Wheat miRNAs Research Articles

FUNCTIONAL GENOMIC PROFILING OF DROUGHT RESPONSIVE Micro-RNA IN WHEAT

MicroRNAs are small non-coding RNAs that play a major regulatory role in post translation regulation either by translation inhibition or mRNA cleavage. Recent studies proved that miRNAs have a regulatory role in both abiotic and biotic stress in plants. In our study, we measure the expression profile of 10 known abiotic stress miRNAs in hexaploid bread wheat (Triticum aestivum L.), under drought stress in the seedling stage. Significant upregulation was observed with osa_miR319a-3p.2-3, miR5048, gmamiR5783, hci-miR156a, bdi-miR159b-3p, zma-miR164g-3, ssl-miR398 and ptcmiR482c- 5p. Among these, hci-miR156a, and osa-miR319a-3p.2-3 showed downregulation after 12 h compared to 2 h treatment. Only osa-miR172b was downregulated. This variant and dynamic expression patterns is good evidence and certain indicator of miRNA correlation to drought tolerance mechanisms present in wheat. This is the first data that provides accurate measurement of some drought related miRNA in wheat in the early exposure to drought stress and in the identification of several genes that could be implicated in drought tolerance and could be used for improving wheat and other cereal crops in this respect.

TaMIR1139: a wheat miRNA responsive to Pi-starvation, acts a critical mediator in modulating plant tolerance to Pi deprivation.

Wheat miRNA member TaMIR1139 targets genes functional in various families and plays crucial roles in regulating plant Pi starvation tolerance. Through regulating target genes at posttranscriptional or translational level, plant miRNAs are involved in mediating diverse biological processes associated with growth, development, and responses to adverse stresses. In this study, we characterized the expression pattern and function of TaMIR1139, a miRNA member of wheat (T. aestivum) under Pi deprivation. TaMIR1139 precursor is also present in N. tabucum, suggesting the conserved nature of miR1139 across monocots and eudicots. TaMIR1139 targets seven genes within different families. The transcripts abundance of TaMIR1139 was induced upon Pi deprivation and the upregulated expression under Pi starvation was downregulated by the Pi recovery treatment, In contrast, the genes targeted by TaMIR1139 exhibited reduced transcripts upon Pi starvation and their downregulated expression was recovered by Pi-recovery condition, suggesting the regulation of them under TaMIR1139 through a cleavage mechanism. TaMIR1139 overexpression conferred the Pi-deprived plants improved phenotype, biomass, photosynthesis, and Pi acquisition. Transcriptome analysis identified numerous genes involving biological process, cellular components, and molecular function were differentially expressed in the TaMIR1139 overexpression lines, which suggests the TaMIR1139-mediated plant Pi starvation tolerance to be associated with the role of miRNA in extensively modulating the transcript profiling. A phosphate transporter (PT) gene NtPT showed significantly upregulated expression in TaMIR1139 overexpression lines; overexpression of it conferred plants improved Pi acquisition upon Pi starvation, suggesting its contribution to the TaMIR1139-mediated plant low-Pi stress resistance. Our investigation indicates that TaMIR1139 is critical in plant Pi starvation tolerance through transcriptionally regulating the target genes and modulating the Pi stress-defensiveness processes.

Wheat miRNA TaemiR408 Acts as an Essential Mediator in Plant Tolerance to Pi Deprivation and Salt Stress via Modulating Stress-Associated Physiological Processes.

