The role of NSm during tomato spotted wilt virus infection

M.M.H. Storms

Research output: Thesisinternal PhD, WU

Abstract

<p>In the past ten years the genome organisation of tomato spotted wilt virus (TSWV) has been intensively studied in our laboratory. Complete genome sequence data revealed that this enveloped plant virus belongs to the Bunyaviridae, a virus family further restricted to animals.</p><p>Hence, TSWV is a splendid model to investigate which viral encoded factors are needed for a virus to successfully infect a plant. Comparison of the genome of TSWV with those of the animal-infecting members of the Bunyaviridae reveals the presence of an extra cistron in the TSWV genome. This cistron encodes a non-structural protein of 33.6 kDa, denoted NS <sub>M</sub> . Although some animal infecting Bunyaviridae also specify a protein referred to as NS <sub>M</sub> , this polypeptide does not represent a separate gene product. Hence, the extra NS <sub>M</sub> gene may very well represent the key function for genetic adaptation of Bunyaviruses to plant hosts. For a successful infection of plants, viruses have to pass the plant specific cell wall barrier through plasmodesmata. As the size of most viruses, and even their genomes, exceeds the physical space provided by plasmodesmata for intercellular transport of macromolecules (the size exclusion limit or SEL), cell-cell movement requires a structural modification of the plasmodesmata. For a number of plant viruses it has been shown that they encode one or more movement proteins (MPs) which achieve viral cell-cell movement, among others by modification of the plasmodesmata, most likely in co-operation with host factors and/or other viral proteins.</p><p>At the onset of this PhD research, the working hypothesis was that NS <sub>M</sub> could very well represent the MP of TSWV. This was also based on the presence of a conserved sequence in the NS <sub>M</sub> protein, the so-called "D-motif", characteristic for several other plant viral MPs.</p><p>To obtain experimental data for the role of NS <sub>M</sub> in the TSWV infection cycle, first the expression kinetics and intracellular behaviour of the protein were studied in systemically infected <em>Nicotiana rustica</em> leaf tissue. The NS <sub>M</sub> gene was cloned and expressed in <em>E. coli</em> , the protein was purified and a specific antiserum made. Time course analyses of systemically infected <em>N. rustica</em> leaf tissue, using this anti-NS <sub>M</sub> serum, revealed an early and transient presence of the protein in infected cells, a manifestation that is unique for NS <sub>M</sub> and not found for other viral proteins of TSWV (Chapter 2). The early and temporary appearance of NS <sub>M</sub> suggested that the protein has an early function in the infection cycle in concert with the idea that it is involved in cell-cell movement. During infection of a cell, NS <sub>M</sub> was first localised to newly formed viral nucleocapsids in the cytoplasm and plasmodesmata. Moreover, NS <sub>M</sub> was found to assemble into tubular structures that penetrate the plasmodesmata in a unidirectional way. These tubules were also formed upon expression of the cloned NS <sub>M</sub> gene in protoplasts (Chapter 3)</p><p>The formation of similar tubular structures has been shown to be an essential step during cell-to-cell movement of a number of RNA (e.g. como- and nepoviruses) and DNA (caulimoviruses) plant viruses. For these latter viruses, it was shown that mature virions are transported from cell-to-cell through MP-induced tubular structures. Investigations on the contents of the TSWV tubules, formed on synchronously infected protoplasts, revealed the presence of only the nucleoprotein inside the tubular structures (Chapter 4). Based on this, it is tempting to assume that TSWV is translocated into the adjacent non-infected cell as non-enveloped, viral nucleocapsids.</p><p>An important characteristic of several plant virus MPs is that they evoke a modification of the macromolecular diffusion limits (SEL) of plasmodesmata. To investigate whether NS <sub>M</sub> has a similar function, the NS <sub>M</sub> gene was transgenically expressed in <em>Nicotiana tabacum</em> SR1 plants (Chapter 5). The resulting transgenic plants were examined for NS <sub>M</sub> expression, subcellular localisation of this protein, and changes in the diffusion properties of the plasmodesmata. The NS <sub>M</sub> expression levels obtained were invariably low. However, the NS <sub>M</sub> protein could be localised to over 80% of the plasmodesmata in various transgenic tissues, demonstrating its specific targeting to and association with these organelles. Probably as a result of NS <sub>M</sub> accumulation at the plasmodesmata, the plants showed severe aberrations in growth and development (Chapter 5).