A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins.

Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

Specific targeting of a plasmodesmal protein affecting cell-to-cell communication.

Plasmodesmata provide the cytoplasmic conduits for cell-to-cell communication throughout plant tissues and participate in a diverse set of non-cell-autonomous functions. Despite their central role in growth and development and defence, resolving their modus operandi remains a major challenge in plant biology. Features of protein sequences and/or structure that determine protein targeting to plasmodesmata were previously unknown. We identify here a novel family of plasmodesmata-located proteins (called PDLP1) whose members have the features of type I membrane receptor-like proteins. We focus our studies on the first identified type member (namely At5g43980, or PDLP1a) and show that, following its altered expression, it is effective in modulating cell-to-cell trafficking. PDLP1a is targeted to plasmodesmata via the secretory pathway in a Brefeldin A-sensitive and COPII-dependent manner, and resides at plasmodesmata with its C-terminus in the cytoplasmic domain and its N-terminus in the apoplast. Using a deletion analysis, we show that the single transmembrane domain (TMD) of PDLP1a contains all the information necessary for intracellular targeting of this type I membrane protein to plasmodesmata, such that the TMD can be used to target heterologous proteins to this location. These studies identify a new family of plasmodesmal proteins that affect cell-to-cell communication. They exhibit a mode of intracellular trafficking and targeting novel for plant biology and provide technological opportunities for targeting different proteins to plasmodesmata to aid in plasmodesmal characterisation.

Arabidopsis plant homeodomain finger proteins operate downstream of auxin accumulation in specifying the vasculature and primary root meristem.

In Arabidopsis thaliana, auxin is a key regulator of tissue patterning in the developing embryo. We have identified a group of proteins that act downstream of auxin accumulation in auxin-mediated root and vascular development in the embryo. Combined mutations in OBERON1 (OBE1) and OBERON2 (OBE2) give rise to obe1 obe2 double mutant seedlings that closely phenocopy the monopteros (mp) mutant phenotype, with an absence of roots and defective development of the vasculature. We show that, in contrast to the situation in mp mutants, obe1 obe2 double mutant embryos show auxin maxima at the root pole and in the provascular region, and that the SCF(TIR1) pathway, which translates auxin accumulation into transcriptional activation of auxin-responsive genes, remains intact. Although we focus on the impact of obe mutations on aspects of embryo development, the effect of such mutations on a broad range of auxin-related gene expression and the tissue expression patterns of OBE genes in seedlings suggest that OBE proteins have a wider role to play in growth and development. We suggest that OBE1 and OBE2 most likely control the transcription of genes required for auxin responses through the action of their PHD finger domains.

Arabidopsis cell wall proteome defined using multidimensional protein identification technology.

With the completion of the sequencing of the Arabidopsis genome and the recent advances in proteomic technology, the identification of proteins from highly complex mixtures is now possible. Rather than using gel electrophoresis and peptide mass fingerprinting, we have used multidimensional protein identification technology (MudPIT) to analyse the "tightly-bound" proteome for purified cell walls from Arabidopsis cell suspension cultures. Using bioinformatics for the prediction of signal peptides for targeting to the secretory pathway and for the absence of ER retention signal, 89 proteins were selected as potential extracellular proteins. Only 33% of these were identified in previous proteomic analyses of Arabidopsis cell walls. A functional classification revealed that a large proportion of the proteins were enzymes, notably carbohydrate active enzymes, peroxidases and proteases. Comparison of all the published proteomic analyses for the Arabidopsis cell wall identified 268 non-redundant genes encoding wall proteins. Sixty of these (22%) were derived from our analysis of tightly-bound wall proteins.

Virus induction of heat shock protein 70 reflects a general response to protein accumulation in the plant cytosol.

Different cytoplasmically replicating RNA viruses were shown to induce a specific subset of heat-inducible heat shock protein 70 (HSP70) genes in Arabidopsis (Arabidopsis thaliana). To identify the inducing principle, a promoterreporter system was developed for the facile analysis of differentially responding Arabidopsis HSP70 genes, by infiltration into Nicotiana benthamiana leaves. Through transient expression of individual viral cistrons or through deletion analysis of a viral replicon, we were unable to identify a unique inducer of HSP70. However, there was a positive correlation between the translatability of the test construct and the differential induction of HSP70. Since these data implied a lack of specificity in the induction process, we also expressed a random series of cytosolically targeted Arabidopsis genes and showed that these also differentially induced HSP70. Through a comparison of different promoterreporter constructs and through measurements of the steady-state levels of the individual proteins, it appeared that the HSP70 response reflected the ability of the cytosol to sense individual properties of particular proteins when expressed at high levels. This phenomenon is reminiscent of the unfolded protein response observed when the induced accumulation of proteins in the endoplasmic reticulum also induces a specific suite of chaperones.

Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana.

All of the protein products of Turnip crinkle virus (TCV; Tombusviridae, Carmovirus) were tested for their ability to suppress RNA silencing of a reporter gene after transient expression in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Only the capsid protein, P38, showed suppression activity, although this was not obvious when P38 was expressed as part of a TCV infection of the same tissues. When P38 was expressed from a PVX vector, symptoms with enhanced severity that correlated with increased PVX RNA accumulation were observed. This contradiction between ectopic expression of P38 and TCV infection could be accounted for if the active determinant of suppressor activity within P38 was sequestered within the capsid protein structure. The N-terminal 25 amino acids were shown to be important for this activity. This region forms part of the unexposed R-domain that interacts with the RNA within the virus particle. This observation throws light on some of the complex biology exhibited by TCV.

The potyvirus recessive resistance gene, sbm1, identifies a novel role for translation initiation factor eIF4E in cell-to-cell trafficking.

From the characterization of the recessive resistance gene, sbm1, in pea we have identified the eukaryotic translation initiation factor, eIF4E, as a susceptibility factor required for infection with the Potyvirus, Pea seed-borne mosaic virus. A functional analysis of the mode of action of the product of the dominant allele revealed a novel function for eIF4E in its support for virus movement from cell-to-cell, in addition to its probable support for viral RNA translation, and hence replication. Different resistance specificities in two independent pea lines were explained by different mutations in eIF4E. On the modelled structure of eIF4E the coding changes were in both cases lying in and around the structural pocket involved in binding the 5'-m7G cap of eukaryotic mRNAs. Protein expression and cap-binding analysis showed that eIF4E encoded by a resistant plant could not bind to m7G-Sepharose, a result which may point to functional redundancy between eIF4E and the paralogous eIF(iso)4E in resistant peas. These observations, together with related findings for other potyvirus recessive resistances, provide a more complete picture of the potyvirus life cycle.