Cocoa Apoplastome Contains Defense Proteins Against Pathogens.

The apoplast performs important functions in the plant, such as defense against stress, and compounds present form the apoplastic washing fluid (AWF). The fungus Moniliophthora perniciosa, the causal agent of witches' broom disease (WBD) in Theobroma cacao, initially colonizes the apoplast in its biotrophic phase. In this period, the fungus can remain for approximately 60 days, until it changes to its second phase, causing tissue death and consequently large loss in the production of beans. To better understand the importance of the apoplast in the T. cacao-M. perniciosa interaction, we performed the first apoplastic proteomic mapping of two contrasting genotypes for WBD resistance (CCN51-resistant and Catongo-susceptible). Based on two-dimensional gel analysis, we identified 36 proteins in CCN-51 and 15 in Catongo. We highlight PR-proteins, such as peroxidases, ß-1,3-glucanases, and chitinases. A possible candidate for a resistance marker of the CCN-51 genotype, osmotin, was identified. The antioxidative metabolism of the superoxide dismutase (SOD) enzyme showed a significant increase (P < 0.05) in the AWF of the two genotypes under field conditions (FD). T. cacao AWF inhibited the germination of M. perniciosa basidiospores (>80%), in addition to causing morphological changes. Our results shed more light on the nature of the plant's defense performed by the apoplast in the T. cacao-M. perniciosa interaction in the initial (biotrophic) phase of fungal infection and therefore make it possible to expand WBD control strategies based on the identification of potential targets for resistance markers and advance scientific knowledge of the disease.

BASIDIN as a New Protein Effector of the Phytopathogen Causing Witche's Broom Disease in Cocoa.

The fungus Moniliophthora perniciosa secretes protein effectors that manipulate the physiology of the host plant, but few effectors of this fungus have had their functions confirmed. We performed functional characterization of a promising candidate effector of M. perniciosa. The inoculation of rBASIDIN at 4 µmol L-1 in the mesophyll of leaflets of Solanum lycopersicum caused symptoms of shriveling within 6 h without the presence of necrosis. However, when sprayed on the plant at a concentration of 11 µmol L-1, it caused wilting symptoms only 2 h after application, followed by necrosis and cell death at 48 h. rBASIDIN applied to Theobroma cacao leaves at the same concentration caused milder symptoms. rBASIDIN caused hydrogen peroxide production in leaf tissue, damaging the leaf membrane and negatively affecting the photosynthetic rate of Solanum lycopersicum plants. Phylogenetic analysis indicated that BASIDIN has orthologs in other phytopathogenic basidiomycetes. Analysis of the transcripts revealed that BASIDIN and its orthologs are expressed in different fungal species, suggesting that this protein is differentially regulated in these basidiomycetes. Therefore, the results of applying BASIDIN allow the inference that it is an effector of the fungus M. perniciosa, with a strong potential to interfere in the defense system of the host plant.

Viability of Moniliophthora roreri on Cocoa Beans Under Microfermentation and Long-Term Survival on Carrier Materials.

The viability of Moniliophthora roreri inoculum was evaluated during the microfermentation process of diseased and healthy pulp-seed masses and on a range of carrier materials: aluminum, cloth, glass, paper, plastic, raffia, and rubber tire. Fungal survival was assessed before the microfermentation (0 h) and every 24 to 96 h by the growth of colonies in potato-dextrose-agar (PDA) and sporulation in seed shells. Colonies of M. roreri and sporulation on seed shells were observed from seeds not submitted to microfermentation. No growth was recovered from diseased cocoa beans after 48 h under the microfermentation. The viability of M. roreri spores recovered from carrier materials was evaluated at 7, 15, 30, 45, and 100 days after inoculation (DAI) by collecting spores and plating them on Sabouraud dextrose yeast extract agar amended with chloramphenicol (50 mg l1). The viability was determined by counting germinated and ungerminated spores under a light microscope (40×) after incubating in a moist chamber at 26 ± 2°C for 72 h. Spores maintained long-term viability on all tested carrier materials toward the end of the experiment (overall 26%) with significant differences (<0.05) among them. Maximum spore viability occurred at 7 and 15 DAI, with cloth and plastic carrier materials considered at high risk of acting as vehicles for the fungal spread. Mathematical models of spore viability over time were fit to the data using the Bayesian information criterion. Findings confirmed the importance of the fermentation process to hamper M. roreri growth and the potential of carrier materials for fungal dispersal.

