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Witches' broom disease (WBD) affects cocoa trees (Theobroma cacao L.) and is caused by the fungus Moniliophthora perniciosa that grows in the apoplast in its biotrophic phase and later progresses into the tissues, causing serious losses in the production of cocoa beans. Therefore, the apoplast of T. cacao can provide important defense responses during the interaction with M. perniciosa. In this work, the protein profile of the apoplast of the T. cacao genotypes Catongo, susceptible to WBD, and CCN-51, resistant one, was evaluated. The leaves of T. cacao were collected from asymptomatic plants grown in a greenhouse (GH) and from green witches' brooms grown under field (FD) conditions for extraction of apoplastic washing fluid (AWF). AWF was used in proteomic and enzymatic analysis. A total of 14 proteins were identified in Catongo GH and six in Catongo FD, with two proteins being common, one up-accumulated, and one down-accumulated. In CCN-51, 19 proteins were identified in the GH condition and 13 in FD, with seven proteins being common, one up-accumulated, and six down-accumulated. Most proteins are related to defense and stress in both genotypes, with emphasis on pathogenesis-related proteins (PR): PR-2 (β-1,3-glucanases), PR-3 and PR-4 (chitinases), PR-5 (thaumatine), PR-9 (peroxidases), and PR-14 (lipid transfer proteins). Furthermore, proteins from microorganisms were detected in the AWF. The enzymatic activities of PR-3 showed a significant increase (p < 0.05) in Catongo GH and PR-2 activity (p < 0.01) in CCN-51 FD. The protein profile of the T. cacao apoplastome offers insight into the defense dynamics that occur in the interaction with the fungus M. perniciosa and offers new insights in exploring future WBD control strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 mm2. 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 × 109; 5.2×108, and 6.7 × 108 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. [ABSTRACT FROM AUTHOR]

Background The production and accumulation of pathogenesis-related proteins (PR proteins) in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. A pathogenesis-related protein 4 cDNA was identified from a cacao- Moniliophthora perniciosa interaction cDNA library and named TcPR-4b. Results TcPR-4b presents a Barwin domain with six conserved cysteine residues, but lacks the chitinbinding site. Molecular modeling of TcPR-4b confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. In the cacao genome, TcPR-4b belonged to a small multigene family organized mainly on chromosome 5. TcPR-4b RT-qPCR analysis in resistant and susceptible cacao plants infected by M. perniciosa showed an increase of expression at 48 hours after infection (hai) in both cacao genotypes. After the initial stage (24-72 hai), the TcPR-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection (45-90 days after infection). The recombinant TcPR-4b protein showed RNase, and bivalent ions dependent-DNase activity, but no chitinase activity. Moreover, TcPR-4b presented antifungal action against M. perniciosa, and the reduction of M. perniciosa survival was related to ROS production in fungal hyphae. Conclusion To our knowledge, this is the first report of a PR-4 showing simultaneously RNase, DNase and antifungal properties, but no chitinase activity. Moreover, we showed that the antifungal activity of TcPR-4b is directly related to RNase function. In cacao, TcPR-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the PCD mechanism, in resistant and susceptible cacao genotypes, respectively. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]