Imunolocalização de produtos secretados-excretados de Meloidogyne spp. usando anticorpos policlonais e monoclonais

Moléculas expressadas na cutícula dos nematóides parasitas de plantas representam o primeiro contato planta-nematóide e, junto com produtos secretados/excretados (S-E), são, provavelmente, os primeiros sinais percebidos pelo hospedeiro. Essas moléculas, as quais são liberadas dentro do tecido da planta, podem ter papel importante na interação parasita-hospedeiro. A caracterização desses antígenos pode ajudar na identificação de alvos para novas estratégias que interfiram na infecção de plantas por nematóides. Três anticorpos monoclonais e três anticorpos policlonais produzidos para secreções-excreções de Meloidogyne spp. e Heterodera avenae foram usados para examinar a reatividade a juvenis de segundo estádio (J2) e/ou fêmeas de M. incognita e M. arenaria. Os três anticorpos policlonais mostraram reação tanto para M. incognita quanto para M. arenaria. Roth-PC 373 reconheceu fortemente moléculas presentes na cutícula, anfídios e intestino, Roth-PC 389 reconheceu anfidios e metacorpo, enquanto Roth-PC 419 ligou-se a moléculas presentes nas glândulas subventrais. A reatividade dos MAbs foi testada somente com M. Arenaria. Roth-MAb T116C1.1 reagiu intensamente com moléculas presentes nas glândulas anfidial e fasmidial. Roth-MAb T46.2 e T42D.2 marcaram anfídios e moléculas presentes no esôfago (metacorpo) do nematóide, respectivamente.Molecules expressed at the surface cuticle (SC) of plant parasitic nematodes represent the primary plant-nematode interface, and together with secreted-excreted (S-E) products are probably the first signals perceived by the host. These molecules, which are released into plant tissue, probably play important roles in the host-parasite interactions. Characterisation of these antigens will help in the identification of nematode targets useful for novel control strategies, which interfere with the nematode infection of plants. Three monoclonal (MAbs) and three polyclonal (PAbs) antibodies produced to S-E products of Meloidogyne spp. and Heterodera avenae were used to examine their reactivity towards M. incognita and/or M. arenaria second stage juveniles and adult females. The three PAbs showed cross-reactivity with M. incognita and M. arenaria. Antibody Roth-PC 373 strongly recognised molecules present in the SC, amphids and intestine, antibody Roth-PC 389 recognised the nematode amphids and metacorpus, while antibody Roth-PC 419 bound to molecules present in the subventral glands. Reactivity of the MAbs was only tested against M. arenaria. Monoclonal antibody Roth-MAb T116C1.1 showed intense reactivity with molecules present in the amphidial and phasmidial glands. Monoclonal antibodies Roth-MAb T46.2 and T42D.2 labeled the nematode amphids and molecules present in the nematode oesophagus (metacorpus), respectively

Proteomic Analysis and Functional Validation of a Brassica oleracea Endochitinase Involved in Resistance to Xanthomonas campestris

Black rot is a severe disease caused by the bacterium Xanthomonas campestris pv. campestris (Xcc), which can lead to substantial losses in cruciferous vegetable production worldwide. Although the use of resistant cultivars is the main strategy to control this disease, there are limited sources of resistance. In this study, we used the LC-MS/MS technique to analyze young cabbage leaves and chloroplast-enriched samples at 24 h after infection by Xcc, using both susceptible (Veloce) and resistant (Astrus) cultivars. A comparison between susceptible Xcc-inoculated plants and the control condition, as well as between resistant Xcc-inoculated plants with the control was performed and more than 300 differentially abundant proteins were identified in each comparison. The chloroplast enriched samples contributed with the identification of 600 additional protein species in the resistant interaction and 900 in the susceptible one, which were not detected in total leaf sample. We further determined the expression levels for 30 genes encoding the identified differential proteins by qRT-PCR. CHI-B4 like gene, encoding an endochitinase showing a high increased abundance in resistant Xcc-inoculated leaves, was selected for functional validation by overexpression in Arabidopsis thaliana. Compared to the wild type (Col-0), transgenic plants were highly resistant to Xcc indicating that CHI-B4 like gene could be an interesting candidate to be used in genetic breeding programs aiming at black rot resistance

