Manipulation of endogenous trypsin proteinase inhibitor production in Nicotiana attenuata demonstrates their function as antiherbivore defenses

Evidence for the in planta defensive function of trypsin protease inhibitors (TPIs) comes from observations of enhanced herbivore resistance after heterologous TPI expression or the manipulation of signal cascades that activate numerous defense responses, including TPI production; no studies have altered the expression of an endogenous pi gene to examine defensive function. We isolated two genes with seven- and six-repeat TPI domains from Nicotiana attenuata from the potato (Solanum tuberosum) PI-II family. To determine whether endogenous TPIs in N. attenuata function defensively against the native herbivores, hornworm (Manduca sexta) and mirids (Tupiocoris notatus), we expressed 175 bp of the seven-domain pi from N. attenuata in an antisense orientation in a TPI-producing genotype to reduce TPI expression and expressed the full-length seven-domain pi in a sense orientation under control of a constitutive promoter to restore TPI activity in a natural genotype from Arizona unable to produce TPIs. Constitutive and inducible TPI production in two antisense lines were diminished by 80% to 90% and 33% to 52%, respectively, and sense expression restored 67% of the activity found in the TPI-producing genotype after caterpillar attack in the TPI-deficient A genotype. Hornworm larvae fed on genotypes with low or no TPI activity grew faster, had higher survivorship, and produced heavier pupae than those that fed on genotypes with high TPI activity. T. notatus showed higher preference for genotypes with low or no TPI activity than for genotypes with high TPI levels. We conclude that endogenous TPIs are an effective defense against these native herbivores.     

Nonsense-mediated mRNA decay (NMD) silences the accumulation of aberrant trypsin proteinase inhibitor mRNA in Nicotiana attenuata

In eukaryotes, genes carrying premature termination codons (PTCs) are often associated with decreased mRNA levels compared with their counterparts without PTCs. PTC-harboring mRNA is rapidly degraded through the nonsense-mediated mRNA decay (NMD) pathway to prevent the accumulation of potentially detrimental truncated proteins. In a native ecotype of Nicotiana attenuata collected from Arizona (AZ), the mRNA levels of a trypsin proteinase inhibitor ( TPI ) gene are substantially lower than in plants collected from Utah (UT). Cloning the AZ TPI gene revealed a 6 bp deletion mutation in exon 2 resulting in a PTC and decreased mRNA levels through NMD. Silencing UPF1 , 2 and 3 in N. attenuata AZ plants by virus-induced gene silencing (VIGS) enhanced the levels of PTC-harboring TPI mRNA, demonstrating a conserved role for UPF genes in plants. Furthermore, using cell suspension cultures that express variants of the TPI construct, we demonstrate that both intron-containing and intronless genes are subject to NMD in plants; unlike PTCs in mammals, PTCs downstream of introns activate NMD in plants. However, when a PTC is only 4 bp upstream of an intron, the NMD surveillance mechanism is abrogated. We also demonstrate that, in an intronless TPI gene, a PTC located at the beginning or the end of the coding sequence triggers NMD less efficiently than do PTCs located at the middle of the coding sequence. Taken together, these results highlight the complexity of the NMD activation mechanisms in plants.     

Induced responses in Nicotiana attenuata affect behavior and growth of the specialist herbivore Manduca sexta

Many plants employ induced responses against generalist herbivores. Specialist herbivores, however, may employ several mechanisms to overcome the negative effects of induced plant defenses. Here we test how the behavior and development of specialist Manduca sexta larvae are affected by induced responses in their natural host plant Nicotiana attenuata. On a spatial scale relevant to both the plant and the herbivore, we first determined how methyl jasmonate (MeJA)-induced responses, such as increased nicotine production, affect the tendency of larvae to leave induced plants. When larvae were allowed to move between two plants planted in one pot, they left an MeJA-treated plant faster than a control plant. When both plants in the pot were MeJA-treated, the larvae developed more slowly than when both plants were uninduced, or when the larvae had the opportunity to move to an uninduced neighbor. The sooner larvae moved from an MeJA-treated plant to an untreated neighbor, the larger the body mass they attained. This demonstrates that M. sexta larvae can compensate behaviorally for the deleterious effects of induced plant responses. These effects were observed in plants grown under both low and high N supply rates, though the effects were more pronounced under high N. To examine the consequences of the timing and the direction of the host plant switching behavior for larval development, neonate larvae were fed leaves excised from induced and uninduced plants. Larvae confined to MeJA-treated leaves had higher mortality rates and grew slower than larvae fed only control leaves. This demonstrates that MeJA-induced responses decrease growth and development of specialist herbivores that do not have the behavioral option of moving to an uninduced plant. The sooner the larvae were switched to MeJA-treated leaves, the slower their development compared to larvae fed only uninduced leaves. In contrast, the sooner larvae fed MeJA-treated leaves were switched to control leaves, the faster they developed. Again the effects of MeJA treatment were stronger in plants grown under high N supply. We propose that induced plants growing in close competition with an uninduced conspecific may offset the fitness costs of these induced responses and perhaps obtain a fitness benefit by motivating herbivores to move to their neighboring competitors. Received: 25 March 1999 / Accepted: 8 October 1999     

