Membrane Protein Bcest Is Involved in Hyphal Growth, Virulence and Stress Tolerance of Botrytis cinerea

Botrytis cinerea is a necrotrophic model fungal plant pathogen that causes grey mould, a devastating disease responsible for large losses in the agriculture sector. As important targets of fungicides, membrane proteins are hot spots in the research and development of fungicide products. We previously found that membrane protein Bcest may be closely related to the pathogenicity of Botrytis cinerea. Herein, we further explored its function. We generated and characterised ΔBcest deletion mutants of B. cinerea and constructed complemented strains. The ΔBcest deletion mutants exhibited reduced conidia germination and germ tube elongation. The functional activity of ΔBcest deletion mutants was investigated by reduced necrotic colonisation of B. cinerea on grapevine fruits and leaves. Targeted deletion of Bcest also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted-gene complementation. The role of Bcest in pathogenicity was also supported by reverse-transcriptase real-time quantitative PCR results indicating that melanin synthesis gene Bcpks13 and virulence factor Bccdc14 were significantly downregulated in the early infection stage of the ΔBcest strain. Taken together, these results suggest that Bcest plays important roles in the regulation of various cellular processes in B. cinerea.


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 [1,2]. Moreover, B. cinerea infects over 1400 plant species including many economically important crops, and leads to tremendous economic losses [3]. Due to the lack of resistant varieties, chemical control remains the most effective strategy for grey mould management [4]. Nevertheless, through genetic plasticity, B. cinerea has developed resistance to many types of fungicides. 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, intercellular communication and drug targets. Membrane proteins are recognised and inserted into the lipid bilayer by exquisite cellular machineries, such as the GlpG rhomboid protease, which is thought to allow docking of a transmembrane substrate [5]. However, 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 [6]. Many membrane proteins are primary drug targets, especially those involved in converting extracellular signals into intracellular processes. Among them, G
Conidia were quantified after 10 days of incubation on the PDA medium, washed from plates, diluted to 5 mL with ddH 2 O, and counted with a hemocytometer under a light microscope (×40). Growth tests under different stress conditions were performed on PDA plates supplemented with different agents including H 2 O 2 (10 mM), KCl (1 M) and sodium dodecyl sulphate (SDS; 0.02%) [18]. 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 the control and treatments, respectively [18]. Each experiment was repeated three times.

Gene Deletion and Complementation
To replace the Bcest gene in wild-type (WT) strain B05.10, 1000 bp upstream and 1000 bp downstream flanking sequences of Bcest were amplified by PCR from the genomic DNA of B05. 10. The resulting amplicons were fused with the HPH hygromycin resistance gene using double-joint PCR [19]. Protoplast preparation and transformation were performed as previously described [20]. The resulting hygromycin-resistant transformants were preliminarily screened by PCR with primers (Supplementary Table S1), and further confirmed by Southern blotting analysis. The upstream fragment of Bcest was used as a probe and labelled with digoxigenin (DIG) using High Prime DNA Labelling and Detection Starter Kit I (Roche Diagnostics, Mannheim, Germany) in accordance with the manufacturer's protocol. Genomic DNA was digested with EcoRI endonuclease. For complementation assays, a Bcest green fluorescent protein (GFP) cassette was generated by amplifying the entire open reading frame (ORF) of Bcest (without a stop codon) and cloning it into the pNAN-OGG vector containing the GFP gene and the nourseothricin resistance gene [21]. The resulting construct was confirmed by DNA sequencing and transformed into the Bcest deletion mutant.

