The Role of Aquaporins in pH-Dependent Germination of Rhizopus delemar Spores

Abstract

Rhizopus delemar and associated species attack a wide range of fruit and vegetables after harvest. Host nutrients and acidic pH are required for optimal germination of R. delemar, and we studied how this process is triggered. Glucose induced spore swelling in an acidic environment, expressed by an up to 3-fold increase in spore diameter, whereas spore diameter was smaller in a neutral environment. When suspended in an acidic environment, the spores started to float, indicating a change in their density. Treatment of the spores with HgCl2, an aquaporin blocker, prevented floating and inhibited spore swelling and germ-tube emergence, indicating the importance of water uptake at the early stages of germination. Two putative candidate aquaporin-encoding genes—RdAQP1 and RdAQP2—were identified in the R. delemar genome. Both presented the conserved NPA motif and six-transmembrane domain topology. Expressing RdAQP1 and RdAQP2 in Arabidopsis protoplasts increased the cells' osmotic water permeability coefficient (Pf) compared to controls, indicating their role as water channels. A decrease in R. delemar aquaporin activity with increasing external pH suggested pH regulation of these proteins. Substitution of two histidine (His) residues, positioned on two loops facing the outer side of the cell, with alanine eliminated the pH sensing resulting in similar Pf values under acidic and basic conditions. Since hydration is critical for spore switching from the resting to activate state, we suggest that pH regulation of the aquaporins can regulate the initial phase of R. delemar spore germination, followed by germ-tube elongation and host-tissue infection.

Aspergillus penicillioides differentiation and cell division at 0.585 water activity

“…During conidial swelling, water rehydrates the cytosol and the spore's macromolecular systems, facilitates cell turgor and enables metabolic activity. The process can be partmechanical, and part-ATP-driven, and represents the first stage of germination (Liu et al, 2016;Turgeman et al, 2016). Differentiation of conidia into tapered germination structures (see below) did not take place until after conidial volume had increased by a threshold value of 40%, so this criterion was used as an indicator for the pregermination phase (Fig.…”

“…However, the delay between formation of differentiated germination structures and septate germlings is consistent with fundamental changes known to take place early on during germination of conidia. These include the initiation of respiration, or fermentation followed by respiration; synthesis of proteins, including aquaporins and those involved in substrate uptake; synthesis of DNA and RNA; critical changes in compatible-solute metabolism (assimilation and/or synthesis of trehalose and specific polyols); generation of cell-available energy; and additional changes including upregulation of other genes involved in sensing the environment, substrate uptake, stress responses and developmental processes (Osherov and May, 2001;van Leeuwen et al, 2013;Hagiwara et al, 2016;Novodvorskaa et al, 2016;Turgeman et al, 2016).…”

Environmental interactions with amoebae as drivers of bacterial-fungal endosymbiosis and pathogenicity

“…Upon infection, R. microsporus spores become metabolically active and begin to swell 61,66 . Based on analogy to Aspergillus species, frequently used as a model for Mucorales, swelling would be expected to reveal Pathogen Associated Molecular Patterns (PAMPs) and induce increasing rates of phagocytosis 2,67 .…”

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

“…Regulation of germination in the mucorales remains an under-examined area. Cues for germination include the availability of sufficient water, iron, a suitable carbon source and pH(10, 42, 47), though the mechanisms remain unclear. This study aims to expand current knowledge on the molecular processes which determine germination.…”