Pathogenesis-related proteins (PR-Proteins) are induced in response to environmental stresses such as, osmotic and drought stress, wounding, microbial infections and treatment with specific plant hormones and elicitors. These proteins are presently classified into several groups (PR-1 through PR-17) based on their amino acid sequence and biochemical functions. The present study has been focused on isolation, over-expression and analysis of the physiological activities of the thaumatin-like proteins ( i.e. PR-5) in the model plant Medicago truncatula . Analysis of the M. truncatula genome, available freely on the internet, indicated the presence of at least six PR-5 Open Reading Frames (ORFs), three of which along with two previously reported cDNAs were selected for this study. Highest homologies for the five ORFs/gens (dubbed tlp -1,-2,-3,-4 and -5) to known PR-5 proteins are respectively 87% to Arabidopsis thaliana aad02499, 63% again to A. thaliana aad02499, 78% to A. thaliana NP_173261, 68% to Lycopersicum sculentum L76632, and 56% to either Vitis vinifera Y10992 or Cicer arietinum AJ010501. Using specific primers, the sequences were isolated from the M. truncatula genome and all, but one, were confirmed to match with those previously reported. No sequence was obtained for tlp-4 at time this reported. All the fragments were, then, transformed into Escherichia coli isolate BL21 (DE3), using pET21c(+) plasmids for subsequent overexpression. All five genes were expressed as inclusion bodies (IBs) with masses, estimated by SDS PAGE, corresponding to the theoretical values. As expected none of the protein IBs had any detectable effect on phytopathogenic fungi Rhizoctonia solani ، Alternaria alternata and Fusarium graminearum . However when IBs were refolded, using 4 refolding methods, all showed comparable strong antifungal activity against these fungi. No report on overexpression or analysis of PR-5 family of proteins in M. truncatula had been published prior to this project.
