1.4.5
HOMOLOGOUS GENES OF THE CBEL ELICITOR FROM PHYTOPHTHORA PARASITICA NICOTIANAE ARE PRESENT IN DIFFERENT OOMYCETES

F VILLALBA MATEOS, K PUGET, S CARLIER, M-T ESQUERRE-TUGAYE and M RICKAUER

Centre de Physiologie Végétale, UMR UPS-CNRS 5546, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 4, France

Background and objectives
Phytophthora parasitica nicotianae (Ppn), the causal agent of black shank disease of tobacco, produces in vitro and in planta a cell-wall glycoprotein which elicits defence reactions in its host plant. The elicitor has been purified [1] and the corresponding cDNA has been cloned. Analysis of the deduced peptide sequence unravelled several unusual features and led to the discovery of additional activities of the elicitor [2]. The polypeptide consists of two direct repeats of a cysteine-rich domain (with 54% identity), separated by a Thr/Pro-rich linker. The positions of the cysteine residues in each domain are conserved and part of them occur in a sequence resembling the cellulose-binding domain of fungal hydrolases. The elicitor has no glycolytic activity under experimental conditions, but binds to cellulose and tobacco cell walls. In addition, it agglutinates red blood cells. Because of its multiple activities, the glycoprotein has been named CBEL (for cellulose-binding elicitor lectin). Although a number of fungal elicitors have been characterized, the intrinsic functions of most of them are still unknown. The cellulose-binding and lectin-like activities of CBEL might be related to a role in adhesion phenomena during the saprophytic or parasitic life of the fungus, such as attachment to substrate or to the surface of its host plant. The expression pattern of cbel under different conditions of culture and during infection of the host plant might give further indications of its functions. We therefore cloned the gene in order to use its promoter in future reporter gene expression studies. We also looked for the presence of homologues in different Phytophthora and in Pythium, assuming that a molecule important for adhesion might be present in more than one species.

Results and conclusions
Based on Southern blot analysis of genomic DNA of Ppn race 0, an EcoRI/XhoI digest was size-fractionated, and fragments of ca 5 kb were cloned. After screening of this partial genomic library with the CBEL cDNA, a clone was sequenced and found to contain the entire cDNA sequence plus 5' and 3' regions. The gene is not interrupted by introns. The 5' region does not contain classical promoter motifs such as a TATA box or CAAT box, but these are not mandatory in fungal promoters. However, a 16-nucleotide sequence corresponding to the oomycete transcription start consensus sequence was found at -73 from the ATG.

Homologous genes were detected by Southern blot analysis in Ppn race 1, P. nicotianae nicotianae, P. infestans, P. citricola, P. sojae and Pythium irregularae. DNA fragments corresponding to the open reading frames were amplified by PCR from Ppn race 1, P. infestans and P. nicotianae nicotianae, and sequenced. The deduced polypeptides were found to be either identical to the Ppn race 0 CBEL sequence (P. infestans) or to differ only by two amino acid changes. None of the amplified fragments contained an intron. PCR with DNA from P. sojae and P. citricola was unsuccessful, indicating significant differences in the sequences used for defining the primers. Crude extracts from the four Phytophthora species were analysed by Western blot with an antiserum directed against CBEL. A single band of about 34 kDa was revealed in all cases. Thus very similar polypeptides seem to be produced by different species of Phytophthora and probably also by another oomycete, Pythium. This observation is in accordance with the hypothesis of a general function. However, it cannot be excluded that the glycoproteins differ more significantly in the composition of the carbohydrate part, which accounts for ca 50% in CBEL, as well as in their biological activities. These points still remain to be elucidated. A different approach for testing our hypothesis is also under way and consists of suppressing the expression of cbel by antisense strategy. The study of CBEL-less transformants should allow the role of this interesting molecule to be determined in the future.

References
1. Séjalon-Delmas N, Villalba Mateos F, Bottin A et al., 1997. Phytopathology 87, 899-909.
2. Villalba Mateos F, Rickauer M, Esquerré-Tugayé MT, 1997. Molecular Plant-Microbe Interactions 10, 1045-1053.