1. GUS Reporter Gene Assay
The GUS assay was used to measure the efficiency of delivery and expression
of foreign DNA in legume leaves.
v14 days post inoculation, agro-infiltrated leaveswere collected and vacuum
infiltrated with X-gluc (C14H13BrClNO7). This substrate reacts with GUS
to produce a blue pigment following incubation of leaves for 24-72 hours
(Fig. 6).
v Leaves were destained in absolute ethanol to remove chlorophyll and
excess X-gluc substrate, with a series of washes.
Table 2: Legume varieties that were GUS positive (the appearance of blue) or GUS negative (no blue / no
difference between GUS and control)
*GUS-infiltrated leaves did not stain blue but remained black (while negative controls de-stained)
Figure 6 (Left): GUS positive leaves (bottom) compared to buffer infiltrated leaves (top) of Pinto bush bean;
(right): GUS negative leaves and control leaves of Hairy vetch.
RNA EXTRACTION
vTwo and four weeks post inoculation, totalRNAwas extracted
(OMEGA Biotek Plant RNAKit) (Fig. 7) from petioles of agro-
inoculated plants.
vRNA concentration was measured with the NanoDrop (ThermoFisher
Scientific) and diluted for RT-PCR
Figure 7: Steps of the RNA extraction protocol.
REVERSE TRANSCRIPTION POLYMERASE CHAIN
REACTION (RT-PCR)
vPrimers (Table 3) bridging a splice site in the GRBaV RepA gene were
used in RT-PCR with the Qiagen One-step RT-PCR kit to detect virus
replication.
vPCR products were analyzed by gel electrophoresis and GelRED nucleic
acid staining (Fig. 8)
A pre-requisite for a plant to be considered an alternative host
of GRBaV is for the virus to be delivered through agro-
inoculation and replicate within the plant.
Results of RT-PCR revealed that Vermont cranberry dry bean
and Hi-Style snap bean yielded the spliced GRBaV-RepA
amplicon (Table 4).
Table 4: Cumulative number of plants for each legume variety that tested positive for
GRBaV by RT-PCR at 2 and 4 weeks post inoculation.
Exploration ofAlternative Hosts for
Grapevine red blotch-associated virus
VictoriaPoplaski1 Elizabeth Cieniewicz2 Marc F. Fuchs2
1
Oberlin College, Oberlin, OH 44074; 2
Section of Plant Pathology and Plant Microbe Biology,
School of Integrative Plant Science, Cornell University, New York State Agricultural
Experiment Station, Geneva, NY 14456
Methods
Results
Acknowledgements
References
Conclusions and Discussion
Future Studies
v Agro-inoculation experiments will be replicated in
growth chambers to confidently rule out some legumes
as alternative hosts of GRBaV.
v The three cornered alfalfa treehopper will be allowed to
feed on suspected alternative hosts, e.g. Vermont
cranberry dry bean and Hi-style snap bean. Acquisition
of GRBaV by this insect vector would provide further
evidence of these plants as alternative hosts of GRBaV.
v Transmission experiments will be conducted to
determine the transmissibility of GRBaV from
alternative hosts à insect à new host, including
grapevine.
v Determining the presence of GRBaV in legume cover
crops in vineyard row middles will be important to assess
the epidemiological significance of alternative hosts.
v This and other studies are important for extending
disease management recommendations to grape
growers.
- Yen Mei Cheung
- Dave MacUmber
- Patricia Marsella-Herrick
- Larissa Osterbaan
- Maddison Flasco
MethodsIntroduction
Grapevine red blotch-associated virus (GRBaV) is a recently recognized
virus of grapevine that causes characteristic foliar blotches(Fig. 1). GRBaV
delays fruit ripening, increases titratable acidity, and alters juice chemistry,
affecting the profitability of vineyards. GRBaV has been detected in all
major viticulture regions of the United States, likely due to transmission via
infected propagation material and grafting. GRBaV isalso transmitted by
the three cornered alfalfa treehopper (Spissistilus festinus Say) (Fig. 2). S.
festinus is not generally considered a pest of grapevine, but can cause
economic losses in fabaceous crops, including soybean, alfalfa, and peanut.
These legume species are often sown in vineyard row middles as cover
crops, which warrants their evaluation as potential reservoirs of GRBaV.
Hypothesis: Legumes, which often are sown in vineyard row middles, can be
alternative hostsof GRBaVin vineyards.
Objective: Evaluate a variety of legumesfor the ability to host GRBaV.
Fourteen varieties of fabaceous plants were inoculated with an infectious
bitmer clone of GRBaV via Agrobacterium tumefaciens-mediated
inoculation (Table 1). After two and four weeks, petiolesof agro-
inoculated plants were collected and tested for GRBaV by reverse
transcription polymerase chain reaction (RT-PCR).
