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PubMed2026

Dual-gRNA CRISPR/Cas9 Deletion of CsDMR6 in Sweet Orange Supported by Improved In Vitro Regeneration.

Huanglongbing (HLB), caused by Candidatus Liberibacter spp., remains the most destructive disease affecting citrus worldwide. To support host-directed genome-editing strategies aimed at reducing susceptibility, we optimized key regeneration steps in Citrus sinensis and validated a dual-gRNA CRISPR/Cas9 approach targeting the susceptibility gene CsDMR6. Juvenile explants of 'Valencia' and hybrid genotypes (CsH1-CsH3) were successfully established in vitro, and shoot elongation was markedly improved by supplementing Citrus Shoot Multiplication (CiSM) medium with 1 mg L-1 GA3. Callus induction was most efficient in Citrus Callus Induction (CiCM) medium under dark conditions, while a 48 h NAA pulse (100 µM) significantly enhanced rooting, increasing efficiencies to 37.1% in 'Valencia' and 52.9% in CsH1. Two guide RNAs targeting conserved regions of CsDMR6 were designed and shown to be identical across all evaluated genotypes. The dual-gRNA cassette was assembled into a CRISPR/Cas9 geminivirus-based vector and transiently delivered into sweet orange leaf tissue via Agrobacterium. GFP fluorescence verified construct expression, and PCR amplification across the target region produced a diagnostic ~447 bp fragment corresponding to the expected ~5.8 kb deletion. Sanger sequencing confirmed precise junction formation between the two cut sites. These results demonstrate efficient large-fragment deletion of CsDMR6 in sweet orange and establish an experimentally validated, genotype-compatible regeneration and editing platform. This study provides a transient validation of the dual-gRNA system and establishes the technical foundation required for future stable, non-transgenic edited lines. Together, these advances support the downstream functional evaluation of CsDMR6 loss-of-function alleles under HLB pressure.

CRISPR/Cas9↗
PubMed1988

Genetic analysis of the tomato golden mosaic virus. II. The product of the AL1 coding sequence is required for replication.

Tomato golden mosaic virus (TGMV) belongs to the geminivirus subgroup that is characterized by a split genome consisting of two single-stranded circular DNAs. The TGMV A genome component encodes the virus coat protein as well as all of the functions necessary for viral DNA replication. Analysis of the nucleotide sequence indicates that the TGMV A component has, in addition to the coat protein encoding ORF, four overlapping open reading frames (ORFs) with the potential to encode proteins of greater than 10 kD. We have investigated the functions of these putative proteins in both symptom formation and DNA replication by creating mutations in each of the ORFs. Our results show that the AL4 ORF, which is encoded within the N-terminal region of ORF AL1, is not essential for normal virus infection. In contrast, we find that disruption of the AL3 ORF results in delay and attenuation of symptom formation. We also report that the products of the AL1 and AL2 ORFs are absolutely required for symptom formation. Studies of DNA replication show that only the AL1 open reading frame is essential for viral DNA synthesis. The significance of these results for the development of vectors from the geminiviruses is discussed.

DNA Mutational Analysis↗
PubMed1994

Transposition of the maize Ds element from a viral vector to the rice genome.

The geminivirus miscanthus streak virus (MiSV) was used as a gene vector to study the transposition of the maize Ds element in rice protoplasts. Efficient excision of the Ds from the MiSV vector was observed only when the MiSV vector was allowed to replicate and the plasmid expressing the transposase gene encoded by Ac was co-transfected. Under the same condition, the Ds carrying a hygromycin phosphotransferase gene (Ds::HPT) was also efficiently excised. Hygromycin-resistant calli were obtained by culturing these transfected protoplasts in order to examine the transposition of the excised Ds::HPT into the rice genome. In five out of 16 calli examined, the Ds::HPT, but not the vector sequence, was integrated into the rice genome and 8 bp target site duplication typical of Ac/Ds transposition was generated. These results show that the Ds::HPT inserted in the MiSV vector transposed directly into the rice genome. This demonstrates the direct transposition of a cloned plant transposable element into the plant genome. Implications of these finding are discussed.

Base Sequence↗
PubMed2000

Host range and symptom variation of pseudorecombinant virus produced by two distinct bipartite geminiviruses.

Within the whitefly group only the species Bemisia tabaci (Gennadius) is the vector. Most whitefly-transmitted geminiviruses possess bipartite DNA genomes, DNAs A and B. Although they are closely related to each other, the production of viable pseudorecombinants between bipartite geminiviruses by reassortment of infectious cloned components is generally limited to strains of a particular virus. Following exchange of cloned genomic components of Sida golden mosaic virus (SiGMV/Hoyv) and Abutilon mosaic virus (AbMV), the pseudorecombinant viruses were infectious in various host plants. The symptom type of pseudorecombinant virus was in most cases determined by DNA B. However, in some host plants also DNA A of the pseudorecombinant virus was involved in the symptom phenotype.

