Production of Recombinant Active Human TGFß1 in Nicotiana benthamiana.

The production of recombinant proteins in plant systems is receiving wider attention. Indeed, various plant-produced pharmaceuticals have been shown to be biologically active. However, the production of human growth factors and cytokines in heterologous systems is still challenging because they often act as complex forms, such as homo- or hetero-dimers, and their production is tightly regulated in vivo. In this study, we demonstrated that the mature form of human TGFß1 produced and purified from Nicotiana benthamiana shows biological activity in animal cells. To produce the mature form of TGFß1, various recombinant genes containing the mature form of TGFß1 were generated and produced in N. benthamiana. Of these, a recombinant construct, BiP:M:CBM3:LAP[C33S]:EK:TGFß1, was expressed at a high level in N. benthamiana. Recombinant proteins were one-step purified using cellulose-binding module 3 (CBM3) as an affinity tag and microcrystalline cellulose (MCC) beads as a matrix. The TGFß1 recombinant protein bound on MCC beads was proteolytically processed with enterokinase to separate mature TGFß1. The mature TGFß1 still associated with Latency Associated Protein, [LAP(C33S)] that had been immobilized on MCC beads was released by HCl treatment. Purified TGFß1 activated TGFß1-mediated signaling in the A549 cell line, thereby inducing phosphorylation of SMAD-2, the expression of ZEB-2 and SNAIL1, and the formation of a filopodia-like structure. Based on these results, we propose that active mature TGFß1, one of the most challenging growth factors to produce in heterologous systems, can be produced from plants at a high degree of purity via a few steps.

Comparison of CD20 Binding Affinities of Rituximab Produced in Nicotiana benthamiana Leaves and Arabidopsis thaliana Callus.

Plants are promising drug-production platforms with high economic efficiency, stability, and convenience in mass production. However, studies comparing the equivalency between the original antibodies and those produced in plants are limited. Amino acid sequences that constitute the Fab region of an antibody are diverse, and the post-transcriptional modifications that occur according to these sequences in animals and plants are also highly variable. In this study, rituximab, a blockbuster antibody drug used in the treatment of non-Hodgkin's lymphoma, was produced in Nicotiana benthamiana leaves and Arabidopsis thaliana callus, and was compared to the original rituximab produced in CHO cells. Interestingly, the epitope recognition and antigen-binding abilities of rituximab from N. benthamiana leaves were almost lost. In the case of rituximab produced in A. thaliana callus, the specific binding ability and CD20 capping activity were maintained, but the binding affinity was less than 50% of that of original rituximab from CHO cells. These results suggest that different plant species exhibit different binding affinities. Accordingly, in addition to the differences in PTMs between mammals and plants, the differences between the species must also be considered in the process of producing antibodies in plants.

Frequent asymptomatic infection with tobacco ringspot virus on melon fruit.

Melon is one of the most popular fruits worldwide and has been bred into various cultivars. RNA-sequencing using healthy melon fruit was performed to determine differences in gene expression among cultivars. Unexpected RNA-seq results revealed that viruses asymptomatically infected fruits at a high frequency (16 of 21 fruits examined were infected) and that viral transcripts highly accumulated in comparison with host transcripts (15 %-75 % of total reads). Their nucleotide sequences and phylogenetic analyses indicated that more than 10 novel isolates of tobacco ringspot virus (TRSV) were found in melon fruits. Asymptomatic infection with TRSV on melon fruits was confirmed by both immunoblot and RT-PCR analyses. Numerous isolates of TRSV generated and maintained in melon fields, and this is likely due to their asymptomatic infections. This TRSV melon isolate infected Nicotiana benthamiana plants with stunting and yellowing symptoms. This is the first report of frequent and asymptomatic infection of TRSV in consumable melon fruits.

Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.

KETCH1 imports HYL1 to nucleus for miRNA biogenesis in Arabidopsis.

