C Ability of ΔBcsdr2, B05.10 and ΔBcsdr2-C strains to produce polygalacturonase, cellulases and proteases
Bcsdr2 deletion affects transcription and pathogenicity-related genes
We performed an RNA-Seq analysis to identify genes that might exhibit changes in regulation affected by Bcsdr2 in B. cinerea. Three biological replicates with mRNA isolated from WT B05.10 and ΔBcsdr2 strains were performed, and 319 downregulated and 88 upregulated (fold change > 2, p < 0.05) genes were identified in ΔBcsdr2 compared with B05.10 (Fig. 6A).
Functional annotation of DEGs via GO analysis was performed to identify genes belonging to molecular function, cellular component and biological process categories (Supplemental Fig. S1). Among them, oxidoreductase activity/acting on paired donors, with incorporation or reduction of molecular oxygen/iron ion binding, organelle envelope/mitochondrial outer membrane/organelle outer membrane and oxidation–reduction process/ribosome biogenesis/ribonucleoprotein complex biogenesis, was the main molecular function, cellular composition and biological process subcategories, respectively. These results showed that growth and pathogenicity defects caused by the absence of Bcsdr2 in the B05.10 strain were closely related to these functions.
In addition, enriched DEGs can be functionally classified into metabolism, cellular processes and genetic information processing. Among the metabolic pathways belonging to these three categories, the top ten metabolic pathways of enriched DEGs were metabolic pathways, ribosome biogenesis in eukaryotes, biosynthesis of secondary metabolites, biosynthesis of antibiotics, purine metabolism, RNA polymerase, propanoate metabolism, 2-oxocarboxylic acid metabolism, valine, leucine and isoleucine degradation and amino sugar and nucleotide sugar metabolism, respectively (Supplemental Fig. S2). These metabolic pathways may be closely related to the growth and pathogenicity defects of ΔBcsdr2.
To verify the reliability of DEGs identified from transcriptome sequencing, qRT-PCR was performed on the remaining ΔBcsdr2 RNA samples used for transcriptome analysis. qRT-PCR validation was performed by randomly selecting 16 growth- and pathogenicity-associated genes. Their melt curves are attached to the supplement figures (Fig.S3-Fig. 19). These genes encode proteins that participate in growth regulation (Bcin01g10130, Bcin01g06930, Bcin11g02110), the synthesis of substances and catalytic reactions of enzymes (Bcin02g03350, Bcin01g10150, Bcin12g00420) and virulence and stress factors (Bcin01g04230, Bcin01g00110, Bcin07g01300, Bcin14g03730). The qRT-PCR results were consistent with the RNA-seq results (Fig. 6B), confirming the reliability of the RNA-seq data.
Discussion
Short-chain dehydrogenases (SDRs) are NAD(P)-dependent oxidoreductases that participate in the metabolism of various specific substrates in organisms through oxidation–reduction, isomerisation and cleavage, thereby regulating biochemical reactions and physiological processes (Roth et al. 2018; Cui et al. 2019). SDRs are closely associated with plant growth and development and stress response mechanisms (Stavrinides et al. 2018), as well as pathogenic processes of pathogens. Studies have found that the SDR MoSDR1 inhibits spore formation and germination of rice blast fungus, suppresses the development of invasive structures and reduces the pathogenicity of the rice blast fungus (Kwon et al. 2010). SDRs can also induce systemic resistance in plants and hinder the invasion of plant fruits by pathogens (Hwang et al. 2012). Recent discoveries have revealed that SDRs are capable of sensing the redox state in metabolism and participating in transcription or RNA processing, further extending the functional scope of this superfamily of proteins (Zhao 2018).
