Genetic Determinism and Evolutionary Reconstruction of a Host Jump in a Plant Virus.

In spite of their widespread occurrence, only few host jumps by plant viruses have been evidenced and the molecular bases of even fewer have been determined. A combination of three independent approaches, 1) experimental evolution followed by reverse genetics analysis, 2) positive selection analysis, and 3) locus-by-locus analysis of molecular variance (AMOVA) allowed reconstructing the Potato virus Y (PVY; genus Potyvirus, family Potyviridae) jump to pepper (Capsicum annuum), probably from other solanaceous plants. Synthetic chimeras between infectious cDNA clones of two PVY isolates with contrasted levels of adaptation to C. annuum showed that the P3 and, to a lower extent, the CI cistron played important roles in infectivity toward C. annuum. The three analytical approaches pinpointed a single nonsynonymous substitution in the P3 and P3N-PIPO cistrons that evolved several times independently and conferred adaptation to C. annuum. In addition to increasing our knowledge of host jumps in plant viruses, this study illustrates also the efficiency of locus-by-locus AMOVA and combined approaches to identify adaptive mutations in the genome of RNA viruses.

Effects of implementing a clinical pharmacist service in a mixed Norwegian ICU.

OBJECTIVES: An unacceptably high proportion of patients admitted to intensive care units (ICUs) develop drug-related problems (DRPs). DRPs might cause harm and increase costs and length of stay. The implementation of a clinical pharmacist service has been shown to detect a high number of DRPs and contributes effectively to solving these across different healthcare systems. However, this has not been prospectively studied in a mixed tertiary Norwegian ICU. METHODS: During a 12-month period from October 2012, a clinical pharmacist was dedicated to review medications 3 h daily (Monday to Friday). DRPs were reported at the ICU conference and included advice by the pharmacist for each case. All DRPs were categorised and the clinical impact was documented for later analysis. Drug-related questions from the staff were categorised and answered. RESULTS: 363 of 549 patients admitted to the ICU received medication reviews. 641 DRPs were detected in 194 of these patients (mean 1.8 DRPs per patient, range 0-25). Too high a dose, significant drug interactions and unnecessary or inappropriate drugs were among the most frequently detected DRPs. 87% of advice given by the pharmacist was accepted or taken into consideration. Typical questions from the nursing staff were related to drug preparation, generic equivalents and drug administration. Questions from doctors were most frequently related to drug dosage, efficiency and adverse effects. CONCLUSIONS: The addition of a dedicated clinical pharmacist to the ICU team improves the quality and safety of medication in a mixed Norwegian ICU.

Two euAGAMOUS genes control C-function in Medicago truncatula.

C-function MADS-box transcription factors belong to the AGAMOUS (AG) lineage and specify both stamen and carpel identity and floral meristem determinacy. In core eudicots, the AG lineage is further divided into two branches, the euAG and PLE lineages. Functional analyses across flowering plants strongly support the idea that duplicated AG lineage genes have different degrees of subfunctionalization of the C-function. The legume Medicago truncatula contains three C-lineage genes in its genome: two euAG genes (MtAGa and MtAGb) and one PLENA-like gene (MtSHP). This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily. We have studied the respective functions of each euAG genes in M. truncatula employing expression analyses and reverse genetic approaches. Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern. MtAGa and MtAGb are the only genes showing a full C-function activity, concomitant with their ancestral expression profile, early in the floral meristem, and in the third and fourth floral whorls during floral development. In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function. Furthermore, the redundant MtAGa and MtAGb paralogs have been retained which provides the overall dosage required to specify the C-function in M. truncatula.

Narrow bottlenecks affect Pea seedborne mosaic virus populations during vertical seed transmission but not during leaf colonization.

The effective size of populations (Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Populations having high Ne adapt faster, as selection acts more intensely, than populations having low Ne, where random effects of genetic drift dominate. Estimating Ne for various steps of plant virus life cycle has been the focus of several studies in the last decade, but no estimates are available for the vertical transmission of plant viruses, although virus seed transmission is economically significant in at least 18% of plant viruses in at least one plant species. Here we study the co-dynamics of two variants of Pea seedborne mosaic virus (PSbMV) colonizing leaves of pea plants (Pisum sativum L.) during the whole flowering period, and their subsequent transmission to plant progeny through seeds. Whereas classical estimators of Ne could be used for leaf infection at the systemic level, as virus variants were equally competitive, dedicated stochastic models were needed to estimate Ne during vertical transmission. Very little genetic drift was observed during the infection of apical leaves, with Ne values ranging from 59 to 216. In contrast, a very drastic genetic drift was observed during vertical transmission, with an average number of infectious virus particles contributing to the infection of a seedling from an infected mother plant close to one. A simple model of vertical transmission, assuming a cumulative action of virus infectious particles and a virus density threshold required for vertical transmission to occur fitted the experimental data very satisfactorily. This study reveals that vertically-transmitted viruses endure bottlenecks as narrow as those imposed by horizontal transmission. These bottlenecks are likely to slow down virus adaptation and could decrease virus fitness and virulence.

