Small circular single-stranded DNA satellites, termed DNAbeta, have recently been found associated with some geminivirus infections. The DNA beta associated with Cotton leaf curl virus is responsible for symptom expression of a devastating disease in Pakistan. Mutagenesis of DNA beta revealed that the complementary-sense open reading frame (ORF) betaC1 is required for inducing disease symptoms in Nicotiana tabacum. An ORF present on the virion-sense strand betaV1 appeared to have no role in pathogenesis. Tobacco plants transformed with a betaC1 ORF under the control of the Cauliflower mosaic virus 35S promoter or with a dimeric DNA beta exhibited severe disease-like phenotypes, while plants transformed with a mutated version of betaC1 appeared normal. Northern blot analysis of RNA from the transgenic plants, using strand-specific probes, identified a single complementary-sense transcript. The transcript carries the full betaC1 ORF encoding a 118-amino acid product. It maps to the DNA beta at nucleotide position 186 to 563 and contains a polyadenylation signal 18 nt upstream of the stop codon. A TATA box is located 43 nt upstream of the start codon. Our results indicate that betaC1 protein is responsible for DNA beta-induced disease symptoms.

The 682-nt satellite DNA (sat-DNA) of Tomato leaf curl virus (TLCV) depends on the helper virus for its replication. In contrast to the strict specificity that exists in each geminivirus for its cognate replication associated protein (Rep), TLCV sat-DNA can utilize Rep encoded by distinct geminiviruses. We have used a combination of protein-binding assays and mutagenesis to show that repeat motifs in TLCV and sat-DNA are essential for Rep-binding in vitro. Surprisingly, mutants of TLCV and sat-DNA impaired in their ability to bind TLCV Rep in vitro were infectious in tomato. Thus, in contrast to other geminiviruses reported, TLCV and sat-DNA replication is independent of the high-affinity in vitro Rep binding. These results prompt a reassessment of the current model of geminivirus replication where Rep/DNA interaction is a highly specific step in the initiation of rolling circle replication.

DNA b is a circular single-stranded satellite DNA associated with certain monopartite begomoviruses (family Geminiviridae) which causes economically important diseases such as cotton leaf curl disease. DNA b contains a single gene, bC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the b-glucuronidase reporter gene driven by a bC1 promoter-deletion series of the DNA b associated with cotton leaf curl Multan virus identified a 68 nt region (between "139 and "207) which is important for bC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the bC1 start codon. Mutation of the G-box resulted in a significant reduction in bC1 promoter activity and DNA b replication efficiency. In addition, the G-box motif was found to bind specifically to a protein(s) in nuclear extracts prepared from tobacco leaf tissues. Our results indicate that interaction of the G-box motif with host nuclear factors is important for efficient gene expression and replication of DNA b. INTRODUCTIONThe genus Begomovirus (type strain, bean golden yellow mosaic virus) is the largest group within the family Geminiviridae. The members of this genus have either monopartite or bipartite single-stranded DNA genomes, are transmitted by whiteflies and infect only dicotyledonous plants (Harrison & Robinson, 2002). Begomoviruses are considered one of the largest and most successful groups of plant viruses and are responsible for numerous diseases of economically important crops, such as cassava, cotton, bean, pepper and tomato (Brown, 1992;Polston et al., 1994;Moriones & Navas-Castillo, 2000).Transcriptional regulation of geminivirus genes has been widely studied in both transient and stable transgenic systems (Eagle & Hanley-Bowdoin, 1997;Eagle et al., 1994;Haley et al., 1992; Ruiz-Medrano et al., 1999;Shivaprasad et al., 2005;Sunter & Bisaro, 1991, 1992Zhan et al., 1991). Geminiviruses rely on host factors for transcription. Virus genes are transcribed by host RNA polymerase II and their corresponding mRNAs are polyadenylated and initiated downstream of either a TATA box motif or initiator elements (Eagle & Hanley-Bowdoin, 1997).Several cis-elements and transcription factor binding sites have been characterized in geminiviruses. Sequence analysis of the common region of a range of geminiviruses has shown that there is little sequence homology except for several conserved motifs including a G-box and TATA box (Arguello-Astorga et al., 1994;Eagle & Hanley-Bowdoin, 1997;Fenoll et al., 1990). The G-box and TATA box mutant studies in tomato golden mosaic virus (TGMV) demonstrated that these motifs are important for transcription and also affect origin of replication activity (Eagle & Hanley-Bowdoin, 1997).Small circular single-stranded DNA satellites are associated with some monopartite begomoviruses, including cotton leaf curl Multan virus (CLCuMV), Ageratum yellow vein virus, tomato yellow leaf curl China virus (TYLCCNV) and Bhendi yellow ve...

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