Localization of Tobacco Yellow Dwarf Virus Replication Using the In Plant Activation (INPACT) Expression Platform.

Geminiviruses and their diseases are a considerable economic threat to a vast number of crops worldwide. Investigating how and where these viruses replicate and accumulate in their hosts may lead to novel molecular resistance strategies. In this study, we used the Rep-inducible In Plant Activation (INPACT) expression platform, based on the genome of tobacco yellow dwarf virus (TYDV), to determine where this model mastrevirus replicates in its host tobacco. By developing an infectious clone of TYDV and optimizing its delivery by agroinfiltration, we first established an efficient artificial infection process. When delivered into transgenic tobacco plants containing a TYDV-based INPACT cassette encoding the ß-glucuronidase (GUS) reporter, we showed the virus activates GUS expression. Histology revealed that reporter gene expression was limited to phloem-associated cell types suggesting TYDV replication has a restricted tissue tropism.

Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection.

Histone posttranslational modifications (PTMs) impart information that regulates chromatin structure and activity. Their effects are mediated by histone reader proteins that bind specific PTMs to modify chromatin and/or recruit appropriate effectors to alter the chromatin landscape. Despite their crucial juxtaposition between information and functional outcome, relatively few plant histone readers have been identified, and nothing is known about their impact on viral chromatin and pathogenesis. We used the geminivirus Cabbage leaf curl virus (CaLCuV) as a model to functionally characterize two recently identified reader proteins, EMSY-LIKE 1 (EML1) and EML3, which contain Tudor-like Agenet domains predictive of histone PTM binding function. Here, we show that mutant Arabidopsis plants exhibit contrasting hypersusceptible (eml1) and tolerant (eml3) responses to CaLCuV infection and that EML1 deficiency correlates with RNA polymerase II (Pol II) enrichment on viral chromatin and upregulated viral gene expression. Consistent with reader activity, EML1 and EML3 associate with nucleosomes and with CaLCuV chromatin, suggesting a direct impact on pathogenesis. We also demonstrate that EML1 and EML3 bind peptides containing histone H3 lysine 36 (H3K36), a PTM usually associated with active gene expression. The interaction encompasses multiple H3K36 PTMs, including methylation and acetylation, suggesting nuanced regulation. Furthermore, EML1 and EML3 associate with similar regions of viral chromatin, implying possible competition between the two readers. Regions of EML1 and EML3 association correlate with sites of trimethylated H3K36 (H3K36me3) enrichment, consistent with regulation of geminivirus chromatin by direct EML targeting.IMPORTANCE Histone PTMs convey information that regulates chromatin compaction and DNA accessibility. Histone reader proteins bind specific PTMs and translate their effects by modifying chromatin and/or by recruiting effectors that alter chromatin structure or activity. In this study, CaLCuV was used to characterize the activities of two Arabidopsis Agenet domain histone readers, EML1 and EML3. We show that eml1 mutants are hypersusceptible to CaLCuV, whereas eml3 plants are more tolerant of infection than wild-type plants. We also demonstrate that EML1 and EML3 associate with histones and viral chromatin in planta and that both proteins bind peptides containing H3K36, a PTM associated with active gene expression. Consistent with antiviral activity, EML1 suppresses CaLCuV gene expression and reduces Pol II access to viral chromatin. By linking EML1 and EML3 to pathogenesis, these studies have expanded our knowledge of histone reader proteins and uncovered an additional level of viral chromatin regulation.

Spatiotemporal spread of grapevine red blotch-associated virus in a California vineyard.

