Multimodal protein constructs for herbivore insect control.

Transgenic plants expressing combinations of microbial or plant pesticidal proteins represent a promising tool for the efficient, durable control of herbivorous insects. In this review we describe current strategies devised for the heterologous co-expression of pesticidal proteins in planta, some of which have already shown usefulness in plant protection. Emphasis is placed on protein engineering strategies involving the insertion of single DNA constructs within the host plant genome. Multimodal fusion proteins integrating complementary pesticidal functions along a unique polypeptide are first considered, taking into account the structural constraints associated with protein or protein domain grafting to biologically active proteins. Strategies that allow for the co- or post-translational release of two or more pesticidal proteins are then considered, including polyprotein precursors releasing free proteins upon proteolytic cleavage, and multicistronic transcripts for the parallel translation of single protein-encoding mRNA sequences.

Stress-responsive mitogen-activated protein kinases interact with the EAR motif of a poplar zinc finger protein and mediate its degradation through the 26S proteasome.

Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors.

Recombinant protease inhibitors for herbivore pest control: a multitrophic perspective.

Protease inhibitors are a promising complement to Bt toxins for the development of insect-resistant transgenic crops, but their limited specificity against proteolytic enzymes and the ubiquity of protease-dependent processes in living organisms raise questions about their eventual non-target effects in agroecosystems. After a brief overview of the main factors driving the impacts of insect-resistant transgenic crops on non-target organisms, the possible effects of protease inhibitors are discussed from a multitrophic perspective, taking into account not only the target herbivore proteases but also the proteases of other organisms found along the trophic chain, including the plant itself. Major progress has been achieved in recent years towards the design of highly potent broad-spectrum inhibitors and the field deployment of protease inhibitor-expressing transgenic plants resistant to major herbivore pests. A thorough assessment of the current literature suggests that, whereas the non-specific inhibitory effects of recombinant protease inhibitors in plant food webs could often be negligible and their 'unintended' pleiotropic effects in planta of potential agronomic value, the innocuity of these proteins might always remain an issue to be assessed empirically, on a case-by-case basis.

Plant cystatins.

Plant cystatins have been the object of intense research since the publication of a first paper reporting their existence more than 20 years ago. These ubiquitous inhibitors of Cys proteases play several important roles in plants, from the control of various physiological and cellular processes in planta to the inhibition of exogenous Cys proteases secreted by herbivorous arthropods and pathogens to digest or colonize plant tissues. After an overview of current knowledge about the evolution, structure and inhibitory mechanism of plant cystatins, we review the different roles attributed to these proteins in plants. The potential of recombinant plant cystatins as effective pesticidal proteins in crop protection is also considered, as well as protein engineering approaches adopted over the years to improve their inhibitory potency and specificity towards Cys proteases of biotechnological interest.

A companion protease inhibitor for the protection of cytosol-targeted recombinant proteins in plants.

We reported earlier the potential of tomato cathepsin D inhibitor (SlCDI) as an in-built stabilizing agent for the protection of recombinant proteins in transgenic plant leaf crude extracts (Plant Biotechnol J.4, 359-368). Here we document the potential of SlCDI for the in situ protection of proteins in potato leaves. Total protein assays with control and SlCDI-expressing potato lines indicated a positive impact of slcdi transgene expression on leaf protein content, with a mean relative increase of 35%-40% depending on the light regime. Out of approximately 700 proteins detected on 2-D gels, only 20 exhibited a significantly altered level on a protein-specific basis, whereas most proteins were up-regulated on a leaf fresh weight basis, albeit at variable rates. Quantitative reverse trancriptase-PCR assays for rubisco activase showed similar transcript levels in leaves of test and control lines despite protein levels increased by two- to threefold in SlCDI-expressing lines. These observations, along with the unrelated biological functions assigned to MS-identified proteins up-regulated in leaves and protease assays showing slightly increased proteasome activity in protein extracts of SlCDI-expressing lines, suggest a general, proteasome-independent protein stabilizing effect of SlCDI in planta. Transient expression assays with human alpha(1)-antichymotrypsin also showed a stabilizing effect for SlCDI on heterologous proteins, leading to net levels of the human protein increased by approximately 2.5-fold in SlCDI-expressing plants. These data illustrate, overall, the potential of SlCDI as an in vivo protein-stabilizing agent in transgenic plant systems, useful to improve protein levels and recombinant protein accumulation.

Companion protease inhibitors to protect recombinant proteins in transgenic plant extracts.

