Deciphering the transcriptional regulation and response of barley to obligate fungal biotroph invasion

Obligate fungal biotrophs have co-evolved with their plant hosts, a direct result of an intimate interaction that protects the integrity of the plant during pathogenesis, allowing it to obtain essential nutrients. To restrict the establishment of pathogen colonization, plants have evolved complex regulatory mechanisms to control the defense response, the most extreme of which involves Resistance (R) gene-mediated programmed cell death. While it is known that de novo gene expression and subsequent protein synthesis are required for several cell death programs, the primary transcriptional targets of R gene-mediated responses are unknown. Two alternative approaches were used to identify these transcriptional targets. The first approach uses a time-course microarray experiment that contrasts wild-type and loss-of-function mutant alleles of the Mla (powdery mildew) R gene to identify transcripts that distinguish incompatibility from compatibility. Earlier expression and stronger transcriptional responses were observed in compatible plants at 20 hours after inoculation, though this reaction diminished at later time points. In contrast, incompatible interactions exhibited a time-dependent strengthening of the transcriptional response, with increases in both fold change and total number of genes differentially expressed. These results implicate MLA as a repressor of early gene expression response and provides further evidence for a link between basal and R gene-mediated resistance. The second approach uses natural variation present in a doubled-haploid population to identify the regulatory hierarchy of gene expression during the interaction of barley and stem rust. A trans-eQTL hotspot is not associated with the R gene Rpg-TTKSK, but instead an inoculation-dependent expression polymorphism in Adf3 implicates it as a candidate susceptibility gene. In contrast, co-localization of a trans-eQTL hotspot with an enhancer of R gene-mediated resistance to stem rust associates the suppression of gene expression with enhanced resistance. Lastly, Blufensin1 (Bln1) is used as a case study for functional analysis using gene expression, structural features, and phenotype. Although greater expression of Bln1 was previously associated with incompatibility, virus-induced gene silencing and transient overexpression implicates that Bln1 negatively impacts defense. Collectively, these studies suggest that our understanding of gene expression and its phenotypic consequences is more complex than previously thought

Matching safety to access: global actors and pharmacogovernance in Kenya- a case study

Abstract Background The Kenyan government has sought to address inadequacies in its National Pharmaceutical Policy and the Pharmacy and Poisons Board’s (PPB) medicines governance by engaging with global actors (e.g. the World Health Organization). Policy actors have influenced the way pharmacovigilance is defined, how challenges are understood and which norms are requisite to address drug safety issues. In this paper, we investigate the relationship between specific modes of engagement among global (exogenous) and domestic actors at the national and sub-national level to identify the positive or negative effect on pharmacovigilance and pharmacogovernance in Kenya. Pharmacogovernance is defined as the manner in which governing structures; policy instruments; institutional authority (e.g., ability to act, implement and enforce norms, policies and processes) and resources are managed to promote societal interests for patient safety and protection from adverse drug reactions (ADRs). Qualitative research methods that included key informant interviews and document analysis, were employed to investigate the relationship between global actors’ patterns of engagement with national actors and pharmacogovernance in Kenya. Results Global actors’ influence on pharmacogovernance and pharmacovigilance priorities in Kenya (e.g., legislation and adverse drug reaction surveillance) was positively perceived by key informants. We found that global actors’ engagement with state actors produced positive and negative outcomes. Engagement with the PPB and Ministry of Health (MOH) that was characterized as dependent (advocacy, empowerment, delegated) or interdependent (collaborative, cooperative, consultative) was mostly associated with positive outcomes e.g., capacity building; strengthening legislation and stakeholder coordination. Fragmentation (independent engagement) hindered risk communication between public, private, and NGO health programs. Conclusion A framework for assessing pharmacogovernance would support policy makers’ evidence-based decision making regarding investments to strengthen capacity for pharmacovigilance and guide policies regarding the state and exogenous actor relationship pertaining to pharmacogovernance. Ideally, dependency on exogenous actors should be reduced while retaining consultative, collaborative, and cooperative engagement when inter-dependency is appropriate. The use of global actors to address Kenya’s pharmacovigilance inadequacies leaves the country vulnerable to 1) ad hoc drug surveillance; 2) pharmacovigilance fragmentation; 3) shifting priorities; and 4) cross purpose interests

