Genome-wide DNA methylation analysis of Metarhizium anisopliae during tick mimicked infection condition

Background: The Metarhizium genus harbors important entomopathogenic fungi. These species have been widely explored as biological control agents, and strategies to improve the fungal virulence are under investigation. Thus, the interaction between Metarhizium species and susceptible hosts have been explored employing different methods in order to characterize putative virulence determinants. However, the impact of epigenetic modulation on the infection cycle of Metarhizium is still an open topic. Among the different epigenetic modifications, DNA methylation of cytosine bases is an important mechanism to control gene expression in several organisms. To better understand if DNA methylation can govern Metarhizium-host interactions, the genome-wide DNA methylation profile of Metarhizium anisopliae was explored in two conditions: tick mimicked infection and a saprophytic-like control. Results: Using a genome wide DNA methylation profile based on bisulfite sequencing (BS-Seq), approximately 0.60% of the total cytosines were methylated in saprophytic-like condition, which was lower than the DNA methylation level (0.89%) in tick mimicked infection condition. A total of 670 mRNA genes were found to be putatively methylated, with 390 mRNA genes uniquely methylated in the tick mimicked infection condition. GO terms linked to response to stimuli, cell wall morphogenesis, cytoskeleton morphogenesis and secondary metabolism biosynthesis were over-represented in the tick mimicked infection condition, suggesting that energy metabolism is directed towards the regulation of genes associated with infection. However, recognized virulence determinants known to be expressed at distinct infection steps, such as the destruxin backbone gene and the collagen-like protein gene Mcl1, were found methylated, suggesting that a dynamic pattern of methylation could be found during the infectious process. These results were further endorsed employing RT-qPCR from cultures treated or not with the DNA methyltransferase inhibitor 5-Azacytidine. Conclusions: The set of genes here analyzed focused on secondary metabolites associated genes, known to be involved in several processes, including virulence. The BS-Seq pipeline and RT-qPCR analysis employing 5- Azacytidine led to identification of methylated virulence genes in M. anisopliae. The results provided evidences that DNA methylation in M. anisopliae comprises another layer of gene expression regulation, suggesting a main role of DNA methylation regulating putative virulence determinants during M. anisopliae infection cycle

Transcriptomic analysis reveals that mTOR pathway can be modulated in macrophage cells by the presence of cryptococcal cells

Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, a high mortality disease. The development of such disease depends on the interaction of fungal cells with macrophages, in which they can reside and replicate. In order to dissect the molecular mechanisms by which cryptococcal cells modulate the activity of macrophages, a genome-scale comparative analysis of transcriptional changes in macrophages exposed to Cryptococcus spp. was conducted. Altered expression of nearly 40 genes was detected in macrophages exposed to cryptococcal cells. The major processes were associated with the mTOR pathway, whose associated genes exhibited decreased expression in macrophages incubated with cryptococcal cells. Phosphorylation of p70S6K and GSK-3β was also decreased in macrophages incubated with fungal cells. In this way, Cryptococci presence could drive the modulation of mTOR pathway in macrophages possibly to increase the survival of the pathogen

De novo transcriptome analysis of Hevea brasiliensis tissues by RNA-seq and screening for molecular markers

Abstract\ud \ud Background\ud The rubber tree, Hevea brasiliensis, is a species native to the Brazilian Amazon region and it supplies almost all the world’s natural rubber, a strategic raw material for a variety of products. One of the major challenges for developing rubber tree plantations is adapting the plant to biotic and abiotic stress. Transcriptome analysis is one of the main approaches for identifying the complete set of active genes in a cell or tissue for a specific developmental stage or physiological condition.\ud \ud \ud Results\ud Here, we report on the sequencing, assembling, annotation and screening for molecular markers from a pool of H. brasiliensis tissues. A total of 17,166 contigs were successfully annotated. Then, 2,191 Single Nucleotide Variation (SNV) and 1.397 Simple Sequence Repeat (SSR) loci were discriminated from the sequences. From 306 putative, mainly non-synonymous SNVs located in CDS sequences, 191 were checked for their ability to characterize 23 Hevea genotypes by an allele-specific amplification technology. For 172 (90%), the nucleotide variation at the predicted genomic location was confirmed, thus validating the different steps from sequencing to the in silico detection of the SNVs.\ud \ud \ud Conclusions\ud This is the first study of the H. brasiliensis transcriptome, covering a wide range of tissues and organs, leading to the production of the first developed SNP markers. This process could be amplified to a larger set of in silico detected SNVs in expressed genes in order to increase the marker density in available and future genetic maps. The results obtained in this study will contribute to the H. brasiliensis genetic breeding program focused on improving of disease resistance and latex yield.CNPqPROSULCAPESFUNDHER

