Transgenic tobacco and peas expressing a proteinase inhibitor from Nicotiana alata have increased insect resistance

Proteinase inhibitors have been used to increase resistance to insect pests in transgenic plants. A cDNA clone encoding a multi-domain proteinase inhibitor precursor from Nicotiana alata (Na-PI) was transferred into tobacco and peas under the control of a promoter from a ribulose-1, 5-bisphosphate carboxylase small subunit gene. The Na-PI precursor was cleaved in the leaves of transgenic tobacco and peas, and M_r 6000 polypeptides accumulated to levels of 0.3% and 0.1%, respectively, of the total soluble protein. The Na-PI cDNA segregated as a dominant Mendelian trait and was stably transmitted for at least two generations of both species. Helicoverpa armigera larvae that ingested tobacco or pea leaves containing Na-PI exhibited higher mortality or were delayed in growth and development relative to control larvae.     

NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability in Nicotiana alata Pollen Tubes

In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.To avoid low-fitness progeny, many plants have developed a cell-cell interaction mechanism to promote outcrossing, through the recognition and discrimination of both self and nonself pollen. This recognition system is controlled by the highly polymorphic self-incompatibility S-locus, which determines pollination specificity in both the pollen and pistil. Pollen is rejected when male and female S-haplotypes coincide (de Nettancourt, 1977, 2001; Franklin et al., 1995).In Solanaceae, Plantaginaceae, and Rosaceae, the S-locus product in the pistil is an extracellular glycoprotein named S-RNase (Anderson et al., 1986; McClure et al., 1989). During pollination, S-RNase is taken up by both compatible and incompatible pollen tubes (Luu et al., 2000) and targeted to a vacuole (Goldraij et al., 2006). In the later stages of an incompatible cross, the S-RNase-containing vacuole is disrupted and the S-RNases are released to the pollen tube cytoplasm, where RNA degradation can occur (McClure et al., 2011).The S-pollen gene encodes an SLF or SFB (SLF/SFB; for S-locus F-box) protein, which is a member of the F-box protein family (Entani et al., 2003; Sijacic et al., 2004). In vitro binding assays show that PiSLF in Petunia inflata physically interacts with S-RNases, although this interaction is stronger with nonself S-RNases than with self S-RNases (Hua and Kao, 2006). Additional protein-protein interaction assays suggest that SLF/SFB may be a component of an SCF (for Skp1-Cullin1-F-box) or SCF-like complex (Qiao et al., 2004; Hua and Kao, 2006). Notably, data from Zhao et al. (2010) in Petunia hybrida show that reduction of PhSSK1 (for P. hybrida SLF-interacting Skp-like1) and its Antirrhinum hispanicum ortholog, AhSSK1, is also required for cross-pollen compatibility.Although S-RNase and SLF/SFB define pollen rejection S-specificity, modifier genes unlinked to the S-locus are required for self-incompatibility (SI; Martin, 1968; Ai et al., 1991; Murfett et al., 1996; Tsukamoto et al., 1999).To date, only two pistil-modifier genes have been identified: High Top-Band (HT-B) and 120K. In Nicotiana spp., HT-B is an 8.6-kD acidic protein with a domain consisting of 20 Asn and Asp residues toward its C terminus (McClure et al., 1999; Kondo and McClure, 2008). Loss-of-function assays prove HT-B to be essential for pollen rejection in Nicotiana spp., Solanum spp., and Petunia spp. (McClure et al., 1999; Kondo et al., 2002; O’Brien et al., 2002; Sassa and Hirano, 2006; Puerta et al., 2009), although it is not expressed in SI Solanum habrochaites, prompting the speculation that in this species a related gene, HT-A, may function as a substitute (Covey et al., 2010). Immunolocalization shows that HT-B is readily taken up by pollen tubes during pollination. Its steady-state levels decrease slightly in pollen tubes from incompatible pollinations. However, in compatible crosses, HT-B levels decrease 75% to 97%, probably as a result of protein degradation (Goldraij et al., 2006).120K is a style-specific 120-kD arabinogalactan protein (Schultz et al., 1997) that is taken up by pollen tubes (Lind et al., 1996) and appears to be associated with S-RNase-containing vacuoles (Goldraij et al., 2006). 120K forms complexes with S-RNases and other proteins (Cruz-Garcia et al., 2005) in vitro, and suppression of 120K expression prevents S-specific pollen rejection (Hancock et al., 2005). Protein-protein interaction assays demonstrate that 120K interacts with the pollen-specific protein NaPCCP (a pollen C2 domain-containing protein), a protein that binds phosphatidylinositol 3-phosphate and is associated with the pollen tube endomembrane system (Lee et al., 2008, 2009).Two models have been proposed to explain pollen rejection in Solanaceae. (1) The S-RNase degradation model (Hua and Kao, 2006; Hua et al., 2007, 2008; Kubo et al., 2010) focuses on S-RNase-SLF interactions that bring about preferential nonself S-RNase degradation. In this model, strong nonself S-RNase-SLF interactions lead to the degradation of nonself S-RNases by the ubiquitin-26S proteasome system, allowing pollen tubes to escape from its cytotoxic effect. Weak self S-RNase-SLF interactions, in contrast, permit the persistence of sufficient free S-RNase that pollen tube RNA is degraded, resulting in self-pollen rejection. Notably, by functional and protein-protein interaction assays in Petunia spp., Kubo et al. (2010) found at least three types of divergent SLF proteins encoded at the S-locus, each recognizing a subgroup of nonself S-RNases. The authors proposed the collaborative nonself recognition model, where multiple SLF proteins interact with nonself S-RNases to protect nonself pollen from degradation (Kubo et al., 2010). (2) The compartmentalization model incorporates the observations that pollen tubes internalize both self and nonself S-RNases and targets them to vacuoles and that HT-B is degraded in compatible crosses but is stable in incompatible crosses (Goldraij et al., 2006). In incompatible crosses, the S-RNase-containing vacuoles are ultimately disrupted and S-RNases are released to the cytoplasm, where they degrade RNA, leading to rejection of self-pollen. In compatible crosses, the integrity of the S-RNase-containing vacuoles is preserved, allowing pollen tube growth to continue. Thus, in this model, self or nonself S-RNase-SLF interactions determine the specificity of pollen rejection indirectly.Biochemical and genetic data show that pistil-modifier genes apart from HT-B and 120K are required for SI. We recently described NaStEP (for N. alata Stigma-Expressed Protein), an abundant, pistil-specific stigma protein found in SI Nicotiana spp. (Busot et al., 2008). Its abundance in SI species made NaStEP a strong modifier gene candidate. Here, we demonstrate that NaStEP is taken up by pollen tubes, has subtilisin inhibitory activity, and that suppressing its expression in transgenic hybrids disrupts pollen rejection. Moreover, when NaStEP-suppressed hybrids are pollinated, HT-B protein is degraded in both compatible and incompatible pollen tubes, while in wild-type SI N. alata, HT-B is preferentially stabilized in incompatible pollen tubes.     

