Strategies for the control of geminivirus diseases

Geminiviruses are unique plant DNA viruses that frequently cause significant yield reductions in a wide range of cereal, vegetable and fibre crops. Encouraging progress has recently been made towards the control of geminiviruses that infect dicotyledonous plants. Under laboratory conditions, defective interfering (DI) DNA and antisense strategies, both directed against viral DNA replication, have been used to protect Nicotiana spp. against the bipartite geminivirus African cassava mosaic virus and tomato golden mosaic virus. The use of dominant negative mutants of virus systemic movement might also be adapted as a novel strategy for the control of these important pathogens.     

Wheat dwarf virus, a geminivirus of graminaceous plants needs splicing for replication.

By analysing mRNAs with the polymerase chain reaction (PCR) and by studying in vitro generated mutants we have identified an intron in the genome of wheat dwarf virus (WDV), a geminivirus of cereals. Polypeptides whose expression is essential for the replication of the viral DNA have been defined. They are encoded by two distinct overlapping open reading frames (ORFs). The joining of these two ORFs by deletion of the intron as well as the introduction of a frameshift mutation within the intron do not prevent replication of the viral genome in suspension culture cells. In contrast to WDV, the geminiviruses of dicotyledonous plants possess a single continuous ORF, highly homologous to the two individual ones of WDV. We propose that mRNA splicing is a common feature of all geminiviruses of the Gramineae and might contribute to their host class specificity. The existence of a functional intron is a novel finding for the plant viruses.     

Large bottleneck size in Cauliflower Mosaic Virus populations during host plant colonization

The effective size of populations (Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Despite their high mutation rate and rapid evolution, this parameter is poorly documented experimentally in viruses, particularly plant viruses. All available studies, however, have demonstrated the existence of huge within-host demographic fluctuations, drastically reducing Ne upon systemic invasion of different organs and tissues. Notably, extreme bottlenecks have been detected at the stage of systemic leaf colonization in all plant viral species investigated so far, sustaining the general idea that some unknown obstacle(s) imposes a barrier on the development of all plant viruses. This idea has important implications, as it appoints genetic drift as a constant major force in plant virus evolution. By co-inoculating several genetic variants of Cauliflower mosaic virus into a large number of replicate host plants, and by monitoring their relative frequency within the viral population over the course of the host systemic infection, only minute stochastic variations were detected. This allowed the estimation of the CaMV Ne during colonization of successive leaves at several hundreds of viral genomes, a value about 100-fold higher than that reported for any other plant virus investigated so far, and indicated the very limited role played by genetic drift during plant systemic infection by this virus. These results suggest that the barriers that generate bottlenecks in some plant virus species might well not exist, or can be surmounted by other viruses, implying that severe bottlenecks during host colonization do not necessarily apply to all plant-infecting viruses.     

Inhibition of plant viral systemic infection by non‐toxic concentrations of cadmium

Heavy metals, such as cadmium, have a significant impact on plant physiology. However, their potential effect on plant–pathogen interaction, an important biological process, has not been examined. This study shows that exposure of tobacco plants to non-toxic concentrations of cadmium completely blocked viral disease caused by turnip vein clearing virus. Cadmium-mediated viral protection was due to inhibition of the systemic movement of the virus, i.e. its spread from the inoculated into uninoculated leaves. Exposure of plants to cadmium had no effect on viral replication, assembly and local movement within the inoculated leaf. Analysis of the viral presence in different tissues suggested that cadmium treatment inhibited virus exit from the vascular tissue into uninoculated leaves rather than its entry into the host plant vasculature. Higher, toxic levels of cadmium did not produce this inhibitory effect on viral movement, allowing the systemic spread of the virus and development of the viral disease. These observations suggest that cadmium-induced viral protection requires a relatively healthy, unpoisoned plant in which non-toxic levels of cadmium may trigger the production of cellular factors which interfere with the viral systemic movement.     

Genetic analysis of tomato golden mosaic virus: the coat protein is not required for systemic spread or symptom development

The geminiviruses are a unique group of higher plant viruses that are composed of twin isometric particles which contain circular, single-stranded DNA. Tomato golden mosaic virus (TGMV), a whitefly-transmitted agent, belongs to the subgroup of geminiviruses whose members possess a bipartite genome. The TGMV A genome component has the capacity to encode at least four proteins. One of these is the viral coat protein, as inferred by homology with coat-protein, genes of other geminiviruses and by the observation of typical geminate particles in transgenic plants that contain inserts of TGMV A DNA. We have investigated the role of the coat protein in TGMV replication and report here that its coding sequence may be interrupted or substantially deleted without loss of infectivity. However, certain coat-protein mutants showed reproducible delays in time of symptom appearance as well as reduced symptom development, when inoculated onto transgenic Nicotiana benthamiana plants containing the TGMV B component. The most attenuated symptoms were seen with a mutant in which the coat-protein coding sequence was almost entirely deleted. The significance of these findings for the development of plant vectors from TGMV DNA is discussed.     

