NEWS AND COMMENT

LT receptor, TNF-P (or LT) can bind to both pSS and ~75 TNF-a receptors.

It will be attractive to evaluate the response of these TNF-inhibitor- expressing mice in the context of several pathological conditions. The technique described by Kolls et al. will certainly become a very use- ful tool to analyse cell-cell intcr- actions on a molecular level in in vim models. The significance of the method of Kolls et al. may not be as a suggested method for gene ther- apy, but rather as an elegant tech-

nique to replace the USC of knock- out mice, in that it might create a ‘conditional knockout’ for any gene of interest.

1 Kolls, J, et al. (1994) Puoc. Nritl Acnd. ki. USA 91, 215.-219

2 Beutler, B. (1990) 1, Invest. Dcrmatol. 95, S81-S84

3 Grau, G.E. et al. (1993) ht. Rev. Exp. Pathol. 34, 159-172

4 Grau, GE. and Modlill, R.L. (1991) Cuv. Opin. Immunol. 3,480-485

5 Beutler, B. and Grau, GE. (1993) Crit. Care Med. 21, S423-S435

6 Grau, G.E. (1990) Eur. Cytokine Network 1, 203-210

7 Derkx, B. et al. (1993) Lancet 342, 173-174

8 Maini, R.N. et nl. (1993) C/m. Exp. Rheumntol. 11, S173-S17.5

9 Hung, CL. et al. (I 994) Gene Tber. 1, 64-69

10 Randara, G. et al. (1993) Puoc. Nat1 Acad. Sci. USA 90,10764-10768

11 Zhu, N. et al. (1993) Science 261, 209-211

12 de Kmsodo, S. et al. (1992) 1. Exp. Med. 176,1259-1264

13 de Togni, 1’. et al. (1994) Science 264, 703-707

Plant-virus movement: de novo process or redeDloved machinerv?

d I d

Andy 1. Made

T hree papers have appeared recently that provide a basis for the view that cell-to-

cell trafficking of macromolecules through plant plasmodesmata is not unique to virus infection, but that plant viruses exploit an existing mechanism of cellular communi- cation in plants to spread the infec.- tion through the tissues’-“. This view has been expanded recently to in- clude the idea that plasmodesmata mediate a ‘supracellular’ control of plant processes”.

It has long been recognized that plant viruses encode proteins that mediate viral movement through plasmodesmata, and that these pro- teins can modify plasmodesmal structure and function4J. Pioneering work in this field used the tobacco mosaic virus (TMV) movement pro- tein (Ml’) P30 and the technique of microinjection to assess the size exclusion limit (SEL) of plasmo- desmata in the presence and ab- sence of the MP. By microinjecting fluorescently labelled dextrans of different sizes into cells in trans- genie tobacco plants expressing P30 and monitoring their diffusion into adjacent cells, the normal SEL of <I kDa in non-transformed tissue

was shown to increase to >I0 kDa in the presence of P30 (Ref. 6). This increase corresponds to a change in the functional diameter of the microchannels within plasmo- desmata from 1.2-1.8 nm to 2.4- 3.1 nm, which is still too small to allow the passage of the intact virus or of its genomic RNA. Howcvcr, I’30 can bind to and denature single-stranded (ss) RNA to pro- duce long, thin (~2 nm) ribonucleo- protein complexes7, which might be able to pass through the altered microchannels.

Some other viruses have been shown to have similar properties: their MP can bind to nucleic acids and their coat protein is not necessary for cell-to-cell move- ment. However, this is not always the case: cell biological obser- vations of virus particles within structurally modified plasmo- desmata suggest that a further class of viruses, including the como- viruses8 and the caulimoviruses9, move as intact virions.

A.]. Made 1s WI the Dept of Virus Resemch, john lnnes Ccntx, Coinay i.ane,

Norwich, UK NR4 7UH.

0 1994 Plsevicr Science 1 td WGh 842X/94/$07.00

MPs traffic through plasmodesmata TMV and two functionally related viruses, red clover necrotic mosaic virus (RCNMV) and bean dwarf mosaic virus (BDMV), are the sub- jects of the three recent papers men- tioned above’-“. Whereas TMV and RCNMV have ssRNA genomes and single Ml’s (TMV P30; RCNMV 35 kDa protein), BDMV (a gemini- virus) has a genome of ssDNA and two proteins (BLl and BRl) that arc necessary for a spreading infec- tion in plants. These papers all re- port a novel and elegant approach to assess the effect of MI’s on plas- modesmal function. The proteins were expressed in Escherichia coli, and the purified recombinant pro- tcins were directly comicroinjected with fluorescent dextrans into meso- phyll cells of host plants and the SEL assessed. This approach was extended in two cases1x3 by study- ing the fate of fluorescently labelled MI’ and by directly measuring the ability of the Ml’ to transport nucleic acids by co-injecting MP and fluorescently labcllcd RNA or DNA. 1’30, RCNMV 35 kDa pro- tein and BDMV RI,1 protein all in-, crease the SEI, of plasmodcsmata

NEWS AND COMMENT

L_

-_- Table 1. Movement-protein-mediated transport of

macromolecules through plasmodesmata: how long does it take?”