Identification of a novel four-domain member of the proteinase inhibitor II family from the stigmas of Nicotiana alata

Proteinase inhibitors (PIs) of the potato type II family have been identified in a number of solanaceous species. Most family members have two PI domains which are specific for either chymotrypsin or trypsin. More recently family members have been described with three or six repeated PI domains. Here we describe a novel four-domain family member produced in the stigmas and leaves of the ornamental tobacco, Nicotiana alata, which has high sequence identity with a six-domain member from the same species. Both proteins are produced as precursors that enter the secretory pathway and are subsequently processed into a series of 6 kDa PIs. The four- and six-domain precursor proteins were isolated from immature stigmas and characterised by mass spectrometry which revealed that both proteins had been trimmed at the N-terminus, at a position corresponding to the predicted signal peptide cleavage site. Furthermore, no post-translational modifications were apparent.     

The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata

A two-domain portion of the proteinase inhibitor precursor from Nicotiana alata (NaProPI) has been expressed and its structure determined by NMR spectroscopy. NaProPI contains six almost identical 53 amino acid repeats that fold into six highly similar domains; however, the sequence repeats do not coincide with the structural domains. Five of the structural domains comprise the C-terminal portion of one repeat and the N-terminal portion of the next. The sixth domain contains the C-terminal portion of the sixth repeat and the N-terminal portion of the first repeat. Disulphide bonds link these C and N-terminal fragments to generate the clasped-bracelet fold of NaProPI. The three-dimensional structure of NaProPI is not known, but it is conceivable that adjacent domains in NaProPI interact to generate the circular "bracelet" with the N and C termini in close enough proximity to facilitate formation of the disulphide bonds that form the "clasp". The expressed protein, examined in the current study, comprises residues 25-135 of NaProPI and encompasses the first two contiguous structural domains, namely the chymotrypsin inhibitor C1 and the trypsin inhibitor T1, joined by a five-residue linker, and is referred to as C1-T1. The tertiary structure of each domain in C1-T1 is identical to that found in the isolated inhibitors. However, no nuclear Overhauser effect contacts are observed between the two domains and the five-residue linker adopts an extended conformation. The absence of interactions between the domains indicates that adjacent domains do not specifically interact to drive the circularisation of NaProPI. These results are in agreement with recent data which describe similar PI precursors from other members of the Solanaceae having two, three, or four repeats. The lack of strong interdomain association is likely to be important for the function of individual inhibitors by ensuring that there is no masking of reactive sites upon release from the precursor.     

