PNAS Nexus, 2022, 1, 1–12

https://doi.org/10.1093/pnasnexus/pgac274
Research Report

Botrytis cinerea identifies host plants via the recognition

of antifungal capsidiol to induce expression of a specific
detoxification gene
Teruhiko Kuroyanagia , Abriel Salaria Bulasaga,b , Keita Fukushimaa , Akira Ashidaa , Takamasa Suzuki c
, Aiko Tanakaa ,
a a a a a,
Maurizio Camagna , Ikuo Sato , Sotaro Chiba , Makoto Ojika and Daigo Takemoto *
a
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
b
College of Arts and Sciences, University of the Philippines Los Baños, Los Baños, Laguna 4031, Philippines
c
College of Bioscience and Biotechnology, Chubu University, Kasugai, Aichi 478-8501, Japan
∗
To whom correspondence should be addressed: Email: [email protected]
Edited By: Karen E. Nelson

Abstract
The gray mold pathogen Botrytis cinerea has a broad host range, causing disease in >400 plant species, but it is not known how
this pathogen evolved this polyxenous nature. Botrytis cinerea can metabolize a wide range of phytoalexins, including the stilbenoid
resveratrol in grape, and the sesquiterpenoids capsidiol in tobacco and rishitin in potato and tomato. In this study, we analyzed the
metabolism of sesquiterpenoid phytoalexins by B. cinerea. Capsidiol was dehydrogenated to capsenone, which was then further oxi-
dized, while rishitin was directly oxidized to epoxy- or hydroxyrishitins, indicating that B. cinerea has separate mechanisms to detoxify
structurally similar sesquiterpenoid phytoalexins. RNA-seq analysis revealed that a distinct set of genes were induced in B. cinerea
when treated with capsidiol or rishitin, suggesting that B. cinerea can distinguish structurally similar phytoalexins to activate appro-
priate detoxification mechanisms. The gene most highly upregulated by capsidiol treatment encoded a dehydrogenase, designated
Bccpdh. Heterologous expression of Bccpdh in a capsidiol-sensitive plant symbiotic fungus, Epichloë festucae, resulted in an acquired tol-
erance of capsidiol and the ability to metabolize capsidiol to capsenone, while B. cinerea bccpdh mutants became relatively sensitive
to capsidiol. The bccpdh mutant showed reduced virulence on the capsidiol producing Nicotiana and Capsicum species but remained
fully pathogenic on potato and tomato. Homologs of Bccpdh are found in taxonomically distant Ascomycota fungi but not in related
Leotiomycetes species, suggesting that B. cinerea acquired the ancestral Bccpdh by horizontal gene transfer, thereby extending the
pathogenic host range of this polyxenous pathogen to capsidiol-producing plant species.

Keywords: capsidiol, detoxification, phytoalexin, polyxenous pathogen, Solanaceae plants

Significance Statement:
Botrytis cinerea can metabolize a wide range of phytoalexins; however, the extent to which phytoalexin detoxification contributes to
pathogenicity is largely unknown. In this study, we have shown that B. cinerea recognizes structurally resembling sesquiterpenoid
phytoalexins, capsidiol and rishitin, to activate appropriate detoxification mechanisms. We identify Bccpdh, encoding a dehydro-
genase for capsidiol detoxification, which is upregulated in B. cinerea exclusively during the infection of capsidiol producing plant
species, and is required to exert full virulence. Analysis of the Bccpdh locus implicates that the gene was acquired via horizontal
gene transfer. This work highlights that the polyxenous plant pathogen B. cinerea can distinguish its host plants by its antimicrobial
compounds, to activate appropriate mechanisms for enhanced virulence.

Introduction In plants belonging to the Solanaceae family, the major phy-

The antimicrobial secondary metabolites produced in plants toalexins are sesquiterpenoids, such as capsidiol in Nicotiana and
during the induction of resistance against pathogens are collec- Capsicum species and rishitin in Solanum species (2, 7, 8). In Nico-
tively termed phytoalexins (1, 2). Several hundred phytoalexins tiana sp., production of capsidiol is strictly controlled by regulat-
of diverse structures have been identified from a wide range of ing the gene expression for capsidiol biosynthesis, such as EAS
plant species, which include terpenoids, flavonoids, and indoles (5-epi-aristolochene synthase) and EAH (5-epi-aristolochene dihy-
(3, 4). Many of these phytoalexins are considered to exhibit their droxylase), encoding the enzymes dedicated to the production of
antimicrobial activity by targeting the cell wall or cell membrane capsidiol (9, 10). In N. benthamiana, expression of EAS and EAH
of pathogens (5, 6), but their mechanisms of action remain largely genes is hardly detected in healthy tissues, but their expression is
unknown. rapidly induced during infection by pathogens (11, 12). Produced

Competing Interest: The authors declare no competing interest.

Received: June 16, 2022. Accepted: December 14, 2022

C The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences. This is an Open Access article

distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits
unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
2 | PNAS Nexus, 2022, Vol. 1, No. 5