Transcriptome Analyses
The mycelia of WT B05.10 and Bcest gene deletion mutant ∆Bcest were harvested after growth on the PDA medium at 22 • C under 12 h of light and 12 h of darkness for 3 days (with three biological replicates). Total RNA was extracted using a fungal RNA kit (R6840-01; Omega Bio-Tek, Norcross, GA, USA), using Nanodrop (Thermo Scientific, Waltham, MA, USA) for quality testing and using Agilent 2100 Bioanalyzer (Agilgent, Santa Clara, CA, USA) for obtaining the RNA integrity number (RIN) [22]. RNA samples with a RIN of > 7.0, a 260/280 ratio of > 1.8, and a 260/230 ratio of > 1.9 were analysed by Allwegene Technology Co., Ltd. (Beijing, China). Briefly, Trimmomatic (v0.33) software was used to filter the sequencing data [23]. A reference genome index was built and filtered reads were mapped to the reference genome using STAR (v2.5.2b). 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 the value of fragments per kilobase of exon per million mapped reads (FPKM) was used to standardise the expression. DESeq (v1.10.1) was used to analyse differentially expressed genes (DEGs) with an absolute log2 value of > 1 and a p value of < 0.05 as the cutoff criteria. All DEGs are listed in Supplementary  Table S3. Gene ontology (GO) categories of upregulated and downregulated genes were identified using the g:Profiler toolset [24].
Real-time quantitative reverse-transcription PCR (qRT-PCR) was used to measure the expression of disease-related genes in the Bcest disruption mutant ∆Bcest and the WT B05.10 strain. The total RNA remaining from the transcriptome experiment was used. RNA was reverse-transcribed using HiScript III 1st Strand cDNA Synthesis Kit (R312; Vazyme, Nanjing, China). qRT-PCR was performed using Taq Pro Universal SYBR qPCR Master Mix (Q712; Vazyme, Nanjing, China) and using specific primers and a thermal program to determine the expression of Bcactin (reference gene), Bcin14g02370, Bcin10g01030 and other genes. The expression level of each transcript was calculated using the ∆∆C t method [26]. For the normalisation of the data, the transcription level of each gene in the hyphae of B05.10 was given a value of 1.0, and the scale was used to calibrate the transcript levels of genes in the hyphae of ∆Bcest. 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 cuticles of hosts were wounded with a sterilised needle tip to facilitate the 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 10 samples. Infection-related morphogenesis was observed on an onion epidermis using a published method [27].

Morphology and Ultrastructure of Fungal Hyphae
To investigate the role of Bcest on hypha morphology and ultrastructure in ∆Bcest and WT B05.10 strains, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed. The mycelial morphology and ultrastructure were observed by SEM/TEM in accordance with a modified method [28]. The 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 with an SU8000 SEM instrument (Hitachi, Tokyo, Japan). One millilitre of spore suspension (1 × 10 5 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%), 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, ∆Bcest, and the complemented strain ∆Bcest-C to abiotic stresses, including osmotic pressure, H 2 O 2 , SDS, protease, polygalacturonase and cellulase, and their ability to produce an 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), H 2 O 2 (10 mM) and 20 mg/L of SDS (w/v). Cultures were incubated at 20 • C for 2 days. Secretions of proteases, polygalacturonases and cellulases were assessed using nutrient agar (NA; 50 g skimmed milk powder, 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 (NH 4 ) 2 SO 4 , 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 (NH 4 ) 2 SO 4 , 2 g K 2 HPO 4 , 0.5 g MgSO 4 ·7H 2 O, 20 g agar, 1 L water) mediums, 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 [29]: where AD CK 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, H 2 O 2 or SDS). Each assay was repeated three times.

Statistical Analyses
All assays were conducted in triplicate, unless otherwise indicated. Conidia number, colony diameter and lesion diameter analyses were performed using IBM SPSS statistics 20.0 software (IBM Corp., Armonk, NY, USA). The significance of the effects of different treatments on various indices was evaluated with an analysis of variance (ANOVA) with multiple least-significant-difference comparisons at the p ≤ 0.05 level. After analysis, average angular values were individually back-transformed to numerical values.

