Project description:Native host plant insect resistance in the maize inbred line Mp708 was developed by traditional plant breeding. Resistant Mp708 thwarts feeding by fall armyworm (Spodoptera frugiperda [J.E. Smith]; Lepidoptera: Noctuidae), numerous other lepidopteran pests, and the coleopteran western corn rootworm. This broad resistance makes it an excellent model for studying native host plant resistance mechanisms. In response to caterpillar feeding, Mp708 rapidly mobilizes Mir1-CP, a unique cysteine protease that appears to translocate from roots to the maize midwhorl where it accumulates. This accumulation correlates with a significant reduction in caterpillar growth resulting from diminished food utilization. In addition, the peritrophic membrane (PM) that surrounds the food bolus in the mudgut (MG) is severely damaged in caterpillars fed on sweet corn callus transformed to express the gene encoding Mir1-CP or on midwhorl tissue from resistant Mp708 maize. Functions of the PM include assisting digestion and protecting the epithelium of the caterpillar MG from physical and chemical damage. Consequently, the reduced growth of caterpillars that feed on Mp708 is probably due to the action of Mir1-CP on PM physiology. In fact, previous in vitro studies indicated that Mir1-CP was capable of permeabilizing the PM. The present study used both targeted (qRT-PCR) and global (mRNA-seq) transcriptome analyses to explore the effect of eating Mir1-CP expressing Mp708 maize on abundance of transcripts in the MG of fall armyworm larvae in comparison to MGs from larvae fed on susceptible Tx601 maize that does not express Mir1-CP. Expression of genes encoding proteins involved in PM production is upregulated in MGs from fall armyworm fed on Mp708. Also, several digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on Tx601. Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function. Beginning as neonates, fall armyworm larvae used in the mRNA-seq experiment were reared on yellow-green midwhorl foliage from resistant Mp708 maize or susceptible Tx601 maize. Old foliage and frass were removed every other day and replaced with fresh foliage. Larvae were reared in an environmental chamber at 27M-BM-0C, 14:10 (light:dark) photoperiod, and 70% relative humidity. Midguts were dissected from larvae 2 d after molting to the last instar with masses between 300 and 400 mg. Dissections were done with cold anesthetized larvae submerged in Bombyx saline. After removing Malpighian tubules, foregut anterior to the stomodial valve, hindgut and food bolus, the MG was transferred from the body cavity, rinsed well with cold saline, and preserved in RNAlaterM-BM-.. Equal amounts (3 M-BM-5g) of total RNA from an individual MG were randomly pooled into three replicates per treatment (i.e., Mp708 or Tx601) such that each treatment replicate derived from 12-13 MGs. Each pool of total RNA was separately enriched for poly(A+) RNA and submitted to the Penn State Genomics Core Facility (University Park, PA) where barcoded cDNA libraries were prepared and equimolar quantities of each library sequenced on the SOLiD 3 Plus System. Sequence reads were filtered to accept reads whose median score threshold was M-bM-^IM-%12, contained M-bM-^IM-%25 bases and contained one or more bases with a quality score M-bM-^IM-%14. The 138911 Sanger ESTs in SPODOBASE (http://bioweb.ensam.inra.fr/spodobase) were assembled into a reference transcriptome using SeqMan Pro version 8.0.2. Filtered reads from each library representing a replicate within a maize inbred treatment were mapped separately to the reference transcriptome using the Bowtie-like algorithm in NextGENeM-BM-. with the requirement that 85% of 12 or more nucleotides comprising a read must match the reference. A read was allowed to map only once (i.e., no ambiguous mapping). The number of mapped reads per contig (i.e., gene model) in each treatment replicate-library were summed by NextGENeM-BM-. as read counts per gene and subsequently used in differential expression analyses.

Project description:Native host plant insect resistance in the maize inbred line Mp708 was developed by traditional plant breeding. Resistant Mp708 thwarts feeding by fall armyworm (Spodoptera frugiperda [J.E. Smith]; Lepidoptera: Noctuidae), numerous other lepidopteran pests, and the coleopteran western corn rootworm. This broad resistance makes it an excellent model for studying native host plant resistance mechanisms. In response to caterpillar feeding, Mp708 rapidly mobilizes Mir1-CP, a unique cysteine protease that appears to translocate from roots to the maize midwhorl where it accumulates. This accumulation correlates with a significant reduction in caterpillar growth resulting from diminished food utilization. In addition, the peritrophic membrane (PM) that surrounds the food bolus in the mudgut (MG) is severely damaged in caterpillars fed on sweet corn callus transformed to express the gene encoding Mir1-CP or on midwhorl tissue from resistant Mp708 maize. Functions of the PM include assisting digestion and protecting the epithelium of the caterpillar MG from physical and chemical damage. Consequently, the reduced growth of caterpillars that feed on Mp708 is probably due to the action of Mir1-CP on PM physiology. In fact, previous in vitro studies indicated that Mir1-CP was capable of permeabilizing the PM. The present study used both targeted (qRT-PCR) and global (mRNA-seq) transcriptome analyses to explore the effect of eating Mir1-CP expressing Mp708 maize on abundance of transcripts in the MG of fall armyworm larvae in comparison to MGs from larvae fed on susceptible Tx601 maize that does not express Mir1-CP. Expression of genes encoding proteins involved in PM production is upregulated in MGs from fall armyworm fed on Mp708. Also, several digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on Tx601. Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function.

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