Background: Alphasatellites are small coding DNA satellites frequently associated with a begomovirus/betasatellite complex, where they are known to modulate virulence and symptom development. Two distinct alphasatellites, namely, Cotton leaf curl Multan alphasatellite (CLCuMuA), and Gossypium darwinii symptomless alphasatellite (GDarSLA) associated with Cotton leaf curl Multan virus-India (CLCuMuV-IN) and Ludwigia leaf distortion betasatellite (LuLDB) were found to be associated with yellow mosaic disease of hollyhock (Alcea rosea) plants. In this study, we show that alphasatellites CLCuMuA and GDarSLA attenuate and delay symptom development in Nicotiana benthamiana. The presence of either alphasatellites reduce the accumulation of the helper virus CLCuMuV-IN. However, the levels of the associated betasatellite, LuLDB, remains unchanged. These results suggest that the alphasatellites could contribute to the host defence and understanding their role in disease development is important for developing resistance strategies. Methods: Tandem repeat constructs of two distinct alphasatellites, namely, CLCuMuA and GDarSLA associated with CLCuMuV-IN and LuLDB were generated. N. benthamiana plants were co-agroinoculated with CLCuMuV and its associated alphasatellites and betasatellite molecules and samples were collected at 7, 14 and 21 days post inoculation (dpi). The viral DNA molecules were quantified in N. benthamiana plants by qPCR. The sequences were analysed using the MEGA-X tool, and a phylogenetic tree was generated. Genetic diversity among the CLCuMuA and GDarSLA was analysed using the DnaSP tool. Results: We observed a reduction in symptom severity and accumulation of helper virus in the presence of two alphasatellites isolated from naturally infected hollyhock plants. However, no reduction in the accumulation of betasatellite was observed. The phylogenetic and genetic variability study revealed the evolutionary dynamics of these distinct alphasatellites , which could explain the role of hollyhock-associated alphasatellites in plants. Conclusions: This study provides evidence that alphasatellites have a role in symptom modulation and suppress helper virus replication without any discernible effect on the replication of the associated betasatellite.

Tomato (Solanum lycopersicum L.) production in the USA has been severely impacted by the tomato yellow leaf curl virus (TYLCV). Furthermore, a complex association of whitefly-transmitted TYLCV (genus, begomovirus) and tomato chlorosis virus (ToCV, genus, crinivirus) were recently identified in tomato. Several tomato cultivars were developed and commercialized with intermediate resistance (IR) against TYLCV-IL (Israel), the predominant strain of TYLCV found in Georgia, USA. TYLCV-resistant cultivars were tested in open field conditions against multiple whitefly-transmitted viruses in Georgia under natural disease pressure during the fall of 2022. The area under disease progress curve (AUDPC) over time showed a steady increase in disease severity among all cultivars. Further analysis of infected samples using high throughput sequencing (HTS) and quantitative PCR (qPCR) revealed the presence of TYLCV and ToCV in symptomatic upper and lower leaves, respectively. Moreover, the presence of both viruses in upper and lower leaves was determined. A mixed infection of both viruses, TYLCV and ToCV, resulted in severe disease development which may enhance the commercial tomato plants to break resistance and lead to decreased fruit quality and marketable yields.

Geminivirus replication initiator protein (Rep) is a multifunctional viral protein required for replication. During the process of viral replication, Rep acts as a site‐ and strand‐specific endonuclease, ligase, ATPase, and helicase. B' motif and β‐hairpin loop of the geminivirus Rep are conserved and important for Rep‐mediated helicase activity required for viral replication. To dissect the roles of various amino acid residues of the B' motif and β‐hairpin loop of the geminivirus Rep helicase in its process of unwinding DNA, we investigated eight conserved residues near the ATP active site or the ssDNA contact channel. Our strategy was to mutate these residues to alanines and investigate the effects of these mutations on various biochemical activities associated with DNA unwinding. We looked into the ATP binding, ATP hydrolysis, DNA binding, and DNA unwinding activities of the wild‐type and mutant Rep proteins. These investigations showed four residues (Arg279, Asp280, Tyr287, and Pro290) affecting the DNA unwinding activity. A structural model analysis confirmed the B' loop and ssDNA binding loop to be connected through a β‐hairpin structure, suggesting that changes on one loop might affect the other and that these residues function by acting in concert. Viral genomes containing Rep proteins having these mutations in the B' motif did not replicate in planta. Taken together, these results indicated all four residues to be implicated in helicase activity mediated by Rep and demonstrated the significance, for viral replication, of the B' motif and β‐hairpin loop of the C‐terminal region of the Rep protein.