To explore the function of membrane protein Bcsdr2, a member of the SDR family, we first disrupted the Bcsdr2 gene and characterised the resulting mutant, which showed severe defects in hyphae grow and pathogenicity. We therefore speculated that Bcsdr2 might be involved in regulating hyphae growth and pathogenicity-related genes in its fungal host. Bcsdr2 is a vital virulence determinant since deletion of the Bcsdr2 gene also compromised the penetration ability of B. cinerea, indicating that the reduced virulence of the ΔBcsdr2 mutant was likely due, at least in part, to defective penetration of host cells. In addition, the ability of the ΔBcsdr2 mutant strain to produce cellulases and proteases was significantly reduced, which is another important factor of pathogenicity (He 2014; Hou 2019). Based on the above results, the Bcsdr2 protein appears to be an important virulence factor of B. cinerea. However, the regulatory mechanisms involving Bcsdr2 remain poorly understood, and further research such as a comparative analysis of transcription profiles could provide valuable information.
We used transcriptome sequencing technology to compare and analyse transcriptional regulation differences and DEGs of ∆Bcsdr2 mutant strains following culture for 72 h, and 407 DEGs were screened. GO functional analysis showed that in molecular function and biological process categories, DEGs resulting from loss of the Bcsdr2 gene were mostly involved in oxidoreductase activity and oxidation–reduction process, respectively. Among the cellular component terms, the most enriched were linked to cellular structure and membrane, consistent with the fact that deletion of the Bcsdr2 gene affected biofilm integrity based on TEM observations. Our RNA-seq analysis results suggest that global changes in genes involved in metabolic pathways, biosynthesis of secondary metabolites, ribosome biogenesis in eukaryotes and protein processing in endoplasmic reticulum are likely to underlie this defect.
The reliability of transcriptome data was verified by qRT-PCR analysis of nine growth- and pathogenicity-related genes. Mitochondria and essential organelles in organisms and the primary site for energy production and aerobic respiration. Phosphatidylethanolamine (PE), synthesised from phosphatidylserine by the enzyme encoded by the psd gene, is a non-bilayer phospholipid that helps maintain the shape and function of mitochondria and is involved in regulating the growth, development and virulence of Fusarium graminearum (Tang et al. 2021; Gok et al. 2022). Cox17 is an essential protein for cytochrome c oxidase within the mitochondria that plays a regulatory role in the structure of mitochondrial membranes (Vanišová et al. 2019; Ding et al. 2022). In the present study, the relative expression level of Bcpsd was downregulated 0.82-fold while the relative expression level of Bccox17 was upregulated 1.99-fold. TEM revealed that loss of the membrane protein Bcsdr2 led to mitochondrial swelling, disappearance of the outer membrane and dissolution of the cristae. The significant changes in the expression of the aforementioned genes may be important factors affecting mitochondrial morphology and function. The ribosome, an essential molecular machine responsible for protein synthesis, is composed of the 40S small subunit and the 60S large subunit in eukaryotes. Research has found that Nmd3 acts as a structural mimic of eIF5A and activates the cpGTPase Lsg1 during biogenesis of the ribosome 60S large subunit, thereby influencing its synthesis in mitochondria (Malyutin et al. 2017). By contrast, the RNA-binding protein Nob1 is necessary for the synthesis of the ribosome 40S small subunit (Fatica et al. 2004; Lamanna and Karbstein 2009). In the present work, the relative expression levels of Bcnmd3 and Bcnob1 were downregulated 2.27-fold, indicating that the loss of membrane protein Bcsdr2 affects the biogenesis of ribosomal subunits. Normal growth and development of microorganisms are essential for their life activities. Studies have found that deletion or downregulation of BOA11, HMT1 and DUG2 genes can inhibit the growth of strains and reduce conidiation (Porquier et al. 2019; Li et al. 2020; Reza and Sanyal 2022). In this study, expression levels of BcBOA11, Bchmt1 and Bcdug2 were downregulated to varying degrees, indicating a close relationship between the decreased growth rate and morphological changes in ΔBcsdr2 and the aforementioned genes. The ability to respond to stressful environments is an important indicator of microbial vitality. Studies have found that met16, fap7 and carA genes are important factors in microbial response to oxidative stress (Juhnke et al. 2000; Lage et al. 2019; Buvelot et al. 2021; Luo and Xu 2021). In the present study, the relative expression level of Bcmet16 was upregulated 3.43-fold, while expression levels of Bcfap7 and BccarA were downregulated 1.03-fold and 0.84-fold, respectively, consistent with the reduced ability of ΔBcsdr2 to respond to stress conditions. There were also some studies that suggested that knockout genes can influence the sensitivity to abiotic stresses of Botrytis (Schamber, et al. 2010; Tundo et al. 2020). Therefore, the observed changes in pathogenicity, growth and development, stress responses and mitochondrial morphology for the ΔBcsdr2 strain are closely associated with the differential expression of the aforementioned genes.