Identification of Bcest in B. cinerea
The Bcest gene (Bcin15g00520) of B. cinerea was identified via transcriptome data analysis. Bioinformatic analysis showed that this 874 bp gene with three exons and two introns encodes an 81-amino acid protein. Homologs of Bcest were identified via BLASTp and phylogenetic trees of Bcest proteins were constructed vis MEGA 10.0.5 ( Figure 1). Evolutionary history was inferred using the neighbour-joining method with 1000 bootstrap replications [30]. It can be seen from Figure 1 that the Bcest protein A0A384K4A6 is highly homologous to putative EkdA protein M7TXI3 from the B. cinerea BcDW1 strain. Evolutionary history was inferred using the neighbour-joining method with 1000 bootstrap replications [30]. It can be seen from Figure 1 that the Bcest protein A0A384K4A6 is highly homologous to putative EkdA protein M7TXI3 from the B. cinerea BcDW1 strain.

Deletion and Complementation of Bcest in B. cinerea
To investigate the functions of the Bcest protein in B. cinerea, we generated singlegene deletion mutants of ΔBcest using homologous recombination ( Figure 2A). The left and right arms (1000 bp) of the Bcest gene and the hygromycin gene (2145 bp) of plasmid pUCHYG were amplified. The recombinant Bcest gene containing the above fragments was obtained via fusion PCR.
We obtained independent transformants via screening a selection medium supplemented with hygromycin B and PCR verification. After single0spore isolation, transformants were verified as homozygous via PCR and further confirmed to be single-copy insertions via Southern blotting analysis ( Figure 2B,C). To confirm that the phenotypic changes of the mutants were due to gene deletion, ΔBcest mutants were complemented with the full-length Bcest gene to generate complemented strains of ΔBcest-C.

Deletion and Complementation of Bcest in B. cinerea
To investigate the functions of the Bcest protein in B. cinerea, we generated single-gene deletion mutants of ∆Bcest using homologous recombination ( Figure 2A). The left and right arms (1000 bp) of the Bcest gene and the hygromycin gene (2145 bp) of plasmid pUCHYG were amplified. The recombinant Bcest gene containing the above fragments was obtained via fusion PCR.

Deletion and Complementation of Bcest in B. cinerea
To investigate the functions of the Bcest protein in B. cinerea, we generated singlegene deletion mutants of ΔBcest using homologous recombination (Figure 2A). The left and right arms (1000 bp) of the Bcest gene and the hygromycin gene (2145 bp) of plasmid pUCHYG were amplified. The recombinant Bcest gene containing the above fragments was obtained via fusion PCR.
We obtained independent transformants via screening a selection medium supplemented with hygromycin B and PCR verification. After single0spore isolation, transformants were verified as homozygous via PCR and further confirmed to be single-copy insertions via Southern blotting analysis ( Figure 2B,C). To confirm that the phenotypic changes of the mutants were due to gene deletion, ΔBcest mutants were complemented with the full-length Bcest gene to generate complemented strains of ΔBcest-C.  We obtained independent transformants via screening a selection medium supplemented with hygromycin B and PCR verification. After single0spore isolation, transformants were verified as homozygous via PCR and further confirmed to be single-copy insertions via Southern blotting analysis ( Figure 2B,C). To confirm that the phenotypic changes of the mutants were due to gene deletion, ∆Bcest mutants were complemented with the full-length Bcest gene to generate complemented strains of ∆Bcest-C.

Bcest Is Involved in Hyphal Growth and Conidiation
The mycelial growth rate and the conidium of ∆Bcest were significantly different from the those of the WT parent B05.10. The ∆Bcest strain had a slower growth rate than the ∆Bcest-C complemented strains and the B05.10 WT strain did on PDA, CM and MM, but especially on PDA ( Figure 3A,B). Conidia are the primary inoculum for the disease cycle of B. cinerea [31]. After incubating it on PDA for 10 days, the abundance of conidia for ∆Bcest was significantly less than that for B05.10 and ∆Bcest-C ( Figure 3C). Interestingly, the conidia of ∆Bcest showed morphological and size abnormalities, and some spores produced by ∆Bcest had folds and cracks on the surface ( Figure 3D). In addition, when incubated on the PDA medium at 22 • C for 10 h, all spores of B05.10 germinated, whereas the average germination rate of ∆Bcest was only 78.03%.

