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    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Record number 333759
Title Mechanisms underlying Cowpea mosaic virus systemic infection
Author(s) Santos Silva, M.
Source Wageningen University. Promotor(en): R.W. Goldbach, co-promotor(en): Jan van Lent; Joan Wellink. - Wageningen : S.n. - ISBN 9789085040385 - 118
Department(s) Laboratory of Virology
Laboratory of Molecular Biology
EPS-2
Publication type Dissertation, internally prepared
Publication year 2004
Keyword(s) koebonenmozaïekvirus - manteleiwitten - vigna unguiculata - beweging - plantenvirussen - ziekteresistentie - cowpea mosaic virus - coat proteins - vigna unguiculata - movement - plant viruses - disease resistance
Categories Plant Viruses
Abstract Systemic virus infection of plants involves; intracellularreplication, cell-to-cell movement within the inoculated leaf, and subsequently, long-distance spread to other plant parts via the vasculature (vascular movement).Cell-to-cell movement occurs through the plasmodesma (PD), which are regulated channels in the cell wall connecting adjacent cells. These PD are modified by plant viral movement proteins (MPs) to allow passage of a viral RNA-MP complex as happens with Tobacco mosaic virus (TMV),or virions as happens with Cowpea mosaic virus (CPMV). With the latter virus, virions move through tubules built-up from the MP (tubule-guided cell-to-cell movement). For vascular movement, viruses must enter (loading), translocate through, and exit (unloading) from the phloem. Phloem (un)loading occurs through specialized PD, named the pore-plasmodesma-unit (PPU), connecting the companion cell (CC) and sieve element (SE). The PPU allows passage of much larger molecules than mesophyll PD do. Because of the peculiarities inherent to phloem tissue (e.g. PPU), mechanisms of cell-to-cell movement are usually distinct from those of vascular movement (reviewed in Chapter 1) for the same virus. For instance, TMV requires the viral coat protein (CP) for transport of virions through PPU, but the CP is dispensable for cell-to-cell movement. The success of plant virus infection is also the consequence of an antagonistic balance between viral infection and plant host defence mechanisms that specifically target viral replication (e.g. RNA silencing), or movement (e.g. systemic acquired resistance). In this research thesis CPMV was used as a model for investigations on the mechanisms of systemic infection of plants. Since CPMV replication and cell-to-cell movement are well-investigated, the thesis research was concentrated on vascular movement of CPMV and on barriers imposed by different plant species against systemic infection by this virus.To examine the characteristics of vascular movement in Vigna unguiculata (cowpea), GFP-expressing CPMV (CPMV-GFP) was mechanically inoculated to primary leaves and infection was followed over time (Chapter 2). CPMV-GFP was loaded into both major and minor veins of the primary leaves and unloaded exclusively from major veins, preferentially class III, in the secondary leaves similar to the route of photo-assimilates via phloem. Using electron microscopy, virus infection was observed in all vascular cell types of the loading and unloading sites, with the exception of CC and SE. Furthermore, tubules transporting virions were never found in the PD connecting; phloem parenchyma cells (PPC) and CC, or CC and SE (i.e. PPU). Since in cowpea the SE is symplasmically connected only to the CC, these observations suggest that, unlike cell-to-cell movement, CPMV vascular movement is not tubule-guided.Mutational analysis by reverse genetics is the most common approach to the study of viral factors necessary for vascular movement. CPMV requires its MP and both coat proteins (CPs) for tubule-guided cell-to-cell movement, deletion of any of these genes results in impeded local spread and this restriction severely hampers application of reverse genetics on CPMV for this purpose. In Chapter 3, an attempt was made to circumvent this problem by providing the CPs intrans by agroinfiltration in N. benthamiana to complement cell-to-cell movement of a CPMV mutant devoid of CPs (CPMV-DCP). The aim was to observe whether the mutant would exit from vascular tissue in the absence of CPs in the upper leaves. Whiletrans complementation of CPMV-DCP cell-to-cell movement was demonstrated in planta , the extent of spread was not sufficient to allow CPMV-DCP phloem loading, thus the phloem unloading of the mutant within the upper leaves could not be analysed. Immunoblot analysis of vascular sap from infected cowpea plants showed the presence solely of viral CPs. Furthermore, virions were found in the vasculature of CPMV-immune cowpea scions grafted on CPMV-inoculated susceptible rootstocks (Chapter 3). These results indicate that CPMV circulates in the vasculature in form of mature virions. However, it could not be unequivocally determined whether virions were located in the phloem or in the xylem. As systemic spread by xylem has been reported for beetle transmissible viruses like CPMV, beetle transmission was mimicked by gross-wound inoculation (Chapter 3). However, in this case, as with mechanical inoculation using an abrasive, CPMV spread systemically via the phloem, i.e. directed to sink-leaves solely like the flow of photo-assimilates. This confirms that phloem is the prevailing route for CPMV vascular movement.The potential role of RNA silencing during establishment of infection by CPMV was studied in Chapter 4. Using GFP-expressing CPMV constructs and N. benthamiana as host, the number of infection foci was recorded in the absence or presence of different viral suppressors of RNA silencing, i.e. potyviral HC-Pro, tospoviral NSs and cucumoviral 2b. Upon inoculation with CPMV in vitro transcripts, HC-Pro and NSs, but not 2b, significantly increased the number of CPMV primary infection foci. These results indicate that RNA silencing already has an impact on the establishment of infection even at an early stage. Interestingly, the stimulating effect of suppressors was not observed upon inoculation with virions. This effect may be explained by the recent finding (Lomonossoff, personal communication) that the small (S) CP acts as a suppressor of RNA silencing. To assess the effect of RNA silencing on viral local spread, GFP-expressing CPMV constructs impaired in local spread were tested in the presence or absence of HC-Pro or NSs. Neither of these proteins affected the progress of infection, indicating that RNA silencing does not play a major role in this stage. In Chapter 5, N. tabacum , a semi-permissive host of CPMV, was used to further unravel the viral systemic infection process. CPMV does not infect N. tabacum systemically despite extensive local spread in inoculated leaves. It is shown that neither incubation temperature nor RNA silencing-, salicylic acid- or ethylene-mediated resistance mechanisms are the limiting factor for CPMV systemic infection. Although CPMV-infected N. tabacum plants are normally asymptomatic, symptoms (i.e. necrotic lesions) in the inoculated leaves were observed at low (15 °C) temperature, but not systemic movement. Grafting experiments indicate that CPMV is not capable of phloem loading in N. tabacum , a finding that makes this plant species an interesting system for investigations of the host factors involved in CPMV vascular movement.Finally, in Chapter 6 possible mechanisms of vascular movement of CPMV are presented based on the results obtained in this thesis. Moreover, the various virus-host interactions, which contribute to the success or failure of systemic infection, are put into perspective.
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