Novel molecular components involved in callose-mediated Arabidopsis defense against Salmonella enterica and Escherichia coli O157:H7.

BACKGROUND: Food contamination with Salmonella enterica and enterohemorrhagic Escherichia coli is among the leading causes of foodborne illnesses worldwide and crop plants are associated with > 50% of the disease outbreaks. However, the mechanisms underlying the interaction of these human pathogens with plants remain elusive. In this study, we have explored plant resistance mechanisms against these enterobacteria and the plant pathogen Pseudomonas syringae pv. tomato (Pst) DC3118, as an opportunity to improve food safety. RESULTS: We found that S. enterica serovar Typhimurium (STm) transcriptionally modulates stress responses in Arabidopsis leaves, including induction of two hallmark processes of plant defense: ROS burst and cell wall modifications. Analyses of plants with a mutation in the potentially STm-induced gene EXO70H4 revealed that its encoded protein is required for stomatal defense against STm and E. coli O157:H7, but not against Pst DC3118. In the apoplast however, EXO70H4 is required for defense against STm and Pst DC3118, but not against E. coli O157:H7. Moreover, EXO70H4 is required for callose deposition, but had no function in ROS burst, triggered by all three bacteria. The salicylic acid (SA) signaling and biosynthesis proteins NPR1 and ICS1, respectively, were involved in stomatal and apoplastic defense, as well as callose deposition, against human and plant pathogens. CONCLUSIONS: The results show that EXO70H4 is involved in stomatal and apoplastic defenses in Arabidopsis and suggest that EXO70H4-mediated defense play a distinct role in guard cells and leaf mesophyll cells in a bacteria-dependent manner. Nonetheless, EXO70H4 contributes to callose deposition in response to both human and plant pathogens. NPR1 and ICS1, two proteins involved in the SA signaling pathway, are important to inhibit leaf internalization and apoplastic persistence of enterobacteria and proliferation of phytopathogens. These findings highlight the existence of unique and shared plant genetic components to fight off diverse bacterial pathogens providing specific targets for the prevention of foodborne diseases.

A Comprehensive Arabidopsis Yeast Two-Hybrid Library for Protein-Protein Interaction Studies: A Resource to the Plant Research Community.

Yeast-two-hybrid (Y2H) cDNA library screening is a valuable tool to uncover protein-protein interactions and represents a widely used method to investigate protein function. However, low transcript representation in cDNA libraries limits the depth of the screening. We have developed a Y2H library with cDNA made from Arabidopsis leaves exposed to several stressors as well as untreated leaves. The library was built using pooled mRNA extracted from plants challenged with plant and human bacterial pathogens, the flg22 elicitor, the phytotoxin coronatine, and several hormones associated with environmental stress responses. The purpose of such a library is to maximize the discovery of protein-protein interactions that occur under optimum conditions as well as during biotic and abiotic stresses.

Selection and validation of reference genes for functional studies in the Calliphoridae family.

The genera Cochliomyia and Chrysomya contain both obligate and saprophagous flies, which allows the comparison of different feeding habits between closely related species. Among the different strategies for comparing these habits is the use of qPCR to investigate the expression levels of candidate genes involved in feeding behavior. To ensure an accurate measure of the levels of gene expression, it is necessary to normalize the amount of the target gene with the amount of a reference gene having a stable expression across the compared species. Since there is no universal gene that can be used as a reference in functional studies, candidate genes for qPCR data normalization were selected and validated in three Calliphoridae (Diptera) species, Cochliomyia hominivorax Coquerel, Cochliomyia macellaria Fabricius, and Chrysomya albiceps Wiedemann . The expression stability of six genes ( Actin, Gapdh, Rp49, Rps17, α -tubulin, and GstD1) was evaluated among species within the same life stage and between life stages within each species. The expression levels of Actin, Gapdh, and Rp49 were the most stable among the selected genes. These genes can be used as reliable reference genes for functional studies in Calliphoridae using similar experimental settings.

Involvement of microRNA-related regulatory pathways in the glucose-mediated control of Arabidopsis early seedling development.

In plants, sugars such as glucose act as signalling molecules that promote changes in gene expression programmes that impact on growth and development. Recent evidence has revealed the potential importance of controlling mRNA decay in some aspects of glucose-mediated regulatory responses suggesting a role of microRNAs (miRNAs) in these responses. In order to get a better understanding of glucose-mediated development modulation involving miRNA-related regulatory pathways, early seedling development of mutants impaired in miRNA biogenesis (hyl1-2 and dcl1-11) and miRNA activity (ago1-25) was evaluated. All mutants exhibited a glucose hyposensitive phenotype from germination up to seedling establishment, indicating that miRNA regulatory pathways are involved in the glucose-mediated delay of early seedling development. The expression profile of 200 miRNA primary transcripts (pri-miRs) was evaluated by large-scale quantitative real-time PCR profiling, which revealed that 38 pri-miRs were regulated by glucose. For several of them, the corresponding mature miRNAs are known to participate directly or indirectly in plant development, and their accumulation was shown to be co-regulated with the pri-miR by glucose. Furthermore, the expression of several miRNA target genes was found to be deregulated in response to glucose in the miRNA machinery mutants ago1-25, dcl1-11, and hyl1-2. Also, in these mutants, glucose promoted misexpression of genes for the three abscisic acid signalling elements ABI3, ABI4, and ABI5. Thus, miRNA regulatory pathways play a role in the adjustments of growth and development triggered by glucose signalling.