Base Sequence↗
PubMed1997

Coat protein gene replacement results in whitefly transmission of an insect nontransmissible geminivirus isolate.

Geminiviruses are transmitted by whiteflies, leafhoppers, or treehoppers. The whitefly species Bemisia tabaci (Gennadius) is the most efficient vector of Subgroup III geminiviruses. An isolate of Abutilon mosaic virus (AbMV), a bipartite geminivirus, was not detectable in the DNA extract from insects by Southern blot analysis, nor was the isolate transmissible by the B-biotype of B. tabaci, although the virus DNA was amplified (by PCR) from some insects. In contrast, Sida golden mosaic virus (SiGMV-Co), a closely related geminivirus, was acquired and transmitted by B. tabaci to various host plants. The coat protein of AbMV was replaced with that of SiGMV-Co. The resulting chimeric AbMV was acquired and transmitted to various host plants by B. tabaci, indicating that the coat protein plays an essential role in the transmission process by B. tabaci.

Amino Acid Sequence↗
PubMed1992

Excision of a transposable element from a viral vector introduced into maize plants by agroinfection.

The geminivirus maize streak virus (MSV) was used as a vector to introduce the maize transposable element Dissociation (Ds) and to study its excision in maize plants. MSV carrying Ds1 in its genome was introduced into maize plants by agroinfection. Excision of the Ds1 element from the MSV genome was detected only when functions from the transposable element Activator (Ac) were supplied in trans, either endogenously by the recipient maize plant or by co-transformation with Agrobacterium carrying a genomic Ac clone. The excision of Ds1 could easily be visualized by the appearance of viral symptoms induced by the revertant virus. The junction sequences left on the MSV genome after excision revealed 'footprints' typical of transposition as described for maize. From these results, we conclude that transposition functions in our system and that the use of the MSV replicon provides a rapid and simple tool for the investigation of the excision of transposable elements in maize plants.

Agrobacterium tumefaciens↗
PubMed1996

Analysis of the nucleotide sequence of the treehopper-transmitted geminivirus, tomato pseudo-curly top virus, suggests a recombinant origin.

The genome of tomato pseudo-curly top virus (TPCTV), originating from Florida, has been cloned and sequenced. TPCTV is the only geminivirus identified with a vector specificity which falls outside the Cicadellidae (leafhoppers) and Aleyrodidae (whiteflies). Infectivity of the cloned viral genome was demonstrated by Agrobacterium-mediated inoculation of several host species. Progeny virus was transmissible by the treehopper vector of TPCTV, Micrutalis malleifera (Fowler). The genome of TPCTV shows features typical of both subgroups I and III genera of the family Geminiviridae. The coat protein of TPCTV, although distinct from all previously characterized geminiviruses, exhibits features more akin to the leafhopper-transmitted geminiviruses than those transmissible by the whitefly Bemisia tabaci Genn. The relationship of TPCTV to other geminiviruses, particularly beet curly top virus, is discussed in relation to the possible evolutionary origins of this virus.

Amino Acid Sequence↗
PubMed2006

Use of geminiviral vectors for functional genomics.

Virus-induced gene silencing (VIGS) can be used to study the function of a gene by downregulating its expression and analyzing the resulting phenotype. VIGS is a handy tool that is less time consuming and labor intensive than other methods for generating mutants. Geminiviruses are particularly convenient and valuable choices as VIGS vectors in functional genomics. The small size of their DNA genome, the simplicity of the methods for inoculation, their wide host range and their conserved genome organization are just a few of the advantageous characteristics that this group of viruses has to offer. Geminivirus-based vectors have proved to be very efficient in VIGS systems, and further development of these systems will most probably permit their application in studies of the functional genomics of important crops that are recalcitrant to other forms of analysis.

Geminiviridae↗
PubMed1989

Phylogeny of geminiviruses.

Amino acid sequences of 16 geminivirus replication-associated proteins and 15 coat proteins were aligned and a new computer program was used to calculate the minimum mutation distances for all possible pairwise comparisons. These data were used to construct phylogenetic trees. Trees based on coat proteins had two main branches which were positively correlated with vector specificities of the viruses. Trees based on replication-associated proteins also had two main branches which were positively correlated with viral host specificities for either monocotyledonous or dicotyledonous plants. Therefore, evolutionary pressures on coat proteins and replication-associated proteins are probably highly influenced by vectors and hosts, respectively. Geminiviruses that infect dicotyledonous plants may be divided further by geographical origins into Old World and New World viruses. These results suggest the possible geographical origins of some geminiviruses, that new taxa should be erected, and have implications for distinguishing viruses and strains.

