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

Summary

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.  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.

Abstract

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 α-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. α-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus α-amylase enzyme and belongs to the lectin–arcelin–α-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 α-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.  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%–72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%–88% reduction in aflatoxin levels.

Introduction

Mycotoxin contamination by the fungal genera, Aspergillus, Fusarium, Penicillium, and Alternaria is a major problem of food and feed crops grown worldwide^1,2,3. 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 applications^5,6. Besides biocontrol, application of cutting-edge molecular tools such as ‘Host Induced Gene Silencing’ (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 α-amylase expression and activity were observed^7,8. Inhibition of α-amylase activity either through gene knockout or knockdown approaches negatively affects fungal growth and toxin production. An α-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 α-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 α-amylase expression through HIGS significantly reduced A. flavus growth and aflatoxin production during maize kernel infection¹¹.

Specific inhibitors of α-amylase activity have also produced similar results as obtained from down-regulation of α-amylase expression. The first report on the role of an α-amylase inhibitor in fungal resistance came from the isolation and characterization of a 14-kDa trypsin-α-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 α-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–arcelin–α-amylase inhibitor family reported in common bean^14,15. 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 α-amylase can serve as a target in control approaches to restrict pathogens or pests that depend on starch mobilization (through α-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 pathogenicity^9,10,11, and given the importance of AILP as a potent anti-A. flavus agent (α-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 α-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.

Protocol

1. Plasmid constructs and maize transformation

1. PCR amplify Lablab AILP insert using the primers 5’-TATCTAGAACTAGTGATTACCATGGCTCC-3’ and 5'-ATACTGCAGGATTGCATGCAGAGTAGTACTG-3'. The PCR conditions include an initial denaturation step at 98 °C for 30 s (step 1), followed by denaturation at 98 °C for 10 s (step 2), annealing at 55 °C for 30 s (step 3), elongation at 72 °C for 20 s (step 4), 31 cycles of step 2 to step 4, and a final elongation step at 72 °C for 5 min. Clone the PCR product into a modified pCAMBIA 1,300 vector using XbaI and PstI restriction sites. Sequence the final plant destination vector to confirm the orientation and sequence of the cloned AILP gene in the vector.
2. Transform Agrobacterium strain EHA101 with the final vector construct (Figure 1) as earlier described ¹⁶.
3. Use transformed Agrobacterium containing the final plant destination vector to transform immature maize (Zea mays L. Hi-II) embryos (performed at the Plant Transformation Facility of Iowa State University) ¹⁶.
4. Grow T₀ plants in the greenhouse (26–29 °C; 16/8 h photoperiod supplemented with High Pressure Na-lamps) and repeatedly self-pollinate to obtain T₆ generation to achieve homozygosity for the transgenic trait.

2. Spore germination assay

1. Harvest leaf samples and store at -80 °C. Grind leaf samples with a mortar and pestle using liquid nitrogen. Weigh out 0.5 g in 2 mL microcentrifuge tubes, add 17 µL of protease inhibitor, and place tubes on ice.
2. Centrifuge tubes for 10 min at 16,000 x g. Transfer 225 µL of plant extract to 0.5 mL microcentrifuge tubes and place on ice.
3. In a 15 mL centrifuge tube, prepare a 5 mL spore suspension of Aspergillus flavus 70 – GFP in 1% potato dextrose broth (w/v). Vortex and adjust spore concentration to 10⁵ spores/mL with a haemocytometer.
   CAUTION: A. flavus produces aflatoxins, all work with this fungus should be conducted in a biological safety cabinet.
4. Incubate in PDB at 28 °C until culture achieves 50% initiation of spore germination as indicated by bulging of spores.
5. Vortex and aliquot 25 µL spore solution to the 225 µL plant extract. Incubate samples for 20 hours at 28 °C with intermittent shaking.
6. Aliquot 25 µL of spore solution on a microscope slide and measure the length of germ tubes spores using the digital camera software. Use a minimum of 20 replicate measurements for each line.
   NOTE: At this stage, place all cultures at 4 °C to stop colony growth until ready to count.