MicroRNA (miRNA) is processed from primary transcripts with hairpin structures (pri-miRNAs) by microprocessors in the nucleus. How cytoplasmic-borne microprocessor components are transported into the nucleus to fulfill their functions remains poorly understood. Here, we report KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1) as a partner of hyponastic leaves 1 (HYL1) protein, a core component of microprocessor in Arabidopsis and functional counterpart of DGCR8/Pasha in animals. Null mutation of ketch1 is embryonic-lethal, whereas knockdown mutation of ketch1 caused morphological defects, reminiscent of mutants in the miRNA pathway. ketch1 knockdown mutation also substantially reduced miRNA accumulation, but did not alter nuclear-cytoplasmic shuttling of miRNAs. Rather, the mutation significantly reduced nuclear portion of HYL1 protein and correspondingly compromised the pri-miRNA processing in the nucleus. We propose that KETCH1 transports HYL1 from the cytoplasm to the nucleus to constitute functional microprocessor in Arabidopsis This study provides insight into the largely unknown nuclear-cytoplasmic trafficking process of miRNA biogenesis components through eukaryotes.

Specific control of Arabidopsis BAK1/SERK4-regulated cell death by protein glycosylation.

Precise control of cell death is essential for the survival of all organisms. Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and somatic embryogenesis receptor kinase 4 (SERK4) redundantly and negatively regulate cell death through elusive mechanisms. By deploying a genetic screen for suppressors of cell death triggered by virus-induced gene silencing of BAK1/SERK4 on Arabidopsis knockout collections, we identified STT3a, a protein involved in N-glycosylation modification, as an important regulator of bak1/serk4 cell death. Systematic investigation of glycosylation pathway and endoplasmic reticulum (ER) quality control (ERQC) components revealed distinct and overlapping mechanisms of cell death regulated by BAK1/SERK4 and their interacting protein BIR1. Genome-wide transcriptional analysis revealed the activation of members of cysteine-rich receptor-like kinase (CRK) genes in the bak1/serk4 mutant. Ectopic expression of CRK4 induced STT3a/N-glycosylation-dependent cell death in Arabidopsis and Nicotiana benthamiana. Therefore, N-glycosylation and specific ERQC components are essential to activate bak1/serk4 cell death, and CRK4 is likely to be among client proteins of protein glycosylation involved in BAK1/SERK4-regulated cell death.

Arabidopsis CPL4 is an essential C-terminal domain phosphatase that suppresses xenobiotic stress responses.

Eukaryotic gene expression is both promoted and inhibited by the reversible phosphorylation of the C-terminal domain of RNA polymerase II (pol II CTD). More than 20 Arabidopsis genes encode CTD phosphatase homologs, including four CTD phosphatase-like (CPL) family members. Although in vitro CTD phosphatase activity has been established for some CPLs, none have been shown to be involved in the phosphoregulation of pol II in vivo. Here we report that CPL4 is a CTD phosphatase essential for the viability of Arabidopsis thaliana. Mass spectrometry analysis identified the pol II subunits RPB1, RPB2 and RPB3 in the affinity-purified CPL4 complex. CPL4 dephosphorylates both Ser2- and Ser5-PO(4) of the CTD in vitro, with a preference for Ser2-PO(4). Arabidopsis plants overexpressing CPL4 accumulated hypophosphorylated pol II, whereas RNA interference-mediated silencing of CPL4 promoted hyperphosphorylation of pol II. A D128A mutation in the conserved DXDXT motif of the CPL4 catalytic domain resulted in a dominant negative form of CPL4, the overexpression of which inhibited transgene expression in transient assays. Inhibition was abolished by truncation of the phosphoprotein-binding Breast Cancer 1 C-terminal domain of CPL4, suggesting that both catalytic function and protein-protein interaction are essential for CPL4-mediated regulation of gene expression. We were unable to recover a homozygous cpl4 mutant, probably due to the zygotic lethality of this mutation. The reduction in CPL4 levels in CPL4(RNAi) plants increased transcript levels of a suite of herbicide/xenobiotic-responsive genes and improved herbicide tolerance, thus suggesting an additional role for CPL4 as a negative regulator of the xenobiotic detoxification pathway.

Function of Arabidopsis CPL1 in cadmium responses.