Bcest Is Involved in Hyphal Growth and Conidiation
The mycelial growth rate and the conidium of ΔBcest were significantly different from the those of the WT parent B05.10. The ΔBcest strain had a slower growth rate than the ΔBcest-C complemented strains and the B05.10 WT strain did on PDA, CM and MM, but especially on PDA ( Figure 3A,B). Conidia are the primary inoculum for the disease cycle of B. cinerea [31]. After incubating it on PDA for 10 days, the abundance of conidia for ΔBcest was significantly less than that for B05.10 and ΔBcest-C ( Figure 3C). Interestingly, the conidia of ΔBcest showed morphological and size abnormalities , and some spores produced by ΔBcest had folds and cracks on the surface ( Figure 3D). In addition, when incubated on the PDA medium at 22 °C for 10 h, all spores of B05.10 germinated, whereas the average germination rate of ΔBcest was only 78.03%.  SEM results showed that control strain B05.10 hyphae grew upright, with uniform thickness, a smooth surface, and no distortion or deformities, and apical hyphae branches were relatively uniform; by contrast, those of ∆Bcest showed distortion and deformities, with surface folds and depressions, differences in thickness, and uneven apical branches. TEM results showed that compared with control strain B05.10, the amount of endoplasmic reticulum and the number of mitochondria were increased in ∆Bcest, the mitochondrial membrane had disappeared at one end, and mitochondria were larger, looser and more folded. Furthermore, cell membranes were relatively intact, but tended to sink inwards. The above features are indicated by red arrows in Figure 3E.
These results also indicate that Bcest is important for vegetative growth and the conidiation of B. cinerea.

Bcest Participates in Regulating the Pathogenicity of B. cinerea
To determine and visually observe whether or not Bcest is involved in regulating the infection capacity of B. cinerea, an onion epidermis was inoculated with the ∆Bcest mutant spore suspension. At 12 h, unlike WT spores, ∆Bcest spores failed to form an infection structure after germination, and the WT strain successfully infected the onion epidermis by generating attachment cells after spore germination ( Figure 4A).
Microorganisms 2023, 11, x FOR PEER REVIEW 7 abnormal hyphal growth and uneven branching, and in TEM highlight cell membrane inv tions, mitochondrial swelling, and mitochondrial membrane disappearance.
SEM results showed that control strain B05.10 hyphae grew upright, with un thickness, a smooth surface, and no distortion or deformities, and apical hyphae bra were relatively uniform; by contrast, those of ΔBcest showed distortion and deform with surface folds and depressions, differences in thickness, and uneven apical bran TEM results showed that compared with control strain B05.10, the amount of endopl reticulum and the number of mitochondria were increased in ΔBcest, the mitochon membrane had disappeared at one end, and mitochondria were larger, looser and folded. Furthermore, cell membranes were relatively intact, but tended to sink inw The above features are indicated by red arrows in Figure 3E.
These results also indicate that Bcest is important for vegetative growth and th nidiation of B. cinerea.

Bcest Participates in Regulating the Pathogenicity of B. cinerea
To determine and visually observe whether or not Bcest is involved in regulatin infection capacity of B. cinerea, an onion epidermis was inoculated with the ΔBcest m spore suspension. At 12 h, unlike WT spores, ΔBcest spores failed to form an infe structure after germination, and the WT strain successfully infected the onion epid by generating attachment cells after spore germination ( Figure 4A).  To determine whether or not Bcest is involved in regulating pathogenicity in B. cinerea, grapevine leaves and fruits were inoculated with ∆Bcest mutants. Compared with the WT strain, ∆Bcest mutants exhibited reduced virulence in different hosts ( Figure 4B). At 96 h, grape leaves inoculated with ∆Bcest mutants showed no or small lesions, while WT-inoculated leaves showed larger lesions and the average lesion size reached 0.53 cm and 2.13 cm, respectively ( Figure 4C). Similarly, lesion size was considerably decreased on ∆Bcest mutant-inoculated grape fruits compared with WT-inoculated fruits ( Figure 4D). The complemented strain ∆Bcest-C exhibited almost the same level of virulence as the WT strain did. These results suggest that Bcest plays a crucial role in virulence of B. cinerea.

