The sucrose non-fermenting 1-related kinase 2 gene SAPK9 improves drought tolerance and grain yield in rice by modulating cellular osmotic potential, stomatal closure and stress-responsive gene expression

Abstract Background Family members of sucrose non-fermenting 1-related kinase 2 (SnRK2), being plant-specific serine/threonine protein kinases, constitute the central core of abscisic acid (ABA)-dependent and ABA-independent signaling pathways, and are key regulators of abiotic stress adaptation in plants. We report here the functional characterization of SAPK9 gene, one of the 10 SnRK2s of rice, through developing gain-of-function and loss-of-function phenotypes by transgenesis. Results The gene expression profiling revealed that the abundance of single gene-derived SAPK9 transcript was significantly higher in drought-tolerant rice genotypes than the drought-sensitive ones, and its expression was comparatively greater in reproductive stage than the vegetative stage. The highest expression of SAPK9 gene in drought-tolerant Oryza rufipogon prompted us to clone and characterise the CDS of this allele in details. The SAPK9 transcript expression was found to be highest in leaf and upregulated during drought stress and ABA treatment. In silico homology modelling of SAPK9 with Arabidopsis OST1 protein showed the bilobal kinase fold structure of SAPK9, which upon bacterial expression was able to phosphorylate itself, histone III and OsbZIP23 as substrates in vitro. Transgenic overexpression (OE) of SAPK9 CDS from O. rufipogon in a drought-sensitive indica rice genotype exhibited significantly improved drought tolerance in comparison to transgenic silencing (RNAi) lines and non-transgenic (NT) plants. In contrast to RNAi and NT plants, the enhanced drought tolerance of OE lines was concurrently supported by the upgraded physiological indices with respect to water retention capacity, soluble sugar and proline content, stomatal closure, membrane stability, and cellular detoxification. Upregulated transcript expressions of six ABA-dependent stress-responsive genes and increased sensitivity to exogenous ABA of OE lines indicate that the SAPK9 is a positive regulator of ABA-mediated stress signaling pathways in rice. The yield-related traits of OE lines were augmented significantly, which resulted from the highest percentage of fertile pollens in OE lines when compared with RNAi and NT plants. Conclusion The present study establishes the functional role of SAPK9 as transactivating kinase and potential transcriptional activator in drought stress adaptation of rice plant. The SAPK9 gene has potential usefulness in transgenic breeding for improving drought tolerance and grain yield in crop plants.http://deepblue.lib.umich.edu/bitstream/2027.42/134605/1/12870_2016_Article_845.pd

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Not AvailableThe innate immunity serves as the first line of defence against invasion of pathogens in fish. Toll-like receptors (TLRs) play a pivotal role in the innate immunity by specific pathogen-associated molecules that activate immune responses. In the present study, analysis of TLR 20 and TLR 21 in different tissues of healthy catfish, Heteropneustes fossilis showed that the patterns of the gene expression were in the order of spleen > intestine > head kidney > gill and gill > head kidney > liver > spleen > intestine, respectively. Relative transcription of TLR 20 and TLR 21 in different tissues of the fish against bacterial infection investigated by semi- quantitative RT-PCR showed that expression of both TLR 20 and TLR 21 were up-regulated in spleen and intestine whereas that of TLR 21 expression was down-regulated in kidney, gill and liver relative to control fish. Findings of the study suggested that spleen and intestine showed significant alteration in both TLR 20 and TLR 21 expression. Expression of TLR 21 found to be more sensitive than TLR 20 against bacterial infection in fish.ICAR-NAI

Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes.

Drought is one of the major limiting factors for productivity of crops including rice (Oryza sativa L.). Understanding the role of allelic variations of key regulatory genes involved in stress-tolerance is essential for developing an effective strategy to combat drought. The bZIP transcription factors play a crucial role in abiotic-stress adaptation in plants via abscisic acid (ABA) signaling pathway. The present study aimed to search for allelic polymorphism in the OsbZIP23 gene across selected drought-tolerant and drought-sensitive rice genotypes, and to characterize the new allele through overexpression (OE) and gene-silencing (RNAi). Analyses of the coding DNA sequence (CDS) of the cloned OsbZIP23 gene revealed single nucleotide polymorphism at four places and a 15-nucleotide deletion at one place. The single-copy OsbZIP23 gene is expressed at relatively higher level in leaf tissues of drought-tolerant genotypes, and its abundance is more in reproductive stage. Cloning and sequence analyses of the OsbZIP23-promoter from drought-tolerant O. rufipogon and drought-sensitive IR20 cultivar showed variation in the number of stress-responsive cis-elements and a 35-nucleotide deletion at 5'-UTR in IR20. Analysis of the GFP reporter gene function revealed that the promoter activity of O. rufipogon is comparatively higher than that of IR20. The overexpression of any of the two polymorphic forms (1083 bp and 1068 bp CDS) of OsbZIP23 improved drought tolerance and yield-related traits significantly by retaining higher content of cellular water, soluble sugar and proline; and exhibited decrease in membrane lipid peroxidation in comparison to RNAi lines and non-transgenic plants. The OE lines showed higher expression of target genes-OsRab16B, OsRab21 and OsLEA3-1 and increased ABA sensitivity; indicating that OsbZIP23 is a positive transcriptional-regulator of the ABA-signaling pathway. Taken together, the present study concludes that the enhanced gene expression rather than natural polymorphism in coding sequence of OsbZIP23 is accountable for improved drought tolerance and yield performance in rice genotypes

Correction: Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes.

[This corrects the article DOI: 10.1371/journal.pone.0150763.]

