A study of miRNAs and lncRNAs during Lr28-mediated resistance against leaf rust in wheat (Triticum aestivum L.)
Introduction
Bread wheat (Triticum aestivum L.) is one of the most extensively grown crop worldwide, occupying 225 mha land area. It is a hexaploid (2n = 6x = 42) with a complex and massive genome of ~17 Gb, carrying >80% repetitive DNA. The crop suffers major losses due to a variety of abiotic and biotic stresses including leaf rust (Puccinia triticina Eriks. and E. Henn.), which is an important fungal disease, accounting for an yield loss of 10%–20% [1]. Leaf rust pathogen is an obligate parasite and has an inherent capability to evolve rapidly into new pathotypes with virulence against newly deployed resistance (R) genes [2]. Therefore, a continuous search for novel and effective sources of resistance is required. As many as ~100 leaf rust resistance genes (Lr1-Lr100) are now known, with some redundancy. These genes include several genes derived from the following alien species: Aegilops speltoides (Lr28, Lr35, Lr36, Lr47, Lr51, Lr66), Ae. umbellulata (Lr9, Lr76), Ae. ventricosa (Lr37), Ae. tauschii (Lr21, Lr22a, Lr32, Lr39, Lr40, Lr42, Lr43), Secale cereale (Lr25, Lr26), T. dicoccoides (Lr53, Lr64) and Lophopyrum elongatum (Lr19, Lr24, Lr29) [3,4]. Most of these alien genes are still effective against a number of leaf rust races globally. Lr genes exhibit the widely known gene-for-gene relationship, where, for each R gene, there should be a corresponding Avr gene in the pathogen, such that in the absence of this specific Avr gene in the prevalent race, R gene will not be effective. Six of the above ~100 Lr genes have been cloned, and have been shown to encode NBS-LRR, ABC transporter or kinase proteins, which provide resistance. These six genes include the following: Lr1, Lr10, Lr21, Lr22, Lr34, Lr67.
It is now well known that a major fraction of the massive wheat genome is transcribed into non-coding RNAs (ncRNAs), which generally remain untranslated [5]. The genomic DNA transcribed into ncRNA includes both housekeeping ribosomal/transfer RNA genes and the genes transcribed into regulatory ncRNAs, which include small RNAs (sRNAs) like miRNA, siRNA, piRNAs, etc. (<200bp in length) and lncRNAs (>200bp in length) [6]. These regulatory ncRNAs are derived from long primary transcripts, derived from genomic DNA with the help of DNA dependent RNA Polymerase II.
Genome-wide expression studies in wheat conducted so far also included a number of studies involving Lr genes, which included both, the seedling resistance genes, also called all stage resistance genes (ASRs), and adult plant resistance (APR) genes. The ASR Lr genes used in such studies included Lr1 [7], Lr10 [8], Lr24, Lr28, etc. [9,10] and the APR genes included Lr34 [1], Lr 46 [11], Lr47 [12], Lr48 [13], Lr57 [14], Trp-1, Trp-2, etc. [15]. In each of these studies, a cascade of downstream genes was found to be differentially expressed during infection either to facilitate or to combat the pathogen attack. Although substantial progress has been made in understanding the molecular basis of leaf rust resistance, the role of miRNAs in modulating the expression of downstream genes was examined only for a few R genes, which included the following: Lr24 [16], Lr28 [17], Sr24 [18] and, two studies for stripe rust [19,20]. In some of these studies, miRNAs have been shown to play an important role as negative regulators by either repressing translation or degrading target mRNAs of the host and/or pathogen [21].
Thousands of non-coding RNAs in plants have been identified through in silico studies followed by validation through experimental analysis [22,23]. Among ncRNAs, miRNAs are generally conserved making it possible to use miRNAs known in one crop species for identification of orthologous miRNAs in another species through in silico approach [[24], [25], [26]]. A large number of lncRNAs have also been identified, each playing an important role in transcriptional and post-transcriptional regulation of gene expression [27,28]. These lncRNAs may either act as precursors of miRNAs or may act as endogenous target mimics (TMs), which mimic the real targets of miRNAs, thus rendering the corresponding miRNAs ineffective. Particularly in wheat, although the role of miRNAs in disease resistance (including leaf rust resistance involving Lr46 gene) has been studied [11], very few such studies have been conducted for lncRNAs except two studies involving powdery mildew [28,29]. In the present study, we identified leaf rust responsive miRNAs (along with their potential targets) and lncRNAs in a pair of NILs for the gene Lr28 in the background of the susceptible cultivar HD2329 that was widely used for cultivation till recently; the role of these ncRNAs in regulation of the expression of a number of downstream protein-coding genes was also examined.
Section snippets
Plant material and the pathogen
The plant material used in the present study comprised a susceptible (S) wheat cultivar HD2329 and its resistant (R) NIL (HD2329 + Lr28). The race 77-5 of the fungal pathogen Puccinia triticina Eriks. and E. Henn., which causes leaf rust in a wide spectrum of Indian wheat cultivars, was used for inoculation. The plants were grown and inoculated in the National Phytotron Facility, IARI, New Delhi; the details of the procedures were described in an earlier publication involving transcriptome
Identification and characterization of miRNAs
The number of contigs that were assembled from RNA-sequence data (see M & M section) and utilized for in silico detection of miRNA precursors was 1,46,659. Of these, 1522 contigs carried characteristic secondary structures of precursor miRNA; 263 of these sequences were strong candidates to be the precursors of mature miRNAs. These 263 sequences were classified into 50 different miRNA families following miRbase (Fig. 1a). Most of the miRNA families had only one member; only the following miRNA
Discussion
Among ncRNAs, miRNAs and lncRNAs are two classes of important riboregulators of gene expressions, thus influencing a variety of traits including developmental processes and response to abiotic and biotic stresses. This holds good for both plant and animal systems [96]. Therefore, attempts are being made globally to identify miRNAs and lncRNAs which may be utilized for manipulating desirable traits. In particular, miRNAs and lncRNAs involved in response to infection by a variety of pathogens
Conclusions
In the present study, leaf rust responsive miRNAs and lncRNAs were identified using RNA-seq data, which provided valuable insights into the role of ncRNAs in regulation of gene expression. As many as 16 miRNAs and 22 lncRNAs were found to respond to leaf rust disease. Eight miRNAs that were upregulated in resistant NIL were also shown to target a number of important genes needed to stimulate the defense. Three lncRNAs that were identified to be TMs were also found to suppress the function of
Authorship contribution statement
Neelu Jain: Writing - original draft, conceived and designed research, wrote and finalized the manuscript. Nivedita Sinha: Formal analysis, Writing - original draft, carried out data analyses, wrote and finalized the manuscript. Hari krishna: carried out the rust phenotyping work. Pradeep Kumar Singh: carried out the rust phenotyping work. Tinku Gautam: Writing - original draft, wrote and finalized the manuscript. Pramod Prasad: provided inoculum for leaf rust pathotype 77-5. Harindra Singh
Declaration of competing interest
The authors declare that they have no conflicts of interest.
Acknowledgements
We are grateful to NASF, ICAR for providing financial assistance for this study (grant no. NASF/ABP-6006/2016-17). The first author acknowledges the valuable guidance received from Dr. K V Prabhu, Chairman, PPVFRA, India during this study. HSB was awarded positions of INSA Senior Scientist and later INSA Honorary Scientist during the period of the study.
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