The nuclear immune receptor RPS4 is required for RRS1SLH1-dependent constitutive defense activation in Arabidopsis thaliana

Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific ‘‘avirulent’’ pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NBLRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector

An initial event in insect innate immune response: structural and biological studies of interactions between β-1,3-glucan and the N-terminal domain of β-1,3-glucan recognition protein

In response to invading microorganisms, insect β-1,3-glucan recognition protein (βGRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Here we report on the NMR solution structure of the N-terminal domain of βGRP (N-βGRP) from Indian meal moth (Plodia interpunctella), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. NMR and isothermal calorimetric titrations of N-βGRP with laminarihexaose, a glucose hexamer containing β-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~ 6 kDa) containing β-1,3 and β-1,6 links that activates the proPO pathway, to N-βGRP results in the loss of NMR cross-peaks from the backbone 15N-1H groups of the protein, suggesting the formation of a large complex. Analytical ultra centrifugation (AUC) studies of formation of N-βGRP:laminarin complex show that ligand-binding induces sel-fassociation of the protein:carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (~ 102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to sub-micromolar concentrations. The structural model thus derived from the present studies for N-βGRP:laminarin complex in solution differs from the one in which a single N-βGRP molecule has been proposed to bind to a triple helical form of laminarin on the basis of an X-ray crystallographic structure of N-βGRP:laminarihexaose complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. AUC studies and phenoloxidase activation measurements carried out with the designed mutants of N-βGRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of N-βGRP:laminarin macro complex and that a decreased stability is accompanied by a reduced activation of the proPO pathway. Increased β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of βGRP:β-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the initial signal of pathogen recognition for the activation of the proPO pathway

Crystallization and preliminary X-ray diffraction analyses of the TIR domains of three TIR-NB-LRR proteins that are involved in disease resistance in Arabidopsis thaliana

The Toll/interleukin-1 receptor (TIR) domain is a protein-protein interaction domain that is found in both animal and plant immune receptors. The N-terminal TIR domain from the nucleotide-binding (NB)-leucine-rich repeat (LRR) class of plant disease-resistance (R) proteins has been shown to play an important role in defence signalling. Recently, the crystal structure of the TIR domain from flax R protein L6 was determined and this structure, combined with functional studies, demonstrated that TIR-domain homodimerization is a requirement for function of the R protein L6. To advance the molecular understanding of the function of TIR domains in R-protein signalling, the protein expression, purification, crystallization and X-ray diffraction analyses of the TIR domains of the Arabidopsis thaliana R proteins RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1) and the resistance-like protein SNC1 (suppressor of npr1-1, constitutive 1) are reported here. RPS4 and RRS1 function cooperatively as a dual resistance-protein system that prevents infection by three distinct pathogens. SNC1 is implicated in resistance pathways in Arabidopsis and is believed to be involved in transcriptional regulation through its interaction with the transcriptional corepressor TPR1 (Topless-related 1). The TIR domains of all three proteins have successfully been expressed and purified as soluble proteins in Escherichia coli. Plate-like crystals of the RPS4 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.05 angstrom resolution, had the symmetry of space group P1 and analysis of the Matthews coefficient suggested that there were four molecules per asymmetric unit. Tetragonal crystals of the RRS1 TIR domain were obtained using ammonium sulfate as a precipitant; they diffracted X-rays to 1.75 angstrom resolution, had the symmetry of space group P4(1)2(1)2 or P4(3)2(1)2 and were most likely to contain one molecule per asymmetric unit. Crystals of the SNC1 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.20 angstrom resolution and had the symmetry of space group P4(1)2(1)2 or P4(3)2(1)2, with two molecules predicted per asymmetric unit. These results provide a good foundation to advance the molecular and structural understanding of the function of the TIR domain in plant innate immunity

An RxLR effector from phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus

The plant immune system is activated following the perception of exposed, essential and invariant microbial molecules that are recognised as non-self. A major component of plant immunity is the transcriptional induction of genes involved in a wide array of defence responses. In turn, adapted pathogens deliver effector proteins that act either inside or outside plant cells to manipulate host processes, often through their direct action on plant protein targets. To date, few effectors have been shown to directly manipulate transcriptional regulators of plant defence. Moreover, little is known generally about the modes of action of effectors from filamentous (fungal and oomycete) plant pathogens. We describe an effector, called Pi03192, from the late blight pathogen Phytophthora infestans, which interacts with a pair of host transcription factors at the endoplasmic reticulum (ER) inside plant cells. We show that these transcription factors are released from the ER to enter the nucleus, following pathogen perception, and are important in restricting disease. Pi03192 prevents the plant transcription factors from accumulating in the host nucleus, revealing a novel means of enhancing host susceptibility

Reconceptualizing CSR in the media industry as relational accountability

In this paper, we reconceptualize CSR in the media industries by combining empirical data with theoretical perspectives emerging from the communication studies and business ethics literature. We develop a new conception of what corporate responsibility in media organizations may mean in real terms by bringing Bardoel and d’Haenens’ (European Journal of Communication 19 165–194 2004) discussion of the different dimensions of media accountability into conversation with the empirical results from three international focus group studies, conducted in France, the USA and South Africa. To enable a critical perspective on our findings, we perform a philosophical analysis of its implications for professional, public, market, and political accountability in the media, drawing on the insights of Paul Virilio. We come to the conclusion that though some serious challenges to media accountability exist, the battle for responsible media industries is not lost. In fact, the speed characterizing the contemporary media environment may hold some promise for fostering the kind of relational accountability that could underpin a new understanding of CSR in the media

Agrotóxicos e saúde: realidade e desafios para mudança de práticas na agricultura.

