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PubMed1997

Efficient whitefly transmission of African cassava mosaic geminivirus requires sequences from both genomic components.

Clones of two subgroup III geminiviruses, the common strain of tomato golden mosaic virus (csTGMV) and African cassava mosaic virus originating from Kenya (ACMV-K), were shown to be non-transmissible by whitefiles. Lack of transmissibility of cloned ACMV-K was investigated by exchanging genomic components with a whitefly-transmissible ACMV isolate from Nigeria (ACMV-NOg). Neither pseudorecombinant was transmissible, indicating that defects in both genomic components contributed to the lack of transmissibility. Analysis of the acquisition of the pseudorecombinats by Bemisia tabaci indicated that accumulation of virus within the insect was DNA B dependent. Return of virus to plants was determined by DNA A, although the coat protein was essential for acquisition. Repeated passaging of both the wild strain of ACMV-NOg and the cloned virus led to loss of insect transmissibility of the wild isolate but not the cloned virus. Products encoded on both genomic components are required for transmission of bipartite geminiviruses by B. tabaci.

Animals↗

PubMed2006

Tomato yellow spot virus, a tomato-infecting begomovirus from Brazil with a closer relationship to viruses from Sida sp., forms pseudorecombinants with begomoviruses from tomato but not from Sida.

Geminiviruses are characterized by a circular, single-stranded DNA genome and twinned icosahedral particles. Begomoviruses (whitefly-transmitted geminiviruses) are a major constraint to crop production worldwide. In Brazil, tomato-infecting begomoviruses emerged as serious pathogens over the last 10 years, due to the introduction of a new biotype of the insect vector. Tomato yellow spot virus (ToYSV) is a newly described begomovirus originally isolated from tomato, but phylogenetically closer to viruses from Sida sp. A study was performed to determine the viability of pseudorecombinants formed between the DNA components of ToYSV and other weed- and tomato-infecting begomoviruses from Brazil. Despite its closer relationship to weed-infecting viruses, ToYSV was only capable of forming viable pseudorecombinants with tomato viruses. An infectious pseudorecombinant formed between ToYSV DNA-A and tomato crinkle leaf yellows virus (TCrLYV) DNA-B induced severe symptoms in Nicotiana benthamiana. This was attributed, at least in part, to the fact that the origins of replication of both components had identical Rep-binding sequences. However, this was not the case for another infectious pseudorecombinant formed between tomato golden mosaic virus (TGMV) DNA-A and ToYSV DNA-B, which have different Rep-binding sequences. These results reinforce the notion that pseudorecombinant formation cannot be explained solely on the basis of phylogenetic relationships and conserved iteron sequences, and suggest that the TGMV Rep protein may be more versatile in terms of recognizing heterologous DNA components than that of ToYSV.

Amino Acid Sequence↗

PubMed2001

Gene C2 of the monopartite geminivirus tomato yellow leaf curl virus-China encodes a pathogenicity determinant that is localized in the nucleus.

Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent protein (GFP) fused to the C2 protein (C2-GFP) in Nicotiana benthamiana from a Potato virus X (PVX) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. The localization of GFP fluorescence in plant cells infected with PVX/C2-GFP and in insect cells transfected with Baculovirus expressing C2-GFP indicates that the TYLCV-C C2 protein is capable of shuttling GFP into plant and insect cell nuclei. Our data demonstrate that the TYLCV-C C2 protein may contribute to viral pathogenicity in planta and is nuclear localized.

Amino Acid Sequence↗

PubMed2001

An iteron-related domain is associated to Motif 1 in the replication proteins of geminiviruses: identification of potential interacting amino acid-base pairs by a comparative approach.

Geminiviruses encode a replication initiator protein, Rep, which binds in a sequence-specific fashion to iterated DNA motifs (iterons) functioning as essential elements for virus-specific replication. By using the iterons of more than one hundred geminiviruses as heuristic devices, we have identified a Rep subdomain 8 to 10 residues in length, whose primary structure varies among viruses harboring different iterons, but which is similar among viruses with identical iterons, regardless of their differences in host range, insect vector, geographical origin or genome structure. Close analysis of this iteron-related domain (IRD) revealed consistent correlations between specific Rep residues and defined nucleotides of its cognate iteron, thus providing important insights about the molecular code which dictates the Rep preference for specific DNA sequences. A model of potential Rep-iteron contacts is proposed. The identified IRD is adjacent to a conserved motif characteristic of a superfamily of rolling-circle (RC) replication proteins, and secondary structure predictions suggest that those Rep subdomains form together the core of a novel DNA-binding domain possessing a beta-sheet as recognition subdomain, which is apparently conserved in the replication proteins of nanoviruses, circoviruses, microviruses, and a variety of ssDNA plasmids of eubacteria, archaebacteria and red algae. The evolutionary implications of these findings are discussed.

