Plasmodesmatal function is probed using transgenic tobacco plants that express a virus movement protein.

Abstract

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.

Companion-Cell Specific Localization of Sucrose Synthase in Zones of Phloem Loading and Unloading

“…Among them are the overall area of the sieve plate together with the size and number of the pores within the sieve plate itself. Recent evidence (Wolf et al, 1991) confirms earlier studies showing the potential impact of callose ([ 1+3]-/3-~-glucan) accumulation in the regulation of both cell-to-cell and long-distance transport in plants. Deposition of this polysaccharide reduces the size-exclusion limit of the sieve-plate pore in sieve tubes and, hence, constricts the cell-to-cell movement within the strands.…”

“…The enzyme may also have a key role in the synthesis of callose, a (1+3)-/3-~-glucan that is a common structural and/or storage polysaccharide in higher plants (McNairn and Cumer, 1968). Callose has been implicated in the plugging of sieve plates following injury to phloem and in controlling symplastic transport through its deposition in plasmodesmata (Wolf et al, 1991). Previous studies have shown that the preferred substrate for production of callose is UDP-Glc (Morrow and Lucas, 1987).…”

A comparison of two methods of microinjection for assessing altered plasmodesmal gating in tissues expressing viral movement proteins

“…It has been observed that heterologously expressed viral MPs upon microinjection can trigger an increase of the plasmodesmal SEL allowing the passage of dextran molecules with a molecular mass up to 10 kDa (Ding et al, 1995;Fujiwara et al, 1993;Noueiry et al, 1994;Waigmann and Zambrisky, 1995;Waigmann et al, 1994). Similar results were obtained for transgenic plants expressing the MPs of TMV (Deom et al, 1987(Deom et al, , 1990Ding et al, 1992;Wolf et al, 1989Wolf et al, , 1991, cucumber mosaic virus (CMV; Vaquero et al, 1994) and alfalfa mosaic virus (AMV; Poirson et al, 1993).…”

“…fluorescent probes Ͼ1000 Da and viral RNA) to pass from one cell to another. These observations of increase of SEL are based on pressure microinjection studies involving the injection of purified MPs (Ding et al, 1995;Fujiwara et al, 1993;Noueiry et al, 1994;Waigmann and Zambrisky, 1995;Waigmann et al, 1994), and injection of transgenic plants expressing the MP of TMV (Deom et al, 1987(Deom et al, , 1990Ding et al, 1992;Wolf et al, 1989Wolf et al, , 1991, CMV (Vaquero et al, 1994) and of AMV (Poirson et al, 1993). In this paper we have compared this broadly applied pressure injection technique with iontophoresis, which may represent an alternative injection technique to study the effect of plant viral MPЈs on plasmodesmal gating.…”

“…To test whether such tubule-forming viral MP affects the plasmodesmal SEL in a similar way as described for TMV MP (Deom et al, 1990(Deom et al, , 1991Ding et al, 1992;Wolf et al, 1989Wolf et al, , 1991, pressure injection studies were carried out on the transgenic NS M expressor plants, using the fluorescent probe Lucifer Yellow CH (LYCH) linked to dextran molecules of increasing molecular mass. The three NS M transgenic lines described above (A25-13; A25-26; A25-31) were all included in these studies using non-transgenic tobacco lines as negative controls.…”