| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online February 19, 2004; 10.1104/pp.103.035634 Plant Physiology 134:1181-1190 (2004) © 2004 American Society of Plant Biologists Manipulation of Endogenous Trypsin Proteinase Inhibitor Production in Nicotiana attenuata Demonstrates Their Function as Antiherbivore Defenses1,[w]Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Jena 07745, Germany
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.
Plant proteinase inhibitors (PIs) are polypeptides or proteins that occur naturally in a wide range of plants and are considered to be an essential part of the plant's natural defense system against herbivores (Ryan, 1990  ; Jongsma and Bolter, 1997; Schuler et al., 1998). Evidence for the in planta defense function of PIs comes from two types of experiments: heterologous expression of pi genes (Hilder et al., 1987; Johnson et al., 1989; McManus et al., 1994; Duan et al., 1996) and manipulation of signal cascades affecting PI elicitors (e.g. Orozco-Cardenas et al., 1993). PIs have been found to reduce herbivore growth in plants that were transformed with a heterologous pi gene in at least 45 studies (e.g. Hilder et al., 1987; Johnson et al., 1989; McManus et al., 1994; Duan et al., 1996). Surprisingly, we know of no studies that have altered the expression of an endogenous pi gene to examine its defensive function.
Although heterologous expression studies have demonstrated reduced herbivore performance, two complications temper the extrapolation of these results to the conclusion that PIs are natural defenses against herbivores that normally feed on plants (Johnson et al., 1989
The second line of evidence for PI defensive function comes from experiments in which transgenic suppression of the wound signal cascades that elicit PI production enhances the performance of herbivores compared with those herbivores that feed on plants with intact signaling (McGurl et al., 1992
Ideally, one should determine the benefits of an endogenous pi in plants that differ only in a gene that controls the expression of a resistance trait but are otherwise identical (Bergelson and Purrington, 1996
Nicotiana attenuata Torr. Ex Wats., a postfire annual inhabiting the Great Basin Desert, has a number of well-described herbivore-induced direct and indirect defenses (Baldwin, 2001 Here, we report the cloning of two multidomain TPI genes belonging to potato PI-II family from N. attenuata plants collected in Utah, and we provide a critical test of whether endogenous TPIs in N. attenuata function defensively against two specialist herbivores, hornworm and mirids. We conducted bioassay experiments comparing hornworm larval performance and mirid colonization preference for plants with low or no TPI activity. We used two independently transformed N. attenuata lines in which the expression of the pi gene was down-regulated by antisense expression of a 175-bp fragment of the N. attenuata pi gene (AS–and AS-), one line independently transformed with an empty-vector construct (C), and untransformed wild-type plants (WT) of the same genetic background (an inbred line collected from Utah). In addition, we used a natural N. attenuata genotype collected from Arizona that has a mutation in the endogenous seven-domain pi gene and does not produce pi transcripts or TPI activity (A) and that we transformed with the full-length cDNA of the seven-domain pi gene in a sense orientation under control of a constitutive promotor (S++) so that it produced TPIs at 67% of the level found in the WT Utah genotype after caterpillar attack.
Tobacco Produces a Seven-Domain TPI Belonging to the Potato PI-II Family
Earlier results with PI activity assays of N. attenuata plants revealed that TPI activity is strongly increased after methyl jasmonate (MeJA) elicitation (van Dam and Baldwin, 2001
Multiple sequence alignments of a single PI repeat (T2) with the corresponding repeats of potato PI-II family members found in other Solanaceous species reveal a high degree of amino acid identity, including the conserved cysteines involved in the four disulfide bridges and the conserved Pro (Fig. 1) that characterize all 77 known PI-II repeat sequences (Antcheva et al., 2001
Genomic DNA of N. attenuata was completely digested by EcoRI, HindIII, EcoRV, and SspI enzymes and was hybridized with a radiolabeled plasmid containing the repeat domain of the tobacco PI precursor (Supplemental Fig. 2). The analysis revealed at least two bands and suggested that TPIs exist in a multigene family in N. attenuata. To isolate the additional gene(s), primer pairs corresponding to the 5' and 3' regions were synthesized and used in reverse transcriptase-PCR of mRNA extracted from MeJA-elicited leaves. The resultant clone was sequenced and found to encode a six-domain PI highly homologous to the earlier isolated PI. In addition to the deletion of one repeat domain, the "new" PI also differed by a single amino acid in the signal peptide region (Fig. 1).