Amino Acid Motifs↗

PubMed2006

Whitefly (Bemisia tabaci) genome project: analysis of sequenced clones from egg, instar, and adult (viruliferous and non-viruliferous) cDNA libraries.

BACKGROUND: The past three decades have witnessed a dramatic increase in interest in the whitefly Bemisia tabaci, owing to its nature as a taxonomically cryptic species, the damage it causes to a large number of herbaceous plants because of its specialized feeding in the phloem, and to its ability to serve as a vector of plant viruses. Among the most important plant viruses to be transmitted by B. tabaci are those in the genus Begomovirus (family, Geminiviridae). Surprisingly, little is known about the genome of this whitefly. The haploid genome size for male B. tabaci has been estimated to be approximately one billion bp by flow cytometry analysis, about five times the size of the fruitfly Drosophila melanogaster. The genes involved in whitefly development, in host range plasticity, and in begomovirus vector specificity and competency, are unknown. RESULTS: To address this general shortage of genomic sequence information, we have constructed three cDNA libraries from non-viruliferous whiteflies (eggs, immature instars, and adults) and two from adult insects that fed on tomato plants infected by two geminiviruses: Tomato yellow leaf curl virus (TYLCV) and Tomato mottle virus (ToMoV). In total, the sequence of 18,976 clones was determined. After quality control, and removal of 5,542 clones of mitochondrial origin 9,110 sequences remained which included 3,843 singletons and 1,017 contigs. Comparisons with public databases indicated that the libraries contained genes involved in cellular and developmental processes. In addition, approximately 1,000 bases aligned with the genome of the B. tabaci endosymbiotic bacterium Candidatus Portiera aleyrodidarum, originating primarily from the egg and instar libraries. Apart from the mitochondrial sequences, the longest and most abundant sequence encodes vitellogenin, which originated from whitefly adult libraries, indicating that much of the gene expression in this insect is directed toward the production of eggs. CONCLUSION: This is the first functional genomics project involving a hemipteran (Homopteran) insect from the subtropics/tropics. The B. tabaci sequence database now provides an important tool to initiate identification of whitefly genes involved in development, behaviour, and B. tabaci-mediated begomovirus transmission.

Amino Acid Sequence↗

PubMed1995

Genome organization of ageratum yellow vein virus, a monopartite whitefly-transmitted geminivirus isolated from a common weed.

A full-length copy of a single genomic component of the whitefly-transmitted geminivirus ageratum yellow vein virus (AYVV) has been cloned from an extract of infected Ageratum conyzoides originating from Singapore. Sequence analysis shows that the genomic component encodes two virion-sense (V1 and V2) and four complementary-sense open reading frames (C1-C4), typical of DNA A of whitefly-transmitted geminiviruses from the Eastern hemisphere. A genomic component equivalent to DNA B was not detected in extracts of infected A. conyzoides. The cloned genomic component produced a systemic infection in Nicotiana benthamiana, Phaseolus vulgaris and Lycopersicon esculentum when introduced into plants by agroinoculation, and symptoms were identical to those produced by wild-type virus introduced into these hosts using viruliferous whiteflies. However, attempts to re-establish a systemic infection in A. conyzoides either by agroinoculation or by whitefly transmission of the cloned progeny were unsuccessful, suggesting that additional factors are required for infection of the natural host. The significance of A. conyzoides as a reservoir host for the economically important geminivirus diseases is discussed.

Animals↗

PubMed2004

Geminivirus VIGS of endogenous genes requires SGS2/SDE1 and SGS3 and defines a new branch in the genetic pathway for silencing in plants.

