Israel Pagán

Structural and Genetic Requirements for the Biogenesis of Tobacco Rattle Virus-Derived Small Interfering RNAs

ABSTRACT In plants, small RNA-guided processes referred to as RNA silencing control gene expression and serve as an efficient antiviral mechanism. Plant viruses are inducers and targets of RNA silencing as infection involves the production of functional virus-derived small interfering RNAs (siRNAs). Here we investigate the structural and genetic components influencing the formation of Tobacco rattle virus (TRV)-derived siRNAs. TRV siRNAs are mostly 21 nucleotides in length and derive from positive and negative viral RNA strands, although TRV siRNAs of positive polarity are significantly more abundant. This asymmetry appears not to correlate with the presence of highly structured regions of single-stranded viral RNA. The Dicer-like enzyme DCL4, DCL3, or DCL2 targets, alone or in combination, viral templates to promote synthesis of siRNAs of both polarities from all regions of the viral genome. The heterogeneous distribution profile of TRV siRNAs reveals differential contributions throughout the TRV genome to siRNA formation. Indirect evidence suggests that DCL2 is responsible for production of a subset of siRNAs derived from the 3′ end region of TRV. TRV siRNA biogenesis and antiviral silencing are strongly dependent on the combined activity of the host-encoded RNA-dependent RNA polymerases RDR1, RDR2, and RDR6, thus providing evidence that perfectly complementary double-stranded RNA serves as a substrate for siRNA production. We conclude that the overall composition of viral siRNAs in TRV-infected plants reflects the combined action of several interconnected pathways involving different DCL and RDR activities.

Genetic Structure of the Population of Pepino mosaic virus Infecting Tomato Crops in Spain.

ABSTRACT The population structure of Pepino mosaic virus (PepMV), which has caused severe epidemics in tomato in Spain since 2000, was analyzed. Isolates were characterized by the nucleotide sequence of the triple gene block and coat protein gene and, for a subset of isolates, a part of the RNA-dependent RNA polymerase gene. The full-length sequence of the genomic RNA of a Solanum muricatum isolate from Peru also was determined. In spite of high symptom diversity, the Spanish population of PepMV mostly comprised highly similar isolates belonging to the strain reported in Europe (European tomato strain), which has been the most prevalent genotype in Spain. The Spanish PepMV population was not structured spatially or temporally. Also, isolates highly similar to those from nontomato hosts from Peru (Peruvian strain) or to isolate US2 from the United States (US2 strain) were detected at lower frequency relative to the European strain. These two strains were detected in peninsular Spain only in 2004, but the Peruvian strain has been detected in the Canary Islands since 2000. These results suggest that PepMV was introduced into Spain more than once. Isolates from the Peruvian and US2 strains always were found in mixed infections with the European tomato strain, and interstrain recombinants were detected. The presence of different strains of the virus, and of recombinant isolates, should be considered for the development of control strategies based on genetic resistance.

Long-Term Evolution of the Luteoviridae: Time Scale and Mode of Virus Speciation

ABSTRACT Despite their importance as agents of emerging disease, the time scale and evolutionary processes that shape the appearance of new viral species are largely unknown. To address these issues, we analyzed intra- and interspecific evolutionary processes in the Luteoviridae family of plant RNA viruses. Using the coat protein gene of 12 members of the family, we determined their phylogenetic relationships, rates of nucleotide substitution, times to common ancestry, and patterns of speciation. An associated multigene analysis enabled us to infer the nature of selection pressures and the genomic distribution of recombination events. Although rates of evolutionary change and selection pressures varied among genes and species and were lower in some overlapping gene regions, all fell within the range of those seen in animal RNA viruses. Recombination breakpoints were commonly observed at gene boundaries but less so within genes. Our molecular clock analysis suggested that the origin of the currently circulating Luteoviridae species occurred within the last 4 millennia, with intraspecific genetic diversity arising within the last few hundred years. Speciation within the Luteoviridae may therefore be associated with the expansion of agricultural systems. Finally, our phylogenetic analysis suggested that viral speciation events tended to occur within the same plant host species and country of origin, as expected if speciation is largely sympatric, rather than allopatric, in nature.

