Abstract
Aspergillus flavus is an opportunistic plant pathogen that colonizes and produces the toxic and carcinogenic secondary metabolites, aflatoxins, in oil-rich crops such as maize (Zea mays ssp. mays L.). Pathogenesis-related (PR) proteins serve as an important defense mechanism against invading pathogens by conferring systemic acquired resistance in plants. Among these, production of the PR maize seed protein, ZmPRms (AC205274.3_FG001), has been speculated to be involved in resistance to infection by A. flavus and other pathogens. To better understand the relative contribution of ZmPRms to A. flavus resistance and aflatoxin production, a seed-specific RNA interference (RNAi)-based gene silencing approach was used to develop transgenic maize lines expressing hairpin RNAs to target ZmPRms. Downregulation of ZmPRms in transgenic kernels resulted in a ∼250–350% increase in A. flavus infection accompanied by a ∼4.5–7.5-fold higher accumulation of aflatoxins than control plants. Gene co-expression network analysis of RNA-seq data during the A. flavus-maize interaction identified ZmPRms as a network hub possibly responsible for regulating several downstream candidate genes associated with disease resistance and other biochemical functions. Expression analysis of these candidate genes in the ZmPRms–RNAi lines demonstrated downregulation (vs. control) of a majority of these ZmPRms-regulated genes during A. flavus infection. These results are consistent with a key role of ZmPRms in resistance to A. flavus infection and aflatoxin accumulation in maize kernels.
Highlights
Mycotoxin contamination of food and feed crops is a global problem
Based on the screening of fungal growth in the ZmPRms–RNA interference (RNAi) silenced lines, three independent ZmPRms– RNAi lines that showed higher fungal growth were selected for further investigation to understand the impact of seedspecific ZmPRms silencing on A. flavus growth and aflatoxin production
Seed-specific RNAi-mediated silencing of the native ZmPRms gene resulted in a ∼90–99% downregulation in expression of the gene (Figure 2C) in seeds of infected ZmPRms– RNAi lines compared to seeds of infected control lines
Summary
Mycotoxin contamination of food and feed crops is a global problem. Exposure to mycotoxins in humans and livestock primarily occurs through ingestion of contaminated seeds or other edible plant parts. The majority of mycotoxin contamination of crop plants is the result of infection by members of the three fungal genera, Aspergillus, Fusarium, and Penicillium among. Maize is a major crop grown worldwide and is susceptible to aflatoxin contamination by Aspergillus flavus, especially during episodes of severe drought (Kebede et al, 2012; Fountain et al, 2014). Aflatoxin contamination of maize can result in economic losses as high as US$686.6 million/year in the United States based on a recent estimation of economic losses for the year 2013 (Mitchell et al, 2016). With predicted changes in the global climate, it is estimated that aflatoxin contamination could cause losses to the maize industry ranging from US$52.1 million to US$1.68 billion/year in the United States (Mitchell et al, 2016)
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