植物病毒资源属性和应用基础研究进展

病毒作为地球上最简单的生命形式,通过感染人、动物和植物等寄主产生传染性疾病。与其他微生物相比,病毒具有基因组小、复制量大、遗传操作简单等特点,具有很强的生物资源属性。过去几十年,对植物病毒的研究主要集中于解析其致病机制、植物的抗性机制及如何防控植物病害。但是随着研究的深入及概念的革新,人们发现植物病毒还具有很强的生物资源属性。随着分子生物学以及基因组、转录组、蛋白组学等技术的发展,越来越多的植物病毒被发现、改造和利用。本综述着重围绕植物病毒的资源属性与病毒载体的改造利用及其在生物工程方面的应用等最新研究进展,讨论其广泛的应用前景,挖掘其资源化的潜力。     

Emerging geminivirus problems: A serious threat to crop production

Geminiviruses form the second largest family of plant viruses, the Geminiviridae, represented by four genera: Mastrevirus, Curtovirus, Topocuvirus and Begomovirus. During the last two decades these viruses have emerged as devastating pathogens, particularly in the tropics and subtropics, causing huge economic losses and threatening crop production. Epidemics caused by re‐emerging and newly emerging geminiviruses are becoming frequent even in regions that were earlier free from these viruses. Compared to mastreviruses and curtoviruses, begomoviruses have emerged as more serious problems in a variety of crops, for example, cassava, cotton, grain legumes and vegetables. Major contributory factors for the emergence and spread of new geminivirus diseases are the evolution of variants of the viruses, the appearance of the whitefly ‘B’ biotype and the increase in the vector population. Variability in geminiviruses has arisen through mutations, recombination and pseudorecombination. Genomic recombination in geminiviruses, not only between the variants of the same virus but also between species and even between genera, has resulted in rapid diversification. From the disease point of view, most virulent variants have developed through recombination of viral genomes such as those associated with cassava mosaic, cotton leaf curl, and tomato leaf curl diseases. Heterologous recombinants containing parts of the host genome and/or sequences from satellite‐like molecules associated with monopartite begomoviruses provide unlimited evolutionary opportunities. Human activity has also played an important role in the emergence of serious geminivirus diseases across the globe, like the changes in cropping systems, the introduction of new crops, the movement of infected planting materials and the introduction of host susceptibility genes through the exchange of germplasm.     

Limitations on geminivirus genome size imposed by plasmodesmata and virus-encoded movement protein: insights into DNA trafficking

Animals and plants evolved systems to permit non-cell-autonomous trafficking of RNA, whereas DNA plays a cell-autonomous role. In plants, plasmodesmata serve as the conduit for this phenomenon, and viruses have evolved to use this pathway for the spread of infectious nucleic acids. In this study, a plant DNA virus was used to explore the constraints imposed on the movement of DNA through this endogenous RNA trafficking pathway. The combined properties of the geminivirus-encoded movement protein and plasmodesmata were shown to impose a strict limitation on the size of the viral genome at the level of cell-to-cell movement. Size-increased viral genome components underwent homologous and nonhomologous recombination to overcome this strict limitation. Our results provide insights into the genetic mechanisms that underlie viral evolution and provide a likely explanation for why relatively few types of plant DNA viruses have evolved: they would have had to overcome the constraints imposed by an endogenous system operating to ensure that DNA acts in a cell-autonomous manner.     

The polymerase slips and PIPO exists

Plant viruses that contain plus‐sensed single‐stranded RNA genomes are highly abundant in nature. As the equivalents of large mRNAs, these viral genomes utilize a wide variety of gene expression strategies for the production of their encoded proteins. The potyviruses, which are among the most agriculturally important members in this category, contain a single large open reading frame (ORF) coding for a polyprotein that is processed into functional units. For many years, the products derived from the full‐length polyprotein were thought to be the only functional viral proteins. However, this notion was dispelled when an additional essential viral ORF, PIPO, was discovered encoded in an alternative reading frame. Since then, the PIPO protein—P3N‐PIPO, which mediates virus movement in plants—has been intensively studied, but its mode of expression remained elusive until now. Two articles, one in this issue of EMBO Reports, now report that slippage of the viral polymerase during viral genome replication is responsible for shifting PIPO into a translated reading frame, thereby allowing for production of P3N‐PIPO 1 2 . This mechanism of gene expression represents a novel strategy for plant viruses.     