Transcriptional and post-transcriptional responses to external iron (Fe) availability are essential for the cellular Fe homeostasis. Fe deficiency strongly induces Fe utilization-related gene expression; however, little is known about the early Fe signaling that regulates expression of a central transcription factor FIT. In Arabidopsis, mutations in RNA polymerase II CTD-phosphatase-like 1 (CPL1) enhance the expression of Fe utilization-related genes including FIT under Fe deficiency. Fe content is significantly increased in the roots and decreased in the shoots of cpl1-2 plants, and root growth of the cpl1-2 mutant shows higher tolerance to Fe deficiency and to toxicity of cadmium (Cd). The cpl1-2 plants accumulate more Cd in the shoots, suggesting that Cd toxicity in the cpl1-2 roots is circumvented by the transport of excess Cd to the shoots. Here we show data suggesting that the root-to-shoot translocation of Cd in cpl1-2 is mediated by yet uncharacterized Cd transport mechanisms.

Loss of function of Arabidopsis C-terminal domain phosphatase-like1 activates iron deficiency responses at the transcriptional level.

The expression of genes that control iron (Fe) uptake and distribution (i.e. Fe utilization-related genes) is tightly regulated. Fe deficiency strongly induces Fe utilization-related gene expression; however, little is known about the mechanisms that regulate this response in plants. Transcriptome analysis of an Arabidopsis (Arabidopsis thaliana) mutant defective in RNA polymerase II C-terminal domain-phosphatase-like1 (CPL1) revealed significant up-regulation of Fe utilization-related genes (e.g. IRON-REGULATED TRANSPORTER1), suggesting the importance of RNA metabolism in Fe signaling. An analysis using multiple cpl1 alleles established that cpl1 mutations enhanced specific transcriptional responses to low Fe availability. Changes in protein level were less prominent than those in transcript level, indicating that cpl1-2 mainly affects the Fe deficiency response at the transcriptional level. However, Fe content was significantly increased in the roots and decreased in the shoots of cpl1-2 plants, indicating that the cpl1 mutations do indeed affect Fe homeostasis. Furthermore, root growth of cpl1-2 showed improved tolerance to Fe deficiency and cadmium (Cd) toxicity. cpl1-2 plants accumulated more Cd in the shoots, suggesting that Cd toxicity in the roots of this mutant is averted by the transport of excess Cd to the shoots. Genetic data indicate that cpl1-2 likely activates Fe deficiency responses upstream of both FE-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR-dependent and -independent signaling pathways. Interestingly, various osmotic stress/abscisic acid (ABA)-inducible genes were up-regulated in cpl1-2, and the expression of some ABA-inducible genes was controlled by Fe availability. We propose that the cpl1 mutations enhance Fe deficiency signaling and promote cross talk with a branch of the osmotic stress/ABA signaling pathway.

Antagonistic regulation, yet synergistic defense: effect of bergapten and protease inhibitor on development of cowpea bruchid Callosobruchus maculatus.

The furanocoumarin compound bergapten is a plant secondary metabolite that has anti-insect function. When incorporated into artificial diet, it retarded cowpea bruchid development, decreased fecundity, and caused mortality at a sufficient dose. cDNA microarray analysis indicated that cowpea bruchid altered expression of 543 midgut genes in response to dietary bergapten. Among these bergapten-regulated genes, 225 have known functions; for instance, those encoding proteins related to nutrient transport and metabolism, development, detoxification, defense and various cellular functions. Such differential gene regulation presumably facilitates the bruchids' countering the negative effect of dietary bergapten. Many genes did not have homology (E-value cutoff 10(-6)) with known genes in a BlastX search (206), or had homology only with genes of unknown function (112). Interestingly, when compared with the transcriptomic profile of cowpea bruchids treated with dietary soybean cysteine protease inhibitor N (scN), 195 out of 200 coregulated midgut genes are oppositely regulated by the two compounds. Simultaneous administration of bergapten and scN attenuated magnitude of change in selected oppositely-regulated genes, as well as led to synergistic delay in insect development. Therefore, targeting insect vulnerable sites that may compromise each other's counter-defensive response has the potential to increase the efficacy of the anti-insect molecules.

