Selecting Disease Resistant Transgenic Grapevine for Field Tests

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1 Selecting Disease Resistant Transgenic Grapevine for Field Tests D. J. Gray, Z. T. Li, S. A. Dhekney, M. Dutt, M. Van Aman, J. Tattersall & K. T. Kelley Mid-Florida Research & Education Center

2 Pierce s s Disease Endemic to Southeastern coastal plain of US (all of Florida) and California (increasing in incidence) Caused by Xylella fastidiosa Xylem limited bacterium that infects a range of vascular plants Transmitted by xylem-feeding insects Primarily leaf hoppers & sharpshooters Lethal to all Vitis vinifera varieties By blocking water flow through xylem Florida s s the 2 nd 3 rd largest consumer of grape products in the US, but must import nearly all due to PD

3 Transgenes Tested via Agrobacterium-Mediated Transformation Reporter gene Enhanced Green Fluorescent Protein gene (EGFP) Selectable marker - NPTII (kanamycin( resistance) Putative resistance genes Synthetic lytic peptide gene (bacterial resistance) V. vinifera thaumatin-like protein gene 1 (fungal resistance) EGFP & NPTII are fused and expressed with the Resistance gene via a Bi-Directional Promoter System

4 Typical Transformation Vector Based on a Bi- Directional Promoter (BDDP) and an EGFP/NPTII Fusion Gene Functional Gene CsVMV-BDPC Reporter/Marker Gene Term Lyt Pep or PR EGFP/NPTII Term Core Promoter Core Promoter Modified Enhancer Plant Science 160: , 887, 2001; Transgenic Research 13: , 154, 2004

5 Transgene Expression

6 A B C D GFP Expression in Thompson Seedless Grape The EGFP gene is linked to an antimicrobial lytic peptide gene GFP expressing plants are tested for resistance to Xylella fastidiosa A Leaves & Stems B Shoot Tip C Tendril D Roots

7 Testing Xylem Sap for Lytic Peptide Pure xylem sap is exuded from decapitated plants due to root pressure CK The presence of lytic peptide is determined by ELISA Transgenic Vines Test Samples Purified Protein

8 Detection of Transpeptide in Xylem Sap of Nontransgenic Scion Percent increase in absorbance over non transgenic control Non Transgenic control CS -1 CS -2 TS -1 TS -2 Transgenic control Treatments Detection of Shiva-1 protein in xylem sap by ELISA. Absorbance values from non transgenic Cabernet Sauvignon (CS) and Thompson Seedless (TS) scions, grafted onto transgenic Thompson Seedless Shiva-1 producing vines. The standard error values ranged from to

9 Screening Transgenic Plants

10 Testing Transgenic Plants for Resistance to Pierce s s Disease Plants inferred to express lytic peptide in xylem tissue, based upon GFP expression patterns, are selected for inoculation. Xylella bacterial suspension is injected directly into xylem tissue of transgenic and control plants. Plants are evaluated over time for resistance, based upon symptomology and endogenous bacterial levels.

11 Development of PD In Typical Screening Test Six Weeks Carignane Control Transgenic Thompson Seedless 10 Weeks

12 Pierce s s Disease Resistant Transgenic Grapevines Vines were inoculated July Only the resistant transgenic vines were re-inoculated Nov 8 Since even resistant controls developed symptoms, the PD test is considered to be stringent Lack of symptoms in transgenic plants suggests high level of resistance Susceptible Control Vines Transgenic Vines w Lytic Peptide Resistant Control

13 Testing Transgenic Grapevines for Powdery Mildew-Resistance Transgenic vines that express Vitis vinifera thaumatin-like protein (VVTL-1) were compared with susceptible and resistance controls in an area of high powdery mildew pressure. Powdery Mildew Test Site in Greenhouse See Poster 3021 Dhekney et al. Resistant Tampa Control Susceptible Thompson Control Transgenic Thompson

14 Testing Transgenic Grapevines for Powdery Mildew-Resistance Comparison of PM lesions 8 D after first onset of symptoms Selected transgenic vines that express Vitis vinifera thaumatin-like protein (VVTL-1) exhibit delayed symptom development. See Poster 3021 Dhekney et al. Susceptible Thompson Seedless Transgenic Thompson Seedless

15 Grapevines Transformed with Lytic Peptide Genes Vitis vinifera Cabernet Franc Chardonnay Merlot Pinot Noir Sauvignon Blanc Semillion Thompson Seedless White Riesling V. rotundifolia Alachua Vitis rupestris Vitis hybrids St. George (rootstock) Conquistador Seyval Blanc Freedom & Tampa (rootstock)

16 Grapevines Transformed with VVTL-1 1 Gene Vitis vinifera Merlot Shiraz Thompson Seedless Vitis hybrid Seyval Blanc

17 Applications of Grafting to use of Transgenic Grapevine Use of a transgenic rootstock to transmit peptides into a non-transgenic scion controls gene flow Allows widespread use of elite scions with few transgenic rootstocks Transgenic scions grafted onto transgenic rootstocks could confer multiple traits without problems encountered in gene stacking Control xylem-limited limited PD via rootstock and foliar fungal diseases with transgenic scion

18 Component Plants for a Florida Field Test Resistant and susceptible control varieties Non-grafted transgenics Study lines with either lytic peptide or VVTL-1 Use of seedless varieties eliminates concerns of seed dispersal Grafted transgenics Use adapted rootstocks selected to transmit lytic peptide in xylem sap Graft to non-transgenic scions and test for PD resistance No gene flow & a generic approach to PD resistance Graft to VVTL-1 1 scions and test for PD and fungal disease resistance