Edited by: Maria Eugenia Zanetti, National University of La Plata, Argentina
Reviewed by: Nicolas Rispail, Consejo Superior de Investigaciones Científicas (CSIC), Spain; Izabela Makałowska, Adam Mickiewicz University in Poznań, Poland
*Correspondence: Rajeev K. Varshney
This article was submitted to Plant Genetics and Genomics, a section of the journal Frontiers in Plant Science
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Biotic stress in legume crops is one of the major threats to crop yield and productivity. Being sessile organisms, plants have evolved a myriad of mechanisms to combat different stresses imposed on them. One such mechanism, deciphered in the last decade, is small RNA (sRNA) mediated defense in plants. Small RNAs (sRNAs) have emerged as one of the major players in gene expression regulation in plants during developmental stages and under stress conditions. They are known to act both at transcriptional and post-transcriptional levels. Dicer-like (DCL), Argonaute (AGO), and RNA dependent RNA polymerase (RDR) constitute the major components of sRNA biogenesis machinery and are known to play a significant role in combating biotic and abiotic stresses. This study is, therefore, focused on identification and characterization of sRNA biogenesis proteins in three important legume crops, namely chickpea, pigeonpea, and groundnut. Phylogenetic analysis of these proteins between legume species classified them into distinct clades and suggests the evolutionary conservation of these genes across the members of Papillionidoids subfamily. Variable expression of sRNA biogenesis genes in response to the biotic stresses among the three legumes indicate the possible existence of specialized regulatory mechanisms in different legumes. This is the first ever study to understand the role of sRNA biogenesis genes in response to pathogen attacks in the studied legumes.
Among the legume crops, chickpea (
Pigeonpea is another important legume crop, which provides the protein in the low quality diets of people living in developing countries of Asia and Africa. Pigeonpea is the sixth most important legume food crop with global cultivation on ~5 M ha. In India, the crop is cultivated on 4.04 M ha with an annual production of about ~3 Mt (FAO,
Groundnut, on the other hand, is mainly cultivated for its high quality edible oil and high protein content in the seed. It is an excellent cash crop, used in confectionery preparations, as well as cooking oil, and serves as a rich source of protein feed for animals. Cultivated groundnut is an allotetraploid (AABB), carrying half genome (AA) from
Small RNAs (sRNAs) have emerged as one of the most versatile performers in the regulation of gene expression in both plants and animals, and also orchestrate the defense responses against several biotic stresses. sRNAs are involved in both transcriptional and post-transcriptional gene silencing. They are usually 20–24 nucleotides in length, and can be broadly classified into microRNAs (miRNAs) and short interfering RNAs (siRNAs) on the basis of their origin and biogenesis. sRNAs act at the core of RNA interference which has been implemented as a technology to not only understand the role of genes in plants but also to develop robust crops by gene manipulation. For example, genetic manipulation for virus resistance in plants has been achieved by silencing the coat protein (CP) gene utilizing the artificially synthesized exogenous sRNAs. Likewise, such approaches have also been used to improve the crop's nutritional quality and production (Kamthan et al.,