phytoalexins are secreted locally via ATP-binding cassette (ABC) not always correlate with their host range (Figs. S1–S3 and Supple-
transporters at the site of pathogen attack (13, 14). In N. benthami- mentary Notes 1 and 2). Botrytis cinerea and Sclerotinia sclerotiorum
ana, silencing of genes involved in capsidiol production and secre- are well known as pathogens with a wide host range and are ca-
tion significantly reduces the resistance to the oomycete pathogen pable of metabolizing both capsidiol and rishitin. In this study, B.
Phytophthora infestans, suggesting that the inhibition of pathogen cinerea was selected as the pathogen to further investigate the role
growth by capsidiol plays an important role in disease resistance of detoxification of phytoalexins in the virulence of this polyxe-
of capsidiol-producing plants (10, 12, 13). For plants, temporal and nous pathogen.
spatial control of phytoalexins is probably critical as the toxicity
of phytoalexins is often not specific to microorganisms but is also Metabolization of capsidiol and rishitin by B.
harmful to plant cells (13, 15, 16). For example, rishitin affects the cinerea
permeability of plant liposomal membranes and disrupts chloro- Metabolization of capsidiol and rishitin by B. cinerea was evaluated
plasts (17). Therefore, timely production and efficient transport by LC/MS. Under the experimental conditions of this study, cap-
of phytoalexins to the site of pathogen attack are important for sidiol was metabolized to capsenone by dehydrogenation within
plants to apply these double-edged weapons effectively. 12 h, and oxidized capsenone was detected at 24 h, whereas oxi-
To counter the production of antimicrobial substances in dized capsidiol was not detected (Fig. 1C and Fig. S4). In contrast,
plants, various phytopathogenic fungi can metabolize and detox- rishitin was directly oxidized within 6 h, and at least four differ-
ify phytoalexins (18). Although many studies have shown an ap- ent forms of oxidized rishitin were detected within 24 h (Fig. 1C
proximate correlation between the ability of strains to detox- and Fig. S5), indicating that despite the structural similarity of
ify phytoalexins and their virulence (19–21), the importance of these phytoalexins, B. cinerea detoxifies/metabolizes capsidiol and
phytoalexin detoxification for pathogen virulence is largely un- rishitin by different mechanisms (Fig. 1C).
proven for most plant–pathogen interactions. The best-studied
phytoalexin metabolizing enzyme is pisatin demethylase (PDA)
Unique sets of genes are upregulated in B. cinerea
of Nectria haematococca. Deletion of the PDA gene resulted in re-
treated with capsidiol, rishitin, or resveratrol
duced virulence of N. haematococca on pea, directly proving the
importance of PDA for the virulence of this pathogen (22). It has To identify B. cinerea genes upregulated during the detoxification
also been reported that sesquiterpenoid phytoalexins are metab- of sesquiterpenoid phytoalexins, RNA-seq analysis was performed
olized by pathogenic fungi. Capsidiol is metabolized to less toxic for mycelia of B. cinerea cultured in minimal media supplemented
capsenone via dehydration by pathogens such as the gray mold with capsidiol or rishitin. A stilbenoid phytoalexin, resveratrol,
pathogen Botrytis cinerea and Fusarium oxysporum (23). Gibberella produced in grape was also used for comparison. Mycelia of B.
pulicaris, the dry rot pathogen of potato tubers, oxidizes rishitin cinerea were grown in liquid CM media supplemented with ei-
to 13-hydroxyrishitin and 11,12-epoxyrishitin (24). However, the ther 500 μM rishitin, 500 μM resveratrol, or 100 μM capsidiol, as
enzymes involved in the detoxification of sesquiterpenoid phy- 500 μM capsidiol caused significant growth defects in B. cinerea
toalexins have not been identified to date, and their importance (Fig. 1A and Fig. S6). The mycelial tissue was then used to per-
for pathogenicity has not been demonstrated. form an RNA-seq analysis. Among 11,707 predicted B. cinerea genes
In this study, we first investigated some pathogens isolated (25), 25, 27, or 23 genes were significantly upregulated (log2 fold
from Solanaceae and non-Solanaceae plants on their toler- change > 2, P < 0.05) by the treatment with capsidiol, rishitin,
ance to capsidiol and rishitin, as well as their ability to detox- or resveratrol, respectively. Unexpectedly, distinctive sets of genes
ify/metabolize these phytoalexins. Among the pathogens that can were upregulated even between B. cinerea treated with capsid-
metabolize both capsidiol and rishitin, we chose B. cinerea for iol and rishitin, which resemble each other structurally (Fig. 2A
further analysis to investigate the importance of its ability to and Tables S1–S3), indicating that B. cinerea can either distinguish
detoxify capsidiol for the virulence on plant species that produce the structural difference of capsidiol and rishitin or the dam-
capsidiol. age caused by these sesquiterpenoid phytoalexins. For instance,
two genes Bcin08g00930 and Bcin12g01750 encoding hypotheti-
cal proteins containing a motif for dehydrogenases were specifi-
cally induced by capsidiol, while the treatment of rishitin signif-
Results icantly induced Bcin07g05430, encoding a cytochrome P450 gene
Metabolization of sesquiterpenoid phytoalexins (Fig. 2B and C). Expression of BcatrB encoding an ABC transporter
capsidiol and rishitin by fungal plant pathogens involved in the tolerance of B. cinerea against structurally un-
Four oomycetes and twelve fungal species were evaluated for related phytoalexins resveratrol and camalexin and fungicides
their ability to detoxify/metabolize sesquiterpenoid phytoalexins fenpiclonil and fludioxonil (26, 27) is upregulated by treatment
capsidiol and rishitin, produced by Solanaceae plant species. Four with rishitin and resveratrol, but not by capsidiol (Table S2). In
Phytophthora species isolated from Solanaceae host plants, includ- contrast, Bcin15g00040, encoding a major facilitator superfamily
ing potato late blight pathogen P. infestans, P. nicotiana isolated from (MFS)-type transporter, was upregulated specifically by capsidiol
tobacco, P. capsici isolated from green pepper, and P. cryptogea iso- (Fig. 2B). Interestingly, treatment of B. cinerea with phytoalexins
lated from nipplefruit (Solanum mammosum), are all sensitive to also activated genes predicted to be involved in pathogenicity to
capsidiol and rishitin. The amount of capsidiol and rishitin after plants. For example, capsidiol treatment induced expression of a
the incubation with these oomycete pathogens did not decrease, hydrophobin gene Bcin06g00510.1 (Bhp3), while rishitin treatment
indicating that they are neither capable of metabolizing nor toler- activated the expression of Bcin01g00080.1 (Bcboa8), encoding an
ant to capsidiol and rishitin (Fig. 1 and Fig. S1). Among 12 fungal enzyme for biosynthesis of a phytotoxin, botcinic acid (Fig. 2B and
plant pathogens (eight isolated from Solanaceae plants), 7 and 8 C). Given that capsidiol is metabolized to capsenone by a dehydro-
fungal strains can metabolize capsidiol and rishitin, respectively, genation reaction in B. cinerea (Fig. 1C and Fig. S4), the function of
and showed increased resistance to phytoalexins. In some cases, Bcin08g00930 and Bcin12g01750, both encoding a predicted short-
pathogens can metabolize phytoalexins that are not produced by chain dehydrogenase/reductase (SDR), was further analyzed in
their host, indicating that the ability to detoxify phytoalexins does this study.
 Kuroyanagi et al. | 3

Fig. 1. Sensitivities and metabolic capacities of B. cinerea and P. infestans to sesquiterpenoid phytoalexins. (A) Mycelial blocks (∼1 mm3 ) of indicated
pathogen were incubated in 50 μl water, 500 μM capsidiol, or 500 μM rishitin. Outgrowth of hyphae from the mycelial block (outlined by dotted red
lines) was measured after 24 h of incubation (n = 6). Bars = 100 μm. (B) Residual capsidiol and rishitin were quantified by GC/MS 2 d after the
incubation. (C) Predicted metabolism of capsidiol and rishitin by B. cinerea. Note that the structure of oxidized capsenone was determined in this study.
Data marked with asterisks are significantly different from control as assessed by the two-tailed Student’s t-test: ∗∗P < 0.01, ∗P < 0.05.

