BACKGROUND
moniliophthora perniciosa, previously known as crinipellis perniciosa  stahel, is a hemibiotrophic basidiomycete  fungus that causes witches' broom disease  in cacao   <cit> .
wbd and frosty pod rot , caused by moniliophthora roreri, are the most devastating diseases of cacao in the americas  <cit> .
cacao production in southeastern bahia, the main production area in brazil, was severely affected by the introduction of wbd at the end of 1980's  <cit> .
this disease damaged bahian agribusiness, caused major social problems and has contributed to the degradation of the atlantic rainforest .
this is because cacao producing areas, typically, maintained old-growth native tree species as shade for the cacao plantations, which were converted to pasture  <cit> .
the symptoms displayed by cacao plants during wbd parallel the hemibiotrophic development of m. perniciosa  <cit> .
briefly, the disease begins when fungal spores germinate and infect meristematic tissues, developing into monokaryotic biotrophic hyphae without clamp connections that slowly occupy the intercellular space.
this stage of wbd is characterized by the emergence of hypertrophic and hyperplasic anomalous branches, and the formation of parthenocarpic fruits.
infected branches, known as "green brooms", grow without apical dominance, with a phototropic orientation and displaying epinastic leaves  <cit> .
after two to three months the infected tissue turns necrotic  and the hyphae become saprotrophic with two nuclei per cell and clamp connections, invading the inter and intracellular spaces of the infected tissue  <cit> .
this fungal species exhibits primary homothalism as its reproductive strategy  <cit> ; thus, the change from the monokaryotic to the dikaryotic mycelium occurs without the prerequisite of mating between compatible individuals.
after alternate wet and dry periods, basidiomes produced by the saprotrophic hyphae release basidiospores that are spread by wind or rain, thus completing the m. perniciosa life cycle  <cit> .
the witches' broom genome project  involving several brazilian laboratories was initiated to increase the knowledge of this disease.
the genome size, chromosomal polymorphism, genetic variability and the m. perniciosa mitochondrial genome have already been described  <cit> .
additionally, a biochemical study revealed the metabolic modifications that occur in cacao plantlets during wbd development  <cit> .
technical improvements have been achieved in the manipulation of cacao  <cit>  and m. perniciosa  <cit> .
one of the main bottlenecks in m. perniciosa research was solved with the development of the in vitro production of biotrophic-like cultures  <cit> .
necrotic inducing proteins expressed by m. perniciosa have been characterized  <cit> , and the analysis of est libraries and dna microarrays have identified differentially expressed genes during its development  and for the interaction of the fungus with cacao  <cit> .
despite the substantial progression in understanding wbd, many questions remain unsolved, mainly those concerning the mechanisms controlling processes such as:  the fungal switch from biotrophism to saprotrophism;  the drastic phenotypic alteration of cacao during disease development and  the death of infected tissues.
in vitro cultures in our laboratory demonstrate that this fungus has a great capacity to adapt to media containing different sources of carbon and nitrogen and it is able to grow in extremely nutrient-poor media.
these results together with the fact that hemibiotrophic fungi, such as m. perniciosa, display complex lifestyles  <cit>  suggest that this fungus has a significant genomic and transcriptomic plasticity that contributes to the successful pathogenic mechanisms expressed during its life cycle.
in a recent review, meinhardt et al., give a historical account and summarize the current state of knowledge about wbd  <cit> .
around two thirds of the known basidiomycota species are included in the order agaricales  <cit> .
in addition to being an interesting group for carrying out developmental genetic studies concerning fungal development and reproduction, the agaricales contain many important industrial species , and species with unique lifestyles: saprophytes , symbionts , leaf-litters decomposers  and root pathogens .
interestingly, m. perniciosa and m. roreri are members of a group of agaricales species that are able to infect aerial parts of plants, an uncommon characteristic among this basidiomycota order.
genome sequencing and analysis is an important strategy to obtain comprehensive information concerning the metabolism and development of organisms.
the initial objective of the m. perniciosa genome project was to obtain a genome survey sequence using a whole shotgun strategy to provide genomic information for the wbd research community.
recently, the bioinformatics and genomic communities have been debating about the benefits and costs of finishing a complete genome as compared to applying a genome survey strategy  <cit> .
it has been proposed that a two-fold genome sequence coverage is sufficient to support a high percentage of est alignments and exon similarity matches  <cit> .
additionally, gene models resulting from a genome survey can be predicted accurately by the comparison with complete genomes of phylogenetically related organisms  <cit> .
this strategy has been used for genomic surveys of dog   <cit>  and of the wine spoilage yeast dekkera bruxellensis   <cit> .
recently, the genomes of the basidiomycete species laccaria bicolor, cryptococcus neoformans, ustilago maydis, coprinopsis cinerea  and phanerochaete chrysosporium have been determined and many additional basidiomycota genome projects are ongoing or about to be released ; , which could provide a rich database for additional basidiomycota genomic survey evaluations.
with the existence of several complete basidiomycota genomes, and both m. perniciosa ests libraries, and a  <dig>  × genome coverage, we decided to conduct a genome survey of m. perniciosa to obtain further information about this important phytopathogen.
this report describes a survey of the genome sequences of m. perniciosa, with specific emphasis on the genes potentially involved in disease development such as a cytochrome p <dig> monooxygenases, transposable elements, putative plant defense elicitors, pathogenicity effectors, cell wall degrading enzymes, proteins related to methylotrophy and the biosynthesis of plant hormones by the fungus.
the identification of such genes in the m. perniciosa genomic data lead us to hypothesize a connection between molecular processes involved in the growth phases of the fungus and the progression of wbd.
RESULTS
genome assembly and estimation of genome size
a diagram flow describing the bioinformatic procedures applied in the m. perniciosa genome survey are depicted in figure  <dig>  after sequencing, a total of  <dig>  reads were obtained and assembled using the whole genome shotgun strategy.
during the initial assembly process, a large contig including  <dig>  reads was found.
this contig was identified as the mitochondrial genome of the fungus , which comprised approximately 6% of m. perniciosa sequences  <cit> .
the remaining genome sequences were assembled resulting in  <dig>  contigs and  <dig>  singlets with average lengths of  <dig>  bp and  <dig> bp, respectively.
the largest contig consisted of  <dig>  bp, and was formed by  <dig> reads.
the sum of all reads was close to  <dig> mbp and the total assembly consisted of  <dig>  mbp .
previous feulgen-image analysis experiments estimated m. perniciosa genome size to be  <dig>  ±  <dig>  mbp  <cit> .
due to the large error in this estimation we decided to assess the genome size using the genome estimation protocol established in the dog genome survey  <cit>  .
this analysis resulted in a genome size ranging from  <dig>  to  <dig>  mbp, a value similar to the genome length of another fungus belonging the order agaricales, c. cinerea , and that agrees with the previous size estimated by feulgen-image analysis  <cit> .
the lander waterman theory   <cit>  and its applications  <cit>  were used to confirm the estimate of the m. perniciosa genome size.
the theoretical values for the expected number of clusters , contigs, gaps, average cluster length and average gap length can be calculated using the lw theory from the effective average read length, the number of reads and the genome size as parameters.
if the calculated values derived from the genome assembly were close to the theoretical values derived from lw calculations, then it is possible to confirm the estimated genome size.
a summary of the results obtained from the assembly data and from the estimation using the lw theory is shown in table  <dig>  the experimental values agreed with the theoretical calculations, thus supporting our genome size estimation.
according to lw analysis the average gap size was  <dig> pb.
to have more information about the distribution of the gap size, we performed a comparison between a set of eukaryotic core proteins  and m. perniciosa contigs .
using this methodology, we detected that the average gap size was around  <dig> ±  <dig> bp, corroborating with lw average gap size estimative .
considering that most of the gaps are around  <dig> bp and that c. cinerea average gene size is  <dig>  bp, the majority of m. perniciosa genes or partial gene regions are likely to be included in the contigs.
corroborating this notion, we have found all genes encoding proteins of essential metabolic pathways such as glycolysis, gluconeogenesis, pentose phosphate pathway, and several others, which are available in the website  .
in order to estimate the number of sequences misassembled due to repeat regions, we applied the integrated pipeline for assembly validation, called amosvalidate  <cit>  .
this analysis resulted in  <dig> contigs with overrepresented regions totaling  <dig>  mbp.
multiplying the number of bases in overrepresented regions by over-coverage estimate resulted in  <dig>  mbp of repeat regions in the genome.
since reads from repetitive regions were eliminated from the estimation of the genome length using dog genome survey protocol , this misassembling does not invalidates the genome size estimate.
gene content
the initial step to uncover the gene content of m. perniciosa was performed using the genomic sequences together with a library of  <dig>  ests, previously annotated and partially published  <cit> .
genes were identified by comparing these two libraries using the program exalin  <cit> .
this analysis allowed us to identify expressed genes and intron structure.
the result was  <dig>  ests aligned to the genome contigs.
based on this est-genome sequence alignment, the average intron length was estimated as  <dig> bp.
this information was then used in the next steps of the process.
a more detailed investigation of the gene content was carried out using a combination of ab initio  and comparative gene prediction .
for ab initio gene prediction, we first applied the methodology described for the augustus gene predictor  <cit> .
this methodology essentially trains the augustus program using a combination of sequences from the species of interest together with sequences of a phylogenetically related species, specifically with similarities in intron and exon length distributions.
using this approach, the coding content sensors  are trained with sequences of the species of interest, and signal sensors  are trained with the sequences of the related species.
sequences from c. cinerea were used in the training process; together with selected m. perniciosa ests with e-value in blastx-nr ≤ 1e- <dig>  the sequences of the ests that aligned with proteins in the databank were concatenated, giving rise to a  <dig> kbp sequence.
ten copies of this m. perniciosa est concatamers  together with a dataset of genes from c. cinerea , comprised of  <dig>  mbp, were used to "pre-train" augustus.
the m. perniciosa predictions that came out of this pre-training were compared with the protein databank nr using blastp.
the resulting predictions with similarities in the nr databank, and with a coverage ≥ 90%, were selected.
after eliminating redundancies,  <dig> complete  and  <dig>  partial  m. perniciosa gene models were used to train augustus  <cit>  and two other gene predictors .
predictions with less than  <dig> amino acids were eliminated.
the remaining predictions were grouped into  <dig>  overlapping clusters; that is, genomic regions covered by at least one prediction.
the predictions in each overlapping cluster were ranked according to the criteria used by the fungal genome initiative at the broad institute , with some adaptations.
the ab initio gene finding pipeline generated  <dig>  gene models,  <dig>  of which contained significant similarity to genbank sequences .
the extrinsic prediction methodology consisted of a combination of genomic similarity searches  using contigs and singlets, with the alignment of m. perniciosa ests in the genomic clusters.