Given the nutritional and economic importance of these legumes, and considering the constraints in yield and biomass imposed by biotic stresses, it is imperative to identify the potential sRNA biogenesis genes exploiting the recently available genome sequences of pigeonpea (Varshney et al.,
Two different approaches were used for genome-wide identification of the DCL, AGO, and RDR proteins: (i) In the first approach, the previously identified sRNA biogenesis protein sequences from Arabidopsis, rice, and soybean (Liu et al.,
A non-redundant set of putative sRNA biogenesis proteins identified from the two approaches were further confirmed for the presence of domains specific to each family using SMART and Pfam search. The identified genes were designated based on their phylogenetic relationship with their orthologs in Arabidopsis and soybean. The physio-chemical properties and subcellular localization of the identified proteins were calculated using ProtParam tool (
The genomic coordinates of the above identified DCL, AGO, and RDR genes were used to map their locations on a physical map of the respective legume crops. The exon-intron structure of the genes were determined using the genome annotations which were further represented using Gene Structure Display Server (GSDS; Hu et al.,
The deduced protein sequences of the identified DCL, AGO, and RDR genes of chickpea, pigeonpea, and groundnut along with their counterparts from Arabidopsis and soybean were subjected to phylogenetic analysis. Initially, the multiple sequence alignments of the protein sequences from each family were carried out using ClustalW. The alignments were manually corrected and then used to calculate a
The duplicated genes (paralogs) in each legume were identified using blastp search with an
In chickpea, four AB responsive genotypes, two moderately resistant (ILC 3279 and ICCV 05530) and two susceptible (C 214 and Pb 7) were used for expression analysis at 7th and 11th day post-inoculation (dpi). Seedling raising and inoculum preparation were done as described earlier (Pande et al.,
In the case of pigeonpea, three genotypes including one resistant (ICPL 20096) and two susceptible (ICPL 332-susceptible, ICPL 8863-highly susceptible) to SMD, were grown under glasshouse conditions (25 ± 2°C temperature, 16 h photoperiod). Seedlings emerging after 10 days of sowing were stapled with SMD-infected pigeonpea leaves with at least five live mites (Nene and Reddy,
In the case of groundnut, two resistant (GPBD 4, ICGV 13208) and one susceptible (TAG 24) genotypes for rust and LLS were used in the study. ICGV 13208 was an introgression line with resistance contributed by genomic region from GPBD 4 (Varshney et al.,
Total RNA from chickpea and groundnut leaf tissues was isolated using “NucleoSpin® RNA Plant” kit (Macherey-Nagel, Germany); and for pigeonpea, Plant RNA Miniprep kit, XcelGen (XG661-01, Xcelris, India) was used according to the manufacturer's instructions. The qualitative and quantitative assessment of these total RNA samples were conducted using Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA) and Nano Drop 8000 Spectrophotometer (Thermo Scientific, USA). The RNA samples with RNA integrity value of more than seven and 260/280 ratio of 1.8–2.1 were used to synthesize cDNA samples from the three biological replicates. The expression of sRNA biogenesis (DCL, AGO, and RDR) genes were studied using quantitative real time PCR (qRT-PCR). cDNA was prepared using SuperScript® III First Strand Synthesis System for RT-PCR (Invitrogen, CA, USA) according to the manufacturer's instructions. The gene specific primers were designed using PrimerQuest (Integrated DNA Technologies,
