PHYTO MEDIZIN Mitteilungen der Deutschen ... - Die DPG

cell movement within plants. By use of two-hybrid system, we identified
BC1-interacting plant factors, which are currently under investigation.
Interaction between Cowpea mosaic virus movement tubules and the
plasma membrane
JWM van Lent 1 , J Pouwels 2 , M Rolloos 1,2 , CM Carvalho 1 , J Wellink 2 and RW Goldbach
1; 1 Laboratory of Virology, Wageningen University and 2 Laboratory of Molecular
Biology, Wageningen University
Cowpea mosaic virus (CPMV) virions traverse the cell wall through nanotubules
that are assembled from the viral movement protein (MP). In planta
these tubules are found exclusively in the plasmodesmal canal. In isolated
plant protoplasts, devoid of cell walls and plasmodesmata, a similar polarity
of tubule assembly is observed. The MP anchors somehow at the plasma
membrane (PM) and from there an outward growing tubule is assembled
tightly encased with PM as also occurs within the plasmodesma. Transport
tubules are not only formed in protoplasts of host plants, but also in those
isolated from non-host plants or even animal cells that express the MP. Obviously,
MP interacts with the PM or with PM-associated proteins and such
proteins should be conserved in plant and animal cells. This intriguing interaction
between MP and PM was further analyzed by MP-mutant analysis,
timelapse microscopy and FRET/FRAP studies. It has been shown before
that prior to tubule formation, MP (-GFP) accumulates in so-called peripheral
punctate spots at the PM and it was speculated that these were the nucleation
sites for tubule assembly. Time-lapse fluorescent microscopy was performed
on protoplasts expressing MP-GFP and showed that the punctate spots are
dynamic structures that are immobilized at the PM and that tubules indeed
arise from (a sub-population of) these spots. To investigate the interaction of
tubules with the surrounding PM, the diffusion herein of small and large
fluorescent PM-associated proteins was examined by fluorescence recovery
after photobleaching (FRAP). These studies indicate that tubules made by
CPMV MP do not interact directly with the PM, but most likely via a PM
intrinsic protein (PIP) or otherwise associated host protein. Using affinity
chromatography it was found that purified MP a.o. binds to aquaporin present
in the plasmamembrane. Transient expression of MP-YFP and Arabidopsis
aquaporin P1;4-CFP revealed that these proteins colocalize in punctuate spots
on the surface of cowpea protoplasts. Acceptor photobleaching experiments
indicate that they also physically interact at these sites, suggesting that aquaporin
is used by the virus as a primer for the tubule formation at the plasma
membrane.
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