Amino Acid Sequence↗
PubMed2006

Factors contributing to deletion within Mungbean yellow mosaic virus partial dimers in binary vectors used for agroinoculation.

Mungbean yellow mosaic virus-Vigna (MYMV) sequences cloned as partial dimers within the T-DNA of a binary vector were deleted at a high frequency upon conjugal mobilization from Escherichia coli into Agrobacterium tumefaciens. This deletion involving the genome-length viral DNA did not occur when the binary plasmid was inside E. coli and when the binary plasmid was introduced into Agrobacterium by electroporation. Deletions occurred in both DNA A and DNA B partial dimers. A minimum of 500-nt continuity on either side of the nonanucleotide in the duplicated common region is required for deletion. A. tumefaciens cells in which deletion was complete, grew as larger colonies reflecting a growth advantage. The small, slow-growing colonies eventually lost the genome-length viral sequences after a few more cycles of growth. Partial dimers in binary plasmids pGA472 and pBin19 with RK2 replicon underwent deletion while those in pPZP with pVS1 replicon did not undergo deletion. Deletion was observed in A. tumefaciens strains C58, A136, A348 and A281 with C58 chromosome background, but not in Ach5 and T37. Interestingly, deletion did not occur in A. tumefaciens strain AGL1 with a recA mutation in C58 chromosome, implying a clear role for recombination in deletion. These observations suggest the choice of Agrobacterium strains and binary vectors for agroinoculation of geminiviruses.

Agrobacterium tumefaciens↗
PubMed2005

Whitefly Bemisia tabaci (Homoptera: Aleyrodidae) infestation on cassava genotypes grown at different ecozones in Nigeria.

Large-scale screening of cassava, Manihot esculenta Crantz, genotypes for resistance to infestation by whitefly Bemisia tabaci Gennadius, the vector of cassava mosaic geminiviruses, is limited. A range of new cassava elite clones were therefore assessed for the whitefly infestation in the 1999/2000 and 2000/2001 cropping seasons in experimental fields of International Institute of Tropical Agriculture, Ibadan, Nigeria. On each scoring day, between 0600 and 0800 hours when the whiteflies were relatively immobile, adult whitefly populations on the five topmost expanded leaves of cassava cultivars were counted. All through the 6-mo scoring period, there was a highly significant difference in whitefly infestation among the new cassava elite clones. Vector population buildup was observed in Ibadan (forest-savanna transition zone) and Onne (humid forest), 2 mo after planting (MAP). Mean infestation across cassava genotypes was significantly highest (16.6 whiteflies per plant) in Ibadan and lowest in Zaria (0.2). Generally, whitefly infestation was very low in all locations at 5 and 6 MAP. During this period, cassava genotypes 96/1439 and 91/02324 significantly supported higher infestations than other genotypes. Plants of 96/1089A and TMS 30572 supported the lowest whitefly infestation across cassava genotypes in all locations. The preferential whitefly visitation, the differences between locations in relation to whitefly population, cassava mosaic disease, and the fresh root yield of cassava genotypes are discussed.

Animals↗
PubMed2004

East African cassava mosaic Zanzibar virus - a recombinant begomovirus species with a mild phenotype.

Cassava plants exhibiting mild symptoms of cassava mosaic disease (CMD) were collected from Unguja island, Zanzibar. Cuttings grown from these plants in the glasshouse produced similar symptoms, which were milder than those caused by other known cassava mosaic geminiviruses (CMGs). The whitefly vector, Bemisia tabaci (Gennadius), transmitted the putative virus to 27.7% (n = 18) of target plants. Total DNA extracted from diseased leaves did not yield diagnostic PCR-bands using virus-specific primers to known CMGs. Degenerate primers, however, produced a diagnostic band indicating the presence of a begomovirus. Full-length DNA-A (2785 nucleotides) and DNA-B (2763 nucleotides) components were subsequently PCR-amplified, cloned and sequenced. Phylogenetic analyses of DNA-A and -B sequences showed that they were most similar to strains of East African cassava mosaic virus from Tanzania and Uganda at 83% and 86% nucleotide identities, respectively. The number and arrangement of open reading frames were similar to those of bipartite begomoviruses from the Old World. DNA-A was predicted to have recombined in the intergenic region (IR), AC1 and AC4 genes, and DNA-B in the IR. A maximum nucleotide identity of 83% in the DNA-A component with other sequenced begomoviruses, together with different biological properties allows this virus to be recognised as belonging to a new species named East African cassava mosaic Zanzibar virus (EACMZV).

Animals↗
PubMed2001

Investigation of the potential of maize streak virus to act as an infectious gene vector in maize plants.

