A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity

Abstract

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H2O2 and O2 −, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H2O2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses.

Reactive oxygen species and plant resistance to fungal pathogens

“…DAB staining has been often used to visualise the generation of H 2 O 2 in planta (Asai et al, 2010;Dubreuil-Maurizi et al, 2010;Kobayashi et al, 2012;L'Haridon et al, 2011;Liao et al, 2012;Rojas et al, 2012;Simon et al, 2013;Torres et al, 2005;Yokawa et al, 2011;Zhang et al, 2012). The Amplex Red hydrogen peroxide/peroxidase activity assay also allows quantifying H 2 O 2 concentrations and consists in a non-fluorescent molecule that is oxidised by H 2 O 2 and becomes fluorescent in presence of peroxidase.…”

“…Yellow, water-soluble NBT is reduced by superoxide radicals to blue, water-insoluble formazan (Grosskinsky et al, 2012;Jabs et al, 1996;Kawarazaki et al, 2013;L'Haridon et al, 2011;Liao et al, 2012;Wang and Higgins, 2006;Xia et al, 2009). Superoxide anion radicals can also be detected using dihydroethidium (DHE), a cell-permeable blue fluorescent stain that forms red fluorescent oxyethidium upon oxidation and intercalates with nucleic acids (see recent applications in Lehotai et al, 2011;Mai et al, 2013;Pet} o et al, 2013).…”

“…They consist in non-fluorescent molecules that become fluorescent when oxidised by ROS, and the emitted fluorescence can be observed by fluorimetry and/or by fluorescent microscopy, an advantage of such probes (Benikhlef et al, 2013;Bulgakov et al, 2012;Fester and Hause, 2005;Guo et al, 2010;Kolla et al, 2007;L'Haridon et al, 2011;Li et al, 2007;Liu et al, 2010;Ma et al, 2013;Peleg-Grossman et al, 2012;Plancot et al, 2013;Tada et al, 2004;Wen et al, 2008;Ye et al, 2013). Luminol or luminol analogues are sensitive chemiluminescent probes used to quantify a relative intensity of ROS by counting the emitted light with a luminometer, CDD camera or a scintillation counter (Dubreuil-Maurizi et al, 2010;Flury et al, 2013;Kunz et al, 2006;L'Haridon et al, 2011;Mersmann et al, 2010).…”

The novel elicitor AsES triggers a defense response against Botrytis cinerea in Arabidopsis thaliana

“…Culture of B. cinerea, inoculation and staining of hyphae B. cinerea strain BMM was provided by Brigitte Mauch-Mani (University of Neuchatel, Switzerland). Growth, preparation of spore suspension and infection procedure were performed as previously described [28]. In all the experiments, mock-and AsEStreated plants were kept in trays covered with a transparent plastic dome to maintain high humidity and incubated under the same plant growth conditions.…”

Interferon-β lipofection II. Mechanisms involved in cell death and bystander effect induced by cationic lipid-mediated interferon-β gene transfer to human tumor cells

“…23 Briefly, a PBS solution of 1 Â 10 6 ml cells of control (untreated), bgal-and hIFNb-lipofected cells was incubated with 0.1 mM luminol final concentration and 1 mM H 2 O 2 . The chemoluminescence of oxidized luminol was evaluated immediately, every 20 s, 25 times.…”