Small RNA sequencing analysis provides novel insights into microRNA-mediated regulation of defense responses in chickpea against Fusarium wilt infection

Small RNA sequencing analysis in two chickpea genotypes, JG 62 (Fusarium wilt-susceptible) and WR 315 (Fusarium wilt-resistant), under Fusarium wilt stress led to identification of 544 miRNAs which included 406 known and 138 novel miRNAs. A total of 115 miRNAs showed differential expression in both the genotypes across different combinations. A miRNA, Car-miR398 targeted copper chaperone for superoxide dismutase (CCS) that, in turn, regulated superoxide dismutase (SOD) activity during chickpea-Foc interaction. Fusarium wilt (FW) of chickpea (Cicer arietinum L.) caused by Fusarium oxysporum f. sp. ciceris (Foc) is a destructive soil-borne disease that severely reduces the chickpea yield and quality globally. In the present study, we have investigated microRNAs and the microRNA/target gene crosstalk involved in chickpea resistance to FW. The control and stress samples from two genotypes, JG 62 (FW-susceptible) and WR 315 (FW-resistant), collected at 10 days post-inoculation (dpi), were selected for small RNA sequencing. A total of 12 libraries were constructed and sequenced using Illumina HiSeq 2500 platform. The sequencing and in silico analyses revealed the identification of 544 miRNAs which included 406 known and 138 novel miRNAs. A total of 50 miRNAs were physically co-localized with Foc-resistance QTLs present on chromosome 2 (also known as Foc hotspot). A total of 115 miRNAs showed differential expression in both the genotypes across different combinations. Prediction and functional annotation of miRNA targets revealed their role in transcription regulation, disease resistance, defense response, metabolism, etc. Ten miRNAs and their targets were validated using poly(A)-based qRT-PCR in two genotypes grown under lab and field conditions. Many miRNAs and their targets showed genotype-specific expression. The expression profiling also highlighted, both, similar and different expression patterns for the same sets of miRNA and mRNA at different stages of Foc infection. A high correlation in expression patterns of the miRNAs and their targets in lab- and field-grown plant samples was observed. Interestingly, Car-miR398 targeted copper chaperone for superoxide dismutase (CCS) that, in turn, regulated superoxide dismutase (SOD) activity during chickpea-Foc interaction. The cleavage site in targets was mapped for three miRNAs by analyzing publicly available degradome data for chickpea. The study, for the first time, provides novel insights into microRNA-mediated regulation of resistance and susceptibility mechanisms in chickpea against FW and opens up avenues for the development of the wilt-resistant cultivars in chickpea.