Multidrug resistance of Botrytis cinerea associated with its adaptation to plant secondary metabolites

ABSTRACT Fungicides are an effective way to control gray mold of grapes, but the pathogen Botrytis cinerea can develop resistance, overcoming the effectiveness of a fungicide that is repeatedly applied. More importantly, the emergence of multidrug resistance (MDR) in the field, where multiple fungicides with different modes of action simultaneously lose their efficacies, is a significant concern. MDR is associated with ATP-binding cassette (ABC) transporters of the pathogen, and certain plant secondary metabolites (PSMs) stimulate the upregulation of ABC transporters, we hypothesized that the pathogen’s preadaptation to PSMs might contribute to MDR development. To test this in B. cinerea, ten PSMs, namely, resveratrol, reserpine, chalcone, flavanone, eugenol, farnesol, anethene, camptothecin, salicylic acid, and psoralen, were selected based on their association with ABC transporters involved in fungicide resistance. B. cinerea strain B05.10 was continuously transferred for 15 generations on potato dextrose agar amended with a PSM (PDAP), and sensitivities to PSMs and fungicides were examined on the 5th, 10th, and 15th generations. RNA was extracted from B. cinerea from the selected generations. After 15 generations of culture transfers, an up-regulation was observed in the expression of ABC transporter-encoding genes BcatrB, BcatrD, and BcatrK using quantitative polymerase chain reaction (qPCR). This upregulation was found to contribute to MDR of B. cinerea against two or more fungicides, among azoxystrobin, boscalid, fludioxonil, difenoconazole, prochloraz, and pyrimethanil. This finding was confirmed through genetic transformation. The decreased sensitivity of B. cinerea to fungicides was confirmed as a subsequent MDR phenotype after exposure to camptothecin, flavanone, and resveratrol. Besides, transcriptome analysis also revealed the upregulation of transcription factors related to ABC expression following resveratrol exposure. This suggests that PSMs contributed to inducing preadaptation of B. cinerea, leading to subsequent MDR. IMPORTANCE The emergence of MDR in plant pathogens is a threat to plant disease management and leads to the use of excessive fungicides. Botrytis cinerea is of particular concern because its MDR has widely emerged in the field. Understanding its genesis is the first step for controlling MDR. In this study, the contribution of PSMs to MDR has been examined. Effective management of this pathogen in agroecosystems relies on a better understanding of how it copes with phytochemicals or fungicides.

field (1,2).This indicates that pathogens can develop resistance to several fungicides that possess different modes of action (MOA) simultaneously.This occurrence may lead to the failure of disease management through a conventional resistance manage ment strategy, such as alternating or tank-mixing of fungicides with different MOAs.The excessive use of fungicides in field crops can multiply environmental pollution and increase the risk of food contamination due to pesticide residues.
There are various known mechanisms of fungicide resistance, including genetic mutation, overexpression of the target protein, xenobiotic metabolism (3), and activation of efflux pumps, such as ATP-binding cassette (ABC) transporters and major facilita tor superfamily (MFS) transporters (4).Efflux-related resistance is the most important mechanism in B. cinerea.The ABC transporter is one of the largest transporter families, which uses the energy produced by ATP hydrolysis (5).The two core structures of ABC transporters usually consist of six trans-membrane domains and two nucleotide-binding fold domains.Fourteen ABC transporters (BcatrA to BcatrN) have been identified (6).Among them, the BcatrB, BcatrD, and BcatrK genes have been reported to be associated with MDR in B. cinerea (7)(8)(9).
Plant secondary metabolites (PSMs) can influence both pathogens and plants in their interactions.While they can help in defending plants against pathogen infections, they may also elicit the pathogen to overcome the defense of plants.Most studies have been focused on PSMs involved in phytoalexin production (10), but how patho gens defend themselves against PSMs has been rarely investigated.Some PSMs have been found to affect ABC transporters of pathogens by enhancing the expression of ABC genes (11)(12)(13)(14)(15)(16).For example, resveratrol induces the overexpression of the atrB gene in B. cinerea (11) and of the PMR5 and Bcmfs1 genes in Penicillium digitatum (12), while psoralen and eugenol induce Bcmfs1 gene overexpression in B. cinerea (13).Camptothecin induces atrB overexpression in Aspergillus nidulans (14).The alkaloid reserpine can cause upregulation of ABC transporters' gene expression in Aspergillus oryzae (15).Chalcone induces the overexpression of P-glycoprotein (P-gp) and multi drug resistance proteins (16).Meanwhile, some other PSMs inhibit the function of ABC transporters.Flavanone, farnesol, chalcone, and anethene are transporter protein inhibitors (16,17), and beauvericin counteracts MDR by intercepting the ABC transport ers (18).Ellagic acid and schisandrins derivatives, which are natural biannual polyphe nols, have therapeutic potential in overcoming MDR in cancer by regulating drug efflux through P-gp and similar transporters (19).Transporters in pathogens are key factors contributing to multidrug efflux, which caused changes in pathogen sensitivity to plant-derived secondary metabolites.Thus, ABC transporters may regulate the adaptabil ity of pathogens to xenobiotic compounds, such as PSMs and fungicides (11).
ABC transporters play a critical role in the adaptability of pathogens to xenobiotic compounds such as PSMs and fungicides (11).These transporters also influence the interaction between pathogens and antibiotics, including 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid, and phenazine-1-carboxamide, broad-spectrum antibiotics produced by Pseudomonas spp.(20) .A plant pathogenic fungi may gradually build up resistance to the antibiotic pyrrolnitrin over time, rendering it less effective against biocontrol agents that target B. cinerea (21).
While it is unclear whether the overexpression of ABC transporter genes induced by PSMs can result in MDR in B. cinerea, it is possible that the preadaptation of plant pathogens may contribute to MDR observed in the field.Previous studies suggest that such preadaptation may be linked to the preadaptive reactions to PSMs existing in human pathogens (22) or insects (23).In our previous study, it was found that the PSMs such as resveratrol accumulated significantly in grape leaves infected by B. cinerea (24).Whether these PSMs participated in the preadaptation of pathogens to fungicides remains unclear.Thus, the objectives of this study were to examine whether PSM preadaptation exists in B. cinerea and whether it even contributes to MDR.The results will help us understand the mechanisms of development of MDR in the field.