Bcin08g00930 encodes a short-chain hibited in the 100 μM capsidiol (Fig. 3A and B). Epichloë festu-
dehydrogenase for the detoxification of capsidiol cae transformants expressing Bcin12g01750 also hardly grew in
To investigate the function of SDR genes induced by capsidiol 100 μM capsidiol; however, E. festucae became tolerant to cap-
treatment, an endophytic fungus Epichloë festucae was employed sidiol by the expression of Bcin08g00930 (Fig. 3A and B). The
for the heterologous expression of these genes. Bcin08g00930 amount of capsidiol was not altered by wild type, DsRed or
and Bcin12g01750 were expressed in E. festucae under the con- Bcin12g01750 expressing E. festucae, while capsidiol was me-
trol of the constitutive TEF promoter (28). The growth of wild- tabolized to capsenone by the E. festucae transformant ex-
type and control E. festucae expressing DsRed was severely in- pressing Bcin08g00930 (Fig. 3C). These results indicated that
4 | PNAS Nexus, 2022, Vol. 1, No. 5

Fig. 2. Unique set of genes are upregulated in B. cinerea treated with capsidiol, rishitin, and resveratrol. (A) Venn diagram showing genes upregulated in
B. cinerea cultured in CM media containing 100 μM capsidiol, 500 μM rishitin, or 500 μM resveratrol for 24 h. The numbers of significantly upregulated
genes by phytoalexin treatment with log2 FC > 2 compared with control (CM without phytoalexin) and P-value < 0.05 are presented. (B–D) Expression
profiles of representative genes upregulated by the treatment with capsidiol (B), rishitin (C), or resveratrol (D). The gene expression (FPKM value) was
determined by RNA-seq analysis of B. cinerea cultured in CM media containing 100 μM capsidiol, 500 μM rishitin, or 500 μM resveratrol for 24 h. Data
are mean ± SE (n = 3). Asterisks indicate a significant difference from the control (CM) as assessed by two-tailed Student’s t-test, ∗∗P < 0.01, ∗P < 0.05.

Bcin08g00930 encodes a dehydrogenase that can detoxify cap- iol 3-acetate, we investigated whether BcCPDH could metabo-
sidiol; thus, the SDR encoded by Bcin08g00930 was desig- lize capsidiol 3-acetate to capsenone 3-acetate. However, capsid-
nated as BcCPDH standing for B. cinerea capsidiol dehydroge- iol 3-acetate was not metabolized by E. festucae expressing Bc-
nase. cpdh (Bcin08g00930) (Fig. S7A). Consistently, capsidiol 3-acetate
was directly oxidized by B. cinerea, and at least two different
Expression of bccpdh is specifically induced by forms of oxidized capsidiol 3-acetate, but not capsenone 3-
capsidiol acetate, were detected (Fig. S7B), indicating that capsidiol and
To further investigate the expression pattern of Bccpdh, B. capsidiol 3-acetate were metabolized in B. cinerea by distinctive
cinerea was transformed with a reporter construct for GFP ex- pathways.
pression under the control of the 1 kb proximal Bccpdh pro- Analysis of different lengths of Bccpdh promoters indicated
moter (P_Bccpdh:GFP). P_Bccpdh:GFP transformants showed no that 250 bp of promoter sequence is sufficient for specific ac-
obvious expression of GFP in water, but a significant in- tivation of the promoter by capsidiol treatment, and the cis-
crease of GFP fluorescence was detected in 500 μM capsidiol element required for capsidiol-specific expression was shown to
(Fig. 4A). The P_Bccpdh:GFP transformant was incubated with be within −250 and −200 bp upstream of the start codon (Figs.
other antimicrobial terpenoids produced by Solanaceae species, S8 and S9). By further analysis using B. cinerea transformant
including rishitin, debneyol (29), sclareol (30), and capsidiol P_Bccpdh:Luc using luciferase as a quantitative marker, it was
3-acetate (31). Among the terpenoids tested, treatment of cap- shown that Bccpdh promoter was activated within 2 h of capsid-
sidiol and capsidiol 3-acetate induced expression of GFP in the iol treatment, and the expression activity decreased as capsid-
P_Bccpdh:GFP transformant. This result further indicated that B. iol was metabolized (Fig. S10, Supplementary Notes 3), suggest-
cinerea specifically reacts to capsidiol and its derivative capsid- ing that the Bccpdh promoter is under strict control of capsidiol
iol 3-acetate. As the Bccpdh promoter was activated by capsid- recognition.
 Kuroyanagi et al. | 5

Fig. 3. Bcin08g00930 encodes a capsidiol detoxification enzyme, capsidiol dehydrogenase BcCPDH. (A) Mycelia of Epichloë festucae wild type (WT) or a
transformant expressing Bcin08g00930 were incubated in water or 100 μM capsidiol and outgrowth of mycelia was observed 7 d after inoculation. Bars
= 100 μm. (B) Hyphal outgrowth of E. festucae WT, and transformants expressing DsRed, Bcin08g00930 (BcCPDH), or Bcin12g01750 in water, and 100 or
500 μM capsidiol was measured after 24 h of incubation. Data are mean ± SE (n = 6). Asterisks indicate a significant difference from WT as assessed by
two-tailed Student’s t-test, ∗∗P < 0.01. (C) Mycelia of E. festucae WT or transformant expressing Bcin08g00930 (Bccpdh) were incubated in 100 μM
capsidiol or for 48 h. Capsidiol and capsenone were detected by LC/MS.

