Regis Pepin

PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves.

Production of isolated somatic embryos from sunflower thin cell layers.

We describe here a two step procedure which allows the easy isolation of somatic embryos from Sunflower (Helianthus annuus L.) hypocotyl tissues. Thin cell layers composed of the epidermis plus 3 to 6 parenchyma cell layers were incubated for 5 days in a basal Murashige and Skoog medium using an auxin to cytokinin weight ratio of 1/1. The epidermis layers were then transferred to a Gamborg medium containing a high level of sucrose. After one week of incubation in this medium, many somatic embryos started to be released from the parental epidermal tissue. Even though the germination of these embryos is difficult, we have been able to induce secondary embryos and regenerate fertile plants.

The solubilization of the basic subunit of sugarbeet seed 11-S globulin during priming and early germination

AbstractThe basic B-subunit of the seed storage protein 11-Sglobulin (an 11-S-legumin type protein) is the majorpolypeptide in soluble protein extracts from primedsugarbeet {Beta vulgaris L.) seeds. In contrast, only asmall amount of this protein is present in correspondingextracts from untreated dry mature seeds. Here, and asfor all 11-S globulins describe B-chaid so far isn, thelinked to other polypeptide(s), corresponding mostpresumably to an acidic A-chain, through the formationof disulphide bridge(s). Polyacrylamide gel electrophoreticanalyses of total and soluble protein extracts fromuntreated and primed seeds strongly indicate that thispriming-induced solubilizatio B-chain of resulte thne dfrom an endoproteolytic attack on the A-chain.Microscopical immunolocalization showed a uniformdistribution of the 11-S globuli B-chain oven r the proteinbodies of the embryonic cells from the untreated seeds.For the primed seeds, however, the B-subunit of 11-Sglobulin diffused out of the protein bodies and invadedthe cytosolic compartment. This phenomenon occurredindependently of the manner of priming, being observedwith hydroprimed and osmoprimed seeds, as well as withsugarbeet seeds that had been primed by a prehydrationtreatment. Quantitative analyses of the amounts ofsoluble 11-S globuli B-chain havn e enabled the primingof sugarbeet seeds to be optimized.Keywords: germination, 11-S globulin mobilization,immunocytochemistry, seed priming, storage proteins,sugarbeet.CorrespondenceAbbreviations: DTT = dithiothreitol; PAGE = polyacrylamidegel electrophoresis; PEG = polyethylene glycol; SDS =sodium dodecyl sulphate.

Imaging surface of gold-immunolabeled thin sections by atomic force microscopy

Atomic force microscopy (AFM) has been used to image the surface of thin sections of fungal infected plant tissue, with or without post‐embedding immunocytochemical labeling with gold conjugates. Plant and fungal cells are easily identified from their size, shape and roughness. The cellular shape is similar to that observed by light or electron microscopy (LM or EM) and some internal organelles can even be individualized. The gold beads are easily observed and counted. Their dimensions varied according to the roughness of the surface, but fit with the expected sizes.