Greenhouse Screening and Field Testing of Transgenic Grapevine for Fungal Resistance SA Dhekney 1, ZT Li 1, M Dutt 2, TW Zimmerman 3 and DJ Gray 4 1 Mid Florida Research and Education Center, University of Florida 2725 Binion Road, Apopka, FL USA 2 Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL University of Virgin Islands Agric. Experiment Research Station, RR1 Box 10,000, Kingshill, St. Croix, VI INTRODUCTION A number of pathogenesis related (PR) proteins are expressed when grapevine is subjected to biotic stresses. The endogenous genes that produce certain PR proteins are considered good candidates for genetic engineering of disease resistance. Five putative PR proteins were identified when grapevine embryogenic cells were exposed to fungal culture filtrate (Jayasankar et al., 2000). Grapevine embryogenic cultures exposed to culture filtrate of Elsinoe ampelina (the causal agent of anthracnose) differentially expressed two Vitis vinifera thaumatin-like proteins (VVTL-1 and VVTL-2). Purified recombinant VVTL-1 protein inhibited spore germination and hyphal growth of E. ampelina, which suggested an active role in disease resistance (Jayasankar et al., 2003). In the current study, the VVTL-1 gene was re-engineered for constitutive expression and inserted into grapevine in order to study fungal disease resistance. MATERIALS AND METHODS Induction of embryogenic cultures Embryogenic cultures of V. vinifera ‘ Cabernet Franc’, ‘Merlot’, ‘Orange Muscat’, ‘Shiraz’, ‘Thompson Seedless’ and Vitis hybrid ‘Seyval Blanc’ were initiated from anthers and leaves (Gray et al., 2005). Somatic embryos at the mid-cotyledonary stage of development were utilized for genetic transformation. Isolation of the VVTL-1 gene and construction of a binary vector The coding sequence of VVTL-1 was isolated from V. vinifera ‘Chardonnay’ by PCR (Jayasankar et al., 2003) and cloned into a binary vector along with a green fluorescent protein/neomycin phosphotransferase II (EGFP/NPT II) fusion gene under control of a CaMV 35S bi-directional duplex promoter (BDDP) complex (Li et al., 2001b; 2004). Genetic transformation of embryogenic cultures and recovery of transgenic plants A pBIN 19-derived binary vector harboring the VVTL-1 gene was introduced into Agrobacterium tumefaciens strain ‘EHA 105’. Transgenic grapevines were regenerated following transformation of embryogenic cultures (Li et al., 2006). PCR analysis of transgenic plant lines Genomic DNA was extracted from leaves of eight transgenic lines and a non-transformed control. EGFP and NPTII genes in transgenic plants were detected using PCR. To differentiate transgenic from native VVTL-1 genes with PCR, forward and reverse primers corresponding to the unique nucleotide sequence of the binary vector were designed. Testing of transgenic plant lines for expression of transgenic VVTL-1 protein An antiserum against purified recombinant VVTL-1 protein (Jayasankar et al., 2003) was used to test leaves of transgenic plants and controls for presence of transgenic VVTL-1 protein via ELISA (Li et al., 2001a). Greenhouse screening of transgenic plant lines for resistance to powdery mildew Transgenic plant lines were screened for resistance to powdery mildew (Uncinula necator) by comparing symptom development with non-transgenic susceptible and resistant control varieties. A minimum of three clones of each line was tested at a time. Each plant was scored three times a week (for 4 weeks) on a scale of 1 to 5 based upon the number of lesions on five leaves. The rate of lesion development was determined for each plant line. Greenhouse screening was repeated four times during three years to confirm resistance response of outstanding plant lines. Field testing of transgenic plant lines Transgenic plant lines and non-transgenic controls were tested both as self-rooted and grafted plants by planting in field sites at the University of Florida and the University of Virgin Islands. RESULTS AND DISCUSSION Transgene integration in independent transgenic plant lines Amplification of a 720 bp fragment corresponding to the EGFP gene (Figure 1A), a 798 bp fragment corresponding to the NPTII gene (Figure 1B) and a 686 bp fragment corresponding to