\u3ci\u3ePhaseolus vulgaris\u3c/i\u3e GENES OBTAINED BY SUBTRACTIVE HYBRIDIZATION UNDER \u3ci\u3eMacrophomina phaseolina\u3c/i\u3e INFECTION

INTRODUCTION The fungus Macrophomina phaseolina (Tassi) Goid. causes charcoal rot in bean (Phaseolus vulgaris L.). The fungus causes dark lesions on epicotyls and hypocotyls of seedlings and they die due to obstruction of the xylem vessels and vascular wilting. In adult plants, the pathogen causes yellowing of roots, the stems show dark longitudinal lesions and the plant was defoliated and withered. The charcoal rot is promoted by high temperatures and drought conditions, which are frequent in beans growing areas in Mexico. M. phaseolina is responsible for the 65% on loss of bean yield (Abawi and Pastor-Corrales, 1990; Mayek-Perez et al., 1995;, Mayek-Perez et al., 1997). The study of genes differentially expressed in plants under M. phaseolina infection, will allow to gain a better understanding about plant defense mechanism. The objective of this study is the identification of P. vulgaris genes related to plant defense after fungal infection by subtractive hybridization (SSH) technique. MATERIALS AND METHODS The HMP05 strain of M. phaseolina and Pinto UI-114 P. vulgaris plants (susceptible variety to M. phaseolina) were grown in MS medium and incubated in a climate chamber at 25º C with 16-h light and 8-h darkness period, during 5 and 15 days for the fungus and plantlets, respectively. For interaction assays, the plants were place on the mature mycelium and incubated in a climate chamber at same conditions of growth. After 48 h three replicates of the interaction samples, and the controls (only fungus and only plants without interaction) were collected. For expressed assays, RNA from interactions and control samples was extracted by TRIzol® (Invitrogen®) method. The SSH was performed with the PCR-Select™ cDNA Subtraction Kit as describe the manufacturer (637401, Clontech®). SSH library products were sent to Eurofins MWG Operon for sequencing. The expressed sequences tag (EST) obtained from the SSH were analyzed using Blastn and Blastx algorithms against the complete transcriptome of P. vulgaris (NCBI repository). Thereafter the ESTs corresponding with P. vulgaris genes that coding to hypothetic or unknown protein were analyzed using Blastp algorithm