<dig>  contigs and  <dig>  singlets were submitted to similarity analysis in a databank containing blastx-nr plus p. chrysosporium proteins.
the genomic regions containing homologues in this databank were selected and exon-intron boundaries were determined.
we also selected the alignments between ests and genomic clusters.
then, we evaluated if there was a superposition of blast alignments and est alignments.
after this analysis, the extrinsic prediction methodology revealed  <dig>  gene models.
most of these gene models  were also predicted by the ab initio gene predictor programs.
of the  <dig>  remaining gene models not predicted by the gene predictor programs,  <dig>  presented significant similarity to sequences deposited in the genbank  and  <dig> did not have any significant similarity in the genbank  .
one of the reasons why these genes were not detected by the gene prediction programs could be the presence of low quality sequences that may have lead to frameshifts, thus making them impossible to detect by these programs.
the total number of gene models obtained using both approaches, ab initio and extrinsic predictions, is  <dig> .
assuming that the average contig length is  <dig>  kbp and the average gap length is  <dig> bp, it is possible that this total number of gene models  represents an overestimation.
for instance, in our assembly a gene with >  <dig> kbp  could be represented by two gene models, with each one in different contigs, which suggests a possible redundancy in this gene model prediction.
thus, it is very likely that the real number of gene models will be less and closer to the ab initio or extrinsic predictions separately.
however, to obtain the maximum amount of information, we decided to evaluate the results of similarity searches in genbank  for all  <dig>  gene models.
as a result,  <dig> %  of the gene models showed significant sequence similarity  to genbank proteins.
the remaining  <dig> % , designated as "no-hits", were sequences with lower e-value scores .
of these gene models  <dig>  were predicted with the ab initio gene predictors and  <dig> with the extrinsic predictions .
as expected, blastx analysis against available complete fungal genomes showed that m. perniciosa gene models present higher similarity to genes from basidiomycota  than those from ascomycota .
* – each m. perniciosa gene model was compared to all the proteins from the organisms listed in the table.
blastx score was defined as 1e-10
in order to find groups of similar proteins in our dataset, we applied a markov clustering  algorithm  <cit>  to the m. perniciosa gene models.
although the output by this method is not highly reliable, they correlate well with "real" gene families and can be applied efficiently to cluster large quantities of genes in a high throughput fashion  <cit> .
mcl also helped to the assemble genes without similarity in the genbank into gene families, which are described in the following sections.
those gene models that were not grouped into gene families by the mcl algorithm have been compared with the genome of c. cinerea in order to discard those genes with parts of their sequences present in more than one contig.
in order to perform this comparison  we assume that the length distribution of m. perniciosa proteins is similar to those from c. cinerea.
in this comparison we evaluated similar genes according to the length of the protein .
this comparison showed that there is a clear correlation between complete genes in c. cinerea and m. perniciosa gene models coding for proteins smaller than  <dig> aa.
however, for larger proteins more than one m. perniciosa gene model showed similarity to a single c. cinerea protein, thus confirming the overestimation of the number of gene models.
given the fact that the genome sequence is incomplete, there is high probability that we are predicting more than one gene model per gene.
in order to have a measurement of the overestimation and use it to correct our database, the corresponding relationship between the protein size and the number of gene models was calibrated with the genome of c. cinerea using the gene models unlinked to gene families identified by the mcl algorithm .
a linear regression formula was used to correct for the overestimation according to the protein size.
for example, proteins smaller than  <dig> amino acids will have only one model representing the gene, while larger proteins will have one model and a fraction of a second model representing its gene.
this fraction represents the overestimation of the number of gene models.
in order to estimate the gene density in m. perniciosa, and deal with the overestimation evident by the previous analysis, we averaged the total number of gene models obtained by the different predictions: ab initio predictions , extrinsic predictions  and total number of gene predictions .
this resulted in  <dig>  gene predictions that were divided by the  <dig> mbp estimated genome size, which gave a gene density of approximately  <dig>  ±  <dig>  kbp/gene or  <dig>  ±  <dig>  gene/kbp for the genome of m. perniciosa.
figure  <dig> depicts the gene density comparison between m. perniciosa, two eubacteria , one archea , an apicomplexan , a primitive chordate  and a series of fungal genomes, and this showed that our gene density evaluation is in agreement with gene density data of other filamentous fungi  <cit> .
the overall m. perniciosa genomic features are summarized in table  <dig>  the genomic clusters of m. perniciosa were submitted to ncbi  and the whole genome shotgun project has been deposited at ddbj/embl/genbank under the project accession abre <dig>  the version described in this paper is the first version, abre <dig>
* – singlet size after lucy quality trimming .
overall functional annotation and metabolic maps
the number of gene models used for the functional annotation and building of the metabolic maps was  <dig> , which represents the total number of gene models obtained using ab initio and extrinsic predictions.
although this number represents an overestimation of the real number of genes , it was used to maximize the information acquired from these sequences.
all data obtained in the gene families and protein domain analyses were corrected according to the normalization procedure developed from the gene models not included in any of the mcl families based on comparisons with the c. cinerea genome .
the program autofact  <cit> , an automated annotation tool, was used to evaluate the putative functions of m. perniciosa gene models.
this program determines the most informative functional description by combining multiple blast reports from a number of user selected databases, and provides a consensus result  <cit> .
autofact classifies proteins as "classified proteins", which can be "assigned"  or "unassigned" , and "unclassified proteins" .
using this software, 73% of gene models  were annotated as previously classified proteins.
from these  <dig>   were assigned proteins, and  <dig>   were classified as unassigned proteins, which can be interpreted as a class of conserved hypothetical proteins .
the remaining  <dig>  gene models  were considered as unclassified proteins.
the number of gene models with e-value scores > 1e- <dig> obtained by blastx-nr  was  <dig>  and represented  <dig> % of the total number of models.
the result obtained with autofact increased the number of classified proteins by  <dig> % .
this indicates that the use of domain classification databases is helpful in the identification and annotation of gene models.
additionally, we correlated the autofact annotation with the gene families assembled by the mcl algorithm.
as depicted in table  <dig> and additional file  <dig>  mcl data indicated that the cytochrome p <dig> monooxygenase family had the largest number of m. perniciosa members, followed by a gypsy-like retrotransposon gene family, and a gene family that has similarity to a c. cinerea hypothetical protein  and to a shitake mushroom lentinula edodes est .
this new gene family appears to be related to specific developmental traits of the agaricales.
also found were new gene families with unknown functions based on their lack of homology with genbank sequences .
these families could possibly be linked to m. perniciosa physiological characteristics.
a – number of gene models present in each family
b – normalization factor used to normalize the number of gene models present in each family
c – normalization number of gene models present in each family
d – manual annotation of mcl families according to blast similarity analysis
a comparison of the gene models with the cdd-pfam databank  <cit>  was performed to obtain information about protein domains present in m. perniciosa proteins.
the data obtained were normalized using the procedure described above.
cytochrome p <dig> monooxygenase was the most prevalent protein domain assigned in m. perniciosa , followed by protein kinases , sugar transporters , short chain dehydrogenases  and carboxylesterases/lipases  .
the prevalence of cytochrome p <dig> monooxygenase domains in the genome agrees with the results of mcl analysis.
cdd-pfam analysis was also used to evaluate the protein domains in other genome fungi .
the comparisons between m. perniciosa and the other fungi analyzed are described below.
a – number of cdd entry
b – number and description of
pfam domain
c – number of gene models containing each cdd-pfam domain
d – percentage of gene models containing each cdd-pfam domain in relation to total number of m. perniciosa gene models .
e – non-normalized cdd-pfam ranking
f – factor used to normalize the number of gene models containing each cdd-pfam domain
g – normalized number of gene models containing a cdd-pfam domain
h – percentage of normalized number of gene models containing each cdd-pfam domain in relation to total number of m. perniciosa gene models
i – normalized m. perniciosa cdd-pfam ranking
a hypothetical metabolic map of m. perniciosa was built using biocyc  <cit> .
this analysis allowed us to annotate  <dig> metabolic pathways.
these include  <dig> enzymatic reactions incorporating a total of  <dig> enzymes .
a comparison of this metabolic map with the fungal model saccharomyces cerevisiae s288c  showed that m. perniciosa has more metabolic pathways than s. cerevisiae; a result that corresponds to the smaller genome size of s. cerevisiae and possibly the more complex lifestyle of m. perniciosa.
interestingly, m. perniciosa has a higher number of reactions involving o <dig>  co <dig>  h2o <dig>  and nad+/nadh than s. cerevisiae, suggesting a greater capacity to use and deal with oxidation-reduction reactions .
we also detected reactions with farnesyl pyrophosphate and dimethylallyl-diphosphate, which are involved in the biosynthesis of the secondary metabolites such as isoprenoids and indoles.
moreover, according to biocyc analysis, m. perniciosa has more amino acid catabolic pathways, alternative carbon sources degradation and biosynthesis routes and c <dig> compounds  utilization and assimilation reactions than s. cerevisiae .
these pathways are under manual annotation and will be published on biocyc web page.
the existence of these pathways in m. perniciosa suggest ecological and physiological adaptations to environmental stresses; to competition present in its native habitat in the amazon basin, and to traits that enable it to colonize cacao and trigger wbd.
detoxification and general resistance mechanisms: cytochrome p <dig> monooxygenases, efflux transporters and anti-oxidative apparatus
based on cdd-pfam and mcl analyses gene members of the cytochrome p <dig> monooxygenase superfamily are prevalent in the genome of m. perniciosa .
cytochrome p <dig> monooxygenases play a role in hydroxylation and oxidation processes involved in biosynthesis and degradation of different compounds  <cit> .
therefore, a large number of gene models similar to cytochrome p <dig> monooxygenases suggest a significant capacity for synthesis of secondary metabolites, such as hormones or toxins, and for detoxification.
among the fungi analyzed, m. perniciosa has the highest number of cytochrome p <dig> monooxygenase genes  representing  <dig> % of the gene models .
saproprotrophic basidiomycetes p. chrysosporium and c. cinerea and the hemibiotrophic ascomycetes magnaporthe grisea and fusarium graminearum also have more than one hundred cytochrome p <dig> monooxygenase genes representing between  <dig> % and  <dig> % of their gene models .
the basidiomycete l. bicolor  and the ascomycete neurospora crassa  follow with fewer genes.
u. maydis , c. neoformans  and s. cerevisiae  have the fewest cytochrome p <dig> monoooxygenase genes of the fungi compared in this study .
this analysis clearly demonstrates the prevalence of cytochrome p <dig> monooxygenases in saprotrophic and hemibiotrophic fungi, which have to hydrolize complex wood polymers and deal with a highly oxidative environment.
as discussed by gonzalez and nebert  <cit> , cytochrome p <dig> monooxygenase polymorphisms may be the product of the "molecular warfare" that occurs during the co-evolution of preys and predators, which produce toxins and detoxifying genes, respectively.
this logic can be extrapolated to the plant-fungus interaction, and in that sense, we believe that the plethora of cytochrome p <dig> monooxygenases in m. perniciosa may be critical to detoxification and environmental adaptation as well as for disease development.
another set of proteins related to detoxification processes are the efflux transporters.
similarity searches in the transport classification database   <cit>  which compare the genome of m. perniciosa with other fungal genomes  verified an extended set of efflux transporters from classes  <dig> a.
