Protein Profile And Protein Interaction Network Of Moniliophthora Perniciosa Basidiospores

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)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 x 10(9); 5.2 x 10(8), and 6.7 x 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.16Bahia Research Foundation (FAPESB) [PNE0005/2011]National Council for Scientific Development (CNPq) [305309/2012-9]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

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

Abstract 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