Interactions of ALY proteins with the Tomato bushy stunt virus P19 silencing suppressor protein

Tomato bushy stunt virus (TBSV) is a plant virus with a positive-strand RNA genome of about 4.8kb that is packaged in icosahedral virus particles of about 30 nm diameter. The virus infects primarily vegetable and ornamental crops although it has a wide experimental host range and is present in soil or contaminated water.

Infection occurs following mechanical damage of roots growing in contaminated soil, apparently without any vector, and transmission can also occur via infected seed. Virus particles are easily purified in high yield, which led to TBSV being the first virus to have its atomic structure determined by X-ray crystallography.

The TBSV genome contains four open reading frames (ORFs). ORF1 encodes the p33 protein and readthrough of the p33 gene termination codon produces a second, C-terminally extended p92 protein. Both p33 and p92 are involved in virus replication. ORF2 encodes p41, the virus coat protein. ORF4 encodes p22, a cell-to-cell movement protein. ORF 3 is nested within ORF4 but in a different reading frame and encodes p19 which is a pathogenicity determinant and suppressor of RNA silencing.

The p19 protein is one of the best studied plant virus silencing suppressor proteins and has strong activity when expressed in plants from Agrobacterium tumefaciens. P19 has been shown to bind small interfering (si)RNAs; the crystal structure of a p19 dimer bound to a siRNA duplex was determined, revealing a mechanism by which the p19 dimer could selectively bind to siRNAs of a particular length. From this and other studies it has been proposed that p19 suppresses RNA silencing by sequestering siRNAs, preventing their amplification and incorporation into RISC.

We screened an Arabidopsis cDNA library by yeast two-hybrid analysis to identify a family of plant proteins that interact with p19. The ALY proteins are 30kDa in size and have a central RNA binding (RRM) domain flanked by regions containing varying numbers of Arg-Gly-Gly (RGG) repeats embedded in non-conserved sequence. Arabidopsis encodes four ALY proteins, and we have also identified four ALY genes in Nicotiana benthamiana (although the complete genome sequence is not available for this plant, and other ALY genes could be present).

Function of ALY

The function of ALY in plants has not been experimentally determined, however, in yeast, Drosophila and other higher organisms ALY is known to be part of the system for export of spliced RNAs out of the nucleus. There is also some evidence that ALY might have transcription activation activity. It may be significant that Drosophila and mammals have only one or two ALY homologues. Perhaps the four (or more) plant ALYs have additional, unique functions?

In animals (and we expect also in plants) the ALYs are nucleocytoplasmic shuttle proteins that exit the nuclear pore together with their cargo RNA but then rapidly re-enter the nucleus. The p19 protein is primarily cytoplasmic, where siRNA-mediated RISC cleavage of RNAs takes place, but small amounts of fluorescent protein (GFP, mRFP)-tagged p19 also pass into the nucleus (but not the nucleolus). When each of the ALY proteins was fused to GFP and expressed in N. benthamiana plants from A. tumefaciens all were nuclear and all except AtALY2 were also nucleolar. This contrasts with ALY proteins from animals which are not found in the nucleolus.

However, when expressed transiently in plants together with p19, or in plants infected with TBSV, several of the ALY proteins (AtALY2-GFP, AtALY4-GFP, NbALY617-GFP and NbALY916-GFP) were no longer nuclear but were relocalised to the cytoplasm. The possibility is that either p19 recruits specific ALYs to the cytoplasm perhaps to play a role in the capture of siRNAs or that p19 disrupts the function of these ALYs perhaps to influence normal processing of host RNAs.

A further complication was noticed when fluorescent protein-tagged p19, which retains its silencing suppression activity, was co-expressed in plants with untagged ALY proteins. In these experiments four ALY proteins (AtALY1, AtALY3, NbALY615 and NbALY1693) relocalised p19 from the cytoplasm into the nucleus and nucleolus. Whether there is any purpose to this from the perspective of the virus is not known, however, when p19 was moved into the nucleus the degree of silencing suppression in the infiltrated tissue was significantly reduced. This might be expected to weaken the invading virus.

Figure: P19 is normally mostly cytoplasmic (see Field view, lower left panel). When co-expressed with Arabidopsis ALY1, P19 is moved out of the cytoplasm (see Field view, lower right panel) and becomes confined to the nucleolus (see Nucleus view, upper right panel.

From these experiments it seems that the various ALY proteins interact with p19 and affect its silencing suppression activity in different ways. The precise effect may depend on the relative level of expression of each individual ALY protein in the particular tissue that the virus invades, and we are doing more work to examine this possibility.

References

Uhrig, J.F., Canto, T., Marshall, D. and MacFarlane, S.A. 2004. Relocalisation of nuclear ALY proteins to the cytoplasm by the Tomato bushy stunt virus P19 pathogenicity protein. Plant Physiology 135, 2411-2423.

Canto, T., Uhrig, J.F., Swanson, M., Wright, K.M. and MacFarlane, S.A. 2006. Translocation of the TBSV P19 protein into the nucleus by ALY compromises its silencing suppressor activity. Journal of Virology 80, 9064-9072.

This work has been done in collaboration with Dr Joachim Uhrig, Botanisches Institut der Universität zu Köln, Germany.