To determine the constitutive and caterpillar-inducible levels of TPI mRNA of the transformed lines, northern-blot analysis was performed on total RNA from transformed lines (AS-, AS–, S+, S++, C, and AC) and untransformed genotypes (WT and A). Analysis of unattacked leaves by larvae from untransformed WT and the line transformed with empty vector construct (C) revealed a 1.4-kb TPI transcript that increased 4-fold 24 h after the larvae started to feed on the leaf (Fig. 2A). Although TPI transcripts were not detectable in AS–, not even after caterpillar damage, intermediate levels were found in the AS-line (Fig. 2A). TPI mRNA in A as well as in the AC line (A independently transformed with an empty-vector construct), which lacks the ability to produce TPIs (Glawe et al., 2003
Endogenous leaf TPI activity was determined before and 3 d after larvae started to feed on the node +1 leaf of plants from transformed and untransformed genotypes. Compared with the constitutive levels of TPI activity in the WT and C plants (which did not differ significantly; F1,38= 0.04; P = 0.8434), levels in AS–and AS-plants were 90% and 33% lower, respectively (Fig. 2A; F3,76= 90.640; P < 0.0001). Caterpillar damage increased TPI activity 4-fold in WT and C plants, whereas AS–and AS-TPI levels were also increased after caterpillar damage and TPI levels in these two genotypes were 17% and 48% of those found in attacked WT plants (Fig. 2A; F3,76= 49.434; P < 0.0001). Caterpillar attack did not alter TPI activity in S++ and S+ plants (F1,38-S++= 3.744; P = 0.06; F1,38-S+= 0.015; P = 0.9044), which remained at approximately 67% and 4% of the induced WT plants, respectively (Fig. 2, A and B; F2,57= 122.655; P < 0.0001). As expected, the untransformed A and the transformed AC genotypes showed no TPI activity even after caterpillars had fed on the plant for 24 h (Fig. 2B). Protein levels were not significantly different among genotypes.
Because chymotrypsin can be inhibited by TPI (Moura and Ryan, 2001 Real-time PCR analysis was used to quantify the increases in the TPI transcripts in response to caterpillar elicitation. WT and C plants showed similar responses to caterpillar attack; TPI mRNA expression increased 7-fold after 24 h of caterpillar damage in the attacked leaf (+1) and 4-fold in the undamaged systemic leaf (-1), compared with undamaged WT plants (Fig. 3A). Constitutive expression of TPI mRNA was lower in AS–(30% of the undamaged WT) than in AS-(50% of the undamaged WT; Fig. 3B). After caterpillars fed on antisense plants for 24 h, the expression of TPI mRNA increased 2- and 1.5-fold in AS–and 5-and 2-fold in AS-lines in the local and systemic leaves, respectively, compared with undamaged WT (Fig. 3B). Caterpillar attack had a small effect on TPI mRNA transcripts in S++ (2-fold) and S+ (0.5-fold) genotypes during the 48-h feeding period, compared with the levels found in undamaged WT leaves growing at the same nodal positions (Fig. 3C). Surprisingly, 24 h after caterpillar damage, the relative expression of TPI mRNA in the systemic leaves decreased transiently in the S++ genotype to only 1.1-fold of that found in the undamaged WT (Fig. 3C), perhaps due to general metabolic stress resulting from caterpillar attack.
