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. 1. Introduction Bread wheat (Triticum aestivum L.) is the second most important food crop after rice based on international commodity price and third in order of production after maize and rice. Wheat is extensively grown throughout the world and provides protein content, as well as 20% of basic caloric value to humans [1]. Wheat is the last major cereal crop for which complete genome sequence is not available due to its large genome size (16.9？GB), high proportion of transposable and repetitive DNA element, and presence of three closely related genomes (AABBDD, 2n = 6x = 42) [2]. However, recent technological advances such as next-generation sequencing platforms now offer large scale programs that delivered 5x genomic sequence resources for wheat [3]. Leaf rust is a major disease of wheat caused by the obligate biotrophic fungus Puccinia triticina Eriks that results in 10% yield loss annually [4]. More than 60 leaf rust resistant (Lr) genes had been identified from wheat and its wild relatives that confer resistance in a gene-for-gene manner. Many of them had been introgressed into wheat, but due to continuous evolution of the pathogen genome resistance breaks down quickly [5]. The molecular basis
References
[1]
F.A.O. Food and Agriculture Organization of the United Nations, 2012, http://www.fao.org/home/en/.
[2]
B. S. Gill, R. Appels, A. Botha-Oberholster et al., “A workshop report on wheat genome sequencing: international genome research on wheat consortium,” Genetics, vol. 168, no. 2, pp. 1087–1096, 2004.
[3]
R. Brenchley, M. Spannagl, M. Pfeifer, et al., “Analysis of the bread wheat genome using whole-genome shotgun sequencing,” Nature, vol. 491, pp. 705–710, 2012.
[4]
R. Dean, J. A. L. Van Kan, Z. A. Pretorius et al., “The Top 10 fungal pathogens in molecular plant pathology,” Molecular Plant Pathology, vol. 13, no. 4, pp. 414–430, 2012.
[5]
R. A. McIntosh and Z. A. Pretorius, “Borlaug global rust Initiative provides momentum for wheat rust research,” Euphytica, vol. 179, no. 1, pp. 1–2, 2011.
[6]
E. J. Chapman and J. C. Carrington, “Specialization and evolution of endogenous small RNA pathways,” Nature Reviews Genetics, vol. 8, no. 11, pp. 884–896, 2007.
[7]
S. Li, L. Liu, X. Zhuang et al., “MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis,” Cell, vol. 153, no. 3, pp. 562–574, 2013.
[8]
F. Vazquez, “Arabidopsis endogenous small RNAs: highways and byways,” Trends in Plant Science, vol. 11, no. 9, pp. 460–468, 2006.
[9]
A. A. Millar and P. M. Waterhouse, “Plant and animal microRNAs: similarities and differences,” Functional and Integrative Genomics, vol. 5, no. 3, pp. 129–135, 2005.
[10]
M. Megraw and A. G. Hatzigeorgiou, “MicroRNA promoter analysis,” Methods in Molecular Biology, vol. 592, pp. 149–161, 2010.
[11]
Y. Lee, M. Kim, J. Han et al., “MicroRNA genes are transcribed by RNA polymerase II,” The EMBO Journal, vol. 23, no. 20, pp. 4051–4060, 2004.
[12]
D. P. Bartel, “MicroRNAs: genomics, biogenesis, mechanism, and function,” Cell, vol. 116, no. 2, pp. 281–297, 2004.
[13]
D. Baulcombe, “RNA silencing in plants,” Nature, vol. 431, no. 7006, pp. 356–363, 2004.
[14]
S. Campo, C. Peris-Peris, C. Siré et al., “Identification of a novel microRNA (miRNA) from rice that targets an alternatively spliced transcript of the Nramp6 (Natural resistance-associated macrophage protein 6) gene involved in pathogen resistance,” New Phytologist, vol. 199, no. 1, pp. 212–227, 2013.
[15]
R. Sunkar, V. Chinnusamy, J. Zhu, and J. Zhu, “Small RNAs as big players in plant abiotic stress responses and nutrient deprivation,” Trends in Plant Science, vol. 12, no. 7, pp. 301–309, 2007.
[16]
M. Xin, Y. Wang, Y. Yao et al., “Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.),” BMC Plant Biology, vol. 10, article 123, 2010.
