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      Global analyses of Ceratocystis cacaofunesta mitochondria: from genome to proteome

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          Abstract

          Background

          The ascomycete fungus Ceratocystis cacaofunesta is the causal agent of wilt disease in cacao, which results in significant economic losses in the affected producing areas. Despite the economic importance of the Ceratocystis complex of species, no genomic data are available for any of its members. Given that mitochondria play important roles in fungal virulence and the susceptibility/resistance of fungi to fungicides, we performed the first functional analysis of this organelle in Ceratocystis using integrated “omics” approaches.

          Results

          The C. cacaofunesta mitochondrial genome (mtDNA) consists of a single, 103,147-bp circular molecule, making this the second largest mtDNA among the Sordariomycetes. Bioinformatics analysis revealed the presence of 15 conserved genes and 37 intronic open reading frames in C. cacaofunesta mtDNA. Here, we predicted the mitochondrial proteome (mtProt) of C. cacaofunesta, which is comprised of 1,124 polypeptides - 52 proteins that are mitochondrially encoded and 1,072 that are nuclearly encoded. Transcriptome analysis revealed 33 probable novel genes. Comparisons among the Gene Ontology results of the predicted mtProt of C. cacaofunesta, Neurospora crassa and Saccharomyces cerevisiae revealed no significant differences. Moreover, C. cacaofunesta mitochondria were isolated, and the mtProt was subjected to mass spectrometric analysis. The experimental proteome validated 27% of the predicted mtProt. Our results confirmed the existence of 110 hypothetical proteins and 7 novel proteins of which 83 and 1, respectively, had putative mitochondrial localization.

          Conclusions

          The present study provides the first partial genomic analysis of a species of the Ceratocystis genus and the first predicted mitochondrial protein inventory of a phytopathogenic fungus. In addition to the known mitochondrial role in pathogenicity, our results demonstrated that the global function analysis of this organelle is similar in pathogenic and non-pathogenic fungi, suggesting that its relevance in the lifestyle of these organisms should be based on a small number of specific proteins and/or with respect to differential gene regulation. In this regard, particular interest should be directed towards mitochondrial proteins with unknown function and the novel protein that might be specific to this species. Further functional characterization of these proteins could enhance our understanding of the role of mitochondria in phytopathogenicity.

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          Most cited references72

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          Translocation of proteins into mitochondria.

          About 10% to 15% of the nuclear genes of eukaryotic organisms encode mitochondrial proteins. These proteins are synthesized in the cytosol and recognized by receptors on the surface of mitochondria. Translocases in the outer and inner membrane of mitochondria mediate the import and intramitochondrial sorting of these proteins; ATP and the membrane potential are used as energy sources. Chaperones and auxiliary factors assist in the folding and assembly of mitochondrial proteins into their native, three-dimensional structures. This review summarizes the present knowledge on the import and sorting of mitochondrial precursor proteins, with a special emphasis on unresolved questions and topics of current research.
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            Pfam is a large collection of protein multiple sequence alignments and profile hidden Markov models. Pfam is available on the WWW in the UK at http://www.sanger.ac.uk/Software/Pfam/, in Sweden at http://www.cgr.ki.se/Pfam/ and in the US at http://pfam.wustl.edu/. The latest version (4.3) of Pfam contains 1815 families. These Pfam families match 63% of proteins in SWISS-PROT 37 and TrEMBL 9. For complete genomes Pfam currently matches up to half of the proteins. Genomic DNA can be directly searched against the Pfam library using the Wise2 package.
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              RNA-Seq-quantitative measurement of expression through massively parallel RNA-sequencing.

              The ability to quantitatively survey the global behavior of transcriptomes has been a key milestone in the field of systems biology, enabled by the advent of DNA microarrays. While this approach has literally transformed our vision and approach to cellular physiology, microarray technology has always been limited by the requirement to decide, a priori, what regions of the genome to examine. While very high density tiling arrays have reduced this limitation for simpler organisms, it remains an obstacle for larger, more complex, eukaryotic genomes. The recent development of "next-generation" massively parallel sequencing (MPS) technologies by companies such as Roche (454 GS FLX), Illumina (Genome Analyzer II), and ABI (AB SOLiD) has completely transformed the way in which quantitative transcriptomics can be done. These new technologies have reduced both the cost-per-reaction and time required by orders of magnitude, making the use of sequencing a cost-effective option for many experimental approaches. One such method that has recently been developed uses MPS technology to directly survey the RNA content of cells, without requiring any of the traditional cloning associated with EST sequencing. This approach, called "RNA-seq", can generate quantitative expression scores that are comparable to microarrays, with the added benefit that the entire transcriptome is surveyed without the requirement of a priori knowledge of transcribed regions. The important advantage of this technique is that not only can quantitative expression measures be made, but transcript structures including alternatively spliced transcript isoforms, can also be identified. This article discusses the experimental approach for both sample preparation and data analysis for the technique of RNA-seq.
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                Author and article information

                Contributors
                Journal
                BMC Genomics
                BMC Genomics
                BMC Genomics
                BioMed Central
                1471-2164
                2013
                11 February 2013
                : 14
                : 91
                Affiliations
                [1 ]Laboratório de Genômica e Expressão, Departamento de Genética Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, CEP: 13083-970, Campinas, São Paulo, Brasil
                [2 ]Laboratório Nacional de Biociências-LNBio, Centro Nacional de Pesquisa em Energia e Materiais, CEP: 13083-970, Campinas, São Paulo, Brasil
                [3 ]Centro Nacional de Processamento de Alto Desempenho, Universidade Estadual de Campinas, CEP: 13083-970, Campinas, São Paulo, Brasil
                [4 ]High-Throughput Sequencing Facility, University of North Carolina, 2234 Nelson Highway, Chapel Hill, NC, NC 27516, USA
                [5 ]Sustainable Perennial Crops Laboratory, USDA/ARS, 10300 Baltimore Ave, Beltsville, MD, 20705, USA
                Article
                1471-2164-14-91
                10.1186/1471-2164-14-91
                3605234
                23394930
                f4ad683f-27c0-4e9e-a3fa-18a165cc866c
                Copyright ©2013 Ambrosio et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 9 August 2012
                : 27 January 2013
                Categories
                Research Article

                Genetics
                cacao wilt disease,ceratocystis cacaofunesta,mitogenomics,mitochondrial proteome,fungal virulence

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