Induction, expression and characterisation of laccase genes from the marine-derived fungal strains Nigrospora sp. CBMAI 1328 and Arthopyrenia sp. CBMAI 1330

The capability of the fungi Nigrospora sp. CBMAI 1328 and Arthopyrenia sp. CBMAI 1330 isolated from marine sponge to synthesise laccases (Lcc) in the presence of the inducer copper (110 M) was assessed. In a liquid culture medium supplemented with 5 M of copper sulphate after 5 days of incubation, Nigrospora sp. presented the highest Lcc activity (25.2 UL1). The effect of copper on Lcc gene expression was evaluated by reverse transcriptase polymerase chain reaction. Nigrospora sp. showed the highest gene expression of Lcc under the same conditions of Lcc synthesis. The highest Lcc expression by the Arthopyrenia sp. was detected at 96 h of incubation in absence of copper. Molecular approaches allowed the detection of Lcc isozymes and suggest the presence of at least two undescribed putative genes. Additionally, Lcc sequences from the both fungal strains clustered with other Lcc sequences from other fungi that inhabit marine environments.M. Passarini was supported by Ph.D. grant from FAPESP (2008/06720-7), Sao Paulo, Brazil. The authors thank FAPESP for financial support (BIOTA-FAPESP grant 2010/50190-2 and FAPESP grant 2013/19486-0) and Roberto G.S. Berlinck and CEBIMAR for the support related to samples collecting. L.D. Sette thanks CNPq for Productivity Fellowships 304103/2013-6

Expression of a Serine Protease Gene prC Is Up-Regulated by Oxidative Stress in the Fungus Clonostachys rosea: Implications for Fungal Survival

BACKGROUND: Soil fungi face a variety of environmental stresses such as UV light, high temperature, and heavy metals. Adaptation of gene expression through transcriptional regulation is a key mechanism in fungal response to environmental stress. In Saccharomyces cerevisiae, the transcription factors Msn2/4 induce stress-mediated gene expression by binding to the stress response element. Previous studies have demonstrated that the expression of extracellular proteases is up-regulated in response to heat shock in fungi. However, the physiological significance of regulation of these extracellular proteases by heat shock remains unclear. The nematophagous fungus Clonostachys rosea can secret an extracellular serine protease PrC during the infection of nematodes. Since the promoter of prC has three copies of the stress response element, we investigated the effect of environmental stress on the expression of prC. METHODOLOGY/PRINCIPAL FINDINGS: Our results demonstrated that the expression of prC was up-regulated by oxidants (H(2)O(2) or menadione) and heat shock, most likely through the stress response element. After oxidant treatment or heat shock, the germination of conidia in the wild type strain was significantly higher than that in the prC mutant strain in the presence of nematode cuticle. Interestingly, the addition of nematode cuticle significantly attenuated the production of reactive oxygen species (ROS) induced by oxidants and heat shock in the wild type strain, but not in prC mutant strain. Moreover, low molecule weight (<3 kD) degradation products of nematode cuticle suppressed the inhibitory effect of conidial germination induced by oxidants and heat shock. CONCLUSIONS/SIGNIFICANCE: These results indicate that PrC plays a protective role in oxidative stress in C. rosea. PrC degrades the nematode cuticle to produce degradation products, which in turn offer a protective effect against oxidative stress by scavenging ROS. Our study reveals a novel strategy for fungi to adapt to environmental stress