Plasmodesmata (Gateways for virus invasion)

Biochemical and genetic analysis of plasmodesmata

Plasmodesmata (sing. Plasmodesma) form symplastic channels through plant cell walls. They regulate the trafficking of large molecules from cell to cell through restriction or relaxation of their size exclusion limit (SEL). Since viruses are restricted to the plant intracellular environment (or symplast), these channels are crucial to successful tissue invasion. It is now clear that many functions in plants operate non-cell-autonomously (i.e. function in one cell using molecules generated in another cell/tissue). These include the actions of some transcription factors, some small RNAs involved in RNA-silencing mediated defence and a range of protein and RNA molecules present in the vascular phloem elements. Despite the importance of these structures for growth and development, and defence, virtually nothing is known about plasmodesmal structure and function.

To address this gap in our knowledge of a central element in plant biology, my lab has undertaken a programme of biochemical and genetic analysis of plasmodesmata to identify their functional and structural components.

We have chosen to use Arabidopsis cell suspension cultures as source material for the isolation and proteomic analysis of clean cell walls (Bayer et al 2005; 2006). In these wall preparations, plasmodesmata constitute the only visible membranous structures. Based upon the premise that plasmodesmata are rich in membranes, we have concentrated our attention of membrane proteins from the wall.

We have identified two new families of plasmodesmal proteins that we call plasmodemata-located proteins 1 and 2 (or Pdlp1 and 2).

Pdlp1 comprises a family of eight proteins that have a receptor-like structure. They are type 1 membrane proteins with a single trans-membrane domain, a short C-terminal tail and a larger N-terminal apoplastic domain consisting of two DUF26 (domain of unknown function) domains.

Pdlp2 comprises a small family of three glycosylphopshoinositol (GPI) – anchored proteins. These proteins are anchored at the C-terminus into the extracellular face of the plasma membrane and extend an X8 domain into the apoplast. We have shown that the X8-domain can bind to callose in the near cell wall. Callose is an important molecule in plasmodesmata that is located in the wall adjacent to the neck regions where it is involved in the regulation of the plasmodesmal aperture and molecular gating. Using immunogold labelling techniques, we have shown that Pdlp2 is located precisely in the neck region of plasmodesmata.

 

Intracellular targeting to plasmodesmata

What specifies the molecular targeting of proteins to plasmodesmata was completely unknown. 

Studying Pdlp1, we have identified by deletion analysis the amino acid sequence that directs these proteins to plasmodesmata.

Surprisingly, the only sequence necessary to achieve this is the 21 amino acids that constitute the Pldp1 trans-membrane domain. By fusing just this sequence to YFP we were able to direct the fusion protein to plasmodesmata.

Seed transmission of viruses

Seed transmission of pea seed-borne mosaic virus in pea seeds occurs by invasion of the embryo after fertilisation

This work, which has been brought to completion, throws new light on the importance of plasmodesmata in mediating virus movement to achieve virus seed transmission. The work builds on our extensive study of the seed transmission of PSbMV in pea.

We showed some years ago that PSbMV was transmitted into pea seeds by direct embryo invasion after fertilisation. We did not, however, resolve how this could be achieved when the dogma for embryo development had been that the maternal and filial tissues were symplastically isolated.

Using high resolution electron microscopy and immunogold staining (Roberts et al, 2003) we have obtained evidence that the wall between the maternal testa tissues of the seeds and the endosperm may, in fact have plasmodesmata capable of trafficking the virus. Further, our studies showed that the embryonic suspensor along which the virus must pass to infect the embryo has pores at its base that could allow entry of the virus.