To determine whether endogenous TPIs in tobacco function defensively against hornworm and mirids, we assessed caterpillar performance and colonization preference of mirids on transformed and untransformed genotypes with low or no TPI activity (A, AC, S+, AS–, and AS-) and high TPI activity (WT, C, and S++). Hornworm larval mass gain per day, survivorship, and pupal mass differed significantly between caterpillars fed on genotypes with high or low TPI activity (repeated-measures analysis of variance [ANOVA] on larval mass gain, F4,190-A-AS–AS-WT-C = 5.069; P = 0.0023; F4,170-A-AC-S+-S++-WT = 3.910; P = 0.0102; survivorship analysis in Table I; ANOVA on pupal mass, F4,51-A-AS–AS-WT-C = 32.370; P < 0.0001; F4,51-A-AC-S+-S++-WT = 8.613; P < 0.0001; Fig. 4, A and B). Within the first group of genotype comparisons (A, AS–, AS-, WT, and C), larvae fed on genotypes with low or no TPI activity (repeated-measures ANOVA, F2, 130-A-AS–AS- = 1.234; P = 0.3075) grew faster, had higher survivorship (Table I), and produced heavier pupae than those fed on genotypes with high TPI activity (repeated-measures ANOVA, F1, 60-C-WT = 0.024; P 0.8787; Fig. 4A). Between the 1st and 2nd d after caterpillar attack, the larval mass gain per day was 3.89 ± 0.41 mg d-1 (AS–), 4.22 ± 0.45 mg d-1 (AS-), 1.42 ± 0.31 mg d-1 (WT), 3.71 ± 0.33 mg d-1 (A), and 1.57 ± 0.23 mg d-1 (C); data obscured by symbols (Fig. 4A). Larvae fed on AS-lines showed intermediate percentage survivorship (Table I); during the first 3 d, survivorship was similar to that on AS–lines, but after d 3 when plants had accumulated TPIs in response to larval feeding, survivorship was similar to that of larvae fed on WT and C genotypes (Fig. 4A).
Similar responses were found within the second group of genotype comparisons (A, AC, S+, S++, and WT). Larvae fed on S+ genotypes showed intermediate percentage survivorship, whereas larvae fed on A and AC showed the highest percentage survivorship, and those fed on S++ and WT genotypes the lowest percentage survivorship (Fig. 4B; Table I). Pupal mass and larval mass gain were higher on A, AC, and S+ (repeated-measures ANOVA, F2, 85-A-AC-S+ = 0.249; P = 0.7824) than on S++ and WT genotypes (repeated-measures ANOVA, F1, 85-S++-WT = 0.421; P = 0.5253; Fig. 4B). Between the 1st and 2nd d after caterpillar attack, the larval mass gain per day was 1.88 ± 0.46 mg d-1 (S++), 3.37 ± 0.63 mg d-1 (S+), 1.42 ± 0.23 mg d-1 (WT), 2.80 ± 0.33 mg d-1 (A), and 3.97 ± 0.31 mg d-1 (AC); data obscured by symbols (Fig. 4B).
Mirids are usually the first insect species to colonize tobacco in its native habitat, that is, the first growing season after fires in the Great Basin Desert (Glawe et al., 2003
We isolated two genes from N. attenuata coding for PI precursors that belong to the potato PI-II family. One codes for a 455-amino acid protein with a seven-repeat TPI domain, whereas the other codes for a 396-amino acid protein with a six-repeat TPI domain, both having a N-terminal signal peptide (Nielsen et al., 1997 ) and a C-terminal putative vacuolar targeting sequence (Nielsen et al., 1996; Fig. 1; Supplemental Fig. 1). The N. attenuata PI precursor shares a conserved amino acid sequence with that of N. alata (93%; Atkinson et al., 1993) and Nicotiana glutinosa (84%; Choi et al., 2000) PIs, including the Ala-Lys cleavage site for signal peptide cleavage and the position of the Cys residues in the repeat domains for the formation of disulphide linkages. Previously two-, four-, six-, and eight-domain PI-II precursors have been identified in other Solanaceous plants (Graham et al., 1985; Atkinson et al., 1993; Choi et al., 2000; Miller et al., 2000), and the diversity of repeats is thought to have evolved by crossing-over events within the genomes (Barta et al., 2002). How the seven-domain repeat arose is unclear, but two three-domain PI-II family members have been identified in tomato (Lycopersicon esculentum) and tobacco (Taylor et al., 1993; Balandin et al., 1995), and single repeat members have been identified in other plant families (Barta et al., 2002). It is possible that unequal crossing over among even-numbered members, or fusions of odd- and even-numbered members produced the seven-repeat domain member.
Transgenic manipulation of the ability to produce TPIs allowed us to determine whether endogenous TPIs function defensively in response to attack from native, and presumably adapted, herbivores. We used the seven-repeat TPI gene to down-regulate (1.5-and 3.5-fold) and restore (50% and 2-fold of the uninduced WT) the expression of the pi gene in the WT and A genotypes of N. attenuata, respectively (Fig. 2). TPI activity followed the patterns of TPI mRNA (Figs. 2 and 3) and increased 2-fold, 4 d after caterpillar attack (van Dam et al., 2001
The defensive function of TPIs begins with their affinity for insect proteinases (Laing and McManus, 2002
TPI expression alone is known to increase the mortality rate of herbivores, particularly for neonate larvae (McManus and Burguess, 1995 In summary, this research demonstrates that despite the ongoing evolutionary interaction between N. attenuata and its herbivores, TPIs remain an effective defense against mirids and hornworm.
