Cell-surface copper transporters and superoxide dismutase 1 are essential for outgrowth during fungal spore germination

Abstract

During fungal spore germination, a resting spore returns to a conventional mode of cell division and resumes vegetative growth, but the requirements for spore germination are incompletely understood. Here, we show that copper is essential for spore germination in Germinating spores develop a single germ tube that emerges from the outer spore wall in a process called outgrowth. Under low-copper conditions, the copper transporters Ctr4 and Ctr5 are maximally expressed at the onset of outgrowth. In the case of Ctr6, its expression is broader, taking place before and during outgrowth. Spores lacking Ctr4, Ctr5, and the copper sensor Cuf1 exhibit complete germination arrest at outgrowth. In contrast, deletion only partially interferes with formation of outgrowing spores. At outgrowth, Ctr4-GFP and Ctr5-Cherry first co-localize at the spore contour, followed by re-location to a middle peripheral spore region. Subsequently, they move away from the spore body to occupy the periphery of the nascent cell. After breaking of spore dormancy, Ctr6 localizes to the vacuole membranes that are enriched in the spore body relative to the germ tube. Using a copper-binding tracker, results showed that labile copper is preferentially localized to the spore body. Further analysis showed that Ctr4 and Ctr6 are required for copper-dependent activation of the superoxide dismutase 1 (SOD1) during spore germination. This activation is critical because the loss of SOD1 activity blocked spore germination at outgrowth. Taken together, these results indicate that cell-surface copper transporters and SOD1 are required for completion of the spore germination program.

Pathways of pathogenicity: The transcriptional stages of germination in the fatal fungal pathogen Rhizopus delemar

“…These clusters are enriched (Hypergeometric test, corrected P value < 0.05) for transcripts with predicted functions related to kinase, transferase, transposase and oxidoreductase activity, along with pyrimidine and phosphorous metabolism, stress response, transport and signalling (Figure 4C; Supplementary Table 1). This is consistent with the established roles for these processes in starting and maintaining vegetative growth(28, 39–41). Cluster 5 contains genes that have high expression levels in both ungerminated spores and the hyphal form, but low levels during initial swelling (Figure 4B).…”

Spore Germination Requires Ferrichrome Biosynthesis and the Siderophore Transporter Str1 in Schizosaccharomyces pombe

“…However, little is known about a potential requirement for iron during spore germination. To investigate whether iron deficiency hampered spore germination, purified resting spores containing a CRIB (Cdc42/Rac interactive binding domain)-GFP allele (Tatebe et al 2008;Das et al 2009;Plante et al 2017) were first treated in the presence of calcofluor to stain the spore walls. These spores were synchronously induced to undergo germination in the presence of the iron chelator 2,29-dipyridyl (Dip, 400 mM) or FeCl 3 (100 mM).…”

“…Construction of the integrative pJKCRIB-GFP plasmid has been described previously (Plante et al 2017). The CRIB-GFP protein used as a marker for the tip and, to a lesser extent, septum of cells was expressed under the control of the shk1 + promoter as described previously (Tatebe et al 2008;Plante et al 2017).…”

“…Construction of the integrative pJKCRIB-GFP plasmid has been described previously (Plante et al 2017). The CRIB-GFP protein used as a marker for the tip and, to a lesser extent, septum of cells was expressed under the control of the shk1 + promoter as described previously (Tatebe et al 2008;Plante et al 2017). To generate the integrative pJKstr1 + -GFP plasmid, a 2802-bp Asp718-BamHI PCR-amplified DNA fragment containing the str1 + locus, starting at position 2966 from the translational start codon up to the penultimate codon, was inserted into the Asp718 and BamHI sites of pJK148 (Keeney and Boeke 1994).…”

“…However, the addition of glucose alone is insufficient to drive the subsequent developmental stages such as isotropic swelling, hatching of the OSW, and outgrowth. In this context, a recent study of S. pombe spores has revealed that the essential transition metal copper is dispensable for entry into germination and isotropic swelling but it is strictly required for outgrowth and generation of nascent daughter cells (Plante et al 2017). On the basis of these observations, it is expected that iron, one of the transition metals most utilized as cofactor for the function of cellular enzymes, may also be required for the developmental process of spore germination.…”