Governance and pharmacovigilance in Brazil: A scoping review

BACKGROUND: This scoping review investigates the relationship between governance, pharmacovigilance, and Agencia Nacional de Vigilancia Sanitaria (ANVISA) in Brazil, which has authority over Brazil's national pharmaceutical policy, drug registration and coordination of the national pharmacovigilance system. The purpose is to investigate opportunities for effective pharmacovigilance. METHODS: Sixty-three terms pertaining to pharmacovigilance in Brazil and ANVISA, global institutions, pharmaceutical industry, and civil society were searched in thirteen relevant databases on November 17-18, 2013. Using a pharmacogovernance framework we analyzed ANVISA's pharmacogovernance: the manner in which governing structures, policy instruments, and institutional authority are managed to promote societal interests for patient safety due to medication use. The integration of transnational policy ideas for regulatory governance into pharmacogovernance in Brazil was also investigated. RESULTS: Brazil's policy, laws, and regulations support ANVISA's authority to ensure access to safe medicines and health products however ANVISA's broad mandate and gaps in pharmacogovernance account for regional disparities in monitoring and assessing drug safety. Gaps in pharmacogovernance include: equity and inclusiveness; stakeholder coordination; effectiveness and efficiency; responsiveness; and intelligence and information. CONCLUSIONS: Pharmacogovernance that addresses 1) regional resource disparities, 2) federal and state lack of coordination of pharmacovigilance regulations, 3) asymmetric representation in the pharmaceutical regulatory agenda and which 4) disaggregates regulatory authority over health and commercial sectors would strengthen pharmacovigilance in Brazil. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40545-016-0053-y) contains supplementary material, which is available to authorized users

Transcript Profiling in Host–Pathogen Interactions

Using genomic technologies, it is now possible to address research hypotheses in the context of entire developmental or biochemical pathways, gene networks, and chromosomal location of relevant genes and their inferred evolutionary history. Through a range of platforms, researchers can survey an entire transcriptome under a variety of experimental and field conditions. Interpretation of such data has led to new insights and revealed previously undescribed phenomena. In the area of plant-pathogen interactions, transcript profiling has provided unparalleled perception into the mechanisms underlying gene-for-gene resistance and basal defense, host vs nonhost resistance, biotrophy vs necrotrophy, and pathogenicity of vascular vs nonvascular pathogens, among many others. In this way, genomic technologies have facilitated a system-wide approach to unifying themes and unique features in the interactions of hosts and pathogens

Identification of a locus conferring dominant susceptibility to Pyrenophora tritici-repentis in barley

