Tomato golden mosaic virus (TGMV) belongs to the geminivirus subgroup that is characterized by a split genome consisting of two single-stranded circular DNAs. The TGMV A genome component encodes the virus coat protein as well as all of the functions necessary for viral DNA replication. Analysis of the nucleotide sequence indicates that the TGMV A component has, in addition to the coat protein encoding ORF, four overlapping open reading frames (ORFs) with the potential to encode proteins of greater than 10 kD. We have investigated the functions of these putative proteins in both symptom formation and DNA replication by creating mutations in each of the ORFs. Our results show that the AL4 ORF, which is encoded within the N-terminal region of ORF AL1, is not essential for normal virus infection. In contrast, we find that disruption of the AL3 ORF results in delay and attenuation of symptom formation. We also report that the products of the AL1 and AL2 ORFs are absolutely required for symptom formation. Studies of DNA replication show that only the AL1 open reading frame is essential for viral DNA synthesis. The significance of these results for the development of vectors from the geminiviruses is discussed.
We have adapted the "agroinfection" procedure of Grimsley and co-workers [4,5] to develop a simple, efficient, reproducible infectivity assay for the insect-transmitted, split-genome geminivirus, tomato golden mosaic virus (TGMV). Agrobacterium T-DNA vectors provide efficient delivery of both components of TGMV when used in mixed inoculation of wild-type host plants. A greater increase in infection efficiency can be obtained by Agrobacterium delivery of the TGMV A component to "permissive" transgenic plants. These "permissive" plants contain multiple tandem copies of the B component integrated into the host genome. An inoculum containing as few as 2000 Agrobacterium cells can produce 100% infection under these conditions. Further, our results show that there is a marked effect of the configuration of the TGMV A components within the T-DNA vector on time of symptom development. We have also found that transgenic plants carrying tandem copies of the A component do not complement the B component. Possible mechanisms to explain these results and the potential use of this system to further study the functions of the geminivirus components in infection are discussed.
The A component of the bipartite genome of the geminivirus tomato golden mosaic virus (TGMV) encodes the viral protein (ALl) that is required for viral DNA replication. We have constructed transgenic Nicotiana benthamiana plants in which the ALI open reading frame is transcribed under the control of the cauliflower mosaic virus 35S promoter.The transgenic plants, which were phenotypically normal, produced a single transcript from the 35S-ALI construct and a 40-kDa protein that cross-reacted with a polyclonal antiserum raised against ALl protein overproduced in Escherichia coli. Six of nine transgenic lines complemented a TGMV A variant with a mutation in ALI when coinoculated with the B component of the TGMV genome. Single-and double-stranded forms of the B component were synthesized in leaf discs from a complementing, transgenic line in the absence of TGMV A. These results establish that the transgenic plants express functional ALl protein and show that this viral protein is not only required, but sufficient, for single-and double-stranded replication of TGMV DNA in the presence of host proteins. These results also show that the ALl protein is not by itself a determinant of disease or pathogenesis.Geminiviruses are a family of plant viruses characterized by their single-stranded circular DNA genomes and small, twinned isometric particle morphology (for review, see refs. 1 and 2). Geminiviruses can be separated into two major groups by their genomic organization and insect vector. Maize streak virus is representative of the single-component genome, leafhopper-transmitted group. Tomato golden mosaic virus (TGMV) is a member of the two-component genome, whitefly-transmitted group. The two DNA components of TGMV, designated A and B, have been cloned (3), sequenced (4), and shown to be required for productive infection (5). Agrobacterium-mediated transformation has been used to create plants with stably integrated copies of TGMV A-or B-component DNAs in their genomes. The presence of freely replicating viral DNA in transgenic plants containing linear tandem copies of TGMV A DNA demonstrated that the A component encodes all of the information necessary for viral replication (6). Bipartite viral particles were also detected in these plants, demonstrating that TGMV A also encodes all of the functions required for encapsidation (7). The B component cannot replicate in the absence of the A component and, instead, contributes functions essential for systemic spread and symptom development (6). Agrobacterium-mediated transformation has been adapted for the inoculation of cloned TGMV DNAs onto plants at high efficiency (8, 9). This procedure, referred to as "agroinoculation," has provided additional information on the functions of the various open reading frames of the TGMV A and B components (10-12). These studies established that the ALl open reading frame encodes the viral protein required for replication of TGMV DNA in plants (10).The ALI open reading frame of TGMV A and the equivalent open reading frames of oth...
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