Insect feeding mobilizes a unique plant defense protease that disrupts the peritrophic matrix of caterpillars

Abstract

Plants frequently respond to herbivorous insect attack by synthesizing defense proteins that deter insect feeding and prevent additional herbivory. Maize (Zea mays L.) lines, resistant to feeding by a number of lepidopteran species, rapidly mobilize a unique 33-kDa cysteine protease in response to caterpillar feeding. The accumulation of the 33-kDa cysteine protease in the maize mid-whorl was correlated with a significant reduction in caterpillar growth that resulted from impaired nutrient utilization. Black Mexican Sweetcorn callus transformed with mir1, the gene encoding the 33-kDa cysteine protease, expressed the protease and growth of caterpillars reared on the transgenic callus was reduced 60-80%. Scanning electron microscopy was used to examine the effect of plant material expressing the 33-kDa cysteine protease on the structure of the caterpillar peritrophic matrix. Because the peritrophic matrix surrounds the food bolus, assists in digestive processes, and protects the caterpillar midgut from physical and chemical damage, disruption of peritrophic matrix may reduce caterpillar growth. The results indicated that the peritrophic matrix was severely damaged when caterpillars fed on resistant maize plants or transgenic Black Mexican Sweetcorn. The accumulation of the 33-kDa cysteine protease in response to caterpillar feeding, and its ability to damage the insect peritrophic matrix, represents an unusual host-plant resistance mechanism that may have applications in agricultural biotechnology.

Figure 1

Scanning electron micrographs showing fall armyworm midgut and PM. (A) Excised midgut (mg) from a caterpillar reared on a resistant plant showing the exposed microvilli (mv), PM containing the food bolus (PM + FB), and empty PM (magnification = ×50). (B) PM from a caterpillar reared on a susceptible plant (magnification = ×38). (C) Ectoperitrophic layer (1), midperitrophic layers (2, 3) and endoperitrophic layer (4) of a caterpillar reared on a resistant plant (magnification = ×500). The scale is indicated on each micrograph.

Figure 2

Scanning electron micrographs showing peritrophic matrix (PM) of fall armyworm caterpillars that fed on susceptible and resistant corn plants. (A and B) Ectoperitrophic and endoperitrophic layers, respectively, from caterpillars reared on a susceptible plant. (C) Ectoperitrophic layer from a caterpillar reared on a resistant plant. (D) Midperitrophic layer from a caterpillar reared on resistant plant. (E and F) Endoperitrophic layer from a caterpillar reared on a resistant plant. In all cases, the magnification = ×3,000. In this experiment, caterpillars reared on susceptible and resistant plants weighed 89.0 ± 19.6 and 37.3 ± 15.0 mg, respectively. The scale is indicated in each micrograph.

Figure 3

Scanning electron micrographs showing peritrophic matrix (PM) of fall armyworm caterpillars reared on BMS-33 callus overexpressing the 33 kDa cysteine protease. (A) Ectoperitrophic from caterpillars reared on nontransformed BMS (magnification = ×500). (B) Ectoperitrophic from caterpillars reared on nontransformed BMS (magnification = ×3,000). (C) Midperitrophic layers from a larva reared on BMS-33 callus (magnification = ×3,500). (D) Endoperitrophic layer from a caterpillar reared on BMS-33 callus (magnification = ×3,500). In this experiment, caterpillars reared on BMS and BMS-33 callus weighed 264.3 ± 2.5 and 68.6 ± 7.7 mg, respectively. The scale is indicated on each micrograph.