Agroinfiltrationof Legumes
vAgrobacterium tumefaciens transformed with an infectious bitmer
construct of GRBaV isolate NY358 was grown on LB agar plates
containing antibiotics (kanamycin + rifampicin) as selectable markers
vAgrobacterium tumefaciens with a β-glucuronidase (GUS) intron was
used as a control.
vHealthy legumes were infiltrated with agro/GRBaV via vacuum
infiltration or syringe infiltration (Fig. 3 & 4)
Legume Varieties
Table 1: Legume varieties inoculated with GRBaV via agro-infiltration.
Figure 2. Three cornered alfalfa treehopper
(Spissistilus festinus), a vector of GRBaV
Figure 1. Symptoms of red blotch disease on Vitis
vinifera cv. Cabernet franc.
Species Common Name
1. Phaseolusvulgaris‘Vermont
Cranberry’
Vermont cranberry dry
bean
2. Phaseolusvulgaris‘Pinto’ Pinto bush bean
3. Pisum sativum‘Maxum’ Maxum field peas
4. Pisum sativum‘Austrian’ Austrian pea
5. Trifoliumincarnatum Crimson clover
6. Trifoliumrepens New Zealand white clover
7. Vicia faba ‘Windsor’ Windsor fava beans
8. Vicia faba ‘Sweet Lorane’ Sweet Lorane fava bean
9. Vigna unguiculata Cowpeas
10. Phaseolusvulgaris‘Hi Style’ Hi Style snap bean
11. Medicago sativa Alfalfa
12. Trifolium pratense Red clover
13. Lotus corniculatus Birdsfoot trefoil
14. Vicia villosa Hairy vetch
Legume Variety 2 wpi 4 wpi
Pisum sativum ‘Maxum’ 0/10 0/10
Phaseolus vulgaris ‘Pinto’ 0/10 0/10
Vicia faba ‘Sweet Lorane’ 0/10 0/10
Vicia faba ‘Windsor’ 0/10 0/10
Phaseolus vulgaris
‘Vermont Cranberry’
1/8 1/8
Pisum sativum ‘Austrian’ 0/10 0/10
Pisum sativum ‘Maxum’ 0/10 0/10
Medicago sativa 0/10 0/10
Viciav illosa 0/10 0/10
Trifolium incarnatum 0/10 0/10
Trifolium pratense 0/10 0/10
Lotus corniculatus 0/10 0/10
Phaseolus vulgaris ‘Hi
Style’
1/10 1/10
Vigna unguiculata 0/10 0/10
•Fullner, K. J., & Nester, E. W. (1996). Temperature affectsthe T-DNA transfer machinery of Agrobacterium
tumefaciens. Journal of Bacteriology, 178(6), 1498-1504.
•Husk, A., Hamorsky, K. T., & Matoba, N. Monoclonal Antibody Purification (Nicotiana benthamiana plants).
•Santos-Rosa, M., Poutaraud, A., Merdinoglu, D., & Mestre, P. (2008). Development of a transient expression system in
grapevine via agro-infiltration. Plant cell reports, 27(6), 1053-1063.
•Sudarshana, M. R., Perry, K. L., & Fuchs, M. F. (2015). Grapevine red blotch-associated virus,an emerging threat to
the grapevine industry. Phytopathology, 105(7), 1026-1032.
GUS Positive Varieties GUS Negative Varieties
Crimson clover
Red clover
Alfalfa
Vermont cranberry dry bean
Pinto bush bean
Hi-Style snap bean
Cowpeas
Birdsfoot trefoil
Hairy vetch
New Zealand White clover
Windsor fava bean*
Sweet Lorane fava bean*
Maxum field pea
Austrian pea
P1533 [Reverse Primer] P1376 [Forward Primer]
5'-CAAAACGAACTCTACGTGGAAG -3' 5'-TTACAAGGCAAATATTGGAATG-3’
Table 3: GRBaV splice-bridging primers used for RT- PCR
v GRBaV replicates in two Phaseolus vulgaris varieties.
v The role of Vermont cranberry dry bean and Hi-style
snap bean as virus reservoir for transmission by insect
vectors will need to be evaluated.
v No replication of GRBaV was detectable in most plants
that were agro-inoculated. We speculate that high
temperatures (>29°C) in the greenhouse in the summer
months did prevent or slow the transfer of T-DNA by A.
tumefaciens, as well as virus replication. Follow up
experiments to test alternative hosts of GRBaV will be
completed in growth chambers, where lower
temperatures can be maintained.
v Plants that were GUS-negative did not respond well to
vacuum infiltration. In future experiments the syringe
infiltration method will be used to inoculate tissue of
these plants.
Figure 8: Amplification of
GRBaV spliced and
unspliced bands by RT-
PCR and electrophoresis on
a 2% agarose gel. Presence
of spliced amplicon
signifies replicating virus.
210 bp (spliced)
366 bp (unspliced)
Figure 3: Agroinfiltration
via syringe method
Figure 4: The vacuum
chamber and pump used for
vacuum infiltration