The fungus Pyrenophora tritici-repentis (Ptr) causes tan spot, a destructive foliar disease of wheat worldwide. The pathogen produces several necrotrophic effectors, which induce necrosis or chlorosis on susceptible wheat lines. Multiple races of Ptr have been identified, based on their ability to produce one or more of these effectors. Ptr has a wide host range of cereal and non-cereal grasses, but is known to cause damage only on wheat. Previously, we showed that Ptr can interact specifically with cultivated barley (Hordeum vulgare ssp. vulgare), and that the necrotrophic effector Ptr ToxB induces mild chlorosis in a highly selective manner when infiltrated into certain barley genotypes. In the present study, a barley doubled-haploid (DH) population was evaluated for reaction to Ptr race 5, a Ptr ToxB-producer. Then a comprehensive genetic map composed of 381 single nucleotide polymorphism (SNP) markers was used to map the locus conditioning this chlorosis. The F1 seedlings, and 92 DH lines derived from a cross between the resistant Japanese malting barley cultivar Haruna Nijo and the susceptible wild barley (H. vulgare ssp. spontaneum) OUH602 were inoculated with a conidial suspension of Ptr race 5 isolate at the two-leaf stage. The seedlings were monitored daily for symptoms and assessed for chlorosis development on the second leaf, 6 days after inoculation. All tested F1 seedlings exhibited chlorosis symptoms similar to the susceptible parent, and the DH lines segregated 1:1 for susceptible:resistant phenotypes, indicating the involvement of a single locus. Marker-trait linkage analysis based on interval mapping identified a single locus on the distal region of the short arm of chromosome 2H. We designate this locus Susceptibility to P. tritici-repentis1 (Spr1). The region encompassing this locus has 99 high confidence gene models, including membrane receptor-like kinases (RLKs), intracellular nucleotide-binding, leucine-rich repeat receptors (NLRs), and ankyrin-repeat proteins (ANKs). This shows the involvement of a dominant locus conferring susceptibility to Ptr in barley. Further work using high-resolution mapping and transgenic complementation will be required to identify the underlying gene

Differential accumulation of host mRNAs on polyribosomes during obligate pathogen-plant interactions

Plant pathogens elicit dramatic changes in the expression of host genes during both compatible and incompatible interactions. Gene expression profiling studies of plant-pathogen interactions have only considered messenger RNAs (mRNAs) present in total RNA, which contains subpopulations of actively translated mRNAs associated with polyribosomes (polysomes) and non-translated mRNAs that are not associated with polysomes. The goal of this study was to enhance previous gene expression analyses by identifying host mRNAs that become differentially associated with polysomes following pathogen inoculation. Total and polysomal RNA were extracted from barley (Hordeum vulgare) plants at 32 h after inoculation withBlumeria graminis f. sp. hordei, and Arabidopsis thaliana plants at 10 days after inoculation withTurnip mosaic virus. Gene expression profiles were obtained for each pathosystem, which represent diverse plant host-obligate pathogen interactions. Using this approach, host mRNAs were identified that were differentially associated with polysomes in response to pathogen treatment. Approximately 18% and 26% of mRNAs represented by probe sets on the Affymetrix Barley1 and Arabidopsis ATH1 GeneChips, respectively, differentially accumulated in the two populations in one or more combinations of treatment and genotype. Gene ontology analysis of mRNAs sharing the same pattern of accumulation in total and polysomal RNA identified gene sets that contained a significant number of functionally related annotations, suggesting both transcript accumulation and recruitment to polyribosomes are coordinately regulated in these systems

Natural Variation in Brachypodium Links Vernalization and Flowering Time Loci as Major Flowering Determinants

The domestication of plants is underscored by the selection of agriculturally favorable developmental traits, including flowering time, which resulted in the creation of varieties with altered growth habits. Research into the pathways underlying these growth habits in cereals has highlighted the role of three main flowering regulators: VRN1, VRN2, and FT. Previous reverse genetic studies suggested that the roles of VRN1 and FT are conserved in Brachypodium distachyon, yet identified considerable ambiguity surrounding the role of VRN2. To investigate the natural diversity governing flowering time pathways in a non-domesticated grass, the reference B. distachyon accession Bd21 was crossed with the vernalization-dependent accession ABR6. Resequencing of ABR6 allowed the creation of a SNP-based genetic map at the F4 stage of the mapping population. Flowering time was evaluated in F4:5 families in five environmental conditions and three major loci were found to govern flowering time. Interestingly, two of these loci colocalize with the B. distachyon homologs of the major flowering pathway genes VRN2 and FT, whereas no linkage was observed at VRN1. Characterization of these candidates identified sequence and expression variation between the two parental genotypes, which may explain the contrasting growth habits. However, the identification of additional QTLs suggests that greater complexity underlies flowering time in this non-domesticated system. Studying the interaction of these regulators in B. distachyon provides insights into the evolutionary context of flowering time regulation in the Poaeceae, as well as elucidates the way humans have utilized the natural variation present in grasses to create modern temperate cereals

