863:	Isolation and characterization of defense proteins from mashua tubers.

Authors:	Guimaraes, Rejane, L.(A)Savary, Brett(B)Flores, Hector, E.(A)
Affiliations:	(A): Pennsylvania State University, Plant Physiology program (B): Eastern Region Research Center, USDA ARS
Presenter:	Guimaraes, Rejane L., rlg167@psu.edu

Mashua (Tropaeolum tuberosum), a tuber crop from the Andean highlands of Peru, is thought by the farmers to have high resistance against pathogens. Although secondary metabolites such as glucosinolates have been implied in resistance to pathogen attack in Tropaeolum species, bioactive proteins may also contribute to defense mechanisms. To test for putative defense proteins, total protein extracts from tubers of Mashua were assayed against a different array of bacteria and fungi pathogens. Tuber extracts were found to contain very high activity against Trichoderma species especially against Trichoderma harzianum. Antifungal activity was observed as a decrease in hyphae and spore formation after contact with tuber extracts. Inhibition of fungal growth and development was observed with protein extracted from tubers as well as from stems of Mashua plants, although tubers showed higher protein activity. The antifungal activity was found to be associated with the albumin fraction. An antifungal protein has been purified to homogeneity by anion exchange and hydrophobic interaction column chromatography. Molecular weight was determined by SDS-PAGE to be of approximately 32 KDa. N-terminal amino acid sequence of this 32 KDa protein revealed high homology to 1,3-beta-glucanases. Interestingly, the antifungal activity was lower in the purified fraction. A fraction containing an albumin of about 22KDa isolated from Mashua tubers was mixed with the 32 KDa protein fraction and assayed for antifungal activity. This resulted in a significant increase in antifungal activity, indicating synergism. N-terminal amino acid sequence of the 22 KDa protein showed homology to osmotins, such as the Tobacco PR5. Subsequent studies will address the physiological role of these proteins.