</p><p>Possible alterations in plasmodesmal functioning were further analysed by microinjection of fluorescent probes of various sizes into mesophyll cells of the NS <sub>M</sub> transgenic plants. Two different methods of microinjection were used and compared, one by which the influx of probes was achieved by a pressure pulse and a second, by diffusion (iontophoresis). Pressure injection studies on NS <sub>M</sub> transgenic plants with fluorescent probes of different molecular mass showed an increase in plasmodesmal SEL similar to that of control transgenic plants expressing the TMV MP. As, in this respect, NS <sub>M</sub> modifies the plasmodesmal function in a similar manner as the TMV MP, NS <sub>M</sub> was concluded to be the tospoviral MP. Strikingly, when iontophoresis was performed on NS <sub>M</sub> - or TMV-MP transgenic plants, the plasmodesmata showed a decrease rather than an increase in SEL. In control (non-transgenic) plants, the outcome of pressure injection and iontophoresis was identical. This strongly suggested that accumulation of viral MP made the cells respond differently to the microinjection methods used.</p><p>A possible interpretation for these remarkable differences is that TSWV and TMV MP, inhibits or partly blocks, the communication pathway for macromolecular trafficking, as recorded by the less invasive iontophoresis method, which also explains the disturbed morphology and physiology of the MP transgenic plants. The protein barrier at the plasmodesmata may be overcome by a pressure pulse, thereby revealing an increase of SEL induced by the plant viral MP (Chapter 6).</p><p>As TSWV also replicates in its thrips vectors, it was of interest to explore whether NS <sub>M</sub> also had a function during the infection cycle in the insect. The expression kinetics and localisation of the NS <sub>M</sub> protein was studied in all developmental stages of the major viral vector, the thrips <em>Frankliniella occidentalis</em> (Chapter 8). Besides the <em>in situ</em> analyses of NS <sub>M</sub> in the vector itself, the behaviour of the protein was also investigated in heterologous <em>Spodoptera frugiperda</em> and <em>Trichoplusia ni</em> insect cell cultures and in baby hamster kidney (BHK21) cells, a mammalian cell system unrelated to the natural hosts of TSWV (plants and insects)(Chapter 7). Upon expression of NS <sub>M</sub> in cultured insect cells, the protein specifically targeted to the cell periphery and formed tubular structures at the cell surface in a similar fashion as found in plant protoplasts. However, in mammalian cells no plasmamembrane targeting or tubular structures were found. The results obtained for insect cells indicate that the NS <sub>M</sub> protein has the potential to form tubular structures in the absence of any plant specific component.</p><p>In the vector <em>F. occidentalis</em> , NS <sub>M</sub> was present at only low levels in mostly midgut epithelium cells of L2 larvae and in salivary gland cells and midgut muscle cells of adult thrips. Characteristic structures or associations as found in infected plants (e.g. tubule formation, association with viral nucleocapsids) could not be discerned in any of the developmental stages of the thrips. Although this does not exclude a function for NS <sub>M</sub> in the TSWV vector, this function would then be unrelated to its activity as MP.</p><p>In summary, the investigations described in this thesis all demonstrate that NS <sub>M</sub> represents the MP of TSWV, and that it mediates the cell-cell movement of presumably non-enveloped viral nucleocapsids through transiently produced tubular structures that penetrate plasmodesmata. As such, the extra NS <sub>M</sub> gene in the genome of TSWV indeed encodes the key protein required for pathogenicity of Bunyaviridae towards plants.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Goldbach, R.W., Promotor, External person
  • van Lent, J.W.M., Promotor, External person
Award date2 Dec 1998
Place of PublicationS.l.
Publisher
Print ISBNs9789054859215
Publication statusPublished - 1998

Keywords

  • bunyaviridae
  • plant viruses
  • plant pathogens
  • infection
  • movement
  • proteins
  • protoplasts

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