Geminivirus disease complexes: the threat is spreading

Symptom-modulating DNA satellites associated with geminiviruses have come to our attention only recently but have proven to be widespread, associated with many diseases throughout the Old World, and economically significant, particularly in developing countries. Recent developments are elucidating the role played by these novel molecules in pathogenicity and in overcoming host plant defense. Further investigation into the promiscuous nature of these satellites and their ability to recruit further begomoviruses indicates that regions not yet affected by such begomovirus-satellite complexes are at great risk.     

Geminiviruses: Genome structure and gene function

The plant pathogenic single‐strand DNA‐containing geminiviruses have been the recent focus of intense investigation, owing both to their agronomic importance and to their potential as vectors for the expression of foreign genes in plants. Molecular genetic studies have provided detailed information on the genomic organization of many of these viruses. A greater genetic complexity has been demonstrated among the members of this viral family than had previously been suspected, as well as an apparently rapid rate of evolution of genetic diversity. We now recognize fundamental differences in the genome structure and organization of the whitefly‐ and leafhopper‐transmitted viruses, as well as among those geminiviruses infecting dicotyledonous or monocotyledonous hosts. This knowledge has provided new insights into the evolution of these viruses. The viral genes involved in replication and in systemic movement in the plant have been defined, and viral origins for single‐strand (ss) and double‐strand (ds) DNA replication have been mapped to small nucleotide regions. With the structural features of the viral genomes now well defined, current efforts are focused on elucidating the molecular aspects of viral gene regulation and interactions with host‐cell components that lead to the production of disease. Recent progress in determining the mechanism of replication and systemic movement and the contributions of these to symptom and disease development are discussed in the context of the potential for genetically engineering disease‐resistant plants.     

Satellite RNAs of plant viruses: structures and biological effects.

Plant viruses often contain parasites of their own, referred to as satellites. Satellite RNAs are dependent on their associated (helper) virus for both replication and encapsidation. Satellite RNAs vary from 194 to approximately 1,500 nucleotides (nt). The larger satellites (900 to 1,500 nt) contain open reading frames and express proteins in vitro and in vivo, whereas the smaller satellites (194 to 700 nt) do not appear to produce functional proteins. The smaller satellites contain a high degree of secondary structure involving 49 to 73% of their sequences, with the circular satellites containing more base pairing than the linear satellites. Many of the smaller satellites produce multimeric forms during replication. There are various models to account for their formation and role in satellite replication. Some of these smaller satellites encode ribozymes and are able to undergo autocatalytic cleavage. The enzymology of satellite replication is poorly understood, as is the replication of their helper viruses. In many cases the coreplication of satellites suppresses the replication of the helper virus genome. This is usually paralleled by a reduction in the disease induced by the helper virus; however, there are notable exceptions in which the satellite exacerbates the pathogenicity of the helper virus, albeit on only a limited number of hosts. The ameliorative satellites are being assessed as biocontrol agents of virus-induced disease. In greenhouse studies, satellites have been known to "spontaneously" appear in virus cultures. The possible origin of satellites will be briefly considered.     

烟粉虱传播双生病毒研究进展

综述了烟粉虱Bemisia tabaci对双生病毒的获取、传播及存留等方面的特性。烟粉虱最短的获毒和接种时间为15～30 min;双生病毒在烟粉虱体内可存留1至数周,有的终身存在。烟粉虱对双生病毒的传毒效率除了随其获毒及传毒时间的延长、传毒烟粉虱个体数量的增加以及病毒体浓度的增加而提高外,还与烟粉虱的龄期及性别有关。双生病毒除了在植物与粉虱之间直接传播外,还可通过烟粉虱交配及经卵携带的途径在烟粉虱个体和代别间进行传播。寄主植物、双生病毒的一些特殊蛋白以及烟粉虱内共生菌产生的GroEL蛋白,都可影响烟粉虱携带的双生病毒种类及传毒的可能性。双生病毒可对烟粉虱的发育、存活和生殖产生不利或有利的影响。雌成虫携带番茄黄化曲叶病毒（tomato yellow leaf curl virus, TYLCV）后,存活力和生殖力均下降; 而携带番茄斑驳病毒（tomato mottle virus, ToMoV）后,生殖力提高。此外,植物感染双生病毒后,其对烟粉虱的适合性可能提高。     