Arabidopsis thaliana as a model for the study of plant–virus co-evolution

Understanding plant–virus coevolution requires wild systems in which there is no human manipulation of either host or virus. To develop such a system, we analysed virus infection in six wild populations of Arabidopsis thaliana in Central Spain. The incidence of five virus species with different life-styles was monitored during four years, and this was analysed in relation to the demography of the host populations. Total virus incidence reached 70 per cent, which suggests a role of virus infection in the population structure and dynamics of the host, under the assumption of a host fitness cost caused by the infection. Maximum incidence occurred at early growth stages, and co-infection with different viruses was frequent, two factors often resulting in increased virulence. Experimental infections under controlled conditions with two isolates of the most prevalent viruses, cauliflower mosaic virus and cucumber mosaic virus, showed that there is genetic variation for virus accumulation, although this depended on the interaction between host and virus genotypes. Comparison of QST-based genetic differentiations between both host populations with FST genetic differentiation based on putatively neutral markers suggests different selection dynamics for resistance against different virus species or genotypes. Together, these results are compatible with a hypothesis of plant–virus coevolution.

Rapid genetic diversification and high fitness penalties associated with pathogenicity evolution in a plant virus

Under the gene-for-gene model of host-pathogen coevolution, recognition of pathogen avirulence factors by host resistance factors triggers host defenses and limits infection. Theory predicts that the evolution of higher levels of pathogenicity will be associated with fitness penalties and that the cost of higher pathogenicity must be much smaller than that of not infecting the host. The analysis of pathogenicity costs is of academic and applied relevance, as these are determinants for the success of resistance genes bred into crops for disease control. However, most previous attempts of addressing this issue in plant pathogens yielded conflicting and inconclusive results. We have analyzed the costs of pathogenicity in pepper-infecting tobamoviruses defined by their ability to infect pepper plants with different alleles at the resistance locus L. We provide conclusive evidence of pathogenicity-associated costs by comparison of pathotype frequency with the fraction of the crop carrying the various resistance alleles, by timescaled phylogenies, and by temporal analyses of population dynamics and selection pressures using nucleotide sequences. In addition, experimental estimates of relative fitness under controlled conditions also provided evidence of high pathogenicity costs. These high pathogenicity costs may reflect intrinsic properties of plant virus genomes and should be considered in future models of host-parasite coevolution.

The Relationship of Within-Host Multiplication and Virulence in a Plant-Virus System

Background Virulence does not represent any obvious advantage to parasites. Most models of virulence evolution assume that virulence is an unavoidable consequence of within-host multiplication of parasites, resulting in trade-offs between within-host multiplication and between-host transmission fitness components. Experimental support for the central assumption of this hypothesis, i.e., for a positive correlation between within-host multiplication rates and virulence, is limited for plant-parasite systems. Methodology/Principal Findings We have addressed this issue in the system Arabidopsis thaliana-Cucumber mosaic virus (CMV). Virus multiplication and the effect of infection on plant growth and on viable seed production were quantified for 21 Arabidopsis wild genotypes infected by 3 CMV isolates. The effect of infection on plant growth and seed production depended of plant architecture and length of postembryonic life cycle, two genetically-determined traits, as well as on the time of infection in the plants life cycle. A relationship between virus multiplication and virulence was not a general feature of this host-parasite system. This could be explained by tolerance mechanisms determined by the host genotype and operating differently on two components of plant fitness, biomass production and resource allocation to seeds. However, a positive relationship between virus multiplication and virulence was detected for some accessions with short life cycle and high seed weight to biomass ratio, which show lower levels of tolerance to infection. Conclusions/Significance These results show that genotype-specific tolerance mechanisms may lead to the absence of a clear relationship between parasite multiplication and virulence. Furthermore, a positive correlation between parasite multiplication and virulence may occur only in some genotypes and/or environmental conditions for a given host-parasite system. Thus, our results challenge the general validity of the trade-off hypothesis for virulence evolution, and stress the need of considering the effect of both the host and parasite genotypes in analyses of host-parasite interactions.

Host responses in life-history traits and tolerance to virus infection in Arabidopsis thaliana.

Knowing how hosts respond to parasite infection is paramount in understanding the effects of parasites on host populations and hence host–parasite co-evolution. Modification of life-history traits in response to parasitism has received less attention than other defence strategies. Life-history theory predicts that parasitised hosts will increase reproductive effort and accelerate reproduction. However, empirical analyses of these predictions are few and mostly limited to animal-parasite systems. We have analysed life-history trait responses in 18 accessions of Arabidopsis thaliana infected at two different developmental stages with three strains of Cucumber mosaic virus (CMV). Accessions were divided into two groups according to allometric relationships; these groups differed also in their tolerance to CMV infection. Life-history trait modification upon virus infection depended on the host genotype and the stage at infection. While all accessions delayed flowering, only the more tolerant allometric group modified resource allocation to increase the production of reproductive structures and progeny, and reduced the length of reproductive period. Our results are in agreement with modifications of life-history traits reported for parasitised animals and with predictions from life-history theory. Thus, we provide empirical support for the general validity of theoretical predictions. In addition, this experimental approach allowed us to quantitatively estimate the genetic determinism of life-history trait plasticity and to evaluate the role of life-history trait modification in defence against parasites, two largely unexplored issues.

