Cloning and Characterization of a Disease Resistance Response Gene in Pea Inducible byFusarium solani

Abstract

Disease resistance response genes (DRRG) of peas are expressed as the tissue is expressing race-specific or nonhost resistance. A pea genomic clone DRRG49-c encompassing one DRRG structural gene, the expression of which is correlated with the expression of disease resistance, was sequenced and characterized. The 2.3-kb genomic segment sequenced encompassed 986 bp 5' to the major transcriptional initiation site, a 474-bp open-reading frame interrupted by one 88-bp AT-rich intron and an additional 574-bp segment 3' from the stop codon. Southern blot analysis indicated that the DRRG structural gene is one of a multigenic family, and an estimated five copies exist within the pea genome. Primer extension analysis of the 5' terminus of the corresponding RNA suggested the presence of one major transcript with possibly two minor transcripts. The major transcript, located 65 bp from the translational initiation site, was expressed when challenged with Fusarium solani f. sp. phaseoli but not with water. The structural gene sequence corresponding to the genomic clone DRRG49-c is not identical with the structural gene of the cDNA clone DRRG49-a used as a probe for northern blot analysis, and thus, a possibility remains that it is not expressed in peas; however, the DRRG49-c promoter was able to express the chloramphenicol acetyltransferase reporter gene in tobacco protoplasts. Western blot analysis using antiserum prepared from a beta-galactosidase-DRRG49-a fusion protein identified the DRRG49 gene product as a major protein accumulating during the host-pathogen interactions.