Genome-wide SNP genotyping as a simple and practical tool to accelerate the development of inbred lines in outbred tree species: An example in cacao (Theobroma cacao L.).

Cacao is a globally important crop with a long history of domestication and selective breeding. Despite the increased use of elite clones by cacao farmers, worldwide plantations are established mainly using hybrid progeny material derived from heterozygous parents, therefore displaying high tree-to-tree variability. The deliberate development of hybrids from advanced inbred lines produced by successive generations of self-pollination has not yet been fully considered in cacao breeding. This is largely due to the self-incompatibility of the species, the long generation cycles (3-5 years) and the extensive trial areas needed to accomplish the endeavor. We propose a simple and accessible approach to develop inbred lines based on accelerating the buildup of homozygosity based on regular selfing assisted by genome-wide SNP genotyping. In this study we genotyped 90 clones from the Brazilian CEPEC´s germplasm collection and 49 inbred offspring of six S1 or S2 cacao families derived from self-pollinating clones CCN-51, PS-13.19, TSH-1188 and SIAL-169. A set of 3,380 SNPs distributed across the cacao genome were interrogated on the EMBRAPA multi-species 65k Infinium chip. The 90 cacao clones showed considerable variation in genome-wide SNP homozygosity (mean 0.727± 0.182) and 19 of them with homozygosity ≥90%. By assessing the increase in homozygosity across two generations of self-pollinations, SNP data revealed the wide variability in homozygosity within and between S1 and S2 families. Even in small families (<10 sibs), individuals were identified with up to ~1.5 standard deviations above the family mean homozygosity. From baseline homozygosities of 0.476 and 0.454, offspring with homozygosities of 0.862 and 0.879 were recovered for clones TSH-1188 and CCN-51 respectively, in only two generations of selfing (81-93% increase). SNP marker assisted monitoring and selection of inbred individuals can be a practical tool to optimize and accelerate the development of inbred lines of outbred tree species. This approach will allow a faster and more accurate exploitation of hybrid breeding strategies in cacao improvement programs and potentially in other perennial fruit and forest trees.

TcTI, a Kunitz-type trypsin inhibitor from cocoa associated with defense against pathogens.

Protease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [DE3]. The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10-7 mol L-1. The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70-80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and ß-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL-1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao, so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops.

Characterization of the microbiota dynamics associated with Moniliophthora roreri, causal agent of cocoa frosty pod rot disease, reveals new viral species.

Introduction: Theobroma cacao, the cocoa tree, is a target for pathogens, such as fungi from the genera Phytophthora, Moniliophthora, Colletotrichum, Ceratocystis, among others. Some cacao pathogens are restricted to specific regions of the world, such as the Cacao swollen shoot virus (CSSV) in West African countries, while others are expanding geographically, such as Moniliophthora roreri in the Americas. M. roreri is one of the most threatening cacao pathogens since it directly attacks the cacao pods driving a significant reduction in production, and therefore economic losses. Despite its importance, the knowledge about the microenvironment of this pathogen and the cocoa pods is still poorly characterized. Methods: Herein we performed RNA sequencing of spores in differential stages of culture in a medium supplemented with cacao pod extract and mycelium collected of the susceptible variety ICT 7121 naturally infected by the pathogen to evaluate the diversity and transcriptional activity of microorganisms associated with the in vitro sporulation of M. roreri. Results: Our data revealed a great variety of fungi and bacteria associated with M. roreri, with an exceptional diversity of individuals from the genus Trichoderma sp. Interestingly, the dynamics of microorganisms from different kingdoms varied proportionally, suggesting they are somehow affected by M. roreri culture time. We also identified three sequences similar to viral genomes from the Narnaviridae family, posteriorly confirmed by phylogenetic analysis as members of the genus Narnavirus. Screening of M. roreri public datasets indicated the virus sequences circulating in samples from Ecuador, suggesting a wide spread of these elements. Of note, we did not identify traces of the viral sequences in the M. roreri genome or DNA sequencing, restricting the possibility of these sequences representing endogenized elements. Discussion: To the best of our knowledge, this is the first report of viruses infecting the fungus of the genus Moniliophthora and only the third description of viruses that are able to parasite elements from the Marasmiaceae family.

The glutathione peroxidase family of Theobroma cacao: Involvement in the oxidative stress during witches' broom disease.