Adaptation of Botrytis cinerea to PSM stress
A series of culture transfers of B. cinerea was performed on potato dextrose agar amended with a PSM (PDAP) to establish a condition for preadaptation.The sensitivity was determined on the parents, and the 5th, the 10th, and the 15th generations of B. cinerea B05.10 were exposed to PSMs, and the decreased inhibition of induced mutants was compared to that of B05.10 (Fig. 1).
There were four distinct sensitivity patterns observed in B. cinerea when exposed to PSMs, with a few exceptions.Pattern 1: sensitivity continuously declined during the initial ten transfers, stabilizing or increasing thereafter.This pattern was observed in treatments with anethene, camptothecin, farnesol, reserpine, and salicylic acid.Pattern 2: sensitivity slightly increased at the 5th transfer, then significantly decreased at the 10th transfer, and eventually returned to the 5th transfer level.Only the resveratrol treatment followed this pattern.Pattern 3: sensitivity declined at the 5th transfer, significantly increased at the 10th transfer, and decreased again at the 15th transfer.This was observed with psoralen treatment, displaying Pattern 1 at lower concentrations (≤25 µg/mL) and Pattern 3 at higher concentrations (≥50 µg/mL).Pattern 4: sensitivity remained relatively stable across all 15 transfers for chalcone, eugenol, and flavanone treatments.

Sensitivities of the PSM-adapted mutant of Botrytis cinerea to multiple fungicides with different modes of action
The parent and the 5th, 10th, and the 15th generations of PSM-adapted B. cinerea were assayed for their sensitivities to fungicides (Table S1) and resistance factors (Fig. 2).The sensitivity of all PSM-adapted strains decreased to at least two fungicides.For instance, for B. cinerea grown on PDA amended with either eugenol, farnesol, or camptothecin, the EC 50 to boscalid continuously increased, indicating a reduced sensitivity.The fluazinam sensitivity of B. cinerea decreased when treated with either salicylic acid, chalcone, or resveratrol.There were some exemptions.The azoxystrobin sensitivity increased in B. cinerea treated with resveratrol in the third generation.With eugenol, farnesol, chalcone, and psoralen treatments, the sensitivity slightly increased in the first generation.The fludioxonil and prochloraz sensitivity had significant decreases in B. cinerea treated with most PSMs.The difenoconazole sensitivity decreased when treated with anethene, reserpine, and resveratrol.Chalcone and farnesol treatment decreased the pyrimethanil sensitivity.Overall, the ten PSMs enhanced B. cinerea resistance to fungicides.
After 10 transfers on PSM-free PDA, the resistance factors (RFs) of some B. cinerea mutants had changed more or less as indicated by factor of sensitivity change (FSC) values.FSC values of the mutants ranged from 0.03 to 11.96 (Table S2).Most of the mutants' resistance to azoxystrobin, fludioxonil, and pyrimethanil was much higher after 10 transfers.On the other hand, most of the mutants lost their resistance to boscalid, prochloraz, and difenoconazole.