There has been a great deal of interest in the possibility that geminiviruses might be used as infectious gene vectors for expression of foreign proteins in plants. However, generic mastreviruses such as Maize streak virus (MSV) have no sequences which are dispensable for systemic infection of plants, and there is a strict limitation on the size of viral DNA which can be moved systemically. We attempted to complement the movement functions deleted from a wild-type-sized, replication-proficient gene replacement vector, by co-infecting plants with it and either wild type MSV, or a replication-deficient but putatively movement-proficient viral construct. While ssDNA formation by the gene replacement vector could be complemented in trans by co-transfected wild type virus, true systemic movement of either the vector, or of co-complementing constructs, did not occur. However, recombination between the two complementing viral constructs frequently occurred to generate wild-type virus genomes. The results therefore suggest that formation of ssDNA and size of the viral replicon are not the sole determinants of whether the MSV movement proteins can mobilise viral sequences and move them systemically in plants.

Blotting, Southern↗
PubMed1997

Long-term association of tomato yellow leaf curl virus with its whitefly vector Bemisia tabaci: effect on the insect transmission capacity, longevity and fecundity.

The association between tomato yellow leaf curl geminivirus (TYLCV, Israeli isolate) and its insect vector, the whitefly Bemisia tabaci, was investigated. Insects that emerged during a 24 h period were caged with TYLCV-infected plants for a 48 h acquisition access period, then with egg-plants--a TYLCV non-host--for the rest of their lives. While TYLCV DNA was associated with the whiteflies during their entire adult life, the amount of capsid protein rapidly decreased and was not detectable in the insect after approximately 12 days of age. The ability of the infected whiteflies to transmit TYLCV to tomato test plants steadily decreased with age but did not disappear completely. Transmission by viruliferous insects decreased from 100% to 10-20% during their adult lifetime, compared with a decrease from 100% to 50% for non-viruliferous insects. The association of TYLCV with adult B. tabaci led to a reduction of 17-23% in their life expectancy compared with insects that had not acquired the virus, and to a 40-50% decrease in the mean number of eggs laid. These results suggest that TYLCV has some features reminiscent of an insect pathogen.

Animals↗
PubMed1995

Effective ribozyme delivery in plant cells.

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.

Base Sequence↗
PubMed1997

Dynamic genome organization and gene evolution by positive selection in geminivirus (Geminiviridae).

Geminiviruses (Geminiviridae) are a diverse group of plant viruses differing from other known plant viruses in possessing circular, single-stranded DNA. Current classification divides the family into three subgroups, defined in part by genome organization, insect vector, and plant host range. Previous phylogenetic assessments of geminiviruses have used DNA and/or amino acid sequences from the replication-associated and coat protein genes and have relied predominantly on distance analyses. We used amino acid and DNA sequence data from the replication-associated and coat protein genes from 22 geminivirus types in distance and parsimony analyses. Although the results of our analyses largely agree with those reported previously, we could not always predict viral relationships based on genome organization, plant host, or insect vector. Loss of correlation of these traits with phylogeny is likely due to improved sampling of geminivirus types. Unrooted parsimony trees suggest multiple independent origins for the monopartite genome. genome organization is therefore a dynamic character. Estimates of nonsynonymous and synonymous nucleotide substitutions for extant and inferred ancestral sequences were used to evaluate hypotheses that the replication-associated and coat protein sequences evolve to accommodate plant host and insect vector specificities, respectively. Results suggest that plant host specificity does not solely direct replication-associated protein-evolution but that coat protein sequence does evolve in response to insect vector specificity. Genome organization and, possibly, plant host specificity are not reliable taxonomic characters.

Animals↗
PubMed1991

Wheat dwarf virus vectors replicate and express foreign genes in cells of monocotyledonous plants.

Wheat dwarf virus (WDV) is a geminivirus that infects monocotyledonous plants. To exploit the potential of WDV as a replicative gene vector, we developed a transient replication and expression system based on the transfection of protoplasts derived from Triticum monococcum suspension culture cells. Cloned genomic copies of various WDV isolates as well as mutants constructed in vitro were introduced into the protoplasts and assayed for their ability to replicate. As a result, regions of the WDV genome necessary or dispensable for the viral DNA replication could be defined. In addition, the gene encoding the viral capsid protein was replaced by three different bacterial marker genes, neomycin phosphotransferase, chloramphenicol acetyltransferase, and beta-galactosidase. The beta-galactosidase gene doubled the size of the WDV genome. The replication of the recombinant WDV genomes and the expression of these genes were monitored in suspension culture cells of T. monococcum. The potential of replicative expression vectors based on the WDV genome is discussed.

Base Sequence↗