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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Green fluorescent protein
Ribonucleic acid interference
Small interfering ribonucleic acid
Ultra-performance liquid chromatography
Abbas HK, Accinelli C, Shier WT (2017) Biological control of aflatoxin contamination in US crops and the use of bioplastic formulations of Aspergillus flavus biocontrol strains to optimize application strategies. J Agric Food Chem 65(33):7081–7087. https://doi.org/10.1021/acs.jafc.7b01452
Arias RS, Dang PM, Sobolev VS (2015) RNAi-mediated control of aflatoxins in peanut: method to analyze mycotoxin production and transgene expression in the peanut/Aspergillus pathosystem. J Vis Exp 106:e53398. https://doi.org/10.3791/53398
Bluhm BH, Woloshuk CP (2005) Amylopectin induces fumonisin B1 production by Fusarium verticillioides during colonization of maize kernels. Mol Plant Microbe Interact 18(12):1333–1339. https://doi.org/10.1094/MPMI-18-1333
Bouras N, Strelkov S (2010) Influence of carbon source on growth and mycotoxin production by isolates of Pyrenophora tritici-repentis from wheat. Can J Microbiol 56:874–882
Brown RL, Menkir A, Chen ZY, Bhatnagar D, Yu J, Yao H, Cleveland TE (2013) Breeding aflatoxin-resistant maize lines using recent advances in technologies—a review. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 30(8):1382–1391. https://doi.org/10.1080/19440049.2013.812808
Chen ZY, Brown RL, Damann KE, Cleveland TE (2010) PR10 expression in maize and its effect on host resistance against Aspergillus flavus infection and aflatoxin production. Mol Plant Pathol 11(1):69–81. https://doi.org/10.1111/j.1364-3703.2009.00574.x
Chen ZY, Warburton ML, Hawkins L, Wei Q, Raruang Y, Brown RL, Zhang L, Bhatnagar D (2016) Production of the 14 kDa trypsin inhibitor protein is important for maize resistance against Aspergillus flavus infection/aflatoxin accumulation. World Mycotoxin J 9(2):215–228. https://doi.org/10.3920/wmj2015.1890
Dahlgren C, Zhang HY, Du Q, Grahn M, Norstedt G, Wahlestedt C, Liang Z (2008) Analysis of siRNA specificity on targets with double-nucleotide mismatches. Nucleic Acids Res 36(9):e53. https://doi.org/10.1093/nar/gkn190
Damalas CA, Eleftherohorinos IG (2011) Pesticide exposure, safety issues, and risk assessment indicators. Int J Environ Res Public Health 8(5):1402–1419. https://doi.org/10.3390/ijerph8051402
Dohroo NP, Bhardwaj SS, Shyram KR (1987) Amylase and invertase activity as influenced by Pythium pleroticum causing rhizome rot of ginger. Plant Dis Res 2:106–107
Doyle J, Doyle J (1987) A rapid procedure for DNA purification from small quantities of fresh leaf tissue. Phytochem Bull 19:11–15
Du Q, Thonberg H, Wang J, Wahlestedt C, Liang Z (2005) A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res 33(5):1671–1677. https://doi.org/10.1093/nar/gki312
Fakhoury AM, Woloshuk CP (1999) Amy1, the alpha-amylase gene of Aspergillus flavus: involvement in aflatoxin biosynthesis in maize kernels. Phytopathology 89(10):908–914. https://doi.org/10.1094/PHYTO.19126.96.36.1998
Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Pegg SE, Li B, Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129(1):13–22. https://doi.org/10.1104/pp.000653
Frame B, Main M, Schick R, Wang K (2011) Genetic transformation using maize immature zygotic embryos. Methods Mol Biol 710:327–341. https://doi.org/10.1007/978-1-61737-988-8_22
Gilbert MK, Mack BM, Payne GA, Bhatnagar D (2016) Use of functional genomics to assess the climate change impact on Aspergillus flavus and aflatoxin production. World Mycotoxin J 9(5):665–672. https://doi.org/10.3920/wmj2016.2049