Transcriptome analysis
To elucidate the mechanisms involved in the resistance phenotype observed in B. cinerea B05.10, a 30th generation resveratrol-induced strain exhibiting an MDR phenotype (Table S3) and a strain with wild-type sensitivity were randomly selected for the transcriptomic analysis through RNA-seq.To identify genes related to resistance, we screened for ABC transporters, MFS transporters, P450, and transcription factors among the differentially expressed genes in group D (the 30th generation of resveratrol-induced B05.10 treated by azoxystrobin) compared to group C (the 30th generation of resvera trol-induced B05.10) (Table 1).Volcano plots were generated to visualize the significantly differentially expressed features (Fig. 3a and b).Gene Ontology (GO) analysis catego   rized the differential genes in 15 functional groups, with "transmembrane transporter activity" (GO: 0022857) and "transporter activity" (GO: 0005215) being enriched in D vs C (Fig. 3d), but not in group B (azoxystrobin-and azoxystrobin-treated B05.10) vs group A (B05.10) (Fig. 3c).Venn diagram analysis showed common and unique differential metabolites in the four comparisons (AB, AC, BD, and CD) to highlight the effects of resveratrol and azoxystrobin on B. cinerea (Fig. 3e).We also examined the expression of ten ABC transporter genes that were shown (Fig. 3f) and identified candidate transcrip tion factors from the reference that were clustered with ABC transporter genes (Fig. 4).AC and BD differential genes include 149 and 219 up-regulation transcription factors coupled with 318 and 237 down-regulation transcription factors, respectively.Among these, eleven transcription factors (BCIN_01g03660, BCIN_03g01840, BCIN_03g06600, BCIN_06g05210, BCIN_09g05540, BCIN_10g03530, BCIN_13g03910, BCIN_13g05610, BCIN_14g00770, BCIN_15g00080, and BCIN_16g03230) were found to be up-regulated in the resveratrol-induced strain compared to the non-induced strain selected from the reference, which was related to drug resistance in microorganisms including Tac1p, Mrr1, and Mrr2 in Candida albicans (25-27) (Table S4).On the contrary, BCIN_02g01900, BCIN_03g02160, and BCIN_13g02900 were found to be down-regulated.Meanwhile, 13g04870, BCIN_13g03910, BCIN_03g00220, BCIN_03g00450, BCIN_03g06600, BCIN_06g05210, BCIN_10g03880, BCIN_04g03220, BCIN_16g03230, BCIN_15g00120, BCIN_01g03660, BCIN_02g01900, BCIN_02g03500, BCIN_11g05340, BCIN_16g03200, and BCIN_03g01840 were up-regulated in group D compared to group C. The RNA-seq results revealed an overexpression of ABC transporter genes in the groups at the 30th generation.To validate these findings, we performed qPCR assays on RNA samples obtained both before and after 4-h azoxystrobin treatment, measuring the expression levels of 10 ABC transporter genes (Fig. 5).Our results showed that overall, the qRT-PCR results were mostly consistent with the RNA-seq data, although there were some minor variations in fold-change between the RNA-seq and qRT-PCR results for some genes.These findings affirm the reliability of our experimental results.Addition ally, BcatrB, BcatrD, and BcatrK were identified as highly upregulated genes following azoxystrobin treatment, as confirmed by qPCR.The transcription factors associated with ABC transporter genes were screened through expression profiles.

ABC gene expression of the PSM-adapted mutant of Botrytis cinerea
The expression of ABC transporter genes BcatrB, BcatrD, and BcatrK was measured through quantitative PCR (qPCR) in the parents and the 5th, 10th, and the 15th generations of B. cinerea B05.10 co-cultured with one of the ten PSMs.The expression levels of BcatrB, BcatrD, and BcatrK in the three generations of B. cinerea were all up-regulated when exposed to salicylic acid, reserpine, chalcone, eugenol, farnesol, and anethene domestication (Fig. 6).PSMs such as resveratrol and flavanone induced the overexpression of one or two of the three ABC transporter genes.Most PSMs stimulated the overexpression of the transporter genes.

