We thank David Meints, University of Arkansas for his assistance in developing and analyzing the transgenic maize during the early generations. This work received the financial support of USDA-ARS CRIS project 6054-42000-025-00D. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA-ARS&#8217; Equal Employment Opportunity (EEO) Policy mandates equal opportunity for all persons and prohibits discrimination in all aspects of the Agency&#8217;s personnel policies, practices, and operations.

1. Plasmid constructs and maize transformation
<ol>
	<li>PCR amplify Lablab AILP insert using the primers 5&#8217;-TATCTAGAACTAGTGATTACCATGGCTCC-3&#8217; and 5&#39;-ATACTGCAGGATTGCATGCAGAGTAGTACTG-3&#39;. The PCR conditions include an initial denaturation step at 98 &#176;C for 30 s (step 1), followed by denaturation at 98 &#176;C for 10 s (step 2), annealing at 55 &#176;C for 30 s (step 3), elongation at 72 &#176;C for 20 s (step 4), 31 cycles of step 2 to step 4, and a final elongation step at 72 &#176;C for...

<ol>
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N., Nagle, M., M&#252;ller, J., Vanlauwe, B., Bandyopadhyay, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+technologies+to+manage+aflatoxins+in+foods+and+feeds+and+the+profitability+of+application+&#8211;+A+review.">Innovative technologies to manage aflatoxins in foods and feeds and the profitability of application &#8211; A review.</a> Food Control. 76, 127-138 (2017).</li><li>Dohroo, N. P., Bhardwaj, S. S., Shyram, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amylase+and+invertase+activity+as+influenced+by+Pythium+pleroticum+causing+rhizome+rot+of+ginger.">Amylase and invertase activity as influenced by Pythium pleroticum causing rhizome rot of ginger.</a> Plant Disease Research. 2, 106-107 (1987).</li><li>Singh, R., Saxena, V. S., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pectinolytic,+cellulolytic,+amylase+and+protease+production+by+three+isolates+of+Fusarium+solani+variable+in+their+virulence.">Pectinolytic, cellulolytic, amylase and protease production by three isolates of Fusarium solani variable in their virulence.</a> Indian Journal of Mycology and Plant Pathology. 19, 22-29 (1989).</li><li>Fakhoury, A. M., Woloshuk, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amy1,+the+&#945;-amylase+gene+of+Aspergillus+flavus:+Involvement+in+aflatoxin+biosynthesis+in+maize+kernels.">Amy1, the &#945;-amylase gene of Aspergillus flavus: Involvement in aflatoxin biosynthesis in maize kernels.</a> Phytopathology. 89 (10), 908-914 (1999).</li><li>Bluhm, B. H., Woloshuk, C. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hevein-an+antifungal+protein+from+rubber-tree+(Hevea+brasiliensis)+latex.">Hevein-an antifungal protein from rubber-tree (Hevea brasiliensis) latex.</a> Planta. 183, 258-264 (1991).</li><li>Gozia, O., Ciopraga, J., Bentia, T., Lungu, M., Zamfirescu, I., Tudor, R., Roseanu, A., Nitu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+properties+of+lectin+and+new+chitinases+from+potato+tubers.">Antifungal properties of lectin and new chitinases from potato tubers.</a> Comptes Rendus de l'Academie des Sciences - Series III. 316 (8), 788-792 (1993).</li><li>Wisessing, A., Choowongkomon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amylase+inhibitors+in+plants:+Structures,+Functions+and+Applications.">Amylase inhibitors in plants: Structures, Functions and Applications.</a> Functional Plant Science and Biotechnology. 6 (1), 31-41 (2012).</li><li>Tyagi, B., Trivedi, N., Dubey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a-amylase+inhibitor:+A+compelling+plant+defense+mechanism+against+insect/pests.">a-amylase inhibitor: A compelling plant defense mechanism against insect/pests.</a> Environment &amp; Ecology. 32 (3), 995-999 (2014).</li><li>Powers, J. 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The authors have no conflict of interest.

Yield losses in agricultural crops due to pathogens and pests is a global problem20. Currently, application of synthetic fungicides and pesticides is the predominant means for controlling plant pathogens and pests, but residual toxicity of these biochemicals in food and feed can pose serious threat to human and animal health21. Considering the economic importance of maize as a food and feed crop, reduction in or elimination of aflatoxin contamination is of utmost importance...

