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

Abstract

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.

Putative Core Transcription Factors Affecting Virulence in Aspergillus flavus during Infection of Maize

“…Two separate KIAs were conducted, both based on modification of the kernel screening assay (KSA) described previously by Gilbert et al [ 8 ] and Castano-Duque et al [ 11 ]. The first KIA to obtain material for RNA sequencing was conducted as described [ 11 ].…”

“…Two separate KIAs were conducted, both based on modification of the kernel screening assay (KSA) described previously by Gilbert et al [ 8 ] and Castano-Duque et al [ 11 ]. The first KIA to obtain material for RNA sequencing was conducted as described [ 11 ]. Briefly, healthy kernels were sterilized with 70% ethanol and soaked in an inoculum of 4 × 10 6 fresh spores/mL of A. flavus NRRL 3357 for 5 min.…”

“…RNA extraction was conducted as described in Castano-Duque et al [ 11 ]. Briefly, kernel samples were ground with 0.5 mm diameter zirconia-silica beads (BioSpec Products, Bartlesville, OK, USA) using a TissueLyser II (Qiagen, Germantown, MD, USA).…”

“…RNA-sequencing of pathogen-susceptible and -resistant maize kernels undergoing infection has provided insight into flavonoid-pathway genes in maize related to host resistance [ 11 ]. In this study, using the same experimental basis, we conducted bioinformatic analysis of fungal genes during maize infection to identify core transcription factors potentially driving virulence-related gene networks.…”

Comprehensive meta-analysis of QTL and gene expression studies identify candidate genes associated with Aspergillus flavus resistance in maize

“…Primers for 12 SSR markers closest to the MQTLs that colocalized with GWAS SNPs (except MQTL2.4 and MQTL8.2) were custom synthesized at Integrated DNA Technologies Inc. ( www.idtdna.com ). Based on the reports on the resistance response of maize genotypes to A. flavus ( Busboom and White, 2004 ; Brooks et al., 2005 ; Menkir et al., 2006 ; Bello, 2007 ; Alwala et al., 2008 ; Warburton et al., 2009 ; Warburton et al., 2011 ; Willcox et al., 2013 ; Williams et al., 2015 ; Brown et al., 2016 ; Smith et al., 2019 ; Womack et al., 2020 ; Castano-Duque et al., 2021 ; Ogunola et al., 2021 ), 14 resistant and eight susceptible maize genotypes were selected for MQTL/marker validation.…”