Michel Petitprez

Characterization of the Interaction Between the Bacterial Wilt Pathogen Ralstonia solanacearum and the Model Legume Plant Medicago truncatula

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt and attacks more than 200 plant species, including some legumes and the model legume plant Medicago truncatula. We have demonstrated that M. truncatula accessions Jemalong A17 and F83005.5 are susceptible to R. solanacearum and, by screening 28 R. solanacearum strains on the two M. truncatula lines, differential interactions were identified. R. solanacearum GMI1000 infected Jemalong A17 line, and disease symptoms were dependent upon functional hrp genes. An in vitro root inoculation method was employed to demonstrate that R. solanacearum colonized M. truncatula via the xylem and intercellular spaces. R. solanacearum multiplication was restricted by a factor greater than 1 x 10(5) in the resistant line F83005.5 compared with susceptible Jemalong A17. Genetic analysis of recombinant inbred lines from a cross between Jemalong A17 and F83005.5 revealed the presence of major quantitative trait loci for bacterial wilt resistance located on chromosome 5. The results indicate that the root pathosystem for M. truncatula will provide useful traits for molecular analyses of disease and resistance in this model plant species.

Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus × domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.

RGD-MEDIATED MEMBRANE-MATRIX ADHESION TRIGGERS AGAROSE-INDUCED EMBRYOID FORMATION IN SUNFLOWER PROTOPLASTS

SummaryAgarose embedding of sunflower (Helianthus annuus L.) hypocotyl protoplasts induces an asymmetric division pattern and subsequent polarized development leading to embryoid formation. We cultured protoplasts in media with different mannitol concentrations. Induction of plasmolysis of agarose-embedded protoplasts by increasing the mannitol concentration lowered the proportion of embryoids formed. This indicates that adhesion sites between the plasma membrane and the agarose matrix are involved in embryoid formation. The involvement of such adhesion sites was tested by incubating embedded protoplasts with RGD peptide. 1 μM RGD heptapeptide reduced embryoid formation by 50% as compared to the control DGR peptide. We also showed that RGD heptapeptide acts on the cytoskeleton by disrupting cortical microtubules. The results are discussed in terms of a model in which the anchorage of the protoplast membrane to the agarose matrix is mediated by RGD-binding proteins connected with microtubules, determining asymmetric division of the cell and polarized development.

Comparative analysis of early embryonic sunflower cDNA libraries.

To gain information concerning cell functions and activities during sunflower embryogenesis, an expressed sequence tag (EST) approach was used to analyse gene expression in the early stages of sunflower embryos development. Confocal microscopy observations of whole-mounted embryos allowed us to identify precisely the major steps of the zygotic embryonic development. A time-course analysis was then employed to collect the embryonic material. Three cDNA libraries were constructed from microdissected embryos, and three other cDNA libraries were created using a classical day after pollination schedule. A total of 7106 ESTs were produced and assembled. The total number of putative different genes represents about 43.1 (3064 tentative contigs and singlets) of the analysed sequences. The unigenes that showed similarity to proteins with known or predicted functions (50.3) were classified into 15 different functional categories. The functional profiles were found to be quite similar for all studied embryo stages but statistical analysis revealed that successive and coordinate sets of genes are expressed at each embryonic stage. The analysis allowed us to identify abundant and differentially expressed genes at the early stages of embryos development as well as some putatively interesting genes, showing strong similarities with genes playing key roles in plant and animal embryogenesis. The data presented in this study not only provide a first global overview of the genes expression profile during sunflower embryogenesis but also represent an original and valuable tool for developmental genomics studies on exalbuminous dicots.

Cytolocalization of ion-channel antagonist binding sites in sunflower protoplasts during the early steps of culture

SummaryA fluorescently labeled phenylalkylamine (PAA), DM-Bodipy PAA, was used as a probe for in vivo labeling of PAA binding sites in sunflower hypocotyl protoplasts in culture. Verapamil, a PAA known as a calcium channel antagonist in plants, lowers the division rate of sunflower protoplasts in culture. The binding specificity of DM-Bodipy PAA was established at various culture times by competition experiments with (−)bepridil. Studies on the Cytolocalization of DM-Bodipy PAA binding sites by confocal imaging showed that in freshly isolated protoplasts PAA receptors were organized into clusters uniformly distributed over the cell surface. During protoplast culture, the fluorescence labeling pattern evolved from peripheral to cytoplasmic. After a few days of culture, PAA binding sites were present inside the cell, along cytoplasmic strands, on the membrane of vesicles and vacuoles, and were highly concentrated around the nucleus. After protoplast division, the labeling was mainly restricted to a zone close to the new cell wall. On symmetrical division, binding sites were uniformly distributed on both sides of the new cell wall. With asymmetrical division, binding sites were concentrated in a ring surrounding the new cell plate.

A new tool for plant cell biology: in vivo antibody uptake in plant protoplasts

We report on the in vivo uptake of antibodies into plant protoplasts. When protoplasts of sunflower, Arabidopsis or tobacco were incubated in vivo with an antibody, this antibody was detected by immunofluorescence in the cytoplasm and/or the nucleus, depending on the location of the target protein. Furthermore, when protoplasts were cultured in the presence of antibodies, specific effects were observed. Incubation with antibodies raised against p34cdc2 led to a strong inhibition of the division rate, and a decrease in the average DNA content of protoplasts. With antibodies against HaWLIM1, a LIM domain protein of the CRP type, a negative effect on actin organisation was observed. We conclude that antibodies can penetrate plant protoplasts in vivo, and thus may be used as powerful tools for the study of protein function.

Agarose-induced embryoid formation in sunflower protoplasts is triggered by RGD-mediated membrane-matrix adhesion

Embedding of plant cells or protoplasts in gelling medium is widely used to improve cell viability and plating efficiency (Smidsrod and Skjak-Braek, 1990). In Arabidopsis thaliana (O’Neill and Mathias, 1993) and Helianthus annuus (Chanabe et al. 1991), protoplast inclusion in a solid matrix induces changes in division pattern and development. In sunflower, protoplasts cultured in liquid medium divide symmetrically and form loose microcolonies. In contrast, when they are embedded in agarose, most of them divide asymmetrically and display a polar organization at the onset of their development giving rise to compact embryo-like structures (Petitprez et al, 1995). These embryoids-progress to the heart-shape stage but do not develop further. Various hypotheses have been proposed to explain the effect of embedding on the fate of protoplasts: (i) limiting oxygen diffusion (Barbotin et al., 1993), (ii) simulating a cell wall, (iii) generating physical constraints that affect the stability of the cells (Asano et al., 1994). We previously showed that an increase of physical constraints such as hydrostatic pressure 0.4–1 MPa had an inhibitory effect on microtubule organization and cell wall synthesis and consequently on cell division (Barthou et al., 1997) but had no effect on division asymetry. Thus, physical constraints cannot solely explain the protoplast division pattern observed in agarose cultures.