Constitutive expression of Vitis vinifera thaumatin-like protein after in vitro selection and its role in anthracnose resistance

Abstract

Anthracnose-resistant grapevine (Vitis vinifera L. cv. Chardonnay) plants were regenerated from embryogenic cultures that had been subjected to in vitro selection with culture filtrate of Elsinoe ampelina (de Bary) Shear. Three secreted proteins differentially expressed by in vitro-selected embryogenic cultures and regenerated plants were identified. An 8-kDa protein was identified as a lipid-transfer protein (LTP) by N-terminal amino acid sequence comparison. Two other differentially expressed proteins, with estimated molecular weights of 21.6 and 22 kDa, immuno-reacted with antiserum raised against a thaumatin-like protein (TLP) protein from pinto bean (Phaseolus vulgaris L.). N-terminal amino acid sequencing of the 21.6-kDa protein showed a high degree of homology to V. vinifera thaumatin-like protein 2 (VVTL-2 = grapevine osmotin; Acc no. CAA71883), and that of the 22-kDa protein was homologous to V. vinifera thaumatin-like protein 1 (VVTL-1; AAB61590). Interestingly, both VVTL-1 and VVTL-2 are pathogenesis-related (PR) proteins, belonging to the PR-5 group. Protein produced from the cloned grapevine VVTL-1 gene significantly inhibited E. ampelina spore germination and hyphal growth in vitro. Plants regenerated from in vitro-selected cultures similarly inhibited fungal growth in vivo. Enhanced expression of antifungal VVTL-1 in anthracnose resistant grapevine strongly suggests that it plays an important role, either alone or in conjunction with other PR proteins, by suppressing pathogen growth.