Expression of bccpdh is specifically induced including nine Solanaceae, six Brassicaceae, six Rosaceae, five
during the infection of capsidiol producing plant Fabaceae, and six Asteraceae plants, and development of disease
species symptoms was observed in all tested plant species. Among the
Leaves of N. benthamiana, which produce capsidiol as the major tested plants, expression of GFP under the control of Bccpdh pro-
phytoalexin (13), were inoculated with conidia of the B. cinerea moter was only detected during the infection in three Nicotiana
transformant P_Bccpdh:GFP. No GFP signal was detected in germi- and two Capsicum species (Fig. 4C, Fig. S12, and Table S4), all of
nating B. cinerea conidia grown on the surface of N. benthamiana which are reported to produce capsidiol (8, 32–34). qRT-PCR con-
leaves 2 h after the inoculation, but obvious GFP expression was firmed that the expression of Bccpdh is hardly detected in potato
detected in the appressoria at 8 h after the inoculation (Fig. 4B). leaves (Fig. S11A and Supplementary Notes 4). These results fur-
Growing hyphae in the leaf tissue of N. benthamiana showed GFP ther indicated that B. cinerea specifically recognizes capsidiol for
fluorescence, and the intensity of GFP fluorescence in hyphae was the induction of Bccpdh.
stronger near the edge of the lesion compared with that of hyphae
growing in the areas of dead tissue within the lesions (Fig. 4B), BcCPDH metabolizes and detoxifies capsidiol to
probably because capsidiol was detoxified in these areas, which capsenone in B. cinerea
are heavily infected with B. cinerea. Induction of Bccpdh during Botrytis cinerea Bccpdh KO mutant (bccpdh) was produced to in-
the early stage of infection (8 h) and reduced expression of Bc- vestigate the function of BcCPDH (Fig. S13). Mycelia of B. cinerea
cpdh in hyphae in the necrotic plant tissue were confirmed by wild type and bccpdh were incubated with capsidiol and the
qRT-PCR (Fig. S11A and Supplementary Notes 4). Accumulation of metabolites were detected by LC/MS. While capsidiol was metab-
capsenone was detected both in necrotic tissue and near the edge olized to capsenone in the wild type strain, most of the capsidiol
of the lesion and only trace amounts of capsidiol were detected in remained unmetabolized 2 d after incubation with the bccpdh
infected tissue (Fig. S11B), suggesting that B. cinerea immediately strain (Fig. 5A). Instead, oxidized capsidiol, which was not de-
metabolizes capsidiol produced by the host plant during the in- tectable in the wild type strain, was detected in bccpdh incu-
fection. bations (Fig. S14 and Supplementary Notes 5). While the growth
To examine the possibility whether polyxenous B. cinerea ac- of B. cinerea hyphae was not affected by the disruption of the Bc-
tivates Bccpdh for detoxification of other phytoalexin or antimi- cpdh gene in 100 μM capsidiol, growth of bccpdh was diminished
crobial compounds produced in other plant species, the B. cinerea compared with that of wild type B. cinerea at higher concentra-
P_Bccpdh:GFP transformant was inoculated on a wide variety of tions of capsidiol (Fig. 5B). Colony growth, conidial gemination,
plants. Over 50 plant species were used for the inoculation test and appressoria-mediated penetration were not significantly af-
6 | PNAS Nexus, 2022, Vol. 1, No. 5

Fig. 4. Specific activation of the B. cinerea Bccpdh promoter by capsidiol and its derivative. (A) Mycelia of B. cinerea transformant containing GFP gene
under the control of 1 kb Bccpdh promoter (P_Bccpdh:GFP) was incubated in CM media containing 500 μM of antimicrobial terpenoids. Bars = 50 μm. (B)
(left and middle) Leaves of N. benthamiana were inoculated with conidia of B. cinerea P_Bccpdh:GFP transformant. Expression of GFP in germinating
conidia on the leaf surface was observed by confocal laser microscopy 2 or 8 h after inoculation (hpi). CW, stained with calcofluor white. Arrowheads
indicate the appressoria of B. cinerea. Bars = 20 μm. (right) Leaves of N. benthamiana were inoculated with mycelia of B. cinerea P_Bccpdh:GFP
transformant and the edge of the lesion was observed by confocal laser microscopy 2 d after the inoculation (2 dpi). Bar = 100 μm. (C) Leaves of
indicated plant were inoculated with mycelia of B. cinerea P_Bccpdh:GFP transformant and the edge of the lesion was observed by confocal laser
microscopy 2 d after the inoculation. Bars = 100 μm.
 Kuroyanagi et al. | 7

Fig. 5. Botrytis cinerea BcCPDH is essential for the detoxification of capsidiol and virulence in the plant species producing capsidiol. (A) Mycelial block
(∼1 mm3 ) of B. cinerea wild type (WT) or Bccpdh KO mutant strain (bccpdh-52) was incubated in 100 μM capsidiol for 4 d. Capsenone and capsidiol was
detected by LC/MS. (B) Mycelial blocks of B. cinerea WT or bccpdh-52 were incubated in capsidiol and outgrowth of hyphae was measured after 36 h of
incubation. Data are mean ± SE (n = 10). Asterisks indicate a significant difference from WT as assessed by two-tailed Student’s t-test, ∗∗P < 0.01. (C)
To investigate the effect of bccpdh disruption on the ability of B. cinerea mycelia to expand lesions, leaves, tubers, or fruits of indicated plant were
inoculated with mycelial plugs (5 × 5 mm) of B. cinerea WT or bccpdh-52 and lesion size was measured between 4 and 7 d after inoculation (dpi).
Asterisks indicate a significant difference from WT as assessed by two-tailed Student’s t-test. ∗∗P < 0.01. Lines and crosses (×) in the columns indicate
the median and mean values, respectively.
8 | PNAS Nexus, 2022, Vol. 1, No. 5