A proteinase inhibitor from Nicotiana alata inhibits the normal development of light-brown apple moth, Epiphyas postvittana in transgenic apple plants

Insecticidal proteins are a potential resource to enhance resistance to insect pests in transgenic plants. Here, we describe the generation and analysis of the apple cultivar ‘Royal Gala’ transgenic for Nicotiana alata (N. alata) proteinase inhibitor (PI) and the impact of this PI on the growth and development of the Epiphyas postvittiana (light-brown apple moth). A cDNA clone encoding a proteinase inhibitor precursor from N. alata (Na-PI) under the control of either a double 35S promoter or a promoter from a ribulose-1,5-bisphosphate carboxylase small sub-unit gene (rbcS-E9 promoter) was stably incorporated into ‘Royal Gala’ apple using Agrobacterium-mediated transformation. A 40.3 kDa Na-PI precursor protein was expressed and correctly processed into 6-kDa proteinase inhibitors in the leaves of transgenic apple lines. The 6-kDa polypeptides accumulated to levels of 0.05 and 0.1% of the total soluble protein under the control of the rbc-E9 promoter and the double 35S promoter, respectively. Light-brown apple moth larvae fed with apple leaves expressing Na-PI had significantly reduced body weight after 7 days of feeding and female pupae were 19–28% smaller than controls. In addition, morphological changes such as pupal cases attached to the wing, deformed wings, deformed body shape, and pupal cases and curled wings attached to a deformed body were observed in adults that developed from larvae fed with apple leaves expressing Na-PI, when compared to larvae fed with the non-transformed apple leaves.     

Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene from Nicotiana alata.

BACKGROUND: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (PIs) that are derived from a 43 kDa precursor protein, NaProPI. NaProPI contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native PIs. An unusual feature of NaProPI is that the structural domains lie across adjacent repeats and that the sixth PI domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPI repeat (aPI1) forms a basis for unravelling the mechanism by which this protein may have evolved. RESULTS: The three-dimensional structure of aPI1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native PIs. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPI, exists as a disordered loop in aPI1. The presence of this loop in aPI1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. CONCLUSIONS: A single repeat from NaProPI is capable of folding into a compact globular domain that displays native-like PI activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPI evolved.     

Arabinogalactan-Proteins of the Female Sexual Tissue of Nicotiana alata: I. Changes during Flower Development and Pollination

Arabinogalactan-proteins (AGPs), isolated from the pistils of Nicotiana alata, an ornamental tobacco, are developmentally regulated. Both the total amount and concentration of AGP in the stigma increase during flower development, reaching 10 micrograms AGP/stigma at maturity. In contrast, AGP concentration in the style remains constant throughout the maturation period reaching 12 micrograms AGP/style at maturity. The classes of AGP present in the stigma and style during flower development, separated according to their charge by crossed-electrophoresis, are different and change during development. Pollination of flowers of N. alata with compatible or incompatible pollen results in a significant and reproducible increase in the amount of AGPs in the stigma, but not the style, compared with control unpollinated pistils. Pollination with ethanol vapor inactivated pollen also results in an increase in the amount of AGP in the stigma, but this is less than half that observed following pollination with viable pollen. There are no significant differences in the classes of AGP, based on crossed-electrophoresis, present in the pistil following pollination.     

Disorganization of F-actin cytoskeleton precedes vacuolar disruption in pollen tubes during the in vivo self-incompatibility response in Nicotiana alata