Using similarity and HMM searches, a total of four DCLs (DCL1, DCL2, DCL3, and DCL4) were found both in chickpea and pigeonpea similar to Arabidopsis (Xie et al.,
CaDCL1 | Ca3:39793013:39804554 (−) | 1,864 | Nuclear | 209706.6 | 20 | |
CaDCL2 | Ca1:7525781:7537015 (−) | 1,410 | Nuclear | 159948.3 | 23 | |
CaDCL3 | Ca5:35534919:35547109 (−) | 1,649 | Nuclear | 184754.8 | 24 | |
CaDCL4 | Ca7:5953535:5974786 (−) | 1,780 | Nuclear | 199784.1 | 28 | |
CcDCL1 | CcLG03:29072965:29085159 (−) | 1,913 | Nuclear | 215155.4 | 22 | |
CcDCL2 | Scaffold000032:233152:246602 (−) | 1,203 | Nuclear | 136996.2 | 17 | |
CcDCL3 | Scaffold126331:336037:350387 (+) | 1,553 | Nuclear | 173766.2 | 24 | |
CcDCL4 | Scaffold135129:302372:316356 (+) | 1,481 | Nuclear | 166998 | 23 | |
AdDCL1 | Aradu.A06:1660935:1671391 (+) | 1,949 | Nuclear | 219036.7 | 21 | |
AdDCL2 | Aradu.A04:9242477:9261536 (−) | 1,376 | Nuclear | 157029.1 | 26 | |
AdDCL3 | Aradu.A10:89258296:89268902 (−) | 1,680 | Nuclear | 188027.6 | 25 | |
AiDCL1 | Araip.B06:20391007:20401632 (−) | 1,947 | Nuclear | 218954.3 | 19 | |
AiDCL2 | Araip.B04:10697651:10706715 (−) | 1,398 | Nuclear | 159701.5 | 23 | |
AiDCL3 | Araip.B10:112301476:112318201 (−) | 1,692 | Nuclear | 189305.7 | 33 | |
AiDCL4 | Araip.B01:134634628:134652430 (+) | 1,642 | Nuclear | 185300.1 | 29 |
A total of 13 AGO genes each in chickpea, pigeonpea and 22 (11 each from
CaAGO1 | Scaffold1006:491140:497352 (+) | 1,094 | Cytoplasmic | 120964.3 | 20 | |
CaAGO3b | Ca1:7035349:7040048 (−) | 1,033 | Extracellular | 117036.9 | 2 | |
CaAGO10d | Ca1:33057594:33065348 (+) | 867 | Cytoplasmic | 98730.4 | 20 | |
CaAGO4a | Ca2:32900469:32906134 (+) | 906 | Nuclear | 101240.7 | 20 | |
CaAGO7 | Ca2:13131307:13136219 (+) | 1,016 | Nuclear | 116210.4 | 2 | |
CaAGO10a | Ca4:13222797:13228230 (+) | 976 | Cytoplasmic | 110048.8 | 20 | |
CaAGO10c | Ca4:32128211:32135531 (−) | 949 | Cytoplasmic | 106625.5 | 20 | |
CaAGO4c | Ca5:40020432:40030536 (+) | 938 | Nuclear | 105263.4 | 22 | |
CaAGO6a | Ca5:41701444:41707829 (+) | 776 | Nuclear | 86435 | 19 | |
CaAGO6b | Ca5:41677400:41685345 (+) | 875 | Nuclear | 97419.7 | 22 | |
CaAGO3a | Ca6:19393544:19397617 (−) | 887 | Extracellular | 100743.4 | 2 | |
CaAGO5 | Ca6:29304385:29309715 (−) | 966 | Cytoplasmic | 108127.8 | 21 | |
CaAGO10e | Ca7:7288639:7294813 (−) | 894 | Cytoplasmic | 101081.6 | 21 | |
CcAGO4a | CcLG02:12753469:12759534 (−) | 910 | Nuclear | 101927.4 | 21 | |
CcAGO6 | CcLG02:13199198:13214696 (+) | 897 | Nuclear | 100807.9 | 21 | |
CcAGO10c | CcLG02:15919454:15927414 (+) | 934 | cytoplasmic | 105466.2 | 21 | |
CcAGO10a | CcLG02:31329417:31336496 (+) | 953 | Cytoplasmic | 107637.4 | 21 | |
CcAGO10d | CcLG10:5900494:5908489 (+) | 905 | cytoplasmic | 102982.4 | 21 | |
CcAGO9 | CcLG10:15320919:15328269 (+) | 885 | nuclear | 100148.7 | 21 | |
CcAGO1 | CcLG08:3847540:3853508 (−) | 1,054 | cytoplasmic | 116584.9 | 21 | |
CcAGO7 | CcLG07:7367814:7372842 (−) | 1,028 | nuclear | 117568.5 | 2 | |
CcAGO5 | CcLG04:7177034:7182639 (+) | 947 | Cytoplasmic | 106187.2 | 21 | |
CcAGO4c | CcLG05:2617913:2625461 (+) | 945 | Nuclear | 106295.4 | 20 | |
CcAGO10f | Scaffold000017:700964:707596 (−) | 905 | Cytoplasmic | 102649.3 | 21 | |
CcAGO3b | Scaffold134686:75450:78912 (−) | 821 | extracellular | 93401.5 | 2 | |
CcAGO3a | Scaffold137616:114442:117800 (−) | 845 | Cytoplasmic | 95803.9 | 1 | |
AdAGO4c | Aradu.A04:621757:629400 (−) | 925 | Nuclear | 103216.9 | 23 | |
AdAGO4a | Aradu.A07:5793658:5804159 (−) | 913 | Nuclear | 102050.4 | 26 | |
AdAGO10f | Aradu.A01:102555563:102567085 (−) | 886 | Cytoplasm | 100146 | 23 | |