The helper component proteinase cistron of Potato virus Y induces hypersensitivity and resistance in Potato genotypes carrying dominant resistance genes on chromosome IV.

The Nc(tbr) and Ny(tbr) genes in Solanum tuberosum determine hypersensitive reactions, characterized by necrotic reactions and restriction of the virus systemic movement, toward isolates belonging to clade C and clade O of Potato virus Y (PVY), respectively. We describe a new resistance from S. sparsipilum which possesses the same phenotype and specificity as Nc(tbr) and is controlled by a dominant gene designated Nc(spl). Nc(spl) maps on potato chromosome IV close or allelic to Ny(tbr). The helper component proteinase (HC-Pro) cistron of PVY was shown to control necrotic reactions and resistance elicitation in plants carrying Nc(spl), Nc(tbr), and Ny(tbr). However, inductions of necrosis and of resistance to the systemic virus movement in plants carrying Nc(spl) reside in different regions of the HC-Pro cistron. Also, genomic determinants outside the HC-Pro cistron are involved in the systemic movement of PVY after induction of necroses on inoculated leaves of plants carrying Ny(tbr). These results suggest that the Ny(tbr) resistance may have been involved in the recent emergence of PVY isolates with a recombination breakpoint near the junction of HC-Pro and P3 cistrons in potato crops. Therefore, this emergence could constitute one of the rare examples of resistance breakdown by a virus which was caused by recombination instead of by successive accumulation of nucleotide substitutions.

Protocol: using virus-induced gene silencing to study the arbuscular mycorrhizal symbiosis in Pisum sativum.

Virus-induced gene silencing (VIGS) is an alternative reverse genetics tool for silencing of genes in some plants, which are difficult to transform. The pea early-browning virus (PEBV) has been developed as a VIGS vector and used in pea for functional analysis of several genes. However, the available PEBV-VIGS protocols are inadequate for studying genes involved in the symbiosis with arbuscular mycorrhizal fungi (AMF).Here we describe a PEBV-VIGS protocol suitable for reverse genetics studies in pea of genes involved in the symbiosis with AMF and show its effectiveness in silencing genes involved in the early and late stages of AMF symbiosis.

Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat.

BACKGROUND: Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species. RESULTS: Here we demonstrate the successful BSMV-mediated virus induced gene silencing (VIGS) of three different genes in barley roots, i.e. the barley homologues of the IPS1, PHR1, and PHO2 genes known to participate in Pi uptake and reallocation in Arabidopsis. Attempts to silence two other genes, the Pi transporter gene HvPht1;1 and the endo-ß-1,4-glucanase gene HvCel1, in barley roots were unsuccessful, probably due to instability of the plant gene inserts in the viral vector. In B. distachyon leaves, significant silencing of the PHYTOENE DESATURASE (BdPDS) gene was obtained as shown by photobleaching as well as quantitative RT-PCR analysis. On the other hand, only very limited silencing of the oat AsPDS gene was observed in both hexaploid (A. sativa) and diploid (A. strigosa) oat. Finally, two modifications of the BSMV vector are presented, allowing ligation-free cloning of DNA fragments into the BSMV-γ component. CONCLUSIONS: Our results show that BSMV can be used as a vector for gene silencing in barley roots and in B. distachyon leaves and possibly roots, opening up possibilities for using VIGS to study cereal root biology and to exploit the wealth of genome information in the new cereal model plant B. distachyon. On the other hand, the silencing induced by BSMV in oat seemed too weak to be of practical use. The new BSMV vectors modified for ligation-free cloning will allow rapid insertion of plant gene fragments for future experiments.

Potyviral resistance derived from cultivars of Phaseolus vulgaris carrying bc-3 is associated with the homozygotic presence of a mutated eIF4E allele.

Eukaryotic translation initiation factors (eIFs) play a central role in potyviral infection. Accordingly, mutations in the gene encoding eIF4E have been identified as a source of recessive resistance in several plant species. In common bean, Phaseolus vulgaris, four recessive genes, bc-1, bc-2, bc-3 and bc-u, have been proposed to control resistance to the potyviruses Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus. In order to identify molecular entities for these genes, we cloned and sequenced P. vulgaris homologues of genes encoding the eIF proteins eIF4E, eIF(iso)4E and nCBP. Bean genotypes reported to carry bc-3 resistance were found specifically to carry non-silent mutations at codons 53, 65, 76 and 111 in eIF4E. This set of mutations closely resembled a pattern of eIF4E mutations determining potyvirus resistance in other plant species. The segregation of BCMV resistance and eIF4E genotype was subsequently analysed in an F(2) population derived from the P. vulgaris all-susceptible genotype and a genotype carrying bc-3. F(2) plants homozygous for the eIF4E mutant allele were found to display at least the same level of resistance to BCMV as the parental resistant genotype. At 6 weeks after inoculation, all F(2) plants found to be BCMV negative by enzyme-linked immunosorbent assay were found to be homozygous for the mutant eIF4E allele. In F(3) plants homozygous for the mutated allele, virus resistance was subsequently found to be stably maintained. In conclusion, allelic eIF4E appears to be associated with a major component of potyvirus resistance present in bc-3 genotypes of bean.

