Phenotype of a Mechanosensitive Channel Mutant, mid - 1 , in a Filamentous Fungus, Neurospora crassa

Abstract

In the yeast Saccharomyces cerevisiae, the MID1 (mating-induced death) gene encodes a stretch-activated channel which is required for successful mating; the mutant phenotype is rescued by elevated extracellular calcium. Homologs of the MID1 gene are found in fungi that are morphologically complex compared to yeast, both Basidiomycetes and Ascomycetes. We explored the phenotype of a mid-1 knockout mutant in the filamentous ascomycete Neurospora crassa. The mutant exhibits lower growth vigor than the wild type (which is not rescued by replete calcium) and mates successfully. Thus, the role of the MID-1 protein differs from that of the homologous gene product in yeast. Hyphal cytology, growth on diverse carbon sources, turgor regulation, and circadian rhythms of the mid-1 mutant are all similar to those of the wild type. However, basal turgor is lower than wild type, as is the activity of the plasma membrane H ؉ -ATPase (measured by cyanide [CN ؊ ]-induced depolarization of the energy-dependent component of the membrane potential). In addition, the mutant is unable to grow at low extracellular Ca 2؉ levels or when cytoplasmic Ca 2؉ is elevated with the Ca 2؉ ionophore A23187. We conclude that the MID-1 protein plays a role in regulation of ion transport via Ca 2؉ homeostasis and signaling. In the absence of normal ion transport activity, the mutant exhibits poorer growth.

Transient responses during hyperosmotic shock in the filamentous fungus Neurospora crassa

“…The rapid drop in turgor was followed by a recovery to near original values within 60 min ( Fig. 2a) (Lew et al, 2004(Lew et al, , 2008. Turgor loss occurred in concert with a decline in relative hyphal volume per unit length (Fig.…”

“…Time-courses of responses to hyperosmotic treatment. The average time to peak depolarization and onset of sustained hyperpolarization are indicated by vertical bars, as are the onset of ion uptake (Lew et al, 2006(Lew et al, , 2008 and glycerol production (Lew & Levina, 2007). Turgor (a) (the turgor data were previously published in Lew et al, 2004;Lew & Levina, 2007) declines rapidly, as does relative hyphal volume per unit length (V/V i , see Fig.…”

Comparative genomics of MAP kinase and calcium–calcineurin signalling components in plant and human pathogenic fungi

“…The role of these upstream sensors in filamentous fungi is largely unknown. Recently, a N. crassa mutant lacking a Mid1 orthologue was found to be affected in calcium homeostasis and vegetative growth (Lew et al, 2008). Moreover, deletion of a Cch1 orthologue affected ascospore discharge and mycelial growth in F. graminearum (Hallen and Trail, 2008).…”

Cch1 and Mid1 Are Functionally Required for Vegetative Growth under Low-Calcium Conditions in the Phytopathogenic Ascomycete Botrytis cinerea

“…Since the mutants displayed wild-type-like growth on minimal medium with small amounts of CaCl 2 , we suggested that sector formation is due to calcium starvation and hyphal polarity effects, as described for mid1 or cch1 deletion mutants in other filamentous fungi (26,27,53). However, when the cell wall was stained with calcofluor white, no differences from the wild type in hyphal morphology were observed, indicating that there were no defects in hyphal polarity or septation.…”

“…Differences between B. cinerea, on the one hand, and N. crassa, C. purpurea, and G. zeae, on the other hand, were seen upon treatment with the ionophore A23187, which was shown to act as a mobile ion carrier (27,28,53). In the latter three fungi, addition of the calcium ionophore A23187 and subsequent increases in the [Ca 2ϩ ] cyt did not result in elevated growth of the mutants.…”