Effects of ∆Bcest Deletion on Sensitivity to Abiotic Stresses and Pathogenicity Factors
The results of the mycelial responsive assays showed that compared with ∆Bcest-C and B05.10, ∆Bcest mutants exhibited suppressed mycelial growth in the presence of KCl and H 2 O 2 , and the transparent zone in the protease and cellulase test medium was significantly reduced. However, the growth diameter under the SDS treatment and the polygalacturonase production capacity of ∆Bcest were significantly higher than those of B05.10 and ∆Bcest-C, respectively ( Figure 5A). The values were not significantly different (p = 0.05) from the inhibition rates for B05.10 and ∆Bcest-C in response to these chemicals, and the enzyme-producing capacity was not significantly different either ( Figure 5B,C). These results suggest that disruption of Bcest may have marginal effects on mycelial growth in response to abiotic stresses, and that pathogenicity factor production capacity may also be affected.
3 strains for the pathogenicity test of the ΔBcest strain. The red circle represents the diameter of the lesion.
To determine whether or not Bcest is involved in regulating pathogenicity in B. cinerea, grapevine leaves and fruits were inoculated with ΔBcest mutants. Compared with the WT strain, ΔBcest mutants exhibited reduced virulence in different hosts ( Figure 4B). At 96 h, grape leaves inoculated with ΔBcest mutants showed no or small lesions, while WT-inoculated leaves showed larger lesions and the average lesion size reached 0.53 cm and 2.13 cm, respectively ( Figure 4C). Similarly, lesion size was considerably decreased on ΔBcest mutant-inoculated grape fruits compared with WT-inoculated fruits ( Figure  4D). The complemented strain ΔBcest-C exhibited almost the same level of virulence as the WT strain did. These results suggest that Bcest plays a crucial role in virulence of B. cinerea.

Effects of ΔBcest Deletion on Sensitivity to Abiotic Stresses and Pathogenicity Factors
The results of the mycelial responsive assays showed that compared with ΔBcest-C and B05.10, ΔBcest mutants exhibited suppressed mycelial growth in the presence of KCl and H2O2, and the transparent zone in the protease and cellulase test medium was significantly reduced. However, the growth diameter under the SDS treatment and the polygalacturonase production capacity of ΔBcest were significantly higher than those of B05.10 and ΔBcest-C, respectively ( Figure 5A). The values were not significantly different (p = 0.05) from the inhibition rates for B05.10 and ΔBcest-C in response to these chemicals, and the enzyme-producing capacity was not significantly different either ( Figure 5B,C). These results suggest that disruption of Bcest may have marginal effects on mycelial growth in response to abiotic stresses, and that pathogenicity factor production capacity may also be affected.

Bcest Deletion Affects Transcription and Pathogenicity-Related Genes
We performed a RNA-seq analysis to identify genes that might exhibit changes in regulation affected by Bcest in B. cinerea. Three biological replicates with mRNA isolated from WT B05.10 and ∆Bcest strains were performed, and 162 downregulated and 189 upregulated (fold change > 2, p < 0.05) genes were identified in ∆Bcest and compared with those of B05.10 ( Figure 6A). polygalacuronic acid or carboxymethyl cellulose at 20 °C for 3 days. (B) Sensitivity of ΔBcest, B05.10 and ΔBcest-C to KCl, H2O2 and SDS. (C) Ability of ΔBcest, B05.10 and ΔBcest-C to produce proteases, polygalacturonase and cellulases. Bars represent standard errors from three replicates. Values on bars followed by different letters indicate significant differences at p = 0.05.

