The role of aquaporins in pH-Dependent germination of Rhizopus delemar spores

Authors: TURGEMAN, TIDHAR - Volcani Center (ARO); SHATIL-COHEN, ARAVA - Hebrew University Of Jerusalem; MOSHELION, MENACHEM - Hebrew University Of Jerusalem; TEPER-BAMNOLKER, PAULA - Volcani Center (ARO); Skory, Christopher - Chris; LICHTER, AMNON - Volcani Center (ARO); ESHEL, DANI - Volcani Center (ARO)

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/3/2016
Publication Date: 3/9/2016
Citation: Turgeman, T., Shatil-Cohen, A., Moshelion, M., Teper-Bamnolker, P., Skory, C.D., Lichter, A., Eshel, D. 2016. The role of aquaporins in pH-dependent germination of Rhizopus delemar spores. PLoS One. 11(3)e0150543. doi: 10.1371/journal.pone.0150543.

Interpretive Summary: The fungus Rhizopus often causes deterioration or rot of fruits and vegetables during postharvest storage, leading to significant loss of yields. Methods to prevent or minimize the growth of the fungus are limited, so new methods to inhibit the infection process are necessary. Rhizopus typically spreads through small particles, called spores, which germinate and grow into the large colonies that degrade the stored crop. Germination of the spores first requires the uptake of water through special transport proteins called aquaporins. This study isolated Rhizopus aquaporins and showed how the functionality of these proteins could be modified by slight alterations of the protein sequence. This work provides important information that can be used to develop new methods that inhibit the germination process and ultimately minimize the growth of fungus. Such technology will lead to improved yields and higher quality products, benefitting the grower and consumer.

Technical 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 water status. 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 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 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.