Effect of biodiversity changes in disease risk: exploring disease emergence in a plant-virus system.

The effect of biodiversity on the ability of parasites to infect their host and cause disease (i.e. disease risk) is a major question in pathology, which is central to understand the emergence of infectious diseases, and to develop strategies for their management. Two hypotheses, which can be considered as extremes of a continuum, relate biodiversity to disease risk: One states that biodiversity is positively correlated with disease risk (Amplification Effect), and the second predicts a negative correlation between biodiversity and disease risk (Dilution Effect). Which of them applies better to different host-parasite systems is still a source of debate, due to limited experimental or empirical data. This is especially the case for viral diseases of plants. To address this subject, we have monitored for three years the prevalence of several viruses, and virus-associated symptoms, in populations of wild pepper (chiltepin) under different levels of human management. For each population, we also measured the habitat species diversity, host plant genetic diversity and host plant density. Results indicate that disease and infection risk increased with the level of human management, which was associated with decreased species diversity and host genetic diversity, and with increased host plant density. Importantly, species diversity of the habitat was the primary predictor of disease risk for wild chiltepin populations. This changed in managed populations where host genetic diversity was the primary predictor. Host density was generally a poorer predictor of disease and infection risk. These results support the dilution effect hypothesis, and underline the relevance of different ecological factors in determining disease/infection risk in host plant populations under different levels of anthropic influence. These results are relevant for managing plant diseases and for establishing conservation policies for endangered plant species.

Differential tolerance to direct and indirect density-dependent costs of viral infection in Arabidopsis thaliana.

Population density and costs of parasite infection may condition the capacity of organisms to grow, survive and reproduce, i.e. their competitive ability. In host–parasite systems there are different competitive interactions: among uninfected hosts, among infected hosts, and between uninfected and infected hosts. Consequently, parasite infection results in a direct cost, due to parasitism itself, and in an indirect cost, due to modification of the competitive ability of the infected host. Theory predicts that host fitness reduction will be higher under the combined effects of costs of parasitism and competition than under each factor separately. However, experimental support for this prediction is scarce, and derives mostly from animal–parasite systems. We have analysed the interaction between parasite infection and plant density using the plant-parasite system of Arabidopsis thaliana and the generalist virus Cucumber mosaic virus (CMV). Plants of three wild genotypes grown at different densities were infected by CMV at various prevalences, and the effects of infection on plant growth and reproduction were quantified. Results demonstrate that the combined effects of host density and parasite infection may result either in a reduction or in an increase of the competitive ability of the host. The two genotypes investing a higher proportion of resources to reproduction showed tolerance to the direct cost of infection, while the genotype investing a higher proportion of resources to growth showed tolerance to the indirect cost of infection. Our findings show that the outcome of the interaction between host density and parasitism depends on the host genotype, which determines the plasticity of life-history traits and consequently, the host capacity to develop different tolerance mechanisms to the direct or indirect costs of parasitism. These results indicate the high relevance of host density and parasitism in determining the competitive ability of a plant, and stress the need to simultaneously consider both factors to understand the selective pressures that drive host–parasite co-evolution.

Vertical transmission selects for reduced virulence in a plant virus and for increased resistance in the host.

For the last three decades, evolutionary biologists have sought to understand which factors modulate the evolution of parasite virulence. Although theory has identified several of these modulators, their effect has seldom been analysed experimentally. We investigated the role of two such major factors—the mode of transmission, and host adaptation in response to parasite evolution—in the evolution of virulence of the plant virus Cucumber mosaic virus (CMV) in its natural host Arabidopsis thaliana. To do so, we serially passaged three CMV strains under strict vertical and strict horizontal transmission, alternating both modes of transmission. We quantified seed (vertical) transmission rate, virus accumulation, effect on plant growth and virulence of evolved and non-evolved viruses in the original plants and in plants derived after five passages of vertical transmission. Our results indicated that vertical passaging led to adaptation of the virus to greater vertical transmission, which was associated with reductions of virus accumulation and virulence. On the other hand, horizontal serial passages did not significantly modify virus accumulation and virulence. The observed increases in CMV seed transmission, and reductions in virus accumulation and virulence in vertically passaged viruses were due also to reciprocal host adaptation during vertical passages, which additionally reduced virulence and multiplication of vertically passaged viruses. This result is consistent with plant-virus co-evolution. Host adaptation to vertically passaged viruses was traded-off against reduced resistance to the non-evolved viruses. Thus, we provide evidence of the key role that the interplay between mode of transmission and host-parasite co-evolution has in determining the evolution of virulence.