MicroRNAs (miRNA) families act as critical regulators for plant growth, development, and responses to abiotic stresses. In this study, we characterized TaemiR408, a miRNA family member of wheat (Triticum aestivum), for the role in mediating plant responses to Pi starvation and salt stress. TaemiR408 targets six genes that encode proteins involving biochemical metabolism, microtubule organization, and signaling transduction. 5′- and 3′-RACE analyses confirmed the mRNA cleavage of target genes mediated by this wheat miRNA. TaemiR408 showed induced expression patterns upon Pi starvation and salt stress and whose upregulated expression was gradually repressed by the normal recovery treatments. The target genes of TaemiR408 exhibited reverse expression patterns to this miRNA, whose transcripts were downregulated under Pi starvation and salt stress and the reduced expression was recovered by the followed normal condition. These results suggest the regulation of the target genes under TaemiR408 through a cleavage mechanism. Tobacco lines with TaemiR408 overexpression exhibited enhanced stress tolerance, showing improved phenotype, biomass, and photosynthesis behavior compared with wild type under both Pi starvation and salt treatments, which closely associate increased P accumulation upon Pi deprivation and elevated osmolytes under salt stress, respectively. Phosphate transporter (PT) gene NtPT2 displays upregulated transcripts in the Pi-deprived TaemiR408 overexpressors; knockdown of this PT gene reduces Pi acquisition under low-Pi stress, confirming its role in improving plant Pi taken up. Likewise, NtPYL2 and NtSAPK3, genes encoding abscisic acid (ABA) receptor and SnRK2 protein, respectively, exhibited upregulated transcripts in salt-challenged TaemiR408 overexpressors; knockdown of them caused deteriorated growth and lowered osmolytes amounts of plants upon salt treatment. Thus, TaemiR408 is crucial for plant adaptations to Pi starvation and salt stress through regulating Pi acquisition under low-Pi stress and remodel ABA signaling pathway and osmoprotects biosynthesis under salt stress.

Assembly and Annotation of Transcriptome Provided Evidence of miRNA Mobility between Wheat and Wheat Stem Sawfly.

Wheat Stem Sawfly (WSS), Cephus Cinctus Norton (Hymenoptera: Cephidae), is one of the most important pests, causing yield and economic losses in wheat and barley. The lack of information about molecular mechanisms of WSS for defeating plant’s resistance prevents application of effective pest control strategies therefore, it is essential to identify the genes and their regulators behind WSS infestations. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are recognized with their regulatory functions on gene expression, tuning protein production by controlling transcriptional and post-transcriptional activities. A transcriptome-guided approach was followed in order to identify miRNAs, lncRNAs, and mRNA of WSS, and their interaction networks. A total of 1,893 were presented here as differentially expressed between larva and adult WSS insects. There were 11 miRNA families detected in WSS transcriptome. Together with the annotation of 1,251 novel mRNAs, the amount of genetic information available for WSS was expanded. The network between WSS miRNAs, lncRNAs, and mRNAs suggested miRNA-mediated regulatory roles of lncRNAs as competing endogenous RNAs. In the light of the previous evidence that small RNA molecules of a pathogen could suppress the immune response of host plant, we analyzed the putative interactions between larvae and wheat at the miRNA level. Overall, this study provides a profile of larva and adult WSS life stages in terms of coding and non-coding elements. These findings also emphasize the potential roles of wheat and larval miRNAs in wheat resistance to infestation and in the suppression of resistance which is critical for the development of effective pest control strategies.

Bioinformatic identification and expression analysis of new microRNAs from wheat (Triticum aestivum L.)

As an essential regulatory component in plants, microRNAs (miRNAs) have been intensively studied over the past decade. Although hundreds of miRNAs have been identified and analyzed in many important crops and model plants, very little is known about the function of common wheat (Triticum aestivum L.) miRNAs. In this study, we performed computational prediction of novel wheat miRNAs based on BLAST searches of the expressed sequence tag database. The expression profiles of all miRNAs were performed for both vegetative and reproductive tissues to identify developmentally regulated miRNAs. A total of 19 new miRNAs belonging to 12 MIR families were identified using stringent criteria for miRNA annotation. For all of the miRNAs, the secondary structures of their precursor sequences were predicted. Two pairs of distinct miRNAs were found to be located on the same precursor. The predicted miRNAs were experimentally verified by a stem-loop qRT-PCR-based assay. The expression profiles were performed in both vegetative and reproductive tissues to find the potential correlations between the developmental phase and miRNA activity. Thirteen out of 19 miRNAs were upregulated at certain phases of plant development, and three of them (miR319, miR395, and miR171) showed the greatest expression in young spikes during microsporogenesis. Our results provide useful information for future studies of miRNA-mediated regulation of flower and grain development in wheat.

Durum wheat miRNAs in response to nitrogen starvation at the grain filling stage.

