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

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.

The role of pre-symbiotic auxin signaling in ectendomycorrhiza formation between the desert truffle Terfezia boudieri and Helianthemum sessiliflorum.

The ectendomycorrhizal fungus Terfezia boudieri is known to secrete auxin. While some of the effects of fungal auxin on the plant root system have been described, a comprehensive understanding is still lacking. A dual culture system to study pre mycorrhizal signal exchange revealed previously unrecognized root-fungus interaction mediated by the fungal auxin. The secreted fungal auxin induced negative taproot gravitropism, attenuated taproot growth rate, and inhibited initial host development. Auxin also induced expression of Arabidopsis carriers AUX1 and PIN1, both of which are involved in the gravitropic response. Exogenous application of auxin led to a root phenotype, which fully mimicked that induced by ectomycorrhizal fungi. Co-cultivation of Arabidopsis auxin receptor mutants tir1-1, tir1-1 afb2-3, tir1-1 afb1-3 afb2-3, and tir1-1 afb2-3 afb3-4 with Terfezia confirmed that auxin induces the observed root phenotype. The finding that auxin both induces taproot deviation from the gravity axis and coordinates growth rate is new. We propose a model in which the fungal auxin induces horizontal root development, as well as the coordination of growth rates between partners, along with the known auxin effect on lateral root induction that increases the availability of accessible sites for colonization at the soil plane of fungal spore abundance. Thus, the newly observed responses described here of the root to Terfezia contribute to a successful encounter between symbionts.

Induction of Rhizopus oryzae germination under starvation using host metabolites increases spore susceptibility to heat stress.

Sweetpotato is a nutritional source worldwide. Soft rot caused by Rhizopus spp. is a major limiting factor in the storage of produce, rendering it potentially unsafe for human consumption. In this study, Rhizopus oryzae was used to develop a concept of postharvest disease control by weakening the pathogen through induction of spore germination under starvation conditions. We isolated the sweetpotato active fractions (SPAFs) that induce spore germination and used them at a low dose to enhance spore weakening caused by starvation. Germination in SPAF at 1 mg/ml weakened the pathogen spores by delaying their ability to form colonies on rich media and by increasing their sensitivity to heat stress. The weakening effect was also supported by reduced metabolic activity, as detected by Alarmar Blue fluorescent dye assays. Spores incubated with SPAF at 1 mg/ml showed DNA fragmentation in some of their nuclei, as observed by TUNEL assay. In addition, these spores exhibited changes in ultrastructural morphology (i.e., shrinkage of germ tubes, nucleus deformation, and vacuole formation) which are hallmarks of programmed cell death. We suggest that induction of spore germination under starvation conditions increases their susceptibility to stress and, therefore, might be considered a new strategy for pathogen control.

Mycorrhizal association between the desert truffle Terfezia boudieri and Helianthemum sessiliflorum alters plant physiology and fitness to arid conditions.

The host plant Helianthemum sessiliflorum was inoculated with the mycorrhizal desert truffle Terfezia boudieri Chatin, and the subsequent effects of the ectomycorrhizal relationship on host physiology were determined. Diurnal measurements revealed that mycorrhizal (M) plants had higher rates of photosynthesis (35%), transpiration (18%), and night respiration (49%) than non-mycorrhizal (NM) plants. Consequently, M plants exhibited higher biomass accumulation, higher shoot-to-root ratios, and improved water use efficiency compared to NM plants. Total chlorophyll content was higher in M plants, and the ratio between chlorophyll a to chlorophyll b was altered in M plants. The increase in chlorophyll b content was significantly higher than the increase in chlorophyll a content (2.58- and 1.52-fold, respectively) compared to control. Calculation of the photosynthetic activation energy indicated lower energy requirements for CO(2) assimilation in M plants than in NM plants (48.62 and 61.56 kJ mol(-1), respectively). Continuous measurements of CO(2) exchange and transpiration in M plants versus NM plants provided a complete picture of the daily physiological differences brought on by the ectomycorrhizal relationships. The enhanced competence of M plants to withstand the harsh environmental conditions of the desert is discussed in view of the mycorrhizal-derived alterations in host physiology.

Fibrosis inhibition and muscle histopathology improvement in laminin-alpha2-deficient mice.

In muscular dystrophies (MD) the loss of muscle and its ability to function are associated with fibrosis. We evaluated the efficacy of halofuginone in reducing fibrosis in the dy(2J)/dy(2J) mouse model of congenital MD. Mice were injected intraperitoneally with 5 microg of halofuginone 3 times a week for 5 or 15 weeks, starting at the age of 3 weeks. Halofuginone caused a reduction in collagen synthesis in hindlimb muscles. This was associated with reductions in the degenerated area, in cell proliferation, in the number of myofibers with central nuclei, with increased myofiber diameter, and with enhanced motor coordination and balance. Halofuginone caused a reduction in infiltrating fibroblasts that were located close to centrally nucleated myofibers. Our results suggest that halofuginone reduced the deleterious effects of fibrosis, thus improving muscle integrity. Halofuginone meets the criteria for a novel antifibrotic therapy for MD patients.

Prevention of muscle fibrosis and improvement in muscle performance in the mdx mouse by halofuginone.

Fibrosis is a known feature of dystrophic muscles, particularly the diaphragm, in the mdx mouse. In this study we evaluated the effect of halofuginone, a collagen synthesis inhibitor, on collagen synthesis in various muscles of young wild-type (C57/BL/6J) and mdx mice. Halofuginone prevented the age-dependent increase in collagen synthesis in the diaphragms of mdx with no effect on wild-type mice (n = 5 for each time point). This was associated with a decrease in the degenerated areas and number of central nuclei. Halofuginone also inhibited collagen synthesis in cardiac muscle. Moreover, enhanced motor coordination, balance and improved cardiac muscle function were observed implying reduced muscle injury. Halofuginone inhibited Smad3 phosphorylation downstream of TGFbeta in the diaphragm and cardiac muscles, in C2 cell line and in primary mouse myoblast cultures representing various muscular dystrophies. We suggest that via its effect on Smad3 phosphorylation, halofuginone inhibits muscle fibrosis and improves cardiac and skeletal muscle functions in mdx mice.