Contribution of BcatrB, BcatrD, and BcatrK overexpression to multidrug resistance in Botrytis cinerea
A pxEH-GFP-plasmid was constructed for overexpression.PCR results indicated the plasmids were successfully transformed into B05.10.The expression of BcatrB, BcatrD, and BcatrK in the transformants was 8 to 18 folds higher than that of the wild-type B05.10 (Fig. 7), indicating overexpression.

Sensitivity of BcatrB, BcatrD, and BcatrK overexpression transformants to fungicides
The sensitivities of B. cinerea transformants to fungicides were determined (Fig. 8; Table S5).The pxEH-GFP-plasmid transformant was a control (CK), which had no significant difference compared with the wild-type B05.10 strain.BcatrB overexpression in transformants BO1 and BO10 resulted in a significant increase in EC 50

DISCUSSION
In this study, we have demonstrated that PSMs including eugenol, farnesol, reserpine, and resveratrol elicited the overexpression of ABC transporter genes, including BcatrB, BcatrD, and BcatrK, in B. cinerea.As a result of continued PSM exposure over multi ple generations, the response of the fungus was adaptive to both PSM stresses and fungicides.Therefore, it was proved that continuous exposure to PSMs can induce a progressive and elevated adaptability to PSMs, which helps B. cinerea to be resistant to multiple fungicides.This type of preadaptation has been observed in other organisms, including insects and bacteria (23,(28)(29)(30)(31)(32).For instance, flavonoids induce the overexpression of the P450 gene in whitefly (Bemisia tabaci), leading to metabolic detoxification and resistance to insecticides thiamethoxam and flufenoxuron (23).Similarly, querce tin induces the expresson of P450 and carboxylesterase, leading to lambda-cyhalo thrin resistance in cotton bollworm (Helicoverpa armigera) (29,33).Moreover, the secondary metabolites from Pseudomonas aeruginosa, such as phenazine 1-carboxylic acid, phenazine 1-carboxamide, paerucumarin, indole, salicylic acid, prothiocyanide, ergothioneine, polyamines, H 2 S, and Pseudomonas quinolone signal can improve the antibiotic resistance of medical pathogens (10).This preadaptation is primarily due to the enhanced efflux (34,35).Otherwise, capsidiol induces high expression of Bccpdh, which encodes a dehydrogenase in B. cinerea.This gene has been predicted to result in decreased sensitivity of B. cinerea to capsodiol, and the resistance is related to plant metabolites (36).
The major sources of preadaptation in insects and bacteria are P450 detoxification of secondary metabolites and the efflux system.Similarly, the adaptation of plant pathogens to xenobiotics, which leads to insensitivity to multiple fungicides, is also due to the efflux (10,(37)(38)(39), including ABC and MFS efflux transporter genes (2,40,41).To explore the main factor responsible for MDR, we performed genetic transfor mation experiments and found that overexpression of three ABC transporter genes, BcatrB, BcatrD, and BcatrK, caused resistance to fungicides having different MOAs in B. cinerea (Fig. 8).This finding is consistent with the previous report (8,10,42).PSM-induced overexpression of three ABC genes contributes to multidrug resistance.Moreover, we observed significant overexpression of these three ABC transporter genes in PSM-induced strains, regardless of the presence or absence of PSM stimulation, after 15 generations of culture on PDA (the latter data not shown in this study).
We hypothesize that MDR that occurred in the field may be stimulated by PSMs.This hypothesis is supported by other reports (10,11,14), which have demonstrated that PSMs can affect ABC efflux proteins.When exposed to PSMs during growth, the sensitivity of B. cinerea to the PSMs decreased, accompanied with the up-regulation of the three ABC efflux genes.It is acknowledged that under experimental conditions, the concentration of some secondary metabolites may be much higher than that in plants.The purpose of having a higher concentration is to create a high screening pressure, allowing the fungus to adapt to the stress in a shorter period.However, in an in vivo test using a living leaf, the selection pressure may be different compared with in vitro conditions due to differences in nutritional conditions and various types of PSMs.The presence of stress helps the pathogen adapt to the fungicide pressure and more likely develop MDR.Therefore, PSMs impacted the pathogen during plant-pathogen interactions and contribute to the development of MDR.