Deletion of Bcsdr2 significantly reduced the ability of hyphae to produce infection structures, and the ability to produce acids, cellulases and proteases was also significantly diminished, which further indicates that Bcsdr2 may reduce the pathogenicity of strains, and transcriptome and quantitative PCR results further supported these conclusions. Therefore, we preliminarily concluded that the absence of Bcsdr2 can reduce the growth rate and pathogenicity of B. cinerea, while increasing sensitivity to environmental stress.
This further indicates that wuyiencin disrupts the integrity of B. cinerea biofilms by regulating the expression of membrane protein Bcsdr2, thereby inhibiting hyphal growth and development, and decreasing the invasive capacity and pathogenicity of the strain.
In summary, membrane protein Bcsdr2 is involved in regulating B. cinerea biofilm integrity, hyphal growth and virulence.
Data availability
Data will be made available on request, and the transcriptome sequencing data was deposited in NCBI with a number of PRJNA1071901.
References
Abbey JA, Percival D, Abbey L, Asiedu SK, Prithiviraj B, Schilder A (2019) Biofungicides as alternative to synthetic fungicide control of grey mould (Botrytis cinerea) – prospects and challenges. Biocontrol Sci Techn 29(3):207–228
Acevedo N, Lozano A, Zakzuk J, Llinás-Caballero K, Brodin D, Nejsum P, Williams AR, Caraballo L (2024) Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells. Front Immunol 15:1328401
Alguel Y, Cameron AD, Diallinas G, Byrne B (2016) Transporter oligomerization: form and function. Biochem Soc Trans 44(6):1737–1744
Athenstaedt K, Daum G (2000) 1-Acyldihydroxyacetone-phosphate reductase (Ayr1p) of the yeast Saccharomyces cerevisiae encoded by the open reading frame YIL124w is a major component of lipid particles. J Biol Chem 275(1):235–240
Bu SW, Munir S, He PF, Li YM, Wu YX, Li XY, Kong BH, He PB, He YQ (2021) Bacillus subtilis L1–21 as a biocontrol agent for postharvest gray mold of tomato caused by Botrytis cinerea. Biol Control 157:104568
Buvelot H, Roth M, Jaquet V, Lozkhin A, Renzoni A, Bonetti E-J, Gaia N, Laumay F, Mollin M, Stasia M-J, Schrenzel J, François P, Krause KH (2021) Hydrogen peroxide affects growth of S. aureus through downregulation of genes involved in pyrimidine biosynthesis. Front Immunol 12:673985
Cui Y, Feng YH, Chen ZF, Wang XM, Wang YP, Du XZ (2019) Cloning of IDP2557 gene encoding maize short-chain dehydrogenase and identification of its drought resistance. Mol Plant Breed 17(22):7300–7305
DafaAlla T, Abdalla M, El-Arabey AA, Eltayb WA, Mohapatra RK (2022) Botrytis cinerea alcohol dehydrogenase mediates fungal development, environmental adaptation and pathogenicity. J Biomol Struct Dyn 40(23):12426–12438
Ding Y, Chen Y, Wu Z, Yang N, Rana K, Meng X, Liu B, Wan H, Qian W (2022) SsCox17, a copper chaperone, is required for pathogenic process and oxidative stress tolerance of Sclerotinia sclerotiorum. Plant Sci 322:111345
Elad Y (eds) (2016) Plant Hosts of Botrytis spp. In: Fillinger S, Elad Y editors. Botrytis – the fungus, the pathogen and its management in agricultural systems. Springer International Publishing p. 413–486.
