Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology ¨ Edita Hol´askov´a, Petr Galuszka, Ivo Fr´ebort, M. Tufan Oz PII: DOI: Reference:

S0734-9750(15)00057-9 doi: 10.1016/j.biotechadv.2015.03.007 JBA 6916

To appear in:

Biotechnology Advances

Received date: Revised date: Accepted date:

30 August 2014 25 February 2015 10 March 2015

Please cite this article as: Hol´askov´a Edita, Galuszka Petr, Fr´ebort Ivo, Tu¨ M, Antimicrobial peptide production and plant-based expression systems fan Oz for medical and agricultural biotechnology, Biotechnology Advances (2015), doi: 10.1016/j.biotechadv.2015.03.007

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ACCEPTED MANUSCRIPT Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology Edita Holásková, Petr Galuszka, Ivo Frébort, M. Tufan Öz†,*

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Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural

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Research, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic *



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Corresponding author: M. T. Öz, tel +1 352 273 3401, e-mail [email protected] Current address: Agronomy Department, University of Florida, Gainesville, FL 32611, USA

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E-mail addresses: [email protected] (E. Holásková), [email protected] (P. Galuszka), [email protected] (I. Frébort), [email protected] (M. T. Öz)

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Abstract

Antimicrobial peptides (AMPs) are vital components of the innate immune system of nearly all living organisms. They generally act in the first line of defense against various pathogenic bacteria, parasites,

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enveloped viruses and fungi. These low molecular mass peptides are considered prospective

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therapeutic agents due to their broad-spectrum rapid activity, low cytotoxicity to mammalian cells and unique mode of action which hinders emergence of pathogen resistance. In addition to medical use,

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AMPs can also be employed for development of innovative approaches for plant protection in agriculture. Conferred disease resistance by AMPs might help us surmount losses in yield, quality and safety of agricultural products due to plant pathogens. Heterologous expression in plant-based systems, also called plant molecular farming, offers cost-effective large-scale production which is

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regarded as one of the most important factors for clinical or agricultural use of AMPs. This review presents various types of AMPs as well as plant-based platforms ranging from cell suspensions to whole plants employed for peptide production. Although AMP production in plants holds great promises for medicine and agriculture, specific technical limitations regarding product yield, function and stability still remain. Additionally, establishment of particular stable expression systems employing plants or plant tissues generally requires extended time scale for platform development compared to certain other heterologous systems. Therefore, fast and promising tools for evaluation of plant-based expression strategies and assessment of function and stability of the heterologously produced AMPs are critical for molecular farming and plant protection. Key words: Antimicrobial peptides; Therapeutic peptides; Plant protection; Plant molecular farming; Agricultural biotechnology; Medical biotechnology; Heterologous expression; Screening tools; Genetically modified plants

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ACCEPTED MANUSCRIPT Abbreviations: AMP, antimicrobial peptide; CaMV35S, cauliflower mosaic virus 35S RNA; ER, endoplasmic reticulum; GM, genetically modified; PMF, plant molecular farming Contents

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1. Introduction

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2. Plant antimicrobial peptides

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3. Antimicrobial peptide utilization for plant protection and medical approaches 3.1. Antimicrobial peptide expression in plants for disease resistance 3.1.1. Constitutive or inducible expression of antimicrobial peptides in plants 3.1.2. Strategies to enhance stability of antimicrobial peptides

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3.1.3. Gene stacking for improved plant protection

3.2. Therapeutic antimicrobial peptide production in plants

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3.2.1. Expression of therapeutic peptides in plant chloroplasts 3.2.2. Antimicrobial peptide expression in cereal grains 3.2.3. Transient expression of antimicrobial peptides using viral vectors

4.1. Whole transgenic plants

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4. Plant-based expression strategies for antimicrobial peptide production

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4.2. Tissue specific expression

4.3. Expression strategies that utilize plant cell or tissue culture

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4.3.1. Callus culture systems

4.3.2. Suspension cultures of plant cells 4.3.3. Hairy root cultures 4.4. Alternative approaches employing transient expression

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4.4.1. Agrobacterium or virus infection 4.4.2. Cell pack technology

5. Tools for screening and evaluation of plant-based expression strategies 5.1. Agroinfiltration of leaf tissues 5.2. Seedling transformation 6. Functional assessment of antimicrobial peptides expressed in plants 7. Considerations on commercial production of antimicrobial peptides in plants 7.1. Selection of a production platform for molecular farming of antimicrobial peptides and regulations on genetically modified plants 7.2. Considerations on target antimicrobial peptides for commercial production 8. Concluding remarks Acknowledgements References

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ACCEPTED MANUSCRIPT 1. Introduction Low molecular mass antimicrobial peptides (AMPs) are active against a broad range of pathogenic organisms attacking plants or animals (Thevissen et al., 2007). During early stages of response to

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pathogens, AMPs together with antimicrobial metabolites and stress related proteins act as

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components of non-specific basal defense mechanisms (Bent and Mackey, 2007). These small peptides, also called peptide antibiotics, present a new generation of biocidal agents for plant

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protection as well as microbial disease treatment in human and animals. Additionally, they have been shown to promote accumulation of immune cells, stimulate angiogenesis and aid wound repair (Bowdish et al., 2005; Elsbach, 2003). Some of the cationic AMPs have also been demonstrated to

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possess cytotoxic activity against cancer cells (Hoskin and Ramamoorthy, 2008). Diverse types of AMPs show broad-spectrum activity, display low cytotoxicity to mammalian cells

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and exhibit a unique mode of action. Although not unraveled completely, proposed modes of AMP action involve interaction of peptides with microbial membrane and pore formation which leads to one or more of many processes including micellization, membrane depolarization, cytoplasmic leakage, internalization of biocidal peptides or damage to intracellular macromolecule synthesis (Melo et al.,

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2009; Straus and Hancock, 2006). Mode of action relies on three dimensional structure and interaction

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of AMPs with the microbial membrane via hydrophobic or electrostatic forces. Although most of the AMPs adopt a similar characteristic of forming an amphipathic structure, the secondary structures may

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vary between classes (i.e., α-helices, β-sheets, disordered loops and extended structures). To date, more than 1700 natural AMPs have been identified, and thousands of derivatives and analogues have been computationally designed, engineered or synthetically generated using natural

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AMPs as templates (Laverty et al., 2011). Web-based resources and databases (Table 1) provide valuable information on both natural and synthetic AMPs. These resources and databases offer convenient search tools for peptides with desired properties, provide various analysis and prediction tools, and help boost the process of discovery and design of novel AMPs with improved therapeutic index or antimicrobial properties. Moreover, certain databases might also be used for determination of a proper expression strategy for heterologous production of a target AMP (Table 1). Overall, various databases and resources not only provide information but also aid research on AMPs and allow better exploitation of biological activities of peptides in pharmaceutical as well as agricultural approaches. Majority of AMPs are encoded by genes, while others are products of secondary metabolism or synthesized by non-ribosomal peptide synthases (Giessen and Marahiel, 2012). Certain AMPs are generated after post-translational modifications, have cyclic structure or contain unusual amino acids (Laverty et al., 2011). Sources of AMPs range from microorganisms to plants, vertebrates and invertebrates. Furthermore, an AMP, independent of its origin, might provide resistance to a fungal or bacterial pathogen in a plant species (Rahnamaeian et al., 2009). By their own nature, AMPs provide 3

