Botrytis cinerea identifies host plants via the recognition of antifungal capsidiol to induce expression of a specific detoxification gene

Abstract

The gray mold pathogen Botrytis cinerea has a broad host range, causing disease in over 400 plant species, but it is not known how this pathogen evolved this polyxenous nature. B. cinerea can metabolize a wide range of phytoalexins, including the stilbenoid, resveratrol, 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 oxidized, while rishitin was directly oxidized to epoxy- or hydroxy-rishitins indicating that B. cinerea has separate mechanisms to detoxify structurally similar sesquiterpenoid phytoalexins. RNAseq 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 appropriate 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 tolerance of capsidiol and the ability to metabolize capsidiol to capsenone, while B. cinerea Δbccpdh mutants became relatively sensitive to capsidiol. The Δbccpdh 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 other Botrytis 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.Significance StatementB. 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, rishitin and capsidiol, to activate appropriate detoxification mechanisms. We identify Bccpdh, encoding a dehydrogenase 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 suggests 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 anti-microbial compounds, to activate appropriate mechanisms for enhanced virulence.

Functional characterization ofBotrytis cinereaABC transporter geneBcatrBin response to phytoalexins produced in plants belonging to families Solanaceae, Brassicaceae and Fabaceae

“…B. cinerea strain AI18 (Kuroyanagi et al, 2022), Epichloë festucae strain Fl1 and their transformants used in this study are listed in Supplementary Table S1. They were grown on potato dextrose agar (PDA) at 23°C.…”

“…For the detection of rishitin and their metabolites after the incubation with B. cinerea or E. festucae transformants, the supernatant (50 μl) was collected, mixed with 50 μl acetonitrile and measured by LC/MS (Accurate-Mass Q-TOF LC/MS 6520, Agilent Technologies, Santa Clara, CA, USA) with ODS column Cadenza CD-C18, 75 × 2 mm (Imtakt, Kyoto, Japan) as previously described (Kuroyanagi et al, 2022).…”

“…Extraction of RNA and RNA sequencing analysis were performed as previously described (Kuroyanagi et al, 2022). The nucleotides of each read with less than 13 quality value were masked and reads shorter than 50 bp in length were discarded, and filtered reads were mapped to annotated cDNA sequences for B. cinerea (ASM83294v1, GenBank accession GCA_000143535) using Bowtie software (Langmead et al, 2009).…”

“…Preparation of E. festucae and B. cinerea protoplasts and transformation was performed as previously reported (Kuroyanagi et al, 2022). Candidate colonies were exposed to BLB blacklight for the induction of sporulation and single spore isolation was performed to obtain purified strains.…”

“…Another significant hurdle confronting pathogens is that several of these plant chemicals act in unison and occur in such diversity to challenge such dexterous pathogens (Pedras and Abdoli, 2017; Allan et al, 2019; Newman et al, 2020; N’Guyen et al, 2021; Westrick et al, 2021). Several studies attribute this dexterity to detoxification of phytochemicals to multi-functional enzymes such as cytochrome P450 (oxidoreductase, monooxygenase and dehydratases) which are quintessential components of robust fungal pathogenic systems (Pedras and Ahiahonu, 2005; Kuroyanagi et al, 2022).…”