Phloem is among the most complex and least experimentally amenable tissues of vascular plants. Viruses, a great majority of which rely on the phloem in their long-distance transport, provide powerful tools to probe this tissue. Viral loading into, passage through, and unloading from the phloem proceed via utilization and modification of normal phloem functions. With a few exceptions, viruses traffic in the phloem in a form of virions. Other recognized key players in viral long-distance transport are MPs and LTFs. The interplay between the virions, MPs, and LTFs largely defines the virus-specific part of the long-distance transport machinery. However, little is known about how virions, MPs, and LTFs work together to ensure successful virus spread through the phloem.
In a previous work we developed the concept of a BYV virion as a morphologically and functionally bipartite entity (3
). One principal part of the virion, the body made up of CP and RNA, carries the function of genome protection. The other part, the tail assembled by CPm, represents a specialized device for cell-to-cell movement. We have also demonstrated that one of the BYV MPs, Hsp70h, is an integral part of the virion (32
) that is required for tail assembly (3
). It was further hypothesized that the virion translocation through the plasmodesma occurs in a directional, tail-first manner and is powered by ATPase activity of Hsp70h. In this work we show that in addition to its central role in BYV cell-to-cell movement, Hsp70h provides a docking site for p20, an accessory virion protein that is critical for long-distance transport of the virus.
Initially, p20 was identified as the BYV protein that interacts with Hsp70h in yeast, in vitro, and in plant cells. Further experiments revealed that each of the principal domains of the Hsp70h was capable of binding p20. This observation suggests that each Hsp70h molecule can bind at least two p20 molecules. Alternatively, the interface between Hsp70h and p20 in a 1:1 complex may overlap each of the Hsp70h domains. Additional p20 molecules could be included in a complex via p20 self-interaction.
To address the significance of Hsp70h-p20 interaction, we investigated the p20 function in the BYV life cycle and revealed its primary role in the long-distance transport of BYV. Indeed, mutation of the p20 gene resulted in profound defects in a phloem-associated virus spread. First, the mutants were unable to spread systemically in a majority of inoculated plants. Second, in those few plants where systemic spread was detected, it was delayed by at least two weeks. Third, the extent and pattern of virus unloading were affected by mutations. Instead of the massive unloading from class III veins seen with parental BYV-GFP (Fig. ) as well as with other viruses (42
), mutant BYV-GFP variants exhibited patchy unloading from class III and/or class II veins. Taken together, these results established p20 as a BYV LTF.
Further analyses revealed that although p20 is not essential for virion assembly or cell-to-cell movement, it is attached to the virions, presumably via interaction with the BYV MP, Hsp70h. This association between virion, MP, and LTF suggests that the processes of the local and systemic translocation of BYV are mechanistically linked to each other via physical interaction between virion-associated MP and LTF. Furthermore, it prompts a novel paradigm of a BYV virion as a dynamic entity that mediates virus transport via recruitment of the MP and LTF.
It should be emphasized that the transport mechanisms may not be uniform among the viruses in the Closteroviridae
family, as BYV is the only known closterovirus that is capable of exiting the phloem. On the other hand, interaction between the virions and MP was recently described for a Potexvirus
, suggesting that recruitment of the transport proteins by virions could be a widespread phenomenon (4
What is a mechanism of p20 function in long-distance transport of BYV? One possibility is that p20 facilitates either entry of virions into or exit from the phloem, e.g., via modification of the plasmodesmatal function of the Hsp70h. This modification may be required to account for the specialized nature of plasmodesmata that interconnect cells within phloem. Another possibility is stabilization of the virions inside the phloem. In particular, p20 may prevent the disassembly of the virions and damage of RNA due to alkaline pH of the phloem sap (33
). Alternatively, p20 could protect virions from inactivation by plant defense proteins that are present in the phloem (11
The third mechanistic possibility is involvement of p20 with suppression of plant RNA silencing response, which is a likely mechanism of action for several LTFs (1
). Our attempt to reveal such suppressor activity of p20 with a model system in which RNA silencing is induced by transient expression of the double-stranded RNA (22
) was unsuccessful (J. Reed, K. Kasschau, J. Carrington, and V. V. Dolja, unpublished results). However, it cannot be excluded that p20 interferes with something other than the double-stranded-RNA-induced facet of the RNA silencing pathway.
Phylogenetically, p20 represents a novel class of viral LTFs. A database search revealed that p20 shows only marginal similarity to other ~20-kDa proteins encoded by related members of the Closterovirus genus (V. V. Dolja, unpublished data). In general, none of the presently characterized LTFs exhibits sequence similarity to any other proteins except for their orthologs within the same virus taxon. Because of that, LTFs appear to be a relatively recent evolutionary invention that may provide valuable mechanistic clues as to virus-host interactions within the phloem.