Dispositif pour determiner ou etudier l'etat de stimulation des defenses naturelles de plantes ou parties de plantes

The present invention relates to a device for determining or studying the state of stimulation of the natural defences of plants or portions of plants, said plants advantageously belonging to the Rosaceae family. For this purpose, the corresponding device includes a means for determining, in a sample of plants or portions of plants, the level of expression of at least one target gene in each of the following groups (a) to (i): (a) PR-1, PR- 2, PR-4, PR-5, PR-8, PR-14, PR-15; (b) PAL, CHS, DFR, ANS, PPO; (c) HMGR, FPPS, Far; (d) CSL; (e) APOX, GST, POX; (f) CalS, Pect, CAD; (g) EDS1, WRKY; (h) LOX2, JAR; and (i) ACCO, EIN3. Said device preferably consists of a kit which contains a determination means in the form of pairs of primers for implementing a quantitative PCR technique

Same ammo, different weapons: Enzymatic extracts from two apple genotypes with contrasted susceptibilities to fire blight (Erwinia amylovora) differentially convert phloridzin and phloretin in vitro

The necrogenic bacterium Erwinia amylovora responsible for the fire blight disease causes cell death in apple tissues to enrich intercellular spaces with nutrients. Apple leaves contain large amounts of dihydrochalcones (DHCs), including phloridzin and its aglycone phloretin. Previous work showed an important decrease in the constitutive DHCs stock in infected leaves, probably caused by transformation reactions during the infection process. At least two flavonoid transformation pathways have been described so far: deglucosylation and oxidation. The aim of the present study was to determine whether DHCs are differentially converted in two apple genotypes displaying contrasted susceptibilities to the disease. Different analyses were performed: i) enzymatic activity assays in infected leaves, ii) identification/quantification of end-products obtained after in vitro enzymatic reactions with DHCs, iii) evaluation of the bactericidal activity of end-products. The results of the enzymatic assays showed that deglucosylation was dominant over oxidation in the susceptible genotype MM106 while the opposite was observed in the resistant genotype Evereste. These data were confirmed by LC–UV/Vis–MS analysis of in vitro reaction mixtures, especially because higher levels of o-quinoid oxidation products of phloretin were measured by using the enzymatic extracts of Evereste infected leaves. Their presence correlated well with a strong bactericidal activity of the reaction mixtures. Thus, our results suggest that a differential transformation of DHCs occur in apple genotypes with a potential involvement in the establishment of the susceptibility or the resistance to fire blight, through the release of glucose or of highly bactericidal compounds respectively

Histolocalization and physico-chemical characterization of dihydrochalcones: Insight into the role of apple major flavonoids

Flavonoids, like other metabolites synthesized via the phenylpropanoid pathway, possess a wide range of biological activities including functions in plant development and its interaction with the environment. Dihydrochalcones (mainly phloridzin, sieboldin, trilobatin, phloretin) represent the major flavonoid subgroup in apple green tissues. Although this class of phenolic compounds is found in very large amounts in some tissues (≈200 mg/g of leaf DW), their physiological significance remains unclear. In the present study, we highlight their tissue-specific localization in young growing shoots suggesting a specific role in important physiological processes, most notably in response to biotic stress. Indeed, dihydrochalcones could constitute a basal defense, in particular phloretin which exhibits a strong broad-range bactericidal and fungicidal activity. Our results also indicate that sieboldin forms complexes with iron with strong affinity, reinforcing its antioxidant properties and conferring to this dihydrochalcone a potential for iron seclusion and/or storage. The importance of localization and biochemical properties of dihydrochalcones are discussed in view of the apple tree defense strategy against both biotic and abiotic stresses

T3SS-dependent differential modulations of the jasmonic acid pathway in susceptible and resistant genotypes of Malus spp. challenged with Erwinia amylovora

Fire blight is a bacterial disease of Maloideae caused by Erwinia amylovora (Ea). This necrogenic enterobacterium uses a type III secretion system (T3SS) to inject type III effectors into the plant cells to cause disease on its susceptible hosts, including economically important crops like apple and pear. The expressions of marker genes of the salicylic acid (SA) and jasmonic acid (JA) defense regulation pathways were monitored by RT-qPCR in leaves of two apple genotypes, one susceptible and one resistant, challenged with a wild type strain, a T3SS-deficient strain or water. The transcriptional data taken together with hormone level measurements indicated that the SA pathway was similarly induced in both apple genotypes during infection by Ea. On the contrary, the data clearly showed a strong T3SS-dependent down-regulation of the JA pathway in leaves of the susceptible genotype but not in those of the resistant one. Accordingly, methyl-jasmonate treated susceptible plants displayed an increased resistance to Ea. Bacterial mutant analysis indicated that JA manipulation by Ea mainly relies on the type III effector DspA/E. Taken together, our data suggest that the T3SS-dependent down-regulation of the JA pathway is a critical step in the infection process of Malus spp. by Ea

Mutational analysis of a predicted double β-propeller domain of the DspA/E effector of Erwinia amylovora

The bacterium Erwinia amylovora causes fire blight, an invasive disease that threatens apple trees, pear trees and other plants of the Rosaceae family. Erwinia amylovora pathogenicity relies on a type III secretion system and on a single effector DspA/E. This effector belongs to the widespread AvrE family of effectors whose biological function is unknown. In this manuscript, we performed a bioinformatic analysis of DspA/E- and AvrE-related effectors. Motif search identified nuclear localization signals, peroxisome targeting signals, endoplasmic reticulum membrane retention signals and leucine zipper motifs, but none of these motifs were present in all the AvrE-related effectors analysed. Protein threading analysis, however, predicted a conserved double β-propeller domain in the N-terminal part of all the analysed effector sequences. We then performed a random pentapeptide mutagenesis of DspA/E, which led to the characterization of 13 new altered proteins with a five amino acids insertion. Eight harboured the insertion inside the predicted β-propeller domain and six of these eight insertions impaired DspA/E stability or function. Conversely, the two remaining insertions generated proteins that were functional and abundantly secreted in the supernatant suggesting that these two insertions stabilized the protein