In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves

Journal of Experimental Botany, Jun 2012

Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5–6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues.

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In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves

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All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of eKpeiydewromradls:ce5l-lMs.etHhiyglchyetorsflinueo-arnetsibcoednyc,ecaindtmeinusmit-ystrwesasscobndsietirovne,dchirnomgautainrdre-ceolnlfigwuarlalstiotnh, aCnHRinOaMnOyMoEtThHeYrLcAoSmE,partment dDuNeA-tmoetinhyclraetiaosne,dMelothcyalaltivoins-cSoesnitsyit.ivIenAamdpdlifitcioantiosntiPlboelynmeorflpuhoisrmes(cMeSnAcPe), wPoassidfoonuiandocienaneicpaid(eL.r)mDaellilec.ell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced Isntitlbreondeuscintihoonst tissues. - cinerea conidia (Langcake and Pryce, 1976; Langcake, 1981; Hoos and Blaich, 1990) or of P. viticola sporangiophores and to reduce the mobility of zoospores (Pezet et al., 1994). These in vitro studies were an ersatz to studies on mycelia because in vitro experiments are not possible on P. viticola mycelium, which is an obligatory biotroph. Susceptible grapevine species infected by P. viticola mainly produce trans-resveratrol (3,5,4-trihydroxystilbene) and trans- and cis-piceid (3-Ob-D-glucoside of resveratrol) (Pezet et al., 1994) whereas resistant species produce trans-resveratrol, trans-pterostilbene (3,5-dimethoxy-4-hydroxystilbene) and cyclic dehydrodimers of resveratrol trans-e-viniferin and trans-d-viniferin (Langcake, 1981; Dercks and Creasy, 1989; Pezet et al., 2003). This result is corroborated by the fact that distinctive toxicities to P. viticola zoospores were reported for these stilbenes (Pezet et al., 2004a, b). The highest concentrations of viniferins appear in resistant cultivars and they correlate with growth inhibition of Erysiphe necator (powdery mildew) (Schnee et al., 2008). Similarly, an inhibition of the growth of P. viticola was observed in vivo on resistant cultivars producing viniferins and/or pterostilbene (Alonso-Villaverde et al., 2011). All these results suggest that stilbenes are implicated in the resistance of grapevine to fungi. Yet, the effect of stilbenes on mycelia of the biotrophic agent P. viticola remains a matter of debate (Chong et al., 2009) and obviously needs more investigation in vivo. No physical, biochemical, or genetic evidence of the direct interaction between plant-induced stilbenes and mycelium in situ is currently available. Despite the lack of direct evidence, the production of stilbenes was reported to correlate well with resistance levels t (...truncated)


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Sébastien Bellow, Gwendal Latouche, Spencer C. Brown, Anne Poutaraud, Zoran G. Cerovic. In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves, Journal of Experimental Botany, 2012, pp. 3697-3707, 63/10, DOI: 10.1093/jxb/ers060