Witches' broom disease of cocoa : genome organization, genetic variability and anlysis of the identity and expression of pathogenicity genes of the fungal pathogen Crinipellis perniciosa

Orientador: Gonçalo Amarante Guimarães PereiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A Vassoura-de-Bruxa do cacaueiro constitui um patosistema complexo e até o momento não foi estabelecido um plano de controle efetivo na região produtora da Bahia. O objetivo principal deste trabalho foi aprimorar os conhecimentos sobre a interação C. perniciosa-Cacaueiro com informações referentes à: 1) tamanho e organização do genoma através da obtenção do cariótipo molecular do fungo; 2) variabilidade genética deste fitopatógeno a nível cromossômico na região produtora da Bahia; e 3) expressão gênica do fungo em presença de extratos do hospedeiro. O cariótipo molecular foi estabelecido por eletroforese em gel de campo pulsado. Esta mesma metodologia foi utilizada em combinação com análise tipo microsatélites para um estudo de variabilidade a nível cromossômico de 38 isolados provenientes de três biótipos e diferentes regiões geográficas. Para análise da expressão gênica foram seqüenciadas quatro bibliotecas de cDNA do fungo sob diferentes condições de cultivo e em diferentes estágios do desenvolvimento, incluindo uma biblioteca subtrativa obtida por SSH. Como resultados, apresentamos o cariótipo molecular de C. perniciosa e a existência de polimorfismos cromossômicos. A análise dos 38 isolados permitiu observar que o cariótipo do biótipo-C é estável ao longo do tempo e sua variabilidade na região da Bahia é baixa, mostrando assim a fragilidade do programa de melhoramento genético do cacaueiro na Bahia, onde os clones resistentes selecionados no campo foram desafiados contra apenas dois genótipos do patógeno. Com relação à análise da expressão do fungo, obtivemos 1427 unigenes e sua análise por similaridade com as bases de dados permitiu a identificação de diversos mecanismos pelos quais o fungo poderia estar manipulando o metabolismo da planta para seu benefício e em detrimento da produção de cacau. Assim, consideramos que a partir deste trabalho foi possível obter uma maior compreensão dos mecanismos utilizados por C. perniciosa no desenvolvimento da Vassoura-de-Bruxa em cacaueiro e estudos mais aprofundados, baseados nos resultados aqui obtidos, deverão auxiliar no desenvolvimento de estratégias de controle para esta importante doençaAbstract: Witches' broom disease of cocoa is a complex pathosystem that has evaded an efficient control program in the cacao-producing region of Bahia, Brazil. The main goal of this work was to acquire a better understanding of the C. perniciosa-Cacao interaction by providing new data regarding: 1) the size and organization of the fungal genome through molecular karyotyping; 2) the genetic variability of this phytopathogen at the chromosomal level in the cacao-producing region of Bahia; and 3) the gene expression of the fungus in the presence of host extracts. The molecular karyotype was obtained through pulsed-field gel electrophoresis. This same technique was applied in combination with microsatellite PCR analysis of 38 isolates from different biotypes and geographic regions in order to study the chromosomal-level genetic variability of this pathogen. For the gene expression analysis, four cDNA libraries of the fungus grown under different culture conditions and developmental stages, including a subtractive library using SSH, were sequenced and searched for similarities in the public databases. As results, we present the molecular karyotype of C. perniciosa and the existence of chromosome-length polymorphism. The analysis of the 38 isolates showed that the karyotype of the C-biotype is very stable in time and that the variability of the pathogen in Bahia is very low, thus indicating the fragility of the current cacao-breeding program in Bahia, where the resistant cacao clones selected in the field were challenged against only two different genotypes of the pathogen. With regards to the gene expression analysis, we obtained 1427 unigenes and the similarity searches allowed the identification of various mechanisms through which the fungus might be manipulating the metabolism of the plant for its own benefit and in detriment of cocoa production. Therefore, we consider that this work allowed us to acquire a better understanding of the molecular mechanisms used by C. perniciosa during witches' broom development in cacao and more detailed studies, based on the results presented here, might aid in the development of novel control strategies for this important diseaseDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthoraperniciosa, the causal agent of witches' broom disease of Theobroma cacao

This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts

The genome sequence of Propionibacterium acidipropionici provides insights into its biotechnological and industrial potential

Abstract Background Synthetic biology allows the development of new biochemical pathways for the production of chemicals from renewable sources. One major challenge is the identification of suitable microorganisms to hold these pathways with sufficient robustness and high yield. In this work we analyzed the genome of the propionic acid producer Actinobacteria Propionibacterium acidipropionici (ATCC 4875). Results The assembled P. acidipropionici genome has 3,656,170 base pairs (bp) with 68.8% G + C content and a low-copy plasmid of 6,868 bp. We identified 3,336 protein coding genes, approximately 1000 more than P. freudenreichii and P. acnes, with an increase in the number of genes putatively involved in maintenance of genome integrity, as well as the presence of an invertase and genes putatively involved in carbon catabolite repression. In addition, we made an experimental confirmation of the ability of P. acidipropionici to fix CO2, but no phosphoenolpyruvate carboxylase coding gene was found in the genome. Instead, we identified the pyruvate carboxylase gene and confirmed the presence of the corresponding enzyme in proteome analysis as a potential candidate for this activity. Similarly, the phosphate acetyltransferase and acetate kinase genes, which are considered responsible for acetate formation, were not present in the genome. In P. acidipropionici, a similar function seems to be performed by an ADP forming acetate-CoA ligase gene and its corresponding enzyme was confirmed in the proteome analysis. Conclusions Our data shows that P. acidipropionici has several of the desired features that are required to become a platform for the production of chemical commodities: multiple pathways for efficient feedstock utilization, ability to fix CO2, robustness, and efficient production of propionic acid, a potential precursor for valuable 3-carbon compounds