Plant Material and Transformation
Nicotiana attenuata Torr. Ex Wats. (Solanaceae) used in this study was grown from seeds collected from Utah (Baldwin, 1998
A genotype of N. attenuata collected from Arizona (A) has MeJA-inducible nicotine production identical to that found in WT plants, but completely lacked the ability to produce TPIs or accumulate TPI mRNA (Glawe et al., 2003
Based on the Nicotiana alata TPI sequence (Atkinson et al., 1993
To determine the effect of down-regulation or restored expression of the pi gene in N. attenuata on caterpillar mass gain, survivorship, and pupal mass, two feeding experiments with different combinations of genotypes were performed with AS lines (AS–and AS-), untransformed genotypes (WT and A) and a transformed WT line with empty vector construct (C), or with A lines transformed to express the functional pi (S+ and S++), untransformed genotypes (WT and A), and a transformed A line with empty vector construct (C). Seeds were germinated in diluted liquid smoke solutions as described in Baldwin et al. (1994
To determine whether N. attenuata TPI inhibits chymotrypsin, 10 rosette stage plants were unwounded or wounded with a pattern wheel over the source-sink transition leaf surface (W), and 20 µL of hornworm OS was applied to the fresh puncture wounds (W + OS; Halitschke et al., 2000
To determine the colonization preference of mirids (Tupiocoris notatus; Hemiptera: Miridae), three pairs of genotypes with low or no TPI activity and high TPI levels (AS–versus WT, AS–versus C, and A versus S++) were placed in a greenhouse adjacent to N. attenuata plants infested with a mirids colony. We monitored the accumulation of the insects on the plants at 24 h and 3 d after the pairs of genotypes placement. These experiments were repeated twice.
Leaves attacked by larvae during the first 24 h (CAT) and leaves from plants without larvae (CON) from the same position (+1) were harvested for northern-blot analysis of TPI mRNA accumulation as described in Winz and Baldwin (2001 For real-time PCR analysis, leaves growing at nodes +1 and -1 (one position younger than the source-sink transition leaf) from CAT and +1 from CON treatments were harvest from three replicate plants at 0, 6, 12, 24, and 48 h after the larvae started to feed. The relative expression of TPI mRNA was compared with that of undamaged WT. The isolated RNA was quantified spectrophotometrically and was diluted to 300 ng µL-1. The diluted RNA was reverse transcribed (Applied Biosystems, Foster City, CA), and 10 ng of the reverse-transcribed template was used in a 25-µL PCR reaction containing 1x universal mix (Eurogentec, Brussels, Belgium), 300 nM forward (5'-TCAGGAGATAGTAAATATGGCTGTTCA-3') and reverse primers (5'-ATCTGCATGTTCCACATTGCTTA-3'), and 300 nM of FAM-(6-carboxyl-fluorescein) labeled Taqman probe (5'-TCCTTGCTCTCCTCCTCTTATTTGGAATGTCT-3') with 18s RNA (Eurogentec) as internal standard. Thermal cycling and detection was performed on a sequence detector (ABI Prism 7700; Applied Biosystems).
Data were analyzed with Stat View, version 5.0 (SAS, Cary, NC). The TPI, protein, and pupal mass were analyzed by ANOVA followed by Fisher's protected LSD post hoc comparisons in all experiments. For the survivorship analyses, we used the log-rank test for the global hypothesis of equality of survival distribution for hornworm, and performed the same test with a pairwise ranking of data using only two groups at a time (Zavala et al., 2001
We thank Michelle Lim for invaluable assistance in plant transformation. Received November 2, 2003; returned for revision November 30, 2003; accepted December 4, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.035634.
1 This work was supported by the Max Planck Gesellschaft.
[w] The online version of this article contains Web-only data. * Corresponding author; e-mail Baldwin{at}ice.mpg.de; fax 49–3641–571102.
