Tomato golden mosaic virus (TGMV), a member of the geminivirus family, encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its plant host. TGMV AL1 is an oligomeric protein that binds double-stranded DNA and catalyzes cleavage and ligation of single-stranded DNA. The oligomerization domain, which is required for DNA binding, maps to a region that displays strong sequence and structural homology to other geminivirus Rep proteins. To assess the importance of conserved residues, we generated a series of site-directed mutations and analyzed their impact on AL1 function in vitro and in vivo. Two-hybrid experiments revealed that mutation of amino acids 157-159 inhibited AL1-AL1 interactions, whereas mutations at nearby residues reduced complex stability. Changes at positions 157-159 also disrupted interaction between the full-length mutant protein and a glutathione S-transferase-AL1 oligomerization domain fusion in insect cells. The mutations had no detectable effect on oligomerization when both proteins contained full-length AL1 sequences, indicating that AL1 complexes can be stabilized by amino acids outside of the oligomerization domain. Nearly all of the oligomerization domain mutants were inhibited or severely attenuated in their ability to support AL1-directed viral DNA replication. In contrast, the same mutants were enhanced for AL1-mediated transcriptional repression. The replication-defective AL1 mutants also interfered with replication of a TGMV A DNA encoding wild type AL1. Full-length mutant AL1 was more effective in the interference assays than truncated proteins containing the oligomerization domain. Together, these results suggested that different AL1 complexes mediate viral replication and transcriptional regulation and that replication interference involves multiple domains of the AL1 protein.Geminiviruses are a large family of plant viruses with circular, single-stranded DNA genomes that replicate in the nuclei of infected cells (reviewed in Ref. 1). The single-stranded genome is converted to a double-stranded DNA that serves as the template for rolling circle replication (2-4) and transcription (5, 6). Geminiviruses do not encode their own polymerases and, instead, rely on host enzymes for viral DNA and RNA synthesis. These characteristics make geminiviruses excellent model systems for studying plant DNA replication and transcription mechanisms.The geminivirus, tomato golden mosaic virus (TGMV), 1 has a bipartite genome that encodes seven open reading frames that are divergently transcribed. The 5Ј-intergenic region separating the transcription units is nearly identical between the two DNA components and includes the plus strand origin of replication (7, 8). The promoter for complementary sense transcription overlaps the replication origin (5, 9) and shares some of the cis-elements involved in origin function (10). A directly repeated sequence, GGTAG, is required for origin recognition (11) and transcriptional repression of the complementary se...

Successful regeneration of genetically modified plants from cell culture is highly dependent on the species, genotype, and tissue-type being targeted for transformation. Studies in some plant species have shown that when expression is altered, some genes regulating developmental processes are capable of triggering plant regeneration in a variety of plant cells and tissue-types previously identified as being recalcitrant to regeneration. In the present research, we report that developmental genes encoding GROWTH-REGULATING FACTORS positively enhance regeneration and transformation in both monocot and dicot species. In sugar beet ( Beta vulgaris ssp. vulgaris ), ectopic expression of Arabidopsis GRF5 ( AtGRF5 ) in callus cells accelerates shoot formation and dramatically increases transformation efficiency. More importantly, overexpression of AtGRF5 enables the production of stable transformants in recalcitrant sugar beet varieties. The introduction of AtGRF5 and GRF5 orthologs into canola ( Brassica napus L.), soybean ( Glycine max L.), and sunflower ( Helianthus annuus L.) results in significant increases in genetic transformation of the explant tissue. A positive effect on proliferation of transgenic callus cells in canola was observed upon overexpression of GRF5 genes and AtGRF6 and AtGRF9 . In soybean and sunflower, the overexpression of GRF5 genes seems to increase the proliferation of transformed cells, promoting transgenic shoot formation. In addition, the transformation of two putative AtGRF5 orthologs in maize ( Zea mays L.) significantly boosts transformation efficiency and resulted in fully fertile transgenic plants. Overall, the results suggest that overexpression of GRF genes render cells and tissues more competent to regeneration across a wide variety of crop species and regeneration processes. This sets GRFs apart from other developmental regulators and, therefore, they can potentially be applied to improve transformation of monocot and dicot plant species.

1AbstractSuccessful regeneration of genetically modified plants from cell culture is highly dependent on the species, genotype, and tissue-type being targeted for transformation. Studies in some plant species have shown that when expression is altered, some genes regulating developmental processes are capable of triggering plant regeneration in a variety of plant cells and tissue-types previously identified as being recalcitrant to regeneration. In the present research, we report that developmental genes encoding GROWTH-REGULATING FACTORS positively enhance regeneration and transformation in both monocot and dicot species. In sugar beet (Beta vulgaris ssp. vulgaris), ectopic expression of Arabidopsis GRF5 (AtGRF5) in callus cells accelerates shoot formation and dramatically increases transformation efficiency. More importantly, overexpression of AtGRF5 enables the production of stable transformants in recalcitrant sugar beet varieties. The introduction of AtGRF5 and GRF5 orthologs into canola (Brassica napus L.), soybean (Glycine max L.), and sunflower (Helianthus annuus L.) results in significant increases in genetic transformation of the explant tissue. A positive effect on proliferation of transgenic callus cells in canola was observed upon overexpression of GRF5 genes and AtGRF6 and AtGRF9. In soybean and sunflower, the overexpression of GRF5 genes seems to increase the proliferation of transformed cells, promoting transgenic shoot formation. In addition, the transformation of two putative AtGRF5 orthologs in maize (Zea mays L.) significantly boosts transformation efficiency and resulted in fully fertile transgenic plants. Overall, the results suggest that overexpression of GRF genes render cells and tissues more competent to regeneration across a wide variety of crop species and regeneration processes. This sets GRFs apart from other developmental regulators and, therefore, they can potentially be applied to improve transformation of monocot and dicot plant species.

Plants were regenerated from callus cultures of maize inbred W182BN with the S(USDA) type of cytoplasmic male sterility (cms). Some regenerates from 16 of 18 separate cultures had fertile tassels. Many other regenerates, whose fertility could not be scored accurately because of abnormal plant morphology, produced fertile progeny after pollination with N cytoplasm W182BN. Revertant plants and/or progeny were obtained from all 18 cultures, which included the CA, D, LBN, and S sources of cmsS. More revertants were recovered from cultures maintained as callus for 12 months than from 3-4 month old cultures. Several types of evidence (absence of segregation for fertility after selfing or pollination of revertants with standard W182BN, pollen viability counts, failure of revertants to restore sterile cmsS lines to fertility, mitochondrial DNA analyses) indicated that the reversion to fertility involved cytoplasmic rather than nuclear alterations. All revertants examined lacked the S1 and S2 plasmid-like DNAs characteristic of the mitochondrial genome of sterile cmsS lines. Most callus cultures lost S1 and S2 after 13-20 months in vitro. No revertants were seen among thousands of W182BN cmsS plants grown from seed in the field or among plants from tissue cultures of W182BN with the C or T types of cms. The cytoplasmic revertants recovered from culture may be useful for the molecular analysis of cmsS.

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