Durum wheat highly depends on nitrogen for seed development and yield, and the obtainment of varieties with a better nitrogen use efficiency is crucial to reduce production costs and environmental pollution. In this study, sequencing of two small RNA libraries obtained from tissues of Ciccio and Svevo cultivars grown under nitrogen starvation conditions produced 84 novel, and 161 conserved miRNAs. Of these, 7 novel and 13 known miRNAs were newly identified in this work. Quantitative PCR analysis of selected miRNAs highlighted that the expression levels of some of them depends on the tissue and on the cultivar, Svevo being the most responsive to nitrogen starvation. A number of target genes were predicted to be involved in nitrogen metabolism. An inverse correlation for the qPCR expression data of miRNA/target pairs miR399b/PHO2, miR393c/AFB2, ttu-novel-61/CCAAT-TF was observed in specific tissues or cultivar. Especially, ttu-novel-61 was down-regulated and its target CCAAT-TF up-regulated in almost all tissues both in Svevo and in Ciccio. Moreover, CCAAT-TF was confirmed to be cleaved by ttu-novel-61 at the expected site. The discovery of miRNAs involved in the response to nitrogen stress represents an important step towards functional analyses, with the final aim to design strategies for improving nitrogen use efficiency in durum wheat.

Genome-Wide Identification and Characterization of Salinity Stress-Responsive miRNAs in Wild Emmer Wheat (Triticum turgidum ssp. dicoccoides).

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs which regulate diverse molecular and biochemical processes at a post-transcriptional level in plants. As the ancestor of domesticated wheat, wild emmer wheat (Triticum turgidum ssp. dicoccoides) has great genetic potential for wheat improvement. However, little is known about miRNAs and their functions on salinity stress in wild emmer. To obtain more information on miRNAs in wild emmer, we systematically investigated and characterized the salinity-responsive miRNAs using deep sequencing technology. A total of 88 conserved and 124 novel miRNAs were identified, of which 50 were proven to be salinity-responsive miRNAs, with 32 significantly up-regulated and 18 down-regulated. miR172b and miR1120a, as well as mi393a, were the most significantly differently expressed. Targets of these miRNAs were computationally predicted, then Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the targets of salinity-responsive miRNAs were enriched in transcription factors and stress-related proteins. Finally, we investigated the expression profiles of seven miRNAs ranging between salt-tolerant and sensitive genotypes, and found that they played critical roles in salinity tolerance in wild emmer. Our results systematically identified the salinity-responsive miRNAs in wild emmer, not only enriching the miRNA resource but also laying the foundation for further study on the biological functions and evolution of miRNAs in wild wheat and beyond.

Virus-Based MicroRNA Silencing and Overexpressing in Common Wheat (Triticum aestivum L.).

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that arise from large RNA precursors with a stem-loop structure and play important roles in plant development and responses to environmental stress. Although a hundred and nineteen wheat miRNAs have been identified and registered in the miRBase (Release 21.0, June, 2014; http://www.mirbase.org), the functional characterization of these miRNAs in wheat growth and development is lagging due to lack of effective techniques to investigate endogenous miRNA functions in wheat. Here we report barley stripe mosaic virus(BSMV)-based miRNA overexpression and silence systems that can be applied to study miRNA functions in wheat. By utilizing the BSMV system, we successfully knocked down endogenous miR156 and miR166 levels and over-expressed endogenous miR156 and artificial miRNA against phytoene desaturase gene PDS (amiR-PDS) in wheat. amiR-PDS expression caused a great reduction in endogenous mRNA abundance of PDS gene in wheat plant, leading to leaf obviously photobleaching. miR156 silencing led to a great increase in the mRNA level of its target gene SPL2, resulting in a leaf-curl phenotype in wheat seedlings. In contrast, overexpression of miR156 led to a significant reduction in the mRNA level of SPL2 in wheat seedlings, resulting in serious delay of the vegetative phase transitions as well as booting and flowering in wheat. These confirmed that miR156 regulates wheat development and booting time through SPL genes. In summary, the BSMV-based miRNA overexpression and silence systems have extraordinary potential not only for functional study of protein-encoding genes but also for miRNA genes in wheat.

Migration of endophytic diazotroph Azorhizobium caulinodans ORS571 inside wheat (Triticum aestivum L) and its effect on microRNAs.