A three-component gene expression system and its application for inducible flavonoid overproduction in transgenic Arabidopsis thaliana.

Inducible gene expression is a powerful tool to study and engineer genes whose overexpression could be detrimental for the host organisms. However, only limited systems have been adopted in plant biotechnology. We have developed an osmotically inducible system using three components of plant origin, RD29a (Responsive to Dehydration 29A) promoter, CBF3 (C-repeat Binding Factor 3) transcription factor and cpl1-2 (CTD phosphatase-like 1) mutation. The osmotic stress responsible RD29a promoter contains the CBF3 binding sites and thus RD29A-CBF3 feedforward cassette enhances induction of RD29a promoter under stress. The cpl1-2 mutation in a host repressor CPL1 promotes stress responsible RD29a promoter expression. The efficacy of this system was tested using PAP1 (Production of Anthocyanin Pigment 1) transgene, a model transcription factor that regulates the anthocyanin pathway in Arabidopsis. While transgenic plants with only one or two of three components did not reproducibly accumulate anthocyanin pigments above the control level, transgenic cpl1 plants containing homozygous RD29a-PAP1 and RD29a-CBF3 transgenes produced 30-fold higher level of total anthocyanins than control plants upon cold treatment. Growth retardation and phytochemical production of transgenic plants were minimum under normal conditions. The flavonoid profile in cold-induced transgenic plants was determined by LC/MS/MS, which resembled that of previously reported pap1-D plants but enriched for kaempferol derivatives. These results establish the functionality of the inducible three-component gene expression system in plant metabolic engineering. Furthermore, we show that PAP1 and environmental signals synergistically regulate the flavonoid pathway to produce a unique flavonoid blend that has not been produced by PAP1 overexpression or cold treatment alone.

Stability of AtVSP in the insect digestive canal determines its defensive capability.

We have previously demonstrated that Arabidopsis vegetative storage protein (AtVSP) is an acid phosphatase that has anti-insect activity in in vitro feeding assays [Liu et al., 2005. Plant Physiology 139, 1545-1556]. To investigate the functionality of AtVSP in planta as an anti-insect defense protein, we produced AtVSP-overexpressing as well as AtVSP-silenced transgenic Arabidopsis lines, and evaluated impact on the polyphagous American grasshopper Schistocerca americana. Grasshoppers showed no significant difference in weight gain and growth rate when feeding on wild type, overexpressing, or silenced lines, respectively. In addition, AtVSP protein was undetectable in either the midgut or frass of grasshoppers reared on transgenic plants suggesting that AtVSP was unable to withstand proteolytic degradation. To determine the stability of the AtVSP protein in grasshopper digestive canal, midgut extracts from various nymphal stages were incubated with bacterially expressed AtVSP for different periods of time. AtVSP was hydrolyzed rapidly by grasshopper midgut extract, in stark contrast with its fate when incubated with cowpea bruchid midgut extract. Multiple proteases have been detected in the midgut of grasshoppers, which may play important roles in determining the insect response to AtVSP. Results indicate that stability of an anti-insect protein in insect guts is a crucial property integral to the defense protein.

Comparative analyses of Arabidopsis complex glycan1 mutants and genetic interaction with staurosporin and temperature sensitive3a.

We compare three Arabidopsis (Arabidopsis thaliana) complex glycan1 (cgl1) alleles and report on genetic interaction with staurosporin and temperature sensitive3a (stt3a). STT3a encodes a subunit of oligosaccharyltransferase that affects efficiency of N-glycan transfer to nascent secretory proteins in the endoplasmic reticulum; cgl1 mutants lack N-acetyl-glucosaminyltransferase I activity and are unable to form complex N-glycans in the Golgi apparatus. By studying CGL1-green fluorescent protein fusions in transient assays, we show that the extra N-glycosylation site created by a point mutation in cgl1 C5 is used in planta and interferes with folding of full-length membrane-anchored polypeptides in the endoplasmic reticulum. Tunicamycin treatment or expression in the stt3a-2 mutant relieved the folding block, and migration to Golgi stacks resumed. Complementation tests with C5-green fluorescent protein and other N-glycosylation variants of CGL1 demonstrated that suppression of aberrant N-glycosylation restores activity. Interestingly, CGL1 seems to be functional also as nonglycosylated enzyme. Two other cgl1 alleles showed splicing defects of their transcripts. In cgl1 C6, a point mutation affects the 3' splice site of intron 14, resulting in frame shifts; in cgl1-T, intron 11 fails to splice due to insertion of a T-DNA copy. Introgression of stt3a-2 did not restore complex glycan formation in cgl1 C6 or cgl1-T but suppressed the N-acetyl-glucosaminyltransferase I defect in cgl1 C5. Root growth assays revealed synergistic effects in double mutants cgl1 C6 stt3a-2 and cgl1-T stt3a-2 only. Besides demonstrating the conditional nature of cgl1 C5 in planta, our observations with loss-of-function alleles cgl1 C6 and cgl1-T in the stt3a-2 underglycosylation background prove that correct N-glycosylation is important for normal root growth and morphology in Arabidopsis.

Protease inhibitors from several classes work synergistically against Callosobruchus maculatus.

Targeting multiple digestive proteases may be more effective in insect pest control than inhibition of a single enzyme class. We therefore explored possible interactions of three antimetabolic protease inhibitors fed to cowpea bruchids in artificial diets, using a recombinant soybean cysteine protease inhibitor scN, an aspartic protease inhibitor pepstatin A, and soybean Kunitz trypsin inhibitor KI. scN and pepstatin, inhibiting major digestive cysteine and aspartic proteases, respectively, significantly prolonged the developmental time of cowpea bruchids individually. When combined, the anti-insect effect was synergistic, i.e., the toxicity of the mixture was markedly greater than that of scN or pepstatin alone. KI alone did not impact insect development even at relatively high concentrations, but its anti-insect properties became apparent when acting jointly with scN or scN plus pepstatin. Incubating KI with bruchid midgut extract showed that it was partially degraded. This instability may explain its lack of anti-insect activity. However, this proteolytic degradation was inhibited by scN and/or pepstatin. Protection of KI from proteolysis in the insect digestive tract thus could be the basis for the synergistic effect. These observations support the concept that cowpea bruchid gut proteases play a dual role; digesting protein for nutrient needs and protecting insects by inactivating dietary proteins that may otherwise be toxic. Our results also suggest that transgenic resistance strategies that involve multigene products are likely to have enhanced efficacy and durability.

Arabidopsis carboxyl-terminal domain phosphatase-like isoforms share common catalytic and interaction domains but have distinct in planta functions.

An Arabidopsis (Arabidopsis thaliana) multigene family (predicted to be more than 20 members) encodes plant C-terminal domain (CTD) phosphatases that dephosphorylate Ser residues in tandem heptad repeat sequences of the RNA polymerase II C terminus. CTD phosphatase-like (CPL) isoforms 1 and 3 are regulators of osmotic stress and abscisic acid (ABA) signaling. Evidence presented herein indicates that CPL3 and CPL4 are homologs of a prototype CTD phosphatase, FCP1 (TFIIF-interacting CTD-phosphatase). CPL3 and CPL4 contain catalytic FCP1 homology and breast cancer 1 C terminus (BRCT) domains. Recombinant CPL3 and CPL4 interact with AtRAP74, an Arabidopsis ortholog of a FCP1-interacting TFIIF subunit. A CPL3 or CPL4 C-terminal fragment that contains the BRCT domain mediates molecular interaction with AtRAP74. Consistent with their predicted roles in transcriptional regulation, green fluorescent protein fusion proteins of CPL3, CPL4, and RAP74 all localize to the nucleus. cpl3 mutations that eliminate the BRCT or FCP1 homology domain cause ABA hyperactivation of the stress-inducible RD29a promoter, whereas RNAi suppression of CPL4 results in dwarfism and reduced seedling growth. These results indicate CPL3 and CPL4 are a paralogous pair of general transcription regulators with similar biochemical properties, but are required for the distinct developmental and environmental responses. CPL4 is necessary for normal plant growth and thus most orthologous to fungal and metazoan FCP1, whereas CPL3 is an isoform that specifically facilitates ABA signaling.

Arabidopsis C-terminal domain phosphatase-like 1 and 2 are essential Ser-5-specific C-terminal domain phosphatases.

Transcription and mRNA processing are regulated by phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II, which consists of tandem repeats of a Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptapeptide. Previous studies showed that members of the plant CTD phosphatase-like (CPL) protein family differentially regulate osmotic stress-responsive and abscisic acid-responsive transcription in Arabidopsis thaliana. Here we report that AtCPL1 and AtCPL2 specifically dephosphorylate Ser-5 of the CTD heptad in Arabidopsis RNA polymerase II, but not Ser-2. An N-terminal catalytic domain of CPL1, which suffices for CTD Ser-5 phosphatase activity in vitro, includes a signature DXDXT acylphosphatase motif, but lacks a breast cancer 1 CTD, which is an essential component of the fungal and metazoan Fcp1 CTD phosphatase enzymes. The CTD of CPL1, which contains two putative double-stranded RNA binding motifs, is essential for the in vivo function of CPL1 and includes a C-terminal 23-aa signal responsible for its nuclear targeting. CPL2 has a similar domain structure but contains only one double-stranded RNA binding motif. Combining mutant alleles of CPL1 and CPL2 causes synthetic lethality of the male but not the female gametes. These results indicate that CPL1 and CPL2 exemplify a unique family of CTD Ser-5-specific phosphatases with an essential role in plant growth and development.

Inorganic cations mediate plant PR5 protein antifungal activity through fungal Mnn1- and Mnn4-regulated cell surface glycans.

Antimicrobial activities of many defense proteins are profoundly altered by inorganic cations, thereby controlling disease pathologies in a number of mammalian systems, such as cystic fibrosis in humans. Protein-based active defense systems in plants also are influenced by cations; however, little is known of how these cation effects are mediated. Cytotoxicity of the pathogenesis-related protein osmotin against the model fungus Saccharomyces cerevisiae was progressively abolished by K+. By the use of S. cerevisiae mannosylation mutants, this effect was shown to require mannosephosphate residues in the cell wall. However, osmotin activity was not suppressed by even high concentrations of Ca2+. Rather, submillimolar levels of Ca2+ specifically facilitated osmotin's activity, as well as its binding to the cell surface. This effect also was dependent on mannosephosphate groups on the cell surface, and appeared to require negative charge on a portion of the osmotin protein. Results suggest that Ca2+ modulates osmotin action by facilitating its binding to the fungal cell surface, but that K+ blocks this interaction by competing for binding to mannosephosphate groups. Therefore, we have identified glycan interaction as a mechanism for antimicrobial protein activity modulation by cations, a pattern that may apply to diverse innate defense responses.

Soyacystatin N inhibits proteolysis of wheat alpha-amylase inhibitor and potentiates toxicity against cowpea weevil.

Genetic engineering may be used to introduce multiple insect resistance genes with different modes of action into crop plants. We explored the possible interactions of two differing gene products fed in the diet of cowpea weevil, Callosobruchus maculates (F.), a stored grain pest. The soybean cysteine protease inhibitor soyacystatin N (scN) and alpha-amylase inhibitor (alphaAI) from wheat have defensive function against this coleopteran. When artificial seeds containing both scN and alpha(AI) were infested with eggs of C. maculatus, the delays in larval development were longer than was predicted by summing the developmental delays seen when larvae were fed a diet containing the individual proteins, indicating that the effects of scN and alpha(AI) are synergistic. Alpha(AI) was readily hydrolyzed when incubated with insect gut extract. This proteolytic degradation was inhibited by scN, but not by Kunitz inhibitor (a serine protease inhibitor). Thus, degradation of alpha(AI) was due to proteolysis by insect digestive cysteine proteases. These data suggest that C. maculatus uses digestive enzymes not only to function in food protein digestion but also to defend the insects themselves by helping reduce the concentration of a toxic dietary protein.