Comparative Functional Analysis of Wheat (<i>Triticum aestivum</i>) Zinc Finger-Containing Glycine-Rich RNA-Binding Proteins in Response to Abiotic Stresses

<div><p>Although the functional roles of zinc finger-containing glycine-rich RNA-binding proteins (RZs) have been characterized in several plant species, including <i>Arabidopsis thaliana</i> and rice (<i>Oryza sativa</i>), the physiological functions of RZs in wheat (<i>Triticum aestivum</i>) remain largely unknown. Here, the functional roles of the three wheat RZ family members, named TaRZ1, TaRZ2, and TaRZ3, were investigated using transgenic <i>Arabidopsis</i> plants under various abiotic stress conditions. Expression of <i>TaRZs</i> was markedly regulated by salt, dehydration, or cold stress. The TaRZ1 and TaRZ3 proteins were localized to the nucleus, whereas the TaRZ2 protein was localized to the nucleus, endoplasmic reticulum, and cytoplasm. Germination of all three TaRZ-expressing transgenic <i>Arabidopsis</i> seeds was retarded compared with that of wild-type seeds under salt stress conditions, whereas germination of TaRZ2- or TaRZ3-expressing transgenic <i>Arabidopsis</i> seeds was retarded under dehydration stress conditions. Seedling growth of TaRZ1-expressing transgenic plants was severely inhibited under cold or salt stress conditions, and seedling growth of TaRZ2-expressing plants was inhibited under salt stress conditions. By contrast, expression of TaRZ3 did not affect seedling growth of transgenic plants under any of the stress conditions. In addition, expression of TaRZ2 conferred freeze tolerance in <i>Arabidopsis</i>. Taken together, these results suggest that different TaRZ family members play various roles in seed germination, seedling growth, and freeze tolerance in plants under abiotic stress.</p></div

TaRZ2 confers freezing tolerance in <i>Arabidopsis</i> plants.

<p>(A) For non cold-acclimated (NA) freezing tolerance tests, 4-week-old plants of the wild-type (WT) and TaRZ2-expressing plants (OX21, OX22, and OX23) were subjected to freezing shock at −6 for 3–6 h directly under continuous light, then transferred to normal growth conditions. (B) For cold-acclimated (CA) freezing tolerance test, 4-week-old plants were first placed at 4°C for 1 day, −1°C for 1 day, and then subjected to freezing shock at −7 for 12–25 h. Surviving plants were counted 7 days after transferring to normal growth conditions. One representative picture among repeated experiments was shown. (C) For electrolyte leakage test, leaves from the NA wild-type and TaRZ2-expresing plants were frozen at −1 to −10°C, and the extent of cellular damage was estimated by measuring electrolyte leakage.</p

Expression levels and stress-responsive expression patterns of <i>TaRZs</i> in wheat.

<p>(A) Transcript levels of <i>TaRZ1</i>, <i>TaRZ2</i>, and <i>TaRZ3</i> were analyzed via real-time RT-PCR and presented as relative expression (fold) of <i>TaRZ2</i> expression level. Two-week-old wheat plants were subjected to (B) salt, (C) dehydration, or (D) cold stress for 6, 12, 24, and 48 h, and the transcript levels of each <i>TaRZ</i> were analyzed via real-time RT-PCR and presented as the relative expression (fold) of the non-stressed controls. Values are means ± SE obtained from five independent experiments. Asterisks above each column indicate values that are statistically different from the control values (p≤0.05).</p

Subcellular localization of TaRZ proteins.

