Rowena Y. Kelley

Identification of maize genes associated with host plant resistance or susceptibility to Aspergillus flavus infection and aflatoxin accumulation.

Background Aspergillus 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. Results Genome 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. Conclusion Maize 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.

Comparison of Aflatoxigenic and Nonaflatoxigenic Isolates of Aspergillus flavus using DNA Amplification Fingerprinting Techniques

Aspergillus flavus is a filamentous fungus that produces mycotoxins in many food and feed crops, such as maize (Zea mays L.). Isolates were analyzed for toxin production by nucleic acid profiles in an attempt to differentiate aflatoxigenic from nonaflatoxigenic isolates. A total of 41 aflatoxigenic and 34 nonalfatoxigenic isolates were included in the study. The isolates were evaluated initially using DNA amplification fingerprinting (DAF) without clear resolution of the groups. A weak association of aflatoxigenic isolates was observed, as evidenced by their clustering in 18 of 81 trees recovered from maximum parsimony analysis of binary characters derived from arbitrary signatures from amplification profiles (ASAP) data; nonaflatoxigenic isolates exhibited a pattern of paraphyletic laddering. Up to five markers unambiguously supported the aflatoxigenic isolate grouping, but the presence of alternative conflicting topologies in equally parsimonious trees precluded the observation of meaningful statistical support. With additional markers for genome of A. flavus, this method could be used to resolve toxigenic from nontoxigenic strains. This additional work could resolve aflatoxigenic isolates of A. flavus present on maize plants using ASAP, which would reduce labor intense costs and potentially lead to faster determination of resistant cultivars in breeding efforts.

Foliar herbivory triggers local and long distance defense responses in maize

Many studies have documented the induction of belowground defenses in plants in response to aboveground herbivory and vice versa, but the genes and signaling molecules mediating systemic induction are not well understood. We performed comparative microarray analysis on maize whorl and root tissues from the insect resistant inbred Mp708 in response to foliar feeding by fall armyworm (Spodoptera frugiperda) caterpillars. Although Mp708 has elevated jasmonic acid (JA) levels prior to herbivory, genes involved in JA biosynthesis were up-regulated in whorls in response to fall armyworm feeding. Alternatively, genes possibly involved in regulating ethylene (ET) perception and signaling were up-regulated in roots following foliar herbivory. Transcript levels of genes encoding proteins involved in direct defenses against herbivores were enhanced both in roots and leaves, but transcriptional factors and genes involved in various biosynthetic pathways were selectively down-regulated in the whorl. The results indicate that foliar herbivory by fall armyworm changes root gene expression pathways suggesting profound long distance signaling. Tissue specific induction and suppression of JA and ET signaling pathway genes provides a clue to their possible roles in signaling between the two distant tissue types that eventually triggers defense responses in the roots in response to foliar herbivory.

Genomic profile of maize response to Aspergillus flavus infection

The opportunistic pathogen Aspergillus flavus infects important seed crops, including corn, peanuts, and cotton. A. flavus is capable of producing mycotoxins called aflatoxins. Aflatoxin B1, the major mycotoxin contaminant of maize, is a potent carcinogen and has been directly linked to hepatocellular carcinoma. Natural sources of maize fungal resistance exist, but efforts to increase resistance through traditional plant breeding have yielded little success. Using the maize Unigene 1-1.05 arrays, a comparison of resistant (Mp313E) and susceptible (Va35) inbred maize lines 48 hours post-A. flavus infection identified 236 genes as significant. During infection, 135 genes were up-regulated in the susceptible maize line Va35, 112 genes were up-regulated in the resistant maize line Mp313E, 12 genes were up-regulated in both lines, and 1 gene was down-regulated in both lines compared to uninfected lines. Comparisons of the biological profile responses of these maize lines revealed a striking difference in reaction to infection. These identified genes will serve as the initial step for developing molecular markers to understand this complex interaction and help with introgression of A. flavus resistance into maize hybrids.

