Dataset Information

The Pathogenesis-Related Maize Seed (PRms) Gene Plays a Role in Resistance to Aspergillus flavus Infection and Aflatoxin Contamination.

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.

SUBMITTER: Majumdar R

PROVIDER: S-EPMC5651032 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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The Pathogenesis-Related Maize Seed (PRms) Gene Plays a Role in Resistance to Aspergillus flavus Infection and Aflatoxin Contamination.

Majumdar Rajtilak R Rajasekaran Kanniah K Sickler Christine C Lebar Matthew M Musungu Bryan M BM Fakhoury Ahmad M AM Payne Gary A GA Geisler Matt M Carter-Wientjes Carol C Wei Qijian Q Bhatnagar Deepak D Cary Jeffrey W JW

Frontiers in plant science 20171017

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 res ...[more]

PMID: 29089952

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Project description:Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus infection and subsequent contamination with aflatoxins, the most potent naturally produced carcinogenic secondary metabolites. This pathogen can pose serious health concerns and cause severe economic losses due to the Food and Drug Administration (FDA) regulations on permissible levels of aflatoxins in food and feed. Although biocontrol has yielded some successes in managing aflatoxin contamination, enhancing crop resistance is still the preferred choice of management for long-term sustainability. Hence, host induced gene silencing (HIGS) strategy was explored in this study. The A. flavus gene aflM encoding versicolorin dehydrogenase, a key enzyme involved in the aflatoxin biosynthetic pathway, was selected as a possible target for suppression through HIGS. An RNAi vector containing a portion of the aflM gene was constructed and introduced into immature B104 maize zygotic embryos through Agrobacterium transformation. PCR analysis of the genomic DNA from T0 leaf tissue confirmed the presence of the transgene in six out of the seven events. The seeds from the lines that showed reduced aflatoxin production in laboratory aflatoxin kernel screening assay (KSA) have been increased from T1 to T4 generation in the past four years. Changes in aflatoxin resistance in these transgenic kernels have been evaluated under both field and laboratory conditions. The T2 generation kernels containing the transgene from two events out of four examined had less aflatoxin (P ≤ 0.01 and P ≤ 0.08) than those without the transgene. Field-inoculated homozygous T3 and T4 transgenic kernels also revealed lower levels of aflatoxins (P ≤ 0.04) than kernels from the null (segregated non-transgenic samples) or B104 controls. A similar result was observed when the harvested T3 and T4 homozygous transgenic kernels were evaluated under KSA conditions without inoculation (P ≤ 0.003-0.05). These two events were crossed with LH195, LH197, LH210, and PHW79 elite breeding lines and the resulting crosses supported less aflatoxin (P ≤ 0.02) than the crosses made with non-transgenic lines. In addition, significantly higher levels of aflM gene-specific small RNAs were detected in the transgenic leaf and kernel tissues, indicating that the enhanced aflatoxin resistance in the homozygous transgenic kernels is likely due to suppression of aflM expression through HIGS.

Project description:BackgroundAspergillus flavus infection and aflatoxin contamination of maize pose negative impacts in agriculture and health. Commercial maize hybrids are generally susceptible to this fungus. Significant levels of host plant resistance have been observed in certain maize inbred lines. This study was conducted to identify maize genes associated with host plant resistance or susceptibility to A. flavus infection and aflatoxin accumulation.ResultsGenome wide gene expression levels with or without A. flavus inoculation were compared in two resistant maize inbred lines (Mp313E and Mp04:86) in contrast to two susceptible maize inbred lines (Va35 and B73) by microarray analysis. Principal component analysis (PCA) was used to find genes contributing to the larger variances associated with the resistant or susceptible maize inbred lines. The significance levels of gene expression were determined by using SAS and LIMMA programs. Fifty candidate genes were selected and further investigated by quantitative RT-PCR (qRT-PCR) in a time-course study on Mp313E and Va35. Sixteen of the candidate genes were found to be highly expressed in Mp313E and fifteen in Va35. Out of the 31 highly expressed genes, eight were mapped to seven previously identified quantitative trait locus (QTL) regions. A gene encoding glycine-rich RNA binding protein 2 was found to be associated with the host hypersensitivity and susceptibility in Va35. A nuclear pore complex protein YUP85-like gene was found to be involved in the host resistance in Mp313E.ConclusionMaize genes associated with host plant resistance or susceptibility were identified by a combination of microarray analysis, qRT-PCR analysis, and QTL mapping methods. Our findings suggest that multiple mechanisms are involved in maize host plant defense systems in response to Aspergillus flavus infection and aflatoxin accumulation. These findings will be important in identification of DNA markers for breeding maize lines resistant to aflatoxin accumulation.

Project description:Aspergillus flavus is a soil-borne saprophyte and an opportunistic pathogen of both humans and plants. This fungus not only causes disease in important food and feed crops such as maize, peanut, cottonseed, and tree nuts but also produces the toxic and carcinogenic secondary metabolites (SMs) known as aflatoxins. Polyamines (PAs) are ubiquitous polycations that influence normal growth, development, and stress responses in living organisms and have been shown to play a significant role in fungal pathogenesis. Biosynthesis of spermidine (Spd) is critical for cell growth as it is required for hypusination-mediated activation of eukaryotic translation initiation factor 5A (eIF5A), and other biochemical functions. The tri-amine Spd is synthesized from the diamine putrescine (Put) by the enzyme spermidine synthase (Spds). Inactivation of spds resulted in a total loss of growth and sporulation in vitro which could be partially restored by addition of exogenous Spd. Complementation of the Δspds mutant with a wild type (WT) A. flavus spds gene restored the WT phenotype. In WT A. flavus, exogenous supply of Spd (in vitro) significantly increased the production of sclerotia and SMs. Infection of maize kernels with the Δspds mutant resulted in a significant reduction in fungal growth, sporulation, and aflatoxin production compared to controls. Quantitative PCR of Δspds mutant infected seeds showed down-regulation of aflatoxin biosynthetic genes in the mutant compared to WT A. flavus infected seeds. Expression analyses of PA metabolism/transport genes during A. flavus-maize interaction showed significant increase in the expression of arginine decarboxylase (Adc) and S-adenosylmethionine decarboxylase (Samdc) genes in the maize host and PA uptake transporters in the fungus. The results presented here demonstrate that Spd biosynthesis is critical for normal development and pathogenesis of A. flavus and pre-treatment of a Δspds mutant with Spd or Spd uptake from the host plant, are insufficient to restore WT levels of pathogenesis and aflatoxin production during seed infection. The data presented here suggest that future studies targeting spermidine biosynthesis in A. flavus, using RNA interference-based host-induced gene silencing approaches, may be an effective strategy to reduce aflatoxin contamination in maize and possibly in other susceptible crops.

Dataset Information

The Pathogenesis-Related Maize Seed (PRms) Gene Plays a Role in Resistance to Aspergillus flavus Infection and Aflatoxin Contamination.

Publications

The Pathogenesis-Related Maize Seed (<i>PRms</i>) Gene Plays a Role in Resistance to <i>Aspergillus flavus</i> Infection and Aflatoxin Contamination.

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