microRNAs In Wheat Research Articles

Влияние малых интерферирующих РНК, синтезированных in vitro, на рост и споруляцию патогенного гриба Stagonospora nodorum

Recently, several RNA interference-based disease prevention mechanisms using small RNAs have been used to protect crops in some countries. Plants secrete small RNAs that directly or indirectly inhibit the virulence of pathogens. In this work, we studied the effect of three wheat microRNAs (miR159, miR166 and miR408) synthesized in vitro on the growth and sporulation of the pathogenic fungus Stagonospora nodorum, as well as on the expression of the genes of the necrotrophic effectors (NEs) S. nodorum SnToxA and SnTox3 (which determine the virulence of the pathogen) and the fungal transcription factors (TFs) Pf2, StuA, Con7 regulating the transcription of NE genes. It was shown that the addition of microRNAs in various concentrations into the fungal cultivation medium affected the growth of colonies and sporulation of the pathogen. miR166 had the greatest impact on these parameters. Using gene-specific primers, PCR analysis was carried out, which showed that the effect of microRNA on the expression of the genes encoding NEs and TFs of the pathogenic fungus S. nodorum depended on the type and concentration of microRNA in the medium. Thus, 0.5 ng/μl of ssRNA408 reduced the expression of all the studied NEs and TFs genes on the 7th day of cultivation. Further comprehensive studies of the mechanisms of cross-species regulation using miRNAs may help in the development of new pesticides for modern agriculture.

Comprehensive molecular dissection of TIFY Transcription factors reveal their dynamic responses to biotic and abiotic stress in wheat (Triticum aestivum L.)

The plant specific TIFY (previously known as ZIM) transcription factor (TF) family plays crucial roles in cross talk between Jasmonic Acid and other phytohormones like gibberellins, salicylic acid, abscisic acid, auxin, and ethylene signaling pathways. Wheat yield is severely affected by rust diseases and many abiotic stresses, where different phytohormone signaling pathways are involved. TIFYs have been studied in many plants yet reports describing their molecular structure and function in wheat are lacking. In the present study, we have identified 23 novel TIFY genes in wheat genome using in silico approaches. The identified proteins were characterized based on their conserved domains and phylogenetically classified into nine subfamilies. Chromosomal localization of the identified TIFY genes showed arbitrary distribution. Forty cis-acting elements including phytohormone, stress and light receptive elements were detected in the upstream regions of TIFY genes. Seventeen wheat microRNAs targeted the identified wheat TIFY genes. Gene ontological studies revealed their major contribution in defense response and phytohormone signaling. Secondary structure of TIFY proteins displayed the characteristic alpha–alpha–beta fold. Synteny analyses indicated all wheat TIFY genes had orthologous sequences in sorghum, rice, maize, barley and Brachypodium indicating presence of similar TIFY domains in monocot plants. Six TIFY genes had been cloned from wheat genomic and cDNA. Sequence characterization revealed similar characteristics as the in silico identified novel TIFY genes. Tertiary structures predicted the active sites in these proteins to play critical roles in DNA binding. Expression profiling of TIFY genes showed their contribution during incompatible and compatible leaf rust infestation. TIFY genes were also highly expressed during the initial hours of phytohormone induced stress. This study furnishes fundamental information on characterization and putative functions of TIFY genes in wheat.

Transgenerational Effects of Water-Deficit and Heat Stress on Germination and Seedling Vigour-New Insights from Durum Wheat microRNAs.

Water deficiency and heat stress can severely limit crop production and quality. Stress imposed on the parents during reproduction could have transgenerational effects on their progeny. Seeds with different origins can vary significantly in their germination and early growth. Here, we investigated how water-deficit and heat stress on parental durum wheat plants affected seedling establishment of the subsequent generation. One stress-tolerant and one stress-sensitive Australian durum genotype were used. Seeds were collected from parents with or without exposure to stress during reproduction. Generally, stress on the previous generation negatively affected seed germination and seedling vigour, but to a lesser extent in the tolerant variety. Small RNA sequencing utilising the new durum genome assembly revealed significant differences in microRNA (miRNA) expression in the two genotypes. A bioinformatics approach was used to identify multiple miRNA targets which have critical molecular functions in stress adaptation and plant development and could therefore contribute to the phenotypic differences observed. Our data provide the first confirmation of the transgenerational effects of reproductive-stage stress on germination and seedling establishment in durum wheat. New insights gained on the epigenetic level indicate that durum miRNAs could be key factors in optimising seed vigour for breeding superior germplasm and/or varieties.

