Analysis of transgenic plants of Vitis vinifera L. expressing genes encoding the pear PGIP and GFPs fused to secretory sequences

The major objective of this research was to develop an efficient method by which to transform Vitis vinifera L. cultivars with the pear PGIP gene in order to analyze its effect on tolerance to Pierce's disease (PD) and botrytis in transgenic plants. A second goal was the transformation of grape with several gfp constructs carrying sequences expected to enhance secretion from the cell in order to evaluate the effect of signal sequences on the targeting of transgene products to xylem tissue. Transgenic plants of cvs. ‘Chardonnay’ and ‘Thompson Seedless’ expressing the pear PGIP or the GFP genes were produced via somatic embryogenesis. Putative PGIP transgenic lines growing in the greenhouse were analyzed by PCR, Western blotting and PGIP activity assays. Western blot analysis demonstrated the presence of the protein in roots, leaves and young stems of the transgenic plants but not in untransformed controls. High levels of enzyme activity were found in crude extracts from leaves and in xylem sap of transgenic lines obtained from independent transformation events but not in untransformed controls. Enzyme activity was also detected in xylem sap of untransformed scions grafted on transgenic rootstocks. Lesion expansion was retarded in leaves of PGIP transgenic plants infected with Botrytis cinerea. The development of PD was delayed in some transgenic lines with high PGIP activity, which exhibited reduced leaf scorching, lower Xylella titers and better re-growth after pruning than the untransformed controls. GFP transgenic plants were transformed with four gene constructs that included the coding sequences for a synthetic GFP and GFP fused with the amino-terminal of the secreted protein trichosanthin (TCS) or the xylem specific protein XSP30, all under the control of the CaMV 35S promoter. In addition, a fourth construct contained gfp under the control of the potato Ubi3 promoter. Strong fluorescence was found in embryos, roots, stems and leaves of plants transformed with gfp and xsp30-gfp but the levels of fluorescence observed in tcs-gfp transformants were very low. In all cases fluorescence was detected only inside the cells. More analysis is necessary to determine whether or not the signal peptides were recognized by the grape secretory machinery. Plants transformed with gfp controlled by Ubi3 promoter exhibited green fluorescence only in the root apex and in meristematic leaves. No fluorescence was detected in these plants after wounding or ethylene treatment.