Transgenic Expression in Arabidopsis of a Polyprotein Construct Leading to Production of Two Different Antimicrobial Proteins

François, Isabelle; Bolle, Miguel F.C. De; Dwyer, Geoff; Goderis, Inge J.W.M.; Woutors, Piet F.J.; Verhaert, Peter; Proost, Paul; Schaaper, W.M.M.; Cammue, Bruno P. A.; Broekaert, Willem F.

doi:10.1104/pp.010794

Cited by 74 publications

(58 citation statements)

References 33 publications

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“…4A). To directly determine whether the cax mutants have an altered apoplastic pH, the pH of apoplastic fluids from wild-type, cax1, cax3, and cax1/cax3 leaves was measured (François et al, 2002). This revealed that cax1/ cax3 mutants have a significantly higher apoplastic pH than wild-type plants (Fig.…”

Section: Discussionmentioning

confidence: 99%

Vacuolar CAX1 and CAX3 Influence Auxin Transport in Guard Cells via Regulation of Apoplastic pH

et al. 2012

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CATION EXCHANGERs CAX1 and CAX3 are vacuolar ion transporters involved in ion homeostasis in plants. Widely expressed in the plant, they mediate calcium transport from the cytosol to the vacuole lumen using the proton gradient across the tonoplast. Here, we report an unexpected role of CAX1 and CAX3 in regulating apoplastic pH and describe how they contribute to auxin transport using the guard cell's response as readout of hormone signaling and cross talk. We show that indole-3-acetic acid (IAA) inhibition of abscisic acid (ABA)-induced stomatal closure is impaired in cax1, cax3, and cax1/cax3. These mutants exhibited constitutive hypopolarization of the plasma membrane, and time-course analyses of membrane potential revealed that IAAinduced hyperpolarization of the plasma membrane is also altered in these mutants. Both ethylene and 1-naphthalene acetic acid inhibited ABA-triggered stomatal closure in cax1, cax3, and cax1/cax3, suggesting that auxin signaling cascades were functional and that a defect in IAA transport caused the phenotype of the cax mutants. Consistent with this finding, chemical inhibition of AUX1 in wild-type plants phenocopied the cax mutants. We also found that cax1/cax3 mutants have a higher apoplastic pH than the wild type, further supporting the hypothesis that there is a defect in IAA import in the cax mutants. Accordingly, we were able to fully restore IAA inhibition of ABA-induced stomatal closure in cax1, cax3, and cax1/cax3 when stomatal movement assays were carried out at a lower extracellular pH. Our results suggest a network linking the vacuolar cation exchangers to apoplastic pH maintenance that plays a crucial role in cellular processes.Stomata are pores at the surface of the leaves, gating water loss and gas exchange between plants and the atmosphere. One stoma is formed by two specialized guard cells that are able to modulate their size and shape to control stomatal aperture in response to various signals, including water status, hormonal stimuli, CO 2 levels, light, or temperature (Kwak et al., 2008). These stomatal movements are regulated by ion fluxes in guard cells, the changes in the osmoticum status being compensated by water movement, which modifies the cell's volume. Ion transport between the cell and ion stores (vacuole, apoplastic space) must be therefore tightly controlled, and any change in the guard cell's ability to regulate this can compromise its faculty to trigger stomatal movement.Calcium ion (Ca 2+

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Section: Discussionmentioning

confidence: 99%

Vacuolar CAX1 and CAX3 Influence Auxin Transport in Guard Cells via Regulation of Apoplastic pH

et al. 2012

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“…A more advanced and durable approach is to engineer transgenic plants that synthesize chimeras of two or more AMPs with different modes of action to develop a broad-spectrum resistance. For example, overexpression of a chimeric cleavable polyprotein precursor containing the mature domains of the plant defensins Dahlia merckii AMP1 (DmAMP1) and RsAFP2 resulted in the efficient release of both bioactive antifungal peptides (François et al, 2002;Thevissen et al, 2004).…”

