Robert Esnault

Expression of grapevine chitinase genes in berries and leaves infected by fungal or bacterial pathogens

In order to study the involvement of chitinases in the interactions between grape berries and fungi, three genomic sequences encoding chitinases were isolated by PCR walking on Vitis vinifera DNA. Two of these belong to class I chitinases with a putative vacuolar (Vvchit1a) and extracellular (Vvchit1b) localization whilst the third sequence belongs to class III (VvchitIII). Transcripts of class III chitinases were shown to accumulate in unripe berries infected with Plasmopara viticola but not at later developmental stages of berries infected with Uncinula necator or Botrytis cinerea. In contrast, class I transcripts were never detected in grape berries. Specificity of chitinase expression in response to pathogens was investigated in leaves infected with B. cinerea or Pseudomonas syringae pv. pisi, a bacterium leading to an incompatible interaction. Expression of the Vvchit1a gene was induced in leaves upon both infections. In contrast, accumulation of class III transcripts was observed in response to P. syringae but not to B. cinerea infection, whereas Vvchit1b transcripts were detected in leaves only in the latter case. Our data indicate that, in V. vinifera, induction of chitinase genes depends on the infecting pathogen.

Molecular characterization of the incompatible interaction of Vitis vinifera leaves with Pseudomonas syringae pv. pisi: Expression of genes coding for stilbene synthase and class 10 PR protein

The interaction between Vitis vinifera and Pseudomonas syringae pv. pisi was examined at the pathological and molecular levels. Leaves infiltrated with the bacterial suspension developed necrotic regions which remained restricted to the infiltrated areas. In the infiltrated zone the number of bacteria decreased around 24h after inoculation whilst no bacteria could be isolated from the non-infiltrated zone. At the molecular level, two genes, stilbene synthase (SS) and a PR10 gene, encoding putative defense proteins, were analyzed. Expression of the SS gene, measured by the analysis of transcript accumulation, was shown to be highly induced and was followed by the accumulation of resveratrol (peaking at approximately 48h post-inoculation), considered as one of the major nobreak phytolaexins in the Vitis species. We report for the first time the isolation of a genomic clone (VvPR10-1) coding for a PR10 protein from this plant. The accumulation of the corresponding mRNA (0.8kb) was observed from 3 to 96h post-inoculation, peaking at 24–48h, and was followed by the accumulation (between 24 and, at least, 96h after inoculation) of the encoded polypeptide as detected by immuno-blotting. These results indicate that our experimental system based on an interaction of the non-host plant V. vinifera leaves with P. s. pv. pisi, has to be considered as an HR-like response and is well suited for the analysis of the defense reaction of this economically important species.

Role of the K-antigen subgroup of capsular polysaccharides in the early recognition process between Rhizobium meliloti and alfalfa leaves

We used a model system to investigate the induction of plant genes by bacterial surface compounds. We have infiltrated alfalfa leaves with wild-type Rhizobium meliloti strain Rm41 and mutant derivatives, which are deficient in the production of exopolysaccharides (EPS), capsular K-polysaccharides (KPS or K-Antigens), or both. We have shown that wild-type R. meliloti was able to induce transcript accumulation of genes encoding enzymes of the iso-flavonoid biosynthetic pathway: chalcone synthase (CHS), chalcone reductase (CHR), and isoflavone reductase (IFR). Kinetics of these transcript accumulations were biphasic, occurring 0.75 to 1.5 h and 6 to 30 h after treatment. The exoB derivative of Rm41 (strain AK631), which is unable to produce either EPS I or EPS II, was still able to induce very rapid (45-min posttreatment) transcript accumulation of CHS and CHR genes. These results suggested that EPS were not involved in this induction. We then tested three R. meliloti fix-23 mutants (strains PP699, PP711, an...

Differential expression of cDNA coding for chalcone reductase, a key enzyme of the 5-deoxyflavonoid pathway, under various stress conditions in Medicago sativa

Abstract In legumes, two classes of flavonoids (5-hydroxy- or 5-deoxyflavonoids) are produced and the key enzyme involved in the 5-deoxyflavonoid synthesis is chalcone reductase (CHR), co-acting with chalcone synthase (CHS). We have isolated seven clones encoding CHR from a cDNA library constructed with transcripts extracted from Medicago sativa ssp. sativa leaves infiltrated with a suspension of Pseudomonas syringae pv. pisi (incompatible interaction). Analysis of the 3′ UTR (untranslated region) as well as Southern blot hybridizations indicated that CHR is encoded by a small multigene family. Due to the pivotal roles played by CHS (involved in the synthesis of both 5-hydroxy- and 5-deoxyflavonoids) we compared the expression of CHR and CHS genes in various organs and under different physiological stresses. Both CHR and CHS were highly expressed in roots and nodules induced by Rhizobium meliloti, but not in leaves, while during flower development only CHS expression was observed. CHR was induced in various organs by UV-B treatment. After leaf wounding, CHR was induced at a higher level than CHS, whereas the kinetics and intensity of their expression during incompatible interaction with P. syringae pv. pisi were similar. Our results indicate that CHR and CHS expression are under the control of different developmental mechanisms.

Differential expression of two peanut peroxidase cDNA clones in peanut plants and cells in suspension culture in response to stress.