<dig>  and  <dig> a.
<dig> .
the majority of abc transporters identified in m. perniciosa belong to the pleiotropic drug resistance  family and the abc conjugate  transporter family.
these proteins have been associated with fungal pathogenesis  <cit>  and heavy metal resistance  <cit> .
the members of p-type atpase superfamily in m. perniciosa include phospholipid translocating atpases family and fungal ena-atpases transporters, which are involved with the efflux of excessive na+, and especially k+, encountered by fungi during colonization of plants  <cit> .
during the plant defense, reactive oxygen species  are produced to limit the pathogen invasion  <cit> .
however, pathogenic fungi produce antioxidant enzymes that enable them to neutralize host ros.
m. perniciosa contains a myriad of o2- and h2o <dig> decomposing enzymes such as superoxide dismutases, catalases, peroxiredoxins, glutathione-system, thioredoxin-system enzymes and manganese dependent peroxidases .
therefore, this fungal genome harbors a complete ros detoxification system.
reports indicate that h2o <dig> favors necrotrophic pathogens infection  <cit> .
furthermore, m. perniciosa produces calcium oxalate crystals   <cit> , and a cacao susceptible genotype accumulates coc during m. perniciosa infection, followed by a programmed cell death   <cit> .
the degradation of coc produces carbon dioxide and h2o <dig>  suggesting that cocs can be important to necrotrophic mycelia development.
in addition, oxalate chelates ca2+, an important secondary plant defense messenger and a key cross-linker of pectin in the middle lamella pectin  <cit> , and was found to be a trigger of pcd in plants  <cit> .
these findings suggest that oxalate favors m. perniciosa infection by disorganizing plant defense and plant cell wall structure, by facilitating the action of fungal pectinases and possibly triggering pcd in the later stages of wbd  <cit> .
genome variability: mating-type genes and transposable elements
seven m. perniciosa gene models were found to be similar to pheromone receptors .
as a primary homothallic fungus, m. perniciosa does not use its mating type system to outcross, but probably to promote the formation of clamp connections, hyphae dikaryotization and for the expression of pathogenicity genes as in u. maydis  <cit> .
previous reports have indicated that m. perniciosa exhibits high genetic variability at the molecular level  <cit> .
this level of variability may be the reason m. perniciosa overcomes resistant genotypes of cacao, such as scavina  <dig>  <cit> .
furthermore, it has been postulated that the genome variability found in homothallic m. perniciosa may be due to transposable elements  and ectopic recombination guided by the numerous copies of these elements found in the genome  <cit> .
the fact that retrotransposons were identified in est libraries and differentially expressed during m. perniciosa development  <cit> , indicates that they are active elements, which could contribute to genetic variability.
among the m. perniciosa te families, gypsy-like retrotransposons were the most abundant, followed by copia-like retrotransposons .
tes similar to p. chrysosporium copia-like elements were found in m. perniciosa genome inserted within putative cytochrome p <dig> monooxygenase genes  <cit> .
curiously, they were also inserted in a p. chrysosporium cytochrome p <dig> monooxygenase subfamily , suggesting that these tes may have a common ancestral origin in basidiomycota.
retroelements of the tyrosine recombinase  order  <cit>  and dna transposons  from cacta  <cit> , hatc  <cit>  and tc/mariner  <cit>  superfamilies were also found.
finally, a previously described boto dna transposon  from the pif/is <dig> superfamily  <cit>  was also identified in a mcl family .
plant hormonal disarrangement: fungal genes related to plant hormones biosynthesis
there is growing evidence of phytohormones being produced by pathogens during some infective processes  <cit> .
for instance, the production of gibberellins ; hormones involved in the regulation of stem elongation, seed germination, flowering and fruit maturation; have been identified in phytopathogenic bacteria and fungi that cause overgrowth symptoms, such as giberella fujikuroi and sphaceloma manhiticola  <cit> .
a search for homologues of the fungal specific bi-functional ent-kaurene synthase  responsible for the two-step cyclation of ggdp in fungi  <cit>  identified gene models similar to the n-terminal domain of g. fujikuroi cps/ks but did not detect any sequence similar to the c-terminal domain of this protein in m. perniciosa.
another gene model similar to cps/ks that lacks the c-terminal domain was found in the aspergillus niger genome .
genes similar to ga- <dig> desaturase and ga oxidases , part of a ga biosynthesis gene cluster present in g. fujikuroi and phaeosphaeria sp.
were detected .
reinforcing our data mining discover, is the fact that a gibberellin-like compound was detected in basidiospores of m. perniciosa  <cit> .
we can theorize that the production of ga by m. perniciosa may confer the hyperplasic phenotype of the green broom that resembles stem hyper-elongation caused by ga-producing phytopathogens.
another interesting discovery in the m. perniciosa genome is the presence of genes encoding enzymes of two biosynthetic pathways of indole-3-acetic acid , the most abundant natural plant auxin.
we found a gene similar to plant nitrilases  which catalyzes the direct conversion of indole-3-acetonitrile into ammonia and iaa.
additionally, potential genes for the iaa-producing tryptamine pathway  were found .
furthermore, a manual annotation of est libraries reveal the presence of an aromatic amino acid aminotransferase, which could make part of indole-3-pyruvate iaa biosynthetic pathway .
iaa regulates many plant biological processes including cell elongation and fruit ripening.
recently, the presence of iaa in m. perniciosa basidiocarps was reported  <cit> .
iaa is produced by other fungal phytopathogens such as biotrophics u. maydis and taphrina deformans and hemibiotrophic colletotrichum gloeosporioides sp.
<cit> , and induces filamentation and invasive growth in s. cerevisiae  <cit> .
curiously, both gibberellin and iaa induce fruit parthenocarpy and act synergistically in plant organ expansion  <cit> , both of which are traits of wbd.
cacao plantlets inoculated with m. perniciosa spores emit higher levels of ethylene during the late stages of infection than non-inoculated plants  <cit> .
the plant hormone ethylene stimulates elongation at low concentrations, and senescence, fruit ripening, and epinasty at high doses  <cit> .
auxins stimulate the synthesis of ethylene  <cit> , which together with gibberellins have integrated actions in plant cell death  <cit>  and stem elongation during phytochrome-mediated shade avoidance, a phenomenon that occurs in response to the low red to far-red light ratios  under dense canopies  <cit> .
we hypothesize that in a dense and shaded environment, such as a cacao plantation, the low r:fr ratio effects can be increased by the action of the aforementioned phytohormones, explaining the shade avoidance, hypertrophy, and elongation of green brooms.
the presence of genes related to plant hormones production in m. perniciosa supports previous data  <cit>  and suggests that this fungus can influence the plant metabolism and defense, by altering hormonal balance during infection  <cit> .
pathogenicity: fungal effectors and pathogenicity associated proteins
as a pathogen that colonizes the plant apoplast during its biotrophic stage, m. perniciosa may release elicitor or effector proteins into the extracellular medium, which in turn could evade or suppress the plant defense response.
throughout wbd, m. perniciosa produces proteins with the potential to kill plant cells, thereby releasing their contents, which are absorbed by the fungus during its saprotrophic stage.
thus, m. perniciosa uses a varied arsenal of effector proteins in order to complete its infection cycle.
although some phytopathogenic fungi deliver effector proteins into the cytoplasm by means of haustoria  <cit>  this type of structure is absent in m. perniciosa.
therefore it is possible that this fungus secretes these proteins into the apoplast, as has been described for other fungi  <cit> .
effector proteins that are recognized by plant resistance  proteins are known as avr proteins.
no orthologues to the known ascomycota avr genes, not even the u. maydis genes contained in "biotrophic clusters"  <cit> , were found in the genome of m. perniciosa.
however, ab initio gene prediction with peptide signal analysis revealed  <dig> "no hits" small proteins containing secretion signals and at least two cysteines , which is a common trait of many proteins that are delivered into the host apoplast by phytopathogens  <cit> .
in order to validate  <dig> selected ab initio predictions, we conducted rt-pcr using rna from m. perniciosa saprotrophic mycelia.
we validated the expression of  <dig> gene models out of  <dig> tested, all of which contained secretion signals .
possibly, the gene models that were not confirmed by rt-pcr are expressed in other developmental stages of the fungus .
additional file  <dig> depicts the amplification of three of these genes.
whether these proteins play a role in the pathogenicity of m. perniciosa or in elicitation of cacao defense remains to be elucidated.
m. perniciosa contains necrosis and ethylene inducing proteins  and cerato platanins  <cit> , which can act in conjunction with a series of proteinases, hemolysin-like proteins and carboxylesterases/lipases found in the genome .
these proteins appear to be part of the destructive arsenal of m. perniciosa.
the most abundant proteinases in the genome of m. perniciosa are deuterolysins, a type of fungal metalloproteinases that are similar to bacterial thermolysin  <cit> .
compared to other fungi indicates that m. perniciosa has a deuterolysin expansion , suggesting an important role for these proteinases during this fungus development.
we also identified a gene family similar to agaricales pleurotus ostreatus and agrocybe aegerita hemolysin-like aegerolysins.
these proteins have cytolitic properties  <cit>  and seems to play an important role at the initial phase of fungal fruiting by making the fungal membranes permeable during cell signaling  <cit> .
m. perniciosa genome contain a family of aegerolysins .
none of the other agaricales fungi analyzed  contain these proteins.
even though we could not assess the genomic data of the basidiomycete containing aegerolysins, we suggest that the diversification of these proteins in wbd causative agent indicates their importance in m. perniciosa development or even in fungi defense and infective process.
carboxyesterases and lipases are overrepresented in m. perniciosa .
according to cdd-pfam, m. perniciosa have  <dig> gene models annotated as carboxylesterases, approximately twice the number of such proteins in other basidiomycete ; saprobes c. cinerea  and p. crhysosporium ; and biotrophic u. maydis ; figure 5a; additional file 3); while the hemibiotrophic ascomycete f. graminearum and m. grisea have  <dig> and  <dig> carboxylesterases, respectively.
we postulate that the great number of carboxylesterases and lipases in m. perniciosa, f. graminearum and m. grisea is related to their hemibiotrophic lifestyle.