Grapevine red blotch-associated virus (GRBaV), the causative agent of red blotch disease, is a member of the genus Grablovirus, in the family Geminiviridae and the first known geminivirus of Vitis spp. Limited information is available on the epidemiology of red blotch disease. A 2-hectare Vitis vinifera cv. 'Cabernet franc' vineyard in Napa County, California, USA was selected for monitoring GRBaV spread over a three-year period (2014-2016) based on an initially low disease incidence and an aggregation of symptomatic vines at the edge of the vineyard proximal to a wooded riparian area. The incidence of diseased plants increased by 1-2% annually. Spatial analysis of diseased plants in each year using ordinary runs analysis within rows and Spatial Analysis by Distance IndicEs (SADIE) demonstrated aggregation. Spatiotemporal analysis between consecutive years within the association function of SADIE revealed a strong overall association among all three years (X=0.874-0.945). Analysis of epidemic spread fitting a stochastic spatiotemporal model using the Monte Carlo Markov Chain method identified strong evidence for localized (within vineyard) spread. A spatial pattern consisting of a combination of strongly aggregated and randomly isolated symptomatic vines within 8-years post-planting suggested unique epidemic attributes compared to those of other grapevine viruses vectored by mealybugs and soft scales or by dagger nematodes for which typical within-row spread and small-scale autocorrelation are well documented. These findings are consistent with the existence of a new type of vector for a grapevine virus.

A nuclear-replicating viroid antagonizes infectivity and accumulation of a geminivirus by upregulating methylation-related genes and inducing hypermethylation of viral DNA.

DNA methylation and post-transcriptional gene silencing play critical roles in controlling infection of single-stranded (ss) DNA geminiviruses and ssRNA viroids, respectively, but both pathogens can counteract these host defense mechanisms and promote their infectivity. Moreover, a specific role of DNA methylation in viroid-host interactions is not yet confirmed. Here, using an experimental system where two nuclear-replicating agents, the geminivirus tomato yellow leaf curl Sardinia virus (TYLCSV) and potato spindle tuber viroid (PSTVd), co-infect their common host tomato, we observed that PSTVd severely interferes with TYLCSV infectivity and accumulation, most likely as a consequence of strong activation of host DNA methylation pathways. In fact, PSTVd alone or in co-infection with TYLCSV significantly upregulates the expression of key genes governing DNA methylation in plants. Using methylation-sensitive restriction and bisulfite conversion assays, we further showed that PSTVd infection promotes a strong hypermethylation of TYLCSV DNA, thus supporting a mechanistic link with the antagonism of the viroid on the virus in co-infected tomato plants. These results describe the interaction between two nuclear-replicating pathogens and show that they differentially interfere with DNA methylation pathways.

Silencing of a Germin-Like Protein Gene (CchGLP) in Geminivirus-Resistant Pepper (Capsicum chinense Jacq.) BG-3821 Increases Susceptibility to Single and Mixed Infections by Geminiviruses PHYVV and PepGMV.

Germin-like proteins (GLPs) are encoded by a family of genes found in all plants, and in terms of function, the GLPs are implicated in the response of plants to biotic and abiotic stresses. CchGLP is a gene encoding a GLP identified in a geminivirus-resistant Capsicum chinense Jacq accession named BG-3821, and it is important in geminivirus resistance when transferred to susceptible tobacco in transgenic experiments. To characterize the role of this GLP in geminivirus resistance in the original accession from which this gene was identified, this work aimed at demonstrating the possible role of CchGLP in resistance to geminiviruses in Capsicum chinense Jacq. BG-3821. Virus-induced gene silencing studies using a geminiviral vector based in PHYVV component A, displaying that silencing of CchGLP in accession BG-3821, increased susceptibility to geminivirus single and mixed infections. These results suggested that CchGLP is an important factor for geminivirus resistance in C. chinense BG-3821 accession.

Studies on differential behavior of cassava mosaic geminivirus DNA components, symptom recovery patterns, and their siRNA profiles.

Cassava mosaic disease caused by cassava mosaic geminiviruses (CMGs) with bipartite genome organization is a major constraint for production of cassava in the African continent and the Indian sub-continent. Currently, there are eleven recognized species of CMGs, and several diverse isolates represent them, with vast amount of sequence variability, reflecting into diversity of symptom severity/phenotypes. Here, we make a systematic effort to study the infection dynamics of several species of CMGs and their isolates. Further, we try to identify the genomic component of CMGs contributing to the manifestation of diverse patterns of symptoms and the molecular basis for the differential behavior of CMGs. The pseudo-recombination studies carried out by swapping of DNA-A and DNA-B components of the CMGs revealed that the DNA-B component significantly contributes to the symptom severity. Past studies had shown that the DNA-A component of Sri Lankan cassava mosaic virus shows monopartite feature. Thus, the ability of DNA-A component alone, to replicate and move systemically in the host plant with inherent monopartite features was investigated for all the CMGs. Geminiviruses are known to trigger gene silencing and are also its target, resulting in recovery of the host plant from viral infection. In the collection of several different CMG species and isolates we had, there was a vast variability in their recovery and non-recovery phenotypes. To understand the molecular basis of this, the origin and distribution of virus-derived small interfering RNAs were mapped across their genome and across the CMG-infected symptomatic Nicotiana benthamiana.