The Arabidopsis bZIP gene AtbZIP63 is a sensitive integrator of transient abscisic acid and glucose signals.

Glucose modulates plant metabolism, growth, and development. In Arabidopsis (Arabidopsis thaliana), Hexokinase1 (HXK1) is a glucose sensor that may trigger abscisic acid (ABA) synthesis and sensitivity to mediate glucose-induced inhibition of seedling development. Here, we show that the intensity of short-term responses to glucose can vary with ABA activity. We report that the transient (2 h/4 h) repression by 2% glucose of AtbZIP63, a gene encoding a basic-leucine zipper (bZIP) transcription factor partially involved in the Snf1-related kinase KIN10-induced responses to energy limitation, is independent of HXK1 and is not mediated by changes in ABA levels. However, high-concentration (6%) glucose-mediated repression appears to be modulated by ABA, since full repression of AtbZIP63 requires a functional ABA biosynthetic pathway. Furthermore, the combination of glucose and ABA was able to trigger a synergistic repression of AtbZIP63 and its homologue AtbZIP3, revealing a shared regulatory feature consisting of the modulation of glucose sensitivity by ABA. The synergistic regulation of AtbZIP63 was not reproduced by an AtbZIP63 promoter-5'-untranslated region::ß-glucuronidase fusion, thus suggesting possible posttranscriptional control. A transcriptional inhibition assay with cordycepin provided further evidence for the regulation of mRNA decay in response to glucose plus ABA. Overall, these results indicate that AtbZIP63 is an important node of the glucose-ABA interaction network. The mechanisms by which AtbZIP63 may participate in the fine-tuning of ABA-mediated abiotic stress responses according to sugar availability (i.e., energy status) are discussed.

Resultados de uma nova técnica para cirurgia do arco aórtico com uso de perfusão encefálica anterógrada bilateral pelo isolamento do tronco braquiocefálico e artéria carótida esquerda / Results of a new technique for aortic arch surgery using bilateral antegrade encephalic perfusion by isolation of brachiocephalic trunk left carotid artery

Fundamentos: perfusão encefálica anterógrada bilateral(PEAB) é o melhor método de neuroproteção nas cirurgias que envolvem o arco aórtico. Objetivos: Analisar os resultados cirúrgicos e em médio prazo da cirurgia no arco aórtico,utilizando a técnica descrita por Carreira et al. Métodos: Avaliaram-seos dados de 16 pacientes operados entre junho2005 e novembro2007 pela seguinte técnica:uso de prótese de dacron de 10mm ou 12mm no tronco braquiocefálico(TBC) para canulação arterial.Clampeamento do TBC permite PCA unilateral durante o isolamento.PEAB começa após remoção do clamp(500-1000ml/min)(50-70mmHg)(20-25ºC).Ao final, a prótese do isolamento é então incorporada à prótese aórtica. Resultados:8 pacientes masculinos,média de idade 59,5+-14,9 anos, 75% hipertensos. Onze operados devido à dissecção aguda e 5 por aneurisma.Médias de tempo de circulação extracorpórea,anóxia,PEA bilateral,PEA unilareral e temperatura na hipotermia foram, respectivamente:175,3+-36,0min, 135,2+-30,7min, 22,4+-3,2ºC,55,5+-20,3min e 9,9+-3,4min.Houve preservação da válvula aórtica em 93,8%; 5 pacientes receberam implante de endoprótese aórtica.Complicações foram: fibrilação atrial-56,3%;insuficiência renal aguda-31,3%;pneumonia e delirium-18,8%mediastinite e paresia temporária-6,3%;e diálise-12,5%.Médias da drenagem, tempo de entubação, permanência hospitalar e CTI foram,respectivamente:1207,2+-600,3ml,17,7+-20,1 hs,14,3+-10,6 diase 6,5+-7,1 dias. Mortalidade em 3 pacientes (18,8%), todos com dissecção aguda. Não aconteceram eventos neurológicos com média de 11,9+-10,2, meses de seguimento. Os parâmetros não apresentaram associção com mortalidade p>0,05.Conclusão: A técnica do isolameto é segura e apresenta bons resultados neurológicos imediatos e tardios.