Repeated loss of the ability of a wild pepper disease resistance gene to function at high temperatures suggests that thermoresistance is a costly trait.

Specificity in plant-pathogen gene-for-gene (GFG) interactions is determined by the recognition of pathogen proteins by the products of plant resistance (R) genes. The evolutionary dynamics of R genes in plant-virus systems is poorly understood. We analyse the evolution of the L resistance locus to tobamoviruses in the wild pepper Capsicum annuum var. glabriusculum (chiltepin), a crop relative undergoing incipient domestication. The frequency, and the genetic and phenotypic diversity, of the L locus was analysed in 41 chiltepin populations under different levels of human management over its distribution range in Mexico. The frequency of resistance was lower in Cultivated than in Wild populations. L-locus genetic diversity showed a strong spatial structure with no isolation-by-distance pattern, suggesting environment-specific selection, possibly associated with infection by the highly virulent tobamoviruses found in the surveyed regions. L alleles differed in recognition specificity and in the expression of resistance at different temperatures, broad-spectrum recognition of P0 + P1 pathotypes and expression above 32°C being ancestral traits that were repeatedly lost along L-locus evolution. Overall, loss of resistance co-occurs with incipient domestication and broad-spectrum resistance expressed at high temperatures has apparent fitness costs. These findings contribute to understand the role of fitness trade-offs in plant-virus coevolution.

Seasonality of Coxiella burnetii among Wild Rabbits (Oryctolagus cuniculus) and the Hyalomma lusitanicum (Acari: Ixodidae) in a Meso-Mediterranean Ecosystem.

(1) Background: Q fever is a worldwide zoonosis caused by Coxiella burnetii that have cases reported in humans and animals almost everywhere. The aim of this study was to describe the seasonality of Coxiella burnetii in the wild rabbit (Oryctolagus cuniculus) and the tick Hyalomma lusitanicum in a meso-Mediterranean ecosystem. (2) Methods: two populations of wild rabbits that differ in whether or not they share habitat with ungulates, mainly red deer (Cervus elaphus) were sampled for a year to collect ticks, blood and vaginal or anal swabs. Presence of C. burnetii DNA in swabs and the tick H. lusitanicum was determined by PCR and serum antibodies by ELISA. (3) Results: C. burnetii DNA was detected in 47.2% of 583 rabbits, in 65.5% of sera, and in more than half of the H. lusitanicum. There were small variations according to sex and age of the rabbits but significant according to the habitat (4) Conclusions: The results indicate that C. burnetii circulates freely between wild rabbits and H. lusitanicum and the sylvatic cycle in meso-Mediterranean environments relies in the presence of wild rabbits and H. lusitanicum above all if sharing habitat with red deer.

Environmental heterogeneity and the evolution of plant-virus interactions: Viruses in wild pepper populations.

Understanding host-pathogen interactions requires analyses to address the multiplicity of scales in heterogeneous landscapes. Anthropogenic influence on plant communities, especially cultivation, is a major cause of environmental heterogeneity. We have approached the analysis of how environmental heterogeneity determines plant-virus interactions by studying virus infection in a wild plant currently undergoing incipient domestication, the wild pepper or chiltepin, across its geographical range in Mexico. We have shown previously that anthropogenic disturbance is associated with higher infection and disease risk, and with disrupted patterns of host and virus genetic spatial structure. We now show that anthropogenic factors, species richness, host genetic diversity and density in communities supporting chiltepin differentially affect infection risk according to the virus analysed. We also show that in addition to these factors, a broad range of abiotic and biotic variables meaningful to continental scales, have an important role on the risk of infection depending on the virus. Last, we show that natural virus infection of chiltepin plants in wild communities results in decreased survival and fecundity, hence negatively affecting fitness. This important finding paves the way for future studies on plant-virus co-evolution.

Optimization of the balance between effort and yield in unilateral surgical transfer of mouse embryos.

Surgical transfer of embryos is carried out daily in animal facilities worldwide for the rederivation of mouse strains/lines, among other purposes. Current protocols described in laboratory manuals recommend using a high number of embryos during transfer, typically in the range of 15 up to 25. To optimize the use of resources it is necessary to estimate and relate the effort required and the yield obtained. Here, we analyse the balance between the number of embryos transferred (the effort), and the yield as the number of born pups obtained from surgical embryo transfer. To accomplish this, we have analyzed data obtained during rederivation of nearly one hundred lines of mice to a new animal facility. Our results confirm that the use of increasing numbers of embryos per transfer increases the yields of born pups, as has been described previously in the literature, but they also highlight the disproportionate effort required, i.e. in the number of embryos that needed to be transferred. An estimate of the mean expected yields of surgical transfers and their comparison with the actual observed yields indicated that the balance between effort and yield is optimized when using lower numbers of embryos than in currently used protocols, in the range of 8 to 12. Given the heterogeneous nature of the data presented and analyzed here, which is from a population of mice that may be considered as representative of any animal facility, our optimization approach should help save resources in similar facilities and improve the yields of embryo transfer procedures.

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.

Impact of human management on the genetic variation of wild pepper, Capsicum annuum var. glabriusculum.