Engberg O, Ulbricht D, Döbel V, Siebert V, Frie C, Penk A, Lemberg MK, Huster D (2022) Rhomboid-catalyzed intramembrane proteolysis requires hydrophobic matching with the surrounding lipid bilayer. Sci Adv 8(38):eabq8303
Fan L, Wei YY, Chen Y, Jiang S, Xu F, Zhang CD, Wang HF, Shao XF (2023) Epinecidin-1, a marine antifungal peptide, inhibits Botrytis cinerea and delays gray mold in postharvest peaches. Food Chem 403:134419
Fatica A, Tollervey D, Dlakić M (2004) PIN domain of Nob1p is required for D-site cleavage in 20S pre-rRNA. RNA 10(11):1698–1701
Fujisawa H, Nagata S, Misono H (2003) Characterization of short-chain dehydrogenase/reductase homologues of Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C). Biochim Biophys Acta 1645(1):89–94
Gok MO, Speer NO, Henne WM, Friedman JR (2022) ER-localized phosphatidylethanolamine synthase plays a conserved role in lipid droplet formation. Mol Biol Cell 33(1):ar11
Goulin E, Boufleur TR, Negrini F, Carneiro GA, Baraldi E, Machado MA, Le Floch G, Baroncelli R (2023) Genome sequence resources of Colletotrichum abscissum, the causal agent of citrus post-bloom fruit drop, and the closely related species C. filicis. Phytopathology 113(1):104–107
Gronover CS, Kasulke D, Tudzynski P, Tudzynski B (2001) The role of G protein alpha subunits in the infection process of the gray mold fungus Botrytis cinerea. Mol Plant Micro Interac 14(11):1293–1302
He JY (2014) Functional analysis of phytochrome Bcphy3 in Botrytis cinerea. Master dissertation. East China Normal University, Shanghai.
Hou J (2019) Functional analysis and pathogenic mechanisms of histone demethylase BcJar1 in Botrytis cinerea. Doctoral dissertation. Lilin University, Changchun
Huang PY, Wang Z, Cai K, Wei LW, Chu YD, Guo MQ, Fan EG (2022) Targeting bacterial membrane proteins to explore the beneficial effects of natural products: new antibiotics against drug resistance. Curr Med Chem 29(12):2109–2126
Hwang SG, Lin NC, Hsiao YY, Kuo CH, Chang PF, Deng WL, Chiang MH, Shen HL, Chen CY, Cheng WH (2012) The Arabidopsis short-chain dehydrogenase/reductase 3, an abscisic acid deficient 2 homolog, is involved in plant defense responses but not in ABA biosynthesis. Plant Physiol Biochem 51:63–73
Juhnke H, Charizanis C, Latifi F, Krems B, Entian K-D (2000) The essential protein fap7 is involved in the oxidative stress response of Saccharomyces cerevisiae. Mol Microbiol 35(4):936–948
Kwon MJ, Kim KS, Lee YH (2010) A short-chain dehydrogenase/reductase gene is required for infection-related development and pathogenicity in Magnaporthe oryzae. Plant Pathol J 26(1):8–16
Lage P, Sampaio-Marques B, Ludovico P, Mire NP, Mendes-Ferreira A (2019) Transcriptomic and chemogenomic analyses unveil the essential role of Com2-regulon in response and tolerance of Saccharomyces cerevisiae to stress induced by sulfur dioxide. Microb Cell 6(11):509–523
Lamanna AC, Karbstein K (2009) Nob1 binds the single-stranded cleavage site D at the 3′-end of 18S rRNA with its PIN domain. Proc Natl Acad Sci USA 106(34):14259–14264
Li Z, Wu L, Wu H, Zhang X, Mei J, Zhou X, Wang GL, Liu W (2020) Arginine methylation is required for remodelling pre-mRNA splicing and induction of autophagy in rice blast fungus. New Phytol 225(1):413–429
Liu N, Zhou SY, Li BH, Ren WC (2021) Involvement of the autophagy protein Atg6 in development and virulence in the gray mold fungus Botrytis cinerea. Front Microbiol 12:798363
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408