the transgenic VVTL-1 sequence (Figure 1C) was observed in all transgenic lines and the positive control plasmid, whereas no amplification was observed in the non-transformed control. Production of transgenic VVTL-1 protein in independent transgenic plant lines ELISA was used to test plant lines for constitutive expression of transgenic VVTL-1 protein. The presence of the transgenic protein was detected in significantly higher levels in four transgenic plant lines compared to the negative control as indicated by absorbance values at 405nm wavelength (Figure 3). Figure 2. Testing transgenic plant lines for expression of transgenic VVTL-1 protein Absorbance values (405nm) Transgenic plant lines B. NPT II gene Control Plasmid Negative control 1 A. EGFP gene Negative control Control plasmid C. VVTL 1 gene Negative control Control Plasmid Figure 1. PCR analysis RESULTS AND DISCUSSION (Continued) Greenhouse screening and field testing of transgenic plant lines for resistance to powdery mildew Transgenic plant lines along with susceptible and resistant controls were screened for powdery mildew resistance in a greenhouse (Figure 3). Susceptible control plants developed severe disease symptoms 7 days after onset of first visible lesions (Figure 4A), receiving an average score of 5.0. Among 71 transgenic ‘Thompson Seedless lines tested, six percent of plant lines exhibited a 7-10 day delay in symptom development (Figure 4B) compared to the susceptible controls and received an average score ranging from 2.7 to 3.5 (Figure 5). The level of VVTL-1 expression in transgenic plants was positively correlated with disease resistance. The high level of disease pressure in the greenhouse test was demonstrated by the resistant control, which began to exhibit disease symptoms 17 days after the susceptible controls (Figure 5). The resistance of transgenic plant lines was confirmed by repeated screening for four times during three years. Resistant plant lines were used as scions and grafted on locally adapted rootstocks. Self rooted as well as grafted plants were planted at the University of Florida (Figure 6) and the University of Virgin Islands to study their response to fungal diseases under field conditions. In addition, transgenic VVTL-1 lines of V. vinifera ‘Cabernet Franc’, ‘Orange Muscat’ ‘Merlot’, ‘Shiraz’ and Vitis hybrid ‘Seyval Blanc’ are currently being screened in the greenhouse. CONCLUSIONS Grapevines resistant to powdery mildew were produced by transformation with a constitutively expressed VVTL-1 gene. The plants are currently being tested under field conditions for a range of fungal diseases endemic to sub-tropical and tropical environments. Figure 5. Powdery mildew symptom development in transgenic VVTL-1 plants following onset of first visible symptoms Figure 6. Field testing of transgenic grapevines in Florida Days Score REFERENCES Gray D.J., Jayasankar, S., Li, Z.T Vitis spp. Grape. In: Biotechnology of Fruit and Nut Crops 2005, Litz R.E. (ed) Wallingford Oxford CAB International. Jayasankar, S., Li, Z., Gray, D. J In vitro selection of Vitis vinifera Chardonnay with Elsinoe ampelina is accompanied by fungal resistance and enhanced secretion of chitinase. Planta, 211: Jayasankar, S., Li, Z., Gray, D. J Constitutive expression of Vitis vinifera thaumatin like protein after in vitro selection and its role in anthracnose resistance. Functional Plant Biology, 30: Li, Z., Jayasankar, S., Gray, D.J. 2001a. An improved enzyme linked immunosorbent assay protocol for the detection of small lytic peptides in transgenic grapevines (Vitis vinifera). Plant Mol. Biol. Rep., 19: Li, Z., Jayasankar, S. and Gray, D.J. 2001b. Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera). Plant Sci. 160: Li, Z., Jayasankar, S., Gray, D.J Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco. Transgenic Res., 13: Li, Z.T., Dhekney, S., Dutt, M., Van Aman, M., Tattersall, J, Kelley, K.T. and Gray, D.J Optimizing Agrobacterium-mediated transformation of grapevine. In Vitro Cell. Dev. Biol. - Plant, 42: Figure 3. Greenhouse screening of transgenic plant lines Figure 4. Powdery mildew symptom development in A. Control plant and B. Transgenic VVTL1 plant B. TS-VVT A. Control