Plant defense against insect herbivores is mediated in part by enzymes that impair digestive processes in the insect gut. Little is known about the evolutionary origins of these enzymes, their distribution in the plant kingdom, or the mechanisms by which they act in the protease-rich environment of the animal digestive tract. One example of such an enzyme is threonine (Thr) deaminase (TD), which in tomato (Solanum lycopersicum) serves a dual role in isoleucine (Ile) biosynthesis in planta and Thr degradation in the insect midgut. Here, we report that tomato uses different TD isozymes to perform these functions. Whereas the constitutively expressed TD1 has a housekeeping role in Ile biosynthesis, expression of TD2 in leaves is activated by the jasmonate signaling pathway in response to herbivore attack. Ingestion of tomato foliage by specialist (Manduca sexta) and generalist (Trichoplusia ni) insect herbivores triggered proteolytic removal of TD2's C-terminal regulatory domain, resulting in an enzyme that degrades Thr without being inhibited through feedback by Ile. This processed form (pTD2) of TD2 accumulated to high levels in the insect midgut and feces (frass). Purified pTD2 exhibited biochemical properties that are consistent with a postingestive role in defense. Shotgun proteomic analysis of frass from tomato-reared M. sexta identified pTD2 as one of the most abundant proteins in the excrement. Among the other tomato proteins identified were several jasmonate-inducible proteins that have a known or proposed role in anti-insect defense. Subtilisin-like proteases and other pathogenesis-related proteins, as well as proteins of unknown function, were also cataloged. We conclude that proteomic analysis of frass from insect herbivores provides a robust experimental approach to identify hyperstable plant proteins that serve important roles in defense.

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ABSTRACT--The scanning electron microscope (SEM) moir~ method for microscopic measurements based on electron beam lithography and an SEM has been well developed. Although it has been a reliable method, some drawbacks exist: reinforcement effects, complicated processing and low sensitivity. To improve the SEM moir6 method, new gratingcasting techniques and a fringe-viewing technique must be developed. In this study, a carbonaceous grating technique and a total imaging technique are introduced. Accordingly, there are two techniques available for grating-casting (i.e., the carbonaceous grating technique and the existing lithography grating technique) and three techniques available for fringe viewing (i.e., the total imaging technique, the existing monitor viewing technique and the existing photographic viewing technique). A total of six new imaging techniques of SEM moir6 methods are available for microscopic measurements by combining one technique from each of the two groups. This study demonstrates the feasibility of the individual techniques and discusses the characteristics and limitations of each. Based on the presented total imaging technique, the sensitivity of the moir6 method is only dependent on the frequency of specimen grating. Because it can be made as high as 10,000 lines/ram, the SEM moir6 method can achieve sensitivity as high as 0.01 percent.

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