The glutathione peroxidases (GPXs) are enzymes which are part of the cell antioxidant system inhibiting the ROS-induced damages of membranes and proteins. In cacao (Theobroma cacao L.) genome, five GPX genes were identified. Cysteine insertion codons (UGU) were found in TcPHGPX, TcGPX2, TcGPX4, TcGPX6 and tryptophan insertion codon (UGG) in TcGPX8. Multiple alignments revealed conserved domains between TcGPXs and other plants and human GPXs. Homology modeling was performed using the Populus trichocarpa GPX5 structure as template, and the molecular modeling showed that TcGPXs have affinity with selenometionine in their active site. In silico analysis of the TcGPXs promoter region revealed the presence of conserved cis-elements related to biotic stresses and hormone responsiveness. The expression analysis of TcGPXs in cacao plantlet meristems infected by M. perniciosa showed that TcGPXs are most expressed in susceptible variety than in resistant one, mainly in disease stages in which oxidative stress and programmed cell death occurred. This data, associated with phylogenetic and location analysis suggested that TcGPXs may play a role in protecting cells from oxidative stress as a try of disease progression reduction. To our knowledge, this is the first study of the overall GPX family from T. cacao.

Moniliophthora perniciosa development: key genes involved in stress-mediated cell wall organization and autophagy.

Moniliophthora perniciosa is a basidiomycete responsible for the witches' broom disease in cacao (Theobroma cacao L.). Chitin synthase (CHS), chitinase (CHIT) and autophagy (ATG) genes have been associated to stress response preceding the formation of basidiocarp. An analysis of literature mining, interactomics and gene expression was developed to identify the main proteins related to development, cell wall organization and autophagy in M. perniciosa. TORC2 complex elements were identified and were involved in the response to the nutrient starvation during the fungus development stages preceding the basidiocarp formation. This complex interacted with target proteins related to cell wall synthesis and to polarization and cell division (FKS1, CHS, CDC42, ROM2). Autolysis and autophagy processes were associated to CHIT2, ATG8 and to the TORC1 complex (TOR1 and KOG1), which is central in the upstream signalization of the stress response due to nutrient starvation and growth regulation. Other important elements that participate to steps preceding basidiocarp formation were also identified (KOG1, SSZ1, GDI1, FKS1, CCD10, CKS1, CDC42, RHO1, AVO1, BAG7). Similar gene expression patterns during fungus reproductive structure formation and when treated by rapamycin (a nutritional related-autophagy stress agent) were observed: cell division related-genes were repressed while those related to autolysis/autophagy were overexpressed.

Involvement of structurally distinct cupuassu chitinases and osmotin in plant resistance to the fungus Moniliophthora perniciosa.

The cupuassu tree (Theobroma grandiflorum) is a crop of great economic importance to Brazil, mainly for its pulp and seeds, which are used in food industry. However, cupuassu fruit production is threatened by witches' broom disease caused by the fungus Moniliophthora perniciosa. As elements of its defense mechanisms, the plant can produce and accumulate pathogenesis-related (PR) proteins such as chitinases and osmotins. Here, we identified three cupuassu PR proteins (TgPR3, TgPR5 and TgPR8) from cupuassu-M. perniciosa interaction RNA-seq data. TgPR3 and TgPR8 corresponded to chitinases, and TgPR5 to osmotin; they are phylogenetically related to cacao and to Arabidopsis PR sequences involved in biotic and abiotic stress. The TgPR proteins' tridimensional structure was obtained through homology modeling, and molecular docking with chitin and chitosan showed that the TgPR proteins can interact with both cell wall molecules and presented a higher affinity for chitosan. TgPR gene expression was analyzed by RT-qPCR on resistant and susceptible cupuassu genotypes infected by M. perniciosa at 8, 24, 48 and 72 h after infection (hai). The TgPR genes showed higher expression in resistant plants compared to the susceptible ones, mainly for TgPR5 at 8 and 24 hai, while the expression was lower in the susceptible cupuassu plants. To our knowledge, this is the first in silico and in vitro reports of cupuassu PR protein. The data suggested that TgPRs could be involved in recognizing mechanisms of the plant's innate immune system through chitin receptors. Our results also suggest a putative role of chitinase/chitosanase for the TgPR5/osmotin.

Hydrosoluble phylloplane components of Theobroma cacao modulate the metabolism of Moniliophthora perniciosa spores during germination.

