Protein profile and protein interaction network of Moniliophthora perniciosa basidiospores

Abstract

BackgroundWitches’ 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.ResultsThe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0753-0) contains supplementary material, which is available to authorized users.

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

“…The shoot apex of the plantlets was inoculated with a basidiospore suspension of inoculum Mp4145, from CEPLAC/CEPEC, Ilhéus, Bahia, Brazil, accession number 4145 (CEPLAC/ CEPEC phytopathological M. perniciosa collection CEGEN N° 109/2013/SECEXCGEN). The inoculum was prepared as described by Mares and colleagues (2016) [25]. Three to four weeks old cacao seedling (plantlets) were subjected to droplet inoculation [5], about 550 seedlings were inoculated in each treatment.…”

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

“…The inoculum 4145 was obtained from "dry brooms" collected at CEPEC, Ilhéus, Bahia, Brazil, and prepared as described by Mares and colleagues (2016) [25]. Inoculations were conducted using the droplet method [54,95].…”

“…The 2D-PAGE (two-dimensional electrophoresis) followed by mass spectrometry was already used in studies involving M. perniciosa, such as the proteomic analyses of in vitro basidiospores germination [24], protein networks of basidiospores [25] and evaluation of M. perniciosa isolates differing in virulence on cacao seedlings [26]. Similarly, cacao proteomic studies such as protocol optimization to protein extraction [27], somatic and zygotic embryogenesis evaluation [28], seeds development and fruit ripening [29] and phylloplane protein identification in different genotypes of cacao [30] were also carried out.…”

Assessing the Proteomic Activity of the Venom of the AntEctatomma tuberculatum(Hymenoptera: Formicidae: Ectatomminae)

“…The 2D Electrophoresis was performed on 12.5% gel in a SE600 Ruby Vertical Electrophoresis System (Hoefer). The running conditions were similar to those used by Mares and collaborators [29], staining in 0.08% colloidal Coomassie [30] for 5 days, followed by destaining in distilled water for 5 days with daily water replacements to remove excess of colloidal Coomassie.…”