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 doublestranded 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.RNA silencing includes a set of small RNA-guided pathways that regulate gene expression in eukaryotes and control processes such as development, genome stability, stress-induced responses, and defense against molecular parasites (4, 6, 10). An essential component of RNA silencing is RNase III Dicer, an enzyme that cleaves RNA with double-stranded (ds) features into 21-to 24-nucleotide (nt) duplex small interfering RNA (siRNA) or microRNA (miRNA). The Arabidopsis thaliana genome encodes four Dicer-like (DCL) enzymes and six RNA-dependent RNA polymerases (RDR), other components of the silencing machinery in plants (26,39,46,55,58). DCL1 produces 21-nt miRNAs derived from RNA polymerase II transcripts with imperfect self-complementary fold-back structures (22). Endogenous siRNAs are likely generated from dsRNA substrates because of the requirement for a specific RDR for the production of each siRNA class (32, 33). The 24-nt cis-acting siRNA class derives mostly from retroelements, transposons, and DNA repeats and depends on DCL3 and RDR2 that use RNA polymerase IV transcripts as a template for dsRNA production (33). Synthesis of 21-nt trans-acting siRNAs is DCL4 dependent and occurs through an initial DCL1-dependent, miRNA-directed cleavage of trans-acting siRNA precursor transcripts, which are subsequently processed into long, perfect dsRNA by RDR6 (5, 32).In plants, RNA interference is guided by two distinct classes of...

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.

BackgroundVirulence 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 FindingsWe 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 plant's 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/SignificanceThese 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.

Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.

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.

Some receptor-like kinases (RLK) control plant development while others regulate immunity. The Arabidopsis ERECTA (ER) RLK regulates both biological processes. To discover specific components of ER-mediated immunity, a genetic screen was conducted to identify suppressors of erecta (ser) susceptibility to Plectosphaerella cucumerina fungus. The ser1 and ser2 mutations restored disease resistance to this pathogen to wild-type levels in the er-1 background but failed to suppress er-associated developmental phenotypes. The deposition of callose upon P. cucumerina inoculation, which was impaired in the er-1 plants, was also restored to near wild-type levels in the ser er-1 mutants. Analyses of er cell walls revealed that total neutral sugars were reduced and uronic acids increased relative to those of wild-type walls. Interestingly, in the ser er-1 walls, neutral sugars were elevated and uronic acids were reduced relative to both er-1 and wild-type plants. The cell-wall changes found in er-1 and the ser er-1 mutants are unlikely to contribute to their developmental alterations. However, they may influence disease resistance, as a positive correlation was found between uronic acids content and resistance to P. cucumerina. We propose a specific function for ER in regulating cell wall-mediated disease resistance that is distinct from its role in development.

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 Q ST -based genetic differentiations between both host populations with F ST 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.

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.