ACCEPTED MANUSCRIPT resistance to various plant pathogens including bacteria, fungi and viruses. Hence, they present innovative approaches for plant protection in agriculture. Genetically modified (GM) plants have provided tremendous advances in crop improvement and

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molecular farming. Various plants including maize, soybean, canola and others have been engineered

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and commercialized over the last few decades (CERA, 2012). Heterologous expression of proteins and peptides in GM plants are mainly performed for improved agricultural practices, cost-efficient crop

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production and/or increased crop yield and quality. Traits engineered in most of the GM plants include herbicide, insect or disease resistance. On the other hand, progress in biotechnology has enabled plantbased expression of various biological macromolecules and polymers bearing critical importance in

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medicine, pharmacy and industry. These applications generally known as plant molecular farming (PMF) has introduced new areas of utilization for plants besides their conventional uses as food and feed. Various effective plant-based expression platforms range from plant cell suspensions to field-

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grown transgenic plants (Twyman et al., 2003). Although new generation of GM plants moves forward via employment in PMF, heterologous expression of AMPs in plants for molecular farming has been limited. Additionally, technical restrictions leading to low product yield and instability still

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await innovative solutions (Obembe et al., 2011). Therefore, strategies of AMP production for medical

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and agricultural practices and potential of plant-based expression systems are discussed here, to provide a more comprehensive insight in AMP production in plants.

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2. Plant antimicrobial peptides

Plants express, either constitutively or in response to microbial attack, a repertoire of multiple AMPs which are active mainly against phytopathogens including bacteria, fungi, and oomycetes as well as

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herbivorous insects. Moreover, some plant AMPs have been shown to be biologically active against neoplastic cells and microorganisms pathogenic to human, reflecting their high potential for use in not only agriculture but also healthcare. Although plants produce a large cocktail of functionally diverse AMPs, there are some similarities between individual classes, including their cationic character in most of the cases, low molecular weight and high content of cysteine residues that form disulphide bonds stabilizing the structure of peptides. According to their tertiary structure plant AMPs can be categorized into different families including thionins, defensins, lipid transfer proteins (LTPs), snakins, hevein-like AMPs, cyclotides and knottins (Nawrot et al., 2014; Stotz et al., 2013). Thionins are subdivided into two distinct groups, α/β thionins and γ thionins. All prothionins possess a C-terminal acidic peptide that neutralizes the basic thionin, and an N-terminal leader peptide that directs them to the endoplasmic reticulum (ER). Removal of leader peptide is essential for thionin

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ACCEPTED MANUSCRIPT activation (Stec, 2006). Mature thionins are deposited in the vacuole (Romero et al., 1997), and suggested to be released after a pathogen attack (Stotz et al., 2013). The first plant defensins isolated from wheat and barley were initially grouped with γ thionins.

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However this group of plant AMPs was later classified as defensins due to their structural properties

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(Bruix et al., 1993). Majority of proproteins contain N-terminal ER targeting signal sequences. Plant defensins are mainly active against diverse fungi (Hegedus and Marx, 2013), and in certain cases

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bacteria (Franco et al., 2006). Some of plant defensins inhibit protein synthesis (Mendez et al., 1990) or possess trypsin protease (Wijaya et al., 2000) and/or α-amylase inhibitor like activities (Pelegrini et al., 2008). Plant defensins exhibit properties of medical importance including antifungal, antiviral and

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anticancer activities (Thevissen et al., 2007; Wong and Ng, 2005).

Antimicrobial LTPs, sometimes referred as nonspecific LTPs due to their broad substrate specificity,

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are categorized into two subgroups according to their sizes. All nonspecific LTPs have a common structure including secretion signal peptide depositing mature products to apoplastic space, where they play important roles in plant defense response (Terras et al., 1992; Segura et al., 1993). Besides their antifungal and antibacterial activities, LTPs might also be involved in other diverse biological

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processes including induction of somatic embryogenesis (Sabala et al., 2000), synthesis of cutin (Pyee

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et al., 1994), lipid metabolism (Tsuboi et al., 1992), and pollen tube adhesion (Park et al., 2000). Some members of LTPs were found to be important allergens in fruits, vegetables, nuts, seeds, and latex as

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well as in pollens of trees and weeds (Egger et al., 2010). Cell wall associated snakins have been originally determined in potato tubers, where they play an important role in plant defense against various bacteria and fungi. Homologous genes coding for

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snakins have been identified in a variety of monocotyledonous and dicotyledonous plant species (Almasia et al., 2010).

Hevein-like peptides are comprised of classes differing in number of disulphide bonds (3, 4, or 5) present in peptides. They possess an N-terminal secretion signal and a C-terminal extension, which needs to be proteolytically cleaved off to activate the peptide (De Bolle et al., 1996; Nielsen et al., 1997). Mature peptides exhibit chitin-binding properties and are active against various fungi and bacteria (Huang et al., 2002a; Nielsen et al. 1997). Knottins having intramolecular bonds known as cystine knots play multiple biological roles in plant protection against various pathogens. Some members of this group have been shown to possess αamylase inhibitor activity (Chagolla-Lopez et al. 1994; Gao et al. 2001; Yokoyama et al. 2009). Knottins are formed as proproteins having a signal peptide at the N-terminus, which directs them for secretion (De Bolle et al., 1996; Liu et al., 2000).

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ACCEPTED MANUSCRIPT Cyclotides represent AMPs with cyclized head-to-tail covalently joined backbone with cystine knots (Padovan et al., 2010). Cyclotides are categorized according to their structural properties into two main subgroups named Möbius and bracelet (Craik et al., 1999). These AMPs are generated from linear precursors with conserved asparagine or aspartic acid residue at the C-terminal end of the

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mature cyclotide domain (Craik, 2010). The cyclic cystine knot structure is largely responsible for

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exceptionally great chemical and thermal stability as well as recalcitrance to enzymatic degradation

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(Craik et al., 2006). Contrary to the vast majority of AMPs, cyclotides do not possess cationic character, but they contain hydrophobic residues forming a patch that is exposed at the surface of the molecule (Pränting et al., 2010). The first described member of this group was Kalata B1 (Saether et al., 1995). This peptide was used by an African tribe to induce and facilitate childbirth, as it

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accelerates contractions. Overall, cyclotides possess diverse biological activities including antimicrobial, insecticidal, uterotonic and anti-HIV activities (Craik, 2010). Hence, there is a growing

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interest in using cyclotides not only for agricultural purposes but also for drug design in medicine (Craik et al., 2006).