The surface of plants forms a defense barrier that directly inhibits the first point of contact of microorganisms with the host. To understand this defense mechanism in Moniliophthora perniciosa interaction with Theobroma cacao cv Catongo, the aim of this study was to compare the changes in protein expression in basidiospores of the fungus M. perniciosa in response the leaf water washes (LWW) of two contrasting cocoa varieties for resistance to witches' broom disease. A total of 8.1 × 108 basidiospores were used for each treatment containing washed leaf material. Germinated basidiospores in the absence of LWW were used as control. The proteomic analysis was performed by the 2D-PAGE technique combined with mass spectrometry (MS). Protein extraction was based on the SDS-dense method followed by sonication for cell disruption and phenol extraction. Sixty-four proteins had accumulation of variation when compared to the control (no LWW). Proteins were identified associated with energy (ATP synthase) and protein (BiP) metabolism, whose accumulation was reduced by basidiospores germinated in leaf wash from Catongo cocoa. The reduction in ATP synthase of the basidiospores germinated the Catongo LWW suggests a shift from aerobic to fermentative metabolism of the fungus in response to components of the LWW. Furthermore, proteins involved in virulence were identified along with fungal resistance to polyketide cyclase, glycoside hydrolase, multidrug transporter protein (SFM) and proteins related to oxidative stress and fermentation, such as catalase A and alcohol dehydrogenase (ADH). The data showed an effect of cocoa phylloplane substances on the germination of fungal basidiospores through differences in protein expression patterns in the presence of LWW of the CCN51 and Catongo genotypes. These results may reveal mechanisms of resistance, host susceptibility and pathogen virulence.

The pathogen Moniliophthora perniciosa promotes differential proteomic modulation of cacao genotypes with contrasting resistance to witches´ broom disease.

BACKGROUND: Witches' broom disease (WBD) of cacao (Theobroma cacao L.), caused by Moniliophthora perniciosa, is the most important limiting factor for the cacao production in Brazil. Hence, the development of cacao genotypes with durable resistance is the key challenge for control the disease. Proteomic methods are often used to study the interactions between hosts and pathogens, therefore helping classical plant breeding projects on the development of resistant genotypes. The present study compared the proteomic alterations between two cacao genotypes standard for WBD resistance and susceptibility, in response to M. perniciosa infection at 72 h and 45 days post-inoculation; respectively the very early stages of the biotrophic and necrotrophic stages of the cacao x M. perniciosa interaction. RESULTS: A total of 554 proteins were identified, being 246 in the susceptible Catongo and 308 in the resistant TSH1188 genotypes. The identified proteins were involved mainly in metabolism, energy, defense and oxidative stress. The resistant genotype showed more expressed proteins with more variability associated with stress and defense, while the susceptible genotype exhibited more repressed proteins. Among these proteins, stand out pathogenesis related proteins (PRs), oxidative stress regulation related proteins, and trypsin inhibitors. Interaction networks were predicted, and a complex protein-protein interaction was observed. Some proteins showed a high number of interactions, suggesting that those proteins may function as cross-talkers between these biological functions. CONCLUSIONS: We present the first study reporting the proteomic alterations of resistant and susceptible genotypes in the T. cacao x M. perniciosa pathosystem. The important altered proteins identified in the present study are related to key biologic functions in resistance, such as oxidative stress, especially in the resistant genotype TSH1188, that showed a strong mechanism of detoxification. Also, the positive regulation of defense and stress proteins were more evident in this genotype. Proteins with significant roles against fungal plant pathogens, such as chitinases, trypsin inhibitors and PR 5 were also identified, and they may be good resistance markers. Finally, important biological functions, such as stress and defense, photosynthesis, oxidative stress and carbohydrate metabolism were differentially impacted with M. perniciosa infection in each genotype.

The selenium-binding protein of Theobroma cacao: A thermostable protein involved in the witches' broom disease resistance.

The selenium-binding proteins are known to be inducers of apoptosis in human and animals, and have been studied as target for the treatment of various types of cancer. In plants, SBP expression has been related to abiotic and biotic stress resistance. The SBP from Theobroma cacao (TcSBP) was first identified from a cocoa-Moniliophthora perniciosa cDNA library. The present study provides details on the TcSBP gene and protein structure. Multiple alignments revealed conserved domains between SBP from plants, human and archea. Homology modeling and molecular docking were performed and showed that the TcSBP has affinity to selenite in the active CSSC site. This result was confirmed by circular dichroism of the recombinant TcSBP, which also presented thermostable behavior. RT-qPCR analysis showed that TcSBP was differentially expressed in resistant vs susceptible cacao varieties inoculated by M. perniciosa and its expression was probably due to hormone induction via cis-regulating elements present in its promotor. The presence of the CSSC domain suggested that TcSBP acted by altering oxidation/reduction of proteins during H2O2 production and programmed cell death in the final stages of the witches' broom disease. To our knowledge, this is the first in silico and in vitro analysis of the SBP from cacao.

