Michel Boulay

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.

Somatic embryogenesis from grapevine cells. I-Improvement of embryo development by changes in culture conditions

In conventional culture conditions without auxin, somatic embryos arising from suspension cultures of grapevine rootstock 41B (Vitis vinifera cv. Chasselas x Vitis berlandieri) are arrested at the heart stage of development. Starting from indications that inhibitors excreted in the culture medium could be responsible for this arrest, new culture conditions based on daily subculturing embryos in fresh medium have been successfully used to obtain full embryo development. From this technique, a microassay was devised for screening small amounts of extracellular molecules as potential inhibitors of embryonic development. Our results show that extracellular macromolecules of molecular weight higher than 10 kDa are likely involved in the inhibition of caulinary meristem initiation. However, other factors obviously cooperate to inhibit embryo development in conventional culture conditions

Extracellular protein patterns of grapevine cell suspensions in embryogenic and non-embryogenic situations

Abstract Somatic embryos produced from suspension cultures of grapevine rootstock 41B (Vitis vinifera cv. Chasselas × Vitis berlandieri) are blocked at the heart stage of development. Previous results have shown that extracellular macromolecules of molecular weight > 10 kDa, are likely involved in this inhibition (P. Coutos-Thevenot et al., Plant Cell Tiss. Org. Cult., 23 (1992) 125–133). The extracellular proteins and glycoproteins secreted by embryos differentiating in the absence of auxin have been compared to those secreted by undifferentiated cells growing in the presence of auxin. Specific proteins and glycoproteins (66, 62, 56, 51, 48, 36, 32, 25 and 10 kDa), under negative control of auxin, are secreted during embryogenesis. Thus, somatic embryogenesis in grapevine presents several similarities with the carrot model system as far as the influence of auxin on extracellular protein patterns and embryo differentiation is considered. This conclusion has been reinforced by showing that, as described in the carrot system, 36-kDa glycoprotein excreted by grapevine embryos is a cationic peroxidase whereas the 10-kDa protein immunoreacts with an antibody directed against a maize lipid transfer protein. Study of extracellular protein patterns of a Chardonnay CH76 cell strain, strongly affected in its embryogenic response, showed significative differences in the excretion of some specific proteins compared to the 41B cell line.

Influence of extracellular proteins, proteases and protease inhibitors on grapevine somatic embryogenesis

An embryogenic grapevine rootstock cell suspension, continuously grown in the presence of auxin, was predominantly composed of proembryogenic masses. When transferred to an auxin-free medium, grapevine somatic embryos developed but were rapidly blocked at the heart stage. This inhibition has been related to the presence of extracellular macromolecules (Coutos-Thévenot et al., 1992a). In this study, the initial cell population density has been found to influence markedly embryo development. Inoculations below 5·103 cells per ml were required to obtain fully grown cotyledonary embryos. Interestingly, extracellular proteins of molecular weights of 32, 34, 48 and 52 kDa accumulated in cultures grown at high population cell densities and disappeared in cultures inoculated at densities below 5·103 cells per ml. Protein fractions partially purified by ion exchange chromatography caused both an early inhibition of embryogenesis and a stimulation of secondary embryogenesis. Moreover, to test for the possibility of modulating embryo development through alterations of extracellular proteins, cultures were supplemented with proteases and protease inhibitors. The addition of trypsin increased the rate of embryo development only in cultures inoculated at a low cell population density. Conversely, the protease inhibitor aprotinin inhibited development, arresting embryos at globular and heart stages. Together, these results provide evidence that extracellular proteins modulate somatic embryogenesis and suggest that an extracellular proteolitic mechanism could be implicated in development.

Phytoalexin production in grapevine protoplasts during isolation and culture

Abstract The production of stilbene phytoalexins was analyzed in Vitis sp. protoplasts during isolation and culture, by HPLC. The results showed that trans -resveratrol could be detected as early as the fourth hour of digestion and accumulated during the digestion process, while the healthy donor material was devoid. Resveratrol production was due to de novo activation of the Vst1 gene encoding for the stilbene synthase (EC 2.3.1.95) responsible for resveratrol formation. When in culture, leaf protoplasts, whatever the genotype, continued to produce large amounts of resveratrol within the first week of culture, whereas, during the second week, a decrease in resveratrol content was observed, in parallel to a decrease in protoplast viability. Other phytoalexins derived from the resveratrol were produced during the culture, mainly an inducible resveratrol dehydrodimer analogous to e-viniferin and, to a lesser extent, pterostilbene. In order to determine the inducer of phytoalexin production, effects of culture conditions were evaluated on resveratrol production and discussed.