Virus-induced gene silencing (VIGS) is a sequence-specific RNA degradation process that can be used to downregulate plant gene expression. Both RNA and DNA viruses have been used for VIGS, but they differ in their mode of replication, gene expression, and cellular location. This study examined silencing mediated by a DNA virus, cabbage leaf curl virus (CaLCuV), in several silencing-deficient Arabidopsis mutants. A DNA VIGS vector derived from CaLCuV, which silenced chlorata42 (ChlI) needed for chlorophyll formation, was used to test endogenous gene silencing responses in suppressor of gene silencing (sgs)1, sgs2, sgs3, and Argonaute (ago)1 mutants defective in sense transgene-mediated post-transcriptional silencing (S-PTGS). SGS2/silencing defective (SDE)1, SGS3, and AGO1 are each dispensable for silencing mediated by transgenes containing inverted repeats (IR-PTGS), and SGS2/SDE1 is dispensable for RNA VIGS. We show that DNA VIGS requires both SGS2/SDE1 and SGS3, regardless of the orientation of 362 nt ChlI transcripts produced from the viral DNA promoter. Viral DNA accumulation is slightly higher, and viral symptoms increase in sgs2 and sgs3, whereas overexpression of SGS2/SDE1 mRNA results in decreased viral symptoms. Mutants affected in SGS1 and AGO1 function are only delayed in the onset of silencing, and have a small effect on chlorophyll accumulation. DNA VIGS is unaffected in defective DNA methylation (ddm)1/somniferous (som)8 and maintenance of methylation (mom)1 mutants, impaired for TGS. These results demonstrate that SGS2/SDE1 and SGS3 are needed for endogenous gene silencing from DNA viruses, and suggest that SGS2/SDE1 may reduce geminivirus symptoms by targeting viral mRNAs.

Arabidopsis↗

PubMed1992

Coordinate regulation of replication and virion sense gene expression in wheat dwarf virus.

We have investigated the relationship between viral DNA replication and virion sense gene expression in wheat dwarf virus (WDV), a member of the geminivirus group, by testing a series of deletion mutants in transfected Triticum monococcum (einkorn) protoplasts. Mutants contained a transcription fusion of the chloramphenicol acetyltransferase coding sequence to the virion sense promoter that replaced the viral coat protein coding sequence. The deletion analysis revealed that WDV replication and virion sense transcription can proceed independently and are controlled in part by nonoverlapping elements in the large intergenic region. These data and those from a C2 open reading frame (ORF) frameshift mutant also showed that the product of the C2 ORF (C1-C2 protein) is independently involved in both DNA replication and activation of the virion sense promoter. The amino acid sequences encoded by C2, which are highly conserved in the geminivirus group, show some homology to the DNA binding domain of the myb-related class of plant transcription factors. The possible involvement of the host in controlling the function of the C1-C2 protein and the implication of these data for the development of WDV-based gene vectors are discussed.

Amino Acid Sequence↗

PubMed1993

Comparative epidemiology of three tropical whitefly-transmitted geminiviruses.

Spatial and temporal patterns of spread of African cassava mosaic, okra leaf curl and tobacco leaf curl viruses in West Africa, East Africa and India share some general characteristics. By comparing the results and running new analyses on available data, it is shown that the epidemiology of these viruses is controlled by the same key variables. For instance, spatial spread is characterised by strong border effects due to accumulation of whitefly vectors (Bemisia tabaci) on the wind-exposed field borders under the influence of the prevailing wind. This results in pronounced environmental gradients of disease. Temporal patterns of virus spread are driven by the sinusoidal fluctuation of temperature over the year which correspond with changes of whitefly populations.

Animals↗

PubMed1989

Replication of tomato golden mosaic virus DNA B in transgenic plants expressing open reading frames (ORFs) of DNA A: requirement of ORF AL2 for production of single-stranded DNA.

Tomato golden mosaic geminivirus has a genome of two single-stranded (ss) DNA components, A and B. An almost identical 'common' region in DNA A and DNA B is thought to contain sequence elements controlling replication and transcription. Hence investigation of sequences important for DNA replication by in vitro mutagenesis is complicated by possible effects on the transcription of genes for replication proteins. To overcome this problem, transgenic plants expressing open reading frames (ORFs) of DNA A from an enhanced cauliflower mosaic virus 35S RNA promoter were constructed and tested for their ability to support the replication of DNA B and DNA B mutants. The results show that plants transgenic for ORF AL1 are able to support the replication of the double-stranded (ds) forms of DNA B, but that ORF AL2 is required in addition to produce ssDNA B. ORFs AL3, BL1 or BR1 were not required for replication of ds or ssDNA B. To the best of our knowledge this is the first time that essential replication proteins of a geminivirus have been expressed constitutively from a plant genome without giving rise to replicating DNA A molecules, thereby allowing DNA B to replicate alone. Such transgenic plants should enable not only the mutational analysis of sequence elements within the replication origin region, but also the construction of a new generation of vectors for gene amplification in plants, based on a minimal virus replicon.