Witches' broom resistant genotype CCN51 shows greater diversity of symbiont bacteria in its phylloplane than susceptible genotype catongo.

BACKGROUND: Theobroma cacao L. (cacao) is a perennial tropical tree, endemic to rainforests of the Amazon Basin. Large populations of bacteria live on leaf surfaces and these phylloplane microorganisms can have important effects on plant health. In recent years, the advent of high-throughput sequencing techniques has greatly facilitated studies of the phylloplane microbiome. In this study, we characterized the bacterial microbiome of the phylloplane of the catongo genotype (susceptible to witch's broom) and CCN51 (resistant). Bacterial microbiome was determined by sequencing the V3-V4 region of the bacterial 16S rRNA gene. RESULTS: After the pre-processing, a total of 1.7 million reads were considered. In total, 106 genera of bacteria were characterized. Proteobacteria was the predominant phylum in both genotypes. The exclusive genera of Catongo showed activity in the protection against UV radiation and in the transport of substrates. CCN51 presented genus that act in the biological control and inhibition in several taxonomic groups. Genotype CCN51 presented greater diversity of microorganisms in comparison to the Catongo genotype and the total community was different between both. Scanning electron microscopy analysis of leaves revealed that on the phylloplane, many bacterial occur in large aggregates in several regions of the surface and isolated nearby to the stomata. CONCLUSIONS: We describe for the first time the phylloplane bacterial communities of T. cacao. The Genotype CCN51, resistant to the witch's broom, has a greater diversity of bacterial microbioma in comparison to Catongo and a greater amount of exclusive microorganisms in the phylloplane with antagonistic action against phytopathogens.

Genetic diversity and gene flow of the threatened Brazilian endemic parrotfish Scarus trispinosus (Valenciennes, 1840).

The greenback parrotfish, Scarus trispinosus, is the largest herbivorous fish inhabiting Southwestern Atlantic reefs, and was recently included in the IUCN red list of threatened species as endangered due to the overexploitation of their populations. The aim of this work was to evaluate the existence of structured populations (i.e. genetic unities) along a coast of approximately 2000 km of the NE Brazilian coast. The transferability of 17 primers synthesized for Scarus rubroviolaceus was tested for S. trispinosus and five transferable loci were validated and used. Two localities within the Abrolhos Bank, off the Central Brazilian coast (Corumbau and Caravelas) and in close proximity to the MPA, which encompasses the largest remnants of the S. trispinosus population, exhibited higher levels of genetic richness. Remaining locations, Pernambuco, Porto Seguro and Rio Grande do Norte exhibited lower genetic diversity. We found no genetic differences among sampled localities however, when those samples were gathered into latitudinal groups (northern vs southern) a subtle but significant genetic substructuring was revealed. It is proposed that a combination of high local individual admixture favoured by habitat connectivity drived genetic homogeneity at regional scales while larval dispersal contributed to heterogeneities observed at large scales maintaining gene flow through oceanographic currents.

Genome sequence and effectorome of Moniliophthora perniciosa and Moniliophthora roreri subpopulations.