Gressel J, Polturak G (2018) Suppressing aflatoxin biosynthesis is not a breakthrough if not useful. Pest Manag Sci 74(1):17–21. https://doi.org/10.1002/ps.4694
Ismaiel A, Papenbrock J (2015) Mycotoxins: producing fungi and mechanisms of phytotoxicity. Agriculture 5(3):492–537. https://doi.org/10.3390/agriculture5030492
Karlovsky P, Suman M, Berthiller F, De Meester J, Eisenbrand G, Perrin I, Oswald IP, Speijers G, Chiodini A, Recker T, Dussort P (2016) Impact of food processing and detoxification treatments on mycotoxin contamination. Mycotoxin Res 32(4):179–205. https://doi.org/10.1007/s12550-016-0257-7
Koch A, Biedenkopf D, Furch A, Weber L, Rossbach O, Abdellatef E, Linicus L, Johannsmeier J, Jelonek L, Goesmann A, Cardoza V, McMillan J, Mentzel T, Kogel KH (2016) An RNAi-based control of Fusarium graminearum infections through spraying of long dsRNAs involves a plant passage and is controlled by the fungal silencing machinery. PLoS Pathog 12(10):e1005901. https://doi.org/10.1371/journal.ppat.1005901
Konakalla NC, Kaldis A, Berbati M, Masarapu H, Voloudakis AE (2016) Exogenous application of double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco. Planta 244(4):961–969. https://doi.org/10.1007/s00425-016-2567-6
Kowalska A, Walkiewicz K, Koziel P, Muc-Wierzgon M (2017) Aflatoxins: characteristics and impact on human health. Postepy Hig Med Dosw (Online) 71:315–327
Lanubile A, Maschietto V, Battilani P, Marocco A (2017) Infection with toxigenic and atoxigenic strains of Aspergillus flavus induces different transcriptional signatures in maize kernels. J Plant Interact 12(1):21–30. https://doi.org/10.1080/17429145.2016.1274062
Liu J, Sun L, Zhang N, Zhang J, Guo J, Li C, Rajput SA, Qi D (2016) Effects of nutrients in substrates of different grains on aflatoxin B production by Aspergillus flavus. BioMed Res Int 2016:7232858
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Majumdar R, Rajasekaran K, Cary JW (2017) RNA interference (RNAi) as a potential tool for control of mycotoxin contamination in crop plants: concepts and considerations. Front Plant Sci 8:200. https://doi.org/10.3389/fpls.2017.00200
Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH (2017) CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res 45(D1):D200–D203
Masanga JO, Matheka JM, Omer RA, Ommeh SC, Monda EO, Alakonya AE (2015) Downregulation of transcription factor aflR in Aspergillus flavus confers reduction to aflatoxin accumulation in transgenic maize with alteration of host plant architecture. Plant Cell Rep 34(8):1379–1387. https://doi.org/10.1007/s00299-015-1794-9
Mathan S, Subramanian V, Nagamony S (2013) Optimization and antimicrobial metabolite production from endophytic fungi Aspergillus terreus KC 582297. Eur J Exp Biol 3(4):138–144
Mitchell NJ, Bowers E, Hurburgh C, Wu F (2016) Potential economic losses to the US corn industry from aflatoxin contamination. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 33(3):540–550. https://doi.org/10.1080/19440049.2016.1138545
Power IL, Dang PM, Sobolev VS, Orner VA, Powell JL, Lamb MC, Arias RS (2017) Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut. Plant Sci 257(Supplement C):106–125. https://doi.org/10.1016/j.plantsci.2016.12.013
Rajasekaran K, Cary JW, Cotty PJ, Cleveland TE (2008) Development of a GFP-expressing Aspergillus flavus strain to study fungal invasion, colonization, and resistance in cottonseed. Mycopathologia 165(2):89–97. https://doi.org/10.1007/s11046-007-9085-9
