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RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels

Abstract

Main conclusion

Expressing an RNAi construct in maize kernels that targets the gene for alpha-amylase in Aspergillus flavus resulted in suppression of alpha-amylase (amy1) gene expression and decreased fungal growth during in situ infection resulting in decreased aflatoxin production.

Aspergillus flavus is a saprophytic fungus and pathogen to several important food and feed crops, including maize. Once the fungus colonizes lipid-rich seed tissues, it has the potential to produce toxic secondary metabolites, the most dangerous of which is aflatoxin. The pre-harvest control of A. flavus contamination and aflatoxin production is an area of intense research, which includes breeding strategies, biological control, and the use of genetically-modified crops. Host-induced gene silencing, whereby the host crop produces siRNA molecules targeting crucial genes in the invading fungus and targeting the gene for degradation, has shown to be promising in its ability to inhibit fungal growth and decrease aflatoxin contamination. Here, we demonstrate that maize inbred B104 expressing an RNAi construct targeting the A. flavus alpha-amylase gene amy1 effectively reduces amy1 gene expression resulting in decreased fungal colonization and aflatoxin accumulation in kernels. This work contributes to the development of a promising technology for reducing the negative economic and health impacts of A. flavus growth and aflatoxin contamination in food and feed crops.

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Abbreviations

GFP:

Green fluorescent protein

RNAi:

Ribonucleic acid interference

siRNA:

Small interfering ribonucleic acid

UPLC:

Ultra-performance liquid chromatography

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Acknowledgements

We thank Darlene Downey for conducting kernel infection assays. Jonte Ellison and Darlene Downey isolated RNA and DNA, and Jonte Ellison conducted genotyping PCR of individual kernels and qPCR. We also thank Carol Carter-Wientjes for her technical expertise with UPLC analysis. We thank Jay Shockey and Subbaiah Chalivendra for critical reading of the manuscript. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement from the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer.

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Correspondence to Matthew K. Gilbert.

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The authors declare that they have no conflict of interest.

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Supplementary material 1 (TIFF 807 kb) Supplemental Fig. S1 Aspergillus flavus amy1 RNAi vector diagram. a amy1 nucleotide sequence showing RNAi target region. b Conserved domains in the amy1 peptide analyzed using NCBI conserved domain database (CDD) search (Marchler-Bauer et al. 2017). c RNAi vector design for maize transformation to silence the A. flavus amy1 gene

Supplementary material 2 (PNG 11 kb) Supplemental Fig. S2 Quantitative PCR analysis showing the relative expression levels of the A. flavus beta-tubulin gene normalized to the maize ribosomal protein L10 gene, GRMZM2G024838. The results indicate that maize lines expressing the amy1 RNAi construct, lines 1-3, 2-2, and 3-4, have reduced levels of detectable A. flavus beta-tubulin compared to the isogenic control maize line not expressing the amy1 RNAi construct. The ribosomal structural gene from maize and A. flavus beta-tubulin gene showed no reaction could be detected by SYBR qPCR when amplification was attempted using A. flavus-GFP and maize cDNA, respectively (data not shown). (*) indicates significance at P ≤ 0.05. Error bars indicate the standard deviation of replicates

Supplementary material 3 (XLSX 9 kb)

Supplementary material 4 (XLSX 10 kb)

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Gilbert, M.K., Majumdar, R., Rajasekaran, K. et al. RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels. Planta 247, 1465–1473 (2018). https://doi.org/10.1007/s00425-018-2875-0

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Keywords

  • Host-induced gene silencing
  • Mycotoxin
  • RNAi
  • Secondary metabolite
  • siRNA
  • Zea mays