Pierre M. Charest

Identification of a 220 kDa membrane‐associated plant cell protein immunologically related to human β‐spectrin

Electrophoretic analysis of low ionic strength extracts of tomato plant leaves revealed the presence of two proteins of apparent molecular weights of 240 kDa and 220 kDa which co‐migrated with purified human erythrocyte α‐ and β‐spectrin subunits. Immunochemical analyses employing an affinity‐purified polyclonal antibody to human erythrocyte β‐spectrin reacted specifically with the 220 kDa plant cell protein. Immunofluorescence microscopy indicated that the β‐spectrin antibody recognized an antigen which was primarily restricted to the peripheral areas of the cells. Collectively, these results suggest that the cells of higher plants contain polypeptides related to the spectrin family of proteins. It is proposed that the plant cell possesses a membrane skeleton which is structurally and perhaps functionally analogous to that of the animal cell.

Carbonic anhydrase III in skeletal muscle fibers: an immunocytochemical and biochemical study.

The objectives of the present study were to determine if carbonic anhydrase III (CA III) demonstrated a specific association for any particular organelle or structure of the skeletal muscle cell and to quantify the activity and content of this enzyme in different types of skeletal muscle fibers. Ultrastructural localization of CA III in the soleus (SOL), deep vastus lateralis (DVL), and superficial vastus lateralis (SVL), composed of predominantly type I, IIa, and IIb fibers, respectively, was performed using a high-resolution immunocytochemical technique and antibody specific for CA III on ultra-thin sections of skeletal muscle embedded in the water-soluble medium polyvinyl alcohol (PVA). The results indicated a uniform distribution of CA III within the sarcomere. Mitochondria, nuclei, triads, Z-, and M-bands were not specifically labeled. Immunoblotting of washed myofibril preparations did not show any detectable CA III associated with this structure. In addition to quantification of the immunogold labeling, CA III activity and content were assayed in the post-mitochondrial supernatant of the three muscles. In the SOL, these values were found to be 3.6-7.6 times higher than in the DVL. The SVL showed a labeling intensity slightly higher than background level, while the enzyme activity and content were indistinguishable from background levels. We therefore conclude that CA III is randomly distributed in the cytoplasm of the three muscle fiber types and that the relative CA III content and activity in the three muscles studied is SOL greater than DVL greater than SVL approximately equal to 0.

Cell wall alterations in hypocotyls of bean seedlings protected from Rhizoctonia stem canker by a binucleate Rhizoctonia isolate

The influence exerted by the non-pathogenic binucleate Rhizoctonia (np-BNR) isolate 232-CG in stimulating plant defence reactions in young bean plants inoculated with the root rot fungus Rhizoctonia solani (AG-4) was examined using light and electron microscopy and further investigated by gold cytochemistry. Severe necrotic lesions on hypocotyles of diseased beans were observed, and the pathogen invaded the cortical tissue causing extensive damage including cell disorganization and cell wall degradation. In contrast, these host reactions were not seen in bean plants inoculated with the non-pathogenic BNR or in plants that were inoculated with BNR and subsequently challenge-inoculated with R. solani. Microscopic examination of hypocotyls inoculated with the non-pathogenic BNR, showed a different host reaction typical of plant defence reactions. In these samples, epidermal and outer cortical cells were impregnated with an electron-dense material. Histochemical assays of this material confirmed the substantial presence of phenols, pectic substances and suberin. Electron microscope observations clearly showed that in non-pathogenic BNR-inoculated plants, fungal cells were confined to the epidermal layer which was darkly stained. Gold cytochemistry confirmed the presence of pectic substances in the electron dense material. The possibility that pectic oligogalacturonides released after hydrolysis by the non-pathogenic BNR enzymes may act as elicitors of defence responses is discussed. The present ultrastructural observations corroborate that non-pathogenic BNR isolates may function as potential inducers of plant disease resistance.