重大外来害虫B型烟粉虱的入侵行为和生态机制

烟粉虱Bemisia tabaci(Gennadius)复合种是热带、亚热带及相邻温带地区的主要害虫之一。其中的B型烟粉虱在近20年来随观赏植物的运输在世界范围内广泛传播扩散,并在许多入侵地迅速取代本地的土著烟粉虱,通过直接取食植物汁液、传播植物双生病毒等方式对当地的农业生产造成极大危害。在B型烟粉虱入侵生物学研究方面,作者课题组研究发现,至少有两个主要机制导致或促进了B型烟粉虱的广泛入侵及其所伴随的双生病毒流行:(1)入侵烟粉虱与土著烟粉虱之间的"非对称交配互作";(2)入侵烟粉虱与所传双生病毒之间的间接互惠共生关系。这些研究结果从一定程度上揭示了B型烟粉虱成功入侵的行为和生态机制,并为进一步探讨烟粉虱的入侵机制提供了思路。     

Viruses and thyroiditis: an update

Background

Despite the demonstration that geminiviruses, like many other single stranded DNA viruses, are evolving at rates similar to those of RNA viruses, a recent study has suggested that grass-infecting species in the genus Mastrevirus may have co-diverged with their hosts over millions of years. This "co-divergence hypothesis" requires that long-term mastrevirus substitution rates be at least 100,000-fold lower than their basal mutation rates and 10,000-fold lower than their observable short-term substitution rates. The credibility of this hypothesis, therefore, hinges on the testable claim that negative selection during mastrevirus evolution is so potent that it effectively purges 99.999% of all mutations that occur.

Results

We have conducted long-term evolution experiments lasting between 6 and 32 years, where we have determined substitution rates of between 2 and 3 × 10^-4 substitutions/site/year for the mastreviruses Maize streak virus (MSV) and Sugarcane streak Réunion virus (SSRV). We further show that mutation biases are similar for different geminivirus genera, suggesting that mutational processes that drive high basal mutation rates are conserved across the family. Rather than displaying signs of extremely severe negative selection as implied by the co-divergence hypothesis, our evolution experiments indicate that MSV and SSRV are predominantly evolving under neutral genetic drift.

Conclusion

The absence of strong negative selection signals within our evolution experiments and the uniformly high geminivirus substitution rates that we and others have reported suggest that mastreviruses cannot have co-diverged with their hosts.     

Virus Satellites Drive Viral Evolution and Ecology

Virus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hosts.     

Molecular evolution of satellite DNA CLsat in lizards of the Darevskia species (Sauria: Lacertidae): correlation with species diversity

The structure and evolution of a satellite DNA family was examined in lizards from the genus Darevskia (family Lacertidae). Comparison of tandem units of repeated DNA (satDNA), CLsat, in all species from the genus Darevskia has shown that their variability is largely based on single-nucleotide substitutions, which constitute about 50 diagnostic positions underlying classification of the family into three subfamilies. Maximum differences between the subfamilies reached 25%. At this level of tandem unit divergence between the subfamilies, no cross-hybridization between them was observed (at 65 degrees C). The individual variability of one subfamily within the species was on average 5% while the variability between species consensuses within a subfamily was 10%. The presence of highly conserved regions in all monomers and some features of their organization show that satellites of all Darevskia species belong to one satDNA family. The organization of unit sequences of satellites CLsat and Agi also detected by us in another lizard genus, Lacerts s. str. was compared. Similarity that was found between these satellites suggests their relatedness and common origin. A possible pathway of evolution of these two satDNA families is proposed. The distribution and content of CLsat repeat subfamilies in all species of the genus was examined by Southern blotting hybridization. Seven species had mainly CLsatI (83 to 96%); three species, approximately equal amounts of CLsatI and CLsatIII (the admixture of CLsatII was 2-3%); and five species, a combination of all three subfamilies in highly varying proportions. Based on these results as well as on zoogeographic views on phylogeny and taxonomy of the Darevskia species, hypotheses on the evolution of molecular-genetic relationships within this genus are advanced.     