DNA Probes↗

PubMed1993

Transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA of tobacco mosaic virus.

We engineered cDNA of tobacco mosaic tobamovirus (TMV) into Agrobacterium tumefaciens for inoculation of plant cells. The resulting bacterial strains were used to transfect tobacco (Nicotiana tabacum cv. Xanthi and Xanthi/nc) with wild type and a defective virus. Lesion formation on Xanthi/nc tobacco was used to measure the timing and efficiency of transfection. Infections mediated by Agrobacterium produced lesions an average of two days later than infections produced by inoculation with virions. The addition of approximately 80 bp of non-viral sequences to the 5'-end of TMV transcripts abolished transfection. Transcripts with non-viral sequences at the 3'-end initiated infections, while precise transcript termination with a synthetic ribozyme sequence increased transfection frequencies two-fold. Culture conditions reported to induce genes of the vir region of the Agrobacterium Ti plasmid also increased the transfection frequency approximately two-fold. Therefore, in addition to the pararetroviruses and geminiviruses previously described, 'agroinoculation' may be used to infect plants with plus-sense RNA viruses.

Agrobacterium tumefaciens↗

PubMed1988

Characterization of potato yellow mosaic virus as a geminivirus with a bipartite genome.

A whitefly-transmitted virus from Venezuela, potato yellow mosaic virus, has been propagated, isolated and characterized as a member of the Geminivirus group. The virus was transmitted to several species of Nicotiana and to Petunia hybrida by mechanical inoculation, and to potato and tomato plants by grafting. Purified virus possessed typical geminate particle morphology and encapsidated both genomic and subgenomic species consisting of single-stranded DNA. The genome of the virus was cloned into both bacteriophage and plasmid vectors following restriction of supercoiled double-stranded DNA species isolated from infected plant extracts. Two distinct classes of cloned DNA were generated, both about 2,500-2,600 base pairs in length and designated A and B, which when inoculated in combination to Nicotiana benthamiana were infectious after excision from the recombinant clones. The subgenomic DNA was shown to be related to DNA B.

Cloning, Molecular↗

PubMed1987

Structure and replication of geminivirus genomes.

The geminiviruses are a group of plant viruses containing single-stranded (ss) DNA in particles comprising two quasi-icosahedral units. Some are transmitted by whiteflies, others by leafhoppers. Comparisons were made of the genome organization and expression of cassava latent virus (CLV) and maize streak virus (MSV) and beet curly top virus (BCTV), each with distinct host range and insect vector species characteristics. From these studies, several indications as to the replication mechanism(s) are suggested.

Base Sequence↗

PubMed2001

Transmission of viral RNA and DNA to maize kernels by vascular puncture inoculation.

Vascular puncture inoculation (VPI) is an effective technique for transmission of maize viruses without using arthropods or other biological vectors. It involves using a jeweler's engraving tool to push minuten pins through a droplet of virus inoculum toward the major vascular bundle in the scutellum of germinating kernels. Here, VPI is shown to be useful for introducing RNA and DNA viral genomes into maize. Maize dwarf mosaic potyvirus (MDMV) virions, MDMV genomic RNA, foxtail mosaic potexvirus (FoMV) genomic RNA and maize streak geminivirus (MSV) DNA were introduced into kernels by VPI, and infection rates determined. At high concentrations, both MDMV virion and genomic RNA preparations produced 100% infection of susceptible maize. However, MDMV genomic RNA was transmitted with about 100-fold lower efficiency than virions. FoMV genomic RNA and MSV DNA were transmitted at lower efficiency than the MDMV RNA, and the highest transmission rates were about 50%. Ribonuclease A pretreatment prevented genomic MDMV and FoMV RNA transmission, but not MDMV virion transmission indicating the viral RNA was the infectious entity. Proteinase K (ProK) pretreatment reduced transmission of MDMV RNA suggesting that integrity of the viral genomic protein bound covalently to the viral RNA may be important for efficient transmission.

Blotting, Western↗

PubMed2006

Post-transcriptional gene silencing in controlling viruses of the Tomato yellow leaf curl virus complex.

