Identification of six chymotrypsin cDNAs from larval midguts of Helicoverpa zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin inhibitor.

Lepidopteran insects like Helicoverpa zea and Agrotis ipsilon produce STI-insensitive trypsins in the midgut following ingestion of dietary plant proteinase inhibitors like STI [Broadway, R. M., J. Insect Physiol. 43(9) (1997) 855-874]. In this paper, the effects of dietary STI on a related family of midgut serine proteinases, the chymotrypsins, were investigated. STI-insensitive midgut chymotrypsins were detected in larvae of H. zea and A. ipsilon feeding on diets containing 1% STI while STI-sensitive chymotrypsins were present in larvae feeding on diets containing 0% STI. These chymotrypsins were unaffected by TPCK, a diagnostic inhibitor of mammalian chymotrypsins but were fully inhibited by chymostatin. Four midgut cDNA libraries were constructed from larvae of each species fed either 0% STI or 1% STI diets. Six full-length cDNAs(1) encoding diverse preprochymotrypsins were isolated (three from H. zea and three from A. ipsilon) with certain sequence motifs that set them apart from their mammalian counterparts. Northern blots showed that some chymotrypsin mRNA were detected at higher levels while others were down-regulated when comparing insects reared on 0% STI and 1% STI diets. Southern hybridizations suggested that (like mammals) both species contained several chymotrypsin genes. A full-length chymotrypsin gene(1) from H. zea was sequenced for the first time and the presence of four introns was deduced. A first time comparison of 5' upstream regions(1) from three chymotrypsin genes and two trypsin genes of A. ipsilon indicated the presence of putative TATA boxes and regulatory elements. However a lack of consensus motifs in these upstream regions suggested the likelihood of multiple trans factors for regulation of genes encoding digestive proteinases and a complex response mechanism linked to ingestion of proteinase inhibitors.

Transcriptional induction of diverse midgut trypsins in larval Agrotis ipsilon and Helicoverpa zea feeding on the soybean trypsin inhibitor.

Midgut trypsins insensitive to inhibition by the soybean trypsin inhibitor (STI) were found to be transcriptionally regulated in A. ipsilon and H. zea larvae feeding on STI, as demonstrated by injections with actinomycin, a transcriptional inhibitor, which abolished the production of these STI-insensitive trypsins. The induced, STI-insensitive trypsins differed from the constitutive, STI-sensitive trypsins in their susceptibility to inhibitors based on sizes, suggesting that the induced enzymes limited access to their active site by blocking bulky inhibitors. Twenty midgut cDNA fragments(1) were amplified using trypsin-specific PCR primers and at least twelve were shown to encode structurally diverse trypsins. High sequence diversity was observed for both the enzymes encoded by STI-induced mRNAs and those from larvae that had not been exposed to STI. Northern blots showed that midgut mRNAs hybridizing to various trypsin cDNA probes were either transcribed de novo or up-regulated following ingestion of STI. Southern hybridizations indicated the presence of multiple trypsin gene families in the insect genomes. The complete sequence of a trypsin gene(1) from A. ipsilon (AiT9) revealed the presence of three introns. Comparison of 5' upstream sequences(1) from AiT9 and AiT6 genes from A. ipsilon revealed putative TATA box and disparate regulatory motifs, within 500 bp of each translational start site.

Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors.

Ingestion of Kunitz soybean trypsin inhibitor (STI) by larval Helicoverpa zea, Agrotis ipsilon, and Trichoplusia ni extended the retention time of food in the digestive tract and increased the level of activity of proteolytic enzymes that were not susceptible to inhibition by STI. The level of enhancement of activity of STI-resistant (STI-R) enzyme(s) was directly influenced by the dosage and timing of exposure to STI. However, not all proteinase inhibitors (PIs) enhanced the level of proteinase inhibitor resistant (PI-R) enzymes, even if those PIs inhibited a significant proportion of enzyme activity. These findings suggest that a complex system may be responsible for the regulation of proteolytic enzymes in the midgut of larval Lepidoptera, and one hypothesis for this regulation is proposed.

Partial characterization of chitinolytic enzymes from Streptomyces albidoflavus.

Streptomyces albidoflavus NRRL B-16746 secreted three types of chitinolytic enzymes: N-acetyl-glucosaminidase, chitobiosidase and endochitinase. Optimal activity for all three types of enzymes occurred at pH 4-6; however 55-74% of the chitobiosidase and endochitinase activity was detectable at pH 8-10. Chitobiosidase activity originated from two strongly acidic (pI < 3.0) proteins with molecular mass of 27 kDa and 34 kDa, while endochitinase activity originated from five major acidic proteins (pI 5.1, 5.3, 5.75, 5.8-5.9 and 6.4) with molecular mass of 59, 45, 38.5, 27 and 25.5 kDa. Purified chitobiosidases significantly reduced spore germination and germ tube elongation of Botrytis cinerea and Fusarium oxysporum. Chitinolytic enzymes with significant activity at pH 4-10 may be used, transgenically, to reduce the growth and/or development of a broad spectrum of insects and fungi that are major economic pests.