Peanut (Arachis hypogea L.) peroxidase gene expression was analyzed by measuring the accumulation of trancripts in cultured cells and various plant parts (leaf, stem, root) and upon their treatment with ethylene or wounding, respectively. Two transcripts (prxPNC1 and prxPNC2) corresponding to two peroxidase genes are expressed at higher levels in cultured cells as compared to various plant organs. Analysis of total poly(A)+ RNA with an oligonucleotide probe corresponding to a highly conserved region of peroxidase genes showed the expression of three peroxidase related sequences (1,000, 1,400 or 2,600 bp) in stem or leaf but barely detectable in roots. The prxPNC2 transcript transiently expressed at high levels in response to ethylene treatment of cells or wounding of leaves. This suggests that the corresponding gene(s) are expressed in response to stress.

Influence of the excision shock on the protein metabolism ofVicia faba L. meristematic root cells

UsingVicia faba root meristems we have shown that protein synthesis was dramatically changed after excision. The amino-acid incorporation dropped to 13% of the level in the unexcised control. This downshift was a direct consequence of the breakdown of polysomes which are converted into monosomes. In order to perform an analysis of the protein pattern by two-dimensional gel electrophoresis, endogenous proteolytic activity, which is high in broad bean root, had to be inhibited. Therefore, several protease inhibitors were tested and a very efficient inhibitor pool was obtained which could be used during the preparation of meristematic cell extracts. Protein-pattern analysis showed important differences between the unexcised control and excised apices. The number of proteins synthesized after excision droped from 250 in the control to 80, as a consequence of polysome breakdown. Futhermore, we present evidence that new and apparently specific proteins are synthesized in response to this excision shock.

Study on plant RNAases. Isolation and properties of several activities from Vicia faba root cells.

Vicia faba root cells contain several nucleolytic activities: phosphomonoesterase and phosphodiesterase (which however were not studied in details), one nuclease and four ribonucleases. These results were obtained by separating the extracted proteins into anionic and cationic species by chromatography on CM-cellulose at pH 5.5 and analysing each kind of proteins. Anionic species were subjected to chromatography on DEAE-cellulose which lead to isolation of one nuclease (A1) and two RNAases (A2, A3), the properties of which were studied. It was shown that the RNAases pH optima are near 6; A2 is more thermolabile than A3; both are endonucleases unable to attack double-stranded structure; studies with homopolymers, i.e. poly(A), poly(I), poly(C), poly(U), showed that their base specificities were analogous to that of already known plant RNAases. The cationic proteins, analysed with CM-cellulose, contain two RNAases (C1, C2). The pH optima were near 6 and 7, respectively; C1 is much more thermolabile than C2; both were endonucleases inactive on double-stranded structures. C1 and C2 hydrolysed poly(C) and poly(U) but not poly(A) and poly(U).

Gene Expression Is Not Systematically Linked to Phytoalexin Production During Alfalfa Leaf Interaction with Pathogenic Bacteria

During an incompatible interaction between alfalfa leaves and Pseudomonas syringae pv. pisi, flavonoids accumulated between 6 and 24 h, whereas they could not be detected during the first 96 h of a compatible interaction with Xanthomonas campestris pv. alfalfae. Three flavonoids accumulated which were identified as 4′,7-dihydroxyflavanone and 4′,7-dihydroxyflavone and 2′,4,4′-trihydroxychalcone. Surprisingly, the phytoalexin medicarpin was found only at a very low level. Analysis of both the infected and noninfected zones revealed that these flavonoids were detectable only in the infiltrated tissue. Northern hybridizations showed that transcripts encoding for chalcone synthase (CHS), chalcone reductase, chalcone isomerase, and isoflavone reductase (IFR) accumulated in both infiltrated and noninfiltrated zones. Measurements of the CHS and IFR activities in the infiltrated and noninfiltrated zones indicated that the levels of CHS activity were highly increased only in the infiltrated zones, whereas the leve...

Changes in the polyadenylated messenger RNA population during differentiation of Vicia faba root cells

Using cDNA probes, the polysomal polyadenylated RNA populations of Vicia faba meristematic, elongating, and mature root cells were analysed and compared, with respect to complexity, abundance distribution, and sequence representation. These cells express 12 300, 15 800, and 15 200 sequences, respectively, of an average size of 1300 nucleotides, distributed in three frequency classes. Transition from the meristematic to the elongating stage is coordinated with the disappearance of 25-30% of the abundant RNA species (2000 copies per cell) and with the appearance of new transcripts corresponding to 3000-3500 genes expressed at an intermediate level (50-60 copies per cell) and belonging to the rare class (about 4 copies per cell). These new transcripts represent 12.5% of the mass of the mRNA during the elongating stage and are quantitatively modulated during the transition to the mature stage. Thus 80 to 85% of the polysomal polyadenylated RNA species expressed in elongating or mature cells are common to the three cell types.

Conditions d'inactivation de ribonucléases liées aux RNA végétaux

Abstract It has been shown that undergraded RNA from root apex of broad beans can be obtained when RNA-bound acid and neutral RNAases are inhibited. Addition, to the homogenising medium, of 0.5% sodium dodecyl sulphate (SDS) (acid activity inhibitor) and 1·10 −2 M KCN (acid and neutral nucleases inhibitor prevented the [ 3 H]uridine labelled RNA degradation, affecting particularly heavy RNA.