3. Kernel Screening Assay (KSA)

1. Construct KSA caps by gluing 4 snap caps (22 mm) in a 60 x 15 mm petri dish. Allow glue to dry for 48 H before using caps. Each KSA cap constitutes one rep. (Figure 2).
2. In a sterile biological safety cabinet, spray a square bioassay tray (24 cm x 24 cm) with 70% ethanol:H₂O (v/v) and let air dry. Add sterile chromatography paper to the tray. Spray 9 KSA caps with 70% ethanol, let air dry, and place in the bioassay tray.
3. For each transgenic maize line being tested, select 20 undamaged kernels and place in a 50 mL centrifuge tube. Add 70% ethanol and let sit for 4 minutes. Gently shake tubes during sterilization. Pour off the ethanol and rinse kernels three times in sterile deionized H₂O.
4. Prepare a spore suspension of Aspergillus flavus 70 – GFP¹⁷ from a 6 day old culture grown on V8 medium (5% V8 Juice, 2% agar, pH 5.2).
   1. Add 20 mL sterile 0.02% Triton X-100/deionized H₂O (v/v) and scrap off spores with a sterile loop. Pipet off the inoculum and place in a 300 mL sterile beaker.
   2. Prepare a 5-fold dilution with 0.02% Triton X-100, then perform a spore count with a haemocytometer. Dilute again if necessary to obtain 100 mL with a concentration of 4 x 10⁶ spores/mL.
5. Place kernels in a sterile 300 mL beaker with a stir bar. Add inoculum to the beaker.
   CAUTION: Adding kernels to inoculum will cause solution to splash. Place beaker on a stir plate for 3 minutes. After 3 minutes, pour off inoculum into an empty beaker.
6. Using a forceps, place kernels in the bioassay dish (1 kernel/cap). Add 30 mL deionized water to the bottom of each bioassay tray and incubate at 31 °C in the dark for 7 days.
7. Prepare kernels for photography.
   1. Set aside 4 kernels from each line for microscopic analysis and photography.
      NOTE: Kernels can be stored at 4 °C for no longer than 5 days before completing photography.
   2. Take pictures of kernels after seven days of inoculation with A. flavus (Figure 3).
   3. Clean exterior of kernels with a soft tissue and deionized water. Perform longitudinal sections of kernels and immediately take photographs under the fluorescent microscope.
8. Prepare kernels for analysis.
   1. Clean exterior of remaining kernels with a soft tissue and deionized water.
   2. Place 4 kernels, constituting 1 rep, in a 15 mL screw cap polycarbonate vial containing 2 stainless steel balls. Immediately freeze vials in liquid nitrogen and store at -80 °C until further processing and analysis.
   3. Remove vials from -80 °C freezer and grind kernels in a homogenizer at 1,500 rpm for 3 minutes.
      NOTE: Keep kernels frozen with liquid nitrogen.

4. PCR screening of transgenic maize kernels

1. Use DNA isolation kit to isolate genomic DNA (gDNA) from pulverized maize kernels infected with A. flavus according to the manufacturer’s instructions. Briefly, add 280 µL buffer F, 20 µL protease, and 3 µL dithiothreitol (DTT) to 10-15 mg of pulverized maize kernels. Incubate and shake in a thermomixer (56 °C, 1,200 rpm, 30 min). Centrifuge at 10,000 x g for 1 min and transfer the clear supernatant to equilibrated column. Incubate at room temperature for 3 min and centrifuge at 700 x g for 1 min to elute gDNA.
2. Take 0.8 µL of extracted gDNA to set up a 20 µL PCR reaction for each sample using a PCR kit. Follow thermocycling conditions as recommended in the manufacturer’s protocol. The PCR conditions include an initial denaturation step at 98 °C for 5 min (step 1) followed by denaturation at 98 °C for 5 s (step 2), annealing at 55 °C for 5 s (step 3), elongation at 72 °C for 20 s (step 4), 40 cycles of step 2 to step 4, and a final elongation step at 72 °C for 1 min (step 5).
3. Use forward primer 5’-TATACCACCCCCATCCGTGT-3’ and reverse primer 5’-AGCTCGGAAGCAAAAGACCA-3’ to confirm the presence of the Lablab AILP gene in the transgenic maize kernels.

5. RNA isolation, cDNA synthesis, and semi-quantitative RT-PCR

1. Take homogenized A. flavus infected maize kernels previously stored at -80°C for RNA extraction. Extract RNA using a total RNA isolation kit according to the manufacturer’s protocol but with slight modification¹⁸. After adding 50 mg of homogenized maize kernel powder in the extraction buffer, mix it well (without vortexing) using a 1 mL pipet tip and leave it on ice for 5-6 minutes before proceeding to subsequent steps as described in the manufacturer’s protocol.
2. Prepare cDNA using a cDNA synthesis kit according to the manufacturer’s protocol ¹⁸.
3. Use 0.5 µL of undiluted cDNA per PCR reaction for semi-quantitative RT-PCR as earlier described¹⁸. Use forward and reverse primers qAILP-F 5’-TCCCAACAAGGCAACTACTG-3’ and qAILP-R 5’- CGGTGTCGAAGAGACCTAGATA-3’ respectively to detect Lablab AILP gene expression, and forward and reverse primers qRib-F 5ʹ-GGCTTGGCTTAAAGGAAGGT-3ʹ and qRib-R 5ʹ-TCAGTCCAACTTCCAGAATGG-3ʹ respectively for expression of maize ribosomal structural gene (Rib), GRMZM2G024838¹⁹ as a house-keeping gene.

