Abutilon mosaic virus (AbMV) and bean dwarf mosaic virus (BDMV) are two phylogenetically related bipartite begomoviruses. While AbMV is restricted to phloem, BDMV spreads to non-phloem tissues. Cell-to-cell and long-distance movement of AbMV and BDMV were investigated after replacing the coat protein (CP) gene with the reporter gene encoding the green fluorescence protein (GFP). The DNA-A and DNA-B genomic components of AbMV and BDMV, and their pseudorecombinants (PR), were delivered to bean (Phaseolus vulgaris) seedlings and detached leaves with DNA-coated microprojectiles. Virus-associated fluorescence was observed with the confocal microscope. Delivery of AbMV and BDMV GFP reporters showed that the epidermal tissue was the main recipient of the viral DNA; the DNA-A of the two viruses was unable to move out of the recipient cells. AbMV DNA-A co-inoculated with AbMV DNA-B did not move from cell to cell in the epidermis and did not reach the phloem. However, co-inoculation of AbMV DNA-A with BDMV DNA-B resulted in PR cell-to-cell movement out of the epidermis and long-distance movement in the phloem. In contrast, BDMV DNA-A moved from cell to cell and over a long distance when co-inoculated with either its own DNA-B or with the DNA-B of AbMV. Thus, the DNA-B of the non-phloem-limited BDMV overcame the phloem limitation of AbMV. In the reciprocal case, the DNA-B of the phloem-limited AbMV did not confine the non-phloem limited BDMV to the phloem. Hence, we assume that the DNA-A component of BDMV includes determinants involved in the movement pattern of the virus in addition to the DNA-B-encoded BC1 and BV1 which have previously been shown to be involved in virus movement. The results also confirm that the CP is not necessary for virus movement; however, replacing the CP of AbMV and BDMV with GFP resulted in a decrease in symptom severity. DNA-B was involved in symptom severity; the B component of BDMV produced symptoms more severe than those induced by that of AbMV, whether in wild-type PRs or in PRs with CP-GFP replacement. It is interesting to note that when the GFP gene under the control of the CaMV 35S promoter (35S-GFP) was delivered to the bean tissue, with or without the DNA-B component of BDMV, GFP was expressed but did not move from cell to cell. However, when the 35S-GFP was delivered together with BDMV DNA-A and DNA-B, GFP showed cell-to-cell movement in the epidermis but was restricted to these cells. Hence, infection of cells with a functional bipartite begomovirus may facilitate cell-to-cell movement of macromolecules.
The symptom intensity in common bean plants depends on the stage of infection. Early infection of susceptible bean seedlings will result in severe stunting and dwarfing, leaf distortion and mottling or mosaic, as well as chlorotic or yellow spots or blotches. BDMV-infected plants usually abort their flowers or produce severely distorted pods. Late infection of susceptible plants or early infection of moderately resistant genotypes may show a mild mosaic, mottle and crumpling or an irregular distribution of variegated patches. BIOLOGICAL PROPERTIES: As a member of the Begomovirus group, BDMV is transmitted from plant to plant by the whitefly Bemisia tabaci. BDMV is a nonphloem-limited virus and can replicate and move in the epidermal, cortical and phloem cells. As a nonphloem-limited virus, it is sap-transmissible.
Agrobacterium tumefaciens delivers its single-stranded transferred DNA (T-strand) into the host cell nucleus, where it can be converted into double-stranded molecules. Various studies have revealed that double-stranded transfer DNA (T-DNA) intermediates can serve as substrates by as yet uncharacterized integration machinery. Nevertheless, the possibility that T-strands are themselves substrates for integration cannot be ruled out. We attempted to block the conversion of T-strands into double-stranded intermediates prior to integration in order to further investigate the route taken by T-DNA molecules on their way to integration. Transgenic tobacco (Nicotiana benthamiana) plants that overexpress three yeast (Saccharomyces cerevisiae) protein subunits of DNA REPLICATION FACTOR A (RFA) were produced. In yeast, these subunits (RFA1-RFA3) function as a complex that can bind single-stranded DNA molecules, promoting the repair of genomic double strand breaks. Overexpression of the RFA complex in tobacco resulted in decreased T-DNA expression, as determined by infection with A. tumefaciens cells carrying the b-glucuronidase intron reporter gene. Gene expression was not blocked when the reporter gene was delivered by microbombardment. Enhanced green fluorescent protein-assisted localization studies indicated that the three-protein complex was predominantly nuclear, thus indicating its function within the plant cell nucleus, possibly by binding naked T-strands and blocking their conversion into double-stranded intermediates. This notion was further supported by the inhibitory effect of RFA expression on the cell-to-cell movement of Bean dwarf mosaic virus, a single-stranded DNA virus. The observation that RFA complex plants dramatically inhibited the transient expression level of T-DNA and only reduced T-DNA integration by 50% suggests that doublestranded T-DNA intermediates, as well as single-stranded T-DNA, play significant roles in the integration process.
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