AdAGO3a | Aradu.A04:31982490:31985928 (+) | 854 | Cytoplasm | 95879.1 | 2 | |
AdAGO7 | Aradu.A10:8971874:8975054 (+) | 910 | Cytoplasm | 103830.2 | 1 | |
AdAGO9 | Aradu.A10:107171742:107178558 (+) | 936 | Nuclear | 105395 | 21 | |
AdAGO6 | Aradu.A10:108178610:108187445 (−) | 915 | Nuclear | 102363.5 | 24 | |
AdAGO1 | Aradu.A05:98685962:98692253 (+) | 1,033 | Cytoplasm | 114347 | 20 | |
AdAGO5 | Aradu.A03:621532:627450 (−) | 962 | Cytoplasm | 107195.8 | 21 | |
AdAGO10a | Aradu.A09:110871552:110876942 (+) | 929 | Cytoplasm | 104210.8 | 19 | |
AdAGO3b | Aradu.A04:7602305:7609000 (−) | 1,027 | Extracellular | 116651.2 | 5 | |
AiAGO4c | Araip.B04:1151800:1159887 (−) | 952 | Nuclear | 106574.8 | 23 | |
AiAGO4a | Araip.B07:5421317:5431673 (−) | 943 | Nuclear | 105474.7 | 26 | |
AiAGO10a | Araip.B09:146081716:146087148 (−) | 965 | Cytoplasm | 108274.8 | 19 | |
AiAGO9 | Araip.B10:133942144:133948674 (+) | 880 | Nuclear | 99179.8 | 21 | |
AiAGO1 | Araip.B05:126022537:126029025 (−) | 1,056 | Cytoplasm | 117204.3 | 21 | |
AiAGO3b | Araip.B04:9235071:9241757 (−) | 1,027 | Extracellular | 116573.1 | 5 | |
AiAGO3a | Araip.B04:30757796:30761710 (+) | 951 | Extracellular | 106169.7 | 3 | |
AiAGO7 | Araip.B10:14593307:14596492 (+) | 910 | Cytoplasm | 103801.2 | 1 | |
AiAGO10f | Araip.B10:7828151:7838826 (+) | 792 | Cytoplasm | 89506 | 22 | |
AiAGO6 | Araip.B10:134978071:134986384 (−) | 918 | Nuclear | 102670.7 | 23 | |
AiAGO5 | Araip.B03:2620052:2626164 (−) | 966 | Cytoplasm | 107602.3 | 21 |
Chickpea, pigeonpea,
CaRDR1a | Ca4:37163561:37169980 (–) | Extracellular | 1,126 | 129266.8 | 3 | |
CaRDR1b | Ca4:37175471:37186589 (–) | Extracellular | 1,228 | 140705.2 | 6 | |
CaRDR2 | Ca7:9440293:9444640 (+) | Extracellular | 1,122 | 127939.2 | 3 | |
CaRDR3 | Ca8:15687606:15699452 (+) | Extracellular | 987 | 111791.6 | 16 | |
CaRDR6 | Ca5:34764715:34768405 (+) | Extracellular | 1,070 | 122471.4 | 1 | |
CcRDR1a | Scaffold132418:90943:97761 (+) | Extracellular | 1,126 | 128947.9 | 3 | |
CcRDR1b | Scaffold132418:64081:70327 (+) | Extracellular | 1,135 | 130368.1 | 3 | |
CcRDR2 | CcLG11:26891564:26896223 (+) | Extracellular | 1,120 | 128072.7 | 3 | |
CcRDR3 | Scaffold136224:56966:71567 (+) | Extracellular | 957 | 108790.3 | 17 | |
CcRDR6 | Scaffold135491:42338:46321 (+) | Extracellular | 1,050 | 119800.6 | 2 | |
AdRDR1a | Aradu.A08:44261916:44269684 (+) | Extracellular | 1,107 | 126425.2 | 4 | |
AdRDR1b | Aradu.A09:83785988:83794273 (–) | Extracellular | 1,112 | 127088 | 5 | |
AdRDR2 | Aradu.A01:70282483:70286955 (–) | Extracellular | 1,131 | 129260.7 | 3 | |
AdRDR3a | Aradu.A05:89873064:89881688 (–) | Extracellular | 795 | 90116 | 17 | |
AdRDR3b | Aradu.A10:4871397:4880329 (+) | Extracellular | 999 | 113701.6 | 17 | |
AiRDR1a | Araip.B08:123098782:123108524 (+) | Extracellular | 1,107 | 126355.1 | 4 | |
AiRDR1b | Araip.B09:102305651:102313983 (+) | Extracellular | 1,116 | 127617.7 | 5 | |
AiRDR2 | Araip.B01:100821579:100825755 (–) | Extracellular | 839 | 95203.1 | 2 | |
AiRDR3a | Araip.B05:138855272:138866105 (+) | Extracellular | 958 | 108703.5 | 18 | |
AiRDR3b | Araip.B10:6934819:6943920 (+) | Extracellular | 981 | 112385.6 | 18 |
DCLs are large proteins with multiple domains, namely, DEAD, Helicase-C, Dicer-dimer, PAZ, two tandem Ribonuclease III, and double stranded RNA binding domain (dsrm). The characteristic DCL domain organization was found in all chickpea, pigeonpea, and peanut DCL homologs except CcDCL2, which lacks the DEAD domain. In the case of chickpea and groundnut, all DCLs contained two copies of dsrm domain except clade II DCLs. The clade II DCLs harbored a single copy of dsrm domain in accordance with clade II DCL proteins of Arabidopsis and soybean. Interestingly, in pigeonpea only clade I DCL was found to contain two copies of dsrm domain (Supplementary Figures
