1.8.11
CLONING OF ELICITIN GENES FROM THE SOYBEAN PATHOGEN PHYTOPHTHORA SOJAE

J BECKER, S NAGEL and R TENHAKEN

Universitšt Kaiserslautern, FB Biologie Geb. 22, D-67653 Kaiserslautern, Germany

Background and objectives
Phytophthora is a genus of phytopathogenic oomycetes which causes serious diseases in many crop plants. Most of the investigated Phytophthora species secrete 10-kDa proteins into their culture media. These proteins, called elicitins, show a highly conserved amino- acid sequence in all analysed Phytophthora species. Elicitin genes mostly exist as a small gene family [1]. The different elicitin protein isoforms vary in their isoelectric point and the ability to induce necrosis in tobacco. Elicitins are shown to be potent elicitors of a hypersensitive-like reaction when they are infiltrated into tobacco leaves. Other plants, including members of the Solanaceae, do not respond to elicitins with necrosis [2].

Material and methods
With primers derived from conserved sequences, we amplified the elicitin genes from genomic Phytophthora sojae race 1 DNA by PCR. The genes were cloned into Escherichia coli and sequenced. In addition, P. sojae mRNA was isolated and reverse transcribed. After PCR amplification the cDNAs were also cloned and sequenced. The different genes were expressed in E. coli as His-tagged fusion proteins and purified by affinity chromatography.

Native elicitin protein was also isolated from the culture fluid of P. sojae and purified by ion-exchange chromatography. For physiological experiments, tobacco and other plants were infiltrated with protein solutions. Necrosis was observed macroscopically and gene induction was analysed by Northern blotting experiments.

Results and conclusions
Elicitin genes were cloned from genomic DNA and reverse-transcribed mRNA of P. sojae by a PCR approach. During sequencing we found four different elicitin isoforms from P. sojae race 1. The same isoforms were identified from genomic DNA and from the cDNA, indicating that all four isoforms are expressed when the fungus is grown in liquid culture. All of them were identified as acidic elicitins and showed similar effects when infiltrated in tobacco leaves. Within 12 h necrosis occurred, and after 3 days the infiltrated parts of the leaves were completely wilted. Infiltration of elicitins into other plants showed no visible effects. The Northern blot analysis showed that different isoforms vary in their ability to induce PR1 mRNA in tobacco.

References
1. Yu LM, 1995. Proceedings of the National Academy of Sciences, USA 92, 4088-4094.
2. Kamouen O et al., 1997. Molecular Plant-Microbe Interactions 10, 13-20.