Flavonoids Modulate the Accumulation of Toxins From Aspergillus flavus in Maize Kernels.

Aspergillus flavus is an opportunistic fungal pathogen capable of producing aflatoxins, potent carcinogenic toxins that accumulate in maize kernels after infection. To better understand the molecular mechanisms of maize resistance to A. flavus growth and aflatoxin accumulation, we performed a high-throughput transcriptomic study in situ using maize kernels infected with A. flavus strain 3357. Three maize lines were evaluated: aflatoxin-contamination resistant line TZAR102, semi-resistant MI82, and susceptible line Va35. A modified genotype-environment association method (GEA) used to detect loci under selection via redundancy analysis (RDA) was used with the transcriptomic data to detect genes significantly influenced by maize line, fungal treatment, and duration of infection. Gene ontology enrichment analysis of genes highly expressed in infected kernels identified molecular pathways associated with defense responses to fungi and other microbes such as production of pathogenesis-related (PR) proteins and lipid bilayer formation. To further identify novel genes of interest, we incorporated genomic and phenotypic field data from a genome wide association analysis with gene expression data, allowing us to detect significantly expressed quantitative trait loci (eQTL). These results identified significant association between flavonoid biosynthetic pathway genes and infection by A. flavus. In planta fungal infections showed that the resistant line, TZAR102, has a higher fold increase of the metabolites naringenin and luteolin than the susceptible line, Va35, when comparing untreated and fungal infected plants. These results suggest flavonoids contribute to plant resistance mechanisms against aflatoxin contamination through modulation of toxin accumulation in maize kernels.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the α-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 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.

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

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.

Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182.

Aspergillus flavus is an opportunistic, saprophytic fungus that infects maize and other fatty acid-rich food and feed crops and produces toxic and carcinogenic secondary metabolites known as aflatoxins. Contamination of maize with aflatoxin poses a serious threat to human health in addition to reducing the crop value leading to a substantial economic loss. Here we report designing a tachyplesin1-derived synthetic peptide AGM182 and testing its antifungal activity both in vitro and in planta. In vitro studies showed a five-fold increase in antifungal activity of AGM182 (vs. tachyplesin1) against A. flavus. Transgenic maize plants expressing AGM182 under maize Ubiquitin-1 promoter were produced through Agrobacterium-mediated transformation. PCR products confirmed integration of the AGM182 gene, while RT-PCR of maize RNA confirmed the presence of AGM182 transcripts. Maize kernel screening assay using a highly aflatoxigenic A. flavus strain (AF70) showed up to 72% reduction in fungal growth in the transgenic AGM182 seeds compared to isogenic negative control seeds. Reduced fungal growth in the AGM182 transgenic seeds resulted in a significant reduction in aflatoxin levels (76-98%). The results presented here show the power of computational and synthetic biology to rationally design and synthesize an antimicrobial peptide against A. flavus that is effective in reducing fungal growth and aflatoxin contamination in an economically important food and feed crop such as maize.

Response of mannitol-producing Arabidopsis thaliana to abiotic stress.

In celery, mannitol is a primary photosynthetic product that is associated with celery's exceptional salt tolerance. Arabidopsis plants transformed with celery's mannose-6-phosphate reductase (M6PR) gene produce mannitol and grow normally in the absence of stress. Daily analysis of the increase in growth (fresh and dry weight, leaf number, leaf area per plant and specific leaf weight) over a 12-day period showed less effect of salt (100 mm NaCl) on the M2 transformant than wild type (WT). Following a 12-day treatment of WT, M2 and M5 plants with 100 or 200 mm NaCl the total shoot fresh weight, leaf number, and leaf area were significantly greater in transformants than in WT plants. The efficiency of use of energy for photochemistry by PSII was measured daily under growth conditions. In WT plants treated with 100 mm NaCl, the PSII yield begin decreasing after 6 days with a 50% loss in yield after 12 days, indicating a severe loss in PSII efficiency; whereas, there was no effect on the transformants. Under atmospheric levels of CO2, growth with 200 mm NaCl caused an increase in the substomatal levels of CO2 in WT plants but not in transformants. It also caused a marked decrease in carboxylation efficiency under limiting levels of CO2 in WT compared with transformants. When stress was imposed and growth reduced by withholding water for 12 days, which resulted in a similar decrease in relative water content to salt-treated plants, there were no differences among the genotypes in PSII yields or growth. The results suggest mannitol, which is known to be a compatible solute and antioxidant, protects photosynthesis against salt-related damage to chloroplasts.