Fungicide resistance is usually determined by genetic variation and mutation (43).In fungi, whether these two concepts reflect genetic or non-genetic change of resistance of pathogens deserves discussion.Non-genetic variation could be a stress response, and we have used the term "mutant" to describe adaptative generations of the fungus in this study.Similarly, bacteria have been shown to develop multiple phenotypic variants in response to various selective pressures, such as immune / defense challenges, antimicro bial therapy, and oxygen limitation (44,45).In this study, salicylic acid-induced mutants had reduced susceptibility to PSM, as well as boscalid and azoxystrobin.The stable inheritance after 10 generations of relay culture on a drug-free medium was also found to have significant up-regulation of BcatrB and BcatrK.In addition, similar results were found in flavanone-induced mutants exposed to fludioxonil through overexpression of BcatrK.However, the exact mechanism of resistance induction by PSMs, whether through stably or unstably inherited changes in the pathogen, remains unclarified.This is an interesting and important issue that is worthy of further investigation.While various fungicides were intentionally selected to represent different MOAs, the observed efflux effect of ABC transporter proteins may be related to the chemical structures of the fungicides or PSMs.It is plausible that different fungicides may engage distinct ABC transporters in efflux mechanisms that contribute to MDR, which needs further in-depth investigations.
It is an interesting phenomenon that the sensitivity of mutants against PSMs and fungicides has a regression at the 15th generation.In the context of secondary metabolite treatment, organisms may survive in a responsive status when encountering stresses, but the long-term maintenance of this status is detrimental to the normal development of the organism, which is called resistance cost in resistant strains (46).That is a reason some mechanisms will appear in the organism that can regulate this stress state, preventing the organism from remaining in this state for a long time to promote better growth.This might explain why the 15th generation is more sensitive compared to the 10th generation.It was reported that nematodes also exhibit reduced resistance under the stress of antimycin and show the same trend of stress tolerance as our results (increased from the F1 to F3 progeny but decreased in the F4 progeny), and these phenotypes may be related to the properties and applied dosage of the drug (47).
The biochemical or genetic propensity for resistance is the base on which the selection pressure operates, and host characteristics and movement are key ecological traits that influence effective selection intensities for resistance (48).Host-pathogen interactions continuously compel species to adapt to each other, making them one of the most complex and fascinating models of the evolutionary interplay between organisms (49).Resveratrol, which is obtained from the host plant, induces intracellular signal transduction pathways, which ultimately lead to changes in the gene expression patterns of the cells by activating the stimulus-responsive transcription factors such as CREB, AP-1, Egr-1, Elk-1, and Nrf2 (50).Farnesol also activated transcription factors Tac1 and Znc1 to up-regulate the ABC transporter CDR1, which was crucial in drug resistance (51,52).Following activation, these transcription factors induce transcription of delayed response genes.Botrytis cinerea MDR1 strains showed higher expression of the ABC transporter BcatrB due to transcription regulation by transcription factor Mrr1 (2).In this research, based on transcriptome data analysis, the induction of plant secondary metabolites, such as resveratrol, can lead to overexpression of ABC transporter genes, which may be contributed to the effect of secondary metabolites on transcription factors in B. cinerea.Moreover, it has been reported that compounds such as pyocyanin (PYO) in Pseudomonas aeruginosa and indole in Escherichia coli, which were structurally similar to antibiotics like ciprofloxacin and chloramphenicol, respectively, contribute to preadaptation via efflux pumps (38,53).It also found that some fungicides and examined secondary metabolites showed complex and abundant chemical structure, including benzene rings, aldehyde, alkene, and oxhydryl in this study.It is inferred that PSMs with structural similarities of fungicides may regulate up-regulation of ABC transporters via responding of transcriptional factors or dircted combination.Candidate transcription factors relative to ABC transporter gene expression were screened using fold change of expression and cluster analysis and will be validated in further studies.The results of this work can be a good reference for studying other plant pathogens.