Influence of cabbage proteinase inhibitorsin situ on the growth of larvalTrichoplusia ni andPieris rapae.

Trypsin and chymotrypsin inhibitors are proteins that are developmentally regulated in foliage of cabbage plants, appearing at high concentrations in young foliage on mature plants. This temporal and spacial regulation of foliar proteinase inhibitors is synchronized with the appearance and distribution of foliar feeding Lepidoptera. When insects were allowed to select their feeding sites, larvalPieris rapae fed on the young foliage of cabbage plants, while larvalTrichoplusia ni fed on the mature foliage on cabbage plants. LarvalP. rapae that fed on mature plants were significantly smaller than larvae feeding on young plants, while there was no significant difference between larvalT. ni feeding on mature plants and those feeding on young plants. Thus, there was a significant inverse correlation between the level of proteinase inhibitory activity in cabbage foliage and larval growth. WhenP. rapae andT. ni were provided with an artificial diet containing total protein (including significant levels of proteinase inhibitors) that was extracted from cabbage foliage, there was a significant reduction in growth and development of both species of Lepidoptera.

Isolation and characterization of a novel, developmentally regulated proteinase inhibitor I protein and cDNA from the fruit of a wild species of tomato.

A novel member of the proteinase Inhibitor I family having a trypsin inhibitor specificity was isolated from the fruit of the wild tomato species Lycopersicon peruvianum (L.) Mill. (LA 107) and characterized. The protein is among the isoinhibitors of Inhibitor I that comprise 50% of the soluble proteins in the fruit of this wild species of tomato. A cDNA corresponding to the inhibitor protein and mRNA was isolated and characterized. The Inhibitor I mRNA represented 0.06% of the poly(A) RNA and gene copy number reconstruction experiments gave an estimate of two to four genes/haploid genome. The open reading frame of the cDNA codes for a protein of 111 amino acids having a 42-amino acid prepropolypeptide. The NH2-terminal sequence of the first 21 amino acids of the purified Inhibitor I protein confirmed that the cDNA was identical to the protein. The amino acid sequence of the L. peruvianum fruit Inhibitor I exhibits 74% identity with the wound-inducible Inhibitor I from tomato leaves. Whereas all previously identified members of the Inhibitor I family have either Met, Leu, or Asp at the P1 site and can inhibit enzymes such as chymotrypsin, subtilisin, and elastase, the fruit Inhibitor I possesses Lys at the P1 position. Thus, this is the first member of the extensive Inhibitor I family from plants and animals that exhibits trypsin inhibitory specificity. The presence of this inhibitor in wild tomato fruit may reflect a functional role to protect the tissues against herbivory.

Characterization and ecological implications of midgut proteolytic activity in larvalPieris rapae andTrichoplusia ni.

In their larval luminal midgut fluid,Trichoplusia ni (Lepidoptera: Noctuidae) andPieris rapae (Lepidoptera: Pieridae) contain endopeptidases as their primary proteases. Neither species has detectable exopeptidase activity. Studies using enzyme-specific substrates and inhibitors demonstrate that the endopeptidases are serine proteinases (both trypsinlike and chymotrypsinlike) with histidine at the active site. Optimal pH for the tryptic and chymotryptic activity is 8.5 and 8.0, respectively, forT. ni. and 8.0 and 9.0, respectively, forP. rapae. The efficiency of proteolytic digestion (as measured by the rate of in vitro digestion of a standard protein by the midgut luminal fluid) is positively correlated with the larval dietary protein requirement and is significantly influenced by the ratios of tryptic to chymotryptic activity present in the gut lumen of these two species of Lepidoptera.

Plant and fungal cell wall fragments activate expression of proteinase inhibitor genes for plant defense.

Plant and fungal cell wall fragments produced by enzymic degradation during pest attacks are hypothesized to be activators of a universal recognition system for locally and systemically activating genes which control the synthesis of plant defense chemicals such as the antibiotic phytoalexins and antinutritive proteinase inhibitors. Proteinase inhibitor cDNAs have been prepared from wound-induced mRNAs, isolated, and characterized. The cDNAs have been utilized to quantify specific proteinase inhibitor mRNAs in leaves following wounding or simulated insect attacks. The cDNAs have also been utilized as hybridization probes to isolate and characterize proteinase inhibitor genes from tomato and potato genomic DNA. Proteinase inhibitor proteins have been induced in tomato leaves by chewing insects and shown to be highly correlated with a systemically mediated reduction in the nutritional quality of the leaves toward the larval noctuidSpodoptera exigua. Thus, the wound-induced proteinase inhibitors, whose genes in tomato leaves can be activated by wounding, insect attacks, and plant and fungal cell wall fragments, can significantly decrease the quality of the leaves for such herbivorous insects. This inducible set of biochemical reactions leading to the de novo biosynthesis of proteinase inhibitors is, therefore, considered to be a potentially important defense of plant leaves that should be considered both in developing general theories on insect-plant interactions and in selecting insect-resistant crop varieties.