Biology subjects

Kuroyanagi, T.

Publications and source records attributed to Kuroyanagi, T..

4 recordsLinked to original sources

bioRxiv2026

Ups and downs of liquid-liquid transitions in GUV membranes from osmolarity and aspiration tensions

Lipid membranes undergo liquid-liquid phase separation to form micron-scale domains. Several physical parameters affect this phase transition. For example, a decrease in temperature causes membranes to demix, as does an increase in hydrostatic pressure. However, measurements to determine how tension affects membrane phase separation have yielded conflicting results. Experiments that have applied osmotic pressure differences to a population of vesicles have reported an increase in the membrane's miscibility transition temperature. Conversely, experiments that have applied micropipette aspiration or substrate stretching to single membranes have reported a decrease. Here, we find that both osmotic pressure and micropipette aspiration can increase transition temperatures. We discuss how membrane pores and hidden areas in membranes present challenges to researchers seeking to quantitatively convert experimentally measured osmolarity differences into membrane tensions. We show that challenges of comparing data from different osmotic pressure experiments can be mitigated by renormalizing osmolarity differences, specifically by dividing by the exterior osmolarity. We discuss our results in the context of existing theoretical predictions and in light of four known effects of increasing tension on vesicle membranes: 1) a reduction in hidden area of tubes and aggregates, 2) a reduction in out-of-plane thermal fluctuations, 3) an increase in the area per lipid, and 4) the formation of pores. First, hidden area can explain why vesicles can sustain high osmolarity differences. Next, suppression of thermal fluctuations and increases in the area per lipid may account for shifts in miscibility transition temperatures. Finally, pores can explain time dependences. Overall, our results highlight the need for new theory and simulation that unify predictions of how transition temperatures vary over all four regimes of membrane tension.

biophysics↗

bioRxiv2024

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.

microbiology↗

bioRxiv2022

Functional characterization of Botrytis cinerea ABC transporter gene BcatrB in response to phytoalexins produced in plants belonging to families Solanaceae, Brassicaceae and Fabaceae

Botrytis cinerea, a generalist fungal pathogen of economically important crop species, has been shown to exhibit reduced sensitivity to fungicides and plant toxins. Specifically, previous reports indicate B. cinereas efficacy in tolerating a wide array of phytoalexins, toxic plant metabolites that play key role in plant immune defense strategies. Previously, we have shown that a distinct set of genes was induced in B. cinerea when treated with phytoalexins derived from different plant species such as rishitin (tomato and potato), capsidiol (tobacco and bell pepper) or resveratrol (grape and blueberry). In this study, we focused on the functional analyses of B. cinerea genes induced by rishitin treatment. B. cinerea can metabolize rishitin to at least 4 oxidized forms. Heterologous expression of rishitin-induced B. cinerea genes in the plant symbiotic fungus, Epichloe festucae, revealed that oxidoreductase (Bcin08g04910) and cytochrome P450 (Bcin16g01490) genes are involved in the oxidation of rishitin. BcatrB is an exporter of structurally unrelated anti-microbial compounds such as resveratrol, camalexin and fungicide fenpicionil. Expression of BcatrB is upregulated by rishitin, but not by structurally resembling capsidiol. BcatrB knock out transformants ({Delta}bcatrB) showed enhanced sensitivity to rishitin, but not to capsidiol. Likewise,{Delta} bcatrB showed reduced virulence on tomato fruits (which produce rishitin), but showed full virulence on Nicotiana benthamiana (which mainly produces capsidiol), suggesting that B. cinerea distinguishes phytoalexins and activates expression of appropriate transporter genes during the infection. Activation of BcatrB promoter was detected using P_BcatrB:GFP transformant during the B. cinerea infection in plant tissues. Surveying of 26 plant species across 13 families revealed that the BcatrB promoter is mainly activated during the infection of plants belonging to the Solanaceae, Fabaceae and Brassicaceae families. The BcatrB promoter is activated by the treatment with Fabaceae phytoalexins medicarpin and glyceollin, and{Delta} bcatrB showed reduced virulence on red clover, which produces medicarpin. These results suggest that 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 families.

microbiology↗

bioRxiv2022

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 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, Epichloe festucae, resulted in an acquired tolerance of capsidiol and the ability to metabolize capsidiol to capsenone, while B. cinerea {Delta}bccpdh mutants became relatively sensitive to capsidiol. The{Delta} 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 not found in taxonomically distant Ascomycota fungi but not in related Leotiomycete 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 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 anti-microbial compounds, to activate appropriate mechanisms for enhanced virulence.

microbiology↗