Cell Surfaces in Plant-Microorganism Interactions

Abstract

Enrichment of the cell wal in hydroxyproline-rich glycoprotein is involved in the defense of muskmelon (Cucumis melo) seedings to CoUeto-triclwm lagenarium, the causative agent of anthracnose. The extent to which this accumulation proceeds may be experimentally modified by treating plants with ethyleneor growing them in the presence of free L-trans-hydroxyproline. It appears that the increase in the wail hydroxypro-line-rich glycoprotein mediated through ethylene is paralleled by an increasing resistance of the host to the pathogen. Inversely, inhibiting the synthesis of this glycoprotein in diseased plants is strictly correlated to an accelerated and more intense colonization of the host by the pathogen. In both cases, the inverse relationship between the accumulation of hydroxyproline-rich glycoproteins and the ability of the pathogen to develop in the host has been checked by the quantification, in infected tissues, of glucosamine, a characteristic component of chitin-containing fungi. It has been shown that hydroxyproline is associated with the cell wall of green plants (23-25). Its level varies somewhat, depending on the plant or organ considered (6, 28) but, with the exception of some organisms like Chlamydomonas (29), it is generally low. This level is markedly increased in a few instances where the integrity of the plant is disrupted by wounding, aeration of tissue slices (8), subculturing, and in melon plants infected by a fungal pathogen (16). The observed increase in diseased plants accounts for a cell wall enrichment of the glycosylated hydroxy-prolyl and seryl residues (15) which typify the hydroxyproline-rich glycoprotein (HRGP)3 called "extensin" by Lamport (23). The question of the significance of this glycoprotein accumulation in diseased plants has been raised, particularly because cell surface glycoconjugates are often involved in cell to cell interactions (4, 10, 35) of which the host-pathogen system is a special case. This paper reports on the correlations between the level of HRGP in the cell wall, and the extent of pathogen spread in melon plants infected by Colletotrichum lagenarium. The approach to this problem consisted of modifying the hydroxyproline level 'This work received financial support from the Centre National de la Recherche Scientifique (L.A. n° 241). 'A preliminary report of this work was presented at a Symposium on "Cell Wall Biochemistry Related to Specificity in Host-Plant Pathogen Interactions", held in Tromso, Norway, August 1976. 3Abbreviations used: HRGP: hydroxyproline rich glycoprotein; Butyl PBD: 2(4'-t-butylphenyl)-5-(4-biphenylyl)-1,3,4-oxadiazole; TCA: trichlo-roacetic acid. of the wall prior to or during infection of the plants, by two treatments. The first one was a treatment of seedlings with ethyl-ene, a plant hormone, which, according to Ridge and Osborne (32), increases the amount of cell wall hydroxyproline when applied to plants. The second treatment consisted of feeding seedlings hydroxyproline; Cleland (9) demonstrated that free hy-droxyproline inhibits the appearance of peptidyl-bound hydrox-yproline in the cell wall. In both cases, the amount of pathogen present in the tissues of the infected seedlings was assessed by determining their glucosamine content (36). MATERIALS AND METHODS Biological Material. The host, Cucumis melo (variety Cantaloup charentais), and the pathogen, C. lagenarium, were grown as already described (12). Ethylene Treatment. Melon seedlings were divided into three batches when they reached the first leaf stage. Two batches were then enclosed in Plexiglas containers where the amount of air available to each seedling was about 2 liters. The seedlings of one of these two groups were given ethylene (N35, l'Air Liquide, high purity grade) for 7 days at a concentration of 500 ,ul/l in the container unless othewise stated, while the seedlings of the other group were enclosed without added ethylene. The containers were aerated daily and, depending on the batch, supplied with (+) or without (-) ethylene daily. The seedlings of the third batch were grown under the atmosphere of the growth room. Seven days later, at stage 2, part of the seedlings in the threegroups were harvested while the remainder were either inoculated with C. lagenarium or left uninoculated and used as controls. Controls and infected plants were then treated again with or without ethylene in the same conditions for an additional 7-to 10-day period. Hydroxyproline Treatment. Plants were inoculated at stage 2, as described above. Treatment with L-trans-hydroxyproline started 1 day after inoculation by making the culture medium 0.5 to 4 mm with respect to this amino acid. This treatment was performed for 8 h a day over 6 days, with a fresh medium every day. Healthy plants also received the same treatment. Controls were obtained by growing healthy and infected plants without this amino acid. In all of the experiments performed on plants treated with ethylene or hydroxyproline, we used the stems plus petioles, abbreviated as "stems," or the petiole of the first leaf for the analyses. Cell Wall Preparation and Protein Measurement. The cell walls were extracted (16) and their protein content assayed through Nessler determination of the wall nitrogen (26). Cell Wail Hydroxyproline and Hydroxyproline Arabinosides Determination. The hydroxyproline content was colorimetrically assessed at 560 nm according to Kivirikko (21), after the cell wall 320 https://plantphysiol.org Downloaded on February 27, 2021.-Published by