Instar-specific sensitivity of specialist Manduca sexta larvae to induced defences in their host plant Nicotiana attenuata

1. The time delay associated with the activation of induced defences is thought to be a liability for this type of defence because it allows herbivores to remove biomass before the defence is fully induced. When defences are costly and plants grow with competitors, however, it may be more advantageous not to induce defences too fast and motivate the herbivore to move to the neighbour when it is most voracious. 2. Such a strategy can only work when the costs for the herbivore of moving to a neighbouring plant are smaller than the costs of staying on a fully induced plant. For lepidopteran herbivores, both the sensitivity to induced defences and the costs of moving may vary considerably between instars and this variation may constrain the plant's defensive opportunities. 3. This study was designed to examine whether the cost of moving, mimicked by a starvation period of 8 h, was larger than the cost of staying on a fully induced plant for each larval instar of the specialist Manduca sexta feeding on induced and control tissues of Nicotiana attenuata. 4. For first‐ and second‐instar larvae, the costs of moving were larger than the costs of staying on a fully induced plant. In contrast, feeding on induced plant material retarded development in third‐instar larvae more than did starvation, indicating that in this instar the costs of leaving are smaller than the costs of staying on an induced plant. More than 98% of the lifetime leaf mass consumed by a M. sexta larva is consumed during the fourth and fifth instars, and during these instars larval development was not affected by either induced defences or starvation. Thus the third instar, the stage just before larvae cause the majority of damage, represents a window of sensitivity to induced defences during which larvae can be motivated to change plants. 5. These results suggest that N. attenuata plants, which commonly compete with conspecifics in nature, have the opportunity to manipulate the behaviour of the specialist herbivore M. sexta to minimise the fitness effects of inducing defences when these defences are most costly, i.e. when plants grow under intraspecific competition.     

Differential elicitation of two processing proteases controls the processing pattern of the trypsin proteinase inhibitor precursor in Nicotiana attenuata

Trypsin proteinase inhibitors (TPIs) of Nicotiana attenuata are major antiherbivore defenses that increase dramatically in leaves after attack or methyl jasmonate (MeJA) elicitation. To understand the elicitation process, we characterized the proteolytic fragmentation and release of TPIs from a multidomain precursor by proteases in MeJA-elicited and unelicited plants. A set of approximately 6-kD TPI peptides was purified from leaves, and their posttranslational modifications were characterized. In MeJA-elicited plants, the diversity of TPI structures was greater than the precursor gene predicted. This elicited structural heterogeneity resulted from differential fragmentation of the linker peptide (LP) that separates the seven-domain TPI functional domains. Using an in vitro fluorescence resonance energy transfer assay and synthetic substrates derived from the LP sequence, we characterized proteases involved in both the processing of the TPI precursor and its vacuolar targeting sequence. Although both a vacuolar processing enzyme and a subtilisin-like protease were found to participate in a two-step processing of LP, only the activity of the subtilisin-like protease was significantly increased by MeJA elicitation. We propose that MeJA elicitation increases TPI precursor production and saturates the proteolytic machinery, changing the processing pattern of TPIs. To test this hypothesis, we elicited a TPI-deficient N. attenuata genotype that had been transformed with a functional NaTPI gene under control of a constitutive promoter and characterized the resulting TPIs. We found no alterations in the processing pattern predicted from the sequence: a result consistent with the saturation hypothesis.     

