Two Tobravirus expression vectors were evaluated for the use as a rapid screening method for anti-nutritional proteins against plant pathogens. Accumulation of green fluorescent protein (GFP) and snowdrop lectin gene (Galanthus nivalis agglutinin, LECGNA2, M55556) in Nicotiana benthamiana by Tobacco rattle virus expression vectors was characterized. Virally expressed proteins were detected in leaves (3-14 days post-inoculatiion) and roots (6-24 dpi) by UV (GFP), western blotting and tissue printing. 25 -50 ng of GNA was detected in root extracts. Cross protection was induced by TRV-GFP. Foreign genes inserted in place of TRV RNA2 non-structural genes (2b and 2c) were stably maintained over serial passages. But recombination at remaining 'cross-over' sites may occur. 2D iso-electricfocusing detected a 50-kDa GNA molecule in root and leaf extract. GNA did not confer resistance to root-knot nematodes, although gall by root-knot nematodes (mixed Meloidogyne spp. and M.javanica Crete line 17) were significantly reduced by 22% in roots infected by TRV-GNA (3.83 sqrt galls and 4.5 sqrt galls respectively) compared to virus-free roots treatment (4.94 sqrt galls, sed 0.398; p<.025 and 5.273 sqrt galls, sed 0.2403; <.003 respectively). Effects of GNA on Aulacorthum solani was delayed to the second nymph generation (N2). Mean N2 weights feeding on TRV-GNA (0.246 mg ±0.0159; p <05) and TRV-fsGNA (0.212 mg ±0.018; p<.001) infected plants were significantly smaller by 15.2% and 26.6% respectively, compared to virus-free treatments (0.290 mg ±0.014). Similar trends were detected for total nymph weights. Low toxicity was related to high quality phloem and ingestion of smaller volumes for normal development (i.e. concentration effect). Decrease in gall by the mixed Meloidogyne population and an unexpected toxicity to A solani indicated that truncated GNA was a protein with merolectin properties. The viability of this system as a rapid 'm planta' expression system is discussed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:251212 |
| Date | January 2002 |
| Creators | Popovich, Alexandra Helen |
| Publisher | Durham University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.dur.ac.uk/3758/ |
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