Plasmodesmatal function is probed using transgenic tobacco plants that express a virus movement protein.
A gene encoding a temperature-sensitive mutant (MPP154A) of the 30-kilodalton movement protein (MP) of tobacco mosaic virus (TMV) was transformed into Nicotiana tabacum cv Xanthi. Transgenic plants expressing the MPP154A gene complemented local and systemic movement of an MP-defective mutant of TMV (U3/12MPfs) at the permissive temperature of 24 degrees C but not at 32 degrees C, the nonpermissive temperature. A microinjection procedure was used to investigate the effects of the modified TMV MP on plasmodesmatal size-exclusion limits. Movement of fluorescein isothiocyanate-labeled dextran (F-dextran), with an average molecular mass of 9.4 kilodaltons, was detected between leaf mesophyll cells of the transgenic plants at 24 degrees C; however, no movement of either 3.9-kilodalton or 9.4-kilodalton F-dextrans was detected when the transgenic plants were held for 6 hours (or longer) at 32 degrees C. When these plants were shifted back to 24 degrees C for 6 hours, cell-to-cell movement of the F-dextrans was again observed. Accumulation of MPP154A was not affected by the temperature regime, nor was the subcellular distribution of the MP altered. These results are consistent with a change in the protein conformation of MPP154A at the nonpermissive temperature, which gives rise to a protein that fails to modify the molecular size-exclusion limits of plasmodesmata to the same extent as wild-type MP. Surprisingly, at 32 degrees C, movement of the F-dextrans was inhibited in transgenic plants expressing the wild-type MP gene; however, the inhibition was transient and was no longer detected after 48 hours at this elevated temperature. This transient inhibition of plasmodesmatal function was alleviated with Sirofluor, an inhibitor of callose ([1----3]-beta-D-glucan) synthesis. This result provides experimental evidence that callose deposition is involved in regulating the molecular size-exclusion limit of plasmodesmata in plants. Sirofluor had no effect on the inhibition of F-dextran movement at 32 degrees C in plants expressing the MPP154A gene, indicating that callose formation was not responsible for the failure of the temperature-sensitive mutant protein to alter the size-exclusion limit of plasmodesmata.
Wolf
S
S
Botany Department, University of California, Davis 95616-8537.
Deom
C M
CM
Beachy
R
R
Lucas
W J
WJ
D001557:Benzenesulfonates / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000502:physiology*; D002473:Cell Wall / Q000502:physiology*; D003911:Dextrans / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D005936:Glucans / Q000378:metabolism; D006358:Hot Temperature; D009154:Mutation; D015388:Organelles / Q000502:physiology*; D053830:Plant Viral Movement Proteins; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D014026:Nicotiana / Q000502:physiology*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
D001557:Benzenesulfonates; D003911:Dextrans; D005456:Fluorescent Dyes; D005936:Glucans; D053830:Plant Viral Movement Proteins; D014764:Viral Proteins; C046065:4,4'-(carbonylbis(benzene-4,1-diyl)bis(imino))bis(benzene sulfonate) sodium salt; C048306:callose