A Public Platform for the Verification of the Phenotypic Effect of Candidate Genes for Resistance to Aflatoxin Accumulation and Aspergillus flavus Infection in Maize

A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of selected maize gene sequences with resistance under field conditions. Resources include a database of genetic and protein sequences associated with the reduction in aflatoxin contamination from previous studies; eight diverse inbred maize lines for polymorphism identification within any maize gene sequence; four Quantitative Trait Loci (QTL) mapping populations and one association mapping panel, all phenotyped for aflatoxin accumulation resistance and associated phenotypes; and capacity for Insertion/Deletion (InDel) and SNP genotyping in the population(s) for mapping. To date, ten genes have been identified as possible candidate genes and put through the candidate gene testing pipeline, and results are presented here to demonstrate the utility of the pipeline

Identification of Maize Genes Associated with Host Plant Resistance or Susceptibility to Aspergillus flavus Infection and Aflatoxin Accumulation

infection and aflatoxin accumulation. 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. infection and aflatoxin accumulation. These findings will be important in identification of DNA markers for breeding maize lines resistant to aflatoxin accumulation

Fundamental Science and Engineering Questions in Planetary Cave Exploration

32 páginas.- 3 figuras.- 2 tablas.- 260 referenciasNearly half a century ago, two papers postulated the likelihood of lunar lava tube caves using mathematical models. Today, armed with an array of orbiting and fly-by satellites and survey instrumentation, we have now acquired cave data across our solar system-including the identification of potential cave entrances on the Moon, Mars, and at least nine other planetary bodies. These discoveries gave rise to the study of planetary caves. To help advance this field, we leveraged the expertise of an interdisciplinary group to identify a strategy to explore caves beyond Earth. Focusing primarily on astrobiology, the cave environment, geology, robotics, instrumentation, and human exploration, our goal was to produce a framework to guide this subdiscipline through at least the next decade. To do this, we first assembled a list of 198 science and engineering questions. Then, through a series of social surveys, 114 scientists and engineers winnowed down the list to the top 53 highest priority questions. This exercise resulted in identifying emerging and crucial research areas that require robust development to ultimately support a robotic mission to a planetary cave-principally the Moon and/or Mars. With the necessary financial investment and institutional support, the research and technological development required to achieve these necessary advancements over the next decade are attainable. Subsequently, we will be positioned to robotically examine lunar caves and search for evidence of life within Martian caves; in turn, this will set the stage for human exploration and potential habitation of both the lunar and Martian subsurface.The following funding sources are recognized for supporting several of the contributing authors: Human Frontiers Science Program grant #RGY0066/2018 (for AAB), NASA Innovative Advanced Concepts Grant #80HQTR19C0034 (HJ, UYW, and WLW), and European Research Council, ERC Consolidator Grant #818602 (AGF), the Spanish Ministry of Science and Innovation (project PID2019-108672RJ-I00) and the "Ramon y Cajal" post-doctoral contract (grant #RYC2019-026885-I (AZM)), and Contract #80NM0018D0004 between the Jet Propulsion Laboratory, California Institute of Technology and the National Aeronautics and Space Administration (AA, MJM, KU, and LK).Peer reviewe

Comparison of Two Inoculation Methods for Evaluating Maize for Resistance to Aspergillus flavus Infection and Aflatoxin Accumulation

Aflatoxin, the most potent carcinogen found in nature, is produced by the fungus Aspergillus flavus and occurs naturally in maize, Zea mays L. Growing maize hybrids with genetic resistance to aflatoxin contamination are generally considered a highly desirable way to reduce losses to aflatoxin. Developing resistant hybrids requires reliable inoculation methods for screening maize germplasm for resistance to A. flavus infection and aflatoxin accumulation. The side-needle technique is a widely used inoculation technique: an A. flavus conidial suspension is injected underneath the husks into the side of the ear. This wounds the ear and limits expression of resistance associated with husk coverage, pericarp thickness, and seed coat integrity. In this investigation, the side-needle technique was compared with a second inoculation method that involved dispensing wheat kernels infected with A. flavus into plant whorls at 35 and 49 days after planting. Results showed that although the side-needle technique produced higher levels of aflatoxin accumulation, differences in A. flavus biomass produced by the two inoculation techniques were not significant. Both inoculation techniques were effective in differentiating resistant and susceptible single cross hybrids irrespective of the use of A. flavus infection or aflatoxin accumulation as a basis to define resistance

Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus Strain in Contaminated Maize Using Quantitative Real-Time PCR

Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard® (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction between different maize lines and these A. flavus strains

Fungal and herbivore elicitation of the novel maize sesquiterpenoid, zealexin A4, is attenuated by elevated CO2.

Main conclusionChemical isolation and NMR-based structure elucidation revealed a novel keto-acidic sesquiterpenoid, termed zealexin A4 (ZA4). ZA4 is elicited by pathogens and herbivory, but attenuated by heightened levels of CO 2 . The identification of the labdane-related diterpenoids, termed kauralexins and acidic sesquiterpenoids, termed zealexins, demonstrated the existence of at least ten novel stress-inducible maize metabolites with diverse antimicrobial activity. Despite these advances, the identity of co-occurring and predictably related analytes remains largely unexplored. In the current effort, we identify and characterize the first sesquiterpene keto acid derivative of β-macrocarpene, named zealexin A4 (ZA4). Evaluation of diverse maize inbreds revealed that ZA4 is commonly produced in maize scutella during the first 14 days of seedling development; however, ZA4 production in the scutella was markedly reduced in seedlings grown in sterile soil. Elevated ZA4 production was observed in response to inoculation with adventitious fungal pathogens, such as Aspergillus flavus and Rhizopus microsporus, and a positive relationship between ZA4 production and expression of the predicted zealexin biosynthetic genes, terpene synthases 6 and 11 (Tps6 and Tps11), was observed. ZA4 exhibited significant antimicrobial activity against the mycotoxigenic pathogen A. flavus; however, ZA4 activity against R. microsporus was minimal, suggesting the potential of some fungi to detoxify ZA4. Significant induction of ZA4 production was also observed in response to infestation with the stem tunneling herbivore Ostrinia nubilalis. Examination of the interactive effects of elevated CO2 (E-CO2) on both fungal and herbivore-elicited ZA4 production revealed significantly reduced levels of inducible ZA4 accumulation, consistent with a negative role for E-CO2 on ZA4 production. Collectively, these results describe a novel β-macrocarpene-derived antifungal defense in maize and expand the established diversity of zealexins that are differentially regulated in response to biotic/abiotic stress

