Disease Resistant Transgenic Grapevine Constitutively Expresses Disease Resistant Transgenic Grapevine Constitutively Expresses Vitis vinifera Vitis vinifera Thaumatin-like ProteinThaumatin-like Protein SA Dhekney, ZT Li, MM Van Aman, M Dutt, SA Dhekney, ZT Li, MM Van Aman, M Dutt,

J Tattersall, KT Kelley and DJ Gray J Tattersall, KT Kelley and DJ Gray Mid Florida Research and Education Center, University of Florida 2725 Binion Road, Apopka, FL 32703 USAMid Florida Research and Education Center, University of Florida 2725 Binion Road, Apopka, FL 32703 USA

INTRODUCTION

Grapevine produces a number of pathogenesis related (PR) proteins in response to biotic stresses. Induction of PR proteins, such as chitinases, has been demonstrated following challenge with fungal culture filtrates in several cell culture systems (Jayasankar et al., 2000). Grapevine embryogenic cultures that were subjected to in vitro selection with 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 (Jayasankar et al., 2003). The current study was conducted to explore the possibility of producing transgenic grapevines that constitutively express the VVTL-1 gene to achieve fungal disease resistance.

MATERIALS AND METHODS

Induction of embryogenic culturesEmbryogenic cultures of V. vinifera ‘Thompson Seedless’ were initiated from leaves (Gray et al., 1995) and maintained on X6 medium (Li et al., 2001b). Somatic embryos at the cotyledonary stage were utilized for genetic transformation.

Isolation of the VVTL-1 gene and construction of a binary vectorThe coding sequence of VVTL-1 was cloned from grapevine by PCR (Jayasankar et al., 2003) and placed into a CaMV 35S bi-directional duplex promoter (BDDP) complex (Li et al., 2004), which also contained a green fluorescent protein/neomycin phosphotransferase II (EGFP/NPT II) fusion gene (Li et al., 2001b). The fusion gene allowed for simultaneous selection of transgenic plants based on kanamycin resistance and GFP fluorescence.

Genetic transformation of embryogenic cultures and recovery of transgenic plantsA pBIN 19-derived binary vector harboring the BDDP was introduced into Agrobacterium tumefaciens ‘EHA 105’. Transgenic grapevines were regenerated following transformation of embryogenic cultures (Li et al., 2006), hardened in a growth chamber, and transferred to a greenhouse.

PCR analysis of transgenic plant linesGenomic DNA was extracted from young leaves of eight transgenic plant lines as well as from a non-transformed control plant. PCR was performed to detect specific DNA sequences in transgenic plants that corresponded to the EGFP and the NPTII genes.

Testing of transgenic plant lines for detection of transgenic VVTL-1 proteinAn antiserum to purified recombinant VVTL-1 protein (Jayasankar et al., 2003) was used to test leaves of transgenic plants and controls via ELISA (Li et al., 2001a).

Greenhouse testing of transgenic plant lines for resistance to powdery mildewTransgenic plant lines were replicated by vegetative propagation and screened for resistance to powdery mildew (Uncinula necator) in a greenhouse along with non-transformed ‘Thompson Seedless’ as a susceptible control and Vitis hybrid ‘Tampa’ as a resistant control. Individual plants were rated by recording the number of lesions on leaves and by assessing overall plant growth. Plants were scored three times a week on a scale of 1 to 5 (1 = no lesions, 2 = <10 lesions, 3 = 10-15 lesions, 4 = 15-20 lesions, 5 = >20 lesions per leaf) based on the number of lesions observed on five randomly selected leaves of an individual plant. The progress of lesion development was recorded over time for clones of each plant line and averaged to derive a score for disease resistance.

RESULTS AND DISCUSSION

PCR analysis of independent transgenic plant lines71 independent transgenic plant lines were regenerated based on resistance to kanamycin and visual assessment of GFP fluorescence. Genomic DNA was extracted from eight transgenic plant lines and used for amplification of the EGFP and NPT II genes. Amplification of a 720 bp fragment corresponding to the EGFP gene (Figure 1), and a 798 bp fragment corresponding to the NPT II gene (Figure 2) was observed in all transgenic lines and the positive control plasmid, whereas no amplification was observed in the non-transformed plant line.

ReferencesGray D.J. 1995. Somatic embryogenesis in grape. In: Jain S.M., Gupta P.K., Newton R.J. (eds) Somatic Embryogenesis in Woody Plants, vol. 2, pp. 191-217. Kluwer Academic Publishers, Dordrecht.Jayasankar, S., Li, Z., Gray, D. J. 2000. In vitro selection of Vitis vinifera Chardonnay with Elsinoe ampelina is accompanied by fungal resistance and enhanced secretion of chitinase. Planta, 211:200-208.Jayasankar, S., Li, Z., Gray, D. J. 2003. Constitutive expression of Vitis vinifera thaumatin like protein after in vitro selection and its role in anthracnose resistance. Functional Plant Biology, 30:1105-1115 . 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., 19:341-355.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: 877-887.Li, Z., Jayasankar, S., Gray, D.J. 2004. Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco. Transgenic Res., 13:143-154Li, Z.T., Dhekney, S., Dutt, M., Van Aman, M., Tattersall, J, Kelley, K.T. and Gray, D.J. 2006. Optimizing Agrobacterium-mediated transformation of grapevine. In Vitro Cell. Dev. Biol. – Plant, In Press.

1 2 3 4 5 6 7 8 Negative control

Plasmid DNA

Figure 1. PCR analysis of EGFP gene

1 2 3 4 5 6 7 8 Negative control

Plasmid DNA

Figure 2. PCR analysis of NPT II gene

Greenhouse testing of transgenic plant lines for resistance to powdery mildew incidenceSusceptible control plants developed severe disease symptoms 7 days after onset of first visible lesions (Figure 4), receiving an average score of 5.0 (Figure 5). Six percent of transgenic plant lines exhibited a 7-10 day delay in symptom development 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 found to be positively co-related with disease resistance. High disease pressure in the greenhouse was demonstrated by the resistant control, Vitis hybrid ‘Tampa’, which began to exhibit disease symptoms 17 days after the susceptible controls (Figure 5). Selected transgenic lines are being propagated, repeatedly re-screened and will be tested under field conditions. In addition, transgenic VVTL-1 lines of V. vinifera ‘Merlot’ and Vitis hybrid ‘Seyval Blanc’ have been regenerated and are currently being screened.

Testing of transgenic plant lines for detection of transgenic VVTL-1 proteinELISA 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 3. Testing transgenic plant lines for expression of transgenic VVTL-1 protein(Figures in parenthesis represent mean absorbance values at 405nm wavelength)

TS-VVT-1(0.4506)

TS-VVT-2(0.3173)

TS-VVT-3(0.5083)

TS-VVT-4(0.7353)

Susceptible Control (0.1686)

RI

RII

RIII

Figure 5. Progression of powdery mildew symptom development in transgenic VVTL-1 grapevines following onset of first visible symptoms

Days

Sco

re

Figure 4. Powdery mildew symptom development in A. Resistant Control ‘Tampa’ B. Susceptible ‘Thompson Seedless’ and C. Resistant transgenic ‘Thompson Seedless’

A B C