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. 2016 Nov 3;8(11):317.
doi: 10.3390/toxins8110317.

RNA Sequencing of Contaminated Seeds Reveals the State of the Seed Permissive for Pre-Harvest Aflatoxin Contamination and Points to a Potential Susceptibility Factor

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RNA Sequencing of Contaminated Seeds Reveals the State of the Seed Permissive for Pre-Harvest Aflatoxin Contamination and Points to a Potential Susceptibility Factor

Josh Clevenger et al. Toxins (Basel). .
Free PMC article

Abstract

Pre-harvest aflatoxin contamination (PAC) is a major problem facing peanut production worldwide. Produced by the ubiquitous soil fungus, Aspergillus flavus, aflatoxin is the most naturally occurring known carcinogen. The interaction between fungus and host resulting in PAC is complex, and breeding for PAC resistance has been slow. It has been shown that aflatoxin production can be induced by applying drought stress as peanut seeds mature. We have implemented an automated rainout shelter that controls temperature and moisture in the root and peg zone to induce aflatoxin production. Using polymerase chain reaction (PCR) and high performance liquid chromatography (HPLC), seeds meeting the following conditions were selected: infected with Aspergillus flavus and contaminated with aflatoxin; and not contaminated with aflatoxin. RNA sequencing analysis revealed groups of genes that describe the transcriptional state of contaminated vs. uncontaminated seed. These data suggest that fatty acid biosynthesis and abscisic acid (ABA) signaling are altered in contaminated seeds and point to a potential susceptibility factor, ABR1, as a repressor of ABA signaling that may play a role in permitting PAC.

Keywords: Arachis hypogaea; Aspergillus; RNA-seq; aflatoxin; non-coding RNA; uHPLC.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Box plots showing daily area under the drought progress curve (AUDPC) of drought tolerance-related traits during the 40-day stress period. Letters indicate significant differences by a Kruskal–Wallis test followed by Dunn’s test for comparisons of mean rank sums. Aflatoxin values were measured one time after harvest and were natural log transformed to stabilize variance. C76 = C76-16; FL07 = Florida07; NC = NC 3033; TG = Tifguard; TR = Tifrunner.
Figure 2
Figure 2
Differential expression of genes affected by Aspergillus flavus colonization and aflatoxin presence. (a): first two principal components of all gene expression profiles across all genotypes with the four identified groups highlighted with a different color. PC1 and PC2 (58% of variation) can define the two gene expression responses; (b): Four groups of differentially expressed genes. Scale is mean-centered relative expression. The color of the titles of the boxplots corresponds to highlighted points in A. TR = Tifrunner; FL07 = Florida-07. Expression is the mean centered normalized z-score of the average FPKM (Fragments per Kilobase of exon per Million reads Mapped) of 2–3 biological replicates each made up of two pooled seeds. Black indicates the presence of Aspergillus spp. and aflatoxin on the seeds. Green indicates no Aspergillus spp. present on the seeds.
Figure 3
Figure 3
An enriched GO term analysis of differentially expressed genes. The left panel of each heatmap shows number of genes with an enriched GO term (top scale). The right panel of each heatmap shows the log transformed adjusted p-value from a hypergeometric enrichment test (bottom scale). GO terms are grouped into meta categories shown at the right of each heatmap. The left heatmap shows genes up-regulated in samples free of infection and contamination. The upper right heatmap shows genes up-regulated in samples contaminated with aflatoxin. Aflatoxin graphs show the natural log transformed average aflatoxin of pooled seeds with standard error. Zero values indicate no detection above detectable limits from those samples.
Figure 4
Figure 4
Groups of genes differentially expressed between aflatoxin contaminated seeds and seeds infected with Aspergillus but not contaminated with aflatoxin ignoring genotype, separated into four groups depending on expression profile across genotypes. (a) 53 genes up-regulated in all three genotypes with no contamination compared to contaminated seeds; (b) 182 genes up-regulated in only NC 3033 and Tifguard non-contaminated, infected seeds; (c) 24 genes up-regulated in only C76-16 and NC 3033 non-contaminated, infected seeds; (d) 29 up-regulated genes in all three genotypes with contaminated seeds compared to non-contaminated, infected seeds. C76 = C76-16; FL07 = Florida07; NC = NC 3033; TG = Tifguard; TR = Tifrunner. Expression is the mean centered normalized z-score of the average FPKM (Fragments per Kilobase of exon per Million reads Mapped) of 2–3 biological replicates each made up of two pooled seeds. Black indicates the presence of Aspergillus spp. and aflatoxin on the seeds. Green indicates Aspergillus spp. present but no aflatoxin contamination on the seeds.
Figure 5
Figure 5
An enriched GO term analysis of differentially expressed genes. The left panel of each heatmap shows number of genes with an enriched GO term (top scale). The right panel of each heatmap shows the log transformed adjusted p-value from a hypergeometric enrichment test (bottom scale). GO terms are grouped into meta categories shown at the right of each heatmap. The left heatmap shows genes up-regulated in Aspergillus infected samples free of contamination. The upper heatmap shows genes up-regulated in samples contaminated with aflatoxin. Aflatoxin graphs show the natural log transformed average aflatoxin of pooled seeds with standard error. Zero values indicate no detection above detectable limits from those samples.
Figure 6
Figure 6
ncRNAs are differentially expressed in contaminated seeds. (a,b) groups of ncRNAs differentially expressed within genotypes (right) and between genotypes (left) differentiated by aflatoxin contamination; (c) Two examples of down-regulated antisense ncRNAs associated with fatty acid biosynthesis genes and their orientation within the predicted gene. Bar graphs below show average expression in FPKM with standard error of two replicates (contaminated) and three replicates (not contaminated); (d) One example of a ncRNA up-regulated in contaminated seeds is intergenic and not associated with a predicted gene. FL07 = Florida-07; TR = Tifrunner. Alignment of ncRNAs is relative to A. duranensis and A. ipaensis pseudomolecules and so is only an approximate alignment for A. hypogaea. Transcripts were assembled de novo and identified as putative non-coding independently.
Figure 7
Figure 7
Pathways down-regulated in aflatoxin contaminated seeds. Heatmaps show the relative expression (Z-score normalized) of each gene shown to the right. Each heatmap is grouped into pathways.

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