Genomic identification and characterization of MYC family genes in wheat (Triticum aestivum L.)

BackgroundMYC transcriptional factors are members of the bHLH (basic helix-loop-helix) superfamily, and play important roles in plant growth and development. Recent studies have revealed that some MYCs are involved in the crosstalk between Jasmonic acid regulatory pathway and light signaling in Arabidopsis, but such kinds of studies are rare in wheat, especially in photo-thermo-sensitive genic male sterile (PTGMS) wheat line.Results27 non-redundant MYC gene copies, which belonged to 11 TaMYC genes, were identified in the whole genome of wheat (Chinese Spring). These gene copies were distributed on 13 different chromosomes, respectively. Based on the results of phylogenetic analysis, 27 TaMYC gene copies were clustered into group I, group III, and group IV. The identified TaMYC genes copies contained different numbers of light, stress, and hormone-responsive regulatory elements in their 1500 base pair promoter regions. Besides, we found that TaMYC3 was expressed highly in stem, TaMYC5 and TaMYC9 were expressed specially in glume, and the rest of TaMYC genes were expressed in all tissues (root, stem, leaf, pistil, stamen, and glume) of the PTGMS line BS366. Moreover, we found that TaMYC3, TaMYC7, TaMYC9, and TaMYC10 were highly sensitive to methyl jasmonate (MeJA), and other TaMYC genes responded at different levels. Furthermore, we confirmed the expression profiles of TaMYC family members under different light quality and plant hormone stimuli, and abiotic stresses. Finally, we predicted the wheat microRNAs that could interact with TaMYC family members, and built up a network to show their integrative relationships.ConclusionsThis study analyzed the size and composition of the MYC gene family in wheat, and investigated stress-responsive and light quality induced expression profiles of each TaMYC gene in the PTGMS wheat line BS366. In conclusion, we obtained lots of important information of TaMYC family, and the results of this study was supposed to contribute novel insights and gene and microRNA resources for wheat breeding, especially for the improvement of PTGMS wheat lines.

The genome-wide impact of cadmium on microRNA and mRNA expression in contrasting Cd responsive wheat genotypes

BackgroundHeavy metal ATPases (HMAs) are responsible for Cd translocation and play a primary role in Cd detoxification in various plant species. However, the characteristics of HMAs and the regulatory mechanisms between HMAs and microRNAs in wheat (Triticum aestivum L) remain unknown.ResultsBy comparative microRNA and transcriptome analysis, a total three known and 19 novel differentially expressed microRNAs (DEMs) and 1561 differentially expressed genes (DEGs) were found in L17 after Cd treatment. In H17, by contrast, 12 known and 57 novel DEMs, and only 297 Cd-induced DEGs were found. Functional enrichments of DEMs and DEGs indicate how genotype-specific biological processes responded to Cd stress. Processes found to be involved in microRNAs-associated Cd response include: ubiquitin mediated proteolysis, tyrosine metabolism, and carbon fixation pathways and thiamine metabolism. For the mRNA response, categories including terpenoid backbone biosynthesis and phenylalanine metabolism, and photosynthesis - antenna proteins and ABC transporters were enriched. Moreover, we identified 32 TaHMA genes in wheat. Phylogenetic trees, chromosomal locations, conserved motifs and expression levels in different tissues and roots under Cd stress are presented. Finally, we infer a microRNA-TaHMAs expression network, indicating that miRNAs can regulate TaHMAs.ConclusionOur findings suggest that microRNAs play important role in wheat under Cd stress through regulation of targets such as TaHMA2;1. Identification of these targets will be useful for screening and breeding low-Cd accumulation wheat lines.

Identification of isoforms of microRNAs in wheat (Triticum aestivum L.) and their role in leaf rust pathogenesis

Identification of isoforms of microRNAs in wheat (Triticum aestivum L.) and their role in leaf rust pathogenesis

Identification and characterization of durum wheat microRNAs in leaf and root tissues.