in fact, these enzymes are induced during carbon and nitrogen starvation  <cit> , and cell wall degradation  <cit> , two events that occur during hemibiotrophism.
other genes associated with the plant-pathogen interaction found in our analysis were similar to the scp-like superfamily proteins, which comprise pathogenesis related  proteins of family  <dig> .
additionally, gene models similar to pr-5/thaumatin superfamily were also detected in the m. perniciosa genome .
pr proteins are well described as associated with defense reactions in plants against various pathogens  <cit> .
for instance, transgenic plants overexpressing pr- <dig> proteins were more resistant to oomycete infection  <cit>  and some thaumatin-like proteins  have β-glucanase activity, inhibit xylanase and have antifungal properties  <cit> .
recently, proteins similar to pr- <dig> and thaumatin have been characterized in animals and fungi  <cit> , indicating a conserved and important role in diverse organisms.
based on cdd-pfam analysis, m. perniciosa contains the largest number of thaumatins of any fungus sequenced, so far .
both pr- <dig> and pr- <dig> are induced by salicylic acid  in plants  <cit> .
curiously, the m. perniciosa necrotrophic  mycelia were found to produce and have tolerance to sa in axenic cultures  <cit> .
plants with wbd have a higher content of sa when compared with healthy plants  <cit> .
m. perniciosa tolerance to sa could be explained by the expression of genes encoding salicylate hydroxylases, which were also detected in the genome .
in this scenario, high levels of sa could block the synthesis of jasmonic acid , a defense compound against necrotrophic pathogens that acts as a necrosis inducer, thus rendering the plant susceptible to the spread of m. perniciosa  <cit> .
in addition, it is possible that sa and sa-induced proteins  may act to limit competition from other microbial competitors during wbd progression, which would be an important component of the m. perniciosa pathogenicity strategy.
colonization: plant cell wall degrading enzymes
degradation of hemicellulose, cellulose, pectin and depolymerization of lignin are some of the mechanisms that necrotrophic fungi use to colonize plant tissues  <cit> .
we identified genes encoding enzymes involved in degradation of hemicellulose and cellulose, including β- <dig>  cellulases, exocellobiohydrolases, endo-beta- <dig> -xylanases and endoglucanases; genes encoding lignolytic enzymes including manganese dependent peroxidase and multicopper polyphenoloxidases ; and genes encoding enzymes involved in pectin degradation, such as pectate lyases, polygalacturonases  and pectin methylesterases  .
m. perniciosa have an arsenal of plant cell wall degrading enzymes that is similar to that found in the hemibiotrophic pathogens f. graminearum and m. grisea .
biotrophic u. maydis and symbiont l. bicolor have a minimal set of pcwdes what is in accordance with their lifestyle .
pectate lyases  cleave pectin, an essential component of plant cell walls.
among the fungi analyzed, f. graminearum and m. perniciosa contain the largest number of pls .
unlike m. grisea, the other hemibiotrophic analyzed, f. graminearum and m. perniciosa does not have specialized structures  for non-enzymatic penetration of plants, and colonizing the apoplast by breaching the middle lamella barrier .
in addition, both are able to infect dicotyledons that contain cell walls with larger amounts of pectin than monocots  <cit> .
this analysis suggests that pls have an important role for pathogens that colonize the apoplast of dicotyledons.
insights into m. perniciosa intermediary metabolism and wbd development
pathogen energy status and the acquisition of host metabolic compounds by the pathogen are factors that determine the outcome of disease onset.
therefore, enzymes of m. perniciosa involved in intermediary metabolism  may have an important role during wbd .
during the first stages of wbd, m. perniciosa may encounter a nutrient-poor and extreme oxidative environment containing host defense molecules, such as nitric oxide , an inhibitor of the cytochrome respiratory pathway  <cit> .
under these conditions, the activity of a no tolerant alternative oxidase  may constitute a critical bypass mechanism for the cytochrome pathway  <cit> .
a single copy of aox was detected in m. perniciosa, and preliminary experiments indicate that this gene has a higher expression in fungi grown in nutrient-poor media .
following this scenario, in the low-energy status represented by the biotrophic phase, aox may provide nad+ for the turnover of the mitochondrial tricarboxylic acid cycle  and the peroxisomal glyoxylate cycle .
many reports have documented the importance of glox for phytopathogens during host infection  <cit> .
all genes coding for proteins of this enzymatic pathway are present in the genomeof m. perniciosa, including the key enzymes isocitrate lyase and malate synthase .
curiously, m. perniciosa is able to use methanol as the only carbon source , indicating that this fungus may have a methylotrophic metabolism.
methylotrophism have been extensively studied in methylotrophic yeasts such as pichia angusta and recently methanol oxidases  have been described in filamentous fungi such as c. fulvum  <cit>  and wood-degrading basidiomycete gloephyllum trabeum  <cit> .
a gene encoding a mox was identified in m. perniciosa genome .
interestingly, this gene was previously detected as being overexpressed in biotrophic-mycelia  <cit> .
the methanol catabolism enzymes formaldehyde dehydrogenase and formate dehydrogenase  were also found in the genome, which provides evidence that m. perniciosa indeed hydrolyzes methanol.
methanol is, possibly, derived from the first step of pectin hydrolyzation performed by the cell wall degrading enzyme pectin methylesterase  <cit> , or from demethylation of lignin that occurs after manganese peroxidase and/or laccase action  <cit> .
we can not discount the possibility that the biotrophic fungi can use the methanol produced by pectin metabolism during normal cell wall synthesis in early stages of plant expansion  <cit> .
the possibility that m. perniciosa is methylotrophic resembles the methylobacteria nutritional strategy, which provides an ecological advantage over non-methylotrophic microorganisms present in the phyllosphere  <cit> .
previous data indicated that the utilization of glycerol, instead of fermentable sugars , is an important environmental clue for the maintenance of the biotrophic stage  <cit> .
accordingly, our group detected higher amounts of glycerol during the biotrophic fungal phase of the green brooms development than in dry brooms  <cit> .
additionally, in vitro assays showed that the shift from glucose to glycerol media increased anti-oxidative defenses of m. perniciosa mycelia  <cit> .
this result correlates well with green broom environment  raising the possibility that glycerol is a critical metabolite during the initial stages of the disease cycle.
genes involved in glycerol metabolism and uptake are present in m. perniciosa, including a biotrophic induced aquaglyceroporin transporter  <cit>  , suggesting that this fungus is able to acquire extracellular glycerol.
after  <dig> days of wbd, glucose levels increase again, concomitant with a reduction in starch levels  <cit> .
we found a gene model similar to a secreted glucoamylase in the m. perniciosa genome .
gibberellin is an inducer of α-amylase production  <cit> .
we can envisage a disease scenario in which the fungus produces an extracellular amylase and hormones  that triggers plant amylolytic activity, which then decrease starch content.
the resulting glucose can be utilized by the plant since at this stage the green brooms/infected tissues are rapidly growing or even be acquired by the pathogen at the transition phase between biotrophic and necrotrophic stages.
nitrogen starvation also appears to be a factor that influences the biotrophic lifestyle  <cit>  and is associated with the expression of pathogenicity genes and pcwde, mainly in hemibiotrophic and biotrophic fungi that deal with nutrient deprivation during early infection  <cit> .
our data from microarray and est analysis suggests that nitrogen catabolite repression  occurs in m. perniciosa by the induction of gaba permease, trna synthetates and arom protein  <cit> .
as reported previously  <cit> m. perniciosa contains a gene homologous to clnr <dig> from the hemibiotrophic fungus c. lindemuthianum , a global nitrogen regulator that belongs to the area/nit <dig> family.
clnr <dig> activates enzymes and transporters that enable uptake and catabolism of secondary nitrogen sources  <cit> .
the depletion of clnr <dig> impaired the fungal switch to necrotrophy  <cit> , emphasizing the importance of nitrogen catabolism in hemibiotrophic development.
our genomic data mining identified a gene similar to npr <dig>  which encodes a regulatory protein that may act upstream of the area/nit <dig> protein .
npr <dig> is required for the expression of the m. grisea pathogenicity gene mpg <dig>  <cit> .
in addition, genes that encode enzymes involved in alternative nitrogen sources uptake  and metabolism  were identified .
the presence of such genes in m. perniciosa genome indicates that this fungus could allocate and utilize alternative nitrogen sources in the absence of preferential nitrogen sources  reinforcing our hypothesis that m. perniciosa suffers ncr during early stages of wbd.
CONCLUSIONS
our analysis of the m. perniciosa genome survey yielded interesting insights and clues into the molecular mechanisms underlying wbd.
as far as we know, this is the first phytopathogen included in the order agaricales sequenced.
therefore, our results support the investigation of pathogenicity mechanisms among agaricales and basidiomycete.
in addition, we provide an approach for normalization of gene family data in a genome survey that can aid the genomics community interested in functional analysis in incomplete genome data.
based on annotated fungal genes from this report and from previous other studies, we designed a hypothetical model for wbd that correlates plant phenotype changes that happen during the disease with the developmental progression of m. perniciosa .
after penetration and adhesion to the plant, the biotrophic fungus slowly grows inside the apoplast causing a series of phenotype changes in cacao, such as hypertrophy and hyperplasia, phototropism and epinasty, by secreting phytohormones that unbalance cacao metabolism .
in addition, m. perniciosa has to deal with nitrogen deprivation in the apoplast, which signals the production of proteins related to the acquisition of alternative nutrient sources, proteolysis and amino acid synthesis.
furthermore, the fungus takes advantage of the increasing content of glycerol in the green broom, an important cue for biotrophic stage maintenance, by expressing aquaglyceroporins.
as previously discussed  <cit> , the starch accumulated in early stages of green brooms seems to be metabolized to glucose, which suggests an amylolytic activity exerted by fungi and/or by plant amylases.
this increase of glucose is not accompanied by an increase of fructose, but by a sucrose augmentation.
since photosynthesis is not increased during wbd, we suggest that sucrose is translocated from other tissues to the green brooms.
therefore, we hypothesize that these mechanisms cause a source-to-sink transition in stem, turning green brooms into a drain of nutrients.
after numerous physiological and biochemical changes in the plant, which may be caused by the fungal infection, there occurs a transition from the biotrophic to the saprotrophic lifestyle .
this change could result from the increase of nutrients in the fungal environment and may be controlled by an area/nit2-like regulator.
during this transition phase, the plant displays the beginning of necrosis at the distal portion of the leaves that could be due to the action of neps and cerato-platanins that are expressed in the biotrophic hyphae.
in addition, m. perniciosa produces pcwdes, such as pectinases, whose action aids the fungus in breaching the middle lamella barrier.
pectin degradation releases methanol, which in turn could be used by m. perniciosa as a carbon source, through the action of a mox and other methanol metabolizing enzymes.