Development of reliable molecular markers to detect non-pathogenic binucleate Rhizoctonia isolates (AG-G) using PCR

Non-pathogenic binucleate Rhizoctonia species (BNR) belonging to the anastomosis group AG-G are commonly associated with members of the Rhizoctonia solani complex. They provide effective protection to young bean seedlings against root rot caused by R. solani AG-4. Both fungi are morphologically similar and it is difficult to differentiate between them without using laborious conventional techniques. RAPD assays were carried out on a large range of isolates of binucleate Rhizoctonia species to identify markers common to all AG-G isolates. Two fragments of 1368 bp and 882 bp were isolated, cloned and used to probe Southern blots of DNA from: AG-G isolates; isolates from other AGs of binucleate and multinucleate Rhizoctonia species; various heterogeneous pathogens known to infect bean plants; and co-inoculated bean plants with BNR AG-G and R. solani AG-4. The fragments hybridized only to DNA from AG-G isolates. Both fragments were nucleotide sequenced and two pairs of SCAR (sequence characterized amplified region) primers (BR1a F/R and BR1b F/R) were generated for use in PCR. Two fragments of anticipated size were generated following PCR of all isolates of AG-G and not from any range of other fungal species associated with root and leaf diseases of beans. The SCAR primers were also used to detect AG-G isolates in DNA extracted from bean and soil samples co-inoculated with binucleate and multinucleate Rhizoctonia species. The assays were capable of detecting as little as 2.6 pg of fungal DNA in extracts of soil samples. This system offers the potential to determine the presence of AG-G isolates in infected soil and plant samples.

Polymerase chain reaction-based assay for specific detection of Rhizoctonia solani AG-3 isolates

Rhizoctonia solani AG-3 causes considerable yield loss in potato fields in eastern Canada and U.S.A. The accurate identification of AG-3 isolates is strategic prior to planting potatoes. To obtain a fast and reliable identification method, RAPD experiments were carried out to obtain specific genetic markers of AG-3 isolates. DNA from various isolates of R. solani was submitted to RAPD amplification using random 10 mers primers. One of the forty primers used yielded a 2.6 kbp fragment present in all isolates of AG-3. The specificity of this fragment was assessed by Southern blot analysis. It was partly sequenced and DNA primers were designed for PCR amplification experiments. A PCR-based restriction mapping method, using one restriction endonuclease, Xho I, was developed for specific detection of AG-3 isolates. Analysis of data showed that AG-3 isolates were distinct to other AGs R. solani. The detection method described here is very specific and reliable; it can be applied on plant tissue and soil infected with R. solani AG-3.

Stachybotrys elegans: a destructive mycoparasite of Rhizoctonia solani

Stachybotrys elegans colonizes hyphae as well as sclerotia of Rhizoctonia solani . Germinability of sclerotia, which had been incubated with the mycoparasite for 49 d, decreased from 84% to 5% as compared with 86% germinability of untreated sclerotia. Light and electron microscopic observations revealed that S. elegans grew toward and coiled around the host cells. In pure culture, cells of S. elegans were surrounded by extracellular fibrillar material. The production of this material was enhanced during contact and colonization of host hyphae only. This fibrillar material was not observed during interaction of the mycoparasite with host sclerotial cells. Penetration of host cells was accomplished by either unspecialized hyphal branches or pseudoappressoria. Penetration of host cells and emergence from them by the mycoparasite is apparently accomplished by both mechanical and enzymatic activity. Trophic hyphae of the parasite developed within the host cytoplasm, resulting in granulation of the cytoplasm and ultimately death of host cells. Papillae of unknown origin and chemical composition were frequently detected in colonized sclerotial cells. The above results indicate that mycoparasitism is the mode of action by which S. elegans attacks its host.

Immunocytochemical localization of vindoline in mesophyll protoplasts of Catharanthus roseus

Abstract Leaves of Catharanthus roseus synthesize the monoterpenoid indole alkaloid, vindoline, while its site of accumulation has not been identified. In order to study the intracellular localization of vindoline, antivindoline antisera were raised in rabbits and used with in situ immunofluorescence and immunogold labelling techniques. While labelling of isolated protoplast preparations showed an intense green fluorescence in the protoplast vacuoles, indicative of the location of vindoline labelled with fluorescein, the high fluorescence intensity did not allow for the recognition of other subcellular compartments. However, the use of cryotechniques coupled with immunogold labelling revealed that the label of vindoline was present in small vesicles and the cytoplasmic area bordering the plasmalemma. A substantial amount of label was also observed in the central vacuole and, to a much lesser extent, in the chloroplasts.

