Eric T. Johnson

Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium

Highlight Reducing the function of PAL, the first enzyme in the phenylpropanoid pathway, in Brachypodium distachyon alters cell wall composition, increases fungal susceptibility, but minimally affects caterpillar herbivory and abiotic stress tolerance.

Colored and White Sectors From Star-Patterned Petunia Flowers Display Differential Resistance to Corn Earworm and Cabbage Looper Larvae

Anthocyanins are likely a visual aid that attract pollinators. However, there is also the possibility that anthocyanins are present in some flowers as defensive molecules that protect them from excess light, pathogens, or herbivores. In this study, resistance due to anthocyanins from commercial petunia flowers (Petunia hybrida) was examined for insecticide/antifeedant activity against corn earworm (CEW, Helicoverpa zea) and cabbage looper (CL, Trichoplusia ni). The petunia flowers studied contained a star pattern, with colored and white sectors. CEW larvae ate significantly less colored sectors than white sectors in no-choice bioassays in most cases. All CEW larvae feeding on blue sectors weighed significantly less after 2xa0days than larvae feeding on white sectors, which was negatively correlated with total anthocyanin levels. CL larvae ate less of blue sectors than white sectors, and blue sectors from one petunia cultivar caused significantly higher CL mortality than white sectors. Partially purified anthocyanin mixtures isolated from petunia flowers, when added to insect diet discs at approximately natural concentrations, reduced both CEW and CL larva weights compared to the controls. These studies demonstrate that the colored sectors of these petunia cultivars slow the development of these lepidopteran larvae and indicate that anthocyanins play some part in flower defense in petunia.

Differential resistance of switchgrass Panicum virgatum L. lines to fall armyworms Spodoptera frugiperda (J. E. Smith)

Six cultivars of switchgrass Panicum virgatum L., a plant native to North America that has potential as a bioenergy source, were evaluated for resistance to feeding by the fall armyworm Spodoptera frugiperda (J. E. Smith). Although no mortality was noted, seedlings of the cultivar ‘Trailblazer’ and older plants of the cultivar ‘Blackwell’ were among the most resistant to feeding by S. frugiperda. Some field-collected samples from natural habitat were fed upon by S. frugiperda as readily as were the cultivars, while others caused high mortality after 2xa0days. Enzyme assays indicated relative differences in expression of two peroxidases thought to be involved in insect resistance in maize, but not in two chitinolytic enzymes. Genomic searches based on maize-sequence templates for the aforementioned genes identified homologs in switchgrass. Sequencing of cDNA coding for these genes identified some differences, especially in the cationic peroxidase, which could influence relative activity. These results indicate switchgrass germplasm has varying resistance to fall armyworms which could be a function of gene sequence diversity, as well as of variation in gene expression due to differences in ploidy levels or other factors.

Differential Activity of Multiple Saponins Against Omnivorous Insects with Varying Feeding Preferences

A variety of glycosylated and unglycosylated saponins from seven different plant families (Aquifoliaceae, Asparagaceae, Caryophyllaceae, Dioscoreaceae, Leguminosae, Rosaceae, Sapindaceae) were tested against the corn earworm, Helicoverpa zea, and the fall armyworm, Spodoptera frugiperda. The corn earworm feeds readily on both monocots and dicots, while the fall armyworm is primarily a grass feeder. Most of the saponins were similarly effective or ineffective against both insect species, with the glycosides being the primary active form (compared to aglycones). However, one aglycone possessed antifeedant properties toward the fall armyworm. Thus, in contrast to many plant secondary metabolites effective against either of these two species where the aglycone is more effective, in the case of the saponins the opposite is generally true. This appears to be a contradictory strategy of plant defenses that requires further consideration. The activity of protodioscin against insects is reported for the first time and may be important in insect defense by the bioenergy crop switchgrass.

Identification and properties of insect resistance-associated maize anionic peroxidases

Previous studies with transgenic plants have indicated a tobacco anionic peroxidase can confer enhanced resistance to a variety of insects when expressed in different plant species. Tissue that expresses high levels of this enzyme often browns rapidly when damaged. Maize roots damaged under sterile conditions browned and had an anionic peroxidase induced. When introduced biolistically, maize callus transformants expressing a maize peroxidase gene with a predicted isoelectric point of ca. 5.1 produced browner callus compared to a corresponding beta-glucuronidase (GUS) transformant as callus aged. Higher production of only one isozyme of ca. pI 4.5 was noted. When the callus was fed to two maize pest caterpillar species, growth rates were slower (as reflected by weights) relative to the GUS callus. Based on examination of published information and electrophoretic properties, this gene appears to code for Px11, a peroxidase isozyme that is primarily produced in root tissue and callus. When sequence of the gene in several inbreds was examined, coding variations were noted, and abilities to utilize ferulic and p-coumaric acids differed. These coding differences may influence the ability of corresponding forms of the peroxidase to promote resistance. In addition to potential use in marker assisted breeding, enhanced expression of this anionic peroxidase through breeding or genetic engineering may lead to enhanced insect or disease resistance.

