Genetic diversity of Xanthomonas axonopodis pv. passiflorae isolates based on rep-PCR and AFLP markers and the construction of specific primers for diagnosis

O patógeno Xanthomonas axonopodis pv. passiflorae causa a mancha oleosa ou bacteriose do maracujazeiro, uma doença que acarreta prejuízos à cultura em decorrência da baixa produção de frutos, podendo causar a morte das plantas. Uma coleção de 87 isolados deste patovar, oriundos de 22 cidades dos Estados de São Paulo, Minas Gerais e Paraná e do Distrito Federal, foi usada para estudar a diversidade genética por rep-PCR e AFLP. Nove isolados de outros patovares foram incluídos nas análises genéticas. A técnica rep-PCR revelou pouca diversidade entre os isolados do patovar passiflorae, mas diferenciou, claramente, os diferentes patovares. Todavia, a técnica AFLP revelou considerável diversidade genética entre os isolados do patovar passiflorae. A análise molecular da variância mostrou que a maior parte da diversidade (49,4%) se encontra entre as cidades de coleta. O agrupamento gerado com base nos coeficientes de similaridade e os resultados do teste de atribuição pelo programa Structure revelaram clusters genotípicos homogêneos. Isto evidencia que a variação está mais associada à geografia, ou seja, às cidades de coleta, e que o fluxo desses isolados é pequeno. Cinco conjuntos de primers foram desenhados para a detecção do patógeno em plantas. Desses, um conjunto de primers foi desenhado a partir da seqüência intergênica 16S-23S rRNA e se mostrou específico para o patovar passiflorae. Nenhum amplicon foi detectado nos patovares controles. O restante dos primers foi desenhado a partir do seqüenciamento de locos de AFLP, monomórficos para o patovar passiflorae e ausentes nos demais patovares. Estes primers não foram totalmente específicos; no entanto, todos podem ser recomendados para o diagnóstico da mancha oleosa, uma vez que não há registros de outras Xanthomonas em pomares de maracujá.The pathogen Xanthomonas axonopodis pv. passiflorae is responsible for the bacterial leaf spot of passion fruits, a disease that provokes commercial losses due to low levels of fruit production and even plant death. A group of 87 isolates of this pathovar, collected from 22 localities of São Paulo, Minas Gerais and Paraná States as in the Federal District was used to evaluate the genetic diversity based on rep-PCR and AFLP. Isolates from other nine pathovars were included in the genetic analyses. Low level of genetic diversity was revealed by the rep- PCR technique, which clearly distinguished the different pathovars. However, considerable diversity between isolates of the pathovar passiflorae was revealed by the AFLP technique. The analysis of molecular variance showed that differences between localities contributed to most part of the variance (49.4%). Groups generated based on similarity coefficients as well as results produced by the software Structure assigning isolates to groups, revealed homogeneous genotypic clusters. This confirms that variance is associated with geographic origin e.g. sampling localities, and that flow of isolates is restricted among localities. Five primer sets were designed for pathogen detection in plants; a primer set was designed for PCR amplification of the intergenic sequence 16-23S rRNA, which was shown to be specific to the pathovar passiflorae. No amplicons were detected in the controls. The remaining primers were designed after sequencing AFLP bands that were monomorphic within the pathovar passiflorae but absent in the other pathovars. These primers were not absolutely specific but all could be recommended for diagnosis of leaf spot as there is no report on the occurrence of other Xanthomonas species in passion fruit orchards

Identification of differentially expressed genes during the yellow passion fruit- Xanthomonas axonopodis interaction

O Brasil é o maior produtor mundial de maracujá azedo (Passiflora edulis f. flavicarpa) sendo esta a espécie de maior expressão comercial dentre as passifloras cultivadas. A bacteriose do maracujazeiro, causada por Xanthomonas axonopodis pv. passiflorae (Xap), é uma das doenças mais severas da cultura, acarretando grandes prejuízos aos produtores. Atualmente, é incipiente o conhecimento sobre a interação maracujá azedo-Xap. Diante disso, a identificação e a caracterização dos genes envolvidos no processo de defesa são passos importantes para dar suporte ao desenvolvimento de variedades resistentes. Assim, o objetivo deste trabalho foi identificar e caracterizar genes de maracujá azedo diferencialmente expressos durante a resposta de defesa à Xap, bem como mensurar a sua expressão. Para isso, foram construídas duas bibliotecas subtrativas de cDNA (forward e reverse) usando o método SSH a partir de transcritos de folhas, que foram inoculadas com o patógeno ou solução salina (controle). Após o sequenciamento dos clones, o processamento e a montagem das sequências, as unisequências foram anotadas através da Plataforma PLAZA e do programa computacional Blast2GO. Genes envolvidos em diversos processos biológicos foram selecionados para a validação das bibliotecas por PCR quantitativo. Usando a Plataforma PLAZA, 78 % (764) das unisequências mostraram similaridade com proteínas de Arabidopsis thaliana, enquanto 87 % (866) delas apresentaram similaridade com proteínas putativas de diversas espécies vegetais, quando se utilizou Blast2GO. Na biblioteca forward, foram identificadas 73 proteínas relacionadas à resposta de defesa, dentre as quais estão proteínas envolvidas na sinalização intracelular, na ativação da transcrição e regulação da expressão de genes de defesa, bem como proteínas de defesa, de resistência e relacionadas à patogênese (PRs). Dentre os 22 transcritos validados, 95 % foram diferencialmente expressos em pelo menos um dos três períodos avaliados; os genes mais expressos em resposta à infecção pelo patógeno são os que codificam as enzimas lipoxigenase, (+)-neomentol desidrogenase e quitinase, as quais participam diretamente nas respostas de defesa vegetal. Dos genes cuja expressão foi mais reprimida, dois codificam proteínas relacionadas à fotossíntese e dois codificam proteínas envolvidas na detoxificação da amônia e do H2O2. Nossos resultados sugerem que a planta utiliza um arsenal de transcritos para responder à infecção; entretanto, este arsenal não é eficiente para impedir a ação do patógeno e, consequentemente, o desenvolvimento da bacteriose nas condições estudadas. Nosso estudo é inédito e gerou informações sobre a reprogramação transcricional durante a interação maracujá azedo-Xap, o que constitui um importante passo para o melhor entendimento sobre este patossistema.Brazil is the main producer of yellow passion fruit (Passiflora edulis f. flavicarpa) worldwide, which is the most widely commercialized crop among the cultivated passifloras. The bacterial leaf spot induced by Xanthomonas axonopodis pv. passiflorae (Xap) is one of the most severe diseases of the crop, causing great losses to producers. Currently, we understand very little about the yellow passion fruit-Xap interaction. Therefore, the identification and characterization of genes involved in the defense process are important steps to support the development of resistant varieties. Thus, the objective of this study was identify and characterize differentially expressed genes during the defense response to Xap, as well as to measure their expression. For that, we constructed two subtractive cDNA libraries (the forward and the reverse) by performing the SSH method from leaf transcripts, which were inoculated with the pathogen or saline solution (control). After sequencing the clones and sequence data processing, sequences were assembled into unique sequences, which were annotated using the PLAZA Platform and the computational program Blast2GO. Genes involved in several biological processes were selected to validate the libraries by quantitative PCR. When PLAZA was used for sequence similarity searches, 78 % (764) of the yellow passion fruit unique sequences showed similarity to proteins of Arabidopsis thaliana; when Blast2GO was used, 87 % (866) of the unique sequences showed similarities to putative proteins of several plant species. For the forward library, 73 proteins related to defense response were identified, such as those involved in intracellular signaling, transcription activation and regulation of defense gene expression, as well as defense and resistance proteins, and pathogenesis-related proteins (PRs). Of the 22 validated transcripts, 95 % were differentially expressed during at least one of the three periods evaluated; the genes up-regulated in response to the pathogen infection were those that code for the enzymes lipoxygenase, (+)-neomenthol dehydrogenase and chitinase, which participate directly in plant-defense responses. Out of down-regulated genes, two code for photosynthesis-related proteins, and two for ammonia and H2O2 detoxification. Our results suggest the plant uses an arsenal of transcripts to respond to infection; however, this arsenal is not effective to prevent pathogen action and consequently the occurrence of bacterial leaf spot under the evaluated conditions. The present study is the first to produce information on the transcriptional reprogramming during the passion fruit-Xap interaction, which represents an important step for a better understanding of this pathosystem

Report on the development of putative functional SSR and SNP markers in passion fruits

Abstract Background Passionflowers Passiflora edulis and Passiflora alata are diploid, outcrossing and understudied fruit bearing species. In Brazil, passion fruit cultivation began relatively recently and has earned the country an outstanding position as the world’s top producer of passion fruit. The fruit’s main economic value lies in the production of juice, an essential exotic ingredient in juice blends. Currently, crop improvement strategies, including those for underexploited tropical species, tend to incorporate molecular genetic approaches. In this study, we examined a set of P. edulis transcripts expressed in response to infection by Xanthomonas axonopodis, (the passion fruit’s main bacterial pathogen that attacks the vines), aiming at the development of putative functional markers, i.e. SSRs (simple sequence repeats) and SNPs (single nucleotide polymorphisms). Results A total of 210 microsatellites were found in 998 sequences, and trinucleotide repeats were found to be the most frequent (31.4%). Of the sequences selected for designing primers, 80.9% could be used to develop SSR markers, and 60.6% SNP markers for P. alata. SNPs were all biallelic and found within 15 gene fragments of P. alata. Overall, gene fragments generated 10,003 bp. SNP frequency was estimated as one SNP every 294 bp. Polymorphism rates revealed by SSR and SNP loci were 29.4 and 53.6%, respectively. Conclusions Passiflora edulis transcripts were useful for the development of putative functional markers for P. alata, suggesting a certain level of sequence conservation between these cultivated species. The markers developed herein could be used for genetic mapping purposes and also in diversity studies

Microsatellite markers: what they mean and why they are so useful

Abstract Microsatellites or Single Sequence Repeats (SSRs) are extensively employed in plant genetics studies, using both low and high throughput genotyping approaches. Motivated by the importance of these sequences over the last decades this review aims to address some theoretical aspects of SSRs, including definition, characterization and biological function. The methodologies for the development of SSR loci, genotyping and their applications as molecular markers are also reviewed. Finally, two data surveys are presented. The first was conducted using the main database of Web of Science, prospecting for articles published over the period from 2010 to 2015, resulting in approximately 930 records. The second survey was focused on papers that aimed at SSR marker development, published in the American Journal of Botany's Primer Notes and Protocols in Plant Sciences (over 2013 up to 2015), resulting in a total of 87 publications. This scenario confirms the current relevance of SSRs and indicates their continuous utilization in plant science

Host Transcriptional Profiling At Early And Later Stages Of The Compatible Interaction Between Phaseolus Vulgaris And Meloidogyne Incognita.

The root-knot nematode (Meloidogyne incognita) is one of most devastating pathogens that attack the common bean crop. Although there is evidence that some cultivars have race-specific resistance against M. incognita, these resistance sources have not proved effective, and nematodes are able to circumvent the host's defense system. We constructed RNA-seq based libraries and used a high-throughput sequencing platform to analyze the plant responses to M. incognita. Assessments were performed at 4 and 10 days after inoculation corresponding to the stages of nematode penetration and giant cell development, respectively. Large-scale transcript mapping to the common bean reference genome (G19833) resulted in the identification of 27,195 unigenes. Of these, 797 host genes were found to be differentially expressed. The functional annotation results confirm the complex interplay between abiotic and biotic stress signaling pathways. High expression levels of the wounding-responsive genes were observed over the interaction. At early response, an overexpression of the N gene, a TIR-NBS-LRR resistance gene, was understood as a host attempt to overcome the pathogen attack. However, the repression of heat shock proteins resulted in a lack of reactive oxygen species accumulation and absence of a hypersensitive response. Furthermore, the host basal response was broken by the repression of the ethylene/jasmonate pathway later in the response, resulting in a continuous compatible process with consequent plant susceptibility.106PHYTO07150160