Nicotiana attenuata LECTIN RECEPTOR KINASE1 suppresses the insect-mediated inhibition of induced defense responses during Manduca sexta herbivory

Nicotiana attenuata has the capacity to respond specifically to herbivory by its natural herbivore, Manduca sexta, through the perception of elicitors in larval oral secretions. We demonstrate that Lectin receptor kinase 1 (LecRK1) functions during M. sexta herbivory to suppress the insect-mediated inhibition of jasmonic acid (JA)-induced defense responses. Gene function analysis performed by reducing LecRK1 expression in N. attenuata by both virus-induced gene silencing and inverted repeated RNA interference (ir-lecRK1 plants) revealed that LecRK1 was essential to mount a full defense response against M. sexta folivory; larvae growing on ir-lecRK1 plants were 40 to 100% larger than those growing on wild-type plants. The insect-induced accumulation of nicotine, diterpene-glucosides, and trypsin protease inhibitors, as well as the expression of Thr deaminase, was severalfold reduced in ir-lecRK1 plants compared with the wild type. The accumulation of JA and JA-Ile was unaffected during herbivory in ir-lecRK1 plants; however, salicylic acid (SA) accumulation was increased by twofold. The expression of nahG in ir-lecRK1 plants prevented the increased accumulation of SA and restored the defense response against M. sexta herbivory. The results suggest that LecRK1 inhibits the accumulation of SA during herbivory, although other mechanisms may also be affected.     

Azadirachtin inhibits secretion of trypsin in midgut of Manduca sexta caterpillars: reduced growth due to impaired protein digestion

When given by injection to tobacco hornworm caterpillars, Manduca sexta, the allelochemical azadirachtin inhibits growth without reducing food intake. The growth reducing effect of azadirachtin is therefore in this case independent of the compound's well-known antifeedant effect. The cause of this reduced rate of growth is an increase in the costs associated with growth. These increased costs are largely a consequence of a decrease in the efficiency of utilisation of dietary nitrogen. This is associated with a drastic reduction in the activity of midgut trypsin. Azadirachtin has no effect on the activity of trypsin in vitro. Thus azadirachtin directly or indirectly inhibits the production of trypsin by the enzyme-secreting cells of the midgut wall; it is suggested that this is the cause of the increased costs and reduced rate of growth. The interesting parallel between this plant defence strategy and that of direct inhibition of herbivore proteinases by allelochemical proteinase inhibitors is discussed.     

Identification of plasma proteinase complexes with serpin-3 in Manduca sexta

Extracellular serine proteinase cascades stimulate prophenoloxidase (proPO) activation and antimicrobial peptide production in insect innate immune responses. Serpins in plasma regulate such cascades by selective inhibition of proteinases, in reactions which result in the formation of covalent serpin-proteinase complexes. We carried out experiments to identify plasma proteinases that are inhibited by Manduca sexta serpin-3, an immune-inducible serpin known to regulate proPO activation. Immunoaffinity chromatography, using antiserum to serpin-3, yielded serpin-3 complexes with proteinases identified by immunoblot analysis as prophenoloxidase-activating proteinase (PAP)-1, PAP-2, PAP-3, and hemolymph proteinase 8 (HP8). HP8 can cleave and activate the Toll ligand, Spätzle, leading to synthesis of antimicrobial peptides. Analysis by mass spectrometry of tryptic peptides derived from the serpin-3 complexes confirmed the presence of PAP-1, PAP-3, and HP8. Purified recombinant serpin-3 and active HP8 formed an SDS-stable complex in vitro. Identification of serpin-3-proteinase complexes in plasma provides insight into proteinase targets of serpin-3 and extends the understanding of serpin/proteinase function in the immune response of M. sexta.     

A comparison of two Nicotiana attenuata accessions reveals large differences in signaling induced by oral secretions of the specialist herbivore Manduca sexta

Genetic variation within and among populations provides the raw material for evolution. Although many studies describe inter- and intraspecific variation of defensive metabolites, little is known about variation among plant populations within early signaling responses elicited by herbivory or by herbivore oral secretions (OS) introduced into wounds during feeding. In this study, we compare the OS-elicited early responses as well as the antiherbivore defensive metabolites in two accessions of the wild tobacco Nicotiana attenuata and show that, compared with an accession collected from Utah, an Arizona accession has lower herbivore-elicited activity of the salicylic acid-induced protein kinase, an important mitogen-activated protein kinase involved in herbivore resistance. These differences in salicylic acid-induced protein kinase activity were associated with substantially different levels of OS-elicited jasmonic acid, jasmonic acid-isoleucine conjugate, and ethylene bursts. Gene expression level polymorphism (ELP) determines phenotypic variation among populations, and we found the two accessions to have significantly different ELPs in the genes involved in early signaling responses to herbivory. In addition, we found differences between the Utah and the Arizona accessions in the concentrations of several secondary metabolites that contribute to N. attenuata's direct and indirect defenses. This study demonstrates significant natural variation in regulatory elements that mediate plant responses to herbivore attack, highlighting the role of ELP in producing a diversity of plant defense phenotypes.     