BACKGROUND: The hemibiotrophic pathogens Moniliophthora perniciosa (witches' broom disease) and Moniliophthora roreri (frosty pod rot disease) are among the most important pathogens of cacao. Moniliophthora perniciosa has a broad host range and infects a variety of meristematic tissues in cacao plants, whereas M. roreri infects only pods of Theobroma and Herrania genera. Comparative pathogenomics of these fungi is essential to understand Moniliophthora infection strategies, therefore the detection and in silico functional characterization of effector candidates are important steps to gain insight on their pathogenicity. RESULTS: Candidate secreted effector proteins repertoire were predicted using the genomes of five representative isolates of M. perniciosa subpopulations (three from cacao and two from solanaceous hosts), and one representative isolate of M. roreri from Peru. Many putative effectors candidates were identified in M. perniciosa: 157 and 134 in cacao isolates from Bahia, Brazil; 109 in cacao isolate from Ecuador, 92 and 80 in wild solanaceous isolates from Minas Gerais (Lobeira) and Bahia (Caiçara), Brazil; respectively. Moniliophthora roreri showed the highest number of effector candidates, a total of 243. A set of eight core effectors were shared among all Moniliophthora isolates, while others were shared either between the wild solanaceous isolates or among cacao isolates. Mostly, candidate effectors of M. perniciosa were shared among the isolates, whereas in M. roreri nearly 50% were exclusive to the specie. In addition, a large number of cell wall-degrading enzymes characteristic of hemibiotrophic fungi were found. From these, we highlighted the proteins involved in cell wall modification, an enzymatic arsenal that allows the plant pathogens to inhabit environments with oxidative stress, which promotes degradation of plant compounds and facilitates infection. CONCLUSIONS: The present work reports six genomes and provides a database of the putative effectorome of Moniliophthora, a first step towards the understanding of the functional basis of fungal pathogenicity.

Deciphering the Theobroma cacao self-incompatibility system: from genomics to diagnostic markers for self-compatibility.

Cocoa self-compatibility is an important yield factor and has been described as being controlled by a late gameto-sporophytic system expressed only at the level of the embryo sac. It results in gametic non-fusion and involves several loci. In this work, we identified two loci, located on chromosomes 1 and 4 (CH1 and CH4), involved in cocoa self-incompatibility by two different processes. Both loci are responsible for gametic selection, but only one (the CH4 locus) is involved in the main fruit drop. The CH1 locus acts prior to the gamete fusion step and independently of the CH4 locus. Using fine-mapping and genome-wide association studies, we focused analyses on restricted regions and identified candidate genes. Some of them showed a differential expression between incompatible and compatible reactions. Immunolocalization experiments provided evidence of CH1 candidate genes expressed in ovule and style tissues. Highly polymorphic simple sequence repeat (SSR) diagnostic markers were designed in the CH4 region that had been identified by fine-mapping. They are characterized by a strong linkage disequilibrium with incompatibility alleles, thus allowing the development of efficient diagnostic markers predicting self-compatibility and fruit setting according to the presence of specific alleles or genotypes. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified, thus allowing screening for self-compatible plants in cocoa populations.

Genome-wide identification and characterization of cacao WRKY transcription factors and analysis of their expression in response to witches' broom disease.

Transcriptional regulation, led by transcription factors (TFs) such as those of the WRKY family, is a mechanism used by the organism to enhance or repress gene expression in response to stimuli. Here, we report on the genome-wide analysis of the Theobroma cacao WRKY TF family and also investigate the expression of WRKY genes in cacao infected by the fungus Moniliophthora perniciosa. In the cacao genome, 61 non-redundant WRKY sequences were found and classified in three groups (I to III) according to the WRKY and zinc-finger motif types. The 61 putative WRKY sequences were distributed on the 10 cacao chromosomes and 24 of them came from duplication events. The sequences were phylogenetically organized according to the general WRKY groups. The phylogenetic analysis revealed that subgroups IIa and IIb are sister groups and share a common ancestor, as well as subgroups IId and IIe. The most divergent groups according to the plant origin were IIc and III. According to the phylogenetic analysis, 7 TcWRKY genes were selected and analyzed by RT-qPCR in susceptible and resistant cacao plants infected (or not) with M. perniciosa. Some TcWRKY genes presented interesting responses to M. perniciosa such as Tc01_p014750/Tc06_p013130/AtWRKY28, Tc09_p001530/Tc06_p004420/AtWRKY40, Tc04_p016130/AtWRKY54 and Tc10_p016570/ AtWRKY70. Our results can help to select appropriate candidate genes for further characterization in cacao or in other Theobroma species.

Proteomic analysis during of spore germination of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao.