AGOs are marked by the presence of four domains: N-terminal domain, PAZ, MID, and PIWI domain. MID domain is known to have a nucleotide specificity loop which makes it a recognition and binding center for sRNAs (Frank et al.,
CaAGO1 | DDH/H | CcAGO1 | DDH/H | AdAGO1 | DDH/H | AiAGO1 | DDH/H |
CaAGO3a | DDD/H | CcAGO3a | DDD/H | AdAGO3a | DDD/H | AiAGO3a | DDD/H |
CaAGO3b | DDD/N | CcAGO3b | DDD/N | AdAGO3b | DDD/N | AiAGO3b | DDD/N |
CaAGO4a | DDH/A | CcAGO4a | DDH/P | AdAGO4a | DDH/P | AiAGO4a | DDH/P |
CaAGO4c | DDH/A | CcAGO4c | DDH/A | AdAGO4c | DDH/A | AiAGO4c | DDH/A |
CaAGO5 | DDH/H | CcAGO5 | DDH/H | AdAGO5 | DDH/H | AiAGO5 | DDH/H |
CaAGO6a | DDH/P | CcAGO6 | DDH/S | AdAGO6 | DDH/S | AiAGO6 | DDH/S |
CaAGO6b | DDH/P | – | – | – | – | – | – |
CaAGO7 | DDH/H | CcAGO7 | DDH/H | AdAGO7 | DDH/H | AiAGO7 | DDH/H |
– | – | CcAGO9 | DDH/S | AdAGO9 | DDH/P | AiAGO9 | DDH/P |
CaAGO10a | DDH/H | CcAGO10a | DDH/H | AdAGO10a | DDH/H | AiAGO10a | DDH/H |
CaAGO10c | DDH/Q | CcAGO10c | DDH/H | – | – | – | – |
CaAGO10d | DDH/H | CcAGO10d | DDH/H | – | – | – | – |
CaAGO10e | DDH/H | – | – | – | – | – | – |
– | – | CcAGO10f | DDH/H | AdAGO10f | DDH/H | AiAGO10f | DD-/H |
All RDR genes are known to have a conserved RdRP domain. However, clade I and clade II in the legumes studied here have an additional conserved RNA Recognition Motif (RRM) as also seen in clades 1 and 2 of Arabidopsis and soybean. In RDRs, among all the motifs analyzed, motif 2 in chickpea and pigeonpea, and motif 6 in groundnut was seen to be conserved among all the RDR members. A short sub-sequence of this motif, DLDGD was seen to be conserved in all RDR clades except clade III where it got converted to DFDGD. This motif is seen conserved in other species as well, and corresponds to the catalytic β′ subunit of DNA-dependent RNA polymerases (Zong et al.,
Chickpea DCLs were distributed on pseudomolecules Ca1, Ca3, Ca5, and Ca7 whereas in pigeonpea, only CcDCL1 was found to be present on pseudomolecule CcLG03 while rest of the DCLs were scattered on unassembled scaffolds (Table
Gene duplication underlines the phenomenon of evolution as it functions as a template for subsequent modifications acted upon by natural selection. Gene families are nothing but true paralogs resulting from gene duplication events. In this study, we identified the duplicated events of sRNA biogenesis genes in the genomes of three SAT legumes. A paralog for an identified gene was considered only if it retained at least one of all the essential domains responsible for defining that gene to be a part of a specific family. Considering the above criteria, no duplicated DCL gene was found in chickpea, whereas two were found in pigeonpea. Likewise, one duplicated DCL gene was found each in
The orthologs of DCL, AGO, and RDR proteins of chickpea, pigeonpea, and groundnut were identified in two closely related legumes, Medicago, and soybean (Figures
Promoter analysis identified a number of
All the DCL genes in chickpea, pigeonpea, and groundnut were recognized as miRNA targets. The DCL1 gene irrespective of the legume species studied in this study was found to be targeted by miR162. One of the miRNAs, miR1515 involved in hyper nodulation in soybean is known to target DCL2 (Li et al.,