3. Antimicrobial peptide utilization for plant protection and medical approaches

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Recently, the interest in expression of AMPs in plants has significantly increased because of two main

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reasons, namely the need for novel approaches in plant protection and the demand for new antimicrobial agents in medicine. Natural and synthetic AMPs are considered prospective candidates

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which might address these demands via various approaches in agricultural and medical biotechnology. Besides AMPs, therapeutic peptides with no antimicrobial activity have also been produced in plants (Lico et al., 2012; Viana et al., 2013). These therapeutic peptides including antigens and antibodies have not been discussed in this work. Although promising, production of AMPs in plants has been

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challenging because of particular physiochemical properties of these peptides. Hence, specific expression strategies leading to higher yield and stability of AMPs are often employed together with optimization of AMP activity against target pathogen groups and alteration of toxicity to host and nontarget organisms. Current progress in the field of AMP production in plants is described in more detail in following sections. 3.1. Antimicrobial peptide expression in plants for disease resistance Crop plants are susceptible to various diseases caused mainly by diverse pathogenic fungi or bacteria. This contributes to substantial loss in yield and quality of agricultural products, which leads to significant economic loss worldwide (Abdallah et al., 2010; Coca et al., 2006; Furman et al., 2013; Rahnamaeian et al., 2009; Rivero et al., 2012; Zakharchenko et al., 2013a, 2013b; Zhou et al., 2011). Crop loss due to plant pathogens and pests reach 30 – 40% in developing countries annually (Flood, 2010). Nowadays, bacterial and fungal disease control mainly relies on agrochemicals including classical antibiotics such as streptomycin and fungicides such as copper containing compounds. These 6

ACCEPTED MANUSCRIPT chemicals may have negative impact on human health and environment. Moreover, extensive use of agrochemicals can induce pathogen resistance which makes conventional pesticides less effective (Komárek et al., 2010; Kümmerer, 2004; McManus et al., 2002). Therefore, strategies based on development of disease resistant plant varieties expressing natural or synthetic AMPs of plant or non-

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plant origin (Table 2) can significantly reduce the use of chemicals in agriculture, which in turn might

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be appreciated by environmentalists and customers.

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To develop protection against plant pathogenic bacteria and fungi, heterologous expression of natural or de novo designed AMPs has been successfully used in an array of diverse agronomically important crops (Table 2). Introduction of genes coding for certain AMPs can also induce resistance to insect

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pathogens. Expression of Brassica rapa defensin 1 transgene in rice resulted in strong resistance to sap-sucking insect, brown planthopper (Choi et al., 2009). Furthermore, AMPs, expressed in plants and integrated in overall protective systems, not only provide defense against biotic stresses, but also

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might induce protection against abiotic stresses. It was demonstrated that expression of a gene coding for cecropin P1 in rapeseed increased tolerance to oxidative stress caused by herbicide paraquat (Zakharchenko et al., 2013a). The same gene was used for transgenesis of camelina, where cecropin

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P1 expressing plants exhibited increased sustainability under salt stress (Zakharchenko et al., 2013b).

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Improved tolerance to both biotic and abiotic stress factors might be explained by probable function of cecropin P1 or certain other AMPs in up-regulation of expression of endogenous genes involved in

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general stress response (Campo et al., 2008). Interestingly, accumulation of AMPs in plants might also improve plant productivity. For example, transgenic tobacco lines expressing an indolicin-derived peptide (Rev4) displayed increased biomass production in field trials (Xing et al., 2006).

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3.1.1. Constitutive or inducible expression of antimicrobial peptides in plants Strong and constitutive promoters that are frequently employed to drive AMP expression for plant protection include cauliflower mosaic virus 35S RNA (CaMV35S) promoter or ubiquitin promoters from various sources (Table 2). The undecapeptide BP100, which was identified from a library of synthetic cecropin A-melittin hybrids (Badosa et al., 2007), was employed as an agent for plant protection (Company et al., 2014; Nadal et al., 2012). Additionally, derivatives of BP100 were designed based on structural requirements for high-level expression in plants (Badosa et al., 2013). Expression of BP100 and derivatives was achieved under the control of CaMV35S or ubiquitin promoters (Table 2). Defensins are a family of naturally occurring cysteine-rich AMPs (Marmiroli and Maestri, 2014). Certain members of defensins were employed in transgenic plants to confer resistance against bacteria and fungi. A putative defensin (NmDef02) from Nicotiana megalosiphon provided enhanced resistance in potato against the oomycete Phytophthora infestans, the causative agent of potato late blight disease (Portieles et al., 2010). The Raphanus sativus antifungal protein 2 (RsAFP2) from radish, expressed under CaMV35S promoter, provided resistance to a fungal pathogen Alternaria 7

ACCEPTED MANUSCRIPT longipes in transgenic tobacco (Terras et al., 1995). The same defensin from radish, i.e., RsAFP2, was expressed in wheat under maize ubiquitin-1 promoter. Transgenic wheat lines displayed enhanced resistance to Fusarium graminearum and Rhizoctonia cerealis (Li et al., 2011).

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On the other hand, use of organ-specific or inducible promoters to precisely control transgene

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expression offer clear advantages. Inducible poplar win3.12T promoter, which exhibited strong systemic activity upon pathogen challenge or wounding, was transcriptionally fused to the plant-

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optimized nucleotide sequences of either MsrA2 or temporin A for development of disease resistance in tobacco (Yevtushenko and Misra, 2007). Transgenic plants had normal phenotype and were resistant to various pathogenic fungi. Moreover, lowest susceptibility to pathogens was observed in

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lines with highest transgene expression and AMP accumulation which were directly correlated to the gene copy number. In a bioassay with detached leaves, lines with multiple copies of the transgenes were found to be more tolerant to pathogens than those with a single copy (Yevtushenko and Misra,

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2007). Similar correlation between transgene copy number, transcript accumulation and strength of disease resistance was reported in other studies (Ntui et al., 2010; Yevtushenko et al., 2005). In another example of transgene expression under an inducible promoter, expression of an insect

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antifungal peptide (metchnikowin) was driven under the control of a pathogen-responsive mannopine

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synthase (mas) promoter in barley plants. Metchnikowin accumulated in plant apoplastic space in response to powdery mildew as well as Fusarium head blight and root rot, and conferred resistance on

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transgenic barley lines (Rahnamaeian et al., 2009). Tissue specific expression can also be used to avoid the phytotoxic effects of AMPs in vegetative tissues. In a recent study, rice glutelin B1 or B4 promoter sequences were used for endosperm specific expression of a bioactive peptide, cecropin A (Bundó et al., 2014). Transgenic lines were resistant to Fusarium verticillioides and Dickeya dadantii,

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and the rice seeds accumulating cecropin A were viable. The amount of accumulated peptide was also determined by an immunoblot assay of protein extracts from transgenic mature seeds, and it was indicated that the study has implications for both molecular farming and plant protection (Bundó et al., 2014). In another study, tuber specific expression of a dermaseptin B1 derivative MsrA2 was driven by BiP Pro1-1 promoter from Douglas-fir, and heterogeneous AMP enabled engineering of resistance to soft rot caused by pathogenic Pectobacterium carotovorum in potato plants (Yevtushenko and Misra, 2012). 3.1.2. Strategies to enhance stability of antimicrobial peptides Evidence on absence of pathogen resistance in transgenic plants expressing AMPs was also reported in couple of studies. Peptides with in vitro biocidal properties did not provide resistance to pathogens when they were expressed in plants (Allefs et al., 2005; Florack et al., 1995; Hightower et al., 1994). One of the possible explanations for low disease resistance is the cellular degradation of foreign AMPs by plant endogenous proteases (Mills et al., 1994; Sharma et al., 2000). Stability and accumulation 8

ACCEPTED MANUSCRIPT levels of peptides are critical for generation of antimicrobial response during pathogen invasion. Hence, researchers employ diverse strategies to achieve stability and accumulation. It has been proposed that one of the most crucial factor influencing not only in vivo stability of the recombinant

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peptide, but also the final yield is subcellular targeting of the product.