Azorhizobium caulinodans ORS571, a novel rhizobium, forms endosymbionts with its nature host Sesbania rostrata, a semi-aquatic leguminous tree. Recent studies showed that A. caulinodans ORS571, as endophytic rhizobium, disseminated and colonized inside of cereal plants. However, how this rhizobium infects monocot plants and the regulatory mechanism remains unknown. MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression at the post-transcriptional levels. In this study, we employed laser scanning confocal microscope to monitor the pathway that rhizobium invade wheat; we also investigated the potential role of miRNAs during A. caulinodans ORS571 infecting wheat. Our results showed that gfp-labeled A. caulinodans ORS571 infected wheat root hairs and emerged lateral roots, then disseminated and colonized within roots and migrated to other plant tissues, such as stems and leaves. Endophytic rhizobium induced the aberrant expression of miRNAs in wheat with a tissue- and time-dependent manner with a peak at 12-24h after rhizobium infection. Some miRNAs, such as miR167 and miR393 responded more in roots than that in shoots. In contrast, miR171 responded higher in shoots than that in roots. These results suggested that miRNAs could be responsive to A. caulinodans ORS571 infection and played important role in plant growth, nutrient metabolisms, and wheat-rhizobium interactions.

A novel comprehensive wheat miRNA database, including related bioinformatics software

MicroRNAs (miRNAs) are emerging as important post-transcriptional regulators that may regulate key genes responsible for agronomic traits such as grain yield and stress tolerance. Several studies identified species and clades specific miRNA families associated with plant stress regulated genes. Here, we propose a novel resource that provides data related to the expression of abiotic stress responsive miRNAs in wheat, one of the most important staple food crops. This database allows the query of small RNA libraries, including in silico predicted wheat miRNA sequences and the expression profiles of small RNAs identified from those libraries. Our database also provides a direct access to online miRNA prediction software tuned to de novo miRNA detection in wheat, in monocotyledon clades, as well as in other plant species. These data and software will facilitate multiple comparative analyses and reproducible studies on small RNAs and miRNA families in plants. Our web portal is available at: http://wheat.bioinfo.uqam.ca.

Uncovering leaf rust responsive miRNAs in wheat (Triticum aestivum L.) using high-throughput sequencing and prediction of their targets through degradome analysis.

Deep sequencing identified 497 conserved and 559 novel miRNAs in wheat, while degradome analysis revealed 701 targets genes. QRT-PCR demonstrated differential expression of miRNAs during stages of leaf rust progression. Bread wheat (Triticum aestivum L.) is an important cereal food crop feeding 30% of the world population. Major threat to wheat production is the rust epidemics. This study was targeted towards identification and functional characterizations of micro(mi)RNAs and their target genes in wheat in response to leaf rust ingression. High-throughput sequencing was used for transcriptome-wide identification of miRNAs and their expression profiling in retort to leaf rust using mock and pathogen-inoculated resistant and susceptible near-isogenic wheat plants. A total of 1056 mature miRNAs were identified, of which 497 miRNAs were conserved and 559 miRNAs were novel. The pathogen-inoculated resistant plants manifested more miRNAs compared with the pathogen infected susceptible plants. The miRNA counts increased in susceptible isoline due to leaf rust, conversely, the counts decreased in the resistant isoline in response to pathogenesis illustrating precise spatial tuning of miRNAs during compatible and incompatible interaction. Stem-loop quantitative real-time PCR was used to profile 10 highly differentially expressed miRNAs obtained from high-throughput sequencing data. The spatio-temporal profiling validated the differential expression of miRNAs between the isolines as well as in retort to pathogen infection. Degradome analysis provided 701 predicted target genes associated with defense response, signal transduction, development, metabolism, and transcriptional regulation. The obtained results indicate that wheat isolines employ diverse arrays of miRNAs that modulate their target genes during compatible and incompatible interaction. Our findings contribute to increase knowledge on roles of microRNA in wheat-leaf rust interactions and could help in rust resistance breeding programs.

Water-deficit stress-responsive microRNAs and their targets in four durum wheat genotypes.