Fungal and herbivore elicitation of the novel maize sesquiterpenoid, zealexin A4, is attenuated by elevated CO2.

Main conclusionChemical isolation and NMR-based structure elucidation revealed a novel keto-acidic sesquiterpenoid, termed zealexin A4 (ZA4). ZA4 is elicited by pathogens and herbivory, but attenuated by heightened levels of CO 2 . The identification of the labdane-related diterpenoids, termed kauralexins and acidic sesquiterpenoids, termed zealexins, demonstrated the existence of at least ten novel stress-inducible maize metabolites with diverse antimicrobial activity. Despite these advances, the identity of co-occurring and predictably related analytes remains largely unexplored. In the current effort, we identify and characterize the first sesquiterpene keto acid derivative of β-macrocarpene, named zealexin A4 (ZA4). Evaluation of diverse maize inbreds revealed that ZA4 is commonly produced in maize scutella during the first 14 days of seedling development; however, ZA4 production in the scutella was markedly reduced in seedlings grown in sterile soil. Elevated ZA4 production was observed in response to inoculation with adventitious fungal pathogens, such as Aspergillus flavus and Rhizopus microsporus, and a positive relationship between ZA4 production and expression of the predicted zealexin biosynthetic genes, terpene synthases 6 and 11 (Tps6 and Tps11), was observed. ZA4 exhibited significant antimicrobial activity against the mycotoxigenic pathogen A. flavus; however, ZA4 activity against R. microsporus was minimal, suggesting the potential of some fungi to detoxify ZA4. Significant induction of ZA4 production was also observed in response to infestation with the stem tunneling herbivore Ostrinia nubilalis. Examination of the interactive effects of elevated CO2 (E-CO2) on both fungal and herbivore-elicited ZA4 production revealed significantly reduced levels of inducible ZA4 accumulation, consistent with a negative role for E-CO2 on ZA4 production. Collectively, these results describe a novel β-macrocarpene-derived antifungal defense in maize and expand the established diversity of zealexins that are differentially regulated in response to biotic/abiotic stress

Linkage map from the F_2:3 mapping population of Mp715 x T173 (n = 192) showing the position of a new QTL on chromosome 1 centered over the SNP within the sequence of GRMZM2G103668 in bin 1.05, a GH19 protein with chitinase activity.

<p>The mapping population was phenotyped and genotyped originally according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126185#pone.0126185.ref025" target="_blank">25</a>].</p

The position of single nucleotide and insertion/ deletion polymorphisms studied within the chitinase gene sequences or within 500 bp of the gene position (Adapted from gramene.org [29]). Schematics of each gene are drawn from information from the MaizeGDB [28]. Other genes in the study not shown here were mapped using further up-or down-stream markers.

<p>Footnotes: (A) GRMZM2G134251; (B) GRMZM2G103668; (C) GRMZM2G162505; (D) GRMZM2G051943; (E) GRMZM2G064360; (F) GRMZM2G145518; (G) GRMZM2G062974; (H) GRMZM2G117405. Orange section is the gene of interest. Red stars and/or arrows indicate position of SNP used. (1) S1_27303546, (2) S1_85545046, (3) S1_240766861, (4) S1_240766882, (5) S2_33534181, (6) S1_63229609, (7) S1_63229636, (8) S6_82813940, (9) S8_88812804, (10) S8_164558329; (11) S8_165558375; (12) S8_164558387. Blue Triangle indicates position of insertion/ deletion polymorphism ChiAMpVa. SNP positions provided as (Maize B73 RefGen_V2) adjusted to reflect correct position on image (Maize B73 RefGen_V3).</p

Dot matrix view of paired alignments of maize chitinase exon sequences with similar gene structures, based on the dendogram in Fig 1.

<p>1 = GRMZM2G051943 (Exon 1) vs. GRMZM2G052175 (exons 1 and 2) (86% identical); 2 = GRMZM2G051943 (Exon 1) vs. GRMZM2G051921 (exon 1) (77% identical); 3 = GRMZM2G051943 (Exon 2) vs. GRMZM2G052175 (exon 3) (85% identical); 4 = GRMZM2G051943 (Exon 2) vs. GRMZM2G051921 (exon 2) (87% identical); 5 = Exons 1 and 2 GRMZM2G412577 vs. GRMZM2G400497 (95% identical); 6 = Exons 4 and 5 of GRMZM2G099454 vs. exons 1–3 of GRMZM2G103668 (78% identical); 7 = GRMZM2G057093 VS GRMZM2G162505 (88% identical); 8 = GRMZM2G162359 VS GRMZM2G328171 (76% identical); 9 = GRMZM2G400999 VS GRMZM2G447795 (81% identical); 10 = GRMZM2G080547 VS GRMZM2G160265 (98% identical).</p