Turnip vein clearing virus movement protein nuclear activity: Do Tobamovirus movement proteins play a role in immune response suppression?

Abstract

Plant viruses' cell-to-cell movement requires the function of virally encoded movement proteins (MPs). The Tobamovirus, Tobacco mosaic virus (TMV) has served as the model virus to study the activities of single MPs. However, since TMV does not infect the model plant Arabidopsis thaliana I have used a related Tobamovirus, Turnip vein-clearing virus (TVCV). I recently showed that, despite belonging to the same genus, the behavior of the 2 viruses MPs differ significantly during infection. Most notably, MP^TVCV, but not MP^TMV, targets the nucleus and induces the formation of F actin-containing filaments that associate with chromatin. Mutational analyses showed that nuclear localization of MP^TVCV was necessary for TVCV local and systemic infection in both Nicotiana benthamiana and Arabidopsis. In this addendum, I propose possible targets for the MP^TVCV nuclear activity, and suggest viewing MPs as viral effector-like proteins, playing a role in the inhibition of plant defense.

Keywords:

• actin
• intercellular communication
• movement protein
• plasmodesmata
• tobamovirus

This article refers to:

Unique Characteristics of the Movement Protein (MP) of Tobamovirus Turnip vein clearing virus (TVCV)

The view of how a single MP coordinates intra/intercellular trafficking of viral genomes is largely based on studies of tobacco mosaic virus (TMV). Upon uncoating, the TMV + RNA genome is translated into replicase to begin viral replication at sites associated with ER, and subgenomic RNAs that encode MP^TMV and coat protein (CP) are synthesized. As the life cycle advances, TMV genomes associate further with ER sites to amplify replication and eventually reach to traffic through plasmodesmata (PD).¹ The MP of TMV and other +RNA viruses with “30K-like” MPs can bind RNA and alter PD gating properties,² suggesting that they play a role in coordinating intracellular viral genome transport to PD and cell-to-cell movement. Among the viruses that have received special attention due to their ability to efficiently infect Brassica species, more specifically the model plant Arabidopsis, is TVCV, a Tobamovirus that is serologically related to TMV.^3,4 However, it remains unclear whether the life cycles of these 2 viruses are identical. To answer this question, we engineered a TVCV replicon that express a functional fusion of MP^TVCV with GFP or RFP, and used these infectious clones to examine the distribution of MP^TVCV during TVCV infection in Arabidopsis Col-0 and N. benthamiana, both susceptible hosts.⁵ Surprisingly, we found that TVCV exhibits 3 distinct differences from TMV: (1) MP^TVCV did not localize to microtubules at any stage of infection; (2) in later stages of infection, as TVCV ER replication sites enlarge and the ER network collapses, actin filaments are distorted and bundle near replication sites; and (3) besides localizing to motile vesicles, ER sites and PD, MP^TVCV also enters the nucleus and localizes to a novel filamentous structure at all stages of infection. We identified a putative NLS (RKRKKK) in MP^TVCV (residues 209-214), which is conserved among subgroup 3 Tobamoviruses, to which TVCV belongs, but not in TMV or other subgroup 1, or the subgroup 2, viruses, and showed that this NLS is necessary for MP^TVCV nuclear localization. The NLS MPK210/212A mutation reduced the rate of TVCV cell-to-cell spread, and produced a significant delay in TVCV systemic infection and attenuated disease symptoms in Arabidopsis and N. benthamiana. Our results show that MP^TVCV activity in the nucleus has a positive effect on virus spread, and, together with the results of Harries et al. (2009),⁶ suggest crucifer-infecting Tobamovirues employ a distinct infection strategy, which differ from that of the Solanaceae-isolated viruses like TMV, and may represent an evolutionary adaptation for the properties of Brassicaceae.

Hypothesized Function of MP^TVCV in the Nucleus

When we infected both N. benthamiana and Arabidopsis with our TVCV replicon, nuclear MP^TVCV localized to a filamentous network that contacted discrete chromatin domains. Using the F-actin probe GFP-UtrCH, which was recently employed in transplanted mouse nuclei in Xenopus GVs,⁷ we determined that actin and MP^TVCV-GFP co-localized in the nuclear filaments. The roles of actin and actin-related proteins (ARP) in the nucleus are complex, with actin playing a role in chromatin remodeling, in intranuclear chromosome movement, and in the transcription process itself (reviewed in Hofmann, 2009⁸). These activities are all tightly related to the regulation of transcription, and to the epigenetic activation or repression of genes. In mammalian cells, different cell stresses, including viral infections, were shown to induce the formation of nuclear actin filaments,⁸ but it is unknown whether similar mechanisms operate in plants. Still, numerous actins and ARPs were shown in Arabidopsis to localize inside the nucleus^9,10 and ARP6 was shown to function as part of chromatin remodeling complexes.¹¹ The tight interaction between MP^TVCV and the chromatin leads us to suggest MP^TVCV interacts with nuclear actin and/or ARPs in chromatin remodeling complexes, to create a filamentous actin network that spatially organizes chromatin to reprogram transcription of host defense-related genes.

