PHYTO MEDIZIN Mitteilungen der Deutschen ... - Die DPG

conserved viral replicase–encoding regions. Ornamental Allium plants
showing diffuse yellow stripes or slight mottle were investigated for virus
infection. ELISA and PCR assay showed the presence of a complex of different
viruses consisting of Potyviruses, Tobacco rattle virus, Tobacco necrosis
virus and a novel (unknown) Potexvirus. Sequence analysis of cloned PCR
amplicon obtained with the primers Potex 4 – Potex 5, showed an 83% identity
with Clover yellow mosaic virus. Testing the method on primarily infected
plants and dormant bulbs of different Allium cultivars has verified the
robustness for application in the routine detection of potexvirus infections.
Detection, performed in microtiter plates, was rapid, specific, contamination
free, reproducible and semiautomated.
The nucleoprotein gene of Tomato spotted wilt virus as a tag protein fusion
for easy purification and enhanced production of recombinant protein
in plants
C Lacorte 1,2 , D Lohuis 1 , R Goldbach 1 and M Prins 1; 1 2Laboratory of Virology,
Wageningen University, Binnenhaven 11, 6709 PD, Wageningen, and EMBRAPA-
Recursos Genéticos e Biotecnologia, Brasília, DF
Upon infection, Tomato spotted wilt virus (TSWV) forms ribonucleoprotein
particles (RNPs) that consist of nucleoprotein (N) and viral RNA. These
aggregates result from the homopolimerization of the N protein, and are
highly stable in plant cells. These properties feature the N protein as a potentially
useful novel plant-based protein fusion system. To evaluate this potential,
we tested whether the N protein and N protein fusions could be
efficiently produced in plants and purified as aggregates, outside the context
of viral infection. To this end the N gene was fused to GFP (Green fluorescent
protein), either at the amino or carboxy terminus, in a binary vector or in
a Potato virus X (PVX) vector. Nicotiana benthamiana leaves were infiltrated
by Agrobacterium tumefaciens transient assay (ATTA) or inoculated
with a PVX viral vector. For both expression methods N and N-GFP fusion
could be detected by Western blot using antisera against N or GFP. Infiltrated
leaves and infected plants expressing N-GFP fusions showed intense fluorescence
under UV light. For purification, a standard method used for viral
RNPs was used, consisting of two low speed centrifugation steps and one
ultracentrifugation on a sucrose cushion. The presence of N and NGFP fusions
after this purification was confirmed by Western blot. Purified N-GFP
retained its fluorescence, indicating no major changes in protein structure due
to the (mild) purification process. These results show that the homopolimerization
properties of the N protein can be used as a fast and simple way to
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