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The most commonly used strategy to enhance stability is inclusion of auxiliary signal sequences from source, host or closely related organisms to target the product to extracellular space (Boscariol et al.,

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2006; Bundó et al., 2014; Chakrabarti et al., 2003; Coca et al., 2006; Jan et al., 2010; Rivero et al., 2012; Vidal et al., 2006; Xing et al., 2006; Zhou et al., 2011). Most of the proteins or peptides lacking a signal peptide accumulate in the cytosol, generally resulting in low yields (Conrad and Fiedler,

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1998). Furthermore, since phytopathogenic microorganisms multiply in the apoplast before attacking plant cells (Alfano and Collmer, 1996), secretion of AMPs should enhance peptide-pathogen interaction and prevent tissue colonization by pathogens (Düring, 1996). For instance, Boscariol et al.

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(2006) used successfully an insect native signal peptide for secretion of the insect-derived attacin A gene product to intercellular space in transgenic sweet orange in order to obtain plants resistant to Xanthomonas axonopodis, the causative agent of citrus canker. In another study, cecropin B gene from

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giant silk moth was fused to barley α-amylase signal sequence, and the chimeric gene was used for

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constitutive expression of cecropin B in tomato (Jan et al., 2010). Transgenic plants were shown to be significantly resistant against bacteria wilt and bacteria spot, diseases caused by two of the major

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pathogens of tomato.

Nevertheless, targeting to apoplastic space is not always necessary to obtain disease resistance (Abdallah et al., 2010; Imamura et al., 2010; Jha and Chattoo, 2010; Li et al., 2011; Osusky et al., 2004). Transformation of creeping bentgrass was performed using two different constructs containing

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either the coding sequence of penaeidin4-1 from shrimp or the penaeidin4-1 fused to secretion signal sequence from tobacco AP24 protein (Zhou et al., 2011). Transgenic lines exhibited similar levels of resistance to brown patch and dollar spot regardless of the construct used for their transformation. In another study, constitutive expression of cecropin A gene designed to secret the encoded peptide to extracellular space had negative effects on fitness and fertility of transgenic rice plants, whereas cecropin A, which had an extra C-terminal KDEL tetrapeptide extension and was retained in ER, had no phenotypic effects on transgenic plants (Coca et al., 2006). Hence, ER retention of AMPs can prevent cytotoxicity in host plant cells. In general, stability of recombinant peptide is largely influenced by its final subcellular destination. Among subcellular targets, ER possesses a favorable environment with low protease activity and presence of molecular chaperones enabling efficient protein folding. Moreover, inclusion of a KDEL sequence does not influence significantly the biological activity of a heterologously produced AMP (Badosa et al., 2013). It has been shown that

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ACCEPTED MANUSCRIPT incorporation of an ER retention signal promotes accumulation of recombinant AMP in seed protein bodies (Bundó et al., 2014). Native seed protein storage organelles (such as ER-derived protein bodies and de novo formed protein

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storage vacuoles) offer tremendous benefits in terms of product protection from degradation.

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Additionally, protein storage bodies aid in purification steps as well as post-harvest encapsulation. Expression of an AMP with certain protein fusion partners, which induce aggregation of protein

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bodies-like organelles (even in plant tissues normally lacking such subcellular compartments), might result in peptide accumulation in a practical and efficient manner. Examples of such fusion partners include natural zeins, elastin-like polypeptides and hydrophobins (Khan et al., 2012).

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Fusion of a target AMP to a carrier protein tag is regarded as an effective strategy to stabilize the peptide, increase its accumulation, and protect the final product from proteolytic degradation.

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Moreover, protein fusions can mask the lethal effects of AMPs on host plant cells (Li, 2011; Viana et al., 2013). Bioactive defense peptides were expressed in hairy roots of tomato for development and evaluation of a strategy to reduce infection caused by pathogenic organisms targeting tomato roots (Fang et al., 2006). The peptides were fused at C-terminus with a secreted enzyme from maize, which

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acted as a display scaffold. The peptide–scaffold fusion products accumulated in the rhizosphere and

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reduced pathogen infection significantly (Fang et al., 2006). In another study it was shown that protein extracts of the same secreted enzyme, responsible for phytohormone degradation in maize, were stable

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over a month without addition of any protective compounds (Galuszka et al., 2001). Hence, this enzyme or its homologs might represent effective fusion partners for peptide display, since the stability of fusion protein in the presence of plant proteases is one of the most important factors for accumulation of product at effective levels. On the other hand, the carrier protein in the peptide–

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scaffold construct might retain its enzymatic activity and expression of a phytohormone-degrading enzyme in intact plants might have negative effects on plant development as a result of a probable phytohormone imbalance (Fang et al., 2006). In addition to the aforementioned carrier protein, there are various tags widely used in heterologous expression of peptides. They can be grouped according to their common features and include easy to detect fusions, fusions to viral coat proteins, immunogenic protein partners, and purification-facilitating proteins (Viana et al., 2013). 3.1.3. Gene stacking for improved plant protection An approach to obtain plants broadly protected against a range of phytopathogens employs pyramiding or stacking of diverse transgenes coding for proteins or peptides with complementary antibacterial and antifungal activities. Such a combinatorial approach has recently been used in potato by expression of three different transgenes, namely AP24, dermaseptin, and lysozyme, products of which exhibit diverse antimicrobial activities (Rivero et al., 2012). When two or three transgenes were co-expressed, transgenic potato lines performed better resistance against bacterial infections than those 10

ACCEPTED MANUSCRIPT expressing a single transgene. Moreover, triple transgene combination provided protection against Streptomyces scabies, Phytophthora infestans, Erwinia carotovora, Rhizoctonia solani, and Fusarium solani, i.e., five potato pathogens causing substantial economic loss in potato production worldwide. In a similar work, Xing et al. (2006) used gene stacking and obtained increased pathogen resistance in

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Arabidopsis thaliana expressing a combination of an analog of magainin (Myp30) and an indolicin-

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derived peptide (Rev4), which not only possesses antimicrobial activity but also inhibits protease

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activity in vitro.

For improvement of crop disease resistance, certain properties of AMPs, target pathogen groups and host plants, as well as interaction of these actors should be considered carefully. There are complex

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associations between AMP phytotoxicity to host, plant stress response due to a transgene, and disease resistance and fitness of the transgenic plant. These relations are also affected by expression level, yield and activity of the heterologous peptide and nature of the disease caused by the phytopathogen.

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Careful consideration of all these interactions is required for sustainable plant protection in agricultural crops.

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3.2. Therapeutic antimicrobial peptide production in plants

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Modern medicine continuously faces incidences of resistance among pathogenic microorganisms to commonly employed antibiotics, thus it encourages the search for novel strategies and new

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antimicrobial agents for treatment of pathogenic diseases. Various AMPs are making their way as potential novel therapeutics for both human and animals (da Rocha Pitta et al., 2010; Hancock, 2001; Peters et al., 2010). Commercial large-scale production of AMPs with high purity is required for practical approaches in microbial disease treatment. However, lack of a suitable production platform in

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terms of product yield, cost and purity represent a barrier for use of AMPs in medicine. Therefore, the interest in PMF where plants are employed as biofactories for AMP production has significantly increased during the last decade (Table 3; Cabanos et al., 2013; Lien and Lowman, 2003; Nadal et al., 2012). Recent advances in biotechnology allowed use of plant bioreactors as attractive platforms for commercial production of peptides, proteins and pharmaceuticals (Cabanos et al., 2013; Fukuzawa et al., 2010; Lee et al., 2011; Oey et al., 2009; Rubio-Infante et al., 2012; Sathish et al., 2011). High-level product expression and accumulation as well as recovery require consideration of certain features of the product and employment of specific strategies. 3.2.1. Expression of therapeutic peptides in plant chloroplasts There are several possibilities, including transient or stable transformation of nuclear or chloroplast genomes of host plants, for heterologous expression of AMPs. Among these, transformation of chloroplast genomes, i.e., generation of transplastomic plants, are considered prospective and efficient 11