The alphaviruses: gene expression, replication, and evolution.

The alphaviruses are a genus of 26 enveloped viruses that cause disease in humans and domestic animals. Mosquitoes or other hematophagous arthropods serve as vectors for these viruses. The complete sequences of the +/- 11.7-kb plus-strand RNA genomes of eight alphaviruses have been determined, and partial sequences are known for several others; this has made possible evolutionary comparisons between different alphaviruses as well as comparisons of this group of viruses with other animal and plant viruses. Full-length cDNA clones from which infectious RNA can be recovered have been constructed for four alphaviruses; these clones have facilitated many molecular genetic studies as well as the development of these viruses as expression vectors. From these and studies involving biochemical approaches, many details of the replication cycle of the alphaviruses are known. The interactions of the viruses with host cells and host organisms have been exclusively studied, and the molecular basis of virulence and recovery from viral infection have been addressed in a large number of recent papers. The structure of the viruses has been determined to about 2.5 nm, making them the best-characterized enveloped virus to date. Because of the wealth of data that has appeared, these viruses represent a well-characterized system that tell us much about the evolution of RNA viruses, their replication, and their interactions with their hosts. This review summarizes our current knowledge of this group of viruses.     

植物病毒表达载体研究进展

利用DNA或RNA植物病毒作载体表达外源蛋白是近几年发展较快的一种新的遗传转化方式,它具有以下几个优点:表达量大,表达速度快,易于进行基因操作和接种以及适用对象广泛。已发展的四种载体构建策略包括:基因取代,基因插入,融合抗原和基因互补。植物病毒表达载体可以用于基因的重组、病毒的移动和基因功能的检测等基础性研究,也可用于商业上表达多种药用蛋白或疫苗。植物病毒表达载体的稳定性主要取决于存在同源序列而引起的基因重组。本文还对病毒载体的生物安全性进行了讨论。     

Genetic Variability of Natural Populations of Cotton Leaf Curl Geminivirus, a Single-Stranded DNA Virus

Reports on the genetic variability and evolution of natural populations of DNA viruses are scarce in comparison with the abundant information on the variability of RNA viruses. Geminiviruses are plant viruses with circular ssDNA genomes that are replicated by the host plant DNA polymerases. Whitefly-transmitted geminiviruses (WTG) are the agents of important diseases of crop plants and best exemplify emerging plant viruses. In this report we have analyzed the genetic diversity of cotton leaf curl geminivirus (CLCuV), a typical emerging WTG. No genetic differentiation was observed between isolates from different host plant species or geographic regions. Thus, the analyzed isolates represented a unique, undifferentiated population. Genetic variability, estimated as nucleotide diversities at synonymous positions in open reading frames (ORFs) for the AC1 (=replication) protein and coat protein (CP = AV1), was very high, exceeding the values reported for different genes in several plant and animal RNA viruses. This was unexpected in a virus that uses the DNA replication machinery of its eukaryotic host. Diversities at nonsynonymous positions, on the other hand, indicated that variability may be constrained in the genome of CLCuV. The ratio of nonsynonymous-to-synonymous substitutions varied for the different ORFs: they were higher for CP than for AC1 and lower still for the AC4 and AV2 ORFs, which overlap AC1 and CP ORFs, respectively. Analysis of nucleotide diversities at synonymous and nonsynonymous positions of the AC4 and AV2 ORFs suggest that their evolution is constrained by AC1 and CP, respectively. Data suggest that AC4 and AV2 are new genes that may have originated by overprinting on the preexistent AC1 and CP genes. Evidence for recombination was found for the AC1 and CP ORFs and for the noncoding intergenic region (IR). Data indicate that the origin of replication is a major recombination point in the IR, but not the only one. Analyses of the IR also suggest that recombinants may be frequent in the population and that recombination may have an important role in the generation of CLCuV variability. Received: 26 February 1999 / Accepted: 31 May 1999     

Molecular phylogeny of geminivirus infecting wild plants in Japan

Few studies have been made on the molecular divergence of plant viruses. To remedy this deficiency, we examined the molecular divergence of the tobacco leaf curl geminivirus (TLCV). TLCV infects not only tobacco but alsoEupatorium andLonicera in the field and causes yellow vein disease. A total of 29 nucleotide sequences of the replication protein gene (ORF C1) of geminiviruses infecting wild plants ofE. makinoi, E. glehni andL. japonica collected from ten localities was determined. Highly divergent sequences were obtained not only among host plant populations but also within a host population. Phylogenetic analyses showed that the TLCVs infectingEupatorium andLonicera were clustered into three different clades, and were either paraphyletic or polyphyletic. This result is the first evidence demonstrating that wild populations of single plant species possess genetically diversified virus strains. Comparison with recently reported genetic variations of tobacco mild green mosaic tobamovirus (TMGMV) revealed three characteristics of TLCV evolution: (1) a higher nucleotide substitution rate, (2) more frequent migration among geographically isolated host populations, and (3) more frequent host changes to different plant families. While TMGMV is an RNA virus, TLCV has DNA genomes. In animal viruses, RNA viruses tend to evolve faster than DNA viruses. Our results indicated that this trend may not hold for plant viruses.     