Tomato yellow leaf curl disease (TYLCD) is caused by a group of geminiviruses that belong to the Tomato yellow leaf curl virus (TYLCV) complex and are transmitted by the whitefly (Bemisia tabaci Genn.). The disease causes great yield losses in many countries throughout the Mediterranean region and the Middle East. In this study, the efficacy of post-transcriptional gene silencing (PTGS) to control the disease caused by TYLCV complex was investigated. Non-coding conserved regions from the genome of TYLCV, Tomato yellow leaf curl virus-mild, tomato yellow leaf curl Sardinia virus, tomato yellow leaf curl Malaga virus, and tomato yellow leaf curl Sardinia virus-Spain [2] were selected and used to design a construct that can trigger broad resistance against different viruses that cause tomato yellow leaf curl disease. The silencing construct was cloned into an Agrobacterium-binary vector in sense and antisense orientation and used in transient assay to infiltrate tomato and Nicotiana benthamiana plants. A high level of resistance was obtained when plants were agro-infiltrated with an infectious clone of the Egyptian isolate of TYLCV (TYLCV-[EG]) or challenge inoculated with TYLCV, TYLCV-Mld, and TYLCSV-ES[2] using whitefly-mediated transmission 16-20 days post infiltration with the silencing construct. Results of the polymerase chain reaction showed that the resistance was effective against all three viruses. Furthermore, dot blot hybridization and PCR failed to detect viral DNA in symptomless, silenced plants. A positive correlation between resistance and the accumulation of TYLCV-specific siRNAs was observed in silenced plants. Together, these data provide compelling evidence that PTGS can be used to engineer geminivirus-resistant plants.

Animals↗

PubMed2003

Geminivirus disease complexes: an emerging threat.

Small circular single-stranded DNA satellites have recently been isolated from plants infected with whitefly-transmitted monopartite begomoviruses. The satellites, named DNA beta, depend on the helper viruses for their proliferation and, in turn, are required for helper virus accumulation and symptom expression. They are highly diverse yet retain an overall conserved structure with respect to potential coding regions and regulatory elements. The begomovirus-satellite disease complexes are associated with economically important diseases, and have been isolated from vegetable and fibre crops, ornamental plants and weeds throughout Africa and Asia. Their widespread distribution and diversity, coupled to the global movement of plant material and the dissemination of the whitefly vector, suggests that these disease complexes pose a serious threat to tropical and sub-tropical agro-ecosystems worldwide.

Africa↗

PubMed2000

Pea embryonic tissues show common responses to the replication of a wide range of viruses.

The response of pea embryonic tissues to the replication of a range of different viruses was investigated using in situ hybridization to analyze changes in the expression of two host genes, heat shock protein 70 (hsp70) and lipoxygenase (lox1). Excised pea embryos were infected using microprojectile bombardment with a nonseed transmissible strain of Pea seed-borne mosaic potyvirus, or with Pea early browning tobravirus (PEBV), White Clover mosaic potexvirus, or Beet curly top geminivirus. Collectively, these examples represent families of viruses with differing genomic features, differing numbers of genomic components and differing replication strategies. In all cases, there was an induction of hsp70 associated with virus replication and, in most cases, a downregulation of lox1. Hence, either each virus has a direct inducer of these common responses or the induction is indirectly the result of a generic feature of virus infection. By exploiting the bipartite nature of the PEBV genome, the coat protein gene and genes involved in vector transmission were excluded as potential inducers.

Cotyledon↗

PubMed1989

Maize streak virus genes essential for systemic spread and symptom development.

The entire genome of single component geminiviruses such as maize streak virus (MSV) consists of a single-stranded circular DNA of ~2.7 kb. Although this size is sufficient to encode only three average sized proteins, the virus is capable of causing severe disease of many monocots with symptoms of chlorosis and stunting. We have identified viral gene functions essential for systemic spread and symptom development during MSV infection. Deletions and gene replacement mutants were created by site-directed mutagenesis and insertion between flanking MSV or reporter gene sequences contained in Agrobacterium T-DNA derived vectors. Following Agrobacterium-mediated inoculation of maize seedlings, the mutated MSV DNAs were excised from these binary vectors by homologous recombination within the flanking sequences. Our analyses show that the capsid gene of MSV, while not required for replication, is essential for systemic spread and subsequent disease development. The ;+' strand open reading frame (ORF) located immediately upstream from the capsid ORF and predicted to encode a 10.9 kd protein was also found to be dispensable for replication but essential for systemic spread. By this analysis, MSV sequences that support autonomous replication were localized to a 1.7 kb segment containing the two viral intergenic regions and two overlapping complementary ;-' strand ORFs. Despite the inability of the gene replacement mutants to spread systemically, both inoculated and newly developed leaves displayed chlorotic patterns similar to the phenotype observed in certain developmental mutants of maize. The similarity of the MSV mutant phenotype to these developmental mutants is discussed.

Journal Article↗