Rajasekaran K, Sickler CM, Brown RL, Cary JW, Bhatnagar D (2013) Evaluation of resistance to aflatoxin contamination in kernels of maize genotypes using a GFP-expressing Aspergillus flavus strain. World Mycotoxin J 6(2):151–158. https://doi.org/10.3920/wmj2012.1497
Rajasekaran K, Majumdar R, Sickler C, Wei Q, Cary J, Bhatnagar D (2017) Fidelity of a simple Liberty leaf-painting assay to validate transgenic maize plants expressing the selectable marker gene, bar. J Crop Improv 31(4):628–636. https://doi.org/10.1080/15427528.2017.1327913
Saleem A, Ebrahim MKH (2014) Production of amylase by fungi isolated from legume seeds collected in Almadinah Almunawwarah, Saudi Arabia. J Taibah Univ Sci 8(2):90–97. https://doi.org/10.1016/j.jtusci.2013.09.002
Savary S, Ficke A, Aubertot J-N, Hollier C (2012) Crop losses due to diseases and their implications for global food production losses and food security. Food Secur 4(4):519–537. https://doi.org/10.1007/s12571-012-0200-5
Sharma KK, Pothana A, Prasad K, Shah D, Kaur J, Bhatnagar D, Chen ZY, Raruang Y, Cary JW, Rajasekaran K, Sudini HK, Bhatnagar-Mathur P (2017) Peanuts that keep aflatoxin at bay: a threshold that matters. Plant Biotechnol J. https://doi.org/10.1111/pbi.12846
Singh R, Saxena VS, Singh R (1989) Pectinolytic, cellulolytic, amylase and protease production by three isolates of Fusarium solani variable in their virulence. J Mycol Plant Pathol 19:22–29
Skuzeski JM, Nichols LM, Gesteland RF (1990) Analysis of leaky viral translation termination codons in vivo by transient expression of improved β-glucuronidase vectors. Plant Mol Biol 15(1):65–79. https://doi.org/10.1007/bf00017725
Thakare D, Zhang J, Wing RA, Cotty PJ, Schmidt MA (2017) Aflatoxin-free transgenic maize using host-induced gene silencing. Sci Adv 3(3):e1602382. https://doi.org/10.1126/sciadv.1602382
Udomkun P, Wiredu AN, Nagle M, Muller J, Vanlauwe B, Bandyopadhyay R (2017) Innovative technologies to manage aflatoxins in foods and feeds and the profitability of application—a review. Food Control 76:127–138. https://doi.org/10.1016/j.foodcont.2017.01.008
Umesha S, Manukumar HM, Chandrasekhar B, Shivakumara P, Shiva Kumar J, Raghava S, Avinash P, Shirin M, Bharathi TR, Rajini SB, Nandhini M, Vinaya Rani GG, Shobha M, Prakash HS (2017) Aflatoxins and food pathogens: impact of biologically active aflatoxins and their control strategies. J Sci Food Agric 97(6):1698–1707. https://doi.org/10.1002/jsfa.8144
Wan HM, Chen CC, Giridhar R, Chang TS, Wu WT (2005) Repeated-batch production of kojic acid in a cell-retention fermenter using Aspergillus oryzae M3B9. J Ind Microbiol Biotechnol 32(6):227–233. https://doi.org/10.1007/s10295-005-0230-5
Williams WP, Krakowsky MD, Scully BT, Brown RL, Menkir A, Warburton ML, Windham GL (2015) Identifying and developing maize germplasm with resistance to accumulation of aflatoxins. World Mycotoxin J 8(2):193–209. https://doi.org/10.3920/wmj2014.1751
Woloshuk CP, Cavaletto JR, Cleveland TE (1997) Inducers of aflatoxin biosynthesis from colonized maize kernels are generated by an amylase activity from Aspergillus flavus. Phytopathology 87(2):164–169
Xiong Y, Wu VW, Lubbe A, Qin L, Deng S, Kennedy M, Bauer D, Singan VR, Barry K, Northen TR, Grigoriev IV, Glass NL (2017) A fungal transcription factor essential for starch degradation affects integration of carbon and nitrogen metabolism. PLoS Genet 13(5):e1006737. https://doi.org/10.1371/journal.pgen.1006737
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.
Conflict of interest
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
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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
- Host-induced gene silencing
- Secondary metabolite
- Zea mays