Contrasting Effects of Wild Arachis Dehydrin Under Abiotic and Biotic Stresses

Plant dehydrins (DNHs) belong to the LEA (Late Embryogenesis Abundant) protein family and are involved in responses to multiple abiotic stresses. DHNs are classified into five subclasses according to the organization of three conserved motifs (K-; Y-; and S-segments). In the present study, the DHN protein family was characterized by molecular phylogeny, exon/intron organization, protein structure, and tissue-specificity expression in eight Fabaceae species. We identified 20 DHN genes, encompassing three (YnSKn, SKn, and Kn) subclasses sharing similar gene organization and protein structure. Two additional low conserved DHN Φ-segments specific to the legume SKn-type of proteins were also found. The in silico expression patterns of DHN genes in four legume species (Arachis duranensis, A. ipaënsis, Glycine max, and Medicago truncatula) revealed that their tissue-specific regulation is associated with the presence or absence of the Y-segment. Indeed, DHN genes containing a Y-segment are mainly expressed in seeds, whereas those without the Y-segment are ubiquitously expressed. Further qRT-PCR analysis revealed that, amongst stress responsive dehydrins, a SKn-type DHN gene from A. duranensis (AdDHN1) showed opposite response to biotic and abiotic stress with a positive regulation under water deficit and negative regulation upon nematode infection. Furthermore, transgenic Arabidopsis lines overexpressing (OE) AdDHN1 displayed improved tolerance to multiple abiotic stresses (freezing and drought) but increased susceptibility to the biotrophic root-knot nematode (RKN) Meloidogyne incognita. This contradictory role of AdDHN1 in responses to abiotic and biotic stresses was further investigated by qRT-PCR analysis of transgenic plants using a set of stress-responsive genes involved in the abscisic acid (ABA) and jasmonic acid (JA) signaling pathways and suggested an involvement of DHN overexpression in these stress-signaling pathways

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

The protease inhibitor chagasin of Trypanosoma cruzi adopts an immunoglobulin-type fold and may have arisen by horizontal gene transfer

Abstract Chagasin, a protein from Trypanosoma cruzi, is the first member of a new family of cysteine protease inhibitors. Despite its lack of significant sequence identity with known proteins, convincing structural models, using variable light chain templates, could be constructed on the basis of threading results. Experimental support for the final structure came from inhibition data for overlapping oligopeptides spanning the chagasin sequence. Chagasin therefore exemplifies a new protease inhibitor structural class and a new natural use for an immunoglobulin-like domain. Limited sequence resemblance suggests that chagasin may represent the result of a rare horizontal gene transfer from host to parasite. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved

Vicilins (7S storage globulins) of cowpea (Vigna unguiculata) seeds bind to chitinous structures of the midgut of Callosobruchus maculatus (Coleoptera: Bruchidae) larvae

The presence of chitin in midgut structures of Callosobruchus maculatus larvae was shown by chemical and immunocytochemical methods. Detection by Western blotting of cowpea (Vigna unguiculata) seed vicilins (7S storage proteins) bound to these structures suggested that C. maculatus-susceptible vicilins presented less staining when compared to C. maculatus-resistant vicilins. Storage proteins present in the microvilli in the larval midgut of the bruchid were recognized by immunolabeling of vicilins in the appropriate sections with immunogold conjugates. These labeling sites coincided with the sites labeled by an anti-chitin antibody. These results, taken together with those previously published showing that the lower rates of hydrolysis of variant vicilins from C. maculatus-resistant seeds by the insect s midgut proteinases and those showing that vicilins bind to chitin matrices, may explain the detrimental effects of variant vicilins on the development of C. maculatus larvae

Proteomic Insights of Cowpea Response to Combined Biotic and Abiotic Stresses

The co-occurrence of biotic and abiotic stresses in agricultural areas severely affects crop performance and productivity. Drought is one of the most adverse environmental stresses, and its association with root-knot nematodes further limits the development of several economically important crops, such as cowpea. Plant responses to combined stresses are complex and require novel adaptive mechanisms through the induction of specific biotic and abiotic signaling pathways. Therefore, the present work aimed to identify proteins involved in the resistance of cowpea to nematode and drought stresses individually and combined. We used the genotype CE 31, which is resistant to the root-knot nematode Meloidogyne spp. And tolerant to drought. Three biological replicates of roots and shoots were submitted to protein extraction, and the peptides were evaluated by LC-MS/MS. Shotgun proteomics revealed 2345 proteins, of which 1040 were differentially abundant. Proteins involved in essential biological processes, such as transcriptional regulation, cell signaling, oxidative processes, and photosynthesis, were identified. However, the main defense strategies in cowpea against cross-stress are focused on the regulation of hormonal signaling, the intense production of pathogenesis-related proteins, and the downregulation of photosynthetic activity. These are key processes that can culminate in the adaptation of cowpea challenged by multiple stresses. Furthermore, the candidate proteins identified in this study will strongly contribute to cowpea genetic improvement programs

Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds

Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to a-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian a-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil