Early development of Moniliophthora perniciosa basidiomata and developmentally regulated genes

<p>Abstract</p> <p>Background</p> <p>The hemibiotrophic fungus <it>Moniliophthora perniciosa </it>is the causal agent of Witches' broom, a disease of <it>Theobroma cacao</it>. The pathogen life cycle ends with the production of basidiocarps in dead tissues of the infected host. This structure generates millions of basidiospores that reinfect young tissues of the same or other plants. A deeper understanding of the mechanisms underlying the sexual phase of this fungus may help develop chemical, biological or genetic strategies to control the disease.</p> <p>Results</p> <p>Mycelium was morphologically analyzed prior to emergence of basidiomata by stereomicroscopy, light microscopy and scanning electron microscopy. The morphological changes in the mycelium before fructification show a pattern similar to other members of the order <it>Agaricales</it>. Changes and appearance of hyphae forming a surface layer by fusion were correlated with primordia emergence. The stages of hyphal nodules, aggregation, initial primordium and differentiated primordium were detected. The morphological analysis also allowed conclusions on morphogenetic aspects. To analyze the genes involved in basidiomata development, the expression of some selected EST genes from a non-normalized cDNA library, representative of the fruiting stage <it>of M. perniciosa</it>, was evaluated. A macroarray analysis was performed with 192 selected clones and hybridized with two distinct RNA pools extracted from mycelium in different phases of basidiomata formation. This analysis showed two groups of up and down-regulated genes in primordial phases of mycelia. Hydrophobin coding, glucose transporter, Rho-GEF, Rheb, extensin precursor and cytochrome p450 monooxygenase genes were grouped among the up-regulated. In the down-regulated group relevant genes clustered coding calmodulin, lanosterol 14 alpha demethylase and PIM1. In addition, 12 genes with more detailed expression profiles were analyzed by RT-qPCR. One aegerolysin gene had a peak of expression in mycelium with primordia and a second in basidiomata, confirming their distinctiveness. The number of transcripts of the gene for plerototolysin B increased in reddish-pink mycelium and indicated an activation of the initial basidiomata production even at this culturing stage. Expression of the glucose transporter gene increased in mycelium after the stress, coinciding with a decrease of adenylate cyclase gene transcription. This indicated that nutrient uptake can be an important signal to trigger fruiting in this fungus.</p> <p>Conclusion</p> <p>The identification of genes with increased expression in this phase of the life cycle of <it>M. perniciosa </it>opens up new possibilities of controlling fungus spread as well as of genetic studies of biological processes that lead to basidiomycete fruiting. This is the first comparative morphologic study of the early development both <it>in vivo </it>and <it>in vitro </it>of <it>M. perniciosa </it>basidiomata and the first description of genes expressed at this stage of the fungal life cycle.</p

Purification, characterization and structural determination of UDP-N-acetylglucosamine pyrophosphorylase produced by Moniliophthora perniciosa

The enzyme UDP-N-acetylglucosamine pyrophosphorylase (PyroMp) from Moniliophthora perniciosa (CCMB 0257), a pathogenic fungal strain and the causative agent of the witches' broom disease in Theobroma cacao, was partially purified by precipitation with ammonium sulfate and gel filtration on Sephacryl S-200. The buffer for enzyme extraction was sodium phosphate, 0.050 mol L-1, pH 7.0, containing 1.0 mol L-1 NaCl. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes (PyroMp I, PyroMp II, PyroMp III and PyroMp IV) were obtained with optimal pH ranging from 6.9-8.4 and optimum temperature ranging from 28 to 68 °C. The 3D structure of pyrophosphorylase of M. perniciosa was determined by comparative modeling. The model obtained showed a good quality, possessing 78.6% of amino acids in energetically allowed regions. The model was then submitted for DM simulation and showed a good geometric quality (91.1% Ramachandran plot). The active site of the enzyme was found to be extremely well conserved. This model will be useful for developing new inhibitors against witches' broom disease.A enzima UDP-N-acetilglicosamina pirofosforilase de Moniliophthora perniciosa (CCMB 0257), o fungo patogênico causador da doença vassoura-de-bruxa do Theobroma cacao, foi parcialmente purificada por precipitação com sulfato de amônio e cromatografia de gel filtração em Sephacryl S-200. O tampão de extração da enzima foi o fosfato de sódio, 0,050 mol L-1, pH 7,0, contendo 1,0 mol L-1 de NaCl. A metodologia de superfície de resposta (MSR) foi usada para a obtenção do pH e temperatura ótima. Os resultados mostraram quatro diferentes isoenzimas (PyroMp I, PyroMp II, PyroMp III e PyroMp IV) que apresentaram pH ótimo na faixa de 6,9-8,4 e temperatura ótima variando entre 28 a 68 °C. A estrutura 3D de pirofosforilase de M. perniciosa foi obtida por modelagem comparativa. O modelo obtido mostrou uma boa qualidade, possuindo 78,6% de aminoácidos nas regiões energeticamente favoráveis. O modelo foi então submetido a simulações de dinâmica molecular (DM). O modelo apresentou uma boa qualidade geométrica após as simulações de DM (91,1% -gráfico de Ramachandran). A procura pelo sítio ativo da enzima mostrou que este é mantido extremamente conservado. Este modelo pode ser útil para desenvolvimento de inibidores contra a doença vassoura de bruxa.FINEPCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)CNPqFAPESBFIOCRUZ - Programa de Pós-Graduação em Biotecnologia UEF

Etiology of bacterial leaf blight of eucalyptus in Brazil

Bacterial leaf blight of eucalyptus is initially characterized by water soaked, angular, amphigenous and interveinal lesions, concentrated along the main vein, at the edges or scattered on the leaf blade. As the disease progresses, the lesions become brown to pale, and when young leaves are infected leaf cut areas at the edges or perforations at the center of the lesions may appear due to abortion of the necrotic area. Eventually, necrosis may be found on petiole and twigs. Leaf fall commonly occurs on highly susceptible genotypes due to the early senescence of diseased leaves. Precise diagnosis is accomplished by bacterial exudation from leaf sections placed in a water drop under light microscope (200 x). Twenty-five bacterial isolates from Amapá (2), Bahia (4), Minas Gerais (2), São Paulo (9), Pará (3), Mato Grosso do Sul (1), and Rio Grande do Sul (4) States, which induced hypersensitive reaction (HR) in non-host plants and were pathogenic to eucalyptus, when inoculated by inoculum injection, were identified by biochemical assays, using carbon sources (MicroLogTM BIOLOG) and sequence analysis (16S rDNA). Ten isolates were identified as Xanthomonas axonopodis, four as X. campestris, four as Pseudomonas syringae, two as P. putida, two as P. cichorii, one as Erwinia sp., and two were similar to bacterial genera of Rhizobiaceae. When spray inoculated on intact plants of eucalyptus, only X. axonopodis, P. cichorii and isolates of the Rhizobiaceae family induced typical symptoms of the disease and were considered pathogenic. In Brazil, X. axonopodis seems to be the most widespread species causing the bacterial leaf blight of Eucalyptus spp.A mancha foliar bacteriana do eucalipto caracteriza-se inicialmente por lesões encharcadas do tipo anasarca, internervurais, angulares e anfígenas, concentradas ao longo da nervura principal, nas margens da folha ou distribuídas aleatoriamente sobre o limbo. Com o progresso da doença, as lesões adquirem aspecto ressecado e coloração marrom a palha, podendo conter orifícios no centro da lesão ou áreas recortadas do limbo em conseqüência do aborto da área necrosada, principalmente em folhas mais jovens. Eventualmente pode haver necrose em pecíolo e ramos. A doença culmina com a desfolha devido à senescência precoce das folhas infectadas. O diagnóstico inequívoco é realizado por meio de exsudação de pus bacteriano a partir de fragmento de folha infectada, sob microscópio óptico de luz (200 x). Vinte e cinco isolados oriundos dos estados do Amapá (2), Bahia (4), Minas Gerais (2), São Paulo (9), Pará (3), Mato Grosso do Sul (1) e Rio Grande do Sul (4) indutores de reação de hipersensibilidade em plantas não-hospedeiras e, patogênicos ao eucalipto em testes de injeção de suspensão bacteriana no mesófilo foliar, foram identificados por meio de testes bioquímicos, utilização de fontes de carbono e seqüenciamento do rDNA16S. Dez foram identificados como Xanthomonas axonopodis, quatro como X. campestris, quatro como Pseudomonas syringae, dois como P. cichorii, dois como P. putida, um como Erwinia sp. e dois foram similares a gêneros da família Rhizobiaceae. Nos testes de inoculação por atomização de suspensão bacteriana, apenas isolados de P. cichorii, X. axonopodis e os similares a membros da família Rhizobiaceae foram patogênicos a eucalipto. X. axonopodis é provavelmente o agente etiológico predominante da mancha foliar bacteriana de Eucalyptus spp., no Brasil

Differential expression of the soybean BiP gene family

The soybean binding protein (BiP) gene family consists of at least four members designated soyBiPA, soyBiPB, soyBiPC and soyBiPD. We have performed immunoblotting of two-dimensional (2D) gels and RT-PCR assays with gene-specific primers to analyze the differential expression of this gene family in various soybean organs. The 2D gel profiles of the BiP forms from different organs were distinct and suggested that the BiP genes are under organ-specific regulation. In fact, while all four BiP transcripts were detected in leaves by gene-specific reverse transcriptase–polymerase chain reaction (RT-PCR) assays, different subsets were detected in the other organs. The soyBiPD was expressed in all organs, whereas the expression of the soyBiPB was restricted to leaves. The soyBiPA transcripts were detected in leaves, roots and seeds and soyBiPC RNA was confined to leaves, seeds and pods. Our data are consistent with organ-specific expression of the soybean BiP gene family

The phosphorylation state and expression of soybean BiP isoforms are differentially regulated following abiotic stresses

The mammalian BiP is regulated by phosphorylation, and it is generally accepted that its unmodified form constitutes the biologically active species. In fact, the glycosylation inhibitor tunicamycin induces dephosphorylation of mammalian BiP. The stress-induced phosphorylation state of plant BiP has not been examined. Here, we demonstrated that soybean BiP exists in interconvertible phosphorylated and nonphosphorylated forms, and the equilibrium can be shift to either direction in response to different stimuli. In contrast to tunicamycin treatment, water stress condition stimulated phosphorylation of BiP species in soybean cultured cells and stressed leaves. Despite their phosphorylation state, we demonstrated that BiP isoforms from water-stressed leaves exhibit protein binding activity, suggesting that plant BiP functional regulation may differ from other eukaryotic BiPs. We also compared the induction of the soybean BiP gene family, which consists of at least four members designated soyBiPA, soyBiPB, soyBiPC, and soyBiPD, by tunicamycin and osmotic stress. Although all soybean BiP genes were induced by tunicamycin, just the soyBiPA RNA was up-regulated by osmotic stress. In addition, these stresses promoted BiP induction with different kinetics and acted synergistically to increase BiP accumulation. These results suggest that the soybean BiP gene family is differentially regulated by abiotic stresses through distinct signaling pathways

PCR amplification and sequence analyses of Reverse Transcriptase-like genes in Crinipellis perniciosa isolates

Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from the gypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.A análise de sequências de transcriptase reversa (RT) é uma etapa importante para descobrir a presença de elementos transponíveis e investigar o seu papel na geração de variabilidade genética em C. perniciosa. Seqüências putativas de TR foram analisadas no genoma do fitopatógeno C. perniciosa, o agente causal da doença vassoura-de-bruxa no cacau. Um fragmento de 394 pb foi amplificado a partir do DNA genômico de diferentes isolados de C. perniciosa, pertencentes aos biótipos C, L e S e a distintas áreas geográficas. A clivagem dos produtos de PCR com diferentes enzimas de restrição e sequenciamento de vários fragmentos de TR indicou a presença de diferentes seqüências mostrando eventos de transição G:C para A:T. A análise por hibridização revelou alto número de sinais sugerindo a presença de cópias de TR com diferentes perfis entre os isolados dos biótipos C, S e L. As comparações de seqüências dos peptídeos preditos indicam uma relação próxima com a proteína TR de retrotransposons-LTR da família gypsy

Comparative Analysis of Expressed Genes from Cacao Meristems Infected by Moniliophthora perniciosa

- Background and Aims Witches' broom disease is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa, and is one of the most important diseases of cacao in the western hemisphere. Because very little is known about the global process of such disease development, expressed sequence tags (ESTs) were used to identify genes expressed during the Theobroma cacao-Moniliophthora perniciosa interaction. - Methods Two cDNA libraries corresponding to the resistant (RT) and susceptible (SP) cacao-M. perniciosa inter-actions were constructed from total RNA, using the DB SMART Creator cDNA library kit (Clontech). Clones were randomly selected, sequenced from the 5' end and analysed using bioinformatics tools including in silico analysis of the differential gene expression. - Key Results A total of 6884 ESTs were generated from the RT and SP cDNA libraries. These ESTs were composed of 2585 singlets and 341 contigs for a total of 2926 non-redundant sequences. The redundancy of the libraries was low and their specificity high when compared with the few other cacao libraries already published. Sequence analysis allowed the assignment of a putative functional category for 54% of sequences, whereas approx. 22% of sequences corresponded to unknown function and approx. 24% of sequences did not show any significant similarity with other proteins present in the database. Despite the similar overall distribution of the sequences in functional categories between the two libraries, qualitative differences were observed. Genes involved during the defence response to pathogen infection or in programmed cell death were identified, such as pathogenesis related-proteins, trypsin inhibitor or oxalate oxidase, and some of them showed an in silico differential expression between the resist-ant and the susceptible interactions. - Conclusions As far as is known this is the first EST resource from the cacao-M. perniciosa interaction and it is believed that it will provide a significant contribution to the understanding of the molecular mechanisms of the resistance and susceptibility of cacao to M. perniciosa, to develop strategies to control witches broom, and as a source of polymorphism for molecular marker development and marker-assisted selection. (Résumé d'auteur