BACKGROUND: Moniliophthora perniciosa is a phytopathogenic fungus responsible for witches' broom disease of cacao trees (Theobroma cacao L.). Understanding the molecular events during germination of the pathogen may enable the development of strategies for disease control in these economically important plants. In this study, we determined a comparative proteomic profile of M. perniciosa basidiospores during germination by two-dimensional SDS-PAGE and mass spectrometry. RESULTS: A total of 316 proteins were identified. Molecular changes during the development of the germinative tube were identified by a hierarchical clustering analysis based on the differential accumulation of proteins. Proteins associated with fungal filamentation, such as septin and kinesin, were detected only 4 h after germination (hag). A transcription factor related to biosynthesis of the secondary metabolite fumagillin, which can form hybrids with polyketides, was induced 2 hag, and polyketide synthase was observed 4 hag. The accumulation of ATP synthase, binding immunoglobulin protein (BiP), and catalase was validated by western blotting. CONCLUSIONS: In this study, we showed variations in protein expression during the early germination stages of fungus M. perniciosa. Proteins associated with fungal filamentation, and consequently with virulence, were detected in basidiospores 4 hag., for example, septin and kinesin. We discuss these results and propose a model of the germination of fungus M. perniciosa. This research can help elucidate the mechanisms underlying basic processes of host invasion and to develop strategies for control of the disease.

Genome size, cytogenetic data and transferability of EST-SSRs markers in wild and cultivated species of the genus Theobroma L. (Byttnerioideae, Malvaceae).

The genus Theobroma comprises several trees species native to the Amazon. Theobroma cacao L. plays a key economic role mainly in the chocolate industry. Both cultivated and wild forms are described within the genus. Variations in genome size and chromosome number have been used for prediction purposes including the frequency of interspecific hybridization or inference about evolutionary relationships. In this study, the nuclear DNA content, karyotype and genetic diversity using functional microsatellites (EST-SSR) of seven Theobroma species were characterized. The nuclear content of DNA for all analyzed Theobroma species was 1C = ~ 0.46 pg. These species presented 2n = 20 with small chromosomes and only one pair of terminal heterochromatic bands positively stained (CMA+/DAPI- bands). The small size of Theobroma ssp. genomes was equivalent to other Byttnerioideae species, suggesting that the basal lineage of Malvaceae have smaller genomes and that there was an expansion of 2C values in the more specialized family clades. A set of 20 EST-SSR primers were characterized for related species of Theobroma, in which 12 loci were polymorphic. The polymorphism information content (PIC) ranged from 0.23 to 0.65, indicating a high level of information per locus. Combined results of flow cytometry, cytogenetic data and EST-SSRs markers will contribute to better describe the species and infer about the evolutionary relationships among Theobroma species. In addition, the importance of a core collection for conservation purposes is highlighted.

Protein profile and protein interaction network of Moniliophthora perniciosa basidiospores.

BACKGROUND: Witches' broom, a disease caused by the basidiomycete Moniliophthora perniciosa, is considered to be the most important disease of the cocoa crop in Bahia, an area in the Brazilian Amazon, and also in the other countries where it is found. M. perniciosa germ tubes may penetrate into the host through intact or natural openings in the cuticle surface, in epidermis cell junctions, at the base of trichomes, or through the stomata. Despite its relevance to the fungal life cycle, basidiospore biology has not been extensively investigated. In this study, our goal was to optimize techniques for producing basidiospores for protein extraction, and to produce the first proteomics analysis map of ungerminated basidiospores. We then presented a protein interaction network by using Ustilago maydis as a model. RESULTS: The average pileus area ranged from 17.35 to 211.24 mm(2). The minimum and maximum productivity were 23,200 and 6,666,667 basidiospores per basidiome, respectively. The protein yield in micrograms per million basidiospores were approximately 0.161; 2.307, and 3.582 for germination times of 0, 2, and 4 h after germination, respectively. A total of 178 proteins were identified through mass spectrometry. These proteins were classified according to their molecular function and their involvement in biological processes such as cellular energy production, oxidative metabolism, stress, protein synthesis, and protein folding. Furthermore, to better understand the expression pattern, signaling, and interaction events of spore proteins, we presented an interaction network using orthologous proteins from Ustilago maydis as a model. Most of the orthologous proteins that were identified in this study were not clustered in the network, but several of them play a very important role in hypha development and branching. CONCLUSIONS: The quantities of basidiospores 7 × 10(9); 5.2 × 10(8), and 6.7 × 10(8) were sufficient to obtain enough protein mass for the three 2D-PAGE replicates, for the 0, 2, and 4 h-treatments, respectively. The protein extraction method that is based on sedimentation, followed by sonication with SDS-dense buffer, and phenolic extraction, which was utilized in this study, was effective, presenting a satisfactory resolution and reproducibility for M. perniciosa basidiospores. This report constitutes the first comprehensive study of protein expression during the ungerminated stage of the M. perniciosa basidiospore. Identification of the spots observed in the reference gel enabled us to know the main molecular interactions involved in the initial metabolic processes of fungal development.