Apoplastic and intracellular plant sugars regulate developmental transitions in witches' broom disease of cacao.

Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation.

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

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 potential role for an extracellular methanol oxidase secreted by Moniliophthora perniciosa in Witches' broom disease in cacao.

The hemibiotrophic basidiomycete fungus Moniliophthora perniciosa, the causal agent of Witches' broom disease (WBD) in cacao, is able to grow on methanol as the sole carbon source. In plants, one of the main sources of methanol is the pectin present in the structure of cell walls. Pectin is composed of highly methylesterified chains of galacturonic acid. The hydrolysis between the methyl radicals and galacturonic acid in esterified pectin, mediated by a pectin methylesterase (PME), releases methanol, which may be decomposed by a methanol oxidase (MOX). The analysis of the M. pernciosa genome revealed putative mox and pme genes. Real-time quantitative RT-PCR performed with RNA from mycelia grown in the presence of methanol or pectin as the sole carbon source and with RNA from infected cacao seedlings in different stages of the progression of WBD indicate that the two genes are coregulated, suggesting that the fungus may be metabolizing the methanol released from pectin. Moreover, immunolocalization of homogalacturonan, the main pectic domain that constitutes the primary cell wall matrix, shows a reduction in the level of pectin methyl esterification in infected cacao seedlings. Although MOX has been classically classified as a peroxisomal enzyme, M. perniciosa presents an extracellular methanol oxidase. Its activity was detected in the fungus culture supernatants, and mass spectrometry analysis indicated the presence of this enzyme in the fungus secretome. Because M. pernciosa possesses all genes classically related to methanol metabolism, we propose a peroxisome-independent model for the utilization of methanol by this fungus, which begins with the extracellular oxidation of methanol derived from the demethylation of pectin and finishes in the cytosol.

The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development.

The tropical pathogen Moniliophthora perniciosa causes witches' broom disease in cacao. As a hemibiotrophic fungus, it initially colonizes the living host tissues (biotrophic phase), and later grows over the dead plant (necrotrophic phase). Little is known about the mechanisms that promote these distinct fungal phases or mediate the transition between them. An alternative oxidase gene (Mp-aox) was identified in the M. perniciosa genome and its expression was analyzed througout the fungal life cycle. In addition, the effects of inhibitors of the cytochrome-dependent respiratory chain (CRC) and alternative oxidase (AOX) were evaluated on the in vitro development of M. perniciosa. Larger numbers of Mp-aox transcripts were observed in the biotrophic hyphae, which accordingly showed elevated sensitivity to AOX inhibitors. More importantly, the inhibition of CRC prevented the transition from the biotrophic to the necrotrophic phase, and the combined use of a CRC and AOX inhibitor completely halted fungal growth. On the basis of these results, a novel mechanism is presented in which AOX plays a role in the biotrophic development of M. perniciosa and regulates the transition to its necrotrophic stage. Strikingly, this model correlates well with the infection strategy of animal pathogens, particularly Trypanosoma brucei, which uses AOX as a strategy for pathogenicity.

Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug.

Heterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA(3)/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2'-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters' edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation.

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 glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthora perniciosa, 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.

Moniliophthora perniciosa, the causal agent of witches' broom disease of cacao: what's new from this old foe?