ACCEPTED MANUSCRIPT for functional expression of AMPs at high levels. Up to 20,000 copies of the transgene per cell can be expressed after gene integration into the chloroplast genome. Production of AMPs, retrocyclin-101 and protegrin-1, which have complex secondary structures stabilized by disulphide bridges, have been achieved by chloroplast transformation (Lee et al., 2011). Both peptides cannot be produced in

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microbial systems because of their complex structures and antimicrobial activities. Retrocyclin-101

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protects human cells from infection by HIV type 1 (Cole et al., 2002) and retains its full activity in

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human cervicovaginal tissue (Cole et al., 2007), whereas protegrin-1 is potent in inactivating Chlamydia trachomatis and Neisseria gonorrhoeae, two causal agents of sexually transmitted diseases in human (Qu et al., 1996; Yasin et al., 1996). When expressed with a green fluorescent protein (GFP) fusion and a histidine tag in tobacco chloroplasts, biologically active retrocyclin-101 and protegrin-1

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accumulated up to 38% and 26% of total soluble protein, respectively. It was also indicated that organic extraction of retrocyclin-101 resulted in almost 11-fold higher yield compared to affinity

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purification using the histidine tag (Lee et al., 2011).

3.2.2. Antimicrobial peptide expression in cereal grains Another expression strategy employs stable expression of AMPs in grains. Since seeds of diverse

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plants are consumed by human as well as animals, accumulation of AMPs in these storage organs

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represents a promising approach for development of plant-derived edible biopharmaceuticals. Moreover, seeds are able to store recombinant products without loss of quality over many years

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because of seed dormancy (Boothe et al., 2010). Canola seeds was proven to be versatile hosts for accumulation of a recombinant protein, hirudin, which was stable in dry seeds for over three years (Boothe et al., 1997). In another study, endosperm specific expression of lactostatin, a peptide with hypocholesterolemic activity, was achieved in rice. Twenty nine copies of lactostatin sequences were

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inserted into the gene sequence of non-conserved regions of a soybean seed storage protein (A1aB1b), and the resulting chimeric gene was used for stable transformation of rice. Transgenic lines accumulated approximately 2 mg of lactostatin per g of dry seeds (Cabanos et al., 2013). Cathelicidin LL-37 is a member of AMPs found in human skin, and is considered a promising template for development of novel antibiotics (Vandamme et al., 2012). It has also been identified in neutrophils and epithelial macrophages (Reinholz et al., 2012). Recently in our research group, heterologous expression of LL-37 fused with a universal secretion signal from maize was driven under the control of an endosperm specific promoter in barley. Our preliminary results show that this approach might represent a reliable and sustainable strategy for AMP production, as we were able to confirm accumulation of LL-37 in transgenic barley grains at protein level (data not shown). Moreover, growth characteristics and morphology of transgenic barley lines, generated using Agrobacterium-mediated transformation (Harwood et al., 2009), were similar to the untransformed control plants (Öz et al., 2014). Barley endosperm tissue as the production platform for molecular 12

ACCEPTED MANUSCRIPT farming of LL-37 is considered a promising strategy. Another report indicated accumulation of a variant of the bioactive LL-37 in extracellular space in Chinese cabbage, even though plant protection, not the PMF of cathelicidin, was the main purpose in the study. The human LL-37 variant provided resistance to various bacterial and fungal phytopathogens in homozygous transgenic lines of Chinese

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cabbage (Jung et al., 2012).

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3.2.3. Transient expression of antimicrobial peptides using viral vectors

Recently, a production system that was based on tobacco mosaic virus (TMV) and use of the full virus as a vector was developed for transient expression of a de novo designed peptide SP1-1 in Nicotiana benthamiana plants (Table 3). Viral coat protein used as a fusion partner enabled presentation of the

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AMP on the surface of viral particles (Zeitler et al., 2013). The amphipathic peptide SP1-1 is active against both plant and human pathogens, including multi-resistant Staphylococcus aureus (Dangel et

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al., 2013). After successful production and subsequent extraction of recombinant virions, the SP1-1 peptide was cleaved off from the fusion partner by bromocyanide, and the target peptide was purified using chromatographic methods. It was indicated that virus-based transient expression of SP1-1

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yielded up to 0.025 mg of pure, biologically active AMP per g of leaf biomass (Zeitler et al., 2013).

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Taken together, published data demonstrate that establishment of viable transgenic plants expressing bioactive AMPs represent a promising strategy for therapeutic peptide production as well as crop

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improvement. However, there is no universal expression strategy. Systematic approaches need to be employed for individual peptides and plant hosts depending on their particular properties. 4. Plant-based expression strategies for antimicrobial peptide production

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As stated in previous sections, AMP production in plants is employed for two main purposes, namely plant protection and molecular farming. Various plant-based expression platforms ranging from cultures of cells or tissues to transgenic plants offer diverse tools for these two applications. Plantbased systems provide cost-effective production and exhibit high scale-up capacity (Fig. 1). These systems are considered promising since they address drawbacks of other biological expression systems (Basaran and Rodriguez-Cerezo, 2008). The main advantages of plant-based systems include high product yield, quality and homogeneity obtained in these systems. Proper folding, glycosylation and disulphide bond formation, which are critical for AMP activity, can also be achieved in plant-based systems (Ma et al., 2003; Ramessar et al., 2008). When expression systems are compared according to costs associated with development, bacterial expression platforms are more desirable (Fig. 1) since bacteria and yeast are easy to transform and maintain while plant-based systems require more effort, time and cost. However, once established plant-based systems offer cost-efficient production, easy, fast and high-capacity scale-up, and low-cost purification and storage (Fig. 1), all of which rank plantbased platforms more desirable among other biological systems. 13

ACCEPTED MANUSCRIPT Various heterologous expression strategies have been used to produce AMPs in large-scale. Among these strategies, microbial systems, i.e., bacteria and yeast, have been the most widely employed hosts for peptide expression (Parachin et al., 2012). However, AMP expression in microorganisms might hinder microbial growth or decrease expression level of the target peptide. Thus, microbial expression

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of an AMP requires controlled or induced transcription and/or fusion to a carrier peptide or protein.

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Plants, on the other hand, present a suitable alternative for production of AMPs without the need for

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controlled expression. Furthermore, plant-based platforms are considered safe since plants are free of animal or human pathogens and endotoxins, and there is almost no risk of product contamination (Daniell et al., 2001; Fischer et al., 2004; Magnusdottir et al., 2013). Although risk of contamination and therapeutic risks are low in plant-based expression systems, public perception of risks are high.