Integrated database for identifying candidate genes for Aspergillus flavus resistance in maize

BackgroundAspergillus flavus Link:Fr, an opportunistic fungus that produces aflatoxin, is pathogenic to maize and other oilseed crops. Aflatoxin is a potent carcinogen, and its presence markedly reduces the value of grain. Understanding and enhancing host resistance to A. flavus infection and/or subsequent aflatoxin accumulation is generally considered an efficient means of reducing grain losses to aflatoxin. Different proteomic, genomic and genetic studies of maize (Zea mays L.) have generated large data sets with the goal of identifying genes responsible for conferring resistance to A. flavus, or aflatoxin.ResultsIn order to maximize the usage of different data sets in new studies, including association mapping, we have constructed a relational database with web interface integrating the results of gene expression, proteomic (both gel-based and shotgun), Quantitative Trait Loci (QTL) genetic mapping studies, and sequence data from the literature to facilitate selection of candidate genes for continued investigation. The Corn Fungal Resistance Associated Sequences Database (CFRAS-DB) (http://agbase.msstate.edu/) was created with the main goal of identifying genes important to aflatoxin resistance. CFRAS-DB is implemented using MySQL as the relational database management system running on a Linux server, using an Apache web server, and Perl CGI scripts as the web interface. The database and the associated web-based interface allow researchers to examine many lines of evidence (e.g. microarray, proteomics, QTL studies, SNP data) to assess the potential role of a gene or group of genes in the response of different maize lines to A. flavus infection and subsequent production of aflatoxin by the fungus.ConclusionsCFRAS-DB provides the first opportunity to integrate data pertaining to the problem of A. flavus and aflatoxin resistance in maize in one resource and to support queries across different datasets. The web-based interface gives researchers different query options for mining the database across different types of experiments. The database is publically available at http://agbase.msstate.edu.

First Report of Plant Regeneration via Somatic Embryogenesis from Shoot Apex-Derived Callus of Hedychium muluense R.M. Smith

ABSTRACT The genus Hedychium consists of about 50 species, with increasing popularity as ornamentals and potential as medicinal crop plants, but there are no reports on somatic embryogenic regeneration of any member of this genus. The objective of this investigation was to establish an in vitro regeneration system based on somatic embryogenesis for Hedychium muluense R.M. Smith using shoot apex-derived callus. Callus was induced and proliferated on a modified Murashige and Skoog (MS) medium (CIPM) supplemented with 9.05 μM 2–4, D, and 4.6 μM kinetin. Friable callus developed into somatic embryos upon transfer to liquid medium (MS basal salts and Gamborgs vitamins) that was supplemented with 0.6 μM thidiazuron (TDZ) and 8.9 μM 6-benzylaminopurine (BA) and shaken for four weeks. The cultures were then transferred to three Hedychium embryo-development media (HEDM) of varying strengths: HEDM, 1/2 HEDM, and 1/4 HEDM. All three media contained both 0.6 μM TDZ and 8.9 μM BA. Somatic embryo production was higher in full strength HEDM, which produced an average of 103 somatic embryos/explant, half of which could be converted into shoots within a month. Regenerated shoots were readily rooted on a medium supplemented with 0.6 μM 3-indoleacetic acid (IAA) and acclimatized before transfer to the greenhouse.

Genetic Analysis of Plant Regenerability in Oats (Avena spp. L.)

Abstract The objective of this study was to estimate genetic effects for transfer of regenerability in oats. Corbit, an agronomically important oat cultivar, was crossed with the highly regenerable, but agronomically undesirable line, GP-1. Callus was induced from mature seeds of each parent (P1 and P2); F1, F2 and their reciprocals; and backcross (BC1 and BC2) generations. The number of somatic embryos was recorded before transfer to regeneration medium and the number of plants regenerated was recorded. Gene effects, using generation mean analysis, were computed when GP-1 was the maternal parent (Set 1) and when Corbit was the maternal parent (Set 2). From this study we conclude that selection for callus weight and plant number would be expected to produce only small gains per cycle because of the substantial negative d X d and dominance effects and these two traits might not be improved simultaneously when selection is practiced for one of them. However, two important characters-callus fresh weight and plant number-were positively correlated when GP-1 was chosen as the maternal parent. Therefore, back-cross strategies for improvement would need to take into consideration the direction of the cross as the highly regenerable plant characteristics observed were considerably influenced by maternal inheritance.