Application of molecular antiviral compounds: novel approach for durable resistance against geminiviruses.

Both transgenic as well as traditional breeding approaches have not been completely successful in inducting resistance against geminiviruses in crop plants. This demands the utilization of non-viral, non-plant compounds possessing antiviral characteristics as an alternate and effective strategy for developing durable resistance against geminiviruses. In recent years, several antiviral molecules have been developed for the treatment of plant virus infections. These molecular antiviral compounds target various geminiviral-DNA and -protein via interacting with them or by cleaving viral RNA fragments. Applications of these proteins such as GroEL, g5g and VirE2 have also provided a convincing evidence of resistance against geminiviruses. Taking advantage of this information, we can generate robust resistance against geminiviruses in diverse crop plants. In this context, the present review provides epigrammatic information on these antiviral compounds and their mode of action in modulating virus infection.

Microarray analysis of tomato plants exposed to the nonviruliferous or viruliferous whitefly vector harboring Pepper golden mosaic virus.

Plants are routinely exposed to biotic and abiotic stresses to which they have evolved by synthesizing constitutive and induced defense compounds. Induced defense compounds are usually made, initially, at low levels; however, following further stimulation by specific kinds of biotic and abiotic stresses, they can be synthesized in relatively large amounts to abate the particular stress. cDNA microarray hybridization was used to identify an array of genes that were differentially expressed in tomato plants 15 d after they were exposed to feeding by nonviruliferous whiteflies or by viruliferous whiteflies carrying Pepper golden mosaic virus (PepGMV) (Begomovirus, Geminiviridae). Tomato plants inoculated by viruliferous whiteflies developed symptoms characteristic of PepGMV, whereas plants exposed to nonviruliferous whitefly feeding or nonwounded (negative) control plants exhibited no disease symptoms. The microarray analysis yielded over 290 spotted probes, with significantly altered expression of 161 putative annotated gene targets, and 129 spotted probes of unknown identities. The majority of the differentially regulated "known" genes were associated with the plants exposed to viruliferous compared with nonviruliferous whitefly feeding. Overall, significant differences in gene expression were represented by major physiological functions including defense-, pathogen-, photosynthesis-, and signaling-related responses and were similar to genes identified for other insect-plant systems. Viruliferous whitefly-stimulated gene expression was validated by real-time quantitative polymerase chain reaction of selected, representative candidate genes (messenger RNA): arginase, dehydrin, pathogenesis-related proteins 1 and -4, polyphenol oxidase, and several protease inhibitors. This is the first comparative profiling of the expression of tomato plants portraying different responses to biotic stress induced by viruliferous whitefly feeding (with resultant virus infection) compared with whitefly feeding only and negative control nonwounded plants exposed to neither. These results may be applicable to many other plant-insect-pathogen system interactions.

Optimización de las condiciones de inoculación por biobalística de un Begomovirus en tomate y tabaco / Optimizing conditions for biolistic inoculation of Begomovirus in tomato and tobacco