moreover, the calcium released from pectin disruption could be scavenged by the oxalate synthesized by the fungus, a compound that triggers cell death.
the release of cell content during necrosis, and the probable aforementioned source-to-sink transition, may influence, or even be indispensable to the m. perniciosa switch from biotrophism to saprotrophism.
thus, the postulated carbon and nitrogen catabolite repression displayed by the biotrophic mycelia would be switched off, thus causing the mycelial change to its invasive dikaryotic/saprotrophic stage.
during disease progression, and mainly during colonization of saprotrophic hyphae, m. perniciosa must deal with an intense oxidative environment.
based on our analysis this stress can be overcome by the action of several anti-oxidative and detoxifying enzymes.
furthermore, we believe that m. perniciosa exerts a negative control on plant defense against necrotrophic/saprotrophic fungi by producing salicylic acid, which would limit competition by other fungi by the action of antifungal proteins.
finally, after alternating wet and dry periods, the formation of the basidiomes produced by saprotrophic hyphae occurs.
latin american cacao crops suffer tremendous damages caused by wbd, which mainly affects small acreage farmers.
such impact in the relatively primitive cacao cultivation system not only affects the socio-economic status of farmers but also the preservation of the rainforest.
the demand for strategies that limit cacao diseases requires an intense effort in understanding the pathogenicity and plant resistance mechanisms.
further sequencing projects of cacao and its pathogens will serve as a background for the integration of transcriptomics, proteomics and metabolomics of these species in a systems biology approach.
such initiatives will provide tools for biological control, crop management and cacao biotechnology to combat cacao diseases.
we believe that our report is the first step towards such an integrative initiative and provides insights into the molecular mechanisms of wbd which can aid the cacao's wbd-concerned community to develop control strategies for this plant-fungus interaction.

 moniliophthora perniciosa, previously known as crinipellis perniciosa stahel, is a hemibiotrophic basidiomycete fungus that causes witches' broom disease in cacao. wbd and frosty pod rot, caused by moniliophthora roreri, are the most devastating diseases of cacao in the americas. cacao production in southeastern bahia, the main production area in brazil, was severely affected by the introduction of wbd at the end of 1980's. this disease damaged bahian agribusiness, caused major social problems and has contributed to the degradation of the atlantic rainforest. this is because cacao producing areas, typically, maintained old-growth native tree species as shade for the cacao plantations, which were converted to pasture. the symptoms displayed by cacao plants during wbd parallel the hemibiotrophic development of m. perniciosa. briefly, the disease begins when fungal spores germinate and infect meristematic tissues, developing into monokaryotic biotrophic hyphae without clamp connections that slowly occupy the intercellular space. this stage of wbd is characterized by the emergence of hypertrophic and hyperplasic anomalous branches, and the formation of parthenocarpic fruits. infected branches, known as "green brooms", grow without apical dominance, with a phototropic orientation and displaying epinastic leaves. after two to three months the infected tissue turns necrotic and the hyphae become saprotrophic with two nuclei per cell and clamp connections, invading the inter and intracellular spaces of the infected tissue. this fungal species exhibits primary homothalism as its reproductive strategy; thus, the change from the monokaryotic to the dikaryotic mycelium occurs without the prerequisite of mating between compatible individuals. after alternate wet and dry periods, basidiomes produced by the saprotrophic hyphae release basidiospores that are spread by wind or rain, thus completing the m. perniciosa life cycle. the witches' broom genome project involving several brazilian laboratories was initiated to increase the knowledge of this disease. the genome size, chromosomal polymorphism, genetic variability and the m. perniciosa mitochondrial genome have already been described. additionally, a biochemical study revealed the metabolic modifications that occur in cacao plantlets during wbd development. technical improvements have been achieved in the manipulation of cacao and m. perniciosa. one of the main bottlenecks in m. perniciosa research was solved with the development of the in vitro production of biotrophic-like cultures. necrotic inducing proteins expressed by m. perniciosa have been characterized, and the analysis of est libraries and dna microarrays have identified differentially expressed genes during its development and for the interaction of the fungus with cacao. despite the substantial progression in understanding wbd, many questions remain unsolved, mainly those concerning the mechanisms controlling processes such as: the fungal switch from biotrophism to saprotrophism; the drastic phenotypic alteration of cacao during disease development and the death of infected tissues. in vitro cultures in our laboratory demonstrate that this fungus has a great capacity to adapt to media containing different sources of carbon and nitrogen and it is able to grow in extremely nutrient-poor media. these results together with the fact that hemibiotrophic fungi, such as m. perniciosa, display complex lifestyles suggest that this fungus has a significant genomic and transcriptomic plasticity that contributes to the successful pathogenic mechanisms expressed during its life cycle. in a recent review, meinhardt et al., give a historical account and summarize the current state of knowledge about wbd. around two thirds of the known basidiomycota species are included in the order agaricales. in addition to being an interesting group for carrying out developmental genetic studies concerning fungal development and reproduction, the agaricales contain many important industrial species, and species with unique lifestyles: saprophytes, symbionts, leaf-litters decomposers and root pathogens. interestingly, m. perniciosa and m. roreri are members of a group of agaricales species that are able to infect aerial parts of plants, an uncommon characteristic among this basidiomycota order. genome sequencing and analysis is an important strategy to obtain comprehensive information concerning the metabolism and development of organisms. the initial objective of the m. perniciosa genome project was to obtain a genome survey sequence using a whole shotgun strategy to provide genomic information for the wbd research community. recently, the bioinformatics and genomic communities have been debating about the benefits and costs of finishing a complete genome as compared to applying a genome survey strategy. it has been proposed that a two-fold genome sequence coverage is sufficient to support a high percentage of est alignments and exon similarity matches. additionally, gene models resulting from a genome survey can be predicted accurately by the comparison with complete genomes of phylogenetically related organisms. this strategy has been used for genomic surveys of dog and of the wine spoilage yeast dekkera bruxellensis. recently, the genomes of the basidiomycete species laccaria bicolor, cryptococcus neoformans, ustilago maydis, coprinopsis cinerea and phanerochaete chrysosporium have been determined and many additional basidiomycota genome projects are ongoing or about to be released;, which could provide a rich database for additional basidiomycota genomic survey evaluations. with the existence of several complete basidiomycota genomes, and both m. perniciosa ests libraries, and a × genome coverage, we decided to conduct a genome survey of m. perniciosa to obtain further information about this important phytopathogen. this report describes a survey of the genome sequences of m. perniciosa, with specific emphasis on the genes potentially involved in disease development such as a cytochrome p monooxygenases, transposable elements, putative plant defense elicitors, pathogenicity effectors, cell wall degrading enzymes, proteins related to methylotrophy and the biosynthesis of plant hormones by the fungus. the identification of such genes in the m. perniciosa genomic data lead us to hypothesize a connection between molecular processes involved in the growth phases of the fungus and the progression of wbd.  genome assembly and estimation of genome size a diagram flow describing the bioinformatic procedures applied in the m. perniciosa genome survey are depicted in figure after sequencing, a total of reads were obtained and assembled using the whole genome shotgun strategy. during the initial assembly process, a large contig including reads was found. this contig was identified as the mitochondrial genome of the fungus, which comprised approximately 6% of m. perniciosa sequences. the remaining genome sequences were assembled resulting in contigs and singlets with average lengths of bp and bp, respectively. the largest contig consisted of bp, and was formed by reads. the sum of all reads was close to mbp and the total assembly consisted of mbp. previous feulgen-image analysis experiments estimated m. perniciosa genome size to be ± mbp. due to the large error in this estimation we decided to assess the genome size using the genome estimation protocol established in the dog genome survey. this analysis resulted in a genome size ranging from to mbp, a value similar to the genome length of another fungus belonging the order agaricales, c. cinerea, and that agrees with the previous size estimated by feulgen-image analysis. the lander waterman theory and its applications were used to confirm the estimate of the m. perniciosa genome size. the theoretical values for the expected number of clusters, contigs, gaps, average cluster length and average gap length can be calculated using the lw theory from the effective average read length, the number of reads and the genome size as parameters. if the calculated values derived from the genome assembly were close to the theoretical values derived from lw calculations, then it is possible to confirm the estimated genome size. a summary of the results obtained from the assembly data and from the estimation using the lw theory is shown in table the experimental values agreed with the theoretical calculations, thus supporting our genome size estimation. according to lw analysis the average gap size was pb. to have more information about the distribution of the gap size, we performed a comparison between a set of eukaryotic core proteins and m. perniciosa contigs. using this methodology, we detected that the average gap size was around ± bp, corroborating with lw average gap size estimative. considering that most of the gaps are around bp and that c. cinerea average gene size is bp, the majority of m. perniciosa genes or partial gene regions are likely to be included in the contigs. corroborating this notion, we have found all genes encoding proteins of essential metabolic pathways such as glycolysis, gluconeogenesis, pentose phosphate pathway, and several others, which are available in the website. in order to estimate the number of sequences misassembled due to repeat regions, we applied the integrated pipeline for assembly validation, called amosvalidate. this analysis resulted in contigs with overrepresented regions totaling mbp. multiplying the number of bases in overrepresented regions by over-coverage estimate resulted in mbp of repeat regions in the genome. since reads from repetitive regions were eliminated from the estimation of the genome length using dog genome survey protocol, this misassembling does not invalidates the genome size estimate. gene content the initial step to uncover the gene content of m. perniciosa was performed using the genomic sequences together with a library of ests, previously annotated and partially published. genes were identified by comparing these two libraries using the program exalin. this analysis allowed us to identify expressed genes and intron structure. the result was ests aligned to the genome contigs. based on this est-genome sequence alignment, the average intron length was estimated as bp. this information was then used in the next steps of the process. a more detailed investigation of the gene content was carried out using a combination of ab initio and comparative gene prediction. for ab initio gene prediction, we first applied the methodology described for the augustus gene predictor. this methodology essentially trains the augustus program using a combination of sequences from the species of interest together with sequences of a phylogenetically related species, specifically with similarities in intron and exon length distributions. using this approach, the coding content sensors are trained with sequences of the species of interest, and signal sensors are trained with the sequences of the related species. sequences from c. cinerea were used in the training process; together with selected m. perniciosa ests with e-value in blastx-nr ≤ 1e- the sequences of the ests that aligned with proteins in the databank were concatenated, giving rise to a kbp sequence. ten copies of this m. perniciosa est concatamers together with a dataset of genes from c. cinerea, comprised of mbp, were