<p>TaRZ-GFP fusion proteins were transiently expressed in tobacco plant, and GFP signals in tobacco leaves were detected using a confocal microscope. DAPI was used to stain the nuclei, and <i>Brassica rapa</i> microsomal delta-12 fatty acid desaturase (BrFAD2) was used as a marker for ER localization. Bar  = 30 mm.</p

Diversity of clinical isolates of <i>Aspergillus terreus</i> in antifungal susceptibilities, genotypes and virulence in <i>Galleria mellonella</i> model: Comparison between respiratory and ear isolates

<div><p>We analyzed the antifungal susceptibility profiles, genotypes, and virulence of clinical <i>Aspergillus terreus</i> isolates from six university hospitals in South Korea. Thirty one isolates of <i>A</i>. <i>terreus</i>, comprising 15 respiratory and 16 ear isolates were assessed. Microsatellite genotyping was performed, and genetic similarity was assessed by calculating the Jaccard index. Virulence was evaluated by <i>Galleria mellonella</i> survival assay. All 31 isolates were susceptible to itraconazole, posaconazole, and voriconazole, while 23 (74.2%) and 6 (19.4%) showed amphotericin B (AMB) minimum inhibitory concentrations (MICs) of ≤ 1 mg/L and > 4 mg/L, respectively. Notably, respiratory isolates showed significantly higher geometric mean MICs than ear isolates to AMB (2.41 <i>vs</i>. 0.48 mg/L), itraconazole (0.40 <i>vs</i>. 0.19 mg/L), posaconazole (0.16 <i>vs</i>. 0.08 mg/L), and voriconazole (0.76 <i>vs</i>. 0.31 mg/L) (all, <i>P</i> <0.05). Microsatellite genotyping separated the 31 isolates into 27 types, but the dendrogram demonstrated a closer genotypic relatedness among isolates from the same body site (ear or respiratory tract); in particular, the majority of ear isolates clustered together. Individual isolates varied markedly in their ability to kill infected <i>G</i>. <i>mellonella</i> after 72 h, but virulence did not show significant differences according to source (ear or respiratory tract), genotype, or antifungal susceptibility. The current study shows the marked diversity of clinical isolates of <i>A</i>. <i>terreus</i> in terms of antifungal susceptibilities, genotypes and virulence in the <i>G</i>. <i>mellonella</i> model, and ear isolates from Korean hospitals may have lower AMB or triazole MICs than respiratory isolates.</p></div

Genetic relatedness by microsatellite analysis and virulence in the <i>G</i>. <i>mellonella</i> model of 15 respiratory and 16 ear isolates of <i>A</i>. <i>terreus</i> from six hospitals.

<p>Genetic relatedness by microsatellite analysis and virulence in the <i>G</i>. <i>mellonella</i> model of 15 respiratory and 16 ear isolates of <i>A</i>. <i>terreus</i> from six hospitals.</p

Antifungal susceptibilities, microsatellite genotypes and virulence of 31 <i>A</i>. <i>terreus</i> isolates from six hospitals in South Korea.

<p>Antifungal susceptibilities, microsatellite genotypes and virulence of 31 <i>A</i>. <i>terreus</i> isolates from six hospitals in South Korea.</p

Genetic relationships of 31 <i>A</i>. <i>terreus</i> isolates according to source.

<p>The dendrogram is based on a categorical analysis of seven microsatellite markers in combination with unweighted pairgroup method using the arithmetic average (UPGMA) clustering. The number on the tree indicates the branch length, showing the difference along a branch. All 31 isolates (R1 to R15 and E1 to E16) comprised 27 distinct genotypes (GT 1 to GT 27) by 7 microsatellite markers. However, when a cluster is defined as the isolation of two or more strains with a branch length distance of < 0.63, ear isolates comprise clusters II and V, and the respiratory isolates comprise clusters I, III, and IV, suggesting a closer genetic relatedness among isolates from the same body site (ear or respiratory tract). Five isolates (R7, R15, E1, E4, and R12) were unique to a single isolate, which did not cluster with other isolate as a branch length distance of < 0.63. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186086#pone.0186086.t002" target="_blank">Table 2</a> for detailed information on each isolate.</p

MICs of AMB and triazoles of 31 <i>A</i>. <i>terreus</i> isolates from respiratory and ear specimens.

<p>MICs of AMB and triazoles of 31 <i>A</i>. <i>terreus</i> isolates from respiratory and ear specimens.</p

Predictive factors for 30-day mortality by multiple logistic regression analysis^a.

<p>Predictive factors for 30-day mortality by multiple logistic regression analysis<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148400#t003fn001" target="_blank">^a</a>.</p