Interplay of selective pressure and stochastic events directs evolution of the MEL172 satellite DNA library in root-knot nematodes

According to the library model, related species can have in common satellite DNA (satDNA) families amplified in differing abundances, but reasons for persistence of particular sequences in the library during long periods of time are poorly understood. In this paper, we characterize 3 related satDNAs coexisting in the form of a library in mitotic parthenogenetic root-knot nematodes of the genus Meloidogyne. Due to sequence similarity and conserved monomer length of 172 bp, this group of satDNAs is named MEL172. Analysis of sequence variability patterns among monomers of the 3 MEL172 satellites revealed 2 low-variable (LV) domains highly reluctant to sequence changes, 2 moderately variable (MV) domains characterized by limited number of mutations, and 1 highly variable (HV) domain. The latter domain is prone to rapid spread and homogenization of changes. Comparison of the 3 MEL172 consensus sequences shows that the LV domains have 6% changed nucleotide positions, the MV domains have 48%, whereas 78% divergence is concentrated in the HV domain. Conserved distribution of intersatellite variability might indicate a complex pattern of interactions in heterochromatin, which limits the range and phasing of allowed changes, implying a possible selection imposed on monomer sequences. The lack of fixed species-diagnostic mutations in each of the examined MEL172 satellites suggests that they existed in unaltered form in a common ancestor of extant species. Consequently, the evolution of these satellites seems to be driven by interplay of selective constraints and stochastic events. We propose that new satellites were derived from an ancestral progenitor sequence by nonrandom accumulation of mutations due to selective pressure on particular sequence segments. In the library of particular taxa, established satellites might be subject to differential amplification at chance due to stochastic mechanisms of concerted evolution.     

Genetic variation of geminiviruses: comparison between sexual and asexual host plant populations

One of the most promising hypotheses for the evolution of sex is that sexual reproduction is advantageous because it increases the rate of adaptive evolution in response to parasites. To investigate this advantage of sex, we compared genetic variation of geminiviruses infecting sexual and asexual populations of Eupatorium (Asteraceae). The infection frequency was 37.5% in the sexual population and 87.8% in the asexual population. The lower infection frequency in the sexual population might be the result of higher genetic diversity of host plants. If geminiviruses have diverged to counter defence systems of genetically variable hosts, genetic diversity of viruses is expected to be higher in sexual host populations than in asexual host populations. To test this expectation, we used single-strand conformation polymorphism (SSCP) analysis to examine genetic diversity of the geminiviruses in a DNA region containing the open-reading frame (ORF) C4 gene, which is known to function as a host range determinant. As predicted, higher genetic diversity of viruses was observed in the sexual population: three SSCP types were found in the asexual population while six types were found in the sexual population. Sequencing of the polymerase chain reaction (PCR) products revealed further genetic diversity. Phylogenetic analysis of the sequences showed that the SSCP types belonged to four different clades. Several SSCP types from the same clade were found in the sexual population, whereas the asexual population included only one SSCP type from each clade. Amino acid replacements of ORF C4 are suggested to be accelerated in the sexual population. This evidence supports the hypothesis that sexual reproduction is advantageous as a defence against epidemic disease.     

Cassava mosaic geminiviruses: actual knowledge and perspectives

Cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses (CMGs) is one of the most devastating crop diseases and a major constraint for cassava cultivation. CMD has been reported only from the African continent and Indian subcontinent despite the large-scale cultivation of cassava in Latin America and several South-East Asian countries. Seven CMG species have been reported from Africa and two from the Indian subcontinent and, in addition, several strains have been recognized. Recombination and pseudo-recombination between CMGs give rise not only to different strains, but also to members of novel virus species with increased virulence and a new source of biodiversity, causing severe disease epidemics. CMGs are known to trigger gene silencing in plants and, in order to counteract this natural host defence, geminiviruses have evolved suppressor proteins. Temperature and other environmental factors can affect silencing and suppression, and thus modulate the symptoms. In the case of mixed infections of two or more CMGs, there is a possibility for a synergistic interaction as a result of the presence of differential and combinatorial suppressor proteins. In this article, we provide the status of recent research findings with regard to the CMD complex, present the molecular biology knowledge of CMGs with reference to other geminiviruses, and highlight the mechanisms by which CMGs have exploited nature to their advantage.     