DCL, AGO, and RDR are broadly known to facilitate the sRNA biogenesis. However, each of them has a precise role in the molecular and biological modus operandi of sRNA biogenesis and regulation of gene expression. Annotation of the identified respective family members revealed that clade IV DCLs were involved in a biological process of mitotic cell cycle and virus induced gene silencing (VIGS; Supplementary Table
In order to understand the role of DCL, AGO, and RDR in post-transcriptional regulation of gene expression in response to AB, we randomly selected 10 candidate genes belonging to DCL (1), AGO (7), and RDR (2) family. At 7th dpi, both moderately resistant genotypes (ILC 3279 and ICCV 05530) showed a general trend of upregulation for all the ten genes, except CaDCL1 and CaAGO3a, which were found to be downregulated in ICCV 05530. The susceptible genotypes (Pb 7 and C 214) showed a general trend of downregulation, except CaAGO1 and CaAGO10e (Figure
In order to investigate the role of sRNA biogenesis genes in viral defense, we studied the expression profile of 21 genes including DCLs (4), AGOs (12), and RDRs (5). The expression of these genes in response to SMD was studied using qRT-PCR at initial (7th dpi) and severe stress (14th dpi) in a resistant (ICPL 20096), a susceptible (ICPL 332), and a highly susceptible (ICPL 8863) genotype of pigeonpea. Significantly, all DCL, AGO, and RDR genes were found to be upregulated at 7th dpi in resistant genotype. General pattern of downregulation of these genes in highly susceptible pigeonpea genotype was observed. A total of 11 out of 21 genes showed stark downregulation of expression in both the susceptible genotypes compared to the resistant ones at 7th dpi, as expected. However, clade III genes of DCL, RDR and clade II of AGO at 7th dpi were found to be induced in all genotypes, along with clade I DCL. At 14th dpi, most of the genes (13) in resistant genotype showed downregulation that were upregulated on 7th dpi. As the infection progressed (14th dpi), all genes, except CcAGO4a and CcAGO6 were seen to be downregulated in a highly susceptible genotype. Interestingly, four genes CcAGO7, CcDCL3, CcAGO3b, and CcAGO3a showed a profound dip in expression, which antagonistically showed an upregulated expression at initial stage of infection in the highly susceptible genotype (Figure
We also observed an up and downregulated expression of CcDCL2 and CcDCL4 at 7th dpi in resistant and susceptible genotypes. However, at 14th dpi (severe stress) both genes showed downregulation irrespective of the genotypes, which could be attributed to senescence and programed cell death (PCD) during the penultimate days of a plant's life cycle. Considering the findings that loss-of-function mutation in DCL2 and DCL4 is necessary and sufficient to make plants susceptible to single stranded viruses (Deleris et al.,
Expression analysis of randomly selected DCL (4), AGO (8), and RDR (8) genes in rust and LLS responsive genotypes of groundnut revealed their role in response to stress imposed by their causal pathogens. Expression studies were conducted at 21st dpi (inception of infection), 35th dpi (systemic spread of infection), and 50th dpi (terminal stage of infection). In course of time, all the genes, except AdDCL2, were consistently downregulated in TAG 24 (susceptible). However, GPBD 4 (resistant) showed an inconsistent expression pattern with a stint of induced expression at all time points. A few genes (4–5) showed upregulation at 21st and 35th dpi, whereas at 50th dpi nearly half of the genes (11) got upregulated in GPBD 4. Out of all the upregulated genes at 50th dpi, AiAGO10a, AdAGO4a, AiAGO4a, clade III RDRs, AdDCL3 showed an initial downregulation at 21st and 35th dpi. AiAGO10a, in particular, showed highly repressed levels at 21st dpi as