Mycotoxin contamination by the fungal genera, Aspergillus, Fusarium, Penicillium, and Alternaria is a major problem of food and feed crops grown worldwide1,2,3. Among these phytopathogenic fungi, Aspergillus has the highest adverse impact on crop value and human and animal health. Aspergillus flavus (A. flavus) is an opportunistic plant pathogen that infects oil rich crops such as maize, cottonseed and peanut and produces the potent carcinogens, aflatoxins, as well as numerous toxic secondary metabolites (SMs). Maize is an important food and feed crop grown worldwide and is highly susceptible to contamination by A. flavus. The economic impact of aflatoxin contamination on loses and reduced value in maize can be as much as $686.6 million/year in the U.S.2 With predicted changes in global climate, the impact of aflatoxins could result in greater economic losses in maize with estimates as high as $1.68 billion/year in the near future2. Given the adverse economic and health effects of aflatoxins in humans and livestock, pre-harvest aflatoxin control in maize might be the most efficient way to prevent aflatoxin contamination in food and feed products.
The major pre-harvest control approach for aflatoxin resistance in maize that has been used extensively in the last few decades is primarily through breeding, which requires a significant amount of time4. Recently, biocontrol has had some success in aflatoxin reduction in large scale field applications5,6. Besides biocontrol, application of cutting-edge molecular tools such as &#8216;Host Induced Gene Silencing&#8217; (HIGS) through RNAi and transgenic expression of resistance-associated proteins has had some success in reduction of A. flavus growth and aflatoxin production in small scale laboratory and field studies. These approaches are currently being optimized in addition to identifying new potential A. flavus gene targets for future manipulation.
Besides genes that are directly involved in mycotoxin production as potential targets of transgenic control strategies, fungal amylases have been shown to play a critical role in maintaining successful pathogenesis and mycotoxin production during early stages of host plant infection. A few examples include Pythium pleroticum (causal agent of ginger rhizome rot), Fusarium solani (causal agent of cauliflower wilt), where positive correlations between pathogenicity and &#945;-amylase expression and activity were observed7,8. Inhibition of &#945;-amylase activity either through gene knockout or knockdown approaches negatively affects fungal growth and toxin production. An &#945;-amylase knockout mutant of A. flavus was unable to produce aflatoxins when grown on starch substrate or degermed maize kernels9. Similarly, in Fusarium verticillioides an &#945;-amylase knockout strain failed to produce fumonisin B1 (mycotoxin) during infection of maize kernels10. In a more recent study, Gilbert et al. (2018) demonstrated that an RNAi-based knock down of A. flavus &#945;-amylase expression through HIGS significantly reduced A. flavus growth and aflatoxin production during maize kernel infection11.
Specific inhibitors of &#945;-amylase activity have also produced similar results as obtained from down-regulation of &#945;-amylase expression. The first report on the role of an &#945;-amylase inhibitor in fungal resistance came from the isolation and characterization of a 14-kDa trypsin-&#945;-amylase inhibitor from maize lines resistant to A. flavus12. Further screening of several hundreds of plant species by Fakhoury and Woloshuk led to the identification of a 36-kDa &#945;-amylase inhibitor-like protein (AILP) from the seeds of hyacinth beans, Lablab purpureus L.13. The peptide sequence of AILP resembled lectins belonging to the lectin&#8211;arcelin&#8211;&#945;-amylase inhibitor family reported in common bean14,15. Purified AILP does not exhibit any inhibitory activity towards mammalian trypsin and further in vitro characterization showed significant inhibition of A. flavus growth and conidial germination13. The reports presented here clearly shows &#945;-amylase can serve as a target in control approaches to restrict pathogens or pests that depend on starch mobilization (through &#945;-amylase activity) and acquisition of soluble sugars as an energy source during their pathogenic interaction with host plants.
Alpha-amylase is known to be critical in A. flavus pathogenicity9,10,11, and given the importance of AILP as a potent anti-A. flavus agent (&#945;-amylase inhibition/antigrowth)13, we generated transgenic maize plants expressing Lablab AILP gene under the constitutive CaMV 35S promoter. The goal was to investigate if heterologous expression of this &#945;-amylase inhibitor in maize is effective against A. flavus pathogenesis and aflatoxin production during maize kernel infection. Our results demonstrate that transgenic maize kernels expressing AILP significantly reduced A. flavus growth and aflatoxin production during kernel infection.