A gene-rich fraction analysis of the Passiflora edulis genome reveals highly conserved microsyntenic regions with two related Malpighiales species

Abstract Passiflora edulis is the most widely cultivated species of passionflowers, cropped mainly for industrialized juice production and fresh fruit consumption. Despite its commercial importance, little is known about the genome structure of P. edulis. To fill in this gap in our knowledge, a genomic library was built, and now completely sequenced over 100 large-inserts. Sequencing data were assembled from long sequence reads, and structural sequence annotation resulted in the prediction of about 1,900 genes, providing data for subsequent functional analysis. The richness of repetitive elements was also evaluated. Microsyntenic regions of P. edulis common to Populus trichocarpa and Manihot esculenta, two related Malpighiales species with available fully sequenced genomes were examined. Overall, gene order was well conserved, with some disruptions of collinearity identified as rearrangements, such as inversion and translocation events. The microsynteny level observed between the P. edulis sequences and the compared genomes is surprising, given the long divergence time that separates them from the common ancestor. P. edulis gene-rich segments are more compact than those of the other two species, even though its genome is much larger. This study provides a first accurate gene set for P. edulis, opening the way for new studies on the evolutionary issues in Malpighiales genomes

Early transplantation of bone marrow mononuclear cells promotes neuroprotection and modulation of inflammation after status epilepticus in mice by paracrine mechanisms.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2015-05-19T17:24:00Z No. of bitstreams: 1 Leal MMT Early....pdf: 781614 bytes, checksum: d9ce1f5869c3b92b18bd5f5dc7f0b8e9 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-05-19T17:50:20Z (GMT) No. of bitstreams: 1 Leal MMT Early....pdf: 781614 bytes, checksum: d9ce1f5869c3b92b18bd5f5dc7f0b8e9 (MD5)Made available in DSpace on 2015-05-19T17:50:20Z (GMT). No. of bitstreams: 1 Leal MMT Early....pdf: 781614 bytes, checksum: d9ce1f5869c3b92b18bd5f5dc7f0b8e9 (MD5) Previous issue date: 2014Hospital São Rafael. Salvador, BA, BrasilHospital São Rafael. Salvador, BA, BrasilHospital São Rafael. Salvador, BA, BrasilHospital São Rafael. Salvador, BA, BrasilHospital São Rafael. Salvador, BA, BrasilHospital São Rafael. Salvador, BA, BrasilHospital São Rafael. Salvador, BA, BrasilHospital São Rafael. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilHospital São Rafael. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilStatus epilepticus (SE) is a severe clinical manifestation of epilepsy associated with intense neuronal loss and inflammation, two key factors involved in the pathophysiology of temporal lobe epilepsy. Bone marrow mononuclear cells (BMMC) attenuated the consequences of pilocarpine-induced SE, including neuronal loss, in addition to frequency and duration of seizures. Here we investigated the effects of BMMC transplanted early after the onset of SE in mice, as well as the involvement of soluble factors produced by BMMC in the effects of the cell therapy. Mice were injected with pilocarpine for SE induction and randomized into three groups: transplanted intravenously with 1 × 10(7) BMMC isolated from GFP transgenic mice, injected with BMMC lysate, and saline-treated controls. Cell tracking, neuronal counting in hippocampal subfields and cytokine analysis in the serum and brain were performed. BMMC were found in the brain 4 h following transplantation and their numbers progressively decreased until 24 h following transplantation. A reduction in hippocampal neuronal loss after SE was found in mice treated with live BMMC and BMMC lysate when compared to saline-treated, SE-induced mice. Moreover, the expression of inflammatory cytokines IL-1ß, TNF-α, IL-6 was decreased after injection of live BMMC and to a lesser extent, of BMMC lysate, when compared to SE-induced controls. In contrast, IL-10 expression was increased. Analysis of markers for microglia activation demonstrated a reduction of the expression of genes related to type 1-activation. BMMC transplantation promotes neuroprotection and mediates anti-inflammatory effects following SE in mice, possibly through the secretion of soluble factors