MicroRNAs are a class of post-transcriptional regulators of plant developmental and physiological processes and responses to environmental stresses. Here, we present the study regarding the annotation and characterization of MIR genes conducted in durum wheat. We characterized the miRNAome of leaf and root tissues at tillering stage under two environmental conditions: irrigated with 100% (control) and 55% of evapotranspiration (early water stress). In total, 90 microRNAs were identified, of which 32 were classified as putative novel and species-specific miRNAs. In addition, seven microRNA homeologous groups were identified in each of the two genomes of the tetraploid durum wheat. Differential expression analysis highlighted a total of 45 microRNAs significantly differentially regulated in the pairwise comparisons leaf versus root. The miRNA families, miR530, miR395, miR393, miR5168, miR396 and miR166, miR171, miR319, and miR167, were the most expressed in leaves in comparison to roots. Putative microRNA targets were predicted for both five and three prime sequences derived from the stem-loop of the MIR gene. Gene ontology analysis showed significant overrepresented gene categories in microRNA targets belonging to transcription factors, phenylpropanoids, oxydases, and lipid binding-protein. This work represents one of the first genome wide characterization of MIR genes in durum wheat, identifying leaf and root tissue-specific microRNAs. This genomic identification of microRNAs together with the analysis of their expression profiles is a well-accepted starting point leading to a better comprehension of the role of MIR genes in the genus Triticum.

Regulation of NBS-LRR Genes by MicroRNAs in Wheat: Computational Identification of Candidate MIR-2118 Genes and Evidence of Flexibility

Plant microRNAs are crucial for post-transcriptional regulation of gene expression, with some of them particularly involved in regulating several disease resistance (R) genes. This study is a computational screening for microRNAs potentially involved in the regulation of disease resistance in bread wheat Triticum aestivum, through the genome and transcriptome of this crop species. We used plant miRNAs of the superfamily miR-482/2118 to find complementarities with the expressed sequence tags (ESTs) coding NBS-LRR proteins of the bread wheat. This analysis revealed a vast recognition potential, highlighting highly variable miRNA-mRNA hybridization schemes. Precursors of miR-2118/482 sequences that showed a potential for recognizing wheat NBS-LRR ESTs were used as input for similarity search in the T. aestivum transcriptome shotgun assemblies (TSA) database, enabling the identification of a couple of wheat TSA sequences (JV952948.1 and JV851699.1) that were predicted to adopt a stable miRNA/miRNA* duplex structure. The genomic regions corresponding to both predicted tae-miR-2118 genes were determined on wheat chromosomes 2DL and 5AS. The findings of the present study will contribute to a better understanding of R gene expression regulation in wheat.

Novel and conserved heat-responsive microRNAs in wheat (Triticum aestivum L.).

MicroRNAs (miRNAs) are small endogenous RNAs of ~22 nucleotides that have been shown to play regulatory role by negatively affecting the expression of genes at the post-transcriptional level. Information of miRNAs on some important crops like soybean, Arabidopsis, and rice, etc. are available, but no study on heat-responsive novel miRNAs has yet been reported in wheat (Triticum aestivum L.). In the present investigation, a popular wheat cultivar HD2985 was used in small RNA library construction and Illumina HiSeq 2000 was used to perform high-throughput sequencing of the library after cluster generation; 110,896,604 and 87,743,861 reads were generated in the control (22 °C) and heat-treated (42 °C for 2 h) samples, respectively. Forty-four precursor and mature miRNAs were found in T. aestivum from miRBase v 19. The frequencies of the miRNA families varied from 2 (tae-miR1117) to 60,672 (tae-miR159b). We identify 1052 and 902 mature miRNA sequences in HD2985 control and HS-treated samples by mapping on reference draft genome of T. aestivum. Maximum identified miRNAs were located on IWGSC_CSS_3B_scaff (chromosome 3B). We could identify 53 and 46 mature miRNA in the control and HS samples and more than 516 target genes by mapping on the reference genome of Oryza sativa, Zea mays, and Sorghum bicolor. Using different pipelines and plant-specific criteria, 37 novel miRNAs were identified in the control and treated samples. Six novel miRNA were validated using qRT-PCR to be heat-responsive. A negative correlation was, however, observed between the expression of novel miRNAs and their targets. Target prediction and pathway analysis revealed their involvement in the heat stress tolerance. These novel miRNAs are new additions to miRNA database of wheat, and the regulatory network will be made use of in deciphering the mechanism of thermotolerance in wheat.