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		<tr>
			<td>C1000 Touch CFX96 Real-Time System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;Falcon Tissue Culture Dishes, 60 mm</td>
			<td>Fisher Scientific</td>
			<td>08-772F</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf&#160;5424 Microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask with stopper, 50 mL</td>
			<td>Ace Glass</td>
			<td>6999-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FluoroQuant Afla</td>
			<td>Romer Labs</td>
			<td>COKFA1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluted Qualitative Filter Paper Circles, 15 cm</td>
			<td>Fisher Scientific</td>
			<td>09-790-14E</td>
			<td></td>
		</tr>
		<tr>
			<td>Force Air Oven</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FQ-Reader</td>
			<td>Romer Labs</td>
			<td>EQFFM3010</td>
			<td></td>
		</tr>
		<tr>
			<td>Geno/Grinder 2010</td>
			<td>OPS Diagnostics</td>
			<td>SP 2010-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Innova 44 Incubator Shaker</td>
			<td>Brunswick Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>1708890</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low Form Griffin Beakers, 100 mL</td>
			<td>DKW Life Sciences</td>
			<td>14000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Chloride</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nexttec 1-step DNA Isolation Kit for Plants</td>
			<td>Nexttec</td>
			<td>47N</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse E600 microscope with Nikon DS-Qi1 camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon SMZ25 stereomicroscope with C-HGFI Episcopic Illuminator and Andor Zyla 4.2 sCMOS camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Square BioAssay Dishes</td>
			<td>ThermoFisher Scientific</td>
			<td>240835</td>
			<td></td>
		</tr>
		<tr>
			<td>Phire Plant Direct PCR Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>F130WH</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate Vials, 15 ml</td>
			<td>OPS Diagnostics</td>
			<td>PCRV 15-100-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato Dextrose Broth</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Snap Cap, 22 mm</td>
			<td>DKW Life Sciences</td>
			<td>242612</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrum Plant Total RNA Kit</td>
			<td>Sigma-Aldrich</td>
			<td>STRN50</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Grinding Balls, 3/8&#39;&#39;</td>
			<td>OPS Diagnostics</td>
			<td>GBSS 375-1000-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir Plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy 4 Fluorometer</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T-9284</td>
			<td></td>
		</tr>
		<tr>
			<td>V8 juice</td>
			<td>Campbell&#39;s</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Qualitative Grade Plain Sheets, Grade 3</td>
			<td>Fisher Scientific</td>
			<td>09-820P</td>
			<td></td>
		</tr>
		<tr>
			<td>Wrist-Action Shaker</td>
			<td>Burrell Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

inhibition of aspergillus flavus growth and aflatoxin production in transgenic maize expressing the &#945;-amylase inhibitor from lablab purpureus l.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of &#945;-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. &#945;-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus &#945;-amylase enzyme and belongs to the lectin&#8211;arcelin&#8211;&#945;-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus &#945;-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values.&#160; The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%&#8211;72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%&#8211;88% reduction in aflatoxin levels.

Maize transformation and molecular screening of transgenic plants
Immature embryos of maize Hi-II lines were transformed using Agrobacterium tumefaciens EHA101 strain containing the final plant destination vector expressing the Lablab purpureus AILP gene under the control of CaMV 35S promoter. Five independently transformed maize lines were advanced to the T6 generation for su...

Watch this Scientific Journal Video about Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the Î±-amylase Inhibitor from Lablab purpureus L. at JoVE.co

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &amp;#945;-amylase Inhibitor from Lablab purpureus L.

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.&#160; Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are ...

