Nano-SPRi Aptasensor for the Detection of Progesterone in Buffer.

Progesterone is a steroid hormone that plays a central role in the female reproductive processes such as ovulation and pregnancy with possible effects on other organs as well. The measurement of progesterone levels in bodily fluids can assist in early pregnancy diagnosis and can provide insight for other reproductive functions. In this work, the detection of progesterone was examined by integrating novel aptamer development with a nanoEnhanced surface plasmon resonance imaging sensor. First, we developed X-aptamers and selected them for binding to progesterone. Then, we took advantage of the multi-array feature of SPRi to develop an optimized biosensor capable of simultaneously screening the 9 X-aptamers developed to determine the binding capabilities of each aptamer. The sensor surface design conditions were further optimized for the sandwich assay, which employed nanoEnhancers (NIR-streptavidin coated quantum dots) for ultrasensitive detection of progesterone molecules. The assay designed was examined over a concentration range of 1.575 ng/mL to 126 µg/mL resulting in a limit of detection (LOD) of 1.575 ng/mL (5 nM) in phosphate buffer.

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.

Aboveground to belowground herbivore defense signaling in maize: a two-way street?

Insect pests that attempt to feed on the caterpillar-resistant maize genotype Mp708 encounter a potent, multipronged defense system that thwarts their invasion. First, these plants are on "constant alert" due to constitutively elevated levels of the phytohormone jasmonic acid that signals the plant to activate its defenses. The higher jasmonic acid levels trigger the expression of defense genes prior to herbivore attack so the plants are "primed" and respond with a faster and stronger defense. The second defense is the rapid accumulation of a toxic cysteine protease called Mir1-CP in the maize whorl in response to caterpillar feeding. When caterpillars ingest Mir1-CP, it damages the insect's midgut and retards their growth. In this article, we discuss a third possible defense strategy employed by Mp708. We have shown that foliar caterpillar feeding causes Mir1-CP and defense gene transcripts to accumulate in its roots. We propose that caterpillar feeding aboveground sends a signal belowground via the phloem that results in Mir1-CP accumulation in the roots. We also postulate that the roots serve as a reservoir of Mir1-CP that can be mobilized to the whorl in response to caterpillar assault.

Plants on constant alert: elevated levels of jasmonic acid and jasmonate-induced transcripts in caterpillar-resistant maize.

This study was conducted to determine if constitutive levels of jasmonic acid (JA) and other octadecanoid compounds were elevated prior to herbivory in a maize genotype with documented resistance to fall armyworm (Spodoptera frugiperda) and other lepidopteran pests. The resistant inbred Mp708 had approximately 3-fold higher levels of jasmonic acid (JA) prior to herbivore feeding than the susceptible inbred Tx601. Constitutive levels of cis-12-oxo-phytodienoic acid (OPDA) also were higher in Mp708 than Tx601. In addition, the constitutive expression of JA-inducible genes, including those in the JA biosynthetic pathway, was higher in Mp708 than Tx601. In response to herbivory, Mp708 generated comparatively higher levels of hydrogen peroxide, and had a greater abundance of NADPH oxidase transcripts before and after caterpillar feeding. Before herbivore feeding, low levels of transcripts encoding the maize insect resistance cysteine protease (Mir1-CP) and the Mir1-CP protein were detected consistently. Thus, Mp708 appears to have a portion of its defense pathway primed, which results in constitutive defenses and the ability to mount a stronger defense when caterpillars attack. Although the molecular mechanisms that regulate the constitutive accumulation of JA in Mp708 are unknown, it might account for its enhanced resistance to lepidopteran pests. This genotype could be valuable in studying the signaling pathways that maize uses to response to insect herbivores.

Mir1-CP, a novel defense cysteine protease accumulates in maize vascular tissues in response to herbivory.

When lepidopteran larvae feed on the insect-resistant maize genotype Mp708 there is a rapid accumulation of a defensive cysteine protease, Maize insect resistance 1-cysteine protease (Mir1-CP), at the feeding site. Silver-enhanced immunolocalization visualized with both light and transmission electron microscopy was used to determine the location of Mir1-CP in the maize leaf. The results indicated that Mir1-CP is localized predominantly in the phloem of minor and intermediate veins. After 24 h of larval feeding, Mir1-CP increased in abundance in the vascular parenchyma cells and in the thick-walled sieve element (TSE); it was also found localized to the bundle sheath and mesophyll cells. In situ hybridization of mRNA encoding Mir1-CP indicated that the primary sites of Mir1-CP synthesis in the whorl are the vascular parenchyma and bundle sheath cells. In addition to the phloem, Mir1-CP was also found in the metaxylem of the leaf and root. After 24 h of foliar feeding, the amount of Mir1-CP in the root xylem increased and it appeared to move from xylem parenchyma into the root metaxylem elements. The accumulation of Mir1-CP in maize vascular elements suggests Mir1-CP may move through these tissues to defend against insect herbivores.

Ethylene signaling mediates a maize defense response to insect herbivory.

The signaling pathways that enable plants to mount defenses against insect herbivores are known to be complex. It was previously demonstrated that the insect-resistant maize (Zea mays L.) genotype Mp708 accumulates a unique defense cysteine proteinase, Mirl-CP, in response to caterpillar feeding. In this study, the role of ethylene in insect defense in Mp708 and an insect-susceptible line Tx601 was explored. Ethylene synthesis was blocked with either cobalt chloride or aminoethoxyvinylglycine. Alternatively, ethylene perception was inhibited with 1-methylcyclopropene. Blocking ethylene synthesis and perception resulted in Mp708 plants that were more susceptible to caterpillar feeding. In addition, fall armyworm (Spodoptera frugiperda) larvae that fed on inhibitor-treated Mp708 plants had signifycantly higher growth rates than those reared on untreated plants. In contrast, these responses were not significantly altered in Tx601. The ethylene synthesis and perception inhibitors also reduced the accumulation of Mirl-CP and its transcript mir1 in response to herbivory. These results indicate that ethylene is a component of the signal transduction pathway leading to defense against insect herbivory in the resistant genotype Mp708.