La transmisión experimental de Begomovirus es problemática. La mayoría de estos virus se pueden transmitir de planta a planta por su vector biológico, Bemisia tabaci. Las inoculaciones experimentales con mosca blanca son problemáticas debido a sus hábitos de alimentación, requerimiento de una planta viva infectada e instalaciones de contención para el vector. Por su parte la inoculación mecánica de Begomovirus es posible, pero generalmente a tasas bajas y no en todos los casos. Por esta razón el bombardeo de partículas (biobalística) de DNA viral como una estrategia de inoculación fue desarrollada. La posibilidad de utilizar el dispositivo de mano Helios Gen Gun System (Biorad®), un equipo de biobalística, para la transmisión de un Begomovirus bipartita a plantas de tomate y tabaco fue ensayado y optimizado. Los parámetros evaluados fueron: número de disparos (1-2), presión de helio (220 y 320 psi) y diámetro de las partículas de oro (0.6 y 1.6µm). Los síntomas característicos de la enfermedad viral (clorosis, mosaico y deformación de la hoja) aparecieron 3 semanas después del bombardeo en las hojas jóvenes no inoculadas. La replicación del DNA viral en las plantas se confirmó por Reacción en cadena de la polimerasa. Plantas infectadas en un 100 se obtuvieron cuando en el bombardeo se emplearon partículas de oro de 1.6 µm recubiertas con DNA viral a una presión de 320psi. A nuestro entender este es el primer reporte en Colombia de la inoculación directa de plantas de tomate y tabaco con un Begomovirus bipartita usando un dispositivo portátil de biobalística.

Experimental transmission of Begomovirus is problematic. Most Begomoviruses can be transmitted readily from plant to plant by the whitefly vector, but this also requires a live infected plant and extensive facilities to maintain the insect. Whitefly inoculations can also be problematic because of their preferential feeding habits on certain plants. Mechanical inoculation of Begomovirus is possible but generally at low rates and for others not at all. For this reason particle bombardment (biolistic) of DNA viral as an inoculum was developed. The possibility of using the Helios Gen Gun System (Biorad®), a biolistic hand-held device, for transmitting Begomovirus bipartite to tomato and tobacco plants was assayed and optimized. Biolistic inoculation was carried out with the hand held device at 220 or 320 psi, applying 1 or 2 shots /plant and using gold particles of 0.6 or 1.6µm in size. Characteristic symptoms of viral disease (chlorosis, mosaic and leaf deformation) appeared 3 weeks post-inoculation in the newly developing leaves. Replication of the viral DNA in plants was confirmed by Polymerase Chain Reaction. All bombarded plants became infected when biolistic inoculation was carried out with the hand held device at 320psi and using 1.6 µm gold particles in size. To our knowledge this is the first report in Colombia of successful direct inoculation of tomato and tobacco plants with Begomovirus bipartite geminivirus using a biolistic hand-held device.

Tropism, compartmentalization and retention of banana bunchy top virus (Nanoviridae) in the aphid vector Pentalonia nigronervosa.

Plant viruses of the families Luteoviridae and Geminiviridae rely on hemipteran vectors for the infection of their hosts. Several lines of evidence have revealed that these viruses are transmitted by competent vectors in a circulative manner, involving entry into the vector's body and the crossing of epithelial tissues forming the alimentary tract and the salivary glands. Similar to luteovirids and geminiviruses, a third family of plant viruses, the family Nanoviridae, have also been reported to be transmitted by aphids in a circulative manner. However, there is limited direct evidence of a possible path of translocation through the aphid vectors. Here, we used time-course experiments and transmission assays coupled with real-time PCR and immunofluorescence assays on dissected tissues to examine the translocation, compartmentalization and retention of banana bunchy top virus (BBTV) into the aphid vector Pentalonia nigronervosa. Our results indicate that BBTV translocates rapidly through the aphid vector; it is internalized into the anterior midgut in which it accumulates and is retained at concentrations higher than either the haemolymph or the principal salivary glands. Despite the large increase in viral concentration, we have failed to detect BBTV transcripts with RT-PCR. When tissues were not permeabilized, BBTV localized as distinct puncta in the proximity of the basal surface of the cells forming the anterior midgut and principal salivary glands, suggesting an on-going process of virion escape and internalization, respectively. Interestingly, we document that those organs can have direct contact within the aphid body, suggesting a possible haemolymph-independent translocation path.

RNA interference-based resistance against a legume mastrevirus.