Cytochemical and immunocytochemical investigation of the mycoparasitic interaction between Stachybotrys elegans and its host Rhizoctonia solani (AG-3)

Hyphal interaction between the mycoparasite Stachybotrys elegans and its host Rhizoctonia solani was investigated cytochemically for the detection of sugar residues using lectin-gold complexes, and immunocytochemically with an antibody raised against purified fimbriae. N -acetyl- d -glucosamine oligomers were revealed in the cell walls of both fungi. The absence of these sugar oligomers at the penetration sites of the host cell wall suggests that extracellular chitinases produced by S. elegans may be involved in the mycoparasitic process. d -galactose residues were also detected throughout the cell wall of S. elegans and may have a role in the recognition process. In R. solani cells, labelling studies revealed that papillae produced in response to attack by the mycoparasite contained N -acetyl- d -glucosamine and to a lesser extent either mannosyl or glucosyl sugar residues. Fimbrial proteins were localized within the extracellular matrix that surrounds S. elegans cells when they were confronted with its host. The present study provides evidence that surface molecules are implicated in the interaction between S. elegans and its host.

Irregular growth forms and cell wall modifications, polygalacturonase detection, and endocell formation in Fusarium oxysporum f. sp. radicis-lycopersici infecting tomato plants, as studied ultrastructurally and cytochemically

Pathogen cells of Fusarium oxysporum f.sp. radicis-lycopersici infecting container-grown tomato plants were characterized ultrastructurally, using gold-complexed probes, chitinase and wheat germ agglutinin to localize chitin, and polyclonal antibodies to a polygalacturonase to localize this enzyme. It was isolated and purified from the pathogen growing in culture. Many fungal cells were of irregular forms (microhyphal, frondose) with modified, thin or imperceptible lucent wall layers, in which were often included components seemingly of host origin. Gold particles of the polygalacturonase probe were concentrated on portions of penetration hyphae and in areas of associated altered host wall. Fine filamentous-like structures, often linked to fungal cells, reached into extracellular matter and into host walls. Examination of 0.2–0.25 μm-thick sections at 120 kV, and tilted at various angles, indicated that fungal cells frequently had a pronounced wavy contour. Labelling of thin walls for chitin was mostly nil, particularly in contact with host walls, as of also thicker walls in similar situations, or it was then associated with the outside opaque layer. Cells of diverse dimensions with thin or thicker walls and with altered or normal content, contained endocells. Walls of the encodcells and of the enclosing cells often labelled differently for chitin with both probes. Endocells mostly did not originate from proliferation of a living into a dead cell but often ensuing as an apparent fragmentation of the cell content or following its retraction. The bearing of these observations on the host-pathogen relationship, particularly concerning the role of thin-walled hyphae and irregular forms, is discussed.

Kinetic of the production of polygalacturonase and pectin lyase by two closely relatedFormae speciales ofFusarium oxysporum

Abstract The kinetic of the in vitro production of polygalacturonase and pectin lyase of two closely related fungi, Fusarium oxysporum f.sp. lycopersici and F. oxysporum f.sp. radicis-lycopersici , was examined under various culture conditions such as the source of carbon, the pH, and the age of cultures. Over a 5-day period, the production of these enzymes by various isolates of the same forma specialis (f. sp.) of F. oxysporum was not significantly different ( P ≥ 0.05). However, the amount of the enzymes produced differed markedly between both f. sp. The different carbon sources added to the culture media, such as citrus pectin, apple pectin, tomato cell wall fragments, and d -galacturonic acid, proved to be higher pectinase inducible substrates than sucrose and glucose. For both fungi, polygalacturonase and pectin lyase activities were optimal at pH 5.0 and 8.0, respectively. Furthermore, pectin lyase production had a partial Ca 2+ requirement in contrast to polygalacturonase production which was limited by Ca 2+ . In most experiments performed, the production of polygalacturonase appeared superior with F. oxysporum f.sp. radicislycopersici than with F. oxysporum f.sp. lycopersici . On the other hand, pectin lyase production of F. oxysporum f.sp. lycopersici was approximately 10-fold greater than that by F. oxysporum f.sp. radicis-lycopersici in media supplemented with d -galacturonic acid.