Bcest Deletion Affects Transcription and Pathogenicity-Related Genes
We performed a RNA-seq analysis to identify genes that might exhibit changes in regulation affected by Bcest in B. cinerea. Three biological replicates with mRNA isolated from WT B05.10 and ΔBcest strains were performed, and 162 downregulated and 189 upregulated (fold change > 2, p < 0.05) genes were identified in ΔBcest and compared with those of B05.10 ( Figure 6A). Functional annotation of DEGs via GO analysis was performed to identify genes belonging to molecular function, cellular component and biological process categories (Figure S1). Among them, catalytic activity/oxidoreductase activity, organelle envelope/mitochondrial outer membrane, the oxidation-reduction process and the monocarboxylic acid metabolic process were the main molecular function, cellular composition, and biological process subcategories, respectively. These results showed that growth and pathogenicity defects caused by the absence of Bcest in the B05.10 strain may be closely related to these six terms.
In addition, enriched DEGs can be functionally classified into metabolism, cellular processes and genetic information processing categories. Among the 45 metabolic pathways belonging to these three categories, the top 10 metabolic pathways of enriched DEGs were metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, carbon metabolism, biosynthesis of amino acids, oxidative phosphorylation, glutathione metabolism, tricarboxylic acid (TCA)/citrate cycle, the pentose phosphate pathway, and ubiquitin-mediated proteolysis ( Figure S2). These metabolic pathways may be closely related to the growth and pathogenicity defects of ΔBcest.
To verify the reliability of DEGs identified from transcriptome sequencing, qRT-PCR was performed on the remaining ΔBcest RNA samples used for transcriptome analysis. Functional annotation of DEGs via GO analysis was performed to identify genes belonging to molecular function, cellular component and biological process categories ( Figure S1). Among them, catalytic activity/oxidoreductase activity, organelle envelope/mitochondrial outer membrane, the oxidation-reduction process and the monocarboxylic acid metabolic process were the main molecular function, cellular composition, and biological process subcategories, respectively. These results showed that growth and pathogenicity defects caused by the absence of Bcest in the B05.10 strain may be closely related to these six terms.
In addition, enriched DEGs can be functionally classified into metabolism, cellular processes and genetic information processing categories. Among the 45 metabolic pathways belonging to these three categories, the top 10 metabolic pathways of enriched DEGs were metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, carbon metabolism, biosynthesis of amino acids, oxidative phosphorylation, glutathione metabolism, tricarboxylic acid (TCA)/citrate cycle, the pentose phosphate pathway, and ubiquitin-mediated proteolysis ( Figure S2). These metabolic pathways may be closely related to the growth and pathogenicity defects of ∆Bcest.
To verify the reliability of DEGs identified from transcriptome sequencing, qRT-PCR was performed on the remaining ∆Bcest RNA samples used for transcriptome analysis. qRT-PCR validation was performed by randomly selecting nine growth-and pathogenicityassociated genes. These genes encode proteins that participate in the synthesis and metabolism of substances (Bcin14g02370, Bcin07g05140, Bcin03g08050, and Bcin11g02040), the catalytic reactions of enzymes (Bcin03g00480, Bcin01g07330, and Bcin10g01030) and virulence and stress factors (Bcin14g04260 and Bcin01g02560). The qRT-PCR results were consistent with the RNA-seq results ( Figure 6B), indicating the reliability of the RNA-seq data in this study.

