The role of a geminiviral DNA β satellite in viral pathogenicity and movement

Abstract

Geminiviruses ( family Geminiviridae ) have circular single - stranded genomes encapsidated in twinned quasi - isometric particles and are responsible for major crop losses worldwide. The largest genus, Begomovirus, comprises viruses transmitted by the whitefly Bemisia tabaci. Most begomoviruses have bipartite genomes, termed DNA A and DNA B. The DNA A component encodes proteins required for viral DNA replication and encapsidation whereas the DNA B encodes two proteins that are essential for systemic movement. A small number of begomoviruses have a monopartite DNA genome that resembles the DNA A of bipartite begomoviruses. This DNA carries all gene functions for replication and pathogenesis. Specific small circular single - stranded DNA satellites containing a single open reading frame ( ORF ), termed DNA β, have recently been found in association with certain monopartite begomovirus infections. They comprise about 1350 nucleotides and require a helper begomovirus for replication and encapsidation. DNA β contributes to the production of symptoms and enhanced helper virus accumulation in certain hosts. This study examines the role of DNA β satellite in viral pathogenicity and movement in the host plant. Infectivity analysis of Tomato leaf curl virus and DNA β having mutation in the C1 and V1 ORF indicated that the complementary - sense ORF, βC1, is responsible for inducing disease symptoms in Nicotiana tabacum. An ORF present on the plus strand, βV1, appeared to have no role in pathogenesis. Tobacco plants transformed with the 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 carried the full βC1 ORF encoding a 118 amino acids product. It mapped to the DNA β nucleotide ( nt ) position 186 - 563 and contained a polyadenylation signal 18 nt upstream of the stop codon. A TATA box was located 43 nt upstream of the start codon. These results indicate that βC1 protein is responsible for DNA β induced disease symptoms. 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 β resulted in systemic infection. The presence of both DNA A and the DNA β in systemically infected tissues and the absence of DNA B was confirmed by probe hybridization. DNA β containing a disrupted βC1 ORF did not mobilize the DNA A for systemic infection. Co - inoculation of plants with DNA A and a construct of βC1 ORF, under the control of the Cauliflower mosaic virus 35S promoter, resulted in the systemic movement of the DNA A. βC1 fused to GFP accumulated around and inside the nucleus, at the periphery of tobacco and onion epidermis cells and co - localized with the endoplasmic reticulum. This distribution would be consistent with βC1 mediating intra cellular transport from the nucleus to the plasma membrane. These results showed that the βC1 protein can replace the functions of DNA B in allowing the systemic movement of a bipartite geminivirus DNA A.