Desenvolvimento de ferramentas para a construção e análise de mutantes funcionais no fungo entomopatogênico Metarhizium anisopliae

O gênero Metarhizium é composto por fungos entomopatogênicos de elevada importância, como Metarhizium anisopliae, e são extensivamente utilizados para o controle de artrópodes praga como Helicoverpa armigera e Spodoptera frugiperda, ambas pragas de diversas culturas como milho, soja e algodão há mais de cem anos. Atualmente, esporos de Metarhizium spp. compõe mais de um terço de todas formulações fúngicas comerciais para o controle biológico. Ainda assim, estas formulações carecem de eficiência quando comparadas ao controle químico. Para mudar este cenário, diversas abordagens estão sendo empregadas, como o estudo de determinantes de virulência e a engenharia genética para a obtenção de linhagens melhoradas para o controle biológico. No entanto, a escassez de ferramentas moleculares padronizadas aplicáveis aos fungos utilizados em controle biológico atrasa o desenvolvimento de linhagens competitivas, e dificulta o estudo do impacto, disseminação e evolução que esses organismos terão na natureza. A fim de ampliar o repertório de ferramentas moleculares para se trabalhar com M. anisopliae, foi construído, no presente trabalho, um cassette para a expressão do sistema repórter Katushka, uma proteína fluorescente far-red de alto brilho, sob o controle do promotor forte gpdA e terminador TrpC de Aspergillus nidulans, o qual foi transformado em M. anisopliae. Katushka é conhecido por ser um sistema eficiente de gene-reporter para técnicas de imageamento in vivo, e ainda não havia sido padronizada para M. anisopliae. Essa construção demonstrou ser geneticamente estável e sua fluorescência pôde ser detectada em besouros adultos de Ulomoides dermestoides após o ciclo completo de infecção de M. anisopliae expressando Katushka. Ainda, a expressão de Katushka em M. anisopliae não influenciou na sua virulência em um bioensaio com larvas de Tenebrio molitor, característica importante para que um gene-reporter possa ser usado em diversos estudos que enfoquem o processo de infecção. A escassez de promotores endógenos descritos em M. anisopliae limita as opções para as análises em nível molecular. Assim, Katushka foi usada como repórter para o screening de sequências promotoras de diferentes tamanhos encontradas à montante dos genes β-tubulina, gliceraldeído-3-fosfato desidrogenase (gpdMa), gamma-actina e trp1 de M. anisopliae. As construções de 1.000 pb e 500 pb da β-tubulina foram capazes de produzir níveis de fluorescência similares quando comparadas ao promotor padrão PgpdA. As construções de PgpdMa não se mostraram eficientes. Não foram obtidos mutantes para as construções dos promotores da gamma-actina e trp1 nos screenings realizados. Pesquisas adicionais são necessárias para aferir estatisticamente a eficiência desses promotores, bem como os elementos genéticos presentes em suas sequências. Este trabalho resultou na construção de modelos eficientes para estudos moleculares em M. anisopliae.The Metarhizium genus is composed by important entomopathogenic fungi species, such as Metarhizium anisopliae, and have been extensively used for controlling arthropod pests such as Helicoverpa armigera and Spodoptera frugiperda, both pests of maize, soy and cotton farms, among others, during the last Century. To date, more than a third of all commercially available fungal formulations for biological control are composed by Metarhizium spp. spores. Still, these formulations lack the effectiveness of chemical control. To change this scenario, many approaches have been made, such as the study of virulence determinants and genetic engineering to achieve enhanced strains for biological control. However, the lack of standardized molecular tools applicable to fungi species used for biological control slows the development of competitive strains, and difficult the study of the impact, dissemination and evolution that the released transgenic organisms can have on nature. In order to increase the repertoire of molecular tools available to work with M. anisopliae, in this work, a cassette for the expression of the reporter system Katushka, a bright far-red fluorescent protein, under the control of the strong gpdA promoter and TrpC terminator from Aspergillus nidulans was constructed and transformed in M. anisopliae. Katushka is known as an efficient gene-reporter system for in vivo imaging techniques, and have not been standardized in M. anisopliae. This construction was shown to be genetically stable and its fluorescence detectable in Ulomoides dermestoides adult beetles after the whole infection cycle of M. anisopliae strains expressing Katushka. Moreover, Katushka expression did not influenced M. anisopliae virulence in a Tenebrio molitor bioassay, which is an important characteristic for a gene-reporter to be used on a variety of studies that focus on the infection process. The lack of endogenous promoters described in M. anisopliae limits the options for molecular scale analysis. As such, Katushka was used as a reporter for the screening of different sized endogenous promoter sequences found upstream of β-tubulin, glyceraldehyde-3-phosphate dehydrogenase (gpdMa), gamma-actin and trp1 genes from M. anisopliae. The 1000 bp and 500 bp β-tubulin promoter constructions were able to produce fluorescence levels similar when compared to the standard promoter PgpdA. The PgpdMa constructions were not efficient. Mutants could not be obtained for gamma-actin and trp1 promoter constructions in the screenings performed. More research is needed to statistically infer promoter efficiency, as well as the genetic elements present in the sequences. This work resulted in the construction of efficient models for molecular studies in M. anisopliae

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