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This misconception ranks plant-based systems, specifically GM plants, among the least desirable in biological expression platforms (Fig. 1). Thus, effort should be invested in public education to build a

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comprehensive and accurate view on use of GM plants for molecular farming. Although a wide range of plant-based platforms for AMP production is available, certain stages of production process and parameters affecting these stages should be considered attentively while

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choosing a platform for AMP molecular farming in plants. Yield of heterologous AMP, cost and time

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required for production, market size of the product, production scale and capacity of the process, and downstream processing should be analyzed in detail for successful applications in molecular farming

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of AMPs in plant-based systems. Time required for transgenic plant development and biosafety concerns related to GM plants are the main disadvantages in plant-based systems (Fig. 1). 4.1. Whole transgenic plants

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Stable constitutive expression of peptides in whole plant body can be achieved with various technologies for transgene integration to plant genome. Transgenic plants which contain the transgene in their genome over many generations are considered stable, and allow easy scale-up and low-cost production. In most of the studies, constitutive expression of AMPs was achieved under CaMV35S or ubiquitin promoters (Abdallah et al., 2010; Jan et al., 2010; Portieles et al., 2010; Rivero et al., 2012; Shukurov et al., 2012; Vidal et al., 2006; Zhou et al., 2011). Expression in whole plant body can be employed for development of disease resistance and/or crop improvement. Potato plants have been transformed with alfalfa antifungal peptide (Gao et al., 2000) and tachyplesin I from horseshoe crab (Allefs et al., 1996), which provided various levels of protection against fungal pathogen Verticillium dahlia and bacterial pathogen Erwinia carotovora, respectively. In another study, constitutive expression of cecropin P1 in transgenic potato conferred resistance to the fungal pathogens Sclerotinium sclerotiorum and Phytophthora infestans, the causative agents of white rot and potato blight (Zakharchenko et al., 2007). Constitutive expression of natural or synthetic

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ACCEPTED MANUSCRIPT AMPs under CaMV35S or maize ubiquitin-1 promoters has also been employed for molecular farming (Company et al., 2014; Nadal et al., 2012). However, constitutive expression and subsequent accumulation of an AMP might negatively affect

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biological functions in a host plant (Coca et al., 2006) and lead to undesirable selection pressure on

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pathogenic microorganisms. Recently, constitutive expression of a tandemly arranged units of a synthetic peptide BP100 was shown to be toxic for rice and Arabidopsis (Company et al., 2014; Nadal

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et al., 2012). Toxicity was avoided by expression of inverted repeats of BP100 whether or not elongated with sequences derived from natural AMPs. It was also indicated that the complex equilibrium between AMP phytotoxicity, antimicrobial activity and transgene-derived plant stress

(Company et al., 2014; Nadal et al., 2012).

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response is critical for fitness and disease resistance of GM plants constitutively expressing AMPs

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Certain wound- or pathogen-inducible promoters which were used for development of disease resistance in plants offer controlled expression, help eliminate phytotoxic effects of AMPs in host plants, and avoid unintended effects on non-target organisms interacting with plants (Rahnamaeian et al., 2009; Yevtushenko et al., 2005). Besides transcriptional control with promoters, product yield can

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also be boosted in many plant host systems with strategies at various stages of product synthesis,

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including translation, post-translational modification and subcellular targeting (Makhzoum et al., 2014; Obembe et al., 2011). Most widely used translational fusions include signal sequences for

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apoplastic secretion, ER retention and intracellular targeting. Additionally, fusion to certain purification tags, such as hexameric histidine tag and maltose binding protein, might also stabilize the product and help boost the expression.

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4.2. Tissue specific expression

Although heterologous expression in whole plant body enables high-level accumulation of recombinant product, the proteins or peptides expressed in all tissues such as leaves, pollen and roots might adversely affect growth and development of the transgenic plants. Expression in all plant parts could also expose herbivores and pollinating insects to the effects of the recombinant peptide, actuate undesired development of resistance among pathogens, and lead to unfavorable environmental consequences. Additionally, various tissues and their complex composition may exert hurdles during isolation and purification of the target AMP. Restriction of peptide accumulation to certain tissues using tissue specific promoters can help reduce these risks. Therefore, desired expression level and functional stability of the product as well as spatial and temporal accumulation should be considered during establishment of stable transgenic plants. Tissue specific expression of peptides might overcome the problems associated with expression and stability of the product as well as downstream processing.

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ACCEPTED MANUSCRIPT Main advantages of tissue specific expression include, but not limited to, contained expression, elimination or relief of AMP cytotoxicity in host plant during certain developmental stages and avoidance of undesired environmental effects. Additionally, AMP stability might be improved and downstream processing and peptide purification might be simplified via expression in certain tissues

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(Xu et al., 2012). Grains of cereals and legumes provide inert and stable environment for peptide

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accumulation and storage (Lau and Sun, 2009). Furthermore, diverse seed specific promoters in

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barley, wheat, maize, bean and others have been identified (De Jaeger et al., 2003; Mrízová et al., 2014; Ramessar et al., 2008). In numerous studies, rice prolamin and glutelin promoters (Bundó et al., 2014; Cabanos et al., 2013; Takagi et al., 2005; Yasuda et al., 2005), barley hordein D, α-amylase, and trypsin inhibitor promoters (Joensuu et al., 2006; Stahl et al., 2002), wheat glutenin promoter

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(Schünmann et al., 2002) and soybean glycinin promoters (Ding et al., 2006; Hudson et al., 2014; Moravec et al., 2007) have been employed for expression of various peptides and proteins in grains.

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These studies generally have implications towards molecular farming of pharmaceutical peptides or proteins, since grains are cheap to produce and harvest, easy to store and distribute, and might be employed as edible vaccines. On the other hand, strict regulations related with GM crops should be considered carefully, and cultivation, harvest and distribution of these crops, specifically GM cereals

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groups for human and animals.

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and legumes, should be isolated from that of unmodified crops, as they provide the main food and feed

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Besides molecular farming, expression restricted to a certain tissue might also be employed for plant protection. Certain phytopathogens attack plants at certain tissues, such as roots, tubers and leaves. Development of disease resistance at the site of attack is critical for durable resistance and sustainable productivity. Additionally, tissue or organ specific accumulation of AMPs might reduce post-harvest

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loss in crop plants (Yevtushenko and Misra, 2012). 4.3. Expression strategies that utilize plant cell or tissue culture There are different types of in vitro cultures where plant cells or tissues are maintained using a welladjusted balance of phytohormones. Among them, cultures of suspension cells, calli and hairy roots represent well-established techniques, in which plant cells or tissues are cultivated under specific conditions absolutely independent of climatic or geographic factors. Common features of these in vitro cultures include simplicity of transformation, product homogeneity and the short period of time required for accumulation of a heterologous product, which is generally days or weeks after transformation. In vitro cultivation methods are also considered powerful tools for testing diverse expression strategies before they are further employed for generation of stable transgenic plants. Moreover, these cultivation techniques are desirable for evaluation of secretion signal sequences, since recombinant products can easily be recovered and purified from the culture medium without large

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ACCEPTED MANUSCRIPT quantities of contaminating macromolecules such as proteins and carbohydrates (Georgiev et al., 2007; Miao et al., 2008; Plasson et al., 2008). 4.3.1. Callus culture systems

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Callus cultures can be obtained from almost every living plant tissue, although young tissues and

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tissues with meristematic or promeristematic activities are preferred (Petersen and Alfermann, 2008).