Increased pathogenicity in a pseudorecombinant bipartite geminivirus correlates with intermolecular recombination.

Most whitefly-transmitted geminiviruses possess bipartite DNA genomes, and this feature may facilitate viral evolution through pseudorecombination and/or recombination. To test this hypothesis, the DNA-A and DNA-B components of the geminiviruses bean dwarf mosaic virus (BDMV) and tomato mottle virus (ToMoV) were exchanged, and the resultant pseudorecombinants were serially passaged through plants. Both pseudorecombinants were infectious in Nicotiana benthamiana but induced attenuated symptoms and had reduced DNA-B levels. Serial passage experiments revealed that the BDMV DNA-A plus ToMoV DNA-B pseudorecombinant could not be maintained beyond three passages. In contrast, the ToMoV DNA-A plus BDMV DNA-B pseudorecombinant was maintained during serial passage through N. benthamiana and Phaseolus vulgaris and, after three to five passages, became highly pathogenic. Furthermore, the increased pathogenicity of this pseudorecombinant was consistently associated with an increased level of DNA-B, which eventuated in equivalent levels of both components. Sequence analysis of the DNA-B component of the more pathogenic pseudorecombinant revealed that intermolecular recombination had taken place in which most of the BDMV DNA-B common region was replaced with the ToMoV DNA-A common region. This recombinant DNA-B component, which contained the ToMoV origin of replication, was the predominant DNA-B component associated with the more pathogenic pseudorecombinant. These results provide the first demonstration of recombination between distinct bipartite geminiviruses and establish that the bipartite genome can facilitate viral evolution through pseudorecombination and intermolecular recombination.     

On the maintenance and origin of alternative strategies in the Ruff Philomachus pugnax

This paper has tried to answer the question of how the independent and the satellite strategy of the Ruff originated and has been maintained during evolution. It is not intended as a report of a piece of completed research but gives a number of tentative, but testable hypotheses. Data are presented that independent males and satellites are equally successful in copulating, provided that almost all copulations occur on leks, and independent males and satellites spend an equal proportion of their time on leks. The extent that males with the different strategies are attached to leks is analysed. Within the group of independent males, considerable differences exist: a resident male is strongly attached to only one particular lek and a marginal male seems to sample many leks. A satellite male behaves intermediately: he is attached to a limited number of leks. The mechanism for the maintenance of both strategies could not be explained by competition for resources (copulations). Contests between independent males and satellites are asymmetric, the satellite being the weaker contestant. The mechanism seems to be due to enlarging the accessibility of the resource by cooperation between both strategies. The ideas on the origin of the system are based mainly on the behaviour of females. There is no relation between the distribution of leks and the size of a female's foraging area, except that most leks are situated along the migration route. It further seems that the number of copulating females in the Netherlands is larger than the number of males on leks, but the number of breeding females is much lower. It is suggested that many females copulate on migration, which is related to extreme breeding conditions in the north of the range. Apart from a change in the sex ratio from south to north, a change in the proportion of satellites is also predicted. The origin of the system is ascribed to the incompatibility between favourable food conditions for chicks and further possibilities for inter-male competition on leks in the same area. The satellite strategy is considered as being derived from a non-competitive strategy accompanying females on migration. In the phase of cooperation between resident and satellite males, individual recognition became important. This could be the factor underlying the evolution of white plumages in satellites and the extreme plumage diversity in independent males.     

Isolation of twelve satellite DNAs from Drosophila hydei

The genome of a Drosophila hydei genotype with a reduced amount of heterochromatin was fractionated by three cycles of preparative gradients: firstly in Ag⁺/Cs₂SO₄, secondly in actimomycin D/CsCl, and finally in neutral CsCl. Using this method, twelve highly repetitive simple-sequence satellites were isolated. Ten of them comprised only a minor amount of the genome in contrast to the two major satellites found earlier¹ (p = 1.696 and 1,714 g/cm³). These minor satellites were characterized by their banding in the gradient systems used, by their density in neutral CsCl, and by their melting point. Using these characteristics, it was found that the fractions of the Ag^?/Cs₂SO₄ gradient do not contain purified single components, because up to five different satellites band in the same position of the Ag^?/Cs₂SO₄ gradient. It was possible to isolate a high number of satellites even from a genome with a reduced amount of heterochromatin. Thus, the D hydei heterochromatin does not domain one unique highly repetitive sequence DNA, but is comprised of many different satellite sequences.     

