Hydrophilins in the filamentous fungus Neosartorya fischeri (Aspergillus fischeri) have protective activity against several types of microbial water stress.

Hydrophilins are proteins that occur in all domains of life and protect cells and organisms against drought and other stresses. They include most of the late embryogenesis abundant (LEA) proteins and the heat shock protein (HSP) Hsp12. Here, the role of a predicted LEA-like protein (LeamA) and two Hsp12 proteins (Hsp12A and Hsp12B) of Neosartorya fischeri was studied. This filamentous fungus forms ascospores that belong to the most stress-resistant eukaryotic cells described to date. Heterologous expression of LeamA, Hsp12A and Hsp12B resulted in increased tolerance against salt and osmotic stress in Escherichia coli. These proteins were also shown to protect lactate dehydrogenase against dry heat and freeze-thaw cycles in vitro. Deletion of leamA caused diminished viability of sexual ascospores after drought and heat. This is the first report on functionality of Hsp12 and putative LeamA proteins derived from filamentous fungi, and their possible role in N. fischeri ascospore resistance against desiccation, high temperature and osmotic stress is discussed.

Mannitol is essential for the development of stress-resistant ascospores in Neosartorya fischeri (Aspergillus fischeri).

The polyol mannitol is one of the main compatible solutes in Neosartorya fischeri and accumulates in conidia and ascospores. Here, it is shown that biosynthesis of mannitol in N. fischeri mainly depends on mannitol 1-phosphate dehydrogenase (MpdA). Reporter studies and qPCR analysis demonstrated that mpdA is moderately expressed in vegetative hyphae and conidiophores, while it is highly expressed during development of ascospores. Deletion of mpdA reduced mannitol in whole cultures as much as 85% of the wild type, while trehalose levels had increased more than 4-fold. Decreased mannitol accumulation had no effect on mycelial growth irrespective of heat- or oxidative stress. Notably, conidia of the ΔmpdA strain had higher mannitol and lower trehalose levels. They were more sensitive to heat stress. The most distinct phenotype of mpdA deletion was the absence of full development of ascospores. Formation of cleistothecia, and asci was not affected. The ascus cell wall, however, did not dissolve and asci contained incompletely formed or aborted ascospores. Addition of the Mpd inhibitor nitrophenide to the wild type strain also resulted in disturbed ascospore formation. Taken together, these results show that mannitol has a role in sexual development of N. fischeri and in stress resistance of conidia.

The effect of natamycin on the transcriptome of conidia of Aspergillus niger.

The impact of natamycin on Aspergillus niger was analysed during the first 8 h of germination of conidia. Polarisation, germ tube formation, and mitosis were inhibited in the presence of 3 and 10 µM of the anti-fungal compound, while at 10 µM also isotropic growth was affected. Natamycin did not have an effect on the decrease of microviscosity during germination and the concomitant reduction in mannitol and trehalose levels. However, it did abolish the increase of intracellular levels of glycerol and glucose during the 8 h period of germination.Natamycin hardly affected the changes that occur in the RNA profile during the first 2 h of germination. During this time period, genes related to transcription, protein synthesis, energy and cell cycle and DNA processing were particularly up-regulated. Differential expression of 280 and 2586 genes was observed when 8 h old germlings were compared with conidia that had been exposed to 3 µM and 10 µM natamycin, respectively. For instance, genes involved in ergosterol biosynthesis were down-regulated. On the other hand, genes involved in endocytosis and the metabolism of compatible solutes, and genes encoding protective proteins were up-regulated in natamycin treated conidia.