CBN-V Video Archives - S5-12
Characterization of Defense Genes to Cassava Bacterial Blight

Santaella M¹., P. Zuluaga¹, E. Suárez¹, C, González¹, C. López², I. Acosta¹, A. Badillo³, S. Restrepo², J. Tohme¹ and V. Verdier²

1. CIAT, Biotechnology Research Unit, CIAT, A.A. 6713, Cali, Colombia
2. Institut de Recherche pour le Développement (IRD) c/o CIAT
3. University of Los Andes, Bogota, Colombia

        Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam) is a major disease causing serious damage to cassava. Deployment of resistant varieties is the major approach to control the disease. We used and developed the cDNA-AFLP technique and the amplification of resistance gene analogs (RGAs) as means of elucidating putative genes involved in the defense response. For the cDNA-AFLP technique, we compared the differential gene expression of a resistant and a susceptible variety over time. RNA was extracted from stem tissues and cDNA was used as template for AFLP. Of ~3600 cDNA fragments screened, 353 fragments were specific to the resistant variety. 202 polymorphic bands were reamplified, cloned and partial sequences were obtained. Sequence analyses showed significant homology with resistance genes, NPK-1 related proteins, senescence-related proteins and other known proteins involved in disease resistance reactions. The cDNA-AFLP technique has proved to be a quick, reliable and powerful way of identifying transcripts showing differential expressions during the host plant reaction. Using degenerate primers designed from the conserved domains Toll-Interleukin 1 Resistance (TIR) and Nucleotide Binding Site (NBS) of the resistance proteins L6 (Flax), RPS2 (Arabidopsis) and N (Tobacco) we identified twelve different classes of resistance-gene analogs (RGAs) from cassava. Two classes are from the TIR domain primers and 10 classes correspond to the NBS domain and were classified in two groups. The first group (classes 1-5) belong to NBS Group I, which is linked to a TIR domain in the N terminus, and is significantly associated with dicot species. The second group (classes 6 to 10) correspond to NBS Group II domains that are linked to putative coiled-coil domains in their N terminus and appear to be present throughout the angiosperms. Screening of 89000 clones in a cassava cDNA library (root and leaf) with class-specific RGAs probes allowed the identification of sixteen expressed clones. Sequence analysis of clone L16 (1690 kb), from the RGA class 10-probe, confirmed the constitutive expression of a protein that shares characteristics with previously reported resistance genes. T he RGA L-16 is a member of the NBS-LRR resistance gene family. The other 15 clones sequenced presented homology with putative ubiquitin-like and ribosomal proteins. Mapping of these RGAs is currently under way.

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