fected by the disruption of bccpdh (Fig. S15). These results con- (another polyxenous pathogen belonging to Leotiomycetes) re-
firmed that BcCPDH is the enzyme responsible for the detoxifi- vealed that the ∼4.9 kb sequence surrounding Bccpdh is unique to
cation of capsidiol in B. cinerea. B. cinerea (Fig. 6). This unique sequence shows a lower GC content
compared to the surrounding region (Fig. S25), suggesting that Bc-
BcCPDH is required for the pathogenicity of B. cpdh might have been obtained via horizontal gene transfer (35). A
cinerea on plant species producing capsidiol blast search using BcCPDH as query sequence revealed that prob-
To investigate the effect of bccpdh disruption on the ability of B. able orthologs can be found only in some Pezizomycotina fungi
cinerea mycelia to expand lesions in host plants producing differ- belonging to Ascomycota (Fig. S23). Orthologs were found from a
ent phytoalexins, leaves, tubers, or fruits of several plant species taxonomically diverse range of fungal species, including animal
were inoculated with mycelial plugs of B. cinerea wild type and and insect pathogens, and there was no correlation between their
bccpdh. On plant species that produce capsidiol, such as N. ben- homology and taxonomic relationship, which might indicate mul-
thamiana, N. tabacum, and C. annuum, the development of dis- tiple horizontal gene transfer events of the cpdh gene in the diver-
ease symptoms by bccpdh was significantly reduced compared sification of Ascomycota fungi (Figs. S23 and S26–S28 and Sup-
with those caused by wild type B. cinerea (Fig. 5C). Consistently, plementary Notes 7). Among plant pathogenic fungi, Bccpdh ho-
capsenone was detected in N. benthamiana leaves inoculated with mologs were found in Fusarium species, all of which are pathogens
wild type, but not that with bccpdh (Fig. S16). In contrast, both that infect plants that do not produce capsidiol. This result is con-
wild type and bccpdh strains caused comparable symptoms in sistent with a previous report showing that some Fusarium species
potato, tomato, grape, and eggplant, which is consistent with the can metabolize capsidiol to capsenone (36).
finding that expression of Bccpdh is not induced during the infec-
tion in these plant species (Fig. 5C and Fig. S17). In the comple- CPDH activity is conserved among B. cinerea
mentation strain, the ability to metabolize capsidiol to capsenone strains
was restored, as was virulence against the capsidiol producing Although B. cinerea is a polyxenous fungus, it has been reported
plants (Fig. S18). These results indicated that BcCPDH is an en- that different strains of B. cinerea exhibit various degrees of vir-
zyme dedicated to virulence of B. cinerea on capsidiol-producing ulence on different host plants (37, 38). Hence, we investigated
plant species. whether BcCPDH is conserved among strains isolated from di-
verse plant species. A total of 24 B. cinerea strains isolated from
Epoxidation of capsidiol and capsenone is 14 different plant species were incubated with capsidiol and re-
mediated via Bcin16g01490 sultant capsenone was detected by GC/MS. All tested B. cinerea
The incubation of the bccpdh knockout strain in capsidiol re- strains can metabolize capsidiol to capsenone, indicating that Bc-
vealed the presence of a potential secondary capsidiol degrada- CPDH is highly conserved among B. cinerea strains even though
tion pathway, which resulted in the accumulation of oxidized cap- their (most recent) host was not a producer of capsidiol (Fig. 7
sidiol. Since we were unable to find any indication for such a path- and Table S5). Similarly, 17 strains of F. oxysporum isolated from
way in our RNA-seq results for 100 μM capsidiol treated incuba- 7 plant species were subjected to the analysis for CPDH activity.
tions, we extended our search to a preliminary RNA-seq analysis, Altogether 8 out of 17 F. oxysporum strains showed CPDH activity,
which used 500 μM capsidiol treatments. We identified the gene and the activity was not related to the natural host of the strains
Bcin16g01490 encoding a cytochrome P450, which was signifi- (Fig. 7 and Table S6). This result is consistent with the finding that
cantly upregulated under these elevated capsidiol concentrations Bccpdh orthologues can be found in 5 out of 14 available genomes
(Table S1 and Fig. S19A). Heterologous expression of Bcin16g01490 of F. oxysporum (Fig. S28). These results indicate that the cpdh gene
in E. festucae resulted in the conversion of capsidiol to oxidized is randomly distributed in F. oxysporum strains, whereas it is highly
capsidiol as detected during bccpdh strain incubations (Figs. S14 conserved among B. cinerea strains.
and S19B and Supplementary Notes 5). Moreover, the sequential
incubation of capsidiol with the Bccpdh expressing E. festucae trans-
formant followed by a Bcin16g01490 expressing transformant re- Discussion
produced the reactions detected in B. cinerea (Figs. S4 and S19B). To survive the exposure to microorganisms in the environment,
Structural analysis of oxidized capsenone indicated that the end different plant species have developed diverse resistance mecha-
product of these reactions is capsenone 11,12-epoxide (Fig. 1, Fig. nisms over the course of evolution. The structural variety of phy-
S19C and S20–S22, and Supplementary Notes 6). toalexins is a prime example of such diversity. Different plant
families produce phytoalexins with relatively similar basic struc-
Distribution of bccpdh homologues in the fungal tures, but their side-chain structures often differ from species to
kingdom species (4). These differences in structure between species may
The distribution of Bccpdh homologs in taxonomically related have contributed to the determination of host specificity between
fungal species (Ascomycota, Leotiomycetes) was investigated. A plants and pathogens, as in some cases a pathogen may have ac-
search for Bccpdh homologs in the genome sequences of six host- quired resistance to a particular phytoalexin, but an analogous
specialized phytopathogenic Botrytis species, such as B. tulipae substance is not overcome by the same resistance mechanism
(pathogen of tulip), B. hyacinthi (hyacinth), and B. paeoniae (peony), (39). However, plant pathogens with a broad host range such as B.
indicated that among Botrytis species, Bccpdh is a unique gene only cinerea must employ strategies to counter a multitude of diverse
found in B. cinerea (Fig. S23). Consistently, four Botrytis species, not phytoalexins. The prompt killing of plant cells, or the presence of
including B. cinerea, were incubated with capsidiol, but no CPDH efflux pumps of extremely low specificity may present effective
activity was detected for the tested strains (Fig. S24). Search in 11 strategies for such pathogens. Indeed, B. cinerea produces host-
draft genomes of Leotiomycete fungi also indicated the absence of nonspecific phytotoxins (such as botrydial and botcinic acid, 40,
Bccpdh homologs in these species. Comparison of the correspond- 41) upon infection, and activates transporters capable of efflux-
ing genome regions between Botrytis species and S. sclerotiorum ing diverse substances such as PDR-type ABC transporter BcatrB
 Kuroyanagi et al. | 9

Fig. 6. Comparison of the B. cinerea Bccpdh gene locus with corresponding genome region of other Botrytis species and S. sclerotiorum. Edge of conserved
region among Botrytis species and specific region for B. cinerea (red line) were indicated by red arrowheads.

Fig. 7. CPDH activity in B. cinerea and F. oxysporum strains isolated form a variety of plants. CPDH activity in B. cinerea and F. oxysporum strains isolated
form a variety of plants. Mycelial blocks (∼1 mm3 ) were incubated in 500 μM capsidiol for 2 d and capsidiol or capsenone were detected by GC/MS.
Each elution profile describes the capsidiol and capsenone content in the solution after incubation of the strain with capsidiol. Strains with a
substantial peak for capsidiol indicate the absence of CPDH activity.
10 | PNAS Nexus, 2022, Vol. 1, No. 5

