Gene expression analysis during microsclerotial development of the entomopathogenic fungus Metarhizium robertsii ARSEF 2575

FLAVIA PAIXÃO; CARLA HUARTE BONNET; EVERTON FERNANDES; NICOLÁS PEDRINI

Simposio; International Symposium of fungal stress 2017; 2017

The main goal of this study was to understand the microsclerotia (MS) metamorphosis in M. robertsii ARSEF 2575, based on the expression pattern of genes markers of oxidative stress (superoxide dismutases, sod, and catalases, cat), peroxisome biogenesis (peroxins, pex) and superficial hydrophobicity (hydrophobin, ssga). ARSEF 2575 was cultured in an agitated (250 rpm) liquid medium with carbon:nitrogen ratio 30:1 for production of MS. At 1, 6, 12, 18, 24, 48, 72 or 96 h, aliquots were collected and examined for observation of MS formation process using optical microscopy. Additional aliquots were stained with 3,3-diaminobenzidine (DAB) for evaluation of peroxidase activity. Samples cultured for 24, 48, 72 or 96 h were macerated with liquid nitrogen to obtain total RNA using the Trizol method. The cDNA was then synthesized. The expression of nine genes was studied by qPCR: Mrsod1, Mrsod2, MrcatA, MrcatB, Mrpex5, Mrpex7, Mrpex14/17, Mrpex19, MrssgA and Mrgpd (housekeeping gene). The results revealed that aggregation of hyphae was not seen before 6 h incubation, and from 6 to 18 h incubation aggregates were compacted and melanized. Mrsod1 (F2,6 = 5.117, P = 0.050); Mrsod2 (F2,6 = 5.203, P = 0.048); MrcatA (F2,6 = 6.900, P = 0.028), and Mrpex5 (F2,6 = 4.597, P = 0.061) were upregulated at 96 h compared to24 h incubation, with expression levels up to 3 fold induction. At 48 or 72 h, no upregulation was detected for any of the genes studied. We concluded that an oxidative stress scenario with an increment of peroxidase activity and peroxisomal proliferation is induced during metamorphosis of ARSEF 2575 in the fourth day of microsclerotial development.