High throughput sequencing reveals modulation of microRNAs in Vigna mungo upon Mungbean Yellow Mosaic India Virus inoculation highlighting stress regulation

Highlights

• miRNA induced regulation of MYMIV-stress response in Vigna mungo has been explored.

• Experimental data revealed altered expression of known and novel miRNAs under MYMIV-stress.

• Differential expression of stress-related target transcripts was validated using qPCR.

• Resistant and susceptible genotypes exhibited differential modulations of miRNA.

• Molecular functions and cellular components of the targets were predicted in this orphan species.

Abstract

MicroRNAs (miRNAs) are 20–24 nucleotides long non-coding RNAs known to play important regulatory roles during biotic and abiotic stresses by controlling gene expression. Blackgram (Vigna mungo), an economically important grain legume is highly susceptible to pathogenic begomovirus Mungbean Yellow Mosaic India Virus (MYMIV) and resulting in high yield loss. In this study two different leaf-small-RNA libraries were prepared from the pooled RNA at three different time points of resistant V. mungo inbred line VM84 inoculated either with viruliferous or non-viruliferous whiteflies carrying MYMIV and performed high-throughput Illumina sequencing. Sequencing followed by bioinformatics analysis of the small RNA reads indicated that the expression patterns of most of the known and novel miRNAs were altered in resistant line over mock-inoculated sample during the plant virus incompatible interaction. Highly altered miRNAs belong to the families of miR156, miR159, miR160, miR166, miR398, miR1511, miR1514, miR2118 and novel vmu-miRn7, vmu-miRn8, vmu-miRn13 and vmu-miRn14. These results were validated using qPCR, and most of the miRNAs showed similar pattern of expression like that of Illumina reads. The expression patterns of some selected known and novel miRNAs were also compared between the infected MYMIV-resistant and -susceptible genotypes and most of these were modulated after MYMIV-inoculation. Target transcripts like NB-LRR, NAC, MYB, Zinc finger, CCAAT-box transcription factor, fructose 2–6 bisphosphate, HDZIP protein that confers immune response were predicted as targets amongst identified miRNAs using psRNATarget server. Some selected target transcripts including NB-LRR, ARF, SOD, SPB, Basic blue copper protein were validated and their differential expression were demonstrated between MYMIV-resistant and −susceptible V. mungo by qPCR data analyses. In the present study we have identified miRNAs that implicate in the regulation of MYMIV-induced stress response in V. mungo; and generated genomic resources for a non-model legume with the aid of bioinformatics tools supplemented by experimental validation.

Introduction

Plant pathogen compatible interaction results in the development of disease symptoms leading to altered phenotype of plants. On the contrary, a resistant plant employs several complex defense mechanisms in response to the pathogenic attack. Central to these defense mechanisms is gene silencing [1], [2], [3], in which small RNAs (sRNA) play a pervasive role in restricting pathogens to damage the host plant [4], [5]. To date, several sub-classes of sRNAs have been identified; the best-characterized being the small, 20–24 nucleotides long, interfering RNAs (siRNAs) and the microRNAs (miRNAs). However, they differ in precursor structures, biogenesis pathways and modes of action [6]. The miRNAs are evolutionarily conserved, non-coding RNAs that modulate gene expression either by post-transcriptional cleavage and degradation of target transcripts or inhibit translation of genes by base complementarity [7]. The miRNA precursors (pre-miRNAs) originate from the intragenic or intergenic regions of the genome and transcribed by RNA polymerase II and associated with a cap binding complex [8]. The stem-loop precursor is then tailored by the cellular proteins to generate a miRNA:miRNA* duplex. After loading into an RNA-induced silencing complex (RISC), the canonical interaction occurs between the miRNA and its cognate mRNA through specific base pairing to induce the mRNA cleavage or hindering translation of mRNA [9], [10].

Since its discovery, a vast majority of studies focused on the role of miRNA in plant-pathogen interaction [11], [12], [13], [14]. In Arabidopsis, the first miRNA discovered to play a role in plant defense was miR393, a flg22-induced miRNA that repressed three F-box proteins (TIR₁, AFB₂ and AFB₃), which are auxin receptors, [15], [16], [17] and contributed to defense against Pseudomonas syringae [18]. Bazzini and co-researchers [11] reported altered accumulation of several miRNA families after infecting tobacco with members of Tobamoviridae, Potyviridae, and Potexviridae. Altered accumulation of miRNAs was also noted in Arabidopsis when infected with TMV and resulted in the discovery of four novel miRNAs miR822, miR823, miR824, miR847 [19]. Since then several other workers reported alteration in miRNA expression during compatible plant begomovirus interaction in different crops like Nicotiana benthamiana [20], tomato [21] soybean [22], etc. Nevertheless, alteration in miRNAs expression was also noted during wheat Puccinia compatible interaction [23]. However, pathogen-induced expression patterns of miRNAs in a resistant background are largely unknown. Recently, aphid responsive miRNAs in resistant Cucumis variety and Phytophthora triggered miRNAs in resistant tomato variety were reported by Sattar et al. [24] and Luan et al. [25], respectively.

In India, recently begomoviral diseases are spreading at an alarming rate among different economically important crop species [26], [27], [28], [29]. Yellow mosaic disease (YMD) caused by the Mungbean Yellow Mosaic India Virus (MYMIV), is one of the destructive members of whitefly-transmitted bipartite begomoviruses. MYMIV infects several edible grain legumes including Vigna mungo that grow throughout the South-Asian countries [30]. Symptomatic manifestations of YMD become prominent in susceptible plants from 7 days post inoculation that includes dispersed yellow patches on leaves, stunted growth and reduced number of seeds/pod resulting in severe yield penalty [30]. Such consequences and high economic impact of YMD render the situation worrisome. Under the circumstance, expression profiles of miRNAs during incompatible interaction between MYMIV and V. mungo were studied to determine whether these are involved in regulating virus-induced gene expression. MYMIV-resistance trait was successfully introgressed in susceptible V. mungo cultivar T9 [30] and a homozygous resistant genotype, VM84, which was selected from a population of recombinant inbred lines [31]. Subsequently, the high-throughput sequencing was carried out in the resistant genotype to explore the small RNA population in the leaves of V. mungo that led to the identification of 45 conserved, 8 non-conserved and 13 novel miRNA candidates [32].

One of the previous studies has provided preliminary molecular insights on transcript modulations in V. mungo upon MYMIV infection [33]. However, profiles of high-throughput miRNA expression during MYMIV-plant interaction remain unexplored. Therefore, in this study we report high-throughput sequencing of small RNA-molecules in MYMIV- and mock-inoculated V. mungo in the resistant background. In addition, qPCR expression profiles of MYMIV-induced miRNAs in the resistant background were compared with those in the susceptible background. Expression profiles of some selected targets that are implicated in plant-pathogen interactions were analyzed in resistant and susceptible background with or without MYMIV inoculation, suggesting their probable role in conferring MYMIV-resistance to the host plant. Overall, the findings will enhance our knowledge of the post-transcriptional regulatory network of the pathogen-induced miRNAs and their targets that determines the outcome of MYMIV-reaction.