A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom Disease of cacao

<p>Abstract</p> <p>Background</p> <p>The basidiomycete fungus <it>Moniliophthora perniciosa </it>is the causal agent of Witches' Broom Disease (WBD) in cacao (<it>Theobroma cacao</it>). It is a hemibiotrophic pathogen that colonizes the apoplast of cacao's meristematic tissues as a biotrophic pathogen, switching to a saprotrophic lifestyle during later stages of infection. <it>M. perniciosa</it>, together with the related species <it>M. roreri</it>, are pathogens of aerial parts of the plant, an uncommon characteristic in the order Agaricales. A genome survey (1.9× coverage) of <it>M. perniciosa </it>was analyzed to evaluate the overall gene content of this phytopathogen.</p> <p>Results</p> <p>Genes encoding proteins involved in retrotransposition, reactive oxygen species (ROS) resistance, drug efflux transport and cell wall degradation were identified. The great number of genes encoding cytochrome P450 monooxygenases (1.15% of gene models) indicates that <it>M. perniciosa </it>has a great potential for detoxification, production of toxins and hormones; which may confer a high adaptive ability to the fungus. We have also discovered new genes encoding putative secreted polypeptides rich in cysteine, as well as genes related to methylotrophy and plant hormone biosynthesis (gibberellin and auxin). Analysis of gene families indicated that <it>M. perniciosa </it>have similar amounts of carboxylesterases and repertoires of plant cell wall degrading enzymes as other hemibiotrophic fungi. In addition, an approach for normalization of gene family data using incomplete genome data was developed and applied in <it>M. perniciosa </it>genome survey.</p> <p>Conclusion</p> <p>This genome survey gives an overview of the <it>M. perniciosa </it>genome, and reveals that a significant portion is involved in stress adaptation and plant necrosis, two necessary characteristics for a hemibiotrophic fungus to fulfill its infection cycle. Our analysis provides new evidence revealing potential adaptive traits that may play major roles in the mechanisms of pathogenicity in the <it>M. perniciosa</it>/cacao pathosystem.</p

Electrophoretic Karyotype Analysis Of Crinipellis Perniciosa, The Causal Agent Of Witches' Broom Disease Of Theobroma Cacao.

Pulse-field gel electrophoresis (PFGE) was used to determine the genome size and characterize karyotypic differences in isolates of the cacao biotype of Crinipellis perniciosa (C-biotype). The karyotype analysis of four isolates from Brazil revealed that this biotype could be divided into two genotypes: one presenting six chromosomal bands and the other presenting eight. The size of the chromosomes ranged from 2.7 to 5.3 Mb. The different genotypes correlate with telomere-based PCR analysis. The isolates with six chromosomal bands had two that appeared to be doublets, as shown by densitometric analysis, indicating that the haploid chromosome number for this biotype is eight. The size of the haploid genomes was estimated at approximately 30 Mb by both PFGE and Feulgen-image analysis. DNA hybridization revealed that the rDNA sequences are clustered on a single chromosome and these sequences were located on different chromosomes in an isolate dependent manner. This is the first report of genome size and chromosomal polymorphism for the C-biotype of C. perniciosa.107452-

In Vitro Production Of Biotrophic-like Cultures Of Crinipellis Perniciosa, The Causal Agent Of Witches' Broom Disease Of Theobroma Cacao.

Witches' broom disease (WBD) of cacao, caused by the hemibiotrophic fungus, Crinipellis perniciosa, exhibits a succession of symptoms that are caused by the biotrophic phase of the fungus. However, the study of this biotrophic phase is limited by its exclusive growth inside the plant or in the presence of callus. Here we report for the first time a method for the growth and maintenance of the biotrophic-like phase of C. perniciosa on a defined medium with metabolites found in the diseased tissues. Our results suggest that glycerol is a key carbon source for this interaction. This is a crucial achievement toward understanding the biology of this fungus during the infectious phase of WBD.52191-

A Method For The Production Of Olefins, An Olefin, A Polyolefin, And Use Of The Polyolefin

PROCESSO PARA PRODUÇÃO DE OLEFINAS, POLIOLEFINA, E, USO DA POLIOLEFINA. A presente invenção refere-se a um processo de produção de uma olefina a partir de pelo menos uma matéria prima natural renovável. Mais especificamente, a presente invenção trata de um processo onde eteno ou propeno é obtida com alto rendimento e alta produtividade através da reação de eletrodescarboxilação anódica de ácidos carboxílicos, respectivamente ácido propiemico ou ácido butírico, produzidos a partir de fermentação, preferencialmente de açúcares. O processo para gerar a olefina é simples, de baixo custo e apresenta baixa emissão de gases de origem fóssil causadores de efeito estufa.BRPI0904979 (A2)C07C11/04C07C11/06C08F10/02C08F10/06BR2009PI04979C07C11/04C07C11/06C08F10/02C08F10/0