Time taken for transport

Macromolecule RCNMV

35 kDa protein I TMV P30 (Ref. 2)

BDMV BLI. (Ref. 3)

10 kDa dextranb <30 s 3-5 min 30s 20 kDa dextranb ND lo-15 min _ MP” 13 min ND <GO s SSRNA~.~ <30 s ND _ SSDNA~,~ _ ND - dsDNAbmd - ND 1.5 s

TMV, tobacco mosaic virus; RCNMV, red clover necrotic mosaic virus; BDMV, bean dwarf mosaic virus; ss, single stranded; ds, double stranded: MP, movement protein; ND, not determined; -, no movement observed. bMolecules co-injected with virus MP. CFluorescently labelled MP injected alone. dlncluding infectious viral nucleic acid.

-

not only in the cells that were micro- injected, but also in cells further from the site of injection. This is a consequence of the movement of the protein itself through plasmo- desmata. The estimated change in the functional size of the micro- channels in plasmodesmata2 is suf- ficient to allow passage of the MI%.

Speed of macromolecular trafficking through plasmodesmata However, it was the speed with which the movement occurred that was noted particularly’-“. In all cases, the movement of large dex- trans, proteins or nucleic acids occurred between 30s and a few minutes after injection, with no apparent difference bctwcen dcx- trans moving by diffusion and the trafficking of proteins and nucleic acids (Table I). The rapidity of the process led the authors to propose that the MPs interacted with or exploited an existing pathway for the transport of macromolecules through plasmodesmata. This is a substantial departure from the dogma that plant cells, although cytoplasmically connected through plasmodesmata, transfer infor- mation through the action of small signal molecules or electrical stimuli.

What do we know of the speed of biological processes? Certainly, the speed at which major intracellular events can take place is frequently

underestimated. For example, the formation of the sperm acrosomal process, which is 60 pm long, can take as little as 7 s, which includes the polymerization of actin and the rapid addition of membrane to cover the elongating processlo. Intuitively, 30 s seems to be too little time for the induction of a complete new pathway for the cell-to-cell trafficking of viral proteins and nu- cleic acids, but how can we make a realistic judgcment? Is it possible that, contrary to the authors’ view, macromolecular trafficking is not a normal plasmodesmal function, but rather either that the MI’s func- tion alone to move macromolecules or that they can redeploy existing cellular proteins (perhaps locally) for this purpose. 7 Since neither of these possibilities require protein synthesis to occur, is 30 s then too short a time? Perhaps the strongest evidence in favour of macromolecu- lar trafficking via plasmodesmata in normal tissues would be to idcnt- ify host proteins that are present at locations distal to the site at which their genes arc cxpressed4.

Future prospects The microinjection of combinations of dcxtrans, proteins and nucleic acids has shown itself to be a power- ful tool in the analysis of virus MPs. It is legitimate, however, to ques- tion whether the microinjcction of

supersaturating qua&tics of MP

might identify activities that do not represent the normal function. Earlier work with related gcmini- viruses’ ‘,12 identified separate func- tions for BE1 and BRl by muta-, genesis, and showed that only one protein was necessary for cell-to-. cell movement, while the other mediated virus movement in the vascular tissues. In contrast, micro- injection of BDMV BRl (Ref. 3) revealed a fascinating activity in the export of ssDNA and doublc- stranded (ds) DNA out of the nu- cleus, while BDMV BLl mediated the trafficking of dsDNA through plasmodesmata. In this case, these separate activities would both be csscntial for movement of BDMV from cell to ce113. Nevertheless, from these three papers alone (and there will doubtless be more), it is clear that a microinjection approach has advanced significantly our knowl- edge of the function of MPs. Furthermore, since the virus MPs remain the only proteins charac- terized that are incontrovertibly associated with plant plasmo- dcsmata, the technique may hold the key to understanding plant-cell communication.

Acknowledgements I thank Licsi Waigmann, Keith Koberts alld Margaret Boulton for discussions of this work.

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