MicroRNAs (miRNAs) guide regulation at the post-transcriptional level by inducing messenger RNA (mRNA) degradation or translational inhibition of their target protein-coding genes. Durum wheat miRNAs may contribute to the genotypic water-deficit stress response in different durum varieties. Further investigation of the interactive miRNA-target regulatory modules and experimental validation of their response to water stress will contribute to our understanding of the small RNA-mediated molecular networks underlying stress adaptation in durum wheat. In this study, a comprehensive genome-wide in silico analysis using the updated Triticum transcriptome assembly identified 2055 putative targets for 113 conserved durum miRNAs and 131 targets for four novel durum miRNAs that putatively contribute to genotypic stress tolerance. Predicted mRNA targets encode various transcription factors, binding proteins and functional enzymes, which play vital roles in multiple biological pathways such as hormone signalling and metabolic processes. Quantitative PCR profiling further characterised 43 targets and 5 miRNAs with stress-responsive and/or genotype-dependent differential expression in two stress-tolerant and two stress-sensitive durum genotypes subjected to pre-anthesis water-deficit stress. Furthermore, a 5' RLM-RACE approach validated nine mRNA targets cleaved by water-deficit stress-responsive miRNAs, which, to our knowledge, has not been previously reported in durum wheat. The present study provided experimental evidence of durum miRNAs and target genes in response to water-deficit stress in contrasting durum varieties, providing new insights into the regulatory roles of the miRNA-guided RNAi mechanism underlying stress adaptation in durum wheat.

Wheat miRNA ancestors: evident by transcriptome analysis of A, B, and D genome donors

MicroRNAs are critical players of post-transcriptional gene regulation with profound effects on the fundamental processes of cellular life. Their identification and characterization, together with their targets, hold great significance in exploring and exploiting their roles on a functional context, providing valuable clues into the regulation of important biological processes, such as stress tolerance or environmental adaptation. Wheat is a hardy crop, extensively harvested in temperate regions, and is a major component of the human diet. With the advent of the next generation sequencing technologies considerably decreasing sequencing costs per base-pair, genomic, and transcriptomic data from several wheat species, including the progenitors and wild relatives have become available. In this study, we performed in silico identification and comparative analysis of microRNA repertoires of bread wheat (Triticum aestivum L.) and its diploid progenitors and relatives, Aegilops sharonensis, Aegilops speltoides, Aegilops tauschii, Triticum monococcum, and Triticum urartu through the utilization of publicly available transcriptomic data. Over 200 miRNA families were identified, majority of which have not previously been reported. Ancestral relationships expanded our understanding of wheat miRNA evolution, while T. monococcum miRNAs delivered important clues on the effects of domestication on miRNA expression. Comparative analyses on wild Ae. sharonensis accessions highlighted candidate miRNAs that can be linked to stress tolerance. The miRNA repertoires of bread wheat and its diploid progenitors and relatives provide important insight into the diversification and distribution of miRNA genes, which should contribute to the elucidation of miRNA evolution of Poaceae family. A thorough understanding of the convergent and divergent expression profiles of miRNAs in different genetic backgrounds can provide unique opportunities to modulation of gene regulation for better crop performance.

Identification of microRNAs and their corresponding targets involved in the susceptibility interaction of wheat response to Puccinia striiformis f. sp. tritici.

MicroRNAs (miRNAs) play very important roles in plant defense responses. However, little is known about their roles in the susceptibility interaction between wheat and Puccinia striiformis f. sp. tritici (Pst). In this study, two miRNA libraries were constructed from the leaves of the cultivar Xingzi 9104 inoculated with the virulent Pst race CYR32 and sterile water, respectively. A total of 1316 miRNA candidates, including 173 known miRNAs that were generated from 98 pre-miRNAs, were obtained. The remaining 1143 miRNA candidates included 145 conserved and 998 wheat-specific miRNAs that were generated from 87 and 1088 pre-miRNAs, respectively. The 173 known and 145 conserved miRNAs were sub-classified into 63 miRNA families. The target genes of wheat miRNAs were also confirmed using degradome sequencing technology. Most of the annotated target genes were related to signal transduction or energy metabolism. Additionally, we found that miRNAs and their target genes form complicated regulation networks. The expression profiles of miRNAs and their corresponding target genes were further analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and the results indicate that some miRNAs are involved in the compatible wheat-Pst susceptibility interaction. Importantly, tae-miR1432 was highly expressed when wheat was challenged with CYR32, and the corresponding target gene, predicted to be a calcium ion-binding protein, also exhibited upregulated expression but a divergent expression trend. PC-3P-7484, a specific wheat miRNA, was highly expressed in the wheat response to Pst infection, while the expression of the corresponding target gene ubiquillin was dramatically downregulated. These data provide the foundation for evaluating the important regulatory roles of miRNAs in wheat-Pst susceptibility interaction.

