Research ArticleAGRICULTURE

Aflatoxin-free transgenic maize using host-induced gene silencing

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Science Advances  10 Mar 2017:
Vol. 3, no. 3, e1602382
DOI: 10.1126/sciadv.1602382
  • Fig. 1 Construction of an RNAi cassette to silence aflatoxin synthesis.

    (A) Overview of the fungal biosynthesis pathway of all four aflatoxin variants (AFB1, AFB2, AFG1, and AFG2) from the common precursor metabolite norsolorinic acid (NOR). The polyketide synthase aflC (pksA) step targeted for silencing in this report is denoted by “*.” (B) Map of transformation vector depicting the RNAi cassette under the control of an endosperm-specific promoter (γ-zein) and with three tandem 200–base pair (bp) fragments homologous to three different regions of the targeted aflatoxin biosynthetic enzyme aflC 6594-bp transcript. En, tobacco etch virus translational enhancer; Vsp, soybean vegetative storage protein terminator; LB, left border; RB, right border; Nos, nopaline synthase gene terminator; CaMV35S, cauliflower mosaic virus promoter; Bar, bialophos resistance gene. The two small arrows depict the annealing site for primers used in the RT-PCR for detecting the expression of the RNAi-inserted cassette.

  • Fig. 2 Expression of RNAi cassette in maize kernels.

    (A) RT-PCR analysis of total RNA isolated from developing kernels to assess whether the RNAiAFL cassette is expressed in transgenic kernels. cDNA produced from RNA isolated from two plants from each of the three transgenic RNAiAFL lines (AFL4, AFL5, and AFL20) and from a segregating null plant was used to amplify a 220-bp segment of the inserted RNAiAFL cassette (upper panel) and amplify a 290-bp endogenous maize gene (GAPDH; lower panel; lanes 1 to 6 cDNA from transgenics and lane 7 cDNA from null). The presence of genomic DNA in the cDNA preparation would be detected with the primers used as amplification from genomic DNA, which would produce a 591-bp band (lane 8 genomic DNA from null). The presence of the RNAi cassette amplicon (upper panel) indicates its expression in the transgenic lines tested (lanes 1 to 6) and not in the null control (lane 7). The differential size of GAPDH control amplification indicates that cDNA was amplified in transgenic samples, not genomic DNA. (B) Leaf-painting assay results using glufosinate ammonium solution (3 mg/ml) and observed after 7 days. Necrotic tissue in null control indicates the lack of expression of the Bar plant selectable marker, whereas RNAiAFL lines (AFL4 and AFL20) were resistant.

  • Fig. 3 Aspergillus infection of transgenic RNAiAFL cobs and toxin assay.

    (A) Toxin-producing A. flavus was injected into 8- to 10-DAP maize cobs and allowed to grow for 30 days. Infected maize cobs were subsequently harvested, and kernels surrounding each infection (denoted by red dots) were combined and assayed for toxin. (B) Aflatoxin levels (as log2 ppb) in three biological replicates of the transgenic RNAiAFL 4 line (AFL4a, AFL4b, and AFL4c), two biological replicates of the transgenic RNAiAFL 20 line (AFL20a and AFL20b), and three Null control (A, B, and C) cobs, as determined by TLC analysis. nd, nondetectable. (C) Transcript levels of the Aspergillus aflC gene relative to the fungal endogenous tubulin gene were determined by qRT-PCR analysis performed on RNA isolated from Aspergillus-infected maize kernels. (D) Relative expression of the Aspergillus chitin synthase gene was determined and normalized to the expression of the maize GAPDH gene. As above, RNA from fungal-infected maize cobs was used to extract RNA and perform qRT-PCR. Analysis showing that only cDNA, not contaminating genomic DNA, was amplified in (C) and (D) reactions is shown in figs. S1 and S4, respectively. All means and SEs were calculated from at least three replicates. Means with the same lowercase letter do not display a significant difference [P < 0.05; analysis of variance (ANOVA)].

  • Fig. 4 Matrix for intersections of differentially expressed transcripts between RNAiAFL transgenic and null kernels.

    The intersection of six pairwise comparisons between two nontransgenic (Null A and Null B) samples and three transgenic (AFL4, AFL5, and AFL20) samples are shown. Set size was sorted by the number of significantly differentially expressed transcripts within a given pairwise comparison (horizontal orange bars). Dark circles in the matrix indicate sets of transcripts that are part of an intersection. The number of intersecting transcripts that are differentially expressed within the given grouping is shown in green. The matrix intersection of the comparison of the three RNAiAFL transgenic lines among themselves and the two Null controls shows similar or larger intersections of the number of differentially expressed transcripts (fig. S3). A generated table shows the significantly expressed transcripts detected in all six pairwise comparisons used to generate this matrix (available online at http://de.iplantcollaborative.org/dl/d/24DC6B1C-EF2C-4A00-9EBF-85B43234E88C/Pairwise_Sig_Transcripts_with_Function.xlsx).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/3/e1602382/DC1

    table S1. Individual aflatoxin quantification reading by TLC on maize kernels after Aspergillus infection.

    table S2. Primer sequences used in this study.

    fig. S1. RT-PCR to detect the expression of the Aspergillus aflC transcript in fungus-infected maize kernels.

    fig. S2. Side-by-side comparisons of RNAiAFL transgenics and null maize plants.

    fig. S3. The intersection matrix of pairwise comparisons of significantly differentially expressed transcripts between three RNAiAFL transgenic plants (AFL4, AFL5, and AFL20) and two nontransgenic controls (Null A and Null B).

    fig. S4. RT-PCR to detect the expression of the Aspergillus chitin synthase C transcript in fungus-infected maize kernels.

  • Supplementary Materials

    This PDF file includes:

    • table S1. Individual aflatoxin quantification reading by TLC on maize kernels after Aspergillus infection.
    • table S2. Primer sequences used in this study.
    • fig. S1. RT-PCR to detect the expression of the Aspergillus aflC transcript in fungus-infected maize kernels.
    • fig. S2. Side-by-side comparisons of RNAiAFL transgenics and null maize plants.
    • fig. S3. The intersection matrix of pairwise comparisons of significantly differentially expressed transcripts between three RNAiAFL transgenic plants (AFL4, AFL5, and AFL20) and two nontransgenic controls (Null A and Null B).
    • fig. S4. RT-PCR to detect the expression of the Aspergillus chitin synthase C transcript in fungus-infected maize kernels.

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