Background and Aims The integrity of actin filaments (F-actin) is essential for pollen-tube growth. In S-RNase-based self-incompatibility (SI), incompatible pollen tubes are inhibited in the style. Consequently, research efforts have focused on the alterations of pollen F-actin cytoskeleton during the SI response. However, so far, these studies were carried out in in vitro-grown pollen tubes. This study aimed to assess the timing of in vivo changes of pollen F-actin cytoskeleton taking place after compatible and incompatible pollinations in Nicotiana alata. To our knowledge, this is the first report of the in vivo F-actin alterations occurring during pollen rejection in the S-RNase-based SI system. Methods The F-actin cytoskeleton and the vacuolar endomembrane system were fluorescently labelled in compatibly and incompatibly pollinated pistils at different times after pollination. The alterations induced by the SI reaction in pollen tubes were visualized by confocal laser scanning microscopy. Key Results Early after pollination, about 70 % of both compatible and incompatible pollen tubes showed an organized pattern of F-actin cables along the main axis of the cell. While in compatible pollinations this percentage was unchanged until pollen tubes reached the ovary, pollen tubes of incompatible pollinations underwent gradual and progressive F-actin disorganization. Colocalization of the F-actin cytoskeleton and the vacuolar endomembrane system, where S-RNases are compartmentalized, revealed that by day 6 after incompatible pollination, when the pollen-tube growth was already arrested, about 80 % of pollen tubes showed disrupted F-actin but a similar percentage had intact vacuolar compartments. Conclusions The results indicate that during the SI response in Nicotiana, disruption of the F-actin cytoskeleton precedes vacuolar membrane breakdown. Thus, incompatible pollen tubes undergo a sequential disorganization process of major subcellular structures. Results also suggest that the large pool of S-RNases released from vacuoles acts late in pollen rejection, after significant subcellular changes in incompatible pollen tubes.     

Evidence for the presence of proteinase inhibitor I in vacuolar protein bodies of plant cells

Summary Protein bodies induced in tomato leaf cells by wounding were shown to contain proteinase Inhibitor I by using ferritin-labelled antibodies, fluorescein-labelled antibodies, and cytochrome C-labelled antibody fragments. Both pre-embedding and postembedding techniques were used. Nonspecific binding was least when p-formaldehyde was used as the initial fixative followed by treatment with cytochrome c-labelled antibody fragments.Abbreviations Fab antibody fragments - BSA bovine serum albumin - GMA glycol methacrylate - THB Tris-HCl buffer Taken in part from a doctoral (Ph.D.) dissertation submitted to Washington State University by Vivian V. Yang. This work was supported largely by NSF Grant GB-29614X (LKS) and in part by the United States Department of Agricultural Cooperative States Research Service Grant 316-15-30 (CAR), the National Science Foundation Grant GB-37972 (CAR), and the College of Agriculture Research Center, Washington State University, Pullman, WA 99163, Scientific Paper No. 4525, Project 1791.Program in Genetics and Department of Botany. To whom reprint requests should be sent.Department of Agricultural Chemistry and Program in Biochemistry and Biophysics.     

Chemical characterization, synthesis and distribution of proteinase inhibitor in newborn rat epidermis

A protein solubilized in Tris-HCl/saline buffer from keratinized cells of newborn rat epidermis exhibited inhibitor activity to papain and ficin, but not to trypsin, cathepsin D and pepsin. This protein was purified from keratinized cells as well as nonkeratinized and germinative cells by means of IgG affinity chromatography. The inhibitors extracted from all cell layers were immunologically identical and had a molecular weight of approximately 12,500 +/- 500. Since amino acid analysis showed that the inhibitor contains about 35 residues of glycine per mol, [3H]glycine was used to investigate synthesis of the protein. The inhibitor from nonkeratinized and germinative cells was radioactively labeled by 2 h after injection and appeared in keratinized cells by 48 h after injection. Indirect immunofluorescence microscopy demonstrated in situ distribution of the protein in the entire epidermis, and the protein localized by the plasma membrane in granular cells and diffusely in keratinized cells was shown to be insoluble in Tris-HCl saline buffer. The results indicate that a thiol-proteinase inhibitor is synthesized in epidermal cells during keratinization and is retained as part of the cytoplasmic structure     

Chemical synthesis, molecular cloning and expression of gene coding for a Bowman-Birk-type proteinase inhibitor

A gene coding for a Bowman-Birk-type proteinase inhibitor was synthesized chemically, cloned and expressed in Escherichia coli as a fusion protein with a beta-galactosidase fragment. The corresponding mutant inhibitor, carrying a P1 = Arg16 instead of Lys and an Ile27 instead of Met was obtained after cyanogen bromide cleavage, refolding and affinity chromatography on trypsin-Sepharose. Dissociation constants of complexes with trypsin of this mutant and wild-type Bowman-Birk inhibitor are identical within experimental error. This is explained by differential patterns of hydrogen bonds between side-chains of Arg or Lys in proteinase inhibitors and the primary specificity pocket of trypsin.     

In vivo metabolism of inter-alpha-trypsin inhibitor and its proteinase complexes: evidence for proteinase transfer to alpha 2-macroglobulin and alpha 1-proteinase inhibitor

Inter-alpha-trypsin inhibitor was purified by a modification of published procedures which involved fewer steps and resulted in higher yields. The preparation was used to study the clearance of the inhibitor and its complex with trypsin from the plasma of mice and to examine degradation of the inhibitor in vivo. Unlike other plasma proteinase inhibitor-proteinase complexes, inter-alpha-trypsin inhibitor reacted with trypsin did not clear faster than the unreacted inhibitor. Studies using 125I-trypsin provided evidence for the dissociation of complexes of proteinase and inter-alpha-trypsin inhibitor in vivo, followed by rapid removal of proteinase by other plasma proteinase inhibitors, particularly alpha 2-macroglobulin and alpha 1-proteinase inhibitor. Studies in vitro also demonstrated the transfer of trypsin from inter-alpha-trypsin inhibitor to alpha 2-macroglobulin and alpha 1-proteinase inhibitor but at a much slower rate. The clearance of unreacted 125I-inter-alpha-trypsin inhibitor was characterized by a half-life ranging from 30 min to more than 1 h. Murine and human inhibitors exhibited identical behavior. Multiphasic clearance of the inhibitor was not due to degradation, aggregation, or carbohydrate heterogeneity, as shown by competition studies with asialoorosomucoid and macroalbumin, but was probably a result of extravascular distribution or endothelial binding. 125I-inter-alpha-trypsin inhibitor cleared primarily in the liver. Analysis of liver and kidney tissue by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis showed internalization and limited degradation of 125I-inter-alpha-trypsin inhibitor in these tissues. No evidence for the production of smaller proteinase inhibitors from 125I-inter-alpha-trypsin inhibitor injected intravenously or intraperitoneally was detected, even in casein-induced peritoneal inflammation. No species of molecular weight similar to that of urinary proteinase inhibitors, 19,000-70,000, appeared in plasma, liver, kidney, or urine following injection of inter-alpha-trypsin inhibitor.     

Interaction between duodenase, a proteinase with dual specificity, and soybean inhibitors of Bowman-Birk and Kunitz type

The interaction between duodenase, which belongs to a group of Janus-faced proteinases, and classical Bowman--Birk (BBI) and Kunitz (STI) type inhibitors from soybean was investigated. Duodenase was shown to interact only with the antichymotrypsin site (Leu-Ser) of BBI, whereas the antitrypsin site (Lys-Ser) of the inhibitor appeared to be vacant and capable of interaction with trypsin. The inhibition constants of duodenase by BBI, the BBI--trypsin complex, and STI were 4, 400, and 40 nM, respectively.     

Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper

Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.     

Localisation and expression of arabinogalactan-proteins in the ovaries of Nicotiana alata Link and Otto

The transmitting-tissue cells of the style of flowering plants secrete a complex extracellular matrix through which pollen tubes grow to the ovary to effect fertilisation. This matrix is particularly rich in a class of proteoglycans, the arabinogalactan-proteins (AGPs). AGPs from the ovary of Nicotiana alata were found to be developmentally regulated, as the different charge classes of AGPs altered during floral development. The AGPs from the mature ovary had charge characteristics that were distinct from those previously reported for the stigma and style. However, the concentration of AGP (0.6 g/ml fresh weight) in the ovary did not change during development, or in response to either compatible or incompatible pollination. The AGPs of the ovary are mainly associated with the epidermis of the placenta.     

Involvement of SLR1 genes in pollen adhesion to the stigmatic surface in Brassicaceae

 The S-locus-related gene SLR1 is highly conserved and highly expressed in several species of the Brassicaceae family. Its function has not been determined, although several features would suggest a fundamental role in pollination. A second related gene (SLR2) is conserved and expressed in a subset of Brassica genotypes. We analysed the stigmatic expression of SLR1 and SLR2 genes among 11 different plants from various species or genera of the Brassicaceae and examined the extent of the pollen-stigma interaction during intraspecific, interspecific and intergeneric pollinations between them. Appropriate statistical tests on these variables (pollen adhesion, germination, penetration into the stigma, style and ovary, and SLR gene expression) showed that expression of SLR1 (but not SLR2) may be a factor in pollen-stigma adhesion. This hypothesis was supported by the observation of reduced pollen-stigma adhesion in transgenic B. napus plants modified for SLR1 expression. Received: 25 February 1997/Revision accepted: 24 June 1997     

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.     

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.