Luo Z, Xu Y (2021) Use of Bcmet16 gene in reducing the pathogenicity of gray mold by knocking out the Bcmet16 gene for reducing the infectivity of gray mold. China:CN112646821-A, CN112646821-B
Malyutin AG, Musalgaonkar S, Patchett S, Frank J, Johnson AW (2017) Nmd3 is a structural mimic of eIF5A, and activates the cpGTPase Lsg1 during 60S ribosome biogenesis. EMBO J 36(7):854–868
Porquier A, Moraga J, Morgant G, Dalmais B, Simon A, Sghyer H, Collado IG, Viaud M (2019) Botcinic acid biosynthesis in Botrytis cinerea relies on a subtelomeric gene cluster surrounded by relics of transposons and is regulated by the Zn2Cys6 transcription factor BcBoa13. Curr Genet 65(4):965–980
Quidde T, Büttner P, Tudzynski P (1999) Evidence for three different specific saponin-detoxifying activities in Botrytis cinerea and cloning and functional analysis of a gene coding for a putative avenacinase. Eur J Plant Pathol 105(3):273–283
Raudvere U, Kolberg L, Kuzmin I, Arak T, Adler P, Peterson H, Vilo J (2019) g:Profiler: a web server for functional enrichment analysis and conversions of gene lists. Nucleic Acids Res 47(W1):W191–W198
Ren WC, Sang CW, Shi DY, Song XS, Zhou MG, Chen CJ (2018) Ubiquitin-like activating enzymes BcAtg3 and BcAtg7 participate in development and pathogenesis of Botrytis cinerea. Curr Genet 64(4):919–930
Reza MH, Sanyal K (2022) Defective in utilizing glutathione 3, DUG3, is required for conidiation and host infection in the rice blast fungus Magnaporthe oryzae. MicroPubl Biol 10:17912
Roth S, Kilgore MB, Kutchan TM, Müller M (2018) Exploiting the catalytic diversity of short-chain dehydrogenases/reductases: versatile enzymes from plants with extended imine substrate scope. ChemBioChem 19(17):1849–1852
Russo CAD, Selvatti AP (2018) Bootstrap and rogue identification tests for phylogenetic analyses. Mol Biol Evol 35(9):2327–2333
Saputro TB, Jakada BH, Chutimanukul P, Comai L, Buaboocha T, Chadchawan S (2023) OsBTBZ1 Confers salt stress tolerance in Arabidopsis thaliana. Int J Mol Sci 24(19):14483
Schamber A, Leroch M, Diwo J, Mendgen K, Hahn M (2010) The role of mitogen-activated protein (MAP) kinase signalling components and the Ste12 transcription factor in germination and pathogenicity of Botrytis cinerea. Mol Plant Pathol 11(1):105–119
Shi LM, Ge BB, Wang JZ, Liu BH, Ma JJ, Wei QH, Zhang KC (2019) iTRAQ-based proteomic analysis reveals the mechanisms of Botrytis cinerea controlled with wuyiencin. BMC Microbiol 19(1):280
Stavrinides AK, Tatsis EC, Dang TT, Caputi L, Stevenson CEM, Lawson DM, Schneider B, O’Connor SE (2018) Discovery of a short-chain dehydrogenase from Catharanthus roseus that produces a new monoterpene indole alkaloid. ChemBioChem 19(9):940–948
Sun YZ, Zeng HM, Shi YP, Li GQ (2003) Mode of action of wuyiencin on Botrytis cinerea. Acta Phytopathol Sin 33(5):434–438
Tang L, Chi H, Li W, Zhang L, Zhang L, Chen L, Zou S, Liu H, Liang Y, Yu J, Dong H (2021) FgPsd2, a phosphatidylserine decarboxylase of Fusarium graminearum, regulates development and virulence. Fungal Genet Biol 146:103483
te Welscher YM, van Leeuwen MR, de Kruijff B, Dijksterhuis J, Breukink E (2012) Polyene antibiotic that inhibits membrane transport proteins. Proc Natl Acad Sci USA 109(28):11156–11159
Tribhuvan KU, Kaila T, Srivastava H, Das A, Kumar K, Durgesh K, Joshi R, Singh BK, Singh NK, Gaikwad K (2022) Structural and functional analysis of CCT family genes in pigeonpea. Mol Biol Rep 49(1):217–226
Tundo S, Paccanaro MC, Elmaghraby I, Moscetti I, D’Ovidio R, Favaron F, Sella L (2020) The xylanase inhibitor TAXI-I increases plant resistance to Botrytis cinerea by inhibiting the BcXyn11a xylanase necrotizing activity. Plants (Basel) 9(5):601