used to "pre-train" augustus. the m. perniciosa predictions that came out of this pre-training were compared with the protein databank nr using blastp. the resulting predictions with similarities in the nr databank, and with a coverage ≥ 90%, were selected. after eliminating redundancies, complete and partial m. perniciosa gene models were used to train augustus and two other gene predictors. predictions with less than amino acids were eliminated. the remaining predictions were grouped into overlapping clusters; that is, genomic regions covered by at least one prediction. the predictions in each overlapping cluster were ranked according to the criteria used by the fungal genome initiative at the broad institute, with some adaptations. the ab initio gene finding pipeline generated gene models, of which contained significant similarity to genbank sequences. the extrinsic prediction methodology consisted of a combination of genomic similarity searches using contigs and singlets, with the alignment of m. perniciosa ests in the genomic clusters. contigs and singlets were submitted to similarity analysis in a databank containing blastx-nr plus p. chrysosporium proteins. the genomic regions containing homologues in this databank were selected and exon-intron boundaries were determined. we also selected the alignments between ests and genomic clusters. then, we evaluated if there was a superposition of blast alignments and est alignments. after this analysis, the extrinsic prediction methodology revealed gene models. most of these gene models were also predicted by the ab initio gene predictor programs. of the remaining gene models not predicted by the gene predictor programs, presented significant similarity to sequences deposited in the genbank and did not have any significant similarity in the genbank. one of the reasons why these genes were not detected by the gene prediction programs could be the presence of low quality sequences that may have lead to frameshifts, thus making them impossible to detect by these programs. the total number of gene models obtained using both approaches, ab initio and extrinsic predictions, is. assuming that the average contig length is kbp and the average gap length is bp, it is possible that this total number of gene models represents an overestimation. for instance, in our assembly a gene with > kbp could be represented by two gene models, with each one in different contigs, which suggests a possible redundancy in this gene model prediction. thus, it is very likely that the real number of gene models will be less and closer to the ab initio or extrinsic predictions separately. however, to obtain the maximum amount of information, we decided to evaluate the results of similarity searches in genbank for all gene models. as a result, % of the gene models showed significant sequence similarity to genbank proteins. the remaining %, designated as "no-hits", were sequences with lower e-value scores. of these gene models were predicted with the ab initio gene predictors and with the extrinsic predictions. as expected, blastx analysis against available complete fungal genomes showed that m. perniciosa gene models present higher similarity to genes from basidiomycota than those from ascomycota. * – each m. perniciosa gene model was compared to all the proteins from the organisms listed in the table. blastx score was defined as 1e-10 in order to find groups of similar proteins in our dataset, we applied a markov clustering algorithm to the m. perniciosa gene models. although the output by this method is not highly reliable, they correlate well with "real" gene families and can be applied efficiently to cluster large quantities of genes in a high throughput fashion. mcl also helped to the assemble genes without similarity in the genbank into gene families, which are described in the following sections. those gene models that were not grouped into gene families by the mcl algorithm have been compared with the genome of c. cinerea in order to discard those genes with parts of their sequences present in more than one contig. in order to perform this comparison we assume that the length distribution of m. perniciosa proteins is similar to those from c. cinerea. in this comparison we evaluated similar genes according to the length of the protein. this comparison showed that there is a clear correlation between complete genes in c. cinerea and m. perniciosa gene models coding for proteins smaller than aa. however, for larger proteins more than one m. perniciosa gene model showed similarity to a single c. cinerea protein, thus confirming the overestimation of the number of gene models. given the fact that the genome sequence is incomplete, there is high probability that we are predicting more than one gene model per gene. in order to have a measurement of the overestimation and use it to correct our database, the corresponding relationship between the protein size and the number of gene models was calibrated with the genome of c. cinerea using the gene models unlinked to gene families identified by the mcl algorithm. a linear regression formula was used to correct for the overestimation according to the protein size. for example, proteins smaller than amino acids will have only one model representing the gene, while larger proteins will have one model and a fraction of a second model representing its gene. this fraction represents the overestimation of the number of gene models. in order to estimate the gene density in m. perniciosa, and deal with the overestimation evident by the previous analysis, we averaged the total number of gene models obtained by the different predictions: ab initio predictions, extrinsic predictions and total number of gene predictions. this resulted in gene predictions that were divided by the mbp estimated genome size, which gave a gene density of approximately ± kbp/gene or ± gene/kbp for the genome of m. perniciosa. figure depicts the gene density comparison between m. perniciosa, two eubacteria, one archea, an apicomplexan, a primitive chordate and a series of fungal genomes, and this showed that our gene density evaluation is in agreement with gene density data of other filamentous fungi. the overall m. perniciosa genomic features are summarized in table the genomic clusters of m. perniciosa were submitted to ncbi and the whole genome shotgun project has been deposited at ddbj/embl/genbank under the project accession abre the version described in this paper is the first version, abre * – singlet size after lucy quality trimming. overall functional annotation and metabolic maps the number of gene models used for the functional annotation and building of the metabolic maps was, which represents the total number of gene models obtained using ab initio and extrinsic predictions. although this number represents an overestimation of the real number of genes, it was used to maximize the information acquired from these sequences. all data obtained in the gene families and protein domain analyses were corrected according to the normalization procedure developed from the gene models not included in any of the mcl families based on comparisons with the c. cinerea genome. the program autofact, an automated annotation tool, was used to evaluate the putative functions of m. perniciosa gene models. this program determines the most informative functional description by combining multiple blast reports from a number of user selected databases, and provides a consensus result. autofact classifies proteins as "classified proteins", which can be "assigned" or "unassigned", and "unclassified proteins". using this software, 73% of gene models were annotated as previously classified proteins. from these were assigned proteins, and were classified as unassigned proteins, which can be interpreted as a class of conserved hypothetical proteins. the remaining gene models were considered as unclassified proteins. the number of gene models with e-value scores > 1e- obtained by blastx-nr was and represented % of the total number of models. the result obtained with autofact increased the number of classified proteins by %. this indicates that the use of domain classification databases is helpful in the identification and annotation of gene models. additionally, we correlated the autofact annotation with the gene families assembled by the mcl algorithm. as depicted in table and additional file mcl data indicated that the cytochrome p monooxygenase family had the largest number of m. perniciosa members, followed by a gypsy-like retrotransposon gene family, and a gene family that has similarity to a c. cinerea hypothetical protein and to a shitake mushroom lentinula edodes est. this new gene family appears to be related to specific developmental traits of the agaricales. also found were new gene families with unknown functions based on their lack of homology with genbank sequences. these families could possibly be linked to m. perniciosa physiological characteristics. a – number of gene models present in each family b – normalization factor used to normalize the number of gene models present in each family c – normalization number of gene models present in each family d – manual annotation of mcl families according to blast similarity analysis a comparison of the gene models with the cdd-pfam databank was performed to obtain information about protein domains present in m. perniciosa proteins. the data obtained were normalized using the procedure described above. cytochrome p monooxygenase was the most prevalent protein domain assigned in m. perniciosa, followed by protein kinases, sugar transporters, short chain dehydrogenases and carboxylesterases/lipases. the prevalence of cytochrome p monooxygenase domains in the genome agrees with the results of mcl analysis. cdd-pfam analysis was also used to evaluate the protein domains in other genome fungi. the comparisons between m. perniciosa and the other fungi analyzed are described below. a – number of cdd entry b – number and description of pfam domain c – number of gene models containing each cdd-pfam domain d – percentage of gene models containing each cdd-pfam domain in relation to total number of m. perniciosa gene models. e – non-normalized cdd-pfam ranking f – factor used to normalize the number of gene models containing each cdd-pfam domain g – normalized number of gene models containing a cdd-pfam domain h – percentage of normalized number of gene models containing each cdd-pfam domain in relation to total number of m. perniciosa gene models i – normalized m. perniciosa cdd-pfam ranking a hypothetical metabolic map of m. perniciosa was built using biocyc. this analysis allowed us to annotate metabolic pathways. these include enzymatic reactions incorporating a total of enzymes. a comparison of this metabolic map with the fungal model saccharomyces cerevisiae s288c showed that m. perniciosa has more metabolic pathways than s. cerevisiae; a result that corresponds to the smaller genome size of s. cerevisiae and possibly the more complex lifestyle of m. perniciosa. interestingly, m. perniciosa has a higher number of reactions involving o co h2o and nad+/nadh than s. cerevisiae, suggesting a greater capacity to use and deal with oxidation-reduction reactions. we also detected reactions with farnesyl pyrophosphate and dimethylallyl-diphosphate, which are involved in the biosynthesis of the secondary metabolites such as isoprenoids and indoles. moreover, according to biocyc analysis, m. perniciosa has more amino acid catabolic pathways, alternative carbon sources degradation and biosynthesis routes and c compounds utilization and assimilation reactions than s. cerevisiae. these pathways are under manual annotation and will be published on biocyc web page. the existence of these pathways in m. perniciosa suggest ecological and physiological adaptations to environmental stresses; to competition present in its native habitat in the amazon basin, and to traits that enable it to colonize cacao and trigger wbd. detoxification and general resistance mechanisms: cytochrome p monooxygenases, efflux transporters and anti-oxidative apparatus based on cdd-pfam and mcl analyses gene members of the cytochrome p monooxygenase superfamily are prevalent in the genome of m. perniciosa. cytochrome p monooxygenases play a role in hydroxylation and oxidation processes involved in biosynthesis and degradation of different compounds. therefore, a large number of gene models similar to cytochrome p monooxygenases suggest a significant capacity for synthesis of secondary metabolites, such as hormones or toxins, and for detoxification. among the fungi analyzed, m. perniciosa has the highest number of cytochrome p monooxygenase genes representing % of the gene models. saproprotrophic basidiomycetes p. chrysosporium and c. cinerea and the hemibiotrophic ascomycetes magnaporthe grisea and fusarium graminearum also have more than one hundred cytochrome p monooxygenase genes representing between % and % of their gene models. the basidiomycete l. bicolor and the ascomycete neurospora crassa follow with fewer genes. u. maydis, c. neoformans and s. cerevisiae have the fewest cytochrome p monoooxygenase genes of the fungi compared in this study. this analysis clearly demonstrates the prevalence of cytochrome p monooxygenases in saprotrophic and hemibiotrophic fungi, which have to hydrolize complex wood polymers and deal with a highly oxidative environment. as discussed by gonzalez and nebert, cytochrome p monooxygenase polymorphisms may be the