Antcheva N, Pintar A, Patthy A, Simonscits A, Barta A, Tchorbanov B, Pongor SN (2001) Proteins of circularly permuted sequence present within the same organism: the major serine proteinase inhibitor from Capsicum annuum seeds. Protein Sci 10: 2280-2290 Atkinson AH, Heath RL, Simpson RJ, Clarke AE, Anderson MA (1993) Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors. Plant Cell 5: 203-213[Abstract] Awmack SC, Leather SR (2002) Host plant quality and fecundity in herbivorous insects. Annu Rev Entomol 47: 817-844[ISI][Medline] Balandin T, Vanderdoes C, Albert JMB, Bol JF, Linthorst HJM (1995) Structure and induction-pattern of a novel proteinase-inhibitor class-I gene of tobacco. Plant Mol Biol 27: 1197-1204[CrossRef][ISI][Medline]
Baldwin IT (1998) Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proc Natl Acad Sci USA 95: 8113-8118
Baldwin IT (2001) An ecology motivated analysis of plant-herbivore interactions in native tobacco. Plant Physiol 127: 1449-1458 Baldwin IT, Staszakkozinki L, Davidson R (1994) Up in smoke: smoke-derived germination cues for the post-fire annual Nicotiana attenuata Torr. Ex Watson. J Chem Ecol 20: 2345-2371 Barta E, Pintar A, Pongor SN (2002) Repeats with variations: accelerated evolution of the Pin2 family of proteinase inhibitors. Trends Genet 18: 600-603[Medline] Bergelson J, Purrington CB (1996) Surveying patterns in the cost of resistance in plants. Am Naturalist 148: 536-558[CrossRef][ISI]
Bergey DR, Howe GA, Ryan CA (1996) Polypeptide signaling for plant defensive genes exhibit analogies to defense signaling in animals. Proc Natl Acad Sci USA 93: 12053-12058 Bouchard E, Cloutier C, Michaud D (2003a) Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant. Mol Ecol 12: 2439-2446[Medline] Bouchard E, Michaud D, Cloutier C (2003b) Molecular interactions between an insect predator and its herbivore prey on transgenic potato expressing a cysteine proteinase inhibitor from rice. Mol Ecol 12: 2429-2437[CrossRef][Medline] Broadway RM (1995) Are insects resistant to plant proteinase inhibitors? J Insect Physiol 41: 107-116[CrossRef] Broadway RM (1996) Dietary proteinase inhibitors alter complement of midgut proteases. Arch Insect Biochm Physiol 32: 39-53 Broadway RM (1997) Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors. J Insect Physiol 43: 855-874[CrossRef][Medline] Charity JA, Anderson MA, Bittisnich DJ, Whitecross M, Higgins TJV (1999) Transgenic tobacco and peas expressing a proteinase inhibitor from Nicotiana alata have increased insect resistance. Mol Breed 5: 357-365 Choi D, Park JA, Seo YS, Chun YJ, Kim WT (2000) Structure and stress-related expression of two cDNAs encoding proteinase inhibitor II of Nicotiana glutinosa L. Biochim Biophys Acta 1492: 211-215[Medline] Cloutier C, Jean C, Fournier M, Yelle S, Michaud D (2000) Adult Colorado potato beetles, Leptinotarsa decemlineata, compensate for nutritional stress on Oryzacystatin I-transgenic potato plants by hypertrophic behavior and over-production of insensitive proteases. Arch Insect Biochm Physiol 44: 69-81[CrossRef] Colebatch G, Cooper P, East P (2002) cDNA cloning of a salivary chymotrypsin-like protease and the identification of six additional cD-NAs encoding putative digestive proteases from the green mirid, Creontiades dilitus (Hemiptera: Miridae). Insect Biochem Mol Biol 32: 1065-1075[Medline] Colebatch G, East P, Cooper P (2001) Preliminary characterization of digestive proteases of the green mirid, Creontiades dilitus (Hemiptera: Miridae). Insect Biochem Mol Biol 31: 415-423[Medline] De Leo F, Bonadé-Bottino M, Ceci LR, Gallerani R, Jouanin L (2001) Effects of mustard trypsin inhibitor expressed in different plants on three lepidopteran pests. Insect Biochem Mol Biol 31: 593-602[CrossRef][ISI][Medline] De Leo F, Gallerani R (2002) The mustard trypsin inhibitor 2 affects the fertility of Spodoptera littoralis larvae fed on transgenic plants. Insect Biochem Mol Biol 32: 489-496[Medline] Duan