Este artigo apresenta os resultados de uma pesquisa que teve como objetivo principal caracterizar o contexto e as práticas relacionadas ao uso de agrotóxicos entre agricultores residentes na Serrinha do Mendanha, comunidade agrícola situada em Campo Grande, município do Rio de Janeiro. A proposta metodológica teve uma abordagem qualitativa, sendo a principal fonte de dados a aplicação de questionário semiestruturado. Participaram da pesquisa 38 agricultores familiares. O perfil socioeconômico mostra uma realidade de idosos no trabalho agrícola, percentuais expressivos de analfabetismo e baixa renda. O uso de agrotóxicos é uma rotina para essas famílias. Apesar de existir uma naturalização do uso, a maioria dos informantes acredita que agrotóxicos podem afetar sua saúde, apresenta preocupação com o consumidor e gostaria de conhecer formas alternativas de cultivo. Tais achados apontam para a possibilidade de busca de mudança de práticas. Não se trata de repasse de conhecimento, pois este tipo de informação de alguma forma eles já possuem. Trata–se de desenvolver estratégias objetivando a minimização dos riscos ou mesmo o desuso de agrotóxicos na lavoura

Substrate integrated waveguide (SIW) bandpass filter with novel microstrip-CPW-SIW input coupling

A Substrate integrated waveguide bandpass filter is presented with a novel CPW-to-SIW transition at both the input and output ports which also served as the input and output couplings into the filter. The CPW-to-SIW transition structures presented here exploited the step impedance between the 50 ohms input/output feedline and the transition to control the input/output couplings of the filter. The SIW filter is also shown to have very minimum milling or etching requirement which reduces the fabrication error. The proposed SIW filter has been validated experimentally and results presented. The results show that a simulated return loss of 15 dB and an initial measured return loss of 16 dB were achieved. An improved measured return loss of 22 dB was later achieved after some tuining adjustments were performed on the filter input and output couplings. A minimum insertion loss of 1.3 dB was also achieved across the band

Atribuciones causales y nivel educativo familiar en la comprensión del desempeño escolar en alumnos portugueses

Este artigo analisa o contributo específico do nível educativo familiar e das atribuições causais para o bom e fraco rendimento escolar dos alunos na explicação do seu desempenho nas disciplinas de Língua Portuguesa e de Matemática numa amostra de 2.082 alunos do ensino público de 11 escolas de Portugal, incluindo as Regiões Autónomas dos Açores e da Madeira. As atribuições causais foram avaliadas por meio do Questionário das Atribuições Causais para os Resultados Escolares (QARE). O nível educativo familiar foi estimado levando em conta o nível escolar do progenitor com maior habilitação escolar. As classificações obtidas pelos alunos nas disciplinas de língua portuguesa e matemática foram também examinadas. Os resultados apontam para correlações estatisticamente significativas, destacando as atribuições na capacidade para a explicação do rendimento escolar, situação que contrasta com a atribuição dos níveis de rendimento a variáveis externas ao aluno. A análise de regressão permite associar 34,5% da variância no rendimento conjunto em Língua Portuguesa e em Matemática no ensino básico, assim como 21,3% no ensino secundário, às dimensões atribucionais e ao nível educativo familiar. Implicações educacionais são derivadas a partir dos achados.This paper analyzes the contribution of both family educational level and causal attributions in explaining students´ academic performance in Mathematics and Portuguese Language in a sample of 2.082 public school students from mainland Portugal and the Azores and Madeira regions. Causal attributions were assessed by the Causal Attributions Questionnaire for School Results (QARE). The family educational level was estimated taking into account the educational level of the parent with higher academic degree. The academic grades obtained by students in mathematics and portuguese language were also considered. The results show statistically significant correlations between variables. Internal causal attributions such as capacity and study methods were predominant to explain school performance in the sample and stood out in contrast to external attributions explanations. Regression analyses revealed that 34.5% of the variance in the combined achievement on Portuguese Language and Mathematics in primary education and 21.3% in secondary education could be explained by both the attributional dimensions and family education level. Some educational implications are present taking these data.Este artículo analiza la contribución específica del nivel educativo familiar y de las atribuciones causales para el buen y el flaco rendimiento escolar de los alumnos en la explicación de su desempeño en las disciplinas de Lengua Portuguesa y Matemáticas en una muestra de 2.082 alumnos de enseñanza pública de 11 escuelas de Portugal, incluyendo las Regiones Autónomas de Açores y Madeira. Las atribuciones causales fueron evaluadas por medio del “Questionário das Atribuições Causais para os Resultados Escolares” (QARE). El nivel educativo familiar fue estimado teniendo en cuenta el nivel escolar del progenitor con mayor habilitación escolar. Las clasificaciones obtenidas por los alumnos en las disciplinas de lengua portuguesa y matemáticas fueron también examinadas. Los resultados señalaron correlaciones estadísticamente significativas, destacando las atribuciones en la capacidad para la explicación del rendimiento escolar, situación que contrasta con la atribución de los niveles de rendimiento a variables externas al alumno. El análisis de regresión permite asociar 34.5% de la variancia en el rendimiento conjunto de la Lengua Portuguesa y las Matemáticas en la enseñanza básica, así como 21.3% en la enseñanza secundaria, a las dimensiones de atribución y al nivel educativo familiar. Implicaciones educacionales son derivadas a partir de los hallazgos.Editora Universitária São Francisc

Functionally Redundant RXLR Effectors from <em>Phytophthora infestans</em> Act at Different Steps to Suppress Early flg22-Triggered Immunity

Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular mechanisms underlying the manipulation of host MAMP-triggered immunity (MTI) by P. infestans and to understand the basis of host versus non-host resistance in plants towards P. infestans