product of the "molecular warfare" that occurs during the co-evolution of preys and predators, which produce toxins and detoxifying genes, respectively. this logic can be extrapolated to the plant-fungus interaction, and in that sense, we believe that the plethora of cytochrome p monooxygenases in m. perniciosa may be critical to detoxification and environmental adaptation as well as for disease development. another set of proteins related to detoxification processes are the efflux transporters. similarity searches in the transport classification database which compare the genome of m. perniciosa with other fungal genomes verified an extended set of efflux transporters from classes a. and a.. the majority of abc transporters identified in m. perniciosa belong to the pleiotropic drug resistance family and the abc conjugate transporter family. these proteins have been associated with fungal pathogenesis and heavy metal resistance. the members of p-type atpase superfamily in m. perniciosa include phospholipid translocating atpases family and fungal ena-atpases transporters, which are involved with the efflux of excessive na+, and especially k+, encountered by fungi during colonization of plants. during the plant defense, reactive oxygen species are produced to limit the pathogen invasion. however, pathogenic fungi produce antioxidant enzymes that enable them to neutralize host ros. m. perniciosa contains a myriad of o2- and h2o decomposing enzymes such as superoxide dismutases, catalases, peroxiredoxins, glutathione-system, thioredoxin-system enzymes and manganese dependent peroxidases. therefore, this fungal genome harbors a complete ros detoxification system. reports indicate that h2o favors necrotrophic pathogens infection. furthermore, m. perniciosa produces calcium oxalate crystals, and a cacao susceptible genotype accumulates coc during m. perniciosa infection, followed by a programmed cell death. the degradation of coc produces carbon dioxide and h2o suggesting that cocs can be important to necrotrophic mycelia development. in addition, oxalate chelates ca2+, an important secondary plant defense messenger and a key cross-linker of pectin in the middle lamella pectin, and was found to be a trigger of pcd in plants. these findings suggest that oxalate favors m. perniciosa infection by disorganizing plant defense and plant cell wall structure, by facilitating the action of fungal pectinases and possibly triggering pcd in the later stages of wbd. genome variability: mating-type genes and transposable elements seven m. perniciosa gene models were found to be similar to pheromone receptors. as a primary homothallic fungus, m. perniciosa does not use its mating type system to outcross, but probably to promote the formation of clamp connections, hyphae dikaryotization and for the expression of pathogenicity genes as in u. maydis. previous reports have indicated that m. perniciosa exhibits high genetic variability at the molecular level. this level of variability may be the reason m. perniciosa overcomes resistant genotypes of cacao, such as scavina. furthermore, it has been postulated that the genome variability found in homothallic m. perniciosa may be due to transposable elements and ectopic recombination guided by the numerous copies of these elements found in the genome. the fact that retrotransposons were identified in est libraries and differentially expressed during m. perniciosa development, indicates that they are active elements, which could contribute to genetic variability. among the m. perniciosa te families, gypsy-like retrotransposons were the most abundant, followed by copia-like retrotransposons. tes similar to p. chrysosporium copia-like elements were found in m. perniciosa genome inserted within putative cytochrome p monooxygenase genes. curiously, they were also inserted in a p. chrysosporium cytochrome p monooxygenase subfamily, suggesting that these tes may have a common ancestral origin in basidiomycota. retroelements of the tyrosine recombinase order and dna transposons from cacta, hatc and tc/mariner superfamilies were also found. finally, a previously described boto dna transposon from the pif/is superfamily was also identified in a mcl family. plant hormonal disarrangement: fungal genes related to plant hormones biosynthesis there is growing evidence of phytohormones being produced by pathogens during some infective processes. for instance, the production of gibberellins; hormones involved in the regulation of stem elongation, seed germination, flowering and fruit maturation; have been identified in phytopathogenic bacteria and fungi that cause overgrowth symptoms, such as giberella fujikuroi and sphaceloma manhiticola. a search for homologues of the fungal specific bi-functional ent-kaurene synthase responsible for the two-step cyclation of ggdp in fungi identified gene models similar to the n-terminal domain of g. fujikuroi cps/ks but did not detect any sequence similar to the c-terminal domain of this protein in m. perniciosa. another gene model similar to cps/ks that lacks the c-terminal domain was found in the aspergillus niger genome. genes similar to ga- desaturase and ga oxidases, part of a ga biosynthesis gene cluster present in g. fujikuroi and phaeosphaeria sp. were detected. reinforcing our data mining discover, is the fact that a gibberellin-like compound was detected in basidiospores of m. perniciosa. we can theorize that the production of ga by m. perniciosa may confer the hyperplasic phenotype of the green broom that resembles stem hyper-elongation caused by ga-producing phytopathogens. another interesting discovery in the m. perniciosa genome is the presence of genes encoding enzymes of two biosynthetic pathways of indole-3-acetic acid, the most abundant natural plant auxin. we found a gene similar to plant nitrilases which catalyzes the direct conversion of indole-3-acetonitrile into ammonia and iaa. additionally, potential genes for the iaa-producing tryptamine pathway were found. furthermore, a manual annotation of est libraries reveal the presence of an aromatic amino acid aminotransferase, which could make part of indole-3-pyruvate iaa biosynthetic pathway. iaa regulates many plant biological processes including cell elongation and fruit ripening. recently, the presence of iaa in m. perniciosa basidiocarps was reported. iaa is produced by other fungal phytopathogens such as biotrophics u. maydis and taphrina deformans and hemibiotrophic colletotrichum gloeosporioides sp., and induces filamentation and invasive growth in s. cerevisiae. curiously, both gibberellin and iaa induce fruit parthenocarpy and act synergistically in plant organ expansion, both of which are traits of wbd. cacao plantlets inoculated with m. perniciosa spores emit higher levels of ethylene during the late stages of infection than non-inoculated plants. the plant hormone ethylene stimulates elongation at low concentrations, and senescence, fruit ripening, and epinasty at high doses. auxins stimulate the synthesis of ethylene, which together with gibberellins have integrated actions in plant cell death and stem elongation during phytochrome-mediated shade avoidance, a phenomenon that occurs in response to the low red to far-red light ratios under dense canopies. we hypothesize that in a dense and shaded environment, such as a cacao plantation, the low r:fr ratio effects can be increased by the action of the aforementioned phytohormones, explaining the shade avoidance, hypertrophy, and elongation of green brooms. the presence of genes related to plant hormones production in m. perniciosa supports previous data and suggests that this fungus can influence the plant metabolism and defense, by altering hormonal balance during infection. pathogenicity: fungal effectors and pathogenicity associated proteins as a pathogen that colonizes the plant apoplast during its biotrophic stage, m. perniciosa may release elicitor or effector proteins into the extracellular medium, which in turn could evade or suppress the plant defense response. throughout wbd, m. perniciosa produces proteins with the potential to kill plant cells, thereby releasing their contents, which are absorbed by the fungus during its saprotrophic stage. thus, m. perniciosa uses a varied arsenal of effector proteins in order to complete its infection cycle. although some phytopathogenic fungi deliver effector proteins into the cytoplasm by means of haustoria this type of structure is absent in m. perniciosa. therefore it is possible that this fungus secretes these proteins into the apoplast, as has been described for other fungi. effector proteins that are recognized by plant resistance proteins are known as avr proteins. no orthologues to the known ascomycota avr genes, not even the u. maydis genes contained in "biotrophic clusters", were found in the genome of m. perniciosa. however, ab initio gene prediction with peptide signal analysis revealed "no hits" small proteins containing secretion signals and at least two cysteines, which is a common trait of many proteins that are delivered into the host apoplast by phytopathogens. in order to validate selected ab initio predictions, we conducted rt-pcr using rna from m. perniciosa saprotrophic mycelia. we validated the expression of gene models out of tested, all of which contained secretion signals. possibly, the gene models that were not confirmed by rt-pcr are expressed in other developmental stages of the fungus. additional file depicts the amplification of three of these genes. whether these proteins play a role in the pathogenicity of m. perniciosa or in elicitation of cacao defense remains to be elucidated. m. perniciosa contains necrosis and ethylene inducing proteins and cerato platanins, which can act in conjunction with a series of proteinases, hemolysin-like proteins and carboxylesterases/lipases found in the genome. these proteins appear to be part of the destructive arsenal of m. perniciosa. the most abundant proteinases in the genome of m. perniciosa are deuterolysins, a type of fungal metalloproteinases that are similar to bacterial thermolysin. compared to other fungi indicates that m. perniciosa has a deuterolysin expansion, suggesting an important role for these proteinases during this fungus development. we also identified a gene family similar to agaricales pleurotus ostreatus and agrocybe aegerita hemolysin-like aegerolysins. these proteins have cytolitic properties and seems to play an important role at the initial phase of fungal fruiting by making the fungal membranes permeable during cell signaling. m. perniciosa genome contain a family of aegerolysins. none of the other agaricales fungi analyzed contain these proteins. even though we could not assess the genomic data of the basidiomycete containing aegerolysins, we suggest that the diversification of these proteins in wbd causative agent indicates their importance in m. perniciosa development or even in fungi defense and infective process. carboxyesterases and lipases are overrepresented in m. perniciosa. according to cdd-pfam, m. perniciosa have gene models annotated as carboxylesterases, approximately twice the number of such proteins in other basidiomycete; saprobes c. cinerea and p. crhysosporium; and biotrophic u. maydis; figure 5a; additional file 3); while the hemibiotrophic ascomycete f. graminearum and m. grisea have and carboxylesterases, respectively. we postulate that the great number of carboxylesterases and lipases in m. perniciosa, f. graminearum and m. grisea is related to their hemibiotrophic lifestyle. in fact, these enzymes are induced during carbon and nitrogen starvation, and cell wall degradation, two events that occur during hemibiotrophism. other genes associated with the plant-pathogen interaction found in our analysis were similar to the scp-like superfamily proteins, which comprise pathogenesis related proteins of family. additionally, gene models similar to pr-5/thaumatin superfamily were also detected in the m. perniciosa genome. pr proteins are well described as associated with defense reactions in plants against various pathogens. for instance, transgenic plants overexpressing pr- proteins were more resistant to oomycete infection and some thaumatin-like proteins have β-glucanase activity, inhibit xylanase and have antifungal properties. recently, proteins similar to pr- and thaumatin have been characterized in animals and fungi, indicating a conserved and important role in diverse organisms. based on cdd-pfam analysis, m. perniciosa contains the largest number of thaumatins of any fungus sequenced, so far. both pr- and pr- are induced by salicylic acid in plants. curiously, the m. perniciosa necrotrophic mycelia were found to produce and have tolerance to sa in axenic cultures. plants with wbd have a higher content of sa when compared with healthy plants. m. perniciosa tolerance to sa could be explained by the expression of genes encoding salicylate hydroxylases, which were also detected in the genome. in this scenario, high levels of sa could block the synthesis of jasmonic acid, a defense compound against necrotrophic pathogens that acts as a necrosis inducer, thus rendering the plant susceptible to the spread of m. perniciosa. in addition, it is possible that sa and sa-induced proteins may act to limit competition from other microbial competitors during wbd progression, which would be an important component of the m. perniciosa pathogenicity strategy. colonization: plant cell wall degrading enzymes degradation of hemicellulose, cellulose, pectin