X, Li X, Xue Q, Abo-El-Saad M, Xu D, Wu R (1996) Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nat Biotechnol 14: 494-498[CrossRef][ISI][Medline] Gatehouse AMR, Norton E, Davison GM, Babbe SM, Newell CA, Gate-house JA (1999) Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleraceae: effects of plant protease inhibitors in vitro and in vivo. J Insect Physiol 45: 545-558[Medline] Glawe AG, Zavala JA, Kessler A, van Dam NM, Baldwin IT (2003) Ecological costs and benefits correlated with trypsin protease inhibitor production in Nicotiana attenuata. Ecology 84: 79-90[CrossRef][ISI]
Graham JS, Pearce G, Merryweather F, Titani K, Ericsson LH, Ryan CA (1985) Wound-induced proteinase inhibitors from tomato leaves: the cDNA-deduced primary structure of the pre-inhibitor II. J Biol Chem 260: 6561-6564
Green TR, Ryan CA (1972) Wound-induced proteinase inhibitor in plant leaves: a possible defense mechanism against insects. Science 175: 776-777 Halitschke R, Kessler A, Kahl J, A. L, Baldwin IT (2000) Ecophysiological comparison of direct and indirect defenses in Nicotiana attenuata. Oecologia 124: 408-417[CrossRef][ISI]
Halitschke R, U. Schittko, G. Pohnert, W. Boland, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata: Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses. Plant Physiol 125: 711-717 Haukioja E, Neuvonen S (1985) The relationship between size and reproductive potential in male and female Epirritia autumnata (Lep., Geometridae). Ecol Entomol 10: 267-270 Heath RL, McDonald G, Christeller JT, Lee M, Bateman K, West J, van Heeswijck R, Anderson MA (1997) Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pest. J Insect Physiol 43: 833-842[Medline]
Hermsmeier D, Schittko U, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata: large-scale changes in the accumulation of growth- and defense-related plant mRNAs. Plant Physiol 125: 683-700 Hilder VA, Gatehouse AMR, Sheerman SE, Barker RF, Boulter D (1987) A novel mechanism of insect resistance engineered in to tobacco. Nature 330: 160-163[CrossRef][ISI] Howe GA, Lightner J, Browse J, Ryan CA (1996) An octadecanoid pathway mutant (JL5) of tomato is compromised in signaling for defense against insect attack. Plant Cell 8: 2067-2077[Abstract]
Johnson R, Narvaez J, An G, Ryan CA (1989) Expression of proteinase inhibitors I and II in transgenic tobacco plants: effects on natural defense against Manduca sexta larvae. Proc Natl Acad Sci USA 86: 9871-9875 Johnston KA, Gatehouse JA, Anstee JH (1993) Effects of soybean protease inhibitor on the growth and development of larval Helicoverpa armigera. J Insect Physiol 39: 657-664[CrossRef]
Jongsma MA, Bakker PL, Peters J, Bosch D, Stiekema WJ (1995) Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 92: 8041-8045 Jongsma MA, Bakker PL, Visser B, Stiekema WJ (1994) Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack, wounding, and virus infection. Planta 195: 29-35[ISI] Jongsma MA, Bolter C (1997) The adaptation of insects to plant protease inhibitors. J Insect Physiol 43: 885-895[CrossRef][ISI][Medline]
Kessler A, Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature. Science 291: 2141-2144 Klemola T, Ruohomaki K, Tanhuanpaa M, P. K (2003) Performance of a spring-feeding moth in relation to time of oviposition and bud-burst phenology of different host species. Ecol Entomol 28: 319-327[CrossRef] Laing W, McManus MT (2002) Proteinase inhibitors. In MT McManus, WA Laing, AC Allan, eds, Protein-Protein Interactions in Plant Biology. CRC Press, Boca Raton, FL, pp 77-119 Marchetti S, Delledonne M, Fogher C, Chiabá C, Chiesa F, Savazzini F, Giordano A (2000) Soybean Kunitz, C-II and PI-IV inhibitor genes confer different levels of insect resistance to tobacco and potato transgenic plants. Theor Appl Genet 101: 519-526[CrossRef]