Pathogen and growth conditions
Botrytis cinerea B05.10 was used as a standard strain.Mycelial plugs (5 mm diameter) of B. cinerea strains were cultivated on potato dextrose agar (PDA) at 18°C in the dark for 3 to 5 days.To produce a conidial suspension, the strains were cultured in the dark at 18°C on carrot agar (CA) for 3 to 5 days and placed under a black light lamp for 5 days.Conidia were washed with sterile water and filtered through three layers of filter paper to eliminate mycelia.Ten PSMs, namely, resveratrol, reserpine, chalcone, flavanone, eugenol, farnesol, anethene, camptothecin, salicylic acid, and psoralen (Fig. 9a), were used to induce B. cinerea for preadaptation at different concentrations (Table 2).The 5th, 10th, and 15th generation strains exposed to PSMs were collected and used for further testing.

Effects of PSMs on Botrytis cinerea growth
The sensitivities of B. cinerea were examined by following Song et al. 's method (54) with some modifications.Briefly, PDA was amended with one of the ten PSMs at final concentrations of 10, 25, 50, and 100 mg/L with three replicates.Mycelial growth of B. cinerea under the PSM treatment was measured for the 5th, 10th, and 15th generations.
Mycelial growth of the 5th, 10th, and 15th generations of PSM-treated B. cinerea was measured under various concentrations of fungicides, and EC 50 was calculated (55).RF was calculated as RF = EC 50 of the mutant / EC 50 of the parental strain.To assess the stability of the resistant mutants, mycelial plugs of ten 15th generation-induced mutants were subjected to 10 successive culture transfers on fungicide-free PDA plates, and EC 50 values were measured on the 1st and 10th transfers.The stability of resistance was calculated by the FSC, where FSC was calculated by dividing the RF of the tenth subculture by that of the first (56).

Transcriptome sequencing and qRT-PCR
The sensitivities of the 20th and 30th generations of resveratrol-induced mutants were examined against seven fungicides as described in preceding section.RNA was extracted from four groups of B. cinerea B05.10: group A (B05.10), group B (azoxy strobin-treated B05.10 mutant induced by resveratrol at the 30th generation), group C (the 30th generation of resveratrol-induced B05.10 mutant), and group D (azoxy strobin-treated B05.10 mutant induced by azoxystrobin).The extraction was carried out using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, United States).Sequencing was performed on AMPure XP beads as 250-300 bp paired-end reads and mapped using the B. cinerea B05.10 reference genome (https:// www.ncbi.nlm.nih.gov/data-hub/genome/GCF_000143535.2/).The Qubit 2.0 fluorometer was used for preliminary quantification, and the insert size of the library was detected by using an Agilent 2100 bioanalyzer (Agilent Technologies, CA, USA) following library construction.The effective concentration of the library was accurately quantified by qRT-PCR, and the differential genes were screened by |log 2 (FoldChange)|> 1 and P < 0.05 as the cut-off criteria.Three independent biological replicates were analyzed in this experiment.
The most significant 15 terms were selected to draw a scatter plot for display in GO enrichment analysis.The volcano plot can visually display the distribution of differentially expressed genes for comparison of each combination.RNA extraction was performed as described previously.The PrimeScript RT reagent Kit with gDNA Eraser (Takara, Beijing, China) was used to synthesize cDNAs.Ten ABC transporter genes were validated by the transcriptome results by qPCR (BcatrA BCIN_11g04460, BcatrB BCIN_13g00710, BcatrC BCIN_04g02930, BcatrD BCIN_13g02720, BcatrE BCIN_02g04760, BcatrF BCIN_05g03610, BcatrG BCIN_10g00650, BcatrH BCIN_15g00830, BcatrI BCIN_16g00820, and BcatrK BCIN_01g05890).qPCR was performed on an ABI7500 sequence detection system (Applied Biosystems, California, United States) using the FastSYBR Mixture kit (Beijing ComWin Biotech Co., Ltd., Beijing, China) (Table S6) and the cDNA as a template.In the qPCR analysis, the reaction system was mixed with 10 µL 2 × FastSYBR, 0.4/0.4µL mixture forward/reverse primer, 1 µL template cDNA, and 8.2 µL ddH 2 O.The settings of the thermocycler included denatura tion at 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, and 60°C for 34 s.The relative expression of genes was calculated using the 2 −ΔΔCt method (57), and the actin genes (BCIN_16g02020) were used as a reference to normalize the quantification of the ABC gene expression levels.The experiment was conducted twice, and each treatment had three replicates.

qPCR of Botrytis cinerea induced by PSMs
Test cultures of gene quantification included B. cinerea B05.10 and its 5th, 10th, and 15th generations shaken at 180 rpm at 18°C under PSM treatments.Biological materials of induced B. cinerea in potato dextrose broth (PDB) were collected in an RNA-free tube.Total RNA was extracted from the samples, and qPCR was performed as described previously.

