IDENTIFICATION OF HR-RELATED GENES INDUCED IN THE SOYBEAN- PSEUDOMONAS SYRINGAE PV. GLYCINEA INTERACTION BY DIFFERENTIAL DISPLAY
ANDREA LUDWIG, KAI SEEHAUS and RAIMUND TENHAKEN
Universität Kaiserlautern, FB Biologie, Geb. 22, D-67653 Kaiserslautern, Germany
Background and objectives
Resistance of soybean (Glycine max) plants to the bacterial pathogen Pseudomonas syringae pv. glycinea (Psg) is mediated by a gene-for-gene interaction. The soybean cultivar Williams 82, which carries the Rpg2 resistance gene, interacts with the avirulence gene avrA from Psg, resulting in a strong hypersensitive response (HR), restricting the pathogen to the site of attempted infection and inhibiting its development. In contrast, bacteria without the avrA gene cause disease symptoms. During HR, several secondary defence responses are induced in adjacent cells, such as the accumulation of PR-proteins or phytoalexins. We were interested in genes which are induced early after Psg-inoculation and may participate in signal transduction during HR.
Materials and methods
3-4-day-old soybean cell-suspension cultures (cv. Williams 82) were inoculated with 107 c.f.u./ml Pseudomonas syringae pv. glycinea (avrA). For differential display  total RNA from cells was isolated 3 and 5 h after inoculation with bacteria using TRI ReagentTM according to the manufacturers instructions (Molecular Research Centre, Cincinnati, Ohio, USA). One µg of the RNA was reverse-transcribed using an NM(T)12 anchored primer without prior DNA digestion. The cDNA was diluted 1:60 and amplified in the presence of [alpha-33P] dATP with the same anchored primer and 30 different arbitrary 10-mer primers. Amplified cDNAs were separated on a 6% denaturating sequencing gel in the presence of 7 M urea, transferred to blotting paper, dried and exposed to Kodak Biomax MR film. cDNA fragments of interest were cut from the gel, eluted by boiling in water and subsequently re-amplified by PCR using the same primer combination. Re-amplified cDNA was subcloned into pKS vector by TA cloning and transformed into E. coli XL-1. Clones were sequenced and compared with the Genbank databases using the Blast tools.
Results and conclusions
After inoculation of soybean cell-suspension cultures with Psg(avrA), HR cell death is observed which does not occur after inoculation with the virulent Psg bacteria . The HR is either dependent on  or largely accelerated by low concentrations of salicylic acid (SA, 50 µM). In the presence of SA, cell death can be measured by vital stain 6 h after inoculation, compared to 12 h in the absence of SA.
To identify genes induced during the early phase of the HR, a differential display analysis was performed with RNA isolated 3 or 5 h after inoculation. Four different sets of probes were compared: control, SA, Psg(avrA) and Psg(avrA)+SA, of which only the latter induces HR. From the isolated cDNA fragments some genes are induced in the HR as verified by Northern blot hybridization. These cDNA clones code for proteins (e.g. a protein kinase) which may be important in the signal transduction pathway leading to HR.
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