Stress activated MAP kinases (SAPKs) of the Hog1/Sty1/p38 family are specialized in transducing stress signals. In contrast to what is seen in animal cells, very few fungal species contain more than one SAPK. Aspergillus nidulans and other Aspergilli contain two SAPKs called SakA/HogA and MpkC. We have shown that SakA is essential for conidia to maintain their viability and to survive high H2O2 concentrations. H2O2 induces SakA nuclear accumulation and its interaction with transcription factor AtfA. Although SakA and MpkC show physical interaction, little is known about MpkC functions. Here we show that ΔmpkC mutants are not sensitive to oxidative stress but in fact MpkC inactivation partially restores the oxidative stress resistance of ΔsakA mutants. ΔmpkC mutants display about twofold increase in the production of fully viable conidia. The inactivation of the SakA upstream MAPKK PbsB or the simultaneous elimination of sakA and mpkC result in virtually identical phenotypes, including decreased radial growth, a drastic reduction of conidiation and a sharp, progressive loss of conidial viability. SakA and to a minor extent MpkC also regulate cell-wall integrity. Given the roles of MpkC in conidiation and oxidative stress sensitivity, we used a functional MpkC::GFP fusion to determine MpkC nuclear localization as an in vivo indicator of MpkC activation during asexual development and stress. MpkC is mostly localized in the cytoplasm of intact conidia, accumulates in nuclei during the first 2 h of germination and then becomes progressively excluded from nuclei in growing hyphae. In the conidiophore, MpkC nuclear accumulation increases in vesicles, metulae and phialides and decreases in older conidia. Oxidative and osmotic stresses induce MpkC nuclear accumulation in both germinating conidia and hyphae. In all these cases, MpkC nuclear accumulation is largely dependent on the MAPKK PbsB. Our results indicate that SakA and MpkC play major, distinct and sometimes opposing roles in conidiation and conidiospore physiology, as well as common roles in response to stress. We propose that two SAPKs are necessary to delay (MpkC) or fully stop (SakA) mitosis during conidiogenesis and the terminal differentiation of conidia, in the highly prolific phialoconidiation process characteristic of the Aspergilli.
The dynamin-like protein Drp1 and its receptor Fis-1 are required for mitochondria and peroxisome fission in animal and yeast cells. Here, we show that in the fungus Aspergillus nidulans the lack of Drp1 and Fis-1 homologs DnmA and FisA has strong developmental defects, leading to a notable decrease in hyphal growth and asexual and sexual sporulation, with some of these defects being aggravated or partially remediated by different carbon sources. Although both DnmA and FisA, are essential for mitochondrial fission, participate in peroxisomal division and are fully required for H 2 O 2-induced mitochondrial division, they also appear to play differential functions. Despite their lack of mitochondrial division, dnmA and fisA mutants segregate mitochondria to conidiogenic cells and produce viable conidia that inherit a single mitochondrion. During sexual differentiation, dnmA and fisA mutants develop fruiting bodies (cleistothecia) that differentiate excessive ascogenous tissue and a reduced number of viable ascospores. dnmA and fisA mutants show decreased respiration and notably high levels of mitochondrial reactive oxygen species (ROS), which likely correspond to superoxide. Regardless of this, dnmA mutants can respond to an external H 2 O 2 challenge by re-localizing the MAP kinase-activated protein kinase (MAPKAP) SrkA from the cytoplasm to the nuclei. Our results show that ROS levels regulate mitochondrial dynamics while a lack of mitochondrial fission results in lower respiration, increased mitochondrial ROS and developmental defects, indicating that ROS, mitochondrial division and development are critically interrelated processes.
This research was conducted to extend the knowledge on the differential regulation of laccase genes in response to dyes. In order to accomplish this, we analyzed both, the expression of five laccase genes by real time RT-qPCR, and also the laccase activity and isoforms patterns during the time-course of a Pleurotus ostreatus submerged fermentation supplemented with either acetyl yellow G (AYG) or remazol brilliant blue R (RBBR) dyes. For the purpose of obtaining a stable reference gene for optimal normalization of RT-quantitative PCR gene expression assays, we tested four candidate reference genes. As a result of this analysis, gpd was selected as reference index for data normalization. The addition of dyes had an induction effect on the enzymatic activity and also modified the zymogram profile. Fermentation with RBBR showed the highest laccase activity and number of isoforms along the course of the fermentation. Laccase gene expression profiles displayed up/down regulation along the fermentation time in four laccase genes (pox4, pox3, poxa1b and pox2), while pox1 was not expressed in either of the fermentation conditions. AYG addition caused the highest induction and repression levels for genes pox3 and poxa1b respectively. The expression level for all genes in the presence of RBBR were lower than in AYG, being in both conditions this response growth time dependent. These results show the influence of the nature of dyes on the induction level of laccase activity and on the differential regulation of the laccase genes expression in P. ostreatus.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0263-3) contains supplementary material, which is available to authorized users.
Aspergillus flavus induced ear rots and subsequent contamination of maize (Zea mays L.) by aflatoxin is a serious food safety issue, especially in developing countries where the crop is mostly cultivated by smallholder famers for own consumption and income generation. A better understanding of the mechanisms of resistance could help breeders to develop resistant maize varieties. In this study, a set of six tropical maize inbred lines previously identified as resistant or susceptible under natural field conditions were evaluated for response to A. flavus colonisation and aflatoxin contamination. Fungal biomass was significantly higher (P<0.05) in susceptible than resistant maize inbred lines, and this was highly correlated (P=0.001) to aflatoxin levels. Maize inbred lines MRI, MR2 and MR3 had low fungal biomass and low aflatoxin levels, suggesting that resistance in these lines was mediated through restricted fungal colonisation and establishment. Among the three putatively resistant inbred lines mentioned above, MR2 had a relatively high colonisation compared to the other two lines, revealing that A. flavus could establish and colonise kernels that were injured during inoculation, but did not contain high levels of aflatoxin. This could signify the presence of host genes that interfere with the aflatoxin biosynthetic pathway.
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