Fall armyworm larvae (FAW), which are serious pests in the southern United States, show retarded growth when they feed on insect-resistant maize inbreds Mp704 and Mp708. These maize genotypes are not only resistant to FAW, but to a number of other lepidopteran pests. In these genotypes, a unique, extracellular, 33-KDa cysteine protease (Mir1-CP) rapidly accumulates in the whorl in response to insect feeding. Initial morphological studies on larvae feeding on resistant maize plants over-expressing the cysteine protease showed severe damage in insect?s first line of defense, the peritrophic matrix (PM). But it is not known whether the cysteine protease has unprecedented effect on insect defense mechanisms. This study focuses on understanding the functional involvement of the cysteine protease (Mir1-CP) in a plant-herbivore defense mechanism. I used purified, recombinant 33-KDa cysteine protease (Mir1-CP) and its two mutated forms (Mut1 and Mut2) to determine their effects on the permeability of PMs from fall armyworm and other lepidopteran larvae. The purified Mir1-CP was also used to determine its minimal effective dosage on lepidopteran larval growth as well as to qualitatively determine their direct morphological effects on PM and gut regions of fall armyworm larvae. In vitro permeability studies demonstrated that the recombinant Mir1-CP directly permeabilized the PM and requires both cysteine at the active site and the terminal 25 amino acids to achieve complete permeabilization. Dose response study suggested that physiologically relevant concentrations of Mir1-CP in the maize whorl would be effective in controlling a broad range of lepidopteran pests. The study also suggested that stacking Mir1-CP and Bt-toxin (Bt-CryIIA) genes in transgenic plants could broaden the normal range of both Mir1-CP and Bt-toxin. Morphological studies using three different microscopic techniques showed damaged PM in larvae fed on Mir1-CP diet. These results suggest that by directly permeabilizing and damaging the PM, the Mir1-CP provides critical defense in host plants against lepidopteran pests.

Maize is an important food crop in most parts of the world including the United States. The plants growing in the field are constantly challenged with various biotic stresses like insect herbivores and fungal pathogens. The physical wounds produced on the growing crops by the insects render the plants more vulnerable to the fungal pathogens. Hence developing both insect and fungal resistant maize varieties is crucial to benefit more from the harvest. Several studies have been in advance in this direction and as a consequence insect, in particular lepidopteran larve resistant maize genotype Mp708 and Aspergillus flavus resistant genotype Mp313E were developed. This study particularly focuses on understanding the functional involvement of the major phytohormones in the signal transduction and expression of the unique defense protein, Maize insect resistance 1-cysteine protease (Mir1-CP) shown to accumulate in response to herbivory by lepidopteran larvae, Spodoptera frugiperda (Fall armyworm, FAW) as a defense mechanism. Using a pharmacological approach involving exogenous hormone and hormone inhibitor treatments and analyzing the expression and accumulation of Mir1-CP protein and mir1 transcript by immunoblot and qRT-PCR analysis respectively, both JA and ET were found to be involved in mediating Mir1-CP accumulation with JA acting upstream of ET. Results also indicate that Mir1-CP accumulation involves both transcriptional and post-transcriptional (or post-translational) regulations. A different part of the study involved in understanding and evaluating the performance of Aspergillus flavus on the resistant and susceptible maize genotypes during infection. As of part of this study I also analyzed and compared the defense response offered by the resistant maize genotype, Mp313E and the susceptible genotype, Va35 by looking at the expression levels of the various defense related genes. The potency of the resistant maize genotype in sustaining the fungal infection in the field was of particular focus. Resistant maize genotype Mp313E was found to potentially oppose A.flavus proliferation and colonization and also delay aflatoxin biosynthesis unlike Va35. The up regulation of the maize defense genes during the early time points of infection, in Mp313E, indicate the potential role of these genes in conferring resistance against fungal pathogens.