Purification and characterization of Manduca sexta serpin-6: a serine proteinase inhibitor that selectively inhibits prophenoloxidase-activating proteinase-3

The proteolytic activation of prophenoloxidase (proPO) is a critical defense mechanism in insects and crustaceans. We have isolated three prophenoloxidase-activating proteinases (PAPs) from cuticular extracts or hemolymph of Manduca sexta pharate pupae, which are negatively regulated by serpin-1J and serpin-3. To test if other serpins may also inhibit the PAPs, we fractionated the induced hemolymph by ammonium sulfate precipitation, gel filtration, and lectin affinity chromatography. A 47 kDa protein, designated M. sexta serpin-6, was identified in concanavalin A-bound fractions, which formed an SDS-stable complex with PAP-3. This inhibitor, not recognized by the serpin-1 or serpin-3 antibodies, was further purified on HPLC anion exchange and hydroxylapatite columns. The molecular mass and isoelectric point of serpin-6 were found to be 46,710 +/- 10 Da and 5.4. While its amino terminus was blocked, we obtained five internal peptide sequences, one of which is highly similar to M. sexta serpins-1, -2, and -3. Serpin-6 strongly inhibited PAP-3 but not PAP-1 or PAP-2, suggesting that the proPO activation by PAPs is differentially regulated by multiple serpins. When included in the reaction mixture containing proPO, PAP-3, and its cofactor, serpin-6 efficiently blocked the cleavage activation of proPO.     

The viral protein Egf1.0 is a dual activity inhibitor of prophenoloxidase-activating proteinases 1 and 3 from Manduca sexta

Some pathogens are capable of suppressing the melanization response of host insects, but the virulence factors responsible are largely unknown. The insect pathogen Microplitis demolitor bracovirus encodes the Egf family of small serine proteinase inhibitors. One family member, Egf1.0, was recently shown to suppress melanization of hemolymph in Manduca sexta in part by inhibiting the enzymatic activity of prophenoloxidase activating proteinase 3 (PAP3). However, other experiments suggested this viral protein suppresses melanization by more than one mechanism. Here we report that Egf1.0 inhibited the amidolytic activity of PAP1 and dose-dependently blocked processing of pro-PAP1 and pro-PAP3. Consistent with its PAP inhibitory activity, Egf1.0 also prevented processing of pro-phenoloxidase, serine proteinase homolog (SPH) 1, and SPH2. Isolation of Egf1.0-protein complexes from plasma indicated that Egf1.0 binds PAPs through its C-terminal repeat domain. Egf1.0 also potentially interacts with SPH2 and two other proteins, ferritin and gloverin, not previously associated with the phenoloxidase cascade. Overall, our results indicate that Egf1.0 is a dual activity PAP inhibitor that strongly suppresses the insect melanization response.     

Growth of Manduca sexta on wounded tomato plants: role of induced proteinase inhibitors

The effects of plant age and growing conditions on the wound-induced responses of tomatoes (Lycopersicon esculentum Mill) was studied. The impact of these variables on the suitability of tomato foliage as food was assessed both by growth rates of neonate Manduca sexta Johannson larvae and by radiometric assay for proteinase inhibitor concentration. Plant quality (as assessed by M. sexta growth rates) was decreased by wounding regardless of plant age or environmental conditions at the time of wounding. The decrease in quality was not necessarily associated with increased concentrations of proteinase inhibitor. When tested directly, high concentrations of proteinase inhibitor did not affect larval growth rates. Induction of foliar proteinase inhibitors in response to wounding was found to peak while the plants were young and decrease as the plants matured.

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Résumé Le feuillage de pieds de tomates (Lycospersicon esculentum Mill) a été endommagé à différents âges et dans différentes conditions écologiques. Les modifications de sa valeur alimentaire ont été testées avec des chenilles néonates de Manduca sexta Johannson et par des dosages radiométriques de sa concentration en inhibiteurs de protéïnase. Des plantes endommagées à 2 semaines provoquent une croissance larvaire plus lente que des plantes endommagées à 2 semaines provoquent une croissance larvaire plus lente que des plantes endommagées à 4 semaines. Des plantes soumises à une forte intensité lumineuse et à des températures élevées lors de leur altération provoquent une croissance larvaire plus lente que des plantes maintenues en serre pendant cette période. Les changements de la qualité des plantes mis en évidence par les taux de croissance larvaire n'étaient pas liés à l'importance de l'induction des inhibiteurs de protéïnase dans le feuillage. Quand les plantes sont endommagées à 3 jours d'intervalle, 10 à 30 jours après la germination, l'induction des inhibiteurs de protéïnase est maximale le 16^e jour et diminue ensuite. M. sexta se développe aussi bien sur les feuilles mûres de tomate, qu'elles contiennent ou non des concentrations élevées en inhibiteurs de protéïnase. Ces résultats montrent que la blessure a un impact négatif sur la valeur comme aliment pour M. sexta du feuillage de tomate, qui ne dépend pas de la quantité d'inhibiteurs de protéïnase induite dans les feuilles.

     

Characterization of two midgut proteinases of Helicoverpa armigera and their interaction with proteinase inhibitors

Two serine proteinases from the midgut of Helicoverpa armigera have been partially purified and characterized. One proteinase, HGP-1, was capable of hydrolyzing a synthetic substrate of elastase and was inhibited by elastatinal. The second proteinase, HGP-2, was inhibited by a trypsin inhibitor. Molecular weights of HGP-1 and HGP-2 were approximately 26.0 and 29.0kDa, respectively. Both the proteinases exhibited alkaline pH optima in the range of 10-11. Furthermore, interaction of HGP-1 and HGP-2 with proteinase inhibitors (PIs) from host and non-host plants was studied. HGP-1 was not only insensitive to a PI from chickpea (host) but was also able to degrade it. The same PI from chickpea was able to inhibit over 50% activity of HGP-2. On the contrary, PIs from potato (non-host) showed strong inhibition of both, HGP-1 and HGP-2 and also demonstrated protection of chickpea seed proteins from digestion by both the HGPs. These results could provide important clues in designing strategies for sustainable use of plant PIs in developing insect-tolerant transgenic plants.     

Human urinary proteinase inhibitor: inhibitory properties and interaction with bovine trypsin

The major urinary trypsin inhibitor (UTI) was found to inhibit bovine chymotrypsin and human leucocyte elastase strongly, cathepsin G weakly. No inhibition of porcine pancreatic elastase was observed. The stoichiometry of the inhibition of bovine trypsin by UTI was determined spectrophotometrically to be 1:2 (I/E molar ratio). After incubation of UTI with this enzyme in various molar ratios, two complexes (C1 and C2) could be visualized in alkaline polyacrylamide gel electrophoresis. C1 was isolated by affinity chromatography on Con-A Sepharose. In dodecyl sulfate polyacrylamide gel electrophoresis, C1 was dissociated to give an inhibitory band with the same electrophoretic mobility as native UTI. C2 released an active inhibitory fragment with Mr near 20000. A time-course study demonstrated that at a molar ratio I/E of 1.5:1, the C2 complex appears after two hours of incubation.     

Inhibition of digestive proteinases of stored grain Coleoptera by oryzacystatin, a cysteine proteinase inhibitor from rice seed

Electrophoresis of midgut extracts from the rice weevil, Sitophilus oryzae, and the red flour beetle, Tribolium castaneum, in polyacrylamide gels containing sodium dodecyl sulfate and gelatin revealed there was one major proteinase (apparent molecular mass = 40,000) in the rice weevil and two major proteinases (apparent molecular masses = 20,000 and 17,000) in the red flour beetle. The pH optima using [3H]casein as substrate were about pH 6.8 for the rice weevil and pH 5.2 for the red flour beetle. Use of specific inhibitors, including L-trans-epoxysuccinyl-leucylamino-(4- guanidino)-butane (E-64), p-chloromercuriphenylsulfonic acid (PCMS), and oryzacystatin, indicated that nearly all of the proteinase activity against casein was contributed by cysteine proteinases. The estimated IC50 values for oryzacystatin were 2 x 10(-6) M and 4 x 10(-7) M when tested against midgut extracts from T. castaneum and S. oryzae, respectively.     

Ecdysone 20-hydroxylation in Manduca sexta midgut: Kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases

The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20-monooxygenase (E20MO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The K_m⁵ (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 × 10⁻⁵ and 3.67 × 10⁻⁷ M, respectively. The V_max was 82.7 pmol/min/mg protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondrial E20MO has the higher V_max, at physiological ecdysone concentrations (10⁻⁷ − 10⁻⁸ M) it is only one-eighth to one-tenth as active as the microsomal enzyme. It is concluded that the microsomal E20MO is the primary, if not the only, enzyme involved in ecdysone 20-hydroxylation in M. sexta midgut. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.