A. M. Idris

Diversity of DNA β, 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.

Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus

Abstract The family Geminiviridae includes plant-infecting circular single-stranded DNA viruses that have geminate particle morphology. Members of this family infect both monocotyledonous and dicotyledonous plants and have a nearly global distribution. With the advent of new molecular tools and low-cost sequencing, there has been a significant increase in the discovery of new geminiviruses in various cultivated and non-cultivated plants. In this communication, we highlight the establishment of three new genera (Becurtovirus, Eragrovirus and Turncurtovirus) to accommodate various recently discovered geminiviruses that are highly divergent and, in some cases, have unique genome architectures. The genus Becurtovirus has two viral species, Beet curly top Iran virus (28 isolates; leafhopper vector Circulifer haematoceps) and Spinach curly top Arizona virus (1 isolate; unknown vector), whereas the genera Eragrovirus and Turncurtovirus each have a single assigned species: Eragrostis curvula streak virus (6 isolates; unknown vector) and Turnip curly top virus (20 isolates; leafhopper vector Circulifer haematoceps), respectively. Based on analysis of all of the genome sequences available in public databases for each of the three new genera, we provide guidelines and protocols for species and strain classification within these three new genera.

CRISPR/Cas9-mediated viral interference in plants

BackgroundThe CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species.ResultsIn this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses.ConclusionsThese data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

The core region of the coat protein gene is highly useful for establishing the provisional identification and classification of begomoviruses

Summary. Polymerase chain reaction (PCR) was applied to detect and establish provisional identity of begomoviruses through amplification of a ∼ 575 bp fragment of the begomoviral coat protein gene (CP), referred to as the ‘core’ region of the CP gene (core CP). The core CP fragment contains conserved and unique regions, and was hypothesized to constitute a sequence useful for begomovirus classification. Virus relationships were predicted by distance and parsimony analyses using the A component (bipartite viruses) or full genome (monopartite viruses), CP gene, core CP, or the 200 5′-nucleotides (nt) of the CP. Reconstructed trees and sequence divergence estimates yielded very similar conclusions for all sequence sets, while the CP 5′-200 nt was the best strain discriminator. Alignment of the core CP region for 52 field isolates with reference begomovirus sequences permitted provisional virus identification based on tree position and extent of sequence divergence. Geographic origin of field isolates was predictable based on phylogenetic separation of field isolates examined here. A ‘closest match’ or genus-level identification could be obtained for previously undescribed begomoviruses using the BLAST program to search a reference core CP database located at our website and/or in GenBank. Here, we describe an informative molecular marker that permits provisional begomovirus identification and classification using a begomoviral sequence that is smaller than the presently accepted, but less accessible CP sequence.

Geminivirus-Mediated Gene Silencing from Cotton Leaf Crumple Virus Is Enhanced by Low Temperature in Cotton

A silencing vector for cotton (Gossypium hirsutum) was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, the magnesium chelatase subunit I gene (ChlI) or the phytoene desaturase gene (PDS). Cotyledons of cotton cultivar ‘Deltapine 5415’ bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in approximately 70% of inoculated plants after 2 to 3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30°C/26°C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22°C/18°C. However, endogenous gene silencing decreased at 30°C/26°C. There was an approximately 7 d delay in the onset of gene silencing at 22°C/18°C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.

An unusual alphasatellite associated with monopartite begomoviruses attenuates symptoms and reduces betasatellite accumulation.

The Oman strain of Tomato yellow leaf curl virus (TYLCV-OM) and its associated betasatellite, an isolate of Tomato leaf curl betasatellite (ToLCB), were previously reported from Oman. Here we report the isolation of a second, previously undescribed, begomovirus [Tomato leaf curl Oman virus (ToLCOMV)] and an alphasatellite from that same plant sample. This alphasatellite is closely related (90 % shared nucleotide identity) to an unusual DNA-2-type Ageratum yellow vein Singapore alphasatellite (AYVSGA), thus far identified only in Singapore. ToLCOMV was found to have a recombinant genome comprising sequences derived from two extant parents, TYLCV-OM, which is indigenous to Oman, and Papaya leaf curl virus from the Indian subcontinent. All possible combinations of ToLCOMV, TYLCV-OM, ToLCB and AYVSGA were used to agro-inoculate tomato and Nicotiana benthamiana. Infection with ToLCOMV yielded mild leaf-curl symptoms in both hosts; however, plants inoculated with TYLCV-OM developed more severe symptoms. Plants infected with ToLCB in the presence of either helper begomovirus resulted in more severe symptoms. Surprisingly, symptoms in N. benthamiana infected with the alphasatellite together with either of the helper viruses and the betasatellite were attenuated and betasatellite DNA accumulation was substantially reduced. However, in the latter plants no concomitant reduction in the accumulation of helper virus DNA was observed. This is the first example of an attenuation of begomovirus-betasatellite symptoms by this unusual class of alphasatellites. This observation suggests that some DNA-2 alphasatellites encode a pathogenicity determinant that may modulate begomovirus-betasatellite infection by reducing betasatellite DNA accumulation.

Sinaloa tomato leaf curl geminivirus: biological and molecular evidence for a new subgroup III virus.