Moniliophthora perniciosa (=Crinipellis perniciosa) causes one of the three main fungal diseases of Theobroma cacao (cacao), the source of chocolate. This pathogen causes Witches' broom disease (WBD) and has brought about severe economic losses in all of the cacao-growing regions to which it has spread with yield reductions that range from 50 to 90%. Cacao production in South America reflects the severity of this pathogen, as the yields in most of the infected regions have not returned to pre-outbreak levels, even with the introduction of resistant varieties. In this review we give a brief historical account and summarize the current state of knowledge focusing on developments in the areas of systematics, fungal physiology, biochemistry, genomics and gene expression in an attempt to highlight this disease. Moniliophthora perniciosa is a hemibiotrophic fungus with two distinct growth phases. The ability to culture a biotrophic-like phase in vitro along with new findings derived from the nearly complete genome and expression studies clearly show that these different fungal growth phases function under distinct metabolic parameters. These new findings have greatly improved our understanding of this fungal/host interaction and we may be at the crossroads of understanding how hemibiotrophic fungal plant pathogens cause disease in other crops. HISTORICAL SUMMARY OF WBD: The first WDB symptoms appear to have been described in the diaries of Alexandre Rodrigues Ferreira (described as lagartão; meaning big lizard) from his observations of cacao trees in 1785 and 1787 in Amazonia, which is consistent with the generally accepted idea that M. perniciosa, like its main host T. cacao, evolved in this region. The disease subsequently arrived in Surinam in 1895. WBD moved rapidly, spreading to Guyana in 1906, Ecuador in 1918, Trinidad in 1928, Colombia in 1929 and Grenada in 1948. In each case, cacao production was catastrophically affected with yield reductions of 50-90%. After the arrival of M. perniciosa in Bahia in 1989, Brazil went from being the world's 3rd largest producer of cacao (347 000 tonnes in 1988-1990; c. 15% of the total world production at that time) to a net importer (141 000 tonnes in 1998-2000). Fortunately for chocolate lovers, other regions of the world such as West Africa and South East Asia have not yet been affected by this disease and have expanded production to meet growing world demand (predicted to reach 3 700 000 tonnes by 2010). CLASSIFICATION: Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora: super-kingdom Eukaryota; kingdom Fungi; phylum Basidiomycota; subphylum Agaricomycotina; class Agaricomycetes; subclass Agaricomycetidae; order Agaricales; family Marasmiaceae; genus Moniliophthora. USEFUL WEBSITES: http://www.lge.ibi.unicamp.br/vassoura/, http://nt.ars-grin.gov/taxadescriptions/keys/TrichodermaIndex.cfm, http://www.worldcocoafoundation.org/info-center/research-updates.asp, http://www.ars.usda.gov/ba/psi/spcl.

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

BACKGROUND: The basidiomycete fungus Moniliophthora perniciosa is the causal agent of Witches' Broom Disease (WBD) in cacao (Theobroma cacao). 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. M. perniciosa, together with the related species M. roreri, are pathogens of aerial parts of the plant, an uncommon characteristic in the order Agaricales. A genome survey (1.9x coverage) of M. perniciosa was analyzed to evaluate the overall gene content of this phytopathogen. RESULTS: 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 M. perniciosa 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 M. perniciosa 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 M. perniciosa genome survey. CONCLUSION: This genome survey gives an overview of the M. perniciosa 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 M. perniciosa/cacao pathosystem.

The mitochondrial genome of the phytopathogenic basidiomycete Moniliophthora perniciosa is 109 kb in size and contains a stable integrated plasmid.

We present here the sequence of the mitochondrial genome of the basidiomycete phytopathogenic hemibiotrophic fungus Moniliophthora perniciosa, causal agent of the Witches' Broom Disease in Theobroma cacao. The DNA is a circular molecule of 109,103 base pairs, with 31.9% GC, and is the largest sequenced so far. This size is due essentially to the presence of numerous non-conserved hypothetical ORFs. It contains the 14 genes coding for proteins involved in the oxidative phosphorylation, the two rRNA genes, one ORF coding for a ribosomal protein (rps3), and a set of 26 tRNA genes that recognize codons for all amino acids. Seven homing endonucleases are located inside introns. Except atp8, all conserved known genes are in the same orientation. Phylogenetic analysis based on the cox genes agrees with the commonly accepted fungal taxonomy. An uncommon feature of this mitochondrial genome is the presence of a region that contains a set of four, relatively small, nested, inverted repeats enclosing two genes coding for polymerases with an invertron-type structure and three conserved hypothetical genes interpreted as the stable integration of a mitochondrial linear plasmid. The integration of this plasmid seems to be a recent evolutionary event that could have implications in fungal biology. This sequence is available under GenBank accession number AY376688.