[17]
T. Unver, D. M. Namuth-Covert, and H. Budak, “Review of current methodological approaches for characterizing MicroRNAs in plants,” International Journal of Plant Genomics, vol. 2009, Article ID 262463, 11 pages, 2009.
[18]
B. H. Zhang, X. P. Pan, Q. L. Wang, G. P. Cobb, and T. A. Anderson, “Identification and characterization of new plant microRNAs using EST analysis,” Cell Research, vol. 15, no. 5, pp. 336–360, 2005.
[19]
E. Bonnet, J. Wuyts, P. Rouzé, and Y. Van De Peer, “Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliania and Oryza sativa identifies important target genes,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 31, pp. 11511–11516, 2004.
[20]
Y. S. Han, F. L. Luan, H. L. Zhu et al., “Computational identification of microRNAs and their targets in wheat (Triticum aestivum L.),” Science in China C, vol. 52, no. 11, pp. 1091–1100, 2009.
[21]
B. Zhang, X. Pan, C. H. Cannon, G. P. Cobb, and T. A. Anderson, “Conservation and divergence of plant microRNA genes,” Plant Journal, vol. 46, no. 2, pp. 243–259, 2006.
[22]
M. Din and M. Y. K. Barozai, “Profiling and characterization of eggplant (Solanum melongena L.) microRNAs and their targets,” Molecular Biology Reports, vol. 41, no. 2, pp. 889–894, 2014.
[23]
J. R. Brown and P. Sanseau, “A computational view of microRNAs and their targets,” Drug Discovery Today, vol. 10, no. 8, pp. 595–601, 2005.
[24]
N. Fahlgren, M. D. Howell, K. D. Kasschau et al., “High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes,” PLoS ONE, vol. 2, no. 2, article e219, 2007.
[25]
R. Sunkar, X. Zhou, Y. Zheng, W. Zhang, and J. Zhu, “Identification of novel and candidate miRNAs in rice by high throughput sequencing,” BMC Plant Biology, vol. 8, article 25, 2008.
[26]
D. Klevebring, N. R. Street, N. Fahlgren et al., “Genome-wide profiling of Populus small RNAs,” BMC Genomics, vol. 10, article 620, 2009.
[27]
V. Pantaleo, G. Szittya, S. Moxon et al., “Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis,” Plant Journal, vol. 62, no. 6, pp. 960–976, 2010.
[28]
G. Szittya, S. Moxon, D. M. Santos et al., “High-throughput sequencing of Medicago truncatula short RNAs identifies eight new miRNA families,” BMC Genomics, vol. 9, article 593, 2008.
[29]
D. Qiu, X. Pan, I. W. Wilson et al., “High throughput sequencing technology reveals that the taxoid elicitor methyl jasmonate regulates microRNA expression in Chinese yew (Taxus chinensis),” Gene, vol. 436, no. 1-2, pp. 37–44, 2009.
[30]
F. Yi, S. Xie, Y. Liu, X. Qi, and J. Yu, “Genome-wide characterization of microRNA in foxtail millet (Setaria italica),” BMC Plant Biology, vol. 13, article 212, 2013.
[31]
B. Wei, T. Cai, R. Zhang et al., “Novel microRNAs uncovered by deep sequencing of small RNA transcriptomes in bread wheat (Triticum aestivum L.) and Brachypodium distachyon (L.) Beauv,” Functional and Integrative Genomics, vol. 9, no. 4, pp. 499–511, 2009.
[32]
B. C. Meyers, M. J. Axtell, B. Bartel et al., “Criteria for annotation of plant microRNAs,” Plant Cell, vol. 20, no. 12, pp. 3186–3190, 2008.
[33]
T. Tanaka, F. Kobayashi, G. P. Joshi, et al., “Next-Generation survey sequencing and the molecular organization of wheat chromosome 6B,” DNA Research, vol. 21, no. 2, pp. 103–114, 2014.
[34]
A. Bipinraj, B. Honrao, M. Prashar, S. Bhardwaj, S. Rao, and S. Tamhankar, “Validation and identification of molecular markers linked to the leaf rust resistance gene Lr28 in wheat,” Journal of Applied Genetics, vol. 52, no. 2, pp. 171–175, 2011.
