The non-enveloped bacilliform viruses are the second group of plant viruses known to possess a genome consisting of circular double-stranded DNA. We have characterized the viral transcript and determined the complete sequence of the genome of Commelina mellow mottle virus (CoYMV), a member of this group. Analysis of the viral transcript indicates that the virus encodes a single terminally-redundant genome-length plus 120 nucleotide transcript. A fraction of the transcripts is polyadenylated, although the majority of the transcript is not polyadenylated. Analysis of the genome sequence indicates that the genome is 7489 bp in size and that the transcribed strand contains three open reading frames capable of encoding proteins of 23, 15 and 216 kd. The function of the 25 and 15 kd proteins is unknown. Similarities between the 216 kd polypeptide and the cauliflower mosaic virus coat protein and protease/reverse transcriptase polyprotein suggest that the 216 kd polypeptide is a polyprotein that is proteolytically processed to yield the virion coat protein, a protease, and replicase (reverse transcriptase and ribonuclease H). Each strand of the CoYMV genome is interrupted by site-specific discontinuities. The locations of the 5'-ends of these discontinuities, and the presence and location of a region on the CoYMV transcript capable of annealing with the 3'-end of cytosolic initiator methionine tRNA are consistent with replication by reverse transcription. We have demonstrated that a construct containing 1.3 CoYMV genomes is infective when introduced into Commelina diffusa, the host for CoYMV, using Agrobacterium-mediated infection.

Chromosomal rearrangements are useful genetic and breeding tools but are often difficult to detect and characterize. To more easily identify and define chromosome deletions and inversions, we have used the bacteriophage P1 Cre-lox site-specific recombination system to generate these events in plants. This involves three steps: (i) the introduction of two lox sites into one locus in a plant genome, including one site within a modified Ds transposon; (ii) Ac transposase-mediated transposition of the Ds-lox element to a new locus on the same chromosome; (iii) Cre-mediated site-specific recombination between the two lox sites that bracket a chromosome segment. We report the production of a deletion and three inversion events in tobacco. The utility of chromosomal segments bracketed by lox sites for targeted manipulation and cloning is discussed.

Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects the monocot Commelina diffusa. Although CoYMV and cauliflower mosaic virus (CaMV; another double-stranded DNA virus) probably replicate by a similar mechanism, the particle morphology and host range of CoYMV place it in a distinct group. We present evidence that a promoter fragment isolated from CoYMV confers a tissue-specific pattern of expression that is different from that conferred by the CaMV 35s promoter. When the CoYMV promoter is used to drive expression of the P-glucuronidase reporter gene in stably transformed tobacco plants, 0-glucuronidase activity occurs primarily in the phloem, the phloemassociated cells, and the axial parenchyma of roots, stems, leaves, and flowers. Activity is also detected throughout the anther, with highest activity in the tapetum. In contrast, the CaMV 35s promoter is active in most cell types. The CoYMV promoter is a strong promoter, and when the activity of the CoYMV promoter is compared with that of a duplicated CaMV 35s promoter, it is 30% as active in tobacco suspension cells and up to 25% as active in maize suspension cells. These properties of the CoYMV promoter make it potentially useful for high-leve1 expression of engineered genes in vascular cells.

Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects the monocot Commelina diffusa. Although CoYMV and cauliflower mosaic virus (CaMV; another double-stranded DNA virus) probably replicate by a similar mechanism, the particle morphology and host range of CoYMV place it in a distinct group. We present evidence that a prompter fragment isolated from CoYMV confers a tissue-specific pattern of expression that is different from that conferred by the CaMV 35S promoter. When the CoYMV promoter is used to drive expression of the beta-glucuronidase reporter gene in stably transformed tobacco plants, beta-glucuronidase activity occurs primarily in the phloem, the phloem-associated cells, and the axial parenchyma of roots, stems, leaves, and flowers. Activity is also detected throughout the anther, with highest activity in the tapetum. In contrast, the CaMV 35S promoter is active in most cell types. The CoYMV promoter is a strong promoter, and when the activity of the CoYMV promoter is compared with that of a duplicated CaMV 35S promoter, it is 30% as active in tobacco suspension cells and up to 25% as active in maize suspension cells. These properties of the CoYMV promoter make it potentially useful for high-level expression of engineered genes in vascular cells.

No abstract

Several DNA site-specific recombination systems have been shown to function in higher eukaryotic cells. These two-component systems consist of a single-polypeptide recombinase and a short recognition sequence of less than 35 bp. Strategic placement of the recognition sites into the plant genome has permitted the deletion, inversion, integration, and translocation of host and introduced DNA fragments. Recombinase-based strategies afford precise and predictable engineering of the plant genome.

Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects a monocot host. A promoter fragment isolated from CoYMV is a strong promoter when assayed after transient introduction into monocot and dicot suspension cells and is highly active in vascular cells of flowers, leaves, stems and roots of stably transformed tobacco plants. Here it is reported that in stably transformed maize calli and transgenic tobacco leaves the CoYMV and CaMV 35S promoters exhibit similar amounts of activity. Deletion of the sequences located distal to nucleotide -230 relative to the start of transcription has no significant effect on promoter strength or tissue specificity. The region between -230 and -200 shares sequence similarity with the as-1 promoter element of the CaMV 35S promoter. Deletion of this as-1-like motif decreases promoter activity in maize suspension cells by 85%. Analysis of deletions affecting the -200 to -52 region indicates that sequences located between -159 and -84 are required for activity in vascular tissues. In addition, this region exhibits properties of a vascular tissue-specific enhancer since it confers vascular expression in an orientation-independent manner when fused to promoters that are not normally active in vascular tissues.

No abstract