Management of wild peppers in Mexico has occurred for a long time without clear phenotypic signs of domestication. However, pre-domestication management could have implications for the population's genetic richness. To test this hypothesis we analysed 27 wild (W), let standing (LS) and cultivated (C) populations, plus 7 samples from local markets (LM), with nine polymorphic microsatellite markers. Two hundred and fifty two alleles were identified, averaging 28 per locus. Allele number was higher in W, and 15 and 40% less in LS and C populations, respectively. Genetic variation had a significant population structure. In W populations, structure was associated with ecological and geographic areas according to isolation by distance. When LM and C populations where included in the analysis, differentiation was no longer apparent. Most LM were related to distant populations from Sierra Madre Oriental, which represents their probable origin. Historical demography shows a recent decline in all W populations. Thus, pre-domestication human management is associated with a significant reduction of genetic diversity and with a loss of differentiation suggesting movement among regions by man. Measures to conserve wild and managed populations should be implemented to maintain the source and the architecture of genetic variation in this important crop relative.

Transcriptional changes and oxidative stress associated with the synergistic interaction between Potato virus X and Potato virus Y and their relationship with symptom expression.

Many virus diseases of economic importance to agriculture result from mixtures of different pathogens invading the host at a given time. This contrasts with the relatively scarce studies available on the molecular events associated with virus-host interactions in mixed infections. Compared with single infections, co-infection of Nicotiana benthamiana with Potato virus X (PVX) and Potato virus Y (PVY) resulted in increased systemic symptoms (synergism) that led to necrosis of the newly emerging leaves and death of the plant. A comparative transcriptional analysis was undertaken to identify quantitative and qualitative differences in gene expression during this synergistic infection and correlate these changes with the severe symptoms it caused. Global transcription profiles of doubly infected leaves were compared with those from singly infected leaves using gene ontology enrichment analysis and metabolic pathway annotator software. Functional gene categories altered by the double infection comprise suites of genes regulated coordinately, which are associated with chloroplast functions (downregulated), protein synthesis and degradation (upregulated), carbohydrate metabolism (upregulated), and response to biotic stimulus and stress (upregulated). The expressions of reactive oxygen species-generating enzymes as well as several mitogen-activated protein kinases were also significantly induced. Accordingly, synergistic infection induced a severe oxidative stress in N. benthamiana leaves, as judged by increases in lipid peroxidation and by the generation of superoxide radicals in chloroplasts, which correlated with the misregulation of antioxidative genes in microarray data. Interestingly, expression of genes encoding oxylipin biosynthesis was uniquely upregulated by the synergistic infection. Virus-induced gene silencing of alpha-dioxygenase1 delayed cell death during PVX-PVY infection.

The multiplicity of infection of a plant virus varies during colonization of its eukaryotic host.

The multiplicity of infection (MOI), i.e., the number of virus genomes that infect a cell, is a key parameter in virus evolution, as it determines processes such as genetic exchange among genomes, selection intensity on viral genes, epistatic interactions, and the evolution of multipartite viruses. In fact, the MOI level is equivalent to the virus ploidy during genome expression. Nevertheless, there are few experimental estimates of MOI, particularly for viruses with eukaryotic hosts. Here we estimate the MOI of Tobacco mosaic virus (TMV) in its systemic host, Nicotiana benthamiana. The progress of infection of two TMV genotypes, differently tagged with the green or red fluorescent proteins GFP and RFP, was monitored by determining the number of leaf cell protoplasts that showed GFP, RFP, or GFP and RFP fluorescence at different times postinoculation. This approach allowed the quantitative analysis of the kinetics of infection and estimation of the generation time and the number of infection cycles required for leaf colonization. MOI levels were estimated from the frequency of cells infected by only TMV-GFP or TMV-RFP. The MOI was high, but it changed during the infection process, decreasing from an initial level of about 6 to a final one of 1 to 2, with most infection cycles occurring at the higher MOI levels. The decreasing MOI can be explained by mechanisms limiting superinfection and/or by genotype competition within double-infected cells, which was shown to occur in coinfected tobacco protoplasts. To our knowledge, this is the first estimate of MOI during virus colonization of a eukaryotic host.

A Single Amino Acid Mutation in the Plum pox virus Helper Component-Proteinase Gene Abolishes Both Synergistic and RNA Silencing Suppression Activities.

ABSTRACT The effects on symptom expression of single amino acid mutations in the central region of the Plum pox virus (PPV) helper component-proteinase (HC-Pro) gene were analyzed in Nicotiana benthamiana using Potato virus X (PVX) recombinant viruses. PVX recombinant virus expressing the wild-type variant of PPV HC-Pro induced the expected enhancement of PVX pathogenicity, manifested as necrosis and plant death. Recombinant virus expressing a variant of PPV HC-Pro containing a single point mutation ( HCL(134)H) was unable to induce this synergistic phenotype. The RNA silencing suppressor activity of PPV HC-Pro was demonstrated in a transient silencing suppression assay. In contrast, the HCL(134)H mutant showed no such activity. These results indicate that a unique point mutation in PPV HC-Pro impaired its ability to suppress RNA silencing and abolished its capacity to induce synergism, and clearly shows for the first time the link between these two functions in potyvirus HC-Pro. Additionally, we compared the effects on virus accumulation in N. benthamiana plants infected with either the PVX recombinant constructs or with native viruses in double infection experiments. PVX (+) and (-) strand genomic RNA accumulated at similar levels in plants infected with PVX recombinants, leading to an increase in PVX pathology, compared with plants infected with PVX alone. This finding confirms that the enhancement of pathogenicity associated with synergistic interaction is not a consequence of more efficient PVX replication due to RNA silencing suppression by PPV HC-Pro.