Botrytis cinerea detoxifies the sesquiterpenoid phytoalexin rishitin through multiple metabolizing pathways.

Botrytis cinerea is a necrotrophic pathogen that infects across a broad range of plant hosts, including high-impact crop species. Its generalist necrotrophic behavior stems from its ability to detoxify structurally diverse phytoalexins. The current study aims to provide evidence of the ability of B. cinerea to tolerate the sesquiterpenoid phytoalexin rishitin, which is produced by potato and tomato. While the growth of potato pathogens Phytophthora infestans (late blight) and Alternaria solani (early blight) was severely inhibited by rishitin, B. cinerea was tolerant to rishitin. After incubation of rishitin with the mycelia of B. cinerea, it was metabolized to at least six oxidized forms. Structural analysis of these purified rishitin metabolites revealed a variety of oxidative metabolism including hydroxylation at C7 or C12, ketone formation at C5, and dihydroxylation at the 10,11-olefin. Six rishitin metabolites showed reduced toxicity to P. infestans and A. solani, indicating that B. cinerea has at least 5 distinct enzymatic reactions to detoxify rishitin. Four host-specialized phytopathogenic Botrytis species, namely B. elliptica, B. allii, B. squamosa, and B. tulipae also had at least a partial ability to metabolize rishitin as B. cinerea, but their metabolic capacity was significantly weaker than that of B. cinerea. These results suggest that the ability of B. cinerea to rapidly metabolize rishitin through multiple detoxification mechanisms could be critical for its pathogenicity in potato and tomato.

Botrytis cinerea tolerates phytoalexins produced by Solanaceae and Fabaceae plants through an efflux transporter BcatrB and metabolizing enzymes.

Botrytis cinerea, a plant pathogenic fungus with a wide host range, has reduced sensitivity to fungicides as well as phytoalexins, threatening cultivation of economically important fruits and vegetable crops worldwide. B. cinerea tolerates a wide array of phytoalexins, through efflux and/or enzymatic detoxification. Previously, we provided evidence that a distinctive set of genes were induced in B. cinerea when treated with different phytoalexins such as rishitin (produced by tomato and potato), capsidiol (tobacco and bell pepper) and resveratrol (grape and blueberry). In this study, we focused on the functional analyses of B. cinerea genes implicated in rishitin tolerance. LC/MS profiling revealed that B. cinerea can metabolize/detoxify rishitin into at least 4 oxidized forms. Heterologous expression of Bcin08g04910 and Bcin16g01490, two B. cinerea oxidoreductases upregulated by rishitin, in a plant symbiotic fungus Epichloë festucae revealed that these rishitin-induced enzymes are involved in the oxidation of rishitin. Expression of BcatrB, encoding an exporter of structurally unrelated phytoalexins and fungicides, was significantly upregulated by rishitin but not by capsidiol and was thus expected to be involved in the rishitin tolerance. Conidia of BcatrB KO (ΔbcatrB) showed enhanced sensitivity to rishitin, but not to capsidiol, despite their structural similarity. ΔbcatrB showed reduced virulence on tomato, but maintained full virulence on bell pepper, indicating that B. cinerea activates BcatrB by recognizing appropriate phytoalexins to utilize it in tolerance. Surveying 26 plant species across 13 families revealed that the BcatrB promoter is mainly activated during the infection of B. cinerea in plants belonging to the Solanaceae, Fabaceae and Brassicaceae. The BcatrB promoter was also activated by in vitro treatments of phytoalexins produced by members of these plant families, namely rishitin (Solanaceae), medicarpin and glyceollin (Fabaceae), as well as camalexin and brassinin (Brassicaceae). Consistently, ΔbcatrB showed reduced virulence on red clover, which produces medicarpin. These results suggest that B. cinerea distinguishes phytoalexins and induces differential expression of appropriate genes during the infection. Likewise, BcatrB plays a critical role in the strategy employed by B. cinerea to bypass the plant innate immune responses in a wide variety of important crops belonging to the Solanaceae, Brassicaceae and Fabaceae.

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

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 oxidized, 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 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 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.

Measuring Secretion of Capsidiol in Leaf Tissues of Nicotiana benthamiana.

Plant species produce a wide variety of antimicrobial metabolites to protect themselves against potential pathogens in natural environments. Phytoalexins are low molecular weight compounds produced by plants in response to attempted attacks of pathogens. Accumulation of phytoalexins in attacked plant tissues can inhibit the growth of penetrating pathogens. Thus phytoalexins play a major role in post-invasion defense against pathogens. Major phytoalexins produced by Solanaceous plants are sesquiterpenoids such as capsidiol produced by Nicotiana and Capsicum species, and rishitin produced by Solanum species, which are synthesized in the cytosol and secreted into the intercellular space of plant tissues. We previously reported that deficiency in capsidiol secretion causes enhanced susceptibility of Nicotiana benthamiana to potato late blight pathogen, Phytophthora infestans. Here, we describe a practical protocol to measure the secreted capsidiol in N. benthamiana.