Discussion
To explore the functions of membrane protein Bcest, we first disrupted the Bcest gene and characterised the resulting mutant, which showed severe defects in hyphal growth and pathogenicity. These results are consistent with those of experiments on BcHBF1 and BcATG6 genes of B. cinerea [32,33]. We thus hypothesised that the Bcest protein might be involved in regulating hyphal growth and pathogenicity-related genes in this fungal species. Bcest is a vital virulence determinant, since deletion of the Bcest gene also compromised the penetration ability of B. cinerea, indicating that the reduced virulence of the ∆Bcest mutant was likely due, as least in part, to defects in the penetration of host cells. The ∆Bcest mutant exhibited increased sensitivity to H 2 O 2 and osmotic stress, which may be due to the absence of membrane protein Bcest, which destroys mitochondrial membrane integrity, reducing tolerance to osmotic pressure and reactive oxygen species (ROS) [34]. However, under SDS-mediated stress, the growth rate of the ∆Bcest mutant strain was higher than that of WT strain B05.10. However, other studies have shown that SDS can inhibit the growth of B. cinerea [33]; hence, the specific mode of action will be explored in our future research. In addition, the ability of the ∆Bcest mutant strain to produce cellulases and proteases was significantly reduced, which is another important factor of pathogenicity [35,36]. Based on the above results, the Bcest protein appears to be an important virulence factor of B. cinerea. However, the regulatory mechanisms involving Bcest 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 ∆Bcest mutant strains in culture for 72 h, and 351 DEGs were screened. GO functional analysis showed that in biological process and molecular function categories, DEGs resulting from the loss of the Bcest gene were mostly involved in metabolic and cellular processes, and catalytic activity and binding, respectively. Among the cellular component terms, the most enriched ones were linked to cellular structure and membrane, which is consistent with the fact that the deletion of the Bcest gene affected the mitochondrial membrane in the TEM experiment. Our RNA-seq analysis results suggest that global changes in genes involved in metabolic pathways, the biosynthesis of secondary metabolites, the pentose phosphate pathway and the TCA cycle are likely to underlie this defect.
The reliability of the transcriptome data was verified by via qRT-PCR of nine growthand pathogenicity-related genes. Sclerotia are an important virulence factor in B. cinerea, and this study found that the melanin synthesis gene Bcpks13 (Bcin03g08050) was significantly downregulated in the ∆Bcest mutant strain, which may explain why this strain failed to form sclerotia [37]. Deletion of CDC14 in several plant pathogen species severely impairs virulence, demonstrating that CDC14 function is important for host infection [38]. Fusarium graminearum, Magnaporthe oryzae and Aspergillus flavus lacking CDC14 gene expression can lead to conidia formation defects and reduced pathogenicity [39][40][41]. Polygalacturonase and β-1,4-endoxylanase BcXyn11A are important virulence factors of B. cinerea, and they mainly promote the virulence of B. cinerea through necrotic activity rather than enzymatic activity [42]. We found that BcXyn11a (Bcin03g00480), Bccdc14 (Bcin01g02560) and endopolygalacturonase-encoding gene Bcpg5 (Bcin01g07330) were significantly downregulated, which suggests that the decrease in the number of conidia and weakening of the pathogenicity of ∆Bcest mutant strains were the result of a combination of polygenes. Bcgas2 (Bcin14g04260) is required for B. cinerea to cope with stress [43,44], and a decrease in the stress capacity of the ∆Bcest mutants was closely related to the downregulation of this gene. In addition, both the mitochondrial membrane and the mitochondrial electron transport chain can induce a burst of ROS, which in turn affects the growth and development of the strain. In this study, the expression of the cytochrome C synthesis gene Bccyc1 (Bcin14g02370), which is involved in the mitochondrial electron transport chain, was significantly upregulated in ∆Bcest mutant strains [45]. Interestingly, we found that the membrane protein Bcest had little effect on cell membranes, but TEM showed that the mitochondrial membrane of the ∆Bcest strain was damaged, enlarged, wrinkled and relaxed. Thus, we hypothesised that loss of Bcest may destroy the integrity of the mitochondrial membrane, affect the transmission of the mitochondrial electron chain, and influence metabolic pathways in the strain. Therefore, we concluded that an absence of Bcest can reduce the growth rate and pathogenicity of B. cinerea, while increasing sensitivity to environmental stress.

Conclusions
In conclusion, membrane protein Bcest can inhibit the growth of B. cinerea, reduce the spore germination rate and pathogenicity, disrupt the integrity of the mitochondrial structure, and increase sensitivity to oxidative and osmotic stress.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/microorganisms11051225/s1. Figure S1: GO functional classification of ∆Bcest transcriptome DEGs. Figure S2: KEGG enrichment analysis bubble diagram of the ∆Bcest transcriptome. Table S1: Mapping statistics in transcriptome analyses. Table S2: Oligonucleotide primers used in this study. Table S3: List of all DEGs in transcriptome analyses. Table S4: Genes and primers used for qRT-PCR.