Colette Breda

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.

T-DNA tagging in the model legume Medicago truncatula allows efficient gene discovery

The annual legume Medicago truncatula has been proposed as a model plant to study various aspects of legume biology including rhizobial and mycorrhizal symbiosis because it is well suited for the genetic analysis of these processes . To facilitate the characterization of M. truncatula genes participating in various developmental processes we have initiated an insertion mutagenesis program in this plant using three different T-DNAs as tags. To investigate which type of vector is the most suitable for mutagenesis we compared the behavior of these T-DNAs. One T-DNA vector was a derivative of pBin19 and plant selection was based on kanamycin resistance. The two other vectors carried T-DNA conferring Basta resistance in the transgenic plants. For each T-DNA type, we determined the copy number in the transgenic lines, the structure of the T-DNA loci and the sequences of the integration sites. The T-DNA derived from pBin19 generated complex T-DNA insertion patterns. The two others generally gave single copy T-DNA inserts that could result in gene fusions for the pGKB5 T-DNA. Analysis of the T-DNA borders revealed that several M. truncatula genes were tagged in these transgenic lines and in vivo gus fusions were also obtained. These results demonstrate that T-DNA tagging can efficiently be used in M. truncatula for gene discovery.

Pathological and molecular characterizations of alfalfa interactions with compatible and incompatible bacteria, Xanthomonas campestris pv. alfalfae and Pseudomonas syringae pv. pisi

We report on the interactions of alfalfa with Xanthomonas campestris pv. alfalfae and Pseudomonas syringae pv. pisi. A hypersensitive response was observed when leaves were infiltrated with P. s. pv. pisi, which remained strictly limited to the injected zone. The compatible interaction with X. c. pv. alfalfae was characterized by water-soaking symptoms and the spreading of the bacterium into the leaf blade. Analyses of transcript accumulation were conducted with cDNAs encoding enzymes involved in phytoalexin synthesis: chalcone synthase (CHS), chalcone isomerase (CHI), and isoflavone reductase (IFR). In incompatible interactions the maximum accumulation of the CHS, CHI, and IFR transcripts was observed 6 hr postinfection. In the compatible interaction, the induction of these transcripts was delayed until 25-30 hr postinfection, and the level of their accumulation was considerably lower. Extending this molecular analysis to the root system showed that the reaction of roots during an incompatible interaction was quite comparable to that of leaves. To complete these analyses, expression of genes encoding pathogenesis-related (PR) proteins in leaves was also analyzed by polymerase chain reaction. High-level accumulation of a 0.8-kb transcript encoding a PR protein was observed 6 to 30 hr postinfection in the incompatible interaction.

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.

Nucleotide sequences of four pathogen-induced alfalfa peroxidase- encoding cDNAs

We constructed an alfalfa cDNA library from mRNA extracted from leaves after infection with Pseudomonas syringae (incompatible interaction). Screening with oligodeoxyribonucleotides designed from regions conserved in all known peroxidases allowed the isolation of four cDNAs (Msprx1A, 1B, 1C and 2). Sequence analysis revealed the presence of open reading frames of 351, 355, 358 and 323 amino acids, respectively, with the characteristic consensus sequences of plant peroxidases. Sequence comparison showed that the Msprx2 product is significantly different from the others and, particularly, lacks a C-terminal propeptide which might be required for vacuolar targeting.

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...

Measurement and detection of peroxidase

Abstract The purity and the concentration based on the RZ value and the molar extinction coefficient was determined for the cationic peroxidase isolated from peanut (Arachis hypogaea L.) suspension culture medium. The maintenance of a concentrated sample and, if necessary, suspension in slightly alakline buffer was found to be the best condition for storage. As little as 3 ng of peroxidase could be immuno-precipitated, subjected to electrophoresis and immunoprobed and still be detected.