Begomovirus coat protein interacts with a small heat-shock protein of its transmission vector (Bemisia tabaci)

Tomato yellow leaf curl Sardinia virus (TYLCSV) is transmitted from plant to plant by the whitefly Bemisia tabaci in a persistent-circulative manner. The coat protein (CP) plays an important role in this transmission cycle. In this study, the CP was used to screen a Bemisia tabaci cDNA library using the yeast two-hybrid system, in a search for interacting partners. A member of the small heat-shock protein family (termed BtHSP16) was identified and its interaction with the CP was verified by an in vitro pull-down assay. The binding domain was located at the variable N-terminal part of the CP, while full-length BtHSP16 is required for the interaction. The putative role for this interaction in the transmission cycle by the whitefly is discussed.     

Diversity of DNA beta,a satellite molecule associated with some monopartite begomoviruses

DNA beta molecules are symptom-modulating, single-stranded DNA satellites associated with monopartite begomoviruses (family Geminiviridae). Such molecules have thus far been shown to be associated with Ageratum yellow vein virus from Singapore and Cotton leaf curl Multan virus from Pakistan. Here, 26 additional DNA beta molecules, associated with diverse plant species obtained from different geographical locations, were cloned and sequenced. These molecules were shown to be widespread in the Old World, where monopartite begomoviruses are known to occur. Analysis of the sequences revealed a highly conserved organization for DNA beta molecules consisting of a single conserved open reading frame, an adenine-rich region, and a region of high sequence conservation [the satellite conserved region (SCR)]. The SCR contains a potential hairpin structure with the loop sequence TAA/GTATTAC; similar to the origins of replication of geminiviruses and nanoviruses. Two major groups of DNA beta satellites were resolved by phylogenetic analyses. One group originated from hosts within the Malvaceae and the second from a more diverse group of plants within the Solanaceae and Compositae. Within the two clusters, DNA beta molecules showed relatedness based both on host and geographic origin. These findings strongly support coadaptation of DNA beta molecules with their respective helper begomoviruses.     

Detection and Identification of the First Viruses in Chia (Salvia hispanica)

Chia (Salvia hispanica), an herbaceous plant native to Latin America, has become important in the last 20 years due to its beneficial effects on health. Here, we present the first record and identification of two viruses in chia plants. The comparison of the complete nucleotide sequences showed the presence of two viral species with the typical genome organization of bipartite New World begomovirus, identified as Sida mosaic Bolivia virus 2 and Tomato yellow spot virus, according to the ICTV taxonomic criteria for begomovirus classification. DNA-A from Sida mosaic Bolivia virus 2 exhibited 96.1% nucleotide identity with a Bolivian isolate of Sida micrantha, and Tomato yellow spot virus showed 95.3% nucleotide identity with an Argentine bean isolate. This is the first report of begomoviruses infecting chia as well as of the occurrence of Sida mosaic Bolivia virus 2 in Argentina.     

Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing

Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions.     

A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus

Mycoviruses are viruses that infect fungi and have the potential to control fungal diseases of crops when associated with hypovirulence. Typically, mycoviruses have double-stranded (ds) or single-stranded (ss) RNA genomes. No mycoviruses with DNA genomes have previously been reported. Here, we describe a hypovirulence-associated circular ssDNA mycovirus from the plant pathogenic fungus Sclerotinia sclerotiorum. The genome of this ssDNA virus, named Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), is 2166 nt, coding for a replication initiation protein (Rep) and a coat protein (CP). Although phylogenetic analysis of Rep showed that SsHADV-1 is related to geminiviruses, it is notably distinct from geminiviruses both in genome organization and particle morphology. Polyethylene glycol-mediated transfection of fungal protoplasts was successful with either purified SsHADV-1 particles or viral DNA isolated directly from infected mycelium. The discovery of an ssDNA mycovirus enhances the potential of exploring fungal viruses as valuable tools for molecular manipulation of fungi and for plant disease control and expands our knowledge of global virus ecology and evolution.     

Genetic variability of East African cassava mosaic Cameroon virus under field and controlled environment conditions

Cassava geminiviruses occur in all cassava growing areas of Africa and are considered to be the most damaging vector-borne plant pathogens. At least seven species of these viruses have been identified. We investigated genetic variation in East African cassava mosaic cassava Cameroon virus (EACMCV) from naturally infected cassava and from experimentally infected Nicotiana benthamiana. Results showed that the populations of EACMCV in cassava and in N. benthamiana were genetically heterogeneous. Mutation frequencies in the order of 10⁻⁴, comparable to that reported for plant RNA viruses, were observed in both hosts. We also produced an EACMCV mutant that induces reversion and second site mutations, thus suggesting that a high mutation frequency facilitates the maintenance of genome structure and function. This is direct experimental evidence showing that cassava geminiviruses exhibit a high mutation frequency and that a single clone quickly transforms into a collection of mutant sequences upon introduction into the host.     

Analysis of the genetic variability of maize streak virus

The nucleotide sequences of the small intergenic region (SIR) and the gene encoding the coat protein of 12 maize streak virus (MSV) isolates from different geographic locations have been determined. These have been used to assess the variability of the virus and to construct evolutionary dendrograms. For the viruses analyzed, the maximum levels of sequence divergence were found to be 10.9% and 2.0% at the nucleotide and amino acid levels, respectively. A genetically distinct strain of MSV was collected from islands in the Indian ocean. The significance of these findings for detection of the virus in epidemiological studies and breeding of resistant plant varieties is discussed.     

Potential gene products of bean golden mosaic virus have higher sequence homologies to those of tomato golden mosaic virus than to those of cassava latent virus

Comparison of the nucleotide sequences of the DNAs of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and cassava latent virus (CLV) revealed a fairly close relationship between BGMV DNA1, TGMV DNA1, and CLV DNA1 and a comparatively distant relationship between BGMV DNA2, TGMV DNA2, and CLV DNA2. The 200-base region common to the two DNAs of each virus had little sequence homology, except for a highly conserved 33–36 base sequence potentially capable of forming a stable hairpin structure. All the potential coding regions in the BGMV DNAs had counterparts in the TGMV and CLV DNAs suggesting an overall similarity in genome organization but two potential coding regions in the BGMV DNAs had no counterparts in the TGMV DNAs. The most highly conserved ORFs, BGMV 1R1, TGMV 1R1, and CLV 1R1, are the putative genes for the coat proteins of BGMV, TGMV, and CLV. BGMV 1R1 has 91.9% and 71.6% homology with respect to TGMV and CLV. The ORFs (BGMV 1L1; CLV 1L1; TGMV 1L1) and the two smaller overlapping ORFs (BGMV 1L2, 1L3; TGMV 1L2, 1L3; CLV 1L5, 1L3) are conserved in the three viruses. BGMV 2R1 and BGMV 2L1 have higher homology with respect to TGMV but not with respect to 2R1 and 2L1 in CLV. From this study we conclude that BGMV is more closely related to TGMV than CLV.