The role of the coat protein region in symptom formation on Physalis floridana varies between PVY strains.

The Potato virus Y (PVY) cDNA full-length clone created by Jakab et al. [Jakab, G., Droz, E., Brigneti, G., Baulcombe, D., Malnoë, P., 1997. Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y. J. Gen. Virol. 78, 3141-3145] was stabilized by inserting three introns into putatively toxic genes. Using this clone, hybrid viruses were constructed by in vitro recombination. The PVY-N/NTN and PVY-N/O chimeras carried the 3' end of NIb, the whole CP and 3'UTR region of PVY(NTN) and PVY(O), respectively, in a PVY(N) genetic background. The clones proved to be stable after several passages by re-sequencing the exchanged region. Both hybrid viruses showed reduced infectivity in particle bombardment experiments, but they were suitable for further mechanical plant inoculation. In five of the six host plant species, inoculated with the two chimeras and three parental strains, the chimeras produced similar symptoms to those of PVY(N). By contrast, Physalis floridana reacted with different pattern of symptoms. In this species, the symptoms caused by the N/O hybrid were similar to those of the 3'NIb-CP-donating PVY(O) strain, and not to those of the background (PVY(N)). The results suggest that symptom determinants may be different even between strains of the same virus species in a particular host.

The potyvirus recessive resistance gene, sbm1, identifies a novel role for translation initiation factor eIF4E in cell-to-cell trafficking.

From the characterization of the recessive resistance gene, sbm1, in pea we have identified the eukaryotic translation initiation factor, eIF4E, as a susceptibility factor required for infection with the Potyvirus, Pea seed-borne mosaic virus. A functional analysis of the mode of action of the product of the dominant allele revealed a novel function for eIF4E in its support for virus movement from cell-to-cell, in addition to its probable support for viral RNA translation, and hence replication. Different resistance specificities in two independent pea lines were explained by different mutations in eIF4E. On the modelled structure of eIF4E the coding changes were in both cases lying in and around the structural pocket involved in binding the 5'-m7G cap of eukaryotic mRNAs. Protein expression and cap-binding analysis showed that eIF4E encoded by a resistant plant could not bind to m7G-Sepharose, a result which may point to functional redundancy between eIF4E and the paralogous eIF(iso)4E in resistant peas. These observations, together with related findings for other potyvirus recessive resistances, provide a more complete picture of the potyvirus life cycle.

Mutations in potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum.

The recessive resistance genes pot-1 and pvr2 in Lycopersicon hirsutum and Capsicum annuum, respectively, control Potato virus Y (PVY) accumulation in the inoculated leaves. Infectious cDNA molecules from two PVY isolates differing in their virulence toward these resistances were obtained using two different strategies. Chimeras constructed with these cDNA clones showed that a single nucleotide change corresponding to an amino acid substitution (Arg119His) in the central part of the viral protein genome-linked (VPg) was involved in virulence toward the pot-1 resistance. On the other hand, 15 nucleotide changes corresponding to five putative amino acid differences in the same region of the VPg affected virulence toward the pvr2(1) and pvr2(2) resistances. Substitution models identified six and five codons within the central and C terminal parts of the VPg for PVY and for the related potyvirus Potato virus A, respectively, which undergo positive selection. This suggests that the role of the VPg-encoding region is determined by the protein and not by the viral RNA apart from its protein-encoding capacity.

Evidence for diversifying selection in Potato virus Y and in the coat protein of other potyviruses.

The modes of evolution of the proteins of Potato virus Y were investigated with a maximum-likelihood method based on estimation of the ratio between non-synonymous and synonymous substitution rates. Evidence for diversifying selection was obtained for the 6K2 protein (one amino acid position) and coat protein (24 amino acid positions). Amino acid sites in the coat proteins of other potyviruses (Bean yellow mosaic virus, Yam mosaic virus) were also found to be under diversifying selection. Most of the sites belonged to the N-terminal domain, which is exposed to the exterior of the virion particle. Several of these amino acid positions in the coat proteins were shared between some of these three potyviruses. Identification of diversifying selection events in these different proteins will help to unravel their biological functions and is essential to an understanding of the evolutionary constraints exerted on the potyvirus genome. The hypothesis of a link between evolutionary constraints due to host plants and occurrence of diversifying selection is discussed.