[35]
D. Singh, G. Bhaganagare, R. Bandopadhyay, K. V. Prabhu, P. K. Gupta, and K. Mukhopadhyay, “Targeted spatio-temporal expression based characterization of state of infection and time-point of maximum defense in wheat NILs during leaf rust infection,” Molecular Biology Reports, vol. 39, no. 10, pp. 9373–9382, 2012.
[36]
G. Hu and F. H. J. Rijkenberg, “Scanning electron microscopy of early infection structure formation by Puccinia recondita f. sp. tritici on and in susceptible and resistant wheat lines,” Mycological Research, vol. 102, no. 4, pp. 391–399, 1998.
[37]
T. E. Coram, M. L. Settles, and X. Chen, “Transcriptome analysis of high-temperature adult-plant resistance conditioned by Yr39 during the wheat Puccinia striiformis f. sp. tritici interaction,” Molecular Plant Pathology, vol. 9, no. 4, pp. 479–493, 2008.
[38]
M. S. Stark, S. Tyagi, D. J. Nancarrow et al., “Characterization of the melanoma miRNAome by deep sequencing,” PLoS ONE, vol. 5, no. 3, Article ID e9685, 2010.
[39]
S. Griffiths-Jones, H. K. Saini, S. Van Dongen, and A. J. Enright, “miRBase: tools for microRNA genomics,” Nucleic Acids Research, vol. 36, no. 1, pp. D154–D158, 2008.
[40]
A. Kozomara and S. Griffiths-Jones, “MiRBase: integrating microRNA annotation and deep-sequencing data,” Nucleic Acids Research, vol. 39, no. 1, pp. D152–D157, 2011.
[41]
Y. Yao, G. Guo, Z. Ni et al., “Cloning and characterization of microRNAs from wheat (Triticum aestivum L.),” Genome Biology, vol. 8, article R96, 2007.
[42]
C. Xue, F. Li, T. He, G. Liu, Y. Li, and X. Zhang, “Classification of real and pseudo microRNA precursors using local structure-sequence features and support vector machine,” BMC Bioinformatics, vol. 6, article 310, 2005.
[43]
M. Zuker, “Mfold web server for nucleic acid folding and hybridization prediction,” Nucleic Acids Research, vol. 31, no. 13, pp. 3406–3415, 2003.
[44]
V. Ambros, B. Bartel, D. P. Bartel et al., “A uniform system for microRNA annotation,” RNA, vol. 9, no. 3, pp. 277–279, 2003.
[45]
B. H. Zhang, X. P. Pan, S. B. Cox, G. P. Cobb, and T. A. Anderson, “Evidence that miRNAs are different from other RNAs,” Cellular and Molecular Life Sciences, vol. 63, no. 2, pp. 246–254, 2006.
[46]
K. Y. Kurtoglu, M. Kantar, S. J. Lucas, and H. Budak, “Unique and conserved MicroRNAs in wheat chromosome 5D revealed by next-generation sequencing,” PLoS ONE, vol. 8, no. 7, Article ID e69801, 2013.
[47]
X. Dai and P. X. Zhao, “PsRNATarget: a plant small RNA target analysis server,” Nucleic Acids Research, vol. 39, no. 2, pp. W155–W159, 2011.
[48]
M. B. Stocks, S. Moxon, D. Mapleson et al., “The UEA sRNA workbench: a suite of tools for analysing and visualizing next generation sequencing microRNA and small RNA datasets,” Bioinformatics, vol. 28, no. 15, pp. 2059–2061, 2012.
[49]
S. G？tz, J. M. García-Gómez, J. Terol et al., “High-throughput functional annotation and data mining with the Blast2GO suite,” Nucleic Acids Research, vol. 36, no. 10, pp. 3420–3435, 2008.
[50]
J. Ye, L. Fang, H. Zheng et al., “WEGO: a web tool for plotting GO annotations,” Nucleic Acids Research, vol. 34, pp. W293–W297, 2006.
[51]
R. Margis, A. F. Fusaro, N. A. Smith et al., “The evolution and diversification of dicers in plants,” FEBS Letters, vol. 580, no. 10, pp. 2442–2450, 2006.
[52]
M. Wang, Q. Wang, and B. Wang, “Identification and characterization of microRNAs in asiatic cotton (Gossypium arboreum L.),” PLoS ONE, vol. 7, no. 4, Article ID e33696, 2012.