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confidence: 99%

ARACINs, Brassicaceae-Specific Peptides Exhibiting Antifungal Activities against Necrotrophic Pathogens in Arabidopsis

et al. 2015

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Plants have developed a variety of mechanisms to cope with abiotic and biotic stresses. In a previous subcellular localization study of hydrogen peroxide-responsive proteins, two peptides with an unknown function (designated ARACIN1 and ARACIN2) have been identified. These peptides are structurally very similar but are transcriptionally differentially regulated during abiotic stresses during Botrytis cinerea infection or after benzothiadiazole and methyl jasmonate treatments. In Arabidopsis (Arabidopsis thaliana), these paralogous genes are positioned in tandem within a cluster of pathogen defense-related genes. Both ARACINs are small, cationic, and hydrophobic peptides, known characteristics for antimicrobial peptides. Their genes are expressed in peripheral cell layers prone to pathogen entry and are lineage specific to the Brassicaceae family. In vitro bioassays demonstrated that both ARACIN peptides have a direct antifungal effect against the agronomically and economically important necrotrophic fungi B. cinerea, Alternaria brassicicola, Fusarium graminearum, and Sclerotinia sclerotiorum and yeast (Saccharomyces cerevisiae). In addition, transgenic Arabidopsis plants that ectopically express ARACIN1 are protected better against infections with both B. cinerea and A. brassicicola. Therefore, we can conclude that both ARACINs act as antimicrobial peptides.

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“…Some of the polyprotein expression systems have exploited endogenous plant protease activity to liberate multiple POIs connected by protease substrate sequences (François et al ., 2002a; Urwin et al ., 1998; Walker and Vierstra, 2007; Zhang et al ., 2011). However, processing of the polyproteins in this case can only occur in a particular cellular compartment within hosts where the specific proteases are located.…”

Section: Introductionmentioning

confidence: 99%

“…However, the ‘remnant’ 2A residues appended to the carboxyl terminus of the processed proteins could hinder protein activity and/or cellular targeting (François et al ., 2004; Randall et al ., 2004; Samalova et al ., 2006). Removal of the extraneous 2A residues using host endogenous proteases has been attempted in plant (François et al ., 2002a) and mammalian systems (Fang et al ., 2005), yet the requirement of specific endogenous proteases and inability to completely avoid appending remnant protease substrate linker residues to the cleaved POIs have significantly limited its general usefulness. To resolve these problems, we have exploited in vivo self‐excision of the 2A sequence extension via intein‐mediated N‐terminal autocleavage, by fusing an engineered mini‐intein with the 2A sequence through a linker to create the ‘IntF2A’ self‐excising domain.…”

Section: Introductionmentioning

confidence: 99%

Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide

Zhang

Rapolu

Kumar³

et al. 2017

Plant Biotechnology Journal

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SummaryA novel approach is developed for coordinated expression of multiple proteins from a single transgene in plants. An Ssp DnaE mini‐intein variant engineered for hyper‐N‐terminal autocleavage is covalently linked to the foot‐and‐mouth disease virus 2A (F2A) peptide with unique ribosome skipping property, via a peptide linker, to create an ‘IntF2A’ self‐excising fusion protein domain. This IntF2A domain acts, in cis, to direct highly effective release of its flanking proteins of interest (POIs) from a ‘polyprotein’ precursor in plants. This is successfully demonstrated in stably transformed cultured tobacco cells as well as in different organs of transgenic tobacco plants. Highly efficient polyprotein processing mediated by the IntF2A domain was also demonstrated in lettuce and Nicotiana benthamiana based on transient expression. Protein constituents released from the polyprotein precursor displayed proper function and accumulated at similar levels inside the cells. Importantly, no C‐terminal F2A extension remains on the released POIs. We demonstrated co‐expression of as many as three proteins in plants without compromising expression levels when compared with those using single‐protein vectors. Accurate differential cellular targeting of released POIs is also achieved. In addition, we succeeded in expressing a fully assembled and functional chimeric anti‐His Tag antibody in N. benthamiana leaves. The IntF2A‐based polyprotein transgene system overcomes key impediments of existing strategies for multiprotein co‐expression in plants, which is particularly important for gene/trait stacking.

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Transgenic Expression in Arabidopsis of a Polyprotein Construct Leading to Production of Two Different Antimicrobial Proteins

Cited by 74 publications

References 33 publications

Vacuolar CAX1 and CAX3 Influence Auxin Transport in Guard Cells via Regulation of Apoplastic pH

Vacuolar CAX1 and CAX3 Influence Auxin Transport in Guard Cells via Regulation of Apoplastic pH

ARACINs, Brassicaceae-Specific Peptides Exhibiting Antifungal Activities against Necrotrophic Pathogens in Arabidopsis

Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide

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