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      Genome-wide identification and characterization of microRNAs by small RNA sequencing for low nitrogen stress in potato

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          Abstract

          Nitrogen is an important nutrient for plant growth and tuber quality of potato. Since potato crop requires high dose of N, improving nitrogen use efficiency (NUE) of plant is an inevitable approach to minimize N fertilization. The aim of this study was to identify and characterize microRNAs (miRNAs) by small RNA sequencing in potato plants grown in aeroponic under two contrasting N (high and low) regimes. A total of 119 conserved miRNAs belonging to 41 miRNAs families, and 1002 putative novel miRNAs were identified. From total, 52 and 54 conserved miRNAs, and 404 and 628 putative novel miRNAs were differentially expressed in roots and shoots, respectively under low N stress. Of total 34,135 predicted targets, the gene ontology (GO) analysis indicated that maximum targets belong to biological process followed by molecular function and cellular component. Eexpression levels of the selected miRNAs and targets were validated by real time-quantitative polymerase chain reaction (RT-qPCR) analysis. Two predicted targets of potential miRNAs (miR397 and miR398) were validated by 5’ RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). In general, predicted targets are associated with stress-related, kinase, transporters and transcription factors such as universal stress protein, heat shock protein, salt-tolerance protein, calmodulin binding protein, serine-threonine protein kinsae, Cdk10/11- cyclin dependent kinase, amino acid transporter, nitrate transporter, sugar transporter, transcription factor, F-box family protein, and zinc finger protein etc. Our study highlights that miR397 and miR398 play crucial role in potato during low N stress management. Moreover, study provides insights to modulate miRNAs and their predicted targets to develop N-use efficient potato using transgenic/genome-editing tools in future.

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          Most cited references41

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          Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis.

          MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are small noncoding RNAs that have recently emerged as important regulators of mRNA degradation, translational repression, and chromatin modification. In Arabidopsis thaliana, 43 miRNAs comprising 15 families have been reported thus far. In an attempt to identify novel and abiotic stress regulated miRNAs and siRNAs, we constructed a library of small RNAs from Arabidopsis seedlings exposed to dehydration, salinity, or cold stress or to the plant stress hormone abscisic acid. Sequencing of the library and subsequent analysis revealed 26 new miRNAs from 34 loci, forming 15 new families. Two of the new miRNAs from three loci are members of previously reported miR171 and miR319 families. Some of the miRNAs are preferentially expressed in specific tissues, and several are either upregulated or downregulated by abiotic stresses. Ten of the miRNAs are highly conserved in other plant species. Fifty-one potential targets with diverse function were predicted for the newly identified miRNAs based on sequence complementarity. In addition to miRNAs, we identified 102 other novel endogenous small RNAs in Arabidopsis. These findings suggest that a large number of miRNAs and other small regulatory RNAs are encoded by the Arabidopsis genome and that some of them may play important roles in plant responses to environmental stresses as well as in development and genome maintenance.
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            Small RNAs and their roles in plant development.

            Small RNAs of 20-30 nucleotides guide regulatory processes at the DNA or RNA level in a wide range of eukaryotic organisms. Many, although not all, small RNAs are processed from double-stranded RNAs or single-stranded RNAs with local hairpin structures by RNase III enzymes and are loaded into argonaute-protein-containing effector complexes. Many eukaryotic organisms have evolved multiple members of RNase III and the argonaute family of proteins to accommodate different classes of small RNAs with specialized molecular functions. Some small RNAs cause transcriptional gene silencing by guiding heterochromatin formation at homologous loci, whereas others lead to posttranscriptional gene silencing through mRNA degradation or translational inhibition. Small RNAs are not only made from and target foreign nucleic acids such as viruses and transgenes, but are also derived from endogenous loci and regulate a multitude of developmental and physiological processes. Here I review the biogenesis and function of three major classes of endogenous small RNAs in plants: microRNAs, trans-acting siRNAs, and heterochromatic siRNAs, with an emphasis on the roles of these small RNAs in developmental regulation.
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              A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs.

              Analysis of tomato (Solanum lycopersicum) small RNA data sets revealed the presence of a regulatory cascade affecting disease resistance. The initiators of the cascade are microRNA members of an unusually diverse superfamily in which miR482 and miR2118 are prominent members. Members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. We confirm, using transient expression in Nicotiana benthamiana, that miR482 targets mRNAs for NBS-LRR disease resistance proteins with coiled-coil domains at their N terminus. The targeting causes mRNA decay and production of secondary siRNAs in a manner that depends on RNA-dependent RNA polymerase 6. At least one of these secondary siRNAs targets other mRNAs of a defense-related protein. The miR482-mediated silencing cascade is suppressed in plants infected with viruses or bacteria so that expression of mRNAs with miR482 or secondary siRNA target sequences is increased. We propose that this process allows pathogen-inducible expression of NBS-LRR proteins and that it contributes to a novel layer of defense against pathogen attack.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: MethodologyRole: Validation
                Role: InvestigationRole: Methodology
                Role: MethodologyRole: Writing – review & editing
                Role: Funding acquisitionRole: Software
                Role: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                19 May 2020
                2020
                : 15
                : 5
                : e0233076
                Affiliations
                [001]Indian Council of Agricultural Research-Central Potato Research Institute, Shimla, Himachal Pradesh, India
                ICAR - National Research Center on Plant Biotechnology, INDIA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-3936-1458
                Article
                PONE-D-19-16783
                10.1371/journal.pone.0233076
                7237020
                32428011
                ae421a16-f75d-49aa-836e-bd79dc2e6159
                © 2020 Tiwari et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 13 June 2019
                : 28 April 2020
                Page count
                Figures: 8, Tables: 4, Pages: 21
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001503, Indian Council of Agricultural Research;
                Award ID: HORTCPRICIL201500300131
                Award Recipient :
                Funded by: ICAR
                Award ID: CABin Scheme
                Award Recipient :
                We received funds under the Biotechnology Programme (HORTCPRICIL201500300131) and CABin scheme (ICAR-IASRI, New Delhi).
                Categories
                Research Article
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Natural antisense transcripts
                MicroRNAs
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                MicroRNAs
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Solanum
                Potato
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Vegetables
                Potato
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                RNA sequencing
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                RNA sequencing
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Transcription Factors
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Transcription Factors
                Biology and Life Sciences
                Biochemistry
                Proteins
                Regulatory Proteins
                Transcription Factors
                Biology and Life Sciences
                Plant Science
                Plant Physiology
                Plant Defenses
                Plant Resistance to Abiotic Stress
                Biology and Life Sciences
                Plant Science
                Plant Pathology
                Plant Resistance to Abiotic Stress
                Biology and Life Sciences
                Ecology
                Plant Ecology
                Plant-Environment Interactions
                Plant Resistance to Abiotic Stress
                Ecology and Environmental Sciences
                Ecology
                Plant Ecology
                Plant-Environment Interactions
                Plant Resistance to Abiotic Stress
                Biology and Life Sciences
                Plant Science
                Plant Ecology
                Plant-Environment Interactions
                Plant Resistance to Abiotic Stress
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Sequence Databases
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Databases
                Biology and Life Sciences
                Agriculture
                Crop Science
                Crops
                Vegetable Crops
                Custom metadata
                The small RNA sequencing data has been deposited with the NCBI Bioproject: PRJNA489150, and SRA: SRR7791655, SRR7791289, SRR7791376 and SRR7786273.

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