BACKGROUND: RNA interference (RNAi) is a homology-dependant gene silencing mechanism and has been widely used to engineer resistance in plants against RNA viruses. However, its usefulness in delivering resistance against plant DNA viruses belonging to family Geminiviridae is still being debated. Although the RNAi approach has been shown, using a transient assay, to be useful in countering monocotyledonous plant-infecting geminiviruses of the genus Mastrevirus, it has yet to be investigated as a means of delivering resistance to dicot-infecting mastreviruses. Chickpea chlorotic dwarf Pakistan virus (CpCDPKV) is a legume-infecting mastrevirus that affects chickpea and other leguminous crops in Pakistan. RESULTS: Here a hairpin (hp)RNAi construct containing sequences encompassing part of replication-associated protein gene, intergenic region and part of the movement protein gene of CpCDPKV under the control of the Cauliflower mosaic virus 35S promoter has been produced and stably transformed into Nicotiana benthamiana. Plants harboring the hairpin construct were challenged with CpCDPKV. All non-transgenic N. benthamiana plants developed symptoms of CpCDPKV infection within two weeks post-inoculation. In contrast, none of the inoculated transgenic plants showed symptoms of infection and no viral DNA could be detected by Southern hybridization. A real-time quantitative PCR analysis identified very low-level accumulation of viral DNA in the inoculated transgenic plants. CONCLUSIONS: The results presented show that the RNAi-based resistance strategy is useful in protecting plants from a dicot-infecting mastrevirus. The very low levels of virus detected in plant tissue of transgenic plants distal to the inoculation site suggest that virus movement and/or viral replication was impaired leading to plants that showed no discernible signs of virus infection.

The spread of tomato yellow leaf curl virus from the Middle East to the world.

The ongoing global spread of Tomato yellow leaf curl virus (TYLCV; Genus Begomovirus, Family Geminiviridae) represents a serious looming threat to tomato production in all temperate parts of the world. Whereas determining where and when TYLCV movements have occurred could help curtail its spread and prevent future movements of related viruses, determining the consequences of past TYLCV movements could reveal the ecological and economic risks associated with similar viral invasions. Towards this end we applied Bayesian phylogeographic inference and recombination analyses to available TYLCV sequences (including those of 15 new Iranian full TYLCV genomes) and reconstructed a plausible history of TYLCV's diversification and movements throughout the world. In agreement with historical accounts, our results suggest that the first TYLCVs most probably arose somewhere in the Middle East between the 1930s and 1950s (with 95% highest probability density intervals 1905-1972) and that the global spread of TYLCV only began in the 1980s after the evolution of the TYLCV-Mld and -IL strains. Despite the global distribution of TYLCV we found no convincing evidence anywhere other than the Middle East and the Western Mediterranean of epidemiologically relevant TYLCV variants arising through recombination. Although the region around Iran is both the center of present day TYLCV diversity and the site of the most intensive ongoing TYLCV evolution, the evidence indicates that the region is epidemiologically isolated, which suggests that novel TYLCV variants found there are probably not direct global threats. We instead identify the Mediterranean basin as the main launch-pad of global TYLCV movements.

Intervention of geminiviral replication in yeast by ribozyme mediated downregulation of its Rep protein.

Geminiviruses pose serious threat to many economically important crops such as mungbean, tomato, cotton, etc. To devise a specific antiviral strategy at the viral DNA replication level, a hammerhead ribozyme was directed against the mRNA of the replication initiator protein (Rep). Rep is the most important viral protein for the DNA replication of the Mungbean yellow mosaic India virus (MYMIV), a member of the Geminiviridae family. The ribozyme showed approximately 33% cleavage activity on synthetic rep transcript within 1h under in vitro conditions, whereas the mutant ribozyme, designed to lack the catalytic activity but target the same site, showed no cleavage. The in vivo efficiency of ribozyme was evaluated in Saccharomyces cerevisiae as it can act as a surrogate host for replication of the MYMIV-DNA and lacks RNAi machinery. In the presence of the ribozyme, growth of the yeast cells that are dependent on geminiviral replication was inhibited by 30% and cellular generation time was increased by 2h. The RT-PCR analysis showed a maximum of about 50% reduction in the rep mRNA level in presence of the ribozyme compared to its noncatalytic mutant control. About 65% decrease in geminiviral DNA replication was observed due to the downregulation of replication initiator protein by the ribozyme. These results raise the possibility of engineering resistance to geminiviruses employing the ribozyme approach.

Transcriptional analysis of complementary sense genes in Spinach curly top virus and functional role of C2 in pathogenesis.

