Peanuts that keep aflatoxin at bay: a threshold that matters

Abstract

Aflatoxin contamination in peanuts poses major challenges for vulnerable populations of sub-Saharan Africa and South Asia. Developing peanut varieties to combat preharvest Aspergillus flavus infection and resulting aflatoxin contamination has thus far remained a major challenge, confounded by highly complex peanut-Aspergilli pathosystem. Our study reports achieving a high level of resistance in peanut by overexpressing (OE) antifungal plant defensins MsDef1 and MtDef4.2, and through host-induced gene silencing (HIGS) of aflM and aflP genes from the aflatoxin biosynthetic pathway. While the former improves genetic resistance to A. flavus infection, the latter inhibits aflatoxin production in the event of infection providing durable resistance against different Aspergillus flavus morphotypes and negligible aflatoxin content in several peanut events/lines well. A strong positive correlation was observed between aflatoxin accumulation and decline in transcription of the aflatoxin biosynthetic pathway genes in both OE-Def and HIGS lines. Transcriptomic signatures in the resistant lines revealed key mechanisms such as regulation of aflatoxin synthesis, its packaging and export control, besides the role of reactive oxygen species-scavenging enzymes that render enhanced protection in the OE and HIGS lines. This is the first study to demonstrate highly effective biotechnological strategies for successfully generating peanuts that are near-immune to aflatoxin contamination, offering a panacea for serious food safety, health and trade issues in the semi-arid regions.

Keywords: Aspergillus flavus; aflatoxin; defensins; food safety; host-induced gene silencing; mycotoxins; peanut.

Figure 1

Transformation vectors and expression analysis of peanut OE‐Def and HIGS lines. (a) Expression vectors used for peanut transformation. The constitutive figwort mosaic virus (FMV) 35S promoter was used for expression of full‐length MsDef1‐Ec, MtDef4.2‐Ec and MtDef4.2‐ER . MsDef1‐Ec and MtDef4.2‐EC constructs targeted each defensin to the apoplast with signal peptide, whereas MtDef4.2‐ER construct retained this defensin in the endoplasmic reticulum. For targeting the aflatoxin pathway genes, the hpRNA cassettes had inverted repeats of respective omtA (aflP) and ver‐1(aflM) regions around the PR10 intron under the control of double CaMV 35S promoter. LB, left border; RB, right border; nos, nopaline synthase gene terminator; CaMV35S, cauliflower mosaic virus promoter; SP, signal peptide. (b) Expression of defensin transgenes in various OE‐Def events (pooled across generations) at different pod development stages (R5, R6 and R7). (c,d) RT‐PCR analyses to detect the expression of hpRNA transcripts in mature cotyledons. A 310‐bp amplicon for omtA (c) and a 330‐bp amplicon for ver‐1 (d). An intron‐spanning peanut ADH3 gene was used as a control (lower panel). A 400‐bp amplicon is expected from a genomic DNA template, whereas 143‐bp amplicon is expected from a cDNA template. Letters B and C represent Blank and WT control, respectively; L stands for marker ladder and P denotes plasmid. (e) Expression of defensin transgenes in various OE‐Def events (pooled across generations) in mature cotyledons after infection with A. flavus AF11‐4 at 72 hpi. The housekeeping gene, G6PD was used for normalization with respect to the WT. Error bars represent the standard error (SE) of at least five replicates.

Figure 2

Fungal assay of OE‐Def and HIGS lines at 72 hpi. (a) Comparison of fungal colonization on cotyledons of MtDef4‐Ec 96 (top row left), MsDef1‐Ec 23 (top row right), HIGS line; hp‐omtA 16 (middle row left), HIGS lines; hp‐ver1‐1 6 (middle row right), WT control (last row left) and resistant check, 55‐437 (last row right); OE‐Def lines show no or very little fungal growth on events generated with extracellularly targeted Def4 and Def1 genes; HIGS lines show no restriction to fungal growth on events generated with omtA and ver‐1; extensive fungal growth and sporulation on WT controls, resistant check‐peanut variety 55‐437. (b) Fungal load of A. flavus on cotyledons of OE‐Def, HIGS and WT lines after 72 hpi. Error bar represents standard error of at least three biological replicates at P = 0.5.

Figure 3

Aflatoxin profile of T₃ seed cotyledons of OE‐DEf and HIGS peanut lines following A. flavus infection at 72 h using HPLC (a) B₁ levels (ppb) in the inoculated cotyledons of OE‐Def, HIGS and WT peanut lines. (b) Number of best homozygous events across five constructs that accumulate ≤20 ppb B₁ toxin across 24 selected events. The colour codes reveal the range of B₁ content based on HPLC. (c) Aflatoxin profiling based on individual toxin types in selected events of OE‐Def and HIGS lines accumulating <4 ppb B₁ and B₂ toxins after A. flavus ( AF11‐4) infection. The events were sorted by the content. (d) Event‐wise comparison of B₁ toxin (ppb) in a subset of homozygous T₃ progenies of OE‐Def, HIGS and WT peanut lines against three different A. flavus morphotypes (AF11‐4, A‐12, A‐191).

Figure 4

Expression profile of host ROS scavenging antioxidative genes, SOD , CAT and APX in the infected peanut cotyledons of (a) OE‐Def events and (b) HIGS lines in comparison with the WT at 72 hpi.

Figure 5

Reduced expression of aflatoxin pathway genes in A. flavus and induced morphological alterations in infected transgenic/HIGS peanut lines. (a,b) Transcript abundance of fungal biosynthetic cluster genes in OE‐Def events (a) and (b) HIGS lines in comparison with the WT at 72 hpi. (c–e) Morphology of A. flavus ( AF11‐4) infecting the OE‐Def, HIGS and WT peanut lines after 40  hpi. (c) Conidial morphology of OE‐Def events with WT. (d) Bright‐field microscopy of A. flavus at 40 hpi. Profuse vesicles (arrows) detected in the cytoplasm of fungus infecting the WT controls (left) compared to HIGS line OMT15‐1 (right; arrows indicate vacuoles) (magnification at 1000×). (e) High‐intensity staining of vesicles (arrows) reflects higher aflatoxin production ability in the A. flavus‐infecting WT (left) compared to HIGS line Ver1 6‐1 (right; arrows indicate vacuoles) (magnification at 1000×).

Figure 6

Trait stability in the selected peanut OE‐Def and HIGS lines over three seed generations. The B₁ content of T2 through T3 seeds remained relatively consistent across selfed generations for lines Def1Ec23, Def4Ec 26, Def4Ec96, Def4Ec97, Def4ER6, Ver‐12, Ver‐13, Ver‐1 6 and OMT16. The box plots show 25%–50% and 50%–75% quartiles (n = 5–13); the mean B₁ toxin content is shown by the bar.