Molecular Evolution of Satellite DNA CLsat in Lizards from the Genus Darevskia (Sauria: Lacertidae): Correlation with Species Diversity

The structure and evolution of a satellite DNA family was examined in lizards from the genus Darevskia(family Lacertidae). Comparison of tandem units of repeated DNA (satDNA), CLsat, in all species from the genus Darevskiahas shown that their variability is largely explained by single-nucleotide substitutions, which form about 50 diagnostic positions underlying classification of the family into three subfamilies. Maximum differences between the subfamilies reached 25%. At this level of tandem unit divergence in the subfamilies, no cross-hybridization between them was observed (at 65°C). The individual variability within one subfamily within the species was on average 5% while the variability between species consensuses within a subfamily was 10%. The presence of highly conserved regions in all monomers and some features of their organization show that satellites of all Darevskia species belong to one satDNA family. The organization of unit sequences of satellites CLsat and Agi160 also detected by us in another lizard genus, Lacerta s. str. was compared. Similarity that was found between these satellites suggests their relatedness and common origin. A possible pathway of evolution of these two satDNA families is proposed. The distribution and content of CLsat repeat subfamilies in all species of the genus was examined by Southern hybridization. Seven species had mainly CLsatI (83 to 96%); three species, approximately equal amounts of CLsatI and CLsatIII (the admixture of CLsatII was 2–5%); and five species, a combination of all three subfamilies in highly varying proportions. Based on these results as well as on zoogeographic views on the taxonomy and phylogeny of theDarevskia species, hypotheses on the evolution of molecular-genetic relationships within this genus are advanced.     

The resident's dilemma: a female choice model for the evolution of alternative mating strategies in lekking male ruffs (Philomachus pugnax)

Previous models for the evolution of alternative male matingbehavior have virtually ignored the role of female choice. Wepresent a model in which female choice favors the evolutionand maintenance of alternative mating strategies in male ruffs,Philomachus pugnax. Resident male ruffe establish and defendcourts on leks against other residents, while non-territorialsatellite males move between leks and among courts on a lek.Residents appear to actively recruit satellites to their courts,even though satellites may mate with females once there. Residentbehavior toward satellites and data on female behavior suggestthat residents benefit from a satellite's presence due to somefemale preference for mating on co-occupied courts. However,if all residents accept satellites, none gains any relativeadvantage, yet all pay the costs of having satellites on theircourt. We present a game theoretical model that shows that therelative nature of female choice places residents in an evolutionarydilemma with respect to satellite acceptance. Although all residentswould benefit if satellites could be cooperatively excludedfrom leks, the only evolutionarily stable strategy for individualresidents is to defect and accept satellites. The model alsodemonstrates that this "resident's dilemma" likely exists onlyin a local sense, since the failure of residents to cooperativelyexclude satellites from leks need not result in globally lowerpayoffs, due to frequency-dependent selection on the proportionof satellites in the population. Our analysis suggests thatthe resident-satellite relationship in ruffs, despite its obviouscompetitive elements, is fundamentally a cooperative associationfavored by female choice. Female choice has also been proposedas the primary mechanism selecting for male association to formleks in ruffe. In this context, resident-satellite associationsmay be thought of as transitory "leks within a lek     

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.     

Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types

We have previously shown that the soil-borne plant pathogen Agrobacterium tumefaciens supports the replication of tomato leaf curl geminivirus (Australian isolate) (TLCV) DNA. However, the reproducibility of this observation with other geminiviruses has been questioned. Here, we show that replicative DNA forms of three other geminiviruses also accumulate at varying levels in Agrobacterium. Geminiviral DNA constructs that lacked the ability to replicate in Agrobacterium were rendered replication-competent by changing their configuration so that two copies of the viral ori were present. Furthermore, we report that low-level replication of TLCV DNA can occur in Escherichia coli containing a dimeric TLCV construct in a high copy number plasmid. These findings were reinforced by expression studies using beta-glucuronidase which revealed that all six TLCV promoters are active in Agrobacterium, and two are functional in E. coli.