Geminivirus replication enhancer (REn) proteins dramatically increase the accumulation of viral DNA species by an unknown mechanism. In this study, we present evidence implicating SlNAC1, a new member of the NAC domain protein family from tomato (Solanum lycopersicum), in Tomato leaf curl virus (TLCV) REn function. We isolated SlNAC1 using yeast (Saccharomyces cerevisiae) two-hybrid technology and TLCV REn as bait, and confirmed the interaction between these proteins in vitro. TLCV induces SlNAC1 expression specifically in infected cells, and this upregulation requires REn. In a transient TLCV replication system, overexpression of SlNAC1 resulted in a substantial increase in viral DNA accumulation. SlNAC1 colocalized with REn to the nucleus and activated transcription of a reporter gene in yeast, suggesting that in healthy cells it functions as a transcription factor. Together, these results imply that SlNAC1 plays an important role in the process by which REn enhances TLCV replication.

DNA b is a circular single-stranded satellite DNA which co-infects with certain monopartite helper begomoviruses to cause economically important diseases, such as cotton leaf curl disease (CLCuD). DNA b encodes a single protein, bC1. Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus in which both DNA A and DNA B are required for systemic infection. Inoculation of tomato plants with ToLCNDV DNA A alone induced local but not systemic infection, whereas co-inoculation with DNA A and the DNA b associated with CLCuD resulted in systemic infection. DNA b containing a disrupted bC1 open reading frame (ORF) did not mobilize DNA A systemically. Co-inoculation of plants with DNA A and a construct of the bC1 ORF, under the control of the cauliflower mosaic virus 35S promoter, resulted in the systemic movement of DNA A. In inoculated tobacco and onion epidermal cells, bC1 fused to GFP was localized at the cell periphery in association with punctate bodies, around and within the cell nucleus and with the endoplasmic reticulum. It is concluded that heterologous bC1 protein can replace the movement function of the DNA B of a bipartite begomovirus. Evidence is also provided that tomato leaf curl virus-encoded C4 protein confers the same movement function to ToLCNDV DNA A. The intracellular distribution of bC1 is consistent with the hypothesis that it has a role in transporting the DNA A from the nuclear site of replication to the plasmodesmatal exit sites of the infected cell.

Viroids are the smallest autonomous infectious nucleic acids known so far. With a small circular RNA genome of about 250-400 nt, which apparently does not code for any protein, viroids replicate and move systemically in host plants. Since the discovery of the first viroid almost forty-five years ago, many different viroids have been isolated, characterized and, frequently, identified as the causal agents of plant diseases. The first viroid classification scheme was proposed in the early 1990s and adopted by the International Committee on Taxonomy of Viruses (ICTV) a few years later. Here, the current viroid taxonomy scheme and the criteria for viroid species demarcation are discussed, highlighting the main taxonomic questions currently under consideration by the ICTV Viroid Study Group. The impact of correct taxonomic annotation of viroid sequence variants is also addressed, taking into consideration the increasing application of nextgeneration sequencing and bioinformatics for known and previously unrecognized viroids.

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