compared to other genes. Interestingly, in the case of ICGV 13208 a pattern of gradual downregulation of gene expression was observed. At 21st dpi half (10) of the genes were downregulated followed by repression of 12 genes at 35th dpi, and at 50th dpi all the genes except AdDCL2 were downregulated. AdDCL2 showed an upregulation at 21st and 35th dpi in all three genotypes. However, at 50th dpi it showed downregulation in susceptible genotype, which could be attributed to senescence as a result of PCD during the later stages of the plant's life cycle (Figures
There have been very limited studies of expression of sRNA biogenesis genes in response to fungal pathogen and those studies have largely demonstrated that the expression of these genes is directly proportional to the resistance of the genotype in response to pathogen attack. In the current study, chickpea and pigeonpea showed similar expression pattern of upregulation in resistant genotype, contradicted by groundnut where resistant genotypes showed downregulation for most of the genes. Susceptible genotypes of all three legumes showed a general pattern of downregulation across different stages of both viral and fungal stress. The contrasting expression patterns in groundnut can be due to the fact that the genome-wide survey was carried out in diploid progenitors of groundnut, and expression analysis was performed on cultivated tetraploids. However, it definitely requires further substantial evidence to elucidate the role of epigenetic pathways operating under such stress in tolerant and susceptible genotypes. It can be inferred from this observation that the sRNA biogenesis machinery gets affected, leading to its highly depleted levels during the terminal stages of the plant's life.
This is the first report on genome-wide identification and characterization of sRNA biogenesis proteins in chickpea, pigeonpea, and groundnut. The number of proteins in each family (DCL, AGO, and RDR) were found to be conserved across all three legumes. Phylogenetic analysis revealed clade and species-specific differences within members of sRNA biogenesis proteins which might reflect their functional divergence. Further, comparative analysis of DCLs, AGOs, and RDRs reflected the prevalence of functional overlapping and compensatory phenomenon in legumes to accomplish the role of these genes by other homologs. Upstream regulatory regions of the sRNA biogenesis genes were marked by the presence of stress hormone related elements. Moreover, differential expression of these genes under biotic stress confirmed their involvement in combating stress. Expression studies under biotic stress also revealed species-specific expression of these genes. Overall, our findings are an indication of structural diversification of DCL, AGO, and RDR and divergences of the studied legumes during the course of evolution. The genes reported in this study can be targeted in the near future for gene manipulation through post-transcriptional gene silencing approaches so as to develop resistant cultivars against biotic stresses in these legumes.
RKV conceived and designed the experiment. VG, GA, LTP, SNN, HK, MS, and PBK performed the experiments. VG, AWK, and DD analyzed the data. VG and GA with support of RKV wrote the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work has been undertaken as a part the CGIAR Research Program on Genetic Gains. ICRISAT is a member of the CGIAR consortium. VG acknowledges the award of Research Fellowship from Department of Science and Technology, Government of India.
The Supplementary Material for this article can be found online at: