Two Wound-Inducible Soybean Cysteine Proteinase Inhibitors Have Greater Insect Digestive Proteinase Inhibitory Activities than a Constitutive Homolog

Abstract

Diverse functions for three soybean (Glycine max L. Merr.) cysteine proteinase inhibitors (CysPls) are inferred from unique characteristics of differential regulation of gene expression and inhibitory activities against specific Cys proteinases. Based on northern blot analyses, we found that the expression in leaves of one soybean CysPl gene (LI) was constitutive and the other two (N2 and R I ) were induced by wounding or methyl jasmonate treatment. Induction of N2 and R I transcript levels in leaves occurred coincidentally with increased papain inhibitory activity. Analyses of kinetic data from bacterial recombinant CysPl proteins indicated that soybean CysPls are noncompetitive inhibitors of papain. The inhibition constants against papain of the CysPls encoded by the wound and methyl jasmonate-inducible genes (57 and 21 nM for N2 and R1, respectively) were 500 to 1000 times lower than the inhibition constant of L1 (19,000 nM). N2 and R1 had substantially greater inhibitory activities than L l against gut cysteine proteinases of the third-instar larvae of western corn rootworm and Colorado potato beetle. Cysteine proteinases were the predominant digestive proteolytic enzymes i n the guts of these insects at this developmental stage. N2 and R1 were more inhibitory than the epoxide trans-epoxysuccinyl-~-leucylamide-(4-guanidino)butane (E-64) against western corn rootworm gut proteinases (50% inhibition concentration = 50, 200, and 7000 nM for N2, R1, and E-64, respectively). However, N2 and R1 were less effective than E-64 against the gut proteinases of Colorado potato beetle. These results indicate that the wound-inducible soybean CysPls, N2 and R1, function in host plant defense against insect predation, and that substantial variation in CysPl activity against insect digestive proteinases exists among plant CysPl proteins.Proteinaceous CysPIs, which specifically inhibit sulfl~y-dryl proteinase activities, are distributed ubiquitously among animal, plant, and microorganism species. The an-

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

“…By contrast, the reported inefficiency of plant cystatins against a number of Coleoptera was typically associated with the presence of cystatin-insensitive cysteine proteases, thought to help the target insects compensating for the loss of sensitive protease activities following cystatin intake (Cloutier et al, , 2000Zhu-Salzman et al, 2003;Gruden et al, 2003Gruden et al, , 2004. In line with previous studies reporting detrimental effects for cystatins or other plant cysteine-type inhibitors against some coleopteran species (Zhao et al, 1996;Koiwa et al, 2000;Fabrick et al, 2002), the negative effects of OsCYS1 and CpCYS1 against the banana Plant cystatins against the banana weevil Page 16 weevil were correlated with a broad inhibitory spectrum of the two cystatins against Z-FR-MCA-(cathepsin L-like) and Z-RR-MCA-hydrolyzing (cathepsin B-like) cysteine proteases in midgut extracts, comparable to the overall inhibitory effect of E-64 against the same enzymes. Our data support, in sum, the hypothesis of a close relationship between the inhibitory spectrum of plant cystatins against E-64-sensitive cysteine proteases in midgut extracts and the potential of these inhibitors as effective candidates for the development of transgenic plant lines resistant to coleopteran pests.…”

“…Zhao et al, 1996;Koiwa et al, 2000;Liu et al, 2004;Alvarez-Alfageme et al, 2007;Ninkovic et al, 2007), our data indicate a dramatic negative impact of plant cystatins on larval growth, associated with the presence of cystatin-sensitive cysteine cathepsins in midgut extracts. As suggested from studies reporting detrimental effects for the broad-spectrum inhibitor of papain-like proteases E-64 against Coleoptera, these negative effects of OsCYS1 and CpCYS1 were likely the result of a broad sensitivity of the insect digestive cathepsins to the ingested inhibitors.…”

Anthropogenic increase in carbon dioxide compromises plant defense against invasive insects

“…Soybean (Glycine max), the world's most widely grown seed legume, has one constitutive CystPI gene (L1) and two inducible CystPI genes (N2 and R1) (13,14). Soybean CystPIs, as well as the synthetic CystPI, transepoxysucciny-L-leucyl-amido (4-guanidino)…”

Comparative analyses of transcriptome and proteome in response to cotton bollworm between a resistant wild soybean and a susceptible soybean cultivar

“…For example, photosystem I psaG/psaK (Glyma5g22780.2) and thioredoxin (Glyma12g35190.1) were down-regulated at the transcript level while up-regulated at protein level in 2-L vs. 2L (Additional file 2 and 17). This discrepancy between transcript and protein levels were also reported in other studies Liang et al 2016;Zhao et al 1996), which might be attributed to the limited capability of iTAQ technology for the detection of small differences in protein abundance, or the posttranscriptional or posttranslational modification of proteins.…”