Experimental virus evolution reveals a role of plant microtubule dynamics and TORTIFOLIA1/SPIRAL2 in RNA trafficking

Abstract

The cytoskeleton is a dynamic network composed of filamentous polymers and regulatory proteins that provide a flexible structural scaffold to the cell and plays a fundamental role in developmental processes. Mutations that alter the spatial orientation of the cortical microtubule (MT) array of plants are known to cause important changes in the pattern of cell wall synthesis and developmental phenotypes; however, the consequences of such alterations on other MT-network-associated functions in the cytoplasm are not known. In vivo observations suggested a role of cortical MTs in the formation and movement of Tobacco mosaic virus (TMV) RNA complexes along the endoplasmic reticulum (ER). Thus, to probe the significance of dynamic MT behavior in the coordination of MT-network-associated functions related to TMV infection and, thus, in the formation and transport of RNA complexes in the cytoplasm, we performed an evolution experiment with TMV in Arabidopsis thaliana tor1/spr2 and tor2 mutants with specific defects in MT dynamics and asked whether TMV is sensitive to these changes. We show that the altered cytoskeleton induced genetic changes in TMV that were correlated with efficient spread of infection in the mutant hosts. These observations demonstrate a role of dynamic MT rearrangements and of the MT-associated protein TORTIFOLIA1/SPIRAL2 in cellular functions related to virus spread and indicate that MT dynamics and MT-associated proteins represent constraints for virus evolution and adaptation. The results highlight the importance of the dynamic plasticity of the MT network in directing cytoplasmic functions in macromolecular assembly and trafficking and illustrate the value of experimental virus evolution for addressing the cellular functions of dynamic, long-range order systems in multicellular organisms.

Figure 1. Layout of the virus evolution experiment.

The initial TMV inoculum (ancestor) was obtained from N. benthamiana plants inoculated with a cDNA clone of TMV and was used for inoculation of tor1/spr2, tor2, and wild type (WT) A. thaliana plants (three sets of five plants each). Following eight passages, nine independent TMV lineages were obtained. The number of infectious particles used for each passage was controlled by local lesion assays with hypersensitive N. tabacum NN plants.

Figure 2. Average absolute fitness (W) of the TMV ancestor and evolved lineages.

W is shown for infection in A. thaliana tor1/spr2, tor2 and WT plants, and in BY-2 protoplasts. Tor2 lineages show adaptation for efficient systemic movement in tor2 plants without consequences on replication efficiency (lack of a change in W compared to ancestor in protoplasts). Tor1 lineages are affected in replication efficiency and show slight adaptation for efficient systemic movement in tor1/spr2 plants. Asterisks indicate statistically significant differences from the ancestor.