We describe a general approach for the use of recombinant protease inhibitors as stabilizing agents for clinically useful proteins extracted from transgenic plant tissues. A procedure is first described to assess the overall (in)stability of heterologous proteins in transgenic plant crude protein extracts. Step-by-step protocols are then presented for the choice and use of companion protease inhibitors inhibiting the host plant proteases during extraction. This strategy, that reproduces the protein-stabilizing effect of low-molecular-weight protease inhibitors often added to protein extraction media, does not require the exogenous addition of such expensive and often toxic compounds. It also presents the advantage of being intrinsically scalable to the amount of biomass processed.

Nucleocytoplasmic transit of human alpha1-antichymotrypsin in tobacco leaf epidermal cells.

Recently, we have observed a nuclear localization for human alpha(1)-antichymotrypsin (AACT) expressed in the cytosol of transgenic Bright Yellow-2 (BY-2) tobacco cultured cells (see accompanying paper: Benchabane, M., Saint-Jore-Dupas, C., Bardor, M., Faye, L., Michaud, D. and Gomord, V. (2008a) Targeting and post-translational processing of human alpha(1)-antichymotrypsin in BY-2 tobacco cultured cells. Plant Biotechnol. J. doi: 10.1111/j.1467-7652.2008.00382.x). In the present article, we assess whether the intrinsic DNA-binding activity of AACT can explain its nuclear localization, and whether this same activity has an impact on its protease inhibitory potency and stability in planta. An engineered form of AACT with no DNA-binding activity, rAACTDeltaK, was compared with the wild-type polypeptide, rAACT, in terms of chymotrypsin inhibitory potency, stability in planta and distribution in tobacco cells. In accordance with available data reporting distinct sites for protease inhibition and DNA binding, rAACT and rAACTDeltaK showed similar antichymotrypsin activity, similar to the activity of native AACT purified from human plasma. As observed for AACT in BY-2 tobacco cells, a green fluorescent protein (GFP)-AACT fusion transiently expressed in the cytosol of tobacco leaf epidermal cells was detected mainly in the nucleus by confocal laser microscopy. By contrast, rAACTDeltaK expressed as a GFP fusion showed a balanced distribution between the cytosol and the nucleus, similar to the distribution pattern of free GFP exhibiting no DNA-binding affinity. In line with immunodetection data showing higher accumulation levels for GFP-AACT in tobacco leaf cells, rAACTDeltaK was more susceptible than rAACT to tryptic digestion in the presence of DNA. Overall, these observations suggest the following: (i) a retention effect of DNA on AACT in the nucleus; and (ii) a stabilizing effect of the AACT-DNA interaction on rAACT challenged with non-target proteases, which, possibly, may be useful in protecting this protein in plant expression platforms.

Targeting and post-translational processing of human alpha1-antichymotrypsin in BY-2 tobacco cultured cells.

The post-translational processing of human alpha(1)-antichymotrypsin (AACT) in Bright Yellow-2 (BY-2) tobacco cells was assessed in relation to the cellular compartment targeted for accumulation. As determined by pulse-chase labelling experiments and immunofluorescence microscopy, AACT sent to the vacuole or the endoplasmic reticulum (ER) was found mainly in the culture medium, similar to a secreted form targeted to the apoplast. Unexpectedly, AACT expressed in the cytosol was found in the nucleus under a stable, non-glycosylated form, in contrast with secreted variants undergoing multiple post-translational modifications during their transit through the secretory pathway. All secreted forms of AACT were N-glycosylated, with the presence of complex glycans as observed naturally on human AACT. Proteolytic trimming was also observed for all secreted variants, both during their intracellular transit and after their secretion in the culture medium. Overall, the targeting of human AACT to different compartments of BY-2 tobacco cells led to the production of two protein products: (i) a stable, non-glycosylated protein accumulated in the nucleus; and (ii) a heterogeneous mixture of secreted variants resulting from post-translational N-glycosylation and proteolytic processing. Overall, these data suggest that AACT is sensitive to resident proteases in the ER, the Golgi and/or the apoplast, and that the production of intact AACT in the plant secretory pathway will require innovative approaches to protect its structural integrity in vivo. Studies are now needed to assess the activity of the different AACT variants, and to identify the molecular determinants for the nuclear localization of AACT expressed in the cytosol.

Hybrid protease inhibitors for pest and pathogen control--a functional cost for the fusion partners?

Fusion proteins integrating dual pesticidal functions have been devised over the last 10 years to improve the effectiveness and potential durability of pest-resistant transgenic crops, but little attention has been paid to the impact of the fusion partners on the actual activity of the resulting hybrids. Here we assessed the ability of the rice cysteine protease inhibitor, oryzacystatin I (OCI), to retain its protease inhibitory potency when used as a template to devise hybrid inhibitors with dual activity against papain-like proteases and carboxypeptidase A (CPA). C-terminal variants of OCI were generated by fusing to its C-terminal end: (i) the primary inhibitory site of the small CPA inhibitor potato carboxypeptidase inhibitor (PCI, amino acids 35-39); or (ii) the complete sequence of PCI (a.a. 1-39). The hybrid inhibitors were expressed in E. coli and tested for their inhibitory activity against papain, CPA and digestive cysteine proteases of herbivorous and predatory arthropods. In contrast with the primary inhibitory site of PCI, the entire PCI attached to OCI was as active against CPA as free, purified PCI. The OCI-PCI hybrids also showed activity against papain, but the presence of extra amino acids at the C terminus of OCI negatively altered its inhibitory potency against cysteine proteases. This negative effect, although not preventing dual binding to papain and CPA, was correlated with an increased binding affinity for papain presumably due to non-specific interactions with the PCI domain. These results confirm the potential of OCI and PCI for the design of fusion inhibitors with dual protease inhibitory activity, but also point out the possible functional costs associated with protein domain grafting to recipient pesticidal proteins.

Preventing unintended proteolysis in plant protein biofactories.

SUMMARY: Numerous reports have been published over the last decade assessing the potential of plants as useful hosts for the heterologous expression of clinically useful proteins. Significant progress has been made, in particular, in optimizing transgene transcription and translation in plants, and in elucidating the complex post-translational modifications of proteins typical of the plant cell machinery. In this article, we address the important issue of recombinant protein degradation in plant expression platforms, which directly impacts on the final yield, homogeneity and overall quality of the resulting protein product. Unlike several more stable and structurally less complex pharmaceuticals, recombinant proteins present a natural tendency to structural heterogeneity, resulting in part from the inherent instability of polypeptide chains expressed in heterologous environments. Proteolytic processing, notably, may dramatically alter the structural integrity and overall accumulation of recombinant proteins in plant expression systems, both in planta during expression and ex planta after extraction. In this article, we describe the current strategies proposed to minimize protein hydrolysis in plant protein factories, including organ-specific transgene expression, organelle-specific protein targeting, the grafting of stabilizing protein domains to labile proteins, protein secretion in natural fluids and the co-expression of companion protease inhibitors.

Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites.

Cysteine proteinase inhibitors of the cystatin superfamily have several important functions in plants, including the inhibition of exogenous cysteine proteinases during herbivory or infection. Here we used a maximum-likelihood approach to assess whether plant cystatins, like other proteins implicated in host-pest interactions, have been subject to positive selection during the course of their evolution. Several amino acid sites were identified as being positively selected in cystatins from either Poaceae (monocots) and Solanaceae (dicots). These hypervariable sites were located at strategic positions on the protein: on each side of the conserved glycine residues in the N-terminal trunk, within the first and second inhibitory loops entering the active site of target enzymes, and surrounding the larfav motif, a sequence of unknown function conserved among plant cystatins. Supporting the assumption that positively selected, hypervariable sites are indicative of amino acid sites implicated in functional diversity, mutants of the 8th cystatin unit of tomato multicystatin including alternative residues at positively selected sites in the N-terminal trunk exhibited highly variable affinities for the cysteine proteases papain, cathepsin B and cathepsin H. Overall, these observations support the hypothesis that plant cystatins have been under selective pressure to evolve in response to predatory challenges by herbivorous enemies. They also indicate the potential of site-directed mutagenesis at positively selected sites for the generation of cystatins with improved binding properties.

Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming.

Plants have become, over the last ten years, a suitable alternative to microbial and animal cell factories for the production of clinically-useful, therapeutic proteins. Besides the well known advantage of low-cost and large-scale production of safe and biologically active mammalian proteins, plants also are able to perform most post-translational maturations required for biological activity and suitable pharmacokinetics of recombinant therapeutic proteins. In this short review we focus on glycosylation and proteolytic processing of plant-made pharmaceuticals during their transport through the plant cell's secretory pathway. We also address the practical implications of these important processes on the effectiveness of plant molecular pharming systems.