Characterization of wheat miRNAs and their target genes responsive to cadmium stress

A increasing number of microRNAs have been shown to play important regulatory roles in plant responses to various metal stresses. However, little information about miRNAs especially miRNAs responsive to cadmium (Cd) stress is available in wheat. To investigate the role of miRNAs in responses to Cd stress, wheat seedlings were subjected to 250 μM Cd solution for 6, 12, 24 and 48 h, and analyses of morphological and physiological changes as well as the expression of five miRNAs and their corresponding targets were carried out. Our results demonstrated that miRNAs and their targets were differentially expressed in leaves and roots of wheat seedlings exposed to Cd stress. Furthermore, miR398 may involve in oxidative stress tolerance by regulating its target CSD to participate in Cd stress. Among ten miRNA-target pairs studied, nine pairs showed complex regulation relationship in leaves and roots of wheat seedlings exposed to Cd stress. These findings suggested that miRNAs are involved in the mediation of Cd stress signaling responses in wheat. The characterization of the miRNAs and the associated targets in responses to Cd exposure provides a framework for understanding the molecular mechanism of heavy metal tolerance in plants.

Wheat microRNA Member TaMIR444a Is Nitrogen Deprivation-Responsive and Involves Plant Adaptation to the Nitrogen-Starvation Stress

microRNAs (miRNAs) are involved in regulating various plant developmental processes and mediating plant-adaptive responses to nutrient deprivation. In this study, the characterization of a wheat miRNA member TaMIR444a and the role of this miRNA in mediating plant tolerance to the N-starvation stress were investigated. Results indicated that the expression levels of TaMIR444a and NtMIR444a, the homologue of TaMIR444a in tobacco, were upregulated in roots and leaves under N deprivation, whereas the transcription of their target genes showed reverse expression patterns in above tissues. These results suggest that miR444a is conserved across plant species of dicots and monocots and can possibly establish the miRNA/target modules for mediating plant response to N deficiency. Overexpression of TaMIR444a in tobacco improved the plant growth feature, biomass, N content, photosynthetic parameters, and antioxidant enzymatic activities under N deprivation. Based on microarray analyses, a large number of genes were identified to be differentially expressed in the TaMIR444a-overexpressing plants; these differential genes are categorized into functional groups of signal perception and transduction, transcription regulation, primary and secondary metabolism, phytohormone response, cellular protection and defensive responsiveness, etc. qPCR analyses revealed that the nitrate transporter (NRT) genes NtNRT1.1-s, NtNET1.1-t, and NtNRT2.1 and the antioxidant enzyme genes (AEEs) NtCAT1;1, NtPOD1;3, and NtPOD4 were significantly upregulated by TaMIR444a, suggesting that the altered transcription of these NRT and AEE genes is associated with the improvement of the N acquisition and the cellular ROS detoxification in the N-deprived transgenic plants. Together, our findings demonstrate that miR444a acts as one critical regulator in mediating plant tolerance to the N-starvation stress through modulation of the regulatory networks associated with N acquisition, cellular ROS homeostasis, and carbon assimilation. Our findings have provided insights into the mechanisms of plant tolerance to N deficiency mediated by the distinct miRNA pathways.

Exploration of microRNAs and their targets engaging in the resistance interaction between wheat and stripe rust.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. miRNAs are important regulators, they play very central roles in plant organ development, vegetable phase change and defense responses. In this study, two miRNA libraries from wheat cultivar Xingzi 9104 (XZ) challenged with the avirulent Pst race CYR32 and sterile water were constructed, respectively. A total of 596 miRNA candidates were obtained. 420 wheat-specific candidate miRNAs were screened in adult plants challenged with Pst using microarray-based analyses. We analyzed the abundance of candidate miRNAs, and the levels of a subset of candidate miRNAs were determined by quantitative real time PCR (qRT-PCR). The qRT-PCR results indicated that some miRNAs were involved in the incompatible interaction between wheat and Pst. In addition, we identified some miRNAs differentially expressed in different leaves. Additionally, the target genes of wheat miRNAs were confirmed by using degradome sequencing technology. Most of the annotated target genes are related to signal transduction, energy metabolism, and other functions. We selected some target genes for relative expression analysis using qRT-PCR, and found that RabGAP/TBC domain-containing protein, zinc finger protein and Cysteine-rich receptor-like protein kinase 41 may play important role in the incompatible interaction between XZ and CYR32. Intriguingly, miRNAs and target gene seem to form a complicated regulation network that regulates the wheat-Pst interaction. Our data provide the foundation for evaluating the important regulatory roles of miRNAs in the wheat-Pst interaction.