Characterization and expression pattern analysis of microRNAs in wheat under drought stress

Plant microRNAs (miRNAs) play important roles in regulating plant growth, development, and responses to abiotic stresses. In this study, 38 miRNAs (TaMIRs) from wheat (Triticum aestivum L.), 36 from the miRBase database, and two from our previous work were characterized and subjected to an expression pattern analysis under normal conditions and a drought stress. A semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), real-time quantitative PCR (qPCR), and small RNA blot analyses revealed that two TaMIRs (TaMIR1120 and TaMIR1123) were root-predominant and two TaMIRs (TaMIR1121 and TaMIR1134) were leaf-predominant. Seven TaMIR precursors showed altered expressions after the drought; of these, TaMIR1136 was upregulated, whereas TaMIR156, TaMIR408, TaMIR1119, TaMIR1129, TaMIR1133, and TaMIR1139 were downregulated. These seven drought-responsive TaMIRs showed dose-dependent and typical temporal expression patterns during drought induction, and they gradually returned back under the normal growth conditions. The drought-responsive and the tissue-predominant TaMIRs had varying numbers of target genes. Randomly selected target genes exhibited opposite expression patterns to their corresponding TaMIRs suggesting that they were regulated by distinct TaMIRs through a post-transcriptional cleavage. The target genes regulated by drought-responsive and tissue-predominant TaMIRs are involved in various cellular processes, such as signal transduction, transcriptional regulation, primary and secondary metabolisms, development, and defense responses. These results provide a novel insight into the miRNA-mediated responses of wheat to drought stress.

Identification and characterization of a subset of microRNAs in wheat (Triticum aestivum L.)

MicroRNAs (miRNAs) represent a class of endogenous regulator for post-transcriptionally modulating gene expression. Elucidating complete miRNA repertoires for individual species is a long-desired goal in miRNA research. So far only 42 have been annotated for common wheat (Triticum aestivum) due to its large genome. Here, we employed miRDeep-P, a program developed previously for retrieving miRNAs from deep sequencing data in plants, to parse 14 sequenced small RNA libraries of wheat using expression sequence tags (ESTs) as the reference in lieu of a complete genome sequence. This effort identified 145 miRNAs along with the corresponding stem-looped precursors with many differentially expressed in development and associated with powdery mildew infection. Examination of the phylogenetic distribution of these miRNAs and their target genes revealed that many are conserved in monocots. The set of miRNAs identified in this study is thus useful to further miRNA research in wheat and other important cereal crop species.

New wheat microRNA using whole-genome sequence

MicroRNAs are post-transcriptional regulators of gene expression, taking roles in a variety of fundamental biological processes. Hence, their identification, annotation and characterization are of great significance, especially in bread wheat, one of the main food sources for humans. The recent availability of 5× coverage Triticum aestivum L. whole-genome sequence provided us with the opportunity to perform a systematic prediction of a complete catalogue of wheat microRNAs. Using an in silico homology-based approach, stem-loop coding regions were derived from two assemblies, constructed from wheat 454 reads. To avoid the presence of pseudo-microRNAs in the final data set, transposable element related stem-loops were eliminated by repeat analysis. Overall, 52 putative wheat microRNAs were predicted, including seven, which have not been previously published. Moreover, with distinct analysis of the two different assemblies, both variety and representation of putative microRNA-coding stem-loops were found to be predominant in the intergenic regions. By searching available expressed sequences and small RNA library databases, expression evidence for 39 (out of 52) putative wheat microRNAs was provided. Expression of three of the predicted microRNAs (miR166, miR396 and miR528) was also comparatively quantified with real-time quantitative reverse transcription PCR. This is the first report on in silico prediction of a whole repertoire of bread wheat microRNAs, supported by the wet-lab validation.

Discovery of Novel Leaf Rust Responsive microRNAs in Wheat and Prediction of Their Target Genes.

MicroRNAs are endogenous small noncoding RNAs which play critical roles in gene regulation. Few wheat (Triticum aestivum L.) miRNA sequences are available in miRBase repertoire and knowledge of their biological functions related to biotic stress is limited. We identified 52 miRNAs, belonging to 19 families, from next-generation transcriptome sequence data based on homology search. One wheat specific novel miRNA was identified but could not be ascribed or assigned to any known miRNA family. Differentially expressed 22 miRNAs were found between susceptible and resistant wheat near-isogenic lines inoculated with leaf rust pathogen Puccinia triticina and compared with mock inoculated controls. Most miRNAs were more upregulated in susceptible NIL compared to resistant NIL. We identified 1306 potential target genes for these 52 miRNAs with vital roles in response to stimuli, signaling, and diverse metabolic and cellular processes. Gene ontology analysis showed 66, 20, and 35 target genes to be categorized into biological process, molecular function, and cellular component, respectively. A miRNA-mediated regulatory network revealed relationships among the components of the targetome. The present study provides insight into potential miRNAs with probable roles in leaf rust pathogenesis and their target genes in wheat which establish a foundation for future studies.