and depolymerization of lignin are some of the mechanisms that necrotrophic fungi use to colonize plant tissues. we identified genes encoding enzymes involved in degradation of hemicellulose and cellulose, including β- cellulases, exocellobiohydrolases, endo-beta- -xylanases and endoglucanases; genes encoding lignolytic enzymes including manganese dependent peroxidase and multicopper polyphenoloxidases; and genes encoding enzymes involved in pectin degradation, such as pectate lyases, polygalacturonases and pectin methylesterases. m. perniciosa have an arsenal of plant cell wall degrading enzymes that is similar to that found in the hemibiotrophic pathogens f. graminearum and m. grisea. biotrophic u. maydis and symbiont l. bicolor have a minimal set of pcwdes what is in accordance with their lifestyle. pectate lyases cleave pectin, an essential component of plant cell walls. among the fungi analyzed, f. graminearum and m. perniciosa contain the largest number of pls. unlike m. grisea, the other hemibiotrophic analyzed, f. graminearum and m. perniciosa does not have specialized structures for non-enzymatic penetration of plants, and colonizing the apoplast by breaching the middle lamella barrier. in addition, both are able to infect dicotyledons that contain cell walls with larger amounts of pectin than monocots. this analysis suggests that pls have an important role for pathogens that colonize the apoplast of dicotyledons. insights into m. perniciosa intermediary metabolism and wbd development pathogen energy status and the acquisition of host metabolic compounds by the pathogen are factors that determine the outcome of disease onset. therefore, enzymes of m. perniciosa involved in intermediary metabolism may have an important role during wbd. during the first stages of wbd, m. perniciosa may encounter a nutrient-poor and extreme oxidative environment containing host defense molecules, such as nitric oxide, an inhibitor of the cytochrome respiratory pathway. under these conditions, the activity of a no tolerant alternative oxidase may constitute a critical bypass mechanism for the cytochrome pathway. a single copy of aox was detected in m. perniciosa, and preliminary experiments indicate that this gene has a higher expression in fungi grown in nutrient-poor media. following this scenario, in the low-energy status represented by the biotrophic phase, aox may provide nad+ for the turnover of the mitochondrial tricarboxylic acid cycle and the peroxisomal glyoxylate cycle. many reports have documented the importance of glox for phytopathogens during host infection. all genes coding for proteins of this enzymatic pathway are present in the genomeof m. perniciosa, including the key enzymes isocitrate lyase and malate synthase. curiously, m. perniciosa is able to use methanol as the only carbon source, indicating that this fungus may have a methylotrophic metabolism. methylotrophism have been extensively studied in methylotrophic yeasts such as pichia angusta and recently methanol oxidases have been described in filamentous fungi such as c. fulvum and wood-degrading basidiomycete gloephyllum trabeum. a gene encoding a mox was identified in m. perniciosa genome. interestingly, this gene was previously detected as being overexpressed in biotrophic-mycelia. the methanol catabolism enzymes formaldehyde dehydrogenase and formate dehydrogenase were also found in the genome, which provides evidence that m. perniciosa indeed hydrolyzes methanol. methanol is, possibly, derived from the first step of pectin hydrolyzation performed by the cell wall degrading enzyme pectin methylesterase, or from demethylation of lignin that occurs after manganese peroxidase and/or laccase action. we can not discount the possibility that the biotrophic fungi can use the methanol produced by pectin metabolism during normal cell wall synthesis in early stages of plant expansion. the possibility that m. perniciosa is methylotrophic resembles the methylobacteria nutritional strategy, which provides an ecological advantage over non-methylotrophic microorganisms present in the phyllosphere. previous data indicated that the utilization of glycerol, instead of fermentable sugars, is an important environmental clue for the maintenance of the biotrophic stage. accordingly, our group detected higher amounts of glycerol during the biotrophic fungal phase of the green brooms development than in dry brooms. additionally, in vitro assays showed that the shift from glucose to glycerol media increased anti-oxidative defenses of m. perniciosa mycelia. this result correlates well with green broom environment raising the possibility that glycerol is a critical metabolite during the initial stages of the disease cycle. genes involved in glycerol metabolism and uptake are present in m. perniciosa, including a biotrophic induced aquaglyceroporin transporter, suggesting that this fungus is able to acquire extracellular glycerol. after days of wbd, glucose levels increase again, concomitant with a reduction in starch levels. we found a gene model similar to a secreted glucoamylase in the m. perniciosa genome. gibberellin is an inducer of α-amylase production. we can envisage a disease scenario in which the fungus produces an extracellular amylase and hormones that triggers plant amylolytic activity, which then decrease starch content. the resulting glucose can be utilized by the plant since at this stage the green brooms/infected tissues are rapidly growing or even be acquired by the pathogen at the transition phase between biotrophic and necrotrophic stages. nitrogen starvation also appears to be a factor that influences the biotrophic lifestyle and is associated with the expression of pathogenicity genes and pcwde, mainly in hemibiotrophic and biotrophic fungi that deal with nutrient deprivation during early infection. our data from microarray and est analysis suggests that nitrogen catabolite repression occurs in m. perniciosa by the induction of gaba permease, trna synthetates and arom protein. as reported previously m. perniciosa contains a gene homologous to clnr from the hemibiotrophic fungus c. lindemuthianum, a global nitrogen regulator that belongs to the area/nit family. clnr activates enzymes and transporters that enable uptake and catabolism of secondary nitrogen sources. the depletion of clnr impaired the fungal switch to necrotrophy, emphasizing the importance of nitrogen catabolism in hemibiotrophic development. our genomic data mining identified a gene similar to npr which encodes a regulatory protein that may act upstream of the area/nit protein. npr is required for the expression of the m. grisea pathogenicity gene mpg. in addition, genes that encode enzymes involved in alternative nitrogen sources uptake and metabolism were identified. the presence of such genes in m. perniciosa genome indicates that this fungus could allocate and utilize alternative nitrogen sources in the absence of preferential nitrogen sources reinforcing our hypothesis that m. perniciosa suffers ncr during early stages of wbd.  our analysis of the m. perniciosa genome survey yielded interesting insights and clues into the molecular mechanisms underlying wbd. as far as we know, this is the first phytopathogen included in the order agaricales sequenced. therefore, our results support the investigation of pathogenicity mechanisms among agaricales and basidiomycete. in addition, we provide an approach for normalization of gene family data in a genome survey that can aid the genomics community interested in functional analysis in incomplete genome data. based on annotated fungal genes from this report and from previous other studies, we designed a hypothetical model for wbd that correlates plant phenotype changes that happen during the disease with the developmental progression of m. perniciosa. after penetration and adhesion to the plant, the biotrophic fungus slowly grows inside the apoplast causing a series of phenotype changes in cacao, such as hypertrophy and hyperplasia, phototropism and epinasty, by secreting phytohormones that unbalance cacao metabolism. in addition, m. perniciosa has to deal with nitrogen deprivation in the apoplast, which signals the production of proteins related to the acquisition of alternative nutrient sources, proteolysis and amino acid synthesis. furthermore, the fungus takes advantage of the increasing content of glycerol in the green broom, an important cue for biotrophic stage maintenance, by expressing aquaglyceroporins. as previously discussed, the starch accumulated in early stages of green brooms seems to be metabolized to glucose, which suggests an amylolytic activity exerted by fungi and/or by plant amylases. this increase of glucose is not accompanied by an increase of fructose, but by a sucrose augmentation. since photosynthesis is not increased during wbd, we suggest that sucrose is translocated from other tissues to the green brooms. therefore, we hypothesize that these mechanisms cause a source-to-sink transition in stem, turning green brooms into a drain of nutrients. after numerous physiological and biochemical changes in the plant, which may be caused by the fungal infection, there occurs a transition from the biotrophic to the saprotrophic lifestyle. this change could result from the increase of nutrients in the fungal environment and may be controlled by an area/nit2-like regulator. during this transition phase, the plant displays the beginning of necrosis at the distal portion of the leaves that could be due to the action of neps and cerato-platanins that are expressed in the biotrophic hyphae. in addition, m. perniciosa produces pcwdes, such as pectinases, whose action aids the fungus in breaching the middle lamella barrier. pectin degradation releases methanol, which in turn could be used by m. perniciosa as a carbon source, through the action of a mox and other methanol metabolizing enzymes. moreover, the calcium released from pectin disruption could be scavenged by the oxalate synthesized by the fungus, a compound that triggers cell death. the release of cell content during necrosis, and the probable aforementioned source-to-sink transition, may influence, or even be indispensable to the m. perniciosa switch from biotrophism to saprotrophism. thus, the postulated carbon and nitrogen catabolite repression displayed by the biotrophic mycelia would be switched off, thus causing the mycelial change to its invasive dikaryotic/saprotrophic stage. during disease progression, and mainly during colonization of saprotrophic hyphae, m. perniciosa must deal with an intense oxidative environment. based on our analysis this stress can be overcome by the action of several anti-oxidative and detoxifying enzymes. furthermore, we believe that m. perniciosa exerts a negative control on plant defense against necrotrophic/saprotrophic fungi by producing salicylic acid, which would limit competition by other fungi by the action of antifungal proteins. finally, after alternating wet and dry periods, the formation of the basidiomes produced by saprotrophic hyphae occurs. latin american cacao crops suffer tremendous damages caused by wbd, which mainly affects small acreage farmers. such impact in the relatively primitive cacao cultivation system not only affects the socio-economic status of farmers but also the preservation of the rainforest. the demand for strategies that limit cacao diseases requires an intense effort in understanding the pathogenicity and plant resistance mechanisms. further sequencing projects of cacao and its pathogens will serve as a background for the integration of transcriptomics, proteomics and metabolomics of these species in a systems biology approach. such initiatives will provide tools for biological control, crop management and cacao biotechnology to combat cacao diseases. we believe that our report is the first step towards such an integrative initiative and provides insights into the molecular mechanisms of wbd which can aid the cacao's wbd-concerned community to develop control strategies for this plant-fungus interaction. 
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. m. perniciosa, together with the related species m. roreri, are pathogens of aerial parts of the plant, an uncommon characteristic in the order agaricales. 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. a genome survey of m. perniciosa was analyzed to evaluate the overall gene content of this phytopathogen. the great number of genes encoding cytochrome p monooxygenases 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. in addition, an approach for normalization of gene family data using incomplete genome data was developed and applied in m. perniciosa genome survey. 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 basidiomycete fungus moniliophthora perniciosa is the causal agent of witches' broom disease in 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.
 the basidiomycete fungus moniliophthora perniciosa is the causal agent of witches' broom disease in 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 of m. perniciosa was analyzed to evaluate the overall gene content of this phytopathogen.  genes encoding proteins involved in retrotransposition, reactive oxygen species resistance, drug efflux transport and cell wall degradation were identified. the great number of genes encoding cytochrome p monooxygenases 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. 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.  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. 