Hexaploid wheat (Triticum aestivum) root miRNome analysis in response to salt stress

Hexaploid bread wheat (Triticum aestivum) is one of the major crops grown and consumed all over the world. Elevated soil salinity causes reduction in crop yield and quality; therefore, several strategies were developed to improve salt-tolerant cultivars. MicroRNAs (miRNAs), small and non-coding RNAs, regulate gene expression at post-transcriptional level and play important roles in stress tolerance. Here, we used a broad-range miRNA-microarray analysis to investigate the root-miRNA profiles of two cultivars, Bezostaja (sensitive) and Seri-82 (tolerant). A total of 44 differentially regulated miRNAs were identified in the 8 × 15K array containing 11 862 plant miRNAs available in the database. Sixteen novel salt-responsive miRNAs were determined in wheat for the first time. The expression of three miRNAs (hvu-miR5049a, ppt-miR1074 and osa-miR444b.2) was up-regulated more than 260-fold in cv. Bezostaja upon salt stress. The target-gene analyses showed that several salt-stress-responsive miRNAs regulate mainly transcription factors such as bHLH135-like, AP2/ERBP, MADS-box and transporters. Gene ontology searches for 565 putative salt-stress-responsive miRNA target-genes revealed 623 processes in 10 different main topics such as metabolic process and response to stimuli. The genome-wide root miRNome study indicates salt-stress-responsive wheat miRNAs and the possible mechanisms behind the tolerance.

Barley Stripe Mosaic Virus (BSMV) Induced MicroRNA Silencing in Common Wheat (Triticum aestivum L.)

MicroRNAs (miRNAs) play important roles in growth, development, and response to environmental changes in plants. Based on the whole-genome shotgun sequencing strategy, more and more wheat miRNAs have been annotated. Now, there is a need for an effective technology to analyse endogenous miRNAs function in wheat. We report here that the modified barley stripe mosaic virus (BSMV)-induced miRNAs silencing system can be utilized to silence miRNAs in wheat. BSMV-based miRNA silencing system is performed through BSMV-based expression of miRNA target mimics to suppress miR159a and miR3134a. The relative expression levels of mature miR159a and miR3134a decrease with increasing transcript levels of their target genes in wheat plants. In summary, the developed approach is effective in silencing endogenous miRNAs, thereby providing a powerful tool for biological function analyses of miRNA molecules in common wheat.

An integrative approach to identify hexaploid wheat miRNAome associated with development and tolerance to abiotic stress.

BackgroundWheat is a major staple crop with broad adaptability to a wide range of environmental conditions. This adaptability involves several stress and developmentally responsive genes, in which microRNAs (miRNAs) have emerged as important regulatory factors. However, the currently used approaches to identify miRNAs in this polyploid complex system focus on conserved and highly expressed miRNAs avoiding regularly those that are often lineage-specific, condition-specific, or appeared recently in evolution. In addition, many environmental and biological factors affecting miRNA expression were not yet considered, resulting still in an incomplete repertoire of wheat miRNAs.ResultsWe developed a conservation-independent technique based on an integrative approach that combines machine learning, bioinformatic tools, biological insights of known miRNA expression profiles and universal criteria of plant miRNAs to identify miRNAs with more confidence. The developed pipeline can potentially identify novel wheat miRNAs that share features common to several species or that are species specific or clade specific. It allowed the discovery of 199 miRNA candidates associated with different abiotic stresses and development stages. We also highlight from the raw data 267 miRNAs conserved with 43 miRBase families. The predicted miRNAs are highly associated with abiotic stress responses, tolerance and development. GO enrichment analysis showed that they may play biological and physiological roles associated with cold, salt and aluminum (Al) through auxin signaling pathways, regulation of gene expression, ubiquitination, transport, carbohydrates, gibberellins, lipid, glutathione and secondary metabolism, photosynthesis, as well as floral transition and flowering.ConclusionThis approach provides a broad repertoire of hexaploid wheat miRNAs associated with abiotic stress responses, tolerance and development. These valuable resources of expressed wheat miRNAs will help in elucidating the regulatory mechanisms involved in freezing and Al responses and tolerance mechanisms as well as for development and flowering. In the long term, it may help in breeding stress tolerant plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1490-8) contains supplementary material, which is available to authorized users.