Additional file 2: Figure S5. of The sucrose non-fermenting 1-related kinase 2 gene SAPK9 improves drought tolerance and grain yield in rice by modulating cellular osmotic potential, stomatal closure and stress-responsive gene expression

Schematic representation of the genetic constructs based on pCAMBIA1301 plasmid used for Agrobacterium-mediated transformation of drought-sensitive indica rice cultivar IR20. (A) The gene overexpression (OE) construct of SAPK9 carrying 1086 bp CDS from O. rufipogon. The developed transgenic rice lines were designated as SAOE#1, 2, 3 etc. (B) The RNAi-mediated gene silencing (RNAi) construct of endogenous SAPK9 gene. The 605 bp 5′-part of SAPK9 CDS from O. rufipogon was cloned in sense and antisense orientation flanking an arbitrary 200 bp DNA linker. The developed transgenic rice lines were designated as RNAi#1, 2, 3 etc. (TIF 453 kb

Additional file 5: Figure S3. of The sucrose non-fermenting 1-related kinase 2 gene SAPK9 improves drought tolerance and grain yield in rice by modulating cellular osmotic potential, stomatal closure and stress-responsive gene expression

Amino acid sequence alignment of the isolated SAPK9 CDS from wild rice Oryza rufipogon (accession no. KT387673) with the reported sequence of japonica rice cultivar (accession no.AB125310). (TIF 487 kb

Additional file 8: Figure S6. of The sucrose non-fermenting 1-related kinase 2 gene SAPK9 improves drought tolerance and grain yield in rice by modulating cellular osmotic potential, stomatal closure and stress-responsive gene expression

Relative expression level of SAPK8 and SAPK10 gene in SAPK9 overexpressed (OE) and gene silenced (RNAi) transgenic rice lines. Analysis of real-time PCR depicting transcript level of (A) SAPK8 and (B) SAPK10 in leaf tissues of three sets of plants, i.e. OE, RNAi and non-transgenic (NT) plants under drought stress. For internal reference, rice polyubiquitin1 (OsUbi1) gene was used. Error bars represent the mean ± SD of triplicate measurements. Student’s t-test was performed to find out statistically significant differences (*P < 0.01). (TIF 339 kb

Elucidation of Novel Structural Scaffold in Rohu TLR2 and Its Binding Site Analysis with Peptidoglycan, Lipoteichoic Acid and Zymosan Ligands, and Downstream MyD88 Adaptor Protein

Toll-like receptors (TLRs) play key roles in sensing wide array of microbial signatures and induction of innate immunity. TLR2 in fish resembles higher eukaryotes by sensing peptidoglycan (PGN) and lipoteichoic acid (LTA) of bacterial cell wall and zymosan of yeasts. However, in fish TLR2, no study yet describes the ligand binding motifs in the leucine rich repeat regions (LRRs) of the extracellular domain (ECD) and important amino acids in TLR2-TIR (toll/interleukin-1 receptor) domain that could be engaged in transmitting downstream signaling. We predicted these in a commercially important freshwater fish species rohu (Labeo rohita) by constructing 3D models of TLR2-ECD, TLR2-TIR, and MyD88-TIR by comparative modeling followed by 40 ns (nanosecond) molecular dynamics simulation (MDS) for TLR2-ECD and 20 ns MDS for TLR2-TIR and MyD88-TIR. Protein (TLR2-ECD)–ligands (PGN, LTA, and zymosan) docking in rohu by AutoDock4.0, FlexX2.1, and GOLD4.1 anticipated LRR16–19, LRR12–14, and LRR20-CT as the most important ligand binding motifs. Protein (TLR2-TIR)—protein (MyD88-TIR) interaction by HADDOCK and ZDOCK predicted BB loop, αB-helix, αC-helix, and CD loop in TLR2-TIR and BB loop, αB-helix, and CD loop in MyD88-TIR as the critical binding domains. This study provides ligands recognition and downstream signaling

Disruption of the quorum sensing regulated pathogenic traits of the biofilm-forming fish pathogen <i>Aeromonas hydrophila</i> by tannic acid, a potent quorum quencher

<p>The quorum sensing (QS) phenomenon regulates a myriad of pathogenic traits in the biofilm forming fish pathogen, <i>Aeromonas hydrophila</i>. Blocking the QS mechanism of <i>A. hydrophila</i> is a novel strategy to prevent disease in fish. This study evaluated the effect of tannic acid, a QS inhibitor, on <i>A. hydrophila</i>-associated QS regulated phenomena. A streaking assay with <i>Chromobacterium violaceum</i> (CVO26) reported the presence of N-acyl homoserine lactone (AHL) in <i>A. hydrophila</i>, which was confirmed by HPLC and GC-MS analysis. Tannic acid-treated <i>A. hydrophila</i> showed a considerable reduction in violacein production, blood haemolysis activity and the pattern of swarming motility. Biofilm formation was significantly reduced (<i>p</i> < 0.001) (up to 95%), after tannic acid treatment for 48 h. Analysis by qRT-PCR revealed significant downregulation (<i>p</i> < 0.001) of AhyI and AhyR transcripts in <i>A. hydrophila</i> after tannic acid treatment. Co-stimulation of <i>Catla catla</i> with <i>A. hydrophila</i> and tannic acid attenuated pathogen-induced skin haemorrhages and increased the relative survival rate up to 86.6%. The study provides a mechanistic basis of tannic acid as a QS blocker and indicates its therapeutic potential against <i>A. hydrophila</i>-induced pathogenesis.</p