1.3.44
EXPRESSION OF ANTIMICROBIAL DEFENCE REACTIONS IN FOUR SOYBEAN CULTIVARS INDUCED BY FUNGAL AND BACTERIAL PATHOGENS

K GROTEN and W BARZ

Institute of Plant Biochemistry and Biotechnology, University of Muenster, Hindenburgplatz 55, D-48143 Muenster, Germany

Background and objectives
Plants react to microbial attack with a number of defence mechanisms including synthesis of phytoalexins, changes in pH value in the growth medium, H2O2 production and insolubilization of cell wall hydroxyproline glycoproteins. These defence responses can also be induced by various biotic and abiotic elicitors. The initial events between elicitors and plant cells leading to the biochemical events are broadly unknown. Comparative studies with various non-specific biotic elicitors and different cultivars of the same species seem to be helpful to further elucidate the link between elicitor recognition and early intracellular responses.

Materials and methods
Suspension cultures of soybean (Glycine max) cvs Kaimit, Lumut, Wilisand Doko RC were subcultivated every 7 days. Cells were elicited 5 days after subculture with 100 pg fungal elicitor per ml or 50 pi bacterial suspension per ml. The fungal elicitor preparations were cell wall fractions of Phytophthora megasperma f.sp. glycinea and Rhizoctonia solani. For elicitation 20-24-h-old Pseudomonas syringae pv. glycinea cells were resuspended in MES-buffer and the suspension adjusted to 0.1 absorbance units at 500 nm. For SDS-PAGE and immunoblotting, cell wall proteins were extracted with SDS sample buffer. These SDS protein extracts were separated by 7.5% or 10% SDS-PAGE. For detection of p100 the 7.5% stacking gel was AgNO3-stained. The 10% gel was blotted onto PVDF membrane, incubated with a p33 antiserum from soybean and determined by phosphatase activity. H2O2 formation was detected by luminot-dependent chemiluminescence. Phenylalanine ammonia-lyase activity was determined by a photometric assay, chalcone synthase activity by a 14C-radioactivity test.

Results and conclusions
Treatments of suspension-cultured soybean (Glycine max) cells with crude cell wall extracts of the fungal pathogens P. megasperma f.sp. glycinea and R. solani (Pmg-, Riso-elicitor, respectively) and with cells of the bacterial pathogen P. syringae pv. glycinea cause a very rapid release of active oxygen species (oxidative burst). Comparing four cultivars (Kaimit, Lumut, Wilis, Doko RC) with regard to the amount of H2O2 induced by the fungal elicitors, three of them produced significantly less H2O2 after elicitation with Pmg-elicitor than with Riso, whereas Doko RC showed equal values for both elicitors. These results correspond with an elicitor-induced rapid alkalization of the culture medium. Elicitors causing a strong oxidative burst are also capable of inducing a greater transient pH increase. In all cultivars the alkalization is followed by a strong acidification (0.6-1.0 pH units) which is probably due to an activation of plasma-membrane H+-ATPases [1]. The elicitor-induced H2O2 maximum for cultivar Wilis is found to be considerably lower (30 Ámol/g fw for Riso-Elicitor) compared with the three other cell lines (100-120 Ámol/g fw). When based on the number of cells per flask, the difference even increases. These results suggest that soybean cultivars differ in the intensity of elicitor-induced defence reactions. It is unclear if these differences are due to a varying recognition of the elicitor at the plasma membrane, or if some lines react with weaker defence signals. Elicitation also results in an insolubilization of two cell-wall proteins (p33, proline-rich; p100, a putative hydoxyproline-rich glycoprotein) catalysed by peroxidases [2]. There is no direct correlation between the H2O2 production measured as lumino-dependent chemiluminescence and the oxidative cross-linking.

Several hypotheses concerning the possible relation between the oxidative burst analysed in this study and the protein insolubilization are discussed. The induction of phenylalanine ammonia-lyase and chalcone synthase as two key enzymes of glyceollin-phytoalexin biosynthesis upon elicitation is also described.

References
1. Mathieu Y, Lapous D, Thomine S et al., 1996. Planta 199, 416-424.
2. Bradley DJ, Kjellbom P, Lamb IMJ, 1992. Cell 70, 21-30.