It is well established that viral infection causes extensive changes in the transcription of host genes,¹² but the molecular mechanisms that carry out these changes are unknown. Work with non-viral pathogens determined that chromatin remodeling and modifying-complexes play a major role in reconfiguring chromatin and regulating, among others, the defense response.¹³ Arabidopsis PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 (PIE1), a yeast Swr1 homolog, was shown to interact with ARP6 and SERRATED LEAVES AND EARLY FLOWERING (SEF), and to form a complex that is related to the yeast SWR1.^14,15 In yeasts, SWR1 complexes substitute histone H2A-H2B dimmers with H2A.Z-H2B dimmers in euchromatic regions, thus preventing the spread of heterochromatin. There is evidence that strongly suggests that the Arabidopsis PIE1/ARP6/SEF complex possesses a similar function.¹⁶ Interestingly, mutations in PIE1, SEF and 2 H2A.Z loci resulted in increased plant resistance to Pseudomonas syringae, and the PIE1 mutation led to increased expression of defense-related genes such as PATHOGENESIS RELATED 1 (PR1), and the WRKY38 and WRKY18 transcription factors. This suggests that the Arabidopsis SWR1 complex normally acts as a repressor of plant resistance, and that pathogen infection results in the repression release and expression of defense related genes.¹⁴ Formation of nuclear actin filaments during TVCV infection may, in turn, inhibit the SWR1 complex activity, and maintain the defense response in a repressed state.

Tobamovirus MPs as Viral Effectors?

Research with bacterial pathogens has determined that the activation of plant immune responses depends on the ability of a plant to sense pathogen invasion, with the first line of defense response involves receptors that recognize conserved microbial molecules, designated pathogen-associated molecular patterns (PAMPS), and activate PAMP-triggered immunity (PTI). Successful pathogens, in turn, are able to suppress PTI through the activity of effector proteins. Viruses are not generally viewed as encoding effectors, but recent publications start to challenge this assumption.¹⁷ Our results, demonstrating that MP^TVCV functions in the nucleus to enhance infection, imply that MP activity may be similar to bacterial effectors.¹⁸

Support for the defense-suppression activity of Tobamoviruses in Arabidopsis comes from recent work with TMV-Cg,¹⁹ another subgroup 3 virus. WRKY transcription factors family members are the key regulators of plant basal-defense, which function by binding to promoters of defense related genes through a DNA element termed the W box. In addition to WRKY38 and WRKY18 being regulated by the Arabidopsis SWR1 complex (see above), additional members from this family were also shown to be regulated by chromatin regulators such as histone deacetylases²⁰ and histone methytransferases.²¹ Chen et al.(2013) showed that in the absence of WRKY8, TMV-Cg accumulates more rapidly in systemic leaves, indicating WRKY8 acts to inhibit the virus systemic spread.¹⁹ Interestingly, TMV-Cg was shown to suppress the stress-induced up-regulation of WRKY8. These results suggest that TMV-Cg, and maybe additional subgroup 3 Tobamoviruses, inhibits the expression of WRKY8 to promote their own infection, and may point to one possible target of MP^TVCV in the nucleus.

Another example for an ‘effector-like’ activity of Tobamovirus MPs comes from work with TMV. One of the major constituents of a PTI is the cell wall defense response, which includes cell wall thickening and callose deposition. This cell wall defense is inhibited by the Pseudomonas syringae bacterial effector HopM1,^22,23 to prevent callose deposits. Bacterial infection also induces PD callose and PD closure,²⁴ indicating that PD callose is a part of the cell wall defense response. Work with TMV has provided a striking viral parallel to the bacteria interaction, showing that when N. benthamiana plants are infected with TMV replicase alone, callose deposition at PD was induced. However, when the MP was also present, there was little to no callose accumulation.²⁵ These data suggest that the viral replicase or replication induced PTI-like response causing PD closure, while MP acted as an effector, suppressing the basal cell-wall defense. It is this effector-like function that may results in MP's ability to gate PD and enable viral cell-to-cell movement. These findings suggest the intriguing possibility that Tobamoviruses MPs, beyond nucleic acid trafficking, act to inhibit defense responses (and thus prevent Pd closure and allow movement). By modulating gene transcription in the nucleus, subgroup 3 Tobamoviruses has further evolved to counteract additional lines of defense, enabling them to successfully infect Brassicaceae plants.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.