and MFS transporter Bcmfs1 (26, 27, 42). More recently, it has been sitive to rishitin, but not to capsidiol. Moreover, bcatrB KO showed
shown that B. cinerea suppresses the immune response of different reduced virulence on tomato (producing rishitin) but can infect
plant species via gene silencing by delivering various small RNAs N. benthamiana (producing capsidiol) same as the wild type does
into host cells (43, 44). (46). BcatrB not being induced by capsidiol further strengthens the
In addition to effective infection mechanisms that generally fa- notion that B. cinerea relies on specific triggers, rather than on gen-
cilitate infection of a wide range of plants, this study revealed that eral, indirect cues to mount its defense against the various phy-
B. cinerea responds to chemical cues from the host plant to adapt toalexins. Contrary to this notion is, however, that BcatrB is also in-
its infection strategy to overcome specific plant resistance mech- duced by structurally unrelated phytoalexins as well as fungicide
anisms. Botrytis cinerea strictly distinguishes structurally similar treatments (26, 27, 46). It would therefore be reasonable to assume
phytoalexins, such as capsidiol and rishitin, and activates ap- that the perception of phytoalexins may rely on the integration of
propriate detoxification responses. Interestingly, treatment of B. multiple signals, and that specific signals may be attenuated by
cinerea with phytoalexins activated not only genes involved in additional unspecific triggers, such as cell damage. Using the re-
detoxification and efflux of toxic compounds, but also genes pre- porter system developed in this study, mutant strains defective in
dicted to be involved in pathogenicity to plants. These results sup- capsidiol response could be isolated to elucidate the mechanism
port the notion that B. cinerea utilizes phytoalexins as a means to by which B. cinerea distinctly identifies phytoalexins.
identify a given host and adjust the method of infection.
Expression of the Bccpdh gene is activated specifically during in-
fection of capsidiol-producing plants, and the pathogenicity of the Does B. cinerea possess other capsidiol resistance
bccpdh mutant strains is compromised on plants producing cap- mechanism besides detoxification by BcCPDH?
sidiol, but does not suffer any disadvantages on plants that do not Although the bccpdh knockout mutants showed reduced viru-
produce capsidiol. These results suggest that BcCPDH is a critical lence on plant species producing capsidiol, the mutant can de-
component that specifically enables B. cinerea to infect capsidiol- velop the disease symptoms on these plants and showed tol-
producing plants. Despite capsidiol-producing plants represent- erance to 100 μM capsidiol, same as the wild type. In contrast,
ing only a small fraction of >400 host plants of B. cinerea, CPDH Phytophthora spp., Alternaria spp., and E. festucae are sensitive to
activity was maintained in all investigated B. cinerea strains iso- the same concentration of capsidiol. Although we also isolated
lated from plants that do not produce capsidiol. This may hint at Bcin16g01490, which we found to oxidize capsidiol, its activity
the presence of a selection pressure against the loss of CPDH, de- is fairly limited, indicating that it is unlikely to confer suffi-
spite it only affecting a limited number of host plants. This poses cient protection against capsidiol. We therefore presumed that B.
the question of whether B. cinerea is able to maintain acquired re- cinerea has another mechanism for capsidiol tolerance other than
sistances for a prolonged time, which may explain how it evolved detoxification. RNA-seq analysis of B. cinerea genes upregulated
and established itself as the polyxenous pathogen that it is. by capsidiol treatment identified two genes (Bcin15g00040.1 and
Bcin14g02870.1/Bcmfs1) encoding MFS transporters and a gene
(Bcin01g05890.1/Bcbmr1) encoding an ABC transporter (Table S1).
How does B. cinerea recognize phytoalexins with Bcmfs1 has been reported to be involved in the resistance of B.
similar structures? cinerea to structurally different natural toxicants (camptothecin
Although B. cinerea is known to have the ability to metabolize a produced by the plant Camptotheca acuminata and cercosporin pro-
wide variety of phytoalexins (18), only a small portion of these duced by the plant pathogenic fungus Cercospora kikuchii) and
detoxification mechanisms are required for the infection of any fungicides (sterol demethylation inhibitors) (42). The bcbmr1 mu-
particular plant. Thus, B. cinerea needs to strictly control those tants showed an increased sensitivity to fungicides, polyoxin (a
various detoxification mechanisms, since maintaining sufficient chitin synthetase inhibitor) and iprobenfos (a choline biosynthe-
levels of all these detoxification mechanisms represents a consid- sis inhibitor) (47). The expression of one or more of these trans-
erable waste of resources. Elucidating the mechanism by which porters may be involved in capsidiol efflux from B. cinerea cells,
B. cinerea recognizes phytoalexins and activates a specific set of and regulated by signaling that is common to Bccpdh.
genes is one of the important subjects for further research. Two
major scenarios can be envisioned: first, B. cinerea may possess re-
ceptors that can identify the chemical structures of phytoalexins. Why and how can B. cinerea strains stably
In plants, various terpenes with diverse structures are employed maintain BcCPDH?
as hormones recognized by specific receptors, such as gibberellins, Phylogenetic analysis of CPDH orthologs indicates that sequence
cytokinins, and abscisic acid (39). However, the possibility that B. similarity does not necessarily correlate with the taxonomic re-
cinerea maintains receptors to perceive all of the myriad of phy- lationship. Rather, CPDH orthologs of the same cluster type tend
toalexins may not be realistic. The second scenario could be that to form a clade in the phylogenetic tree, which suggest CPDH or-
B. cinerea recognizes the damage caused by phytoalexins. However, thologs and surrounding genes were transferred via multiple hor-
as for capsidiol and rishitin, both are presumed to have cell mem- izontal gene transfer events (Supplementary Note 7). While no
branes as their targets, and these phytoalexins are relatively un- CPDH ortholog was found in other Botrytis species, the Bccpdh gene
specific toxicants that cause damage even to plant cells (5, 13, is conserved in the genomes of published B. cinerea strains, and
17, 45), so it is not certain whether there exists a specific target CPDH activity was detected in all tested B. cinerea strains isolated
by which these two compounds can be distinguished. Immediate from plants that do not produce capsidiol. In F. oxysporum, in con-
downregulation of the bccpdh promoter after the metabolization trast, there were strains with and without CPDH activity regard-
of capsidiol (Fig. S10) also suggests that it is unlikely that bccpdh less of the host plant, and consistently, some publicly available
expression is primarily controlled by cell damage, since the dam- F. oxysporum genomes contain cpdh homologs and others do not.
age signals would remain present even after capsidiol was detox- As shown in this study, metabolic capacity (and tolerance) to cap-
ified. Recently, we have reported that KO transformants of BcatrB sidiol and rishitin can be detected in some fungal strains regard-
(which is activated by rishitin but not by capsidiol) became sen- less of the natural host of these pathogens, and perhaps unused
 Kuroyanagi et al. | 11

detoxification activities are maintained in the population of phy- Authors’ Contributions

