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Disease Resistant Transgenic Grapevine Constitutively Expresses Vitis vinifera Thaumatin-like Protein
SA Dhekney, ZT Li, MM Van Aman, M Dutt,
J Tattersall, KT Kelley and DJ Gray
Mid 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.
Testing of transgenic plant lines for detection of transgenic VVTL-1 protein
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 3. Testing transgenic plant lines for expression of transgenic VVTL-1 protein
(Figures in parenthesis represent mean absorbance values at 405nm wavelength)
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MATERIALS AND METHODS
Induction of embryogenic cultures
Embryogenic 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.
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Isolation of the VVTL-1 gene and construction of a binary vector
The 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 plants
A 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 lines
Genomic 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 protein
An 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).
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TS-VVT-1
(0.4506)
TS-VVT-2
(0.3173)
TS-VVT-3
(0.5083)
TS-VVT-4
(0.7353)
Susceptible
Control (0.1686)
Greenhouse testing of transgenic plant lines for resistance to powdery mildew incidence
Susceptible 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.
Figure 4. Powdery mildew symptom development in A. Resistant Control ‘Tampa’ B. Susceptible ‘Thompson Seedless’ and C. Resistant transgenic ‘Thompson
Greenhouse testing of transgenic plant lines for resistance to powdery mildew
Transgenic 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 lines
71 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.
Figure 1. PCR analysis of EGFP gene
Figure 2. PCR analysis of NPT II gene
A
B
C
Figure 5. Progression of powdery mildew symptom development in transgenic VVTL-1 grapevines following onset of first visible symptoms
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Negative
control
Plasmid
DNA
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2
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Negative
control
Plasmid
DNA
5
TS-VVT-1
4
3
Score
Score
References
Gray 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., 13:143-154
Li, 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.
TS-VVT-2
TS-VVT-3
TS-VVT-4
2
Resistant
Control
1
Susceptible
Control
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