A Novel Function for the Cathepsin D Inhibitor in Tomato

Abstract

Proteinaceous aspartic proteinase inhibitors are rare in nature and are described in only a few plant species. One of them corresponds to a family of cathepsin D inhibitors (CDIs) described in potato (Solanum tuberosum), involving up to 15 isoforms with a high sequence similarity. In this work, we describe a tomato (Solanum lycopersicum) wound-inducible protein called jasmonic-induced protein 21 (JIP21). Sequence analysis of its cDNA predicted a putative function as a CDI. The JIP21 gene, whose protein has been demonstrated to be glycosylated, is constitutively expressed in flowers, stem, and fruit, and is inducible to high levels by wounding and methyl jasmonate in leaves of tomato plants. The genomic sequence of JIP21 shows that the gene is intronless and reveals the presence of both a methyl jasmonate box (TGACT) and a G-box (CACGT) in the promoter. In contrast to the presumed role of JIP21 based on sequence analysis, a detailed biochemical characterization of the purified protein uncovers a different function as a strong chymotrypsin inhibitor, which questions the previously predicted inhibitory activity against aspartic proteinases. Moreover, Egyptian cotton worm (Spodoptera littoralis) larvae fed on transgenic tomato plants overexpressing JIP21 present an increase in mortality and a delay in growth when compared with larvae fed on wild-type plants. These larvae belong to the Lepidoptera family whose main digestive enzymes have been described as being Ser proteases. All these results support the notion that tomato JIP21 should be considered as a chymotrypsin inhibitor belonging to the Ser proteinase inhibitors rather than a CDI. Therefore, we propose to name this protein tomato chymotrypsin inhibitor 21 (TCI21).

Stability of Plant Defense Proteins in the Gut of Insect Herbivores

“…Among the proteins previously implicated in defense against lepidopteran insects were TD2, LAP-A (Gu et al, 1999;Chen et al, 2005), cathepsin D inhibitor (CDI; Lison et al, 2006), and a germin-like protein (GLP) similar to a GLP isozyme from N. attenuata (Lou and Baldwin, 2006). Two stress-inducible proteins of unknown function were also identified.…”

“…CDI is a chymotrypsin inhibitor belonging to the family of Ser PIs (rather than Asp PIs) that exerts potent growth-inhibiting effects on lepidopteran larvae (Li et al, 2004). (Lison et al, 2006). The frass-accumulating GLP is closely related to a MeJA-inducible GLP from N. attenuata that has a role in resistance to M. sexta attack (Lou and Baldwin, 2006).…”

“…Induced expression of the vast majority of these proteins is regulated by the jasmonate signaling pathway (Walling, 2000;Gatehouse, 2002;Kessler and Baldwin, 2002;Howe, 2004;Schilmiller and Howe, 2005). Examples of jasmonate-inducible proteins (JIPs) that have a confirmed or proposed role in postingestive defense include polyphenol oxidase, arginase, Leu amino peptidase A (LAP-A), lipoxygenase, and a battery of PIs (Duffey and Felton, 1991;Felton et al, 1994;Constabel et al, 1995;Felton, 1996;Chen et al, 2005;Walling, 2006;Lison et al, 2006). A jasmonic acid (JA)-inducible acid phosphatase (VSP2) in Arabidopsis (Arabidopsis thaliana) was recently shown to exert insecticidal activity against coleopteran and dipteran insects (Liu et al, 2005).…”

Jasmonates in Plant Defense Responses

“…One such class of proteins is proteinase inhibitors, which disrupt the digestive process in the insect gut, thus thwarting the attack (Ryan 1990;Halitschke et al 2003;Chen et al 2005). These protease inhibitors inhibit gut proteases resulting in amino acid deficiencies that negatively affect the growth of the herbivore (Zavala et al 2004;Lison et al 2006). Another class of plant defense proteins includes enzymes that deplete consumed nutrients in the herbivore's midgut.…”

Plant Resistance to Insect Herbivory

“…Protease inhibitors (PI) are rapidly produced by plants in response to herbivory and are mostly regulated by JA. PI block the degradation of the proteinaceous part of the food and thereby significantly reduce caterpillar growth (Lison et al 2006;Zavala et al 2004). Other plant defensive proteins that interfere with the digestion of proteins and amino acids in the caterpillar gut are arginases and threonine deaminases (Chen et al 2005).…”