Spinach curly top virus (SCTV), the fifth characterized Curtovirus species belonging to the family Geminiviridae, is an agriculturally significant plant pathogen representing an emerging disease threat in the southern United States. The SCTV genome comprises a single DNA chromosome of approximately 3.0 kb, with the potential to code for seven proteins larger than 10 kDa but which relies extensively on the host for replication and transcription of its genome. In this study, we have identified viral and complementary sense transcripts in SCTV-infected plants, confirming a bidirectional transcription strategy for SCTV. The most abundant RNA maps to the virion sense (1.1-kb transcript) and is comparable in size and location to that observed in Beet curly top virus (BCTV). Two complementary sense transcripts (1.7 and 0.7 kb) were identified in SCTV-infected plants. The large, 1.7-kb transcript is comparable in size and position to that identified in BCTV and several begomoviruses and most likely encodes the C1 protein. Both complementary sense RNAs could potentially direct expression of C2 and C3 from polycistronic mRNAs. A mutation in the C2 gene of SCTV results in expression of a truncated protein of 38 amino acids that is capable of interacting with two cellular kinases, AKIN11 and ADK2, and the resulting mutant virus remains highly infectious. A second mutant virus can only express the first three amino acids of the C2 protein and is unable to interact with the same kinases. However, this mutant virus still remains infectious, although a reduction in infectivity and symptom severity was seen in both Arabidopsis and spinach. A possible relationship between the interaction of C2 with AKIN11 and ADK2 and disease severity is presented.

Application of Phi29 DNA polymerase in identification and full-length clone inoculation of tomato yellow leaf curl Thailand virus and tobacco leaf curl Thailand virus.

Tomato plants grown in greenhouses in Thailand developed typical symptoms of a tomato yellow leaf curl Thailand virus (TYLCTHV) infection. After confirmation by ELISA, a Phi29 DNA polymerase approach was chosen for further molecular analysis of TYLCTHV. Total DNA purified from infected tomato leaves was subjected to rolling-circle amplification (RCA) of DNA-A and DNA-B of TYLCVTHV. In addition, a new monopartite geminivirus with a putative recombinant background was identified by RCA and tentatively named tobacco leaf curl Thailand virus (TbLCTHV). To confirm the composition of both geminiviruses, full-length clones were established and used for inoculation of Nicotiana benthamiana by particle bombardment or agroinfection. When TYLCTHV DNA-A and DNA-B were applied together by particle bombardment or agroinfection, severe stunting, yellowing, and leaf curling were observed. Whereas TYLCTHV DNA-A and TbLCTHV revealed no infection after'particle bombardment, similar symptoms in N. benthamiana, like leaf upward curling and yellowing were observed following agroinfection.DNA components of TYLCTHV DNA-A and DNA-B were excised from their respective plasmids, ligated, and amplified by Phi29 DNA polymerase. The ability of viral concatamere inoculation was evaluated in particle co-bombardment experiments on N. benthamiana. Thus, particle bombardment of RCA-derived multimeric products proved to be at least as effective as inoculation with a partial repeat construct and tenfold as effective as inoculation with excised unit-lengths of DNA-A and DNA-B of TYLCVTHV when using each DNA component in an amount of 5 ng.

Molecular characterization of two soybean-infecting begomoviruses from India and evidence for recombination among legume-infecting begomoviruses from South [corrected] South-East Asia.

The complete nucleotide sequences of two soybean-infecting begomoviruses have been determined from central and southern parts of India. Sequence analyses show that the isolate from central India is a strain of Mungbean yellow mosaic India virus (MYMIV) and the southern Indian isolate is a strain of Mungbean yellow mosaic virus (MYMV). Multiple DNA B components could be detected with the soybean strain of Mungbean yellow mosaic virus species. The nucleotide sequence similarity between the DNA A components of the two isolates is higher (82%) than that between the corresponding DNA B components (71%). Analyses of the common region of the genomic components of these two virus isolates indicate considerable divergence in the origin of replication (ori), which did not impair their infectivity as demonstrated for the central Indian isolate by agroinfection with partial tandem repeats (PTRs) of the genomic components. Detailed sequence and phylogenetic analyses reveal the distribution and possible recombination events among legume-infecting begomoviruses from South-East Asia.

