Comparative RNA-Seq analysis unfolds a complex regulatory network imparting yellow mosaic disease resistance in mungbean [Vigna radiata (L.) R. Wilczek]

Abstract

Yellow Mosaic Disease (YMD) in mungbean [Vigna radiata (L.) R. Wilczek] is one of the most damaging diseases in Asia. In the northern part of India, the YMD is caused by Mungbean Yellow Mosaic India Virus (MYMIV), while in southern India this is caused by Mungbean Yellow Mosaic Virus (MYMV). The molecular mechanism of YMD resistance in mungbean remains largely unknown. In this study, RNA-seq analysis was conducted between a resistant (PMR-1) and a susceptible (Pusa Vishal) mungbean genotype under infected and control conditions to understand the regulatory network operating between mungbean-YMV. Overall, 76.8 million raw reads could be generated in different treatment combinations, while mapping rate per library to the reference genome varied from 86.78% to 93.35%. The resistance to MYMIV showed a very complicated gene network, which begins with the production of general PAMPs (pathogen-associated molecular patterns), then activation of various signaling cascades like kinases, jasmonic acid (JA) and brassinosteroid (BR), and finally the expression of specific genes (like PR-proteins, virus resistance and R-gene proteins) leading to resistance response. The function of WRKY, NAC and MYB transcription factors in imparting the resistance against MYMIV could be established. The string analysis also revealed the role of proteins involved in kinase, viral movement and phytoene synthase activity in imparting YMD resistance. A set of novel stress-related EST-SSRs are also identified from the RNA-Seq data which may be used to find the linked genes/QTLs with the YMD resistance. Also, 11 defence-related transcripts could be validated through quantitative real-time PCR analysis. The identified gene networks have led to an insight about the defence mechanism operating against MYMIV infection in mungbean which will be of immense use to manage the YMD resistance in mungbean.

Fig 1. Venn diagram representing the proportion of up-regulated and down-regulated genes in different combinations.

Where MRi: Mungbean Resistant Inoculated; MRc: Mungbean Resistant Control; MSi: Mungbean Susceptible Inoculated; MSc: Mungbean Susceptible Control.

Fig 2. Heat map generated using stress-specific DEG groups like defence enzymes, JA pathway, gene-silencing, transcription factor, kinases and PR proteins.

Fig 3. Gene ontology (GO) categorization of the differentially expressed genes (DEGs) in mungbean resistant infected (MRI) vs resistant control (MRC) combinations.

There are three significantly enriched categories of biological process (BP), cellular component (CC) and molecular function (MF). Where, Y-axis represents unigene percentage.

Fig 4

KEGG pathways of key significantly enriched DGEs in different comparisons (a-d) representing the degree of enriched DGEs in a pathway. Where, gene-ratio is the ratio of input-genes to the total gene-list in the pathway; the number of enriched DGEs in any pathway is indicated by the circle area, and circle color represents the range of corrected p-value.

Fig 5

Protein-protein interaction network of (a-b) resistant genotype using MCODE analysis.

Fig 6. Correlation analysis between RNA-Seq and qPCR data of 11 differentially expressed transcripts.

Fig 7. The complex gene network giving an insight into the probable defence mechanism leading to YMD resistance in mungbean.