Cloning of the rice Xo1 resistance gene and interaction of the Xo1 protein with the defense-suppressing Xanthomonas effector Tal2h

The Xo1 locus in the heirloom rice variety Carolina Gold Select confers resistance to bacterial leaf streak and bacterial blight, caused by Xanthomonas oryzae pv. oryzicola and X. oryzae pv. oryzae, respectively. Resistance is triggered by pathogen-delivered transcription activator-like effectors (TALEs) independent of their ability to activate transcription and is suppressed by truncated variants called truncTALEs, common among Asian strains. By transformation of the susceptible variety Nipponbare, we show that one of 14 nucleotide-binding, leucine-rich repeat (NLR) protein genes at the locus, with a zinc finger BED domain, is the Xo1 gene. Analyses of published transcriptomes revealed that the Xo1-mediated response is more similar to those mediated by two other NLR resistance genes than it is to the response associated with TALE-specific transcriptional activation of the executor resistance gene Xa23 and that a truncTALE dampens or abolishes activation of defense-associated genes by Xo1. In Nicotiana benthamiana leaves, fluorescently tagged Xo1 protein, like TALEs and truncTALEs, localized to the nucleus. And endogenous Xo1 specifically coimmunoprecipitated from rice leaves with a pathogen-delivered, epitope-tagged truncTALE. These observations suggest that suppression of Xo1-function by truncTALEs occurs through direct or indirect physical interaction. They further suggest that effector coimmunoprecipitation may be effective for identifying or characterizing other resistance genes

The genetic architecture of colonization resistance in Brachypodium distachyon to non-adapted stripe rust (Puccinia striiformis) isolates

Multilayered defense responses ensure that plants are hosts to only a few adapted pathogens in the environment. The host range of a plant pathogen depends on its ability to fully overcome plant defense barriers, with failure at any single step sufficient to prevent life cycle completion of the pathogen. Puccinia striiformis, the causal agent of stripe rust (=yellow rust), is an agronomically important obligate biotrophic fungal pathogen of wheat and barley. It is generally unable to complete its life cycle on the non-adapted wild grass species Brachypodium distachyon, but natural variation exists for the degree of hyphal colonization by Puccinia striiformis. Using three B. distachyon mapping populations, we identified genetic loci conferring colonization resistance to wheat-adapted and barley-adapted isolates of P. striiformis. We observed a genetic architecture composed of two major effect QTLs (Yrr1 and Yrr3) restricting the colonization of P. striiformis. Isolate specificity was observed for Yrr1, whereas Yrr3 was effective against all tested P. striiformis isolates. Plant immune receptors of the nucleotide binding, leucine-rich repeat (NB-LRR) encoding gene family are present at the Yrr3 locus, whereas genes of this family were not identified at the Yrr1 locus. While it has been proposed that resistance to adapted and non-adapted pathogens are inherently different, the observation of (1) a simple genetic architecture of colonization resistance, (2) isolate specificity of major and minor effect QTLs, and (3) NB-LRR encoding genes at the Yrr3 locus suggest that factors associated with resistance to adapted pathogens are also critical for non-adapted pathogens

Young Adults, Technology, and Weight Loss: A Focus Group Study

Overweight and obesity are a major concern in young adults. Technology has been integrated into many weight loss interventions; however little is known about the use of this technology in young adults. The purpose of this study was to explore through focus group sessions the opinions of young adults on the use of technology for weight loss. A total of 17 young adults, between 18 and 25 years of age, participated in three focus group sessions. Major results indicated that young adults have very little knowledge on the use of Smartphone technology for weight loss but would like to use this type of technology to help them lose weight. Results also indicated that young adults struggle to make healthy food choices and have priorities that outweigh exercise and they need support and guidance to make better decisions. In conclusion, young adults would be open to using Smartphone technology for weight loss but also need feedback and guidance to help make healthy decisions