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Transgenic callus cultures are generally obtained via direct transformation of calli (Hiei and Komari, 2008), or via transformation of explants and subsequent callus induction from these explants (Carciofi et al., 2012; Imani et al., 2011; Xu et al., 2011). Although cultured calli are not commonly used for production of heterologous compounds, they can be employed as potent testing platforms especially

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for plants, where time-efficient and robust transformation screening technologies are not established. The vast majority of monocotyledonous crop plants are recalcitrant to transformation or in vitro

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regeneration and/or lack a high-throughput screening platform for evaluation of expression strategies. Hence, callus cultures represent alternative screening tools or models which provide identical physiological and genetic background as that of the host in the target expression platform. In a recent study, a probable use of transgenic barley callus as a model system for evaluation of transgenic

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modification strategies for starch bioengineering in cereals was reported. It was indicated that within 9

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weeks, as much as 1.5 g dry weight of fully transgenic calli generated after Agrobacterium-mediated transformation of barley immature embryos could be obtained (Carciofi et al., 2012). In another study,

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a system called STARTS for generation of stable transgenic roots from barley calli was developed. The strategy, which allowed analyses within 6 weeks, was employed for functional analysis of an expressed barley protein (Imani et al., 2011).

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4.3.2. Suspension cultures of plant cells Interest in plant cell suspension cultures as expression platforms has increased over the last decade. Undifferentiated cultured cells of plants bear most of the benefits offered by whole plants and exhibit advantages of microbial systems, such as easy manipulation. Suspension cultures of plant cells are composed of relatively homogeneous suspensions of rapidly dividing cells established by transfer of callus (either transgenic or not) into liquid media. Suspension-cultured transgenic plant cells can also be prepared by their direct transformation (King, 1984; Xu et al., 2011). The most frequently employed plant species for recombinant product expression in suspension cultures is tobacco, since tobacco cells are easy to transform and maintain, and multiply rapidly. On the other hand, various other plant species have been utilized for establishment of cell cultures, including rice, soybean, alfalfa, and tomato (Xu et al., 2011). To date, diverse bioactive pharmaceutical compounds including those with antimicrobial properties have been produced in suspension cultures. A functional human lysozyme was expressed in transgenic 17

ACCEPTED MANUSCRIPT rice cell cultures, and expression levels of the recombinant lysozyme reached approximately 4% of total soluble protein (Huang et al., 2002b). In another study, tobacco Bright Yellow-2 (BY-2) cells were used for characterization of expression and activity of a peptide (ACHE-I-7.1) mimetic of aldicarb, an effective carbamate nematicide. After confirmation of biological activity of the peptide in

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suspension cultures, the construct encoding ACHE-I-7.1 was used for generation of transgenic potato

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plants that exhibited increased resistance to root nematode invasion (Liu et al., 2005).

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Although suspension cell cultures do not offer scale-up capacity as high as transgenic plants, confined expression in bioreactors allow precise control over production process, and provide a platform accordant with regulations and regarded as environmentally and therapeutically safe. Various

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biologically active pharmaceutical proteins have successfully been produced in plant cell cultures. The first commercially available therapeutic protein, that is manufactured using a plant-based system, is actually produced in carrot root cell suspension cultures (Shaaltiel et al., 2007). This plant-made

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recombinant protein (glucocerebrosidase), formulated as Elelyso™ (Protalix Biotherapeutics Inc., Israel), has been approved by the US Food and Drug Administration (FDA) in May 2012 for enzyme replacement therapy of Gaucher's disease (Aviezer et al. 2009; Grabowski et al. 2014). Production of

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glucocerebrosidase in carrot cell suspensions provides cost-efficient and pathogen-free manufacturing

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compared to counterparts produced in Chinese hamster ovary cells or mammalian cell cultures. Additionally, counterparts require post-production modifications in product glycosylation to expose

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terminal α-mannose residues, which are needed for mannose receptor-mediated uptake by target cells. These modifications increase production cost. Plant-made glucocerebrosidase, also called taliglucerase alfa, requires no additional steps to yield a recombinant enzyme with exposed terminal mannose residues (Grabowski et al. 2014). Approval and commercial success of this drug will lead a new era in

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production of pharmaceuticals, including AMPs, in plant-based platforms. Major disadvantages of suspension cultures are their relative instability, low protein productivity and limited scale-up capacity. Moreover, cell cultures are very susceptible to contamination and overgrowth by microbes. Therefore, once transgenic suspension culture is established, it is desirable to prepare stocks in the form of callus cultures (Petersen and Alfermann, 2008; Xu et al., 2011). Efficiency of different stages of production including genetic manipulation, gene expression, culturing, process development, product purification and downstream processing should be maximized in a systematic strategy to increase productivity in suspension cultures (Xu et al., 2012). In a recent study on comparison of various plant-based expression platforms, transgenic cell suspension cultures were suggested as the most promising system for further optimization and large-scale production (Vasilev et al., 2014). Hence, plant cell suspension cultures hold considerable promise for efficient production of therapeutic AMPs for medical approaches. 4.3.3. Hairy root cultures 18

ACCEPTED MANUSCRIPT Hairy root systems are employed in a wide breadth of biotechnological applications such as therapeutic protein production, synthesis of phytochemicals and biotransformation of exogenous substrates (Banerjee et al., 2012; Ono and Tian, 2011). Hairy root cultures are established by infection of plant cells or tissues with A. rhizogenes. Root inducing plasmid of A. rhizogenes is responsible for

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stable incorporation of genetic material (including the gene of interest) into the genome of a host plant

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cell. Generally, one to four weeks after successful transformation of the donor plant tissue, neoplastic

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roots start to grow in a highly branched manner with abundant lateral roots (Pavlov et al., 2002; Sevón and Oksman-Caldentey, 2002). Hairy root cultures can also be employed for rapid evaluation of peptide activity as well as efficiency and yield of peptide production in plant-based platforms.

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Although transgene silencing is a common feature observed in hairy root gene expression strategies (Sivakumar, 2006), long-term genetic and biosynthetic stability (in contrast to undifferentiated cell suspension cultures) together with the fact that more than 400 plant species have already been used for

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establishment of hairy root cultures (Porter, 1991) make expression in hairy root cultures an attractive tool for rapid assessment of diverse aspects of gene expression (Miao et al., 2008; Ron et al., 2014). Hairy root cultures were employed to demonstrate defensive role of AMPs in disease-susceptible

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tissues of plants. Transgenic potato hairy roots, expressing a levamisole-mimetic synthetic peptide

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(LEV-I-7.1), were employed to investigate the effects of LEV-I-7.1 on number of Globodera pallida nematodes that were able to establish in hairy roots. Transgenic hairy roots displayed approximately

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50% reduction in the number of obligate root parasites (Liu et al., 2005). 4.4. Alternative approaches employing transient expression Among plant-based expression platforms, transient expression employing Agrobacterium or virus

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infection attracts attention due to short time scale for development and production, high expression levels and yields obtained, and confined nature of the system. In transient expression process, gene of interest is introduced into plant cells or tissues using an engineered vector, generally Agrobacterium or a plant virus, and protein or peptide production is achieved via extrachromosomal gene expression within plant cells. Heterologous expression starts within one day and lasts for several days to weeks depending on the host, product and the vector employed. During this process host cells or tissues, or plants themselves are maintained in contained environments such as greenhouses or bioreactors. After product purification the host is discarded and a fresh batch of untransformed host is prepared for a second round of production. 4.4.1. Agrobacterium or virus infection All transient expression systems use natural infective properties of Agrobacterium and plant viruses. Various approaches employing intact leaves, cultured hairy roots or suspension cells have been employed for expression of products in transient systems. Most of the studies used transient systems 19

ACCEPTED MANUSCRIPT for evaluation of gene constructs, validation of activity of heterologous product or to determine efficiency of expression platforms (Ben-Amar et al., 2013; Circelli et al., 2010; Company et al., 2014; Tremblay et al., 2010). On the other hand, transient systems have also been used for production of industry-grade pharmaceutical proteins or peptides. In transient systems based on virus infection,

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various proteins and peptides including bovine lysozyme, human interleukin-2, human α-galactosidase

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A, bovine aprotinin and others have been produced (Pogue et al., 2010). Among these, aprotinin is a

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58 amino acid long polypeptide with serine protease inhibitor activity. Functional recombinant aprotinin was produced using tobacco plants infected with a TMV-based vector in greenhouses or field. The recombinant aprotinin exhibited properties highly comparable to an FDA-approved counterpart. Although it does not possess antimicrobial activity, production of aprotinin in a transient

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platform provides an example for possible expression of AMPs in transient systems for large-scale molecular farming.