Vanišová M, Burská D, Křížová J, Daňhelovská T, Dosoudilová Ž, Zeman J, Stibůrek L, Hansíková H (2019) Stable COX17 downregulation leads to alterations in mitochondrial ultrastructure, decreased copper content and impaired cytochrome c oxidase biogenesis in HEK293 cells. Folia Biol (Praha) 65(4):181–187
Weidensdorfer M, Ishikawa M, Hori K, Linke D, Djahanschiri B, Iruegas R, Ebersberger I, Riedel-Christ S, Enders G, Leukert L, Kraiczy P, Rothweiler F, Cinatl J, Berger J, Hipp K, Kempf VAJ, Göttig S (2019) The Acinetobacter trimeric autotransporter adhesin Ata controls key virulence traits of Acinetobacter baumannii. Virulence 10(1):68–81
Yan LY, Yang QQ, Sundin GW, Li HY, Ma ZH (2010) The mitogen-activated protein kinase kinase BOS5 is involved in regulating vegetative differentiation and virulence in Botrytis cinerea. Fungal Genet Biol 47(9):753–760
Yang G, Hu Y, Fasoyin OE, Yue YW, Chen LJ, Qiu Y, Wang XN, Zhuang ZH, Wang SH (2018) The Aspergillus flavus phosphatase CDC14 regulates development, aflatoxin biosynthesis and pathogenicity. Front Cell Infect Microbiol 8:141
Yeliseev A, Iyer MR, Joseph TT, Coffey NJ, Cinar R, Zoubak L, Kunos G, Gawrisch K (2021) Cholesterol as a modulator of cannabinoid receptor CB2 signaling. Sci Rep 11(1):3706
Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Domínguez Y, Scazzocchio C (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41(11):973–981
Zhang ZQ, Liu T, Xu Y, Chen Y, Chen T, Li BQ, Tian SP (2022) Sodium pyrosulfite inhibits the pathogenicity of Botrytis cinerea by interfering with antioxidant system and sulfur metabolism pathway. Postharvest Biol Technol 189:111936
Zhao YT (2018) Analysis of disease resistance mechanism of SDR6 gene in Arabidopsis thaliana. Master dissertation. Agric Univ Heibei Baoding
Zhou YJ, Song JJ, Wang YC, Yang L, Wu MD, Li GQ, Zhang J (2022) Biological characterization of the melanin biosynthesis gene Bcscd1 in the plant pathogenic fungus Botrytis cinerea. Fungal Genet Biol 160:103693
Zou XR, Wei YY, Jiang S, Xu F, Wang HF, Zhan PP, Shao XF (2022) ROS stress and cell membrane disruption are the main antifungal mechanisms of 2-phenylethanol against Botrytis cinerea. J Agric Food Chem 70(45):14468–14479
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We thank the native English-speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript.
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This work was funded by the Major Science and Technology Program of China Tobacco Corporation (grant number 110202201019(LS-03)) and the National Natural Science Foundation of China (grant number 32172493).
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Conceptualization: K.C.Z. and L.M.S.; methodology: L.M.S., W.Z., Y.C. and H.L.; validation: W.Z. and Z.Y.L.; formal analysis: L.M.S., W.Z., B.B.G. and H.L.; investigation: L.M.S., W.Z., B.B.G. and K.P; supervision: K.C.Z., L.M.S. and B.B.G.; writing—original draft preparation; W.Z.: writing—review and editing: W.Z., L.M.S. and H.L.; all authors have read and agreed to the published version of the manuscript.
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Zhang, W., Cao, Y., Li, H. et al. Membrane protein Bcsdr2 mediates biofilm integrity, hyphal growth and virulence of Botrytis cinerea. Appl Microbiol Biotechnol 108, 398 (2024). https://doi.org/10.1007/s00253-024-13238-8
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DOI: https://doi.org/10.1007/s00253-024-13238-8