Functional analysis of gene-silencing suppressors from tomato yellow leaf curl disease viruses

Tomato yellow leaf curl disease (TYLCD) is caused by a complex of phylogenetically related Begomovirus spp. that produce similar symptoms when they infect tomato plants but have different host ranges. In this work, we have evaluated the gene-silencing-suppression activity of C2, C4, and V2 viral proteins isolated from the four main TYLCD-causing strains in Spain in Nicotiana benthamiana. We observed varying degrees of local silencing suppression for each viral protein tested, with V2 proteins from all four viruses exhibiting the strongest suppression activity. None of the suppressors were able to avoid the spread of the systemic silencing, although most produced a delay. In order to test the silencing-suppression activity of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) proteins in a shared (tomato) and nonshared (bean) host, we established novel patch assays. Using these tools, we found that viral proteins from TYLCV were able to suppress silencing in both hosts, whereas TYLCSV proteins were only effective in tomato. This is the first time that viral suppressors from a complex of disease-causing geminiviruses have been subject to a comprehensive analysis using two economically important crop hosts, as well as the established N. benthamiana plant model.     

RNA recombination in animal and plant viruses.

An increasing number of animal and plant viruses have been shown to undergo RNA-RNA recombination, which is defined as the exchange of genetic information between nonsegmented RNAs. Only some of these viruses have been shown to undergo recombination in experimental infection of tissue culture, animals, and plants. However, a survey of viral RNA structure and sequences suggests that many RNA viruses were derived form homologous or nonhomologous recombination between viruses or between viruses and cellular genes during natural viral evolution. The high frequency and widespread nature of RNA recombination indicate that this phenomenon plays a more significant role in the biology of RNA viruses than was previously recognized. Three types of RNA recombination are defined: homologous recombination; aberrant homologous recombination, which results in sequence duplication, insertion, or deletion during recombination; and nonhomologous (illegitimate) recombination, which does not involve sequence homology. RNA recombination has been shown to occur by a copy choice mechanism in some viruses. A model for this recombination mechanism is presented.     

Probing plant cell structure and function with viral movement proteins.

Virus-encoded movement proteins are the principal strategy by which all plant viruses counter the primary physical defense of the plant to infection - the cell wall - to produce systemic infection and disease. Our understanding of how these proteins act at the molecular and cellular level has increased enormously in the past decade and ushered in an exciting new era of plant virology as an approach to investigating plant cell structure and function. The earliest studies focused on how movement proteins interacted with plasmodesmata, and were an important element in demonstrating the dynamic nature of these intercellular channels. Current efforts are focused on the role of movement proteins in coordinating the replication of viral genomes and the vectorial movement of the progeny genomes through the infected cell, as well as into adjacent cells. Movement proteins are thus providing unique approaches to unravel the fundamental mechanisms by which macromolecular transport is directed and integrated within and between plant cells.     

Transgenic expression of pectin methylesterase inhibitors limits tobamovirus spread in tobacco and Arabidopsis

Plant infection by a virus is a complex process influenced by virus‐encoded factors and host components which support replication and movement. Critical factors for a successful tobamovirus infection are the viral movement protein (MP) and the host pectin methylesterase (PME), an important plant counterpart that cooperates with MP to sustain viral spread. The activity of PME is modulated by endogenous protein inhibitors (pectin methylesterase inhibitors, PMEIs). PMEIs are targeted to the extracellular matrix and typically inhibit plant PMEs by forming a specific and stable stoichiometric 1:1 complex. PMEIs counteract the action of plant PMEs and therefore may affect plant susceptibility to virus. To test this hypothesis, we overexpressed genes encoding two well‐characterized PMEIs in tobacco and Arabidopsis plants. Here, we report that, in tobacco plants constitutively expressing a PMEI from Actinidia chinensis (AcPMEI), systemic movement of Tobacco mosaic virus (TMV) is limited and viral symptoms are reduced. A delayed movement of Turnip vein clearing virus (TVCV) and a reduced susceptibility to the virus were also observed in Arabidopsis plants overexpressing AtPMEI‐2. Our results provide evidence that PMEIs are able to limit tobamovirus movement and to reduce plant susceptibility to the virus.