Here we present a simple kernel screening assay to analyze Aspergillus flavus growth and Aflatoxin production in maize kernels expressing an antifungal protein. Using a green fluorescent protein expressing Aspergillus flavus strain, we monitor the infection and spread of the fungus in mature kernels in real-time. This simple assay provides a reliable means of evaluating the maize kernel's ability to resist Aspergillus flavus infection and Aflatoxin production.Results from this lab assay performed under controlled conditions on mature kernels correlates well with results from infection under field conditions. Demonstrating the procedure will be Dr.Rajtilak Majumdar, a postdoc from our laboratory, along with Christine Sickler and David Ambrosio, biological science technicians also from our lab. To begin, glue 20 two mm snap caps in a Petri dish to create KSA caps.Let the glue dry for 48 hours before using the caps. In a biological safety cabinet spray a square bioassay tray with 70%ethanol in water. Let the tray air dry.Add sterile chromatography paper to the dry tray. Spray nine of the KSA caps with 70%ethanol and let them air dry. Then place the caps on the bioassay tray.For each transgenic maize line being tested, select 20 undamaged kernels and place them into a 50 ml centrifuge tube. Using a tabletop stereo microscope select only undamaged kernels for the assay. Damaged kernels are more susceptible for Aspergillus attack and produce more Aflatoxin, thereby skewing the results and not representing the true antifungal or antimicrobial traits of the variety.Add 70%ethanol to each tube. Let the kernels sterilize for four minutes while occasionally gently shaking them. After this gently pour off the ethanol and gently rinse the kernels in sterile deionized water three times.Obtain a six day culture of Aspergillus flavus stain 70 expressing GFP grown on V8 medium. Add 20 ml of sterile 0.02%Triton X-100 in deionized water. And use a sterile loop to scrape off the spores.Pipette off the inoculum and transfer it to a 300 ml sterile beaker. Prepare a five-fold dilution with 0.02%Triton X-100 and use a hemocytometer to perform a spore count. Dilute again if necessary to obtain 100 ml with a concentration of four million spores per ml.Pour off the inoculum into an empty beaker. Place the kernels into a sterile 300 ml beaker with a stir bar and stir for three minutes. Using forceps transfer the kernels to the bioassay dish, making sure to only place one kernel into each cap.Add 30 ml of deionized water to the bottom of each bioassay tray. And incubate at 31 degrees Celsius for seven days. After seven days of inoculation with A.flavus, take pictures of four kernels that were designated for microscopic analysis and photography.Then use a soft tissue and deionized water to clean the exterior of kernels. Perform longitudinal sections of kernels and immediately take photographs under the fluorescent microscope. To prepare the kernels for analysis, clean the outside of the remaining kernels with a soft tissue and deionized water.Place four kernels, which constitute one rep, into a 15 ml screw cap polycarbonate vial containing two stainless steel balls. Immediately freeze the vials in liquid nitrogen and store at 80 degrees Celsius until ready to proceed with further processing and analysis. When ready, remove the kernels from the freezer and use a homogenizer to grind them at 1500 rpm for three minutes.Use the same ground materials for GFP analysis, Aflatoxin determination, and molecular analysis, so that all values are representative of the samples. While a minimum of three replicates are recommended, more is always better depending on the availability of good quality kernels. In this study immature embryos of maize HI-II lines are transformed using Agrobacterium tumefaciens EHA101 strain containing the final plant destination vector expressing the Lablab purpureus AILP gene under the control of the 35S promoter from the cauliflower mosaic virus.PCR amplification of the target AILP gene showed a 548 bp amplicon observed only in the transgenic maize lines. The AILP gene showed expression in the transgenic lines whereas no AILP expression was observed in the control plants. Early stage germinating spores are exposed to crude leaf extracts from young transgenic maize plants for 20 hours.High full length from spores exposed to transgenic leaf extracts show a 58 to 80%reduction compared to the negative control. The A.flavus strain used contains a GFP reporter which enables the monitoring and quantification of fungal growth in real-time. Significant reduction in GFP fluorescence is observed in transgenic AILP kernels compared to the isogenic negative control.A significant reduction is seen in fungal growth for AILP lines four and five, 69%and 72%respectively. The other lines show a reduction of 35 to 60%reduction in comparison to the control kernels. A reduction of 62 to 88%in Aflatoxin content was observed in the AILP kernels as compared to the control.Line four is seen to have the lowest Aflatoxin content at 486 ng per g, followed by other lines ranging which had contents ranging between 640 and 1498 ng per g in the kernels. Following this procedure, additional methods can be performed such as using a fluorometer to quantify GFP and as its fungal growth. Immunobased commercial kits or HPLC or EPLC can be used to perform Aflatoxin quantitation.The availability of KSA provides a quick and simple means of determining resistance to Aflatoxin contamination. The of different maize breeding lines can be assessed quickly using this technique. While the kernel screening assay is not hazardous, the operator should protect himself or herself from exposure to Aspergillus flavus spores, use proper protective equipment.

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10.4K Views.  USDA-ARS, Food and Feed Safety Research Unit. Here we present a simple kernel screening assay to analyze Aspergillus flavus growth and Aflatoxin production in maize kernels expressing an antifungal protein. Using a green fluorescent protein expressing Aspergillus flavus strain, we monitor the infection and spread of the fungus in mature kernels in real-time. This simple assay provides a reliable means of evaluating the maize kernel's ability to resist Aspergillus flavus infection and Aflatoxin production.Results from this lab assa...

Video: Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.