Analysis Of The Ergosterol Biosynthesis Pathway Cloning, Molecular Characterization And Phylogeny Of Lanosterol 14 α-demethylase (erg11) Gene Of Moniliophthora Perniciosa

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches’ broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed.ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). 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Transcriptome analysis reveals putative genes involved in the lipid metabolism of chaulmoogra oil biosynthesis in Carpotroche brasiliensis (Raddi) A.Gray, a tropical tree species

Chaulmoogra oil is found in the seeds of Carpotroche brasiliensis (Raddi) Endl. (syn. Mayna brasiliensis Raddi), an oil tree of the Achariaceae family and native to Brazil’s Atlantic Forest biome, which is considered the fifth most important biodiversity hotspot in the world. Its main constituents are cyclopentenic fatty acids. Chaulmoogra oil has economic potential because of its use in the cosmetics industry and as a drug with anti-tumor activity. The mechanisms related to the regulation of oil biosynthesis in C. brasiliensis seeds are not fully understood, especially from a tissue-specific perspective. In this study, we applied a de novo transcriptomic approach to investigate the transcripts involved in the lipid pathways of C. brasiliensis and to identify genes involved in lipid biosynthesis. Comparative analysis of gene orthology, expression analysis and visualization of metabolic lipid networks were performed, using data obtained from high-throughput sequencing (RNAseq) of 24 libraries of vegetative and reproductive tissues of C. brasiliensis. Approximately 10.4 million paired-end reads (Phred (Q) > 20) were generated and re-assembled into 107,744 unigenes, with an average length of 340 base pairs (bp). The analysis of transcripts from different tissues identified 1131 proteins involved in lipid metabolism and transport and 13 pathways involved in lipid biosynthesis, degradation, transport, lipid bodies, and lipid constituents of membranes. This is the first transcriptome study of C. brasiliensis, providing basic information for biotechnological applications of great use for the species, which will help understand chaulmoogra oil biosynthesis

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. © 2018 The Author(s).This work was done in the frame of the International Consortium in Advanced Biology (CIBA; https://www.ciba-network.org). The authors thank the Molecular Plant Pathology Laboratory and the Plant Pathology Laboratory at INIAP personnel for their assistance in obtaining the DNAs, Dr Carmen Suarez Capello for her kind assistance in Ecuador, and the Núcleo de Biologia Computacional e Gestão de Informações Biotecnológicas - UESC (NBCGIB), and Copenhague University for providing bioinformatics facility. Data sets were processed in sagarana HPC cluster, CPAD-ICB-UFMG. The authors would also like to thank Dr. Claudia Fortes Ferreira (Embrapa CNPMF, Brazil) and Dr. Raul Renné Valle (CEPLAC/CEPEC, Brazil) for English language revision. We are also grateful to Ivanna Michelle Meraz Pérez for helping translating an early version of this manuscript and to the anonymous reviewers who provided helpful comments to our work. KPG, FM and CPP were supported by research fellowship Pq-1 from CNPq. National Council for Scientific Development (CNPq) n° 311759/2014–9. CSB acknowledges FAPESB (Foundation for Research Support of the State of Bahia) for supporting her with a research assistantship during her Master’s Programme

Protein Extraction For Proteome Analysis From Cacao Leaves And Meristems, Organs Infected By Moniliophthora Perniciosa, The Causal Agent Of The Witches' Broom Disease

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