McGurl B, Pearce G, Orozco-Cardenas M, Ryan CA (1992) Structure, expression, and antisense inhibition of the systemin precursor gene. Science 255: 1570-1573 McManus MT, Burguess EPJ (1995) Effects of the soybean (Kunitz) trypsin inhibitor on growth and digestive proteases of larvae of Spodoptera litura. J Insect Physiol 41: 731-738[CrossRef] McManus MT, Burgess EPJ, Philip B, Watson LM, Laing WA, Voisey CR, White DWR (1999) Expression of the soybean (Kunitz) trypsin inhibitor in transgenic tobacco: effects on larval development of Spodoptera litura. Transgenic Res 8: 383-395[CrossRef] McManus MT, White DWR, McGregor PG (1994) Accumulation of the chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests. Transgenic Res 3: 50-58 Miles PW (1972) The saliva of Hemiptera. Adv Insect Physiol 9: 183-255 Miller EA, Lee MCS, Atkinson AHO, Anderson MA (2000) Identification of a novel four-domain member of the proteinase inhibitor II family from the stigmas of Nicotiana alata. Plant Mol Biol 42: 329-333[CrossRef][Medline]
Moura DS, Ryan CA (2001) Wound-inducible proteinase inhibitors in pepper: differential regulation upon wounding, systemin, and methyl jasmonate. Plant Physiol 126: 289-298
Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10: 1-6 Nielsen KJ, Hill JM, Anderson MA, Craik DJ (1996) Synthesis and structure determination by NMR of a putative vacuolar targeting peptide and model of a proteinase inhibitor for Nicotiana alata. Biochemistry 35: 369-378[Medline]
Orozco-Cardenas M, McGurl B, Ryan CA (1993) Expression of an antisense prosystemin gene in tomato plants reduces resistance toward Manduca sexta larvae. Proc Natl Acad Sci USA 90: 8273-8276 Peterson A, Barillas-Mury C, Wells M (1994) Sequence of three cDNA encoding an alkaline midgut trypsin from Manduca sexta. Insect Biochem Mol Biol 24: 463-471[Medline]
Royo J, Leon J, Vancanneyt G, Albar JP, Rosahl S, Ortego F, Castañera P, Sánchez-Serrano JJ (1999) Antisense-mediated depletion of a potato lipooxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pest. Proc Natl Acad Sci USA 96: 1146-1151 Ryan CA (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 28: 425-449[ISI] Schuler TH, Poppy GM, Kerrry BR, Denholm I (1998) Insect-resistant transgenic plants. TIBTECH 16: 168-175 Taylor BH, Young RJ, Scheuring CF (1993) Induction of a proteinase inhibitor II-class gene by auxin in tomato roots. Plant Mol Biol 23: 1005-1014[CrossRef][Medline] van Dam NM, Baldwin IT (2001) Competition mediates costs of jasmonate-induced defenses, N acquisition and transgenerational plasticity in Nicotiana attenuata. Funct Ecol 15: 406-415[CrossRef] van Dam NM, Horn M, Mares M, Baldwin IT (2001) Ontogeny constrains systemic protease inhibitor response in Nicotiana attenuata. J Chem Ecol 27: 547-568[CrossRef][ISI][Medline] Winterer J (2002) The mixed success of protease inhibitors to combat insect pest in transgenic crops. AgBiotechNet 4: 1-7 Winterer J, Bergelson J (2001) Diamondback moth compensatory consumption of protease inhibitor-transformed plants. Mol Ecol 10: 1069-1074[Medline]
Winz RA, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata: Insect-induced ethylene suppresses jasmonate-induced accumulation of nicotine biosynthesis transcripts. Plant Physiol 125: 2189-2202 Wu Y, Llewellyn D, Mathews A, Dennis E (1997) Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Mol Breed 3: 371-380 Zavala JA, Patankar AG, Gase K, Baldwin IT (2004) Constitutive and inducible trypsin protease inhibitor production incurs large fitness cost in Nicotiana attenuata. Proc Natl Acad Sci USA (in press) Zavala JA, Scopel AL, Ballaré CL (2001) Effects of ambient UV-B radiation on soybean crops: impact on leaf herbivory by Anticarsia gemmatalis. Plant Ecol 156 This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
RESULTS
and 
) and systemic (-1; 
and 
) leaves after hornworm elicitation relative to that of unelicited (control; 
and 
) WT leaves from three replicate plants harvested and analyzed separately at 0, 6, 12, 24, and 48 h after elicitation. A, WT and C genotypes. B, AS–and AS- genotypes. C, S++ and S+ genotypes.