Differential gene expression between the biotrophic-like and saprotrophic mycelia of the witches' broom pathogen Moniliophthora perniciosa.

Moniliophthora perniciosa is a hemibiotrophic fungus that causes witches' broom disease (WBD) in cacao. Marked dimorphism characterizes this fungus, showing a monokaryotic or biotrophic phase that causes disease symptoms and a later dikaryotic or saprotrophic phase. A combined strategy of DNA microarray, expressed sequence tag, and real-time reverse-transcriptase polymerase chain reaction analyses was employed to analyze differences between these two fungal stages in vitro. In all, 1,131 putative genes were hybridized with cDNA from different phases, resulting in 189 differentially expressed genes, and 4,595 reads were clusterized, producing 1,534 unigenes. The analysis of these genes, which represent approximately 21% of the total genes, indicates that the biotrophic-like phase undergoes carbon and nitrogen catabolite repression that correlates to the expression of phytopathogenicity genes. Moreover, downregulation of mitochondrial oxidative phosphorylation and the presence of a putative ngr1 of Saccharomyces cerevisiae could help explain its lower growth rate. In contrast, the saprotrophic mycelium expresses genes related to the metabolism of hexoses, ammonia, and oxidative phosphorylation, which could explain its faster growth. Antifungal toxins were upregulated and could prevent the colonization by competing fungi. This work significantly contributes to our understanding of the molecular mechanisms of WBD and, to our knowledge, is the first to analyze differential gene expression of the different phases of a hemibiotrophic fungus.

Characterization of necrosis and ethylene-inducing proteins (NEP) in the basidiomycete Moniliophthora perniciosa, the causal agent of witches' broom in Theobroma cacao.

The hemibiotrophic basidiomycete Moniliophthora perniciosa causes witches' broom disease of Theobroma cacao. Analysis of the M. perniciosa draft genome led to the identification of three putative genes encoding necrosis and ethylene-inducing proteins (MpNEPs), which are apparently located on the same chromosome. MpNEP1 and 2 have highly similar sequences and are able to induce necrosis and ethylene emission in tobacco and cacao leaves. MpNEP1 is expressed in both biotrophic and saprotrophic mycelia, the protein behaves as an oligomer in solution and is very sensitive to temperature. MpNEP2 is expressed mainly in biotrophic mycelia, is present as a monomer in solution at low concentrations (<40 microM) and is able to recover necrosis activity after boiling. These differences indicate that similar NEPs can have distinct physical characteristics and suggest possible complementary roles during the disease development for both proteins. This is the first report of NEP1-like proteins in a basidiomycete.

Genetic variability and chromosome-length polymorphisms of the witches' broom pathogen Crinipellis perniciosa from various plant hosts in South America.

Crinipellis perniciosa has been classified into at least four known biotypes associated with members of unrelated plant families. In this study, genetic variability is shown for 27 C (Cacao), 4 S (Solanum), and 7 L biotype (Liana) isolates of C. perniciosa collected from different regions of Brazil and South America. The objective was to investigate the genetic variability of the pathogen in the cacao-producing region of Bahia, Brazil, and elsewhere, through microsatellite analysis, and attempt to identify possible correlations between host specificity and electrophoretic karyotypes. The PCR-banding patterns were found to vary both within and between the different biotypes, and a correlation was established between the PCR-banding patterns and the chromosomal-banding patterns of each isolate. Microsatellite and chromosomal patterns among all of the L and S biotype isolates were distinctly different from the C biotypes analysed. A higher degree of genetic and chromosomal variability was found among C biotype isolates from the Amazon in comparison with C biotype isolates from Bahia, which seems to be comprised of only two main genotypes. This finding has important implications to the current cacao-breeding programme in Brazil.

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.

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.