Expression pattern analysis of microRNAs in root tissue of wheat (Triticum aestivum L.) under normal nitrogen and low nitrogen conditions

MicroRNAs (miRNAs) are highly conserved non-coding small RNAs involved in regulating plant growth and development, as well as plant responses, to diverse environmental signaling cues. In this study, the expression patterns of 38 wheat microRNAs (TaMIRs) were investigated under normal N and low N stress. Under normal N conditions, the TaMIRs exhibited four expression patterns: high, moderate, low, and undetectable expression. Seven TaMIRs (TaMIR156, TaMIR399, TaMIR444, TaMIR1118, TaMIR1129, TaMIR1133, and TaMIR1136) showed varied expression levels under N deprivation. TaMIR156, TaMIR444, TaMIR1118, TaMIR1129, and TaMIR1136 were upregulated, whereas TaMIR399 and TaMIR1133 were downregulated. The expression patterns of TaMIR444, TaMIR1118, and TaMIR1129 were further analyzed under various low N concentrations and then returned to normal N. The aforementioned TaMIRs exhibited ever higher expression at lower N concentrations and whose expressions returned to those before low N stress after they were restored to normal N conditions, which suggest that N concentration and low N-duration are inversely correlated with their expression. The putative target genes of the low N–responsive TaMIRs were identified and categorized into various functional groups. Expression analysis revealed that the target genes showed opposite pattern to that of their corresponding TaMIRs. Our results suggested that some low N–responsive TaMIRs are involved in regulating plant response to N deprivation by interacting with their target genes. Distinct TaMIRs are potentially involved in plant tolerance to low N stress through microRNA-mediated pathways.

Identification of UV-B-induced microRNAs in wheat

MicroRNAs (miRNAs) play critical roles in post-transcriptional gene regulation and act as important endogenous regulators to various stressors. Ultraviolet-B (UV-B) radiation is a major factor influencing crop growth and development. In this study, we isolated a novel wheat miRNA, named Tae-miR6000, and confirmed its expression diversity after UV-B treatments. Additionally, using the Northern blotting technique, we found that six miRNAs were highly responsive to UV-B stress in wheat. Of these six miRNAs, miR159, miR167a, and miR171 were significantly upregulated, and the remaining three miRNAs were downregulated, at different time points after UV-B treatment. This result indicates that miRNAs may be involved in the regulation of targets after induction by UV-B stress. Furthermore, promoter analysis of the UV-B-responsive miRNA genes revealed some light-relevant cis-elements, such as the I-box and G-box. Taken together, the results of this study suggest that wheat miRNAs play important roles in the response to UV-B stress.

Selection of suitable inner reference genes for relative quantification expression of microRNA in wheat

Quantitative real-time PCR (qRT-PCR) is currently the most accurate and widely applied method to detect differential genes expression, but choosing a suitable gene to be the internal control is a crucial factor for correct analysis of the results. MicroRNAs are fundamental regulatory genes of eukaryotic genomes, acting on several biological functions. Transcription accumulation of microRNAs has been studied using qRT-PCR, while no validated reference genes for microRNAs in wheat are available until now. In this study, nine previous reported housekeeping genes and ten wheat microRNAs were examined with regard to their use as normalizer and data was analyzed using geNorm and NormFind software. Expression stability of candidate inner reference genes was investigated in different conditions. After analysis of all the sample pools and samples after biotic and abiotic stress treatments, it was found that microRNAs had better expression stability than protein-coding genes, and mi167 and mi159 appeared to be the two most suitable reference genes in wheat. To confirm the stable expression of the putative reference genes in wheat, expression of mi171b of wheat was examined with inner reference genes mi167, mi159 and combination of mi157 and mi159 respectively. We provided evidence for that in order to get a more accurate result of gene expression, mi167 and mi159 should be used as inner reference gene for normalization together.