Esteróis e triterpenos de Ganoderma australe (Fr.) Pat. com perspectivas de uso medicinal

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas.Com o objetivo de verificar a presença de triterpenóides bioativos em G. australe foram coletados e identificados 15 basidiomas, dos quais também foram isoladas culturas dicarióticas. A partir de alguns desses basidiomas foi obtido um extrato metanólico, que foi particionado em hexano, clorofórmio e acetato de etila. Esses extratos foram fracionados através de cromatografia em coluna de gel de sílica. O fracionamento foi biomonitorado através do método de difusão em gel, com o qual se verificou a presença de atividade antibacteriana nas frações testadas. As frações foram ativas principalmente contra as cepas Gram-positivas. Nas frações obtidas foram identificados os esteróis: 5a-ergost-7-en-3b-ol, 5a-ergost-7,22-dien-3b-ol, 5,8-epidioxi-5a,8a-ergost-6,22-dien-3b-ol e os triterpenos designados como: ácidos aplanoxídicos A, C, G e F. Este estudo abre diversas perspectivas de investigações, tais como a pesquisa de novas substâncias com propriedades antibacterianas produzidas por esta espécie de fungo, a possibilidade de determinação de outras atividades biológicas para as substâncias isoladas e estudos com as culturas dicarióticas depositadas na coleção do Laboratório de Antibióticos da UFSC

Unravelling the transcriptome profile of the swine respiratory tract mycoplasmas

The swine respiratory ciliary epithelium is mainly colonized by Mycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis. While colonization by M. flocculare is virtually asymptomatic, M. hyopneumoniae and M. hyorhinis infections may cause respiratory disease. Information regarding transcript structure and gene abundance provides valuable insight into gene function and regulation, which has not yet been analyzed on a genome-wide scale in these Mycoplasma species. In this study, we report the construction of transcriptome maps for M. hyopneumoniae, M. flocculare and M. hyorhinis, which represent data for conducting comparative studies on the transcriptional repertory. For each species, three cDNA libraries were generated, yielding averages of 415,265, 695,313 and 93,578 reads for M. hyopneumoniae, M. flocculare and M. hyorhinis, respectively, with an average read length of 274 bp. The reads mapping showed that 92%, 98% and 96% of the predicted genes were transcribed in the M. hyopneumoniae, M. flocculare and M. hyorhinis genomes, respectively. Moreover, we showed that the majority of the genes are co-expressed, confirming the previously predicted transcription units. Finally, our data defined the RNA populations in detail, with the map transcript boundaries and transcription unit structures on a genome-wide scale

Summary of adhesins associated to pathogenicity, genome organization and transcription profile comparison.

<p>NA - not analyzed; NP - not present.</p><p>*MHP - <i>M. hyopneumoniae</i> 7448; MFL - <i>M. flocculare</i> ATCC 27716.</p><p>Summary of adhesins associated to pathogenicity, genome organization and transcription profile comparison.</p

Genes mapped with the highest number of transcript reads in <i>M. hyorhinis</i> genome.

<p>Genes mapped with the highest number of transcript reads in <i>M. hyorhinis</i> genome.</p

Genes mapped with the highest number of transcript reads in the <i>M. hyopneumoniae</i> genome.

<p>Genes mapped with the highest number of transcript reads in the <i>M. hyopneumoniae</i> genome.</p