[53]
L. Yang and S. He, “A bioinformatics-based update on microRNAs and their targets in rainbow trout (Oncorhynchus mykiss),” Gene, vol. 533, no. 1, pp. 261–269, 2014.
[54]
R. Schwab, J. F. Palatnik, M. Riester, C. Schommer, M. Schmid, and D. Weigel, “Specific effects of microRNAs on the plant transcriptome,” Developmental Cell, vol. 8, no. 4, pp. 517–527, 2005.
[55]
I. L. Hofacker, M. Fekete, and P. F. Stadler, “Secondary structure prediction for aligned RNA sequences,” Journal of Molecular Biology, vol. 319, no. 5, pp. 1059–1066, 2002.
[56]
J. Krol, I. Loedige, and W. Filipowicz, “The widespread regulation of microRNA biogenesis, function and decay,” Nature Reviews Genetics, vol. 11, no. 9, pp. 597–610, 2010.
[57]
M. W. Rhoades, B. J. Reinhart, L. P. Lim, C. B. Burge, B. Bartel, and D. P. Bartel, “Prediction of plant microRNA targets,” Cell, vol. 110, no. 4, pp. 513–520, 2002.
[58]
V. Eldem, S. Okay, and T. Unver, “Plant microRNAs: new players in functional genomics,” Turkish Journal of Agriculture and Forestry, vol. 37, pp. 1–21, 2012.
[59]
R. Sunkar, Y. F. Li, and G. Jagadeeswaran, “Functions of microRNAs in plant stress responses,” Trends in Plant Science, vol. 17, no. 4, pp. 196–203, 2012.
[60]
B. Zhang, X. Pan, G. P. Cobb, and T. A. Anderson, “Plant microRNA: a small regulatory molecule with big impact,” Developmental Biology, vol. 289, no. 1, pp. 3–16, 2006.
[61]
R. L. R. Pilcher, S. Moxon, N. Pakseresht et al., “Identification of novel small RNAs in tomato (Solanum lycopersicum),” Planta, vol. 226, no. 3, pp. 709–717, 2007.
[62]
T. Unver, I. Parmaksiz, and E. Dündar, “Identification of conserved micro-RNAs and their target transcripts in opium poppy (Papaver somniferum L.),” Plant Cell Reports, vol. 29, no. 7, pp. 757–769, 2010.
[63]
L. Williams, C. C. Carles, K. S. Osmont, and J. C. Fletcher, “A database analysis method identifies an endogenous trans-acting short-interfering RNA that targets the Arabidopsis ARF2, ARF3, and ARF4 genes,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 27, pp. 9703–9708, 2005.
[64]
E. Allen, Z. Xie, A. M. Gustafson, G. Sung, J. W. Spatafora, and J. C. Carrington, “Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana,” Nature Genetics, vol. 36, no. 12, pp. 1282–1290, 2004.
[65]
J. T. Cuperus, N. Fahlgren, and J. C. Carrington, “Evolution and functional diversification of MIRNA genes,” Plant Cell, vol. 23, no. 2, pp. 431–442, 2011.
[66]
L. Zhou, Y. Liu, Z. Liu, D. Kong, M. Duan, and L. Luo, “Genome-wide identification and analysis of drought-responsive microRNAs in Oryza sativa,” Journal of Experimental Botany, vol. 61, no. 15, pp. 4157–4168, 2010.
[67]
F. Meng, H. Liu, K. Wang et al., “Development-associated microRNAs in grains of wheat (Triticum aestivum L.),” BMC Plant Biology, vol. 13, p. 140, 2013.
[68]
V. Eldem, U. ？elikkol Ak？ay, E. Ozhuner, Y. Bakir, S. Uranbey, and T. Unver, “Genomewide identification of miRNAs responsive to drought in peach (Prunus persica) by high-throughput deep sequencing,” PLoS ONE, vol. 7, no. 12, Article ID e50298, 2012.
[69]
S. Lu, Y. H. Sun, H. Amerson, and V. L. Chiang, “MicroRNAs in loblolly pine (Pinus taeda L.) and their association with fusiform rust gall development,” Plant Journal, vol. 51, no. 6, pp. 1077–1098, 2007.