Fighting geminiviruses by RNAi and vice versa.

Geminiviruses have recently emerged not only as the cause of devastating diseases of important crop plants but also as a tool to study fundamental aspects of RNA interference (RNAi) and virus-induced gene silencing. RNA silencing is an evolutionary conserved mechanism protecting cell from pathogenic RNA and DNA, which is increasingly viewed as an adaptive immune system of plants against viruses. Here we summarize recent developments in the field of geminivirology presented by several leading groups at the Meeting "Gemini2004" (a total of 85 participants from all over the world) with the main focus on the anti-viral strategies that exploit RNAi and related silencing phenomena.

Tête à tête of tomato yellow leaf curl virus and tomato yellow leaf curl sardinia virus in single nuclei.

Since 1997 two distinct geminivirus species, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), have caused a similar yellow leaf curl disease in tomato, coexisted in the fields of southern Spain, and very frequently doubly infected single plants. Tomatoes as well as experimental test plants (e.g., Nicotiana benthamiana) showed enhanced symptoms upon mixed infections under greenhouse conditions. Viral DNA accumulated to a similar extent in singly and doubly infected plants. In situ tissue hybridization showed TYLCSV and TYLCV DNAs to be confined to the phloem in both hosts, irrespective of whether they were inoculated individually or in combination. The number of infected nuclei in singly or doubly infected plants was determined by in situ hybridization of purified nuclei. The percentage of nuclei containing viral DNA (i.e., 1.4% in tomato or 6% in N. benthamiana) was the same in plants infected with either TYLCSV, TYLCV, or both. In situ hybridization of doubly infected plants, with probes that discriminate between both DNAs, revealed that at least one-fifth of infected nuclei harbored DNAs from both virus species. Such a high number of coinfected nuclei may explain why recombination between different geminivirus DNAs occurs frequently. The impact of these findings for epidemiology and for resistance breeding concerning tomato yellow leaf curl diseases is discussed.

Geminivirus DNA replication and cell cycle interactions.

The Geminiviridae family includes a large number of viruses that infect plants and have a unique geminate virion particle, a single-stranded genome of approximately 2.6-3.0 kb, and replicate through a rolling-circle mechanism. Since they encode for just a few proteins (4-6 depending on the members that belong to four different genera), a rich variety of interactions has evolved between viral proteins and host factors to develop the virus replicative cycle. Among them, we have been particularly interested so far: (i). in the interference with cell cycle regulatory proteins of the retinoblastoma-related (RBR)/E2F pathway and (ii). in the interaction with host DNA replication factors necessary for the assembly of a functional replication complex at the viral origin of DNA replication during the rolling-circle stage. Yeast two-hybrid assays revealed that wheat dwarf virus RepA protein, but nor Rep protein, interacts with plant RBR protein. Interestingly, deletion of the C-terminal domain of Rep confers the truncated protein the ability to interact with RBR, suggesting that this domain may hinder the LXCXE RBR-binding motif. Secondary structure predictions support such a possibility.

Cassava mosaic geminiviruses in Africa.

Cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses (CMGs) (Geminiviridae:Begomovirus) is undoubtedly the most important constraint to the production of cassava in Africa at the outset of the 21st century. Although the disease was recorded for the first time in the latter part of the 19th century, for much of the intervening period it has been relatively benign in most of the areas where it occurs and has generally been considered to be of minor economic significance. Towards the end of the 20th century, however, the inherent dynamism of the causal viruses was demonstrated, as a recombinant hybrid of the two principal species was identified, initially from Uganda, and shown to be associated with an unusually severe and rapidly spreading epidemic of CMD. Subsequent spread throughout East and Central Africa, the consequent devastation of production of the cassava crop, a key staple in much of this region, and the observation of similar recombination events elsewhere, has once again demonstrated the inherent danger posed to man by the capacity of these viruses to adapt to their environment and optimally exploit their relationships with the whitefly vector, plant host and human cultivator. In this review of cassava mosaic geminiviruses in Africa, we examine each of these relationships, and highlight the ways in which the CMGs have exploited them to their own advantage.