topathogenic filamentous fungi for future use.
T.K., A.S.B., K.F., I.S., S.C., M.O., and D.T. designed research; T.K.,
It is theoretically implausible that all B. cinerea strains maintain
A.S.B., K.F., A.A., T.S., M.O., and D.T. performed research; T.K., K.F.,
an enzyme required only upon infection of capsidiol-producing
T.S., A.T., M.C., M.O., and D.T. analyzed data; T.S., M.C., M.O., and
plants, even if the said gene is completely repressed in its ex-
D.T. contributed new reagents/analytic tools; and M.C. and D.T.
pression under normal circumstances. Given that B. cinerea has an
wrote the paper.
extremely wide host range, some strains will go for long periods
where capsidiol detoxification ability is not a selection pressure.
One possibility is that BcCPDH has functions other than capsidiol
Preprints
degradation. For example, BcCPDH might be involved in the detox-
ification of antimicrobial substances produced by insects, because This manuscript was posted on a preprint: https://doi.org/10.110
it has been reported that spores of B. cinerea are transmitted be- 1/2022.05.11.490027.
tween plants via insects such as thrips (48). Since homologous
genes for Bccpdh are found in several insect-infecting fungi, it is
expected that CPDH-like enzymes in these species can metabolize
Data Availability
insect-derived substances. We, therefore, performed preliminary RNA-seq data reported in this work are available in GenBank un-
experiments to determine whether Bccpdh expression can be in- der the accession number DRA013980.
duced by inoculation of B. cinerea P_Bccpdh:GFP transformant with
several insects, but to date, no induction of GFP expression has
been detected. Alternatively, B. cinerea may have acquired the trait References
of having many hosts because of its ability to maintain rarely used 1. Müller KO, Börger H. 1940. Experimentelle untersuchungen über
virulence factors. Future clarification of the phytoalexin recogni- die Phytophthora: resistenz der kartoffel. Arb Biol Anst Reich-
tion mechanisms and comparative analysis of the genome with sanst. 23:189–231.
that of closely related Botrytis species are anticipated to elucidate 2. Kuc J. 1995. Phytoalexins, stress metabolism, and disease resis-
unknown features of B. cinerea that have led to its evolution as a tance in plants. Annu Rev Phytopathol. 33:275–297.
polyxenous fungi. 3. Brooks CJ, Watson DG. 1985. Phytoalexins. Nat Prod Rep. 2:427–
459.
4. Hammerschmidt R. 1999. Phytoalexins: what have we learned
after 60 years? Annu Rev Phytopathol. 37:285–306.
Materials and methods 5. Turelli M, Coulomb C, Coulomb PJ, Roggero JP, Bounias M. 1984.
Comprehensive descriptions of the materials and methods used Effects of capsidiol on the lipid and protein content of isolated
in this study, including biological materials (Tables S7–S9 and membranes of Phytophthora capsici. Physiol Plant Pathol. 24:211–
S13), vector construction (Table S10), primer sequences (Tables 221.
S11 and S12), transformation of E. festucae and B. cinerea, confo- 6. Rogers EE, Glazebrook J, Ausubel FM. 1996. Mode of action of
cal microscopy, pathogenicity tests, and detection and structural the Arabidopsis thaliana phytoalexin camalexin and its role in
analysis of phytoalexins and their derivatives, are available in sup- Arabidopsis–pathogen interactions. Mol Plant Microbe Interact.
plementary material. 9:748–757.
7. Katsui N, et al. 1968. The structure of rishitin, a new antifungal
compound from diseased potato tubers. Chem Commun. 1:43–
44.
Acknowledgments 8. Bailey JA, Burden RS, Vincent GG. 1975. Capsidiol: an antifun-
We thank Emeritus Prof. Barry Scott (Massey University, New gal compound produced in Nicotiana tabacum and Nicotiana cleve-
Zealand) for providing E. festucae strain Fl1 and critical reading landii following infection with tobacco necrosis virus. Phyto-
of the manuscript. We also thank Emeritus Prof. David Jones (The chemistry. 14:597.
Australian National University, Australia) for N. benthamiana seeds, 9. Vögeli U, Chappell J. 1988. Induction of sesquiterpene cyclase
Ms. Kayo Shirai (Hokkaido Central Agricultural Experiment Sta- and suppression of squalene synthetase activities in plant cell
tion, Japan) and Dr. Seishi Akino (Hokkaido University, Japan) for cultures treated with fungal elicitor. Plant Physiol. 88:1291–1296.
providing P. infestans isolate 08YD1, Prof. Takashi Tsuge (Chubu 10. Shibata Y, Kawakita K, Takemoto D. 2010. Age-related resis-
University, Japan) for providing Fusarium strains, Dr. Haruhisa tance of Nicotiana benthamiana against hemibiotrophic pathogen
Suga for providing F. graminearum strain, Mr. Masashi Matsusaki Phytophthora infestans requires both ethylene- and salicylic
(Aichi Prefectural Agricultural Research Center, Japan) for provid- acid-mediated signaling pathways. Mol Plant Microbe Interact.
ing F. oxysporum f. sp. lycopersici strain, and Mr. Taku Kawakami 23:1130–1142.
(Mie Prefecture Agricultural Research Institute, Japan) for pro- 11. Rin S, et al. 2020. Expression profiles of genes for enzymes in-
viding B. cinerea strains. We are also grateful to Prof. Kazuhito volved in capsidiol production in Nicotiana benthamiana. J Gen
Kawakita for valuable suggestions, and Dr. Kenji Asano and Mr. Plant Pathol. 86:340–349.
Seiji Tamiya (National Agricultural Research Center for Hokkaido 12. Imano S, et al. 2021. AP2/ERF transcription factor NbERF-IX-33 is
Region, Japan) and Mr. Yasuki Tahara (Nagoya University, Japan) involved in the regulation of phytoalexin production for the re-
for providing tubers of potato cultivars. sistance of Nicotiana benthamiana to Phytophthora infestans. Front
Plant Sci. 12:821574.
13. Shibata Y, et al. 2016. The full-size ABCG transporters Nb-
ABCG1 and Nb-ABCG2 function in pre- and postinvasion defense
Supplementary Material against Phytophthora infestans in Nicotiana benthamiana. Plant Cell.
Supplementary material is available at PNAS Nexus online. 28:1163–1181.
12 | PNAS Nexus, 2022, Vol. 1, No. 5