6. GFP quantitation

1. Prepare 50 mL each of 0.2 M monobasic sodium phosphate (NaH₂PO₄) and 0.2 M dibasic sodium phosphate (Na₂HPO₄·7H₂O) in deionized H₂O.
2. Make up 100 mL of pH 7.0 Sorenson’s phosphate buffer. For pH 7.0, add 19.5 mL NaH₂PO₄ stock, 30.5 mL NaHPO₄·7H₂O stock, and 50 mL deionized H₂O.
3. Weigh out 25 mg ground kernel material (fresh weight, FW) and place in a 1.0 mL microcentrifuge tube. Add 500 µL phosphate buffer to the centrifuge tube and vortex for 30 s.
4. Centrifuge samples for 15 minutes at 16,000 x g. Pipet 100 µL supernatant into a black 96 well plate. Read the samples with a fluorometer (excitation 485 nm, emission 528 nm).

7. Total aflatoxin analysis

1. Sample processing and aflatoxin extraction
   1. Dry kernel samples in a forced air oven for 2 days at 60 °C. Weigh samples and place in a 50 mL Erlenmeyer flask with a glass stopper.
   2. In a fume hood, add 25 mL methylene chloride to each flask.
      CAUTION: Methylene chloride is highly volatile and is considered hazardous (irritant, carcinogen). Neither latex nor nitrile gloves are safe for working with Methylene Chloride. Use IMPROVED organic solvent resistant PolyVinylAlcohol gloves.
   3. Shake samples for 30 min on a wrist action shaker. After 30 min, slowly pour the extract through a fluted filter paper circle into an 80 mL beaker. Let the beakers dry overnight in a fume hood.
   4. The next day, squirt methylene chloride (approximately 5 mL) around the inside rim of the dry beakers, swirl around, and pour into 2 dram glass vials. Leave vials open to dry in a fume hood overnight.
2. Aflatoxin analysis
   1. Add 4.0 mL 80% methanol:deionized H₂O (v/v) to vials.
   2. Use an aflatoxin extraction kit to filter purify methanol solution with the provided column. Analyze total aflatoxin levels with a fluorometer, according to the manufacturer’s instructions.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Agar	Caisson
Amazing Marine Goop	Eclectic Products
C1000 Touch CFX96 Real-Time System	Bio-Rad
Corning Falcon Tissue Culture Dishes, 60 mm	Fisher Scientific	08-772F
Eppendorf 5424 Microcentrifuge	Fisher Scientific
Erlenmeyer flask with stopper, 50 mL	Ace Glass	6999-10
Ethanol
FluoroQuant Afla	Romer Labs	COKFA1010
Fluted Qualitative Filter Paper Circles, 15 cm	Fisher Scientific	09-790-14E
Force Air Oven	VWR
FQ-Reader	Romer Labs	EQFFM3010
Geno/Grinder 2010	OPS Diagnostics	SP 2010-115
Improved PolyVinylAlcohol Organic Solvent Resistant Gloves	Ansell	012-15-554
Innova 44 Incubator Shaker	Brunswick Scientific
iScript cDNA Synthesis Kit	Bio-Rad	1708890
liquid Nitrogen
Low Form Griffin Beakers, 100 mL	DKW Life Sciences	14000-100
Methanol
Methylene Chloride
Nexttec 1-step DNA Isolation Kit for Plants	Nexttec	47N
Nikon Eclipse E600 microscope with Nikon DS-Qi1 camera	Nikon
Nikon SMZ25 stereomicroscope with C-HGFI Episcopic Illuminator and Andor Zyla 4.2 sCMOS camera	Nikon
Nunc Square BioAssay Dishes	ThermoFisher Scientific	240835
Phire Plant Direct PCR Kit	ThermoFisher Scientific	F130WH
Polycarbonate Vials, 15 ml	OPS Diagnostics	PCRV 15-100-23
Potato Dextrose Broth
Snap Cap, 22 mm	DKW Life Sciences	242612
Sodium Phosphate dibasic heptahydrate	Sigma-Aldrich
Sodium Phosphate monobasic	Sigma-Aldrich
Spectrum Plant Total RNA Kit	Sigma-Aldrich	STRN50
Stainless Steel Grinding Balls, 3/8''	OPS Diagnostics	GBSS 375-1000-02
Stir Plate
Synergy 4 Fluorometer	Biotek
T100 Thermal Cycler	Bio-Rad
Triton X-100	Sigma-Aldrich	T-9284
V8 juice	Campbell's
Whatman Qualitative Grade Plain Sheets, Grade 3	Fisher Scientific	09-820P
Wrist-Action Shaker	Burrell Scientific