Comparative transcription profiling analyses of maize reveals candidate defensive genes for seedling resistance against corn earworm

As maize seedlings germinate into the soil, they encounter an environment teeming with insects seeking rich sources of nutrition. Maize presumably has developed a number of molecular mechanisms to ensure survival at the beginning of its life cycle. Comparative transcription analysis using microarrays was utilized to document the expression of a number of genes with potential defensive functions in seedling tissue. In addition to elevated levels of the genes involved in the biosynthesis of DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), an anti-insect resistance molecule, other highly expressed genes in the seedling encode the following putative defensive proteins: defensin, hydroxyproline and proline-rich protein, thaumatin-like protein, lipase, cystatin, protease inhibitor, and a variety of proteases. The potential resistance genes identified occurred mainly on chromosomes 1 and 5 in the B73 genome. Analysis of promoters of seven DIMBOA biosynthetic genes identified three transcription factor binding sites that are possibly involved in regulation of the DIMBOA biosynthetic pathway. The results indicate that maize employs a wide variety of potential resistance mechanisms in seedling tissue to resist a possible insect attack.

Field incidence of mycotoxins in commercial popcorn and potential environmental influences

Popcorn ear damage by insects and mycotoxin levels in kernels were monitored in several commercial popcorn fields in central Illinois over a 4-year period. Aflatoxin was rare, but fumonisin and deoxynivalenol (DON) were commonly encountered each year, and occurred at mean levels in fields up to 1.7xa0mg/kg (sample max. 2.77xa0mg/kg) and 1.9xa0mg/kg (sample max. 2.66xa0mg/kg), respectively. Neither fumonisin nor DON levels were significantly correlated with the percent of ears with visibly moldy insect-damaged kernels. Significant correlations were noted for the percent of ears with early caterpillar damage and both fumonisin and DON levels overall for some years and at specific sites in other years. Fumonisin levels were generally more highly correlated with insect damage than DON levels. Insect damaged kernels had 100- to 500-fold or greater levels of fumonisin compared to noninsect-damaged kernels, while DON levels were closer to 10- to 30-fold higher in insect damaged versus nondamaged kernels. A high percentage of DON-contaminated kernels were not insect damaged in 2007 and 2008. In some cases, differing mycotoxin levels for the same hybrid and same year planted at different locations appeared to be due to the prior crop. Higher DON levels in 2008 than other years were most likely associated with higher levels of rainfall and cooler temperatures than average during ear fill. While kernel sorters are reported to remove mycotoxin-contaminated popcorn kernels to acceptible levels, consideration of environmental factors that promote mycotoxins in popcorn should result in more effective control measures in the field.

Effects of Elevated Peroxidase Levels and Corn Earworm Feeding on Gene Expression in Tomato

Microarray analysis was used to measure the impact of herbivory by Helicoverpa zea, (corn earworm caterpillar) on wild-type and transgenic tomato, Solanum lycopersicum, plants that over-express peroxidase. Caterpillar herbivory had by far the greatest affect on gene expression, but the peroxidase transgene also altered the expression of a substantial number of tomato genes. Particularly high peroxidase activity resulted in the up-regulation of genes encoding proteinase inhibitors, pathogenesis-related (PR) proteins, as well as proteins associated with iron and calcium transport, and flowering. In a separate experiment conducted under similar conditions, real-time quantitative polymerase chain reaction (qPCR) analysis confirmed our microarray results for many genes. There was some indication that multiple regulatory interactions occurred due to the interaction of the different treatments. While herbivory had the greatest impact on tomato gene expression, our results suggest that levels of expression of a multifunctional gene, such as peroxidase and its products, can influence other gene expression systems distinct from conventional signaling pathways, further indicating the complexity of plant defensive responses to insects.

Maize peroxidase Px5 has a highly conserved sequence in inbreds resistant to mycotoxin producing fungi which enhances fungal and insect resistance

Mycotoxin presence in maize causes health and economic issues for humans and animals. Although many studies have investigated expression differences of genes putatively governing resistance to producing fungi, few have confirmed a resistance role, or examined putative resistance gene structure in more than a couple of inbreds. The pericarp expression of maize Px5 has previously been associated with resistance to Aspergillus flavus growth and insects in a set of inbreds. Genes from 14 different inbreds that included ones with resistance and susceptibility to A. flavus, Fusarium proliferatum, F. verticillioides and F. graminearum and/or mycotoxin production were cloned using high fidelity enzymes, and sequenced. The sequence of Px5 from all resistant inbreds was identical, except for a single base change in two inbreds, only one of which affected the amino acid sequence. Conversely, the Px5 sequence from several susceptible inbreds had several base variations, some of which affected amino acid sequence that would potentially alter secondary structure, and thus enzyme function. The sequence of the maize peroxidase Px5 common to inbreds resistant to mycotoxigenic fungi was overexpressed in maize callus. Callus transformants overexpressing the gene caused significant reductions in growth for fall armyworms, corn earworms, and F. graminearum compared to transformant callus with a β-glucuronidase gene. This study demonstrates rarer transcripts of potential resistance genes overlooked by expression screens can be identified by sequence comparisons. A role in pest resistance can be verified by callus expression of the candidate genes, which can thereby justify larger scale transformation and regeneration of transgenic plants expressing the resistance gene for further evaluation.

Leaf axil sampling of midwest U.S. maize for mycotoxigenic Fusarium fungi using PCR analysis

PCR analysis was used to detect Fusarium species generically, as well as the mycotoxin-producing species F.␣subglutinans, F. proliferatum, and F. verticillioides in leaf axil and other maize tissues during ear fill in a multiyear study in central Illinois. The frequency of Fusarium detected varied from site to site and year to year. Fusarium was generically detected more frequently in leaf axil material than in leaf/husk lesions. In two growing seasons, the leaf axil samples were also tested for the presence of the mycotoxin producing species F. proliferatum, F. subglutinans, and F. verticillioides. Overall, F. proliferatum and F. verticillioides were detected less often than F. subglutinans. Fusarium was generically and specifically detected most commonly where visible fungal growth was present in leaf axil material.