Construction of the gene expression cassette
The pxEH-GFP plasmid was constructed by inserting the OliC promoter and GFP tag into the pxEH vector (58) using BamHI and HindIII restriction sites, which served as an overexpression vector.The ABC transporter genes BcatrB, BcatrD, and BcatrK were amplified from B05.10 cDNA using polymerase chain reaction (PCR) with SalI and XbaI restriction enzymes.PCR was performed in a reaction of 20 µL mixture of EasyTaq DNA Polymerase (TransGen, Beijing, China).Thermal cycler settings included an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 61°C for 30 s, and extension at 72°C for 2 min, which was ended with an extension at 72°C for 10 min.The PCR products were retrieved and purified using the Gel Extraction Kit (CWBIO, Beijing, China).

Gene transformation in Botrytis cinerea
The pxEH-GFP plasmid and expression vectors were transformed into Agrobacterium tumefaciens strain AGL-1 and then transformed into B. cinerea B05.10 spores.Transform ants were successfully screened using hygromycin phosphotransferase gene (HPH) resistance methods (58) and identified through PCR (Table S6).The expression of BcatrB, BcatrD, and BcatrK genes in their transformants was measured using qPCR (Table S6).Biological samples from the transformants grown on PDA containing 100 µg/mL hygromycin were collected in an RNA-free tube.Total RNA was extracted from all samples following the manufacturer's instructions.The programs of cDNA synthesis and qPCR of three ABC transporter genes in respectively their over-expression mutants were the same as those in preceding section.

Fungicide sensitivities of Botrytis cinerea transformants
Fungicides were dissolved in dimethyl sulfoxide (DMSO).The B. cinerea transformants were grown on PDA amended with seven fungicides, and EC 50 was calculated as described in preceding section.The inhibition rates were calculated by comparing the growth of B. cinerea transformants on fungicide-amended and to non-amended (control) PDA.To keep the growth conditions consistent, the final concentration of DMSO in PDA was kept at 0.1% (v/v).Each experiment was performed with three replicate plates, and the experiment was conducted twice.

Statistical analysis
Data were analyzed using GraphPad Prism 8.4.3 (GraphPad Software Inc., San Diego, CA, USA).Significance differences of treatments were analyzed by the one-way ANOVA analysis (α = 0.05).

FIG 3 (
FIG 3 (a and b) Volcano plots of differentially expressed genes in azoxystrobin-treated B05.10 compared to non-treated B05.10, with up-regulated genes represented by green dots and down-regulated genes by red dots.(c) Top 15 GO-terms identified in B05.10 exposed to azoxystrobin compared to non-exposed B05.10.(d) Top 15 GO-terms identified in resveratrol-treated B05.10 exposed to azoxystrobin compared to non-exposed resveratrol-treated B05.10.(e) Venn diagram of the number of common and specific differentially expressed genes.AB: group B (azoxystrobin-treated B05.10, 30th generation resveratrol-induced mutant) compared to group A (B05.10); AC: group C (30th generation resveratrol-induced B05.10) compared to group A; BD: group D (azoxystrobin-treated 30th generation resveratrol-induced B05.10) compared to group B; CD: group D compared to group C. (f ) Expression profiles of ABC transporter genes in groups A, B, C, and D. The expression is shown as the log2-fold change (Log 2 FC).

FIG 4
FIG 4 Heat map showing the expression of all ABC transporter genes and related transcription factors in four groups: A (B05.10), B (azoxystrobin-treated B05.10), C (the 30th generation of the B05.10 mutant induced by resveratrol), and D (azoxystrobin-treated B05.10 induced by resveratrol at the 30th generation).Each group has three replicates.

TABLE 1
Differential genes in the transcriptome of resveratrol-induced B05.10 and B05.10 strain a

Differential gene (up-regulation) Differential gene (down-regulation)
a AB: group B (azoxystrobin-treated B05.10 mutant ) compared to group A (B05.10); CD: group D (azoxystrobintreated B05.10 induced by resveratrol at the 30th generation) compared to group C (the 30th generation of resveratrol-induced B05.10); AC: group C compared to group A, BD: group D compared to group B.

TABLE 2
Concentrations of plant secondary metabolites (PSMs) amended in potato dextrose agar for growing Botrytis cinerea B05.10