Small circular single-stranded DNA satellites, termed DNAβ, have recently been found associated with some geminivirus infections. The DNA β associated with Cotton leaf curl virus is responsible for symptom expression of a devastating disease in Pakistan. Mutagenesis of DNA β revealed that the complementary-sense open reading frame (ORF) βC1 is required for inducing disease symptoms in Nicotiana tabacum. An ORF present on the virion-sense strand βV1 appeared to have no role in pathogenesis. Tobacco plants transformed with a βC1 ORF under the control of the Cauliflower mosaic virus 35S promoter or with a dimeric DNA β exhibited severe disease-like phenotypes, while plants transformed with a mutated version of βC1 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 βC1 ORF encoding a 118-amino acid product. It maps to the DNA β 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 βC1 protein is responsible for DNA β-induced disease symptoms.

The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form. The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV. Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region. Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element. This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.

DNA β 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 β contains a single gene, βC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the β-glucuronidase reporter gene driven by a βC1 promoter-deletion series of the DNA β associated with cotton leaf curl Multan virus identified a 68 nt region (between −139 and −207) which is important for βC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the βC1 start codon. Mutation of the G-box resulted in a significant reduction in βC1 promoter activity and DNA β 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 β.

Tomato yellow leaf curl disease is a major constraint for tomato production worldwide and availability of new resistant materials is of great importance for breeding programmes. A phenotypic survey was undertaken to evaluate the level of resistance to the main tomato yellow leaf curl disease-inducing viruses Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus, in several commercial tomato cultivars, never characterised before. Seven weeks post inoculation, two cultivars resulted in high resistant phenotypes to both begomoviruses, and four were tolerant to at least one of them. In the two highly resistant hybrids (SJ12, RFT112), symptoms were completely absent and viral DNA was from 10² to 10⁵ fold lower than in susceptible plants. Molecular marker analysis revealed that these genotypes harbour the resistant genes Ty-1/Ty-3 and Ty-2. Given their high resistance, they can be considered good candidates for cultivation and breeding in areas where incidence of TYLCD is very elevated.