ABSTRACT The biological and molecular properties of Sinaloa tomato leaf curl virus (STLCV) were investigated in line with the hypothesis that STLCV is a previously uncharacterized, whitefly-transmitted geminivirus from North America. STLCV causes yellow leaf curl symptoms in tomato and yellow-green foliar mottle in pepper. Five species belonging to two plant families were STLCV experimental hosts. STLCV had a persistent relationship with its whitefly vector, Bemisia tabaci. Polymerase chain reaction fragments of STLCV common region (CR) sequences of the A or B genomic components and the viral coat protein gene (AV1) were molecularly cloned and sequenced. The STLCV A- and B-component CR sequences (174 nucleotides each) shared 97.9% identity and contained identical cis elements putatively involved in transcriptional regulation and an origin of replication (the AC cleavage site within the loop of the hairpin structure and two direct repeat sequences thought to constitute the Rep binding motif), which collectively are diagnostic for subgroup III geminiviruses. The STLCV CR sequence shared 23.1 to 77.6% identity with CR sequences of representative geminiviridae, indicating the STLCV CR sequence is unique. Molecular phylogenetic analysis of CR or AV1 sequences of STLCV and the respective sequences of 31 familial members supported the placement of STLCV as a unique bipartite, subgroup III virus most closely related to other viruses from the Western Hemisphere. STLCV is provisionally described as a new species within the genus Begomovirus, family Geminiviridae.

ICTV Virus Taxonomy Profile: Geminiviridae

The geminiviruses are a family of small, non-enveloped viruses with single-stranded, circular DNA genomes of 2500–5200 bases. Geminiviruses are transmitted by various types of insect (whiteflies, leafhoppers, treehoppers and aphids). Members of the genus Begomovirus are transmitted by whiteflies, those in the genera Becurtovirus, Curtovirus, Grablovirus, Mastrevirus and Turncurtovirus are transmitted by specific leafhoppers, the single member of the genus Topocuvirus is transmitted by a treehopper and one member of the genus Capulavirus is transmitted by an aphid. Geminiviruses are plant pathogens causing economically important diseases in most tropical and subtropical regions of the world. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Geminiviridae which is available at www.ictv.global/report/geminiviridae.

Sequence analysis and classification of apparent recombinant begomoviruses infecting tomato in the Nile and Mediterranean basins

ABSTRACT Numerous whitefly-transmitted viral diseases of tomato have emerged in countries around the Nile and Mediterranean Basins the last 20 years. These diseases are caused by monopartite geminiviruses (family Gemini viridae) belonging to the genus Begomovirus that probably resulted from numerous recombination events. The molecular biodiversity of these viruses was investigated to better appreciate the role and importance of recombination and to better clarify the phylogenetic relationships and classification of these viruses. The analysis partitioned the tomato-infecting begomoviruses from this region into two major clades, Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus. Phylogenetic and pairwise analyses together with an evaluation for gene conversion were performed from which taxonomic classification and virus biodiversity conclusions were drawn. Six recombination hotspots and three homogeneous zones within the genome were identified among the tomatoinfecting isolates and species examined here, suggesting that the recombination events identified were not random occurrences.

Emergence of a New Cucurbit-Infecting Begomovirus Species Capable of Forming Viable Reassortants with Related Viruses in the Squash leaf curl virus Cluster.

ABSTRACT Cucurbit leaf curl virus (CuLCV), a whitefly-transmitted geminivirus previously partially characterized from the southwestern United States and northern Mexico, was identified as a distinct bipartite begomovirus species. This virus has near sequence identity with the previously partially characterized Cucurbit leaf crumple virus from California. Experimental and natural host range studies indicated that CuLCV has a relatively broad host range within the family Cucurbitaceae and also infects bean and tobacco. The genome of an Arizona isolate, designated CuLCV-AZ, was cloned and completely sequenced. Cloned CuLCV-AZ DNA A and B components were infectious by biolistic inoculation to pumpkin and progeny virus was transmissible by the whitefly vector, Bemisia tabaci, thereby completing Kochs postulates. CuLCV-AZ DNA A shared highest nucleotide sequence identity with Squash leaf curl virus-R (SLCV-R), SLCV-E, and Bean calico mosaic virus (BCaMV) at 84, 83, and 80%, respectively. The CuLCV DNA B component shared highest nucleotide sequence identity with BCaMV, SLCV-R, and SLCV-E at 71, 70, and 68%, respectively. The cis-acting begomovirus replication specificity element, GGTGTCCTGGTG, in the CuLCV-AZ origin of replication is identical to that of SLCV-R, SLCV-E, and BCaMV, suggesting that reassortants among components of CuLCV-AZ and these begomoviruses may be possible. Reassortment experiments in pumpkin demonstrated that both reassortants of CuLCV-AZ and SLCV-E A and B components were viable. However, for CuLCV-AZ and SLCV-R, only one reassortant (SLCV-R DNA A/CuLCV-AZ DNA B) was viable on pumpkin, even though the cognate component pairs of both viruses infect pumpkin. These results demonstrate that reassortment among sympatric begomovirus species infecting cucurbits are possible, and that, if generated in nature, could result in begomoviruses bearing distinct biological properties.