14. Pierman B, et al. 2017. Activity of the purified plant ABC trans- 32. Molot P-M, Mas P, Conus M, Ferrière H, Ricci P. 1981. Relations
porter NtPDR1 is stimulated by diterpenes and sesquiterpenes between capsidiol concentration, speed of fungal invasion and
involved in constitutive and induced defenses. J Biol Chem. level of induced resistance in cultivars of pepper (Capsicum an-
292:19491–19502. nuum) susceptible or resistant to Phytophthora capsici. Physiol
15. Shiraishi T, Oku H, Isono M, Ouchi S. 1975. The injurious effect Plant Pathol. 18:379–389.
of pisatin on the plasma membrane of pea. Plant Cell Physiol. 33. Bohlmann J, et al. 2002. Gene expression of 5-epi-aristolochene
16:939–942. synthase and formation of capsidiol in roots of Nicotiana attenu-
16. Smith DA. 1982. Toxicity of phytoalexins. In: Phytoalexins JAB, ata and N. sylvestris. Phytochemistry. 60:109–116.
Mansfield JW, editors. Phytoalexins. Glasgow: Blackie. 218–252. 34. Matsukawa M, et al. 2013. Nicotiana benthamiana calreticulin 3a is
17. Lyon GD. 1980. Evidence that the toxic effect of rishitin may be required for the ethylene-mediated production of phytoalexins
due to membrane damage. J Exp Bot. 31:957–966. and disease resistance against oomycete pathogen Phytophthora
18. Pedras MSC, Ahiahonu PWK. 2005. Metabolism and detoxifica- infestans. Mol Plant Microbe Interact. 26:880–892.
tion of phytoalexins and analogs by phytopathogenic fungi. Phy- 35. Ravenhall M, Škunca N, Lassalle F, Dessimoz C. 2015. Inferring
tochemistry. 66:391–411. horizontal gene transfer. PLoS Comput Biol. 11:e1004095.
19. Sbaghi M, Jeandet P, Bessis R, Leroux P. 1996. Degradation of 36. Stoessl A, Unwin CH, Ward EWB. 1973. Postinfectional fungus
stilbene-type phytoalexins in relation to the pathogenicity of inhibitors from plants: fungal oxidation of capsidiol in pepper
Botrytis cinerea to grapevines. Plant Pathol. 45:139–144. fruit. Phytopathology. 63:1225–1231.
20. Suleman P, Tohamy AM, Saleh AA, Madkour MA, Straney DC. 37. Mercier A, et al. 2019. The polyphagous plant pathogenic fun-
1996. Variation in sensitivity to tomatine and rishitin among gus Botrytis cinerea encompasses host-specialized and generalist
isolates of Fusarium oxysporum f. sp. lycopersici, and strains not populations. Environ Microbiol. 21:4808–4821.
pathogenic on tomato. Physiol Mol Plant Pathol. 48:131–144. 38. Plesken C, et al. 2021. Genetic diversity of Botrytis cinerea revealed
21. Weltring KM, Loser K, Weimer J. 1998. Genetic instability of by multilocus sequencing, and identification of B. cinerea popu-
rishitin metabolism and tolerance and virulence on potato tu- lations showing genetic isolation and distinct host adaptation.
bers of a strain of Gibberella pulicaris. J Phytopathol. 146:393–398. Front Plant Sci. 12:663027.
22. Wasmann CC, VanEtten HD. 1996. Transformation-mediated 39. Pichersky E, Raguso RA. 2018. Why do plants produce so many
chromosome loss and disruption of a gene for pisatin demethy- terpenoid compounds? New Phytol. 220:692–702.
lase decrease the virulence of Nectria haematococca on pea. Mol 40. Deighton N, Muckenschnabel I, Colmenares AJ, Collado IG,
Plant Microbe Interact. 9:793–803. Williamson B. 2001. Botrydial is produced in plant tissues in-
23. Ward EWB, Stoessl A. 1972. Postinfectional inhibitors from fected by Botrytis cinerea. Phytochemistry. 57:689–692.
plants. III. Detoxification of capsidiol, an antifungal compound 41. Reino JL, Hernández-Galán R, Durán-Patrón R, Collado IG. 2004.
from peppers. Phytopathology. 62:1186. Virulence–toxin production relationship in isolates of the plant
24. Gardner HW, Desjardins AE, McCormick SP, Weisleder D. 1994. pathogenic fungus Botrytis cinerea. J Phytopathol. 152:563–566.
Detoxification of the potato phytoalexin rishitin by Gibberella 42. Hayashi K, jan Schoonbeek H, de Waard MA. 2002. Bcmfs1, a
pulicaris. Phytochemistry. 37:1001–1005. novel major facilitator superfamily transporter from Botrytis
25. van Kan JAL, et al. 2017. A gapless genome sequence of the fun- cinerea, provides tolerance towards the natural toxic compounds
gus Botrytis cinerea. Mol Plant Pathol. 18:75–89. camptothecin and cercosporin and towards fungicides. Appl En-
26. Schoonbeek H, del Sorbo G, de Waard MA. 2001. The ABC trans- viron Microbiol. 68:4996–5004.
porter BcatrB affects the sensitivity of Botrytis cinerea to the phy- 43. Baulcombe D. 2013. Small RNA—the secret of noble rot. Science.
toalexin resveratrol and the fungicide fenpiclonil. Mol Plant Mi- 342:45–46.
crobe Interact. 14:562–571. 44. Wang M, Weiberg A, Dellota E, Yamane D, Jin H. 2017. Botrytis
27. Vermeulen T, Schoonbeek H, de Waard MA. 2001. The ABC trans- small RNA Bc-siR37 suppresses plant defense genes by cross-
porter BcatrB from Botrytis cinerea is a determinant of the activ- kingdom RNAi. RNA Biol. 14:421.
ity of the phenylpyrrole fungicide fludioxonil. Pest Manage Sci. 45. Camagna M, Ojika M, Takemoto D. 2020. Detoxification of
57:393–402. the solanaceous phytoalexins rishitin, lubimin, oxylubimin and
28. vanden Wymelenberg AJ, Cullen D, Spear RN, Schoenike B, An- solavetivone via a cytochrome P450 oxygenase. Plant Signal Be-
drews JH. 1997. Expression of green fluorescent protein in Aure- hav. 15:1707348.
obasidium pullulans and quantification of the fungus on leaf sur- 46. Bulasag AS, et al. 2022. Functional characterization of the ABC
faces. BioTechniques. 23:686–690. transporter gene BcatrB in Botrytis cinerea in response to phy-
29. Burden RS, et al. 1985. Debneyol, a fungicidal sesquiterpene from toalexin produced in Solanaceae, Brassicaceae and Fabaceae
TNV infected Nicotiana debneyi. Phytochemistry. 24:2191–2194. plants. bioRxiv 515369. https://doi.org/10.1101/2022.11.07.51536
30. Guo Z, Wagner GJ. 1995. Biosynthesis of labdenediol and sclareol 9, preprint: not peer reviewed.
in cell-free extracts from trichomes of Nicotiana glutinosa. Planta. 47. Nakajima M, Suzuki J, Hosaka T, Hibi T, Akutsu K. 2001. Func-
197:627–632. tional analysis of an ATP-binding cassette transporter gene in
31. Uegaki R, Kubo S, Fujimori T. 1988. Stress compounds in the Botrytis cinerea by gene disruption. J Gen Plant Pathol. 67:212–214.
leaves of Nicotiana undulata induced by TMV inoculation. Phy- 48. Fermaud M, Gaunt RE. 1995. Thrips obscuratus as a potential
tochemistry. 27:365–368. vector of Botrytis cinerea in kiwifruit. Mycol Res. 99:267–273.