Global Transcriptome Profiling Identified Transcription Factors, Biological Process, and Associated Pathways for Pre-Harvest Aflatoxin Contamination in Groundnut

Abstract

Pre-harvest aflatoxin contamination (PAC) in groundnut is a serious quality concern globally, and drought stress before harvest further exacerbate its intensity, leading to the deterioration of produce quality. Understanding the host-pathogen interaction and identifying the candidate genes responsible for resistance to PAC will provide insights into the defense mechanism of the groundnut. In this context, about 971.63 million reads have been generated from 16 RNA samples under controlled and Aspergillus flavus infected conditions, from one susceptible and seven resistant genotypes. The RNA-seq analysis identified 45,336 genome-wide transcripts under control and infected conditions. This study identified 57 transcription factor (TF) families with major contributions from 6570 genes coding for bHLH (719), MYB-related (479), NAC (437), FAR1 family protein (320), and a few other families. In the host (groundnut), defense-related genes such as senescence-associated proteins, resveratrol synthase, seed linoleate, pathogenesis-related proteins, peroxidases, glutathione-S-transferases, chalcone synthase, ABA-responsive gene, and chitinases were found to be differentially expressed among resistant genotypes as compared to susceptible genotypes. This study also indicated the vital role of ABA-responsive ABR17, which co-regulates the genes of ABA responsive elements during drought stress, while providing resistance against A. flavus infection. It belongs to the PR-10 class and is also present in several plant-pathogen interactions.

Keywords: Aspergillus flavus; RNA-seq; aflatoxin; biotechnology; food safety; gene expression; genomics; pre-harvest aflatoxin contamination (PAC); transcriptome analysis.

Figure 1

Distribution of genes expressed in 16 samples. Based on expression level, genes were grouped into four classes (FPKM < 2, 2 ≤ FPKM < 10, 10 ≤ FPKM < 20 and FPKM > 20) in sixteen samples.

Figure 2

Principal component analysis depicting correlation among samples based on gene expression data. Group 1 (ICGV 91278), highly resistant, group 2 (ICGV 91284) moderately resistant, group 3 (ICGV 91324, ICGV 91315, ICGV 93305, ICGV 94379), moderately susceptible, in comparison to highly susceptible check JL24.

Figure 3

Differential expression of genes (DEGs) of groundnut genotype under control and infected conditions in response to Aspergillus flavus. (A) Group 1: moderately resistant J 11(I) vs. resistant (ICGV 91278, ICGV 91315, ICGV 93305, ICGV 91284, ICGV 94379, ICGV 91324) infected condition, Group 2: susceptible JL 24 vs. resistant (ICGV 91278, ICGV 91315, ICGV 93305, ICGV 91284, ICGV 94379, ICGV 91324) in infected condition, (B) Group 3: resistant J 11(C) vs. resistant (ICGV 91278, ICGV 91315, ICGV 93305, ICGV 91284, ICGV 94379, ICGV 91324) in control conditions, and group 4: susceptible JL 24 vs. resistant (ICGV 91278, ICGV 91315, ICGV 93305, ICGV 91284, ICGV 94379, ICGV 91324) under control conditions. (C) Number of differentially expressed transcripts between group 1, group 2, group 3 and group 4 and (D) group 5: susceptible JL 24 vs. (J 11, ICGV 91278, ICGV 91315, ICGV 93305, ICGV 91284, ICGV 94379, ICGV 91324 under control conditions, group 6: susceptible JL 24 vs. (J 11, ICGV 91278, ICGV 91315, ICGV 93305, ICGV 91284, ICGV 94379, ICGV 91324) in infected condition.

Figure 4

Volcano plot demonstrating the genes that were differentially expressed between susceptible (JL 24) and resistant genotypes (ICGV 91278, ICGV 91284) under infection conditions. Investigation and representation of DEGs were performed for developing Volcano plot. This plot is between log₂ (fold change) and −log₁₀ of the p-value on the x-axis and y-axis, respectively. Here, each dot is representing a gene and black and red coloured dots represent non-significant and significant genes, respectively.

Figure 5

Distribution of gene ontology annotation assigned by Blast2 GO. This figure describes the three GO categories: cellular component (A), biological processes (B), and molecular functions (C).

Figure 6

Spatial transcript expression identified in 16 samples. The figure shows the number of sample or genotype specific transcript in all 16 samples. Tissue specificity index (τ) was calculated to identify the genotype specific transcript. In the present study, transcripts with τ ≥ 0.9 were considered as sample specific. (A) Blue color represents sample specific transcript encoding transcription factors (TF) and orange color represents Non-TF transcripts. (B) The figure shows the distribution of sample specific transcript encoding transcription factors in all 16 samples.

Figure 7

Heat map of different pathways of genes affected in 8 groundnut genotypes, both under control conditions and after infection with A. flavus. Some selected genes altered their expression in all of the pathways due to different pre-harvest aflatoxin contamination. Expression of genes involved in the glutathione-S-transferase, flavonoid biosynthesis, fatty acid biosynthesis, ABA responsive genes, resveratrol synthase, and seed linoleate gene expression is plotted. The color code corresponds to the FPKM value of the transcripts, increase from red to green. Genotype and treatment labels are shown at the top of the figure.

Figure 8

PAC is largely facilitated by moisture and heat stress during pod development in a green house. The study indicated an ABA-responsive ABR17 gene belonging to the PR-10 class has been activated in plant–pathogen interactions during PAC. The components represented in red have an important role in host–pathogen interactions in this study, showing highly up-regulated patterns in resistant genotypes. Several pathways and TFs such as fatty acid biosynthesis, flavonoid biosynthesis, seed linoleate gene expression, resveratrol synthase, and glutathione-S-transferase play an important role in genetic resistance for PAC. The components in black represent the resistant pathways and other components which help in host-plant defense signaling.