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In another approach, efficient DNA delivery by Agrobacterium was combined with rapid replication and high level expression of a plant virus (Gleba et al., 2005; Marillonnet et al., 2003). Plant viral vector carrying gene of interest was introduced into intact tobacco plants via Agrobacterium vacuum

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infiltration. Various heterologous proteins including cytokines, interferons, bacterial and viral

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antigens, and growth hormones have been produced using this transient system (Giritch et al., 2006; Gleba et al., 2007). The approach has also been used for transient expression of an AMP, protegrin-1,

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in leaves of N. tabacum (Patino-Rodriguez et al., 2013). Transgene expression and peptide accumulation in tobacco leaves were demonstrated 10 days post-infection. Additionally, in vitro assays of protein extracts from transiently transformed tobacco showed inhibitory activity of protegrin-1 on growth of various mammalian pathogens including Klebsiella pneumonia,

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Staphylococcus aureus, Candida albicans and others (Patino-Rodriguez et al., 2013). In an attempt to adapt this transient system to large-scale manufacturing formats, viral vectors and Agrobacterium infiltration methods were improved (Pogue et al., 2010). Traditional Agrobacterium vacuum infiltration was performed using automated conveyors and vacuum-rated large chambers. In this robust, large-scale process, the conveyors, loaded with trays holding intact tobacco plants rotate 180° and enter the chamber. The plants are submerged in Agrobacterium solution and vacuum is applied to aid bacterial infection. Plants, removed from chambers and rotated to upright position, are transferred to greenhouses for growth and product accumulation. After a growth period up to two weeks, plants are harvested, and product purification is performed (Gleba et al., 2005; Pogue et al., 2010). A similar approach using transient expression in tobacco leaf tissues was employed by Medicago Inc., a Canada-based biopharmaceutical company, for production of a vaccine against avian H5N1 influenza. The vaccine has been used in a phase I clinical trial (Landry et al., 2010). Overall, it is apparent that advantages offered by viral vectors, Agrobacterium infection and plant-based

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ACCEPTED MANUSCRIPT expression systems might all be combined in a transient, time- and cost-efficient, large-scale manufacturing process for molecular farming of pharmaceuticals. 4.4.2. Cell pack technology

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Recently, a new approach named cell pack technology has been developed for production of

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pharmaceuticals, recombinant proteins and/or secondary metabolites. The technology combines the

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efficiency of methods based on transient expression via Agrobacterium or virus infection, with the advantages of plant cell suspension cultures. This high-throughput, cost-efficient and rapid platform makes use of cells from any plant cell suspension culture (e.g. N. tabacum, A. thaliana, N. benthamiana, Catharanthus roseus, Daucus carota). Initially, a medium-deprived, porous structured

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artificial tissue, called a cell pack or a cell cake, is generated. Cell packs are prepared by removal of liquid media from cell suspensions using simple filters. Subsequently, the cells are transformed, stably

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or transiently, by application of drops or spray of Agrobacterium suspension. Transformed cell packs are incubated one to six days under specific conditions in trays or columns until product recovery. Accumulated product can be harvested by a buffer solution, which allows elution of secreted proteins from packed cells. This rapid technology overcomes the problems associated with handling of large

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volumes of medium and buffers during production and downstream processing. Contrary to systems in

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liquid cultures, there is no need for control of bacterial growth, and the transgene is expressed more rapidly due to higher transformation efficiency. Furthermore, silencing triggered by an individual cell

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does not spread systematically. The method also provides easier product purification since there are less secondary metabolites and host proteins in packed cells compared to leaf-based expression systems. The technology was used for production of heavy and light chains of a human antibody

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translationally fused to a KDEL sequence (Rademacher, 2013, 2014). 5. Tools for screening and evaluation of plant-based expression strategies Although there are diverse heterologous expression systems for production of AMPs, particular properties of these peptides, such as size, folding and glycosylation should be considered carefully during selection of a host platform. Additionally, AMPs exhibit intrinsic instability, low immunogenicity, short half-life and certain level of cytotoxicity to host organism. Hence, researchers have been forced to develop specific expression strategies to address the limitations related to the target peptide and the host organism. These strategies mainly include, but not limited to, fusion of nucleotide sequence of the peptide to sequences of a proper promoter, a carrier protein, purification tags and/or subcellular targeting signals. Additionally, the transgene constructed for peptide production is generally subjected to codon optimization for high level expression in a certain host. All these modifications might alter the activity and stability of the target peptide as well as the yields obtained from an expression system. On the other hand, stable transformation of certain hosts, specifically plants, is a laborious and slow process. Therefore, efforts in the last few decades have 21

ACCEPTED MANUSCRIPT been dedicated to establishment of rapid screening platforms for evaluation of efficiency and yield of expression strategies before they are employed for generation of stably transformed plants. These screening platforms generally employ transient expression of the target peptide in a well-established host. Selection of a screening technology is further critical since a distinct host with a different

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physiological and genetic background than that of the host in the target production platform might lead

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to deceptive results. This section provides an overview of the most potent plant-based screening

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systems or tools with specific emphases on time scale, cost, simplicity of transformation and suitability for diverse plant species. 5.1. Agroinfiltration of leaf tissues

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Infiltration of intact leaf tissues with a suspension of Agrobacterium tumefaciens harboring an expression construct represent nowadays the most commonly used strategy for testing new constructs

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and generating valuable data. This labor-efficient, routine and cost-effective transient expression assay, with high transformation efficiencies, provide data with the analysis performed in several days without a need for selection pressure on leaf tissues. Majority of the analysis in literature have been performed using N. benthamiana leaves (Sparkes et al., 2006; Yang et al., 2000). The procedure has

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also been optimized for other plant species including lettuce, tomato, Arabidopsis and grapevine (Ben-

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Amar et al., 2013; Joh et al., 2005; Tsuda et al., 2012; Wroblewski et al., 2005). Infiltration of tobacco leaves was the screening platform chosen for transient expression of human cathelicidin, LL-37, that

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was fused to a secretion signal sequence. The chimeric product accumulated at high levels in tobacco leaves five days after infiltration (Fig. 2; Öz et al., 2014). In a recent study, production and subcellular localization of a synthetic AMP was demonstrated using a fluorescent tag in a transient expression system employing agroinfiltration. Western blot analysis of proteins extracted from N. benthamiana

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leaves three days after infiltration clearly confirmed the presence of the recombinant product in protein bodies derived from ER (Company et al., 2014). 5.2. Seedling transformation Use of young intact plant seedlings as hosts for transient transformation exhibits a big advantage in terms of time and space otherwise required for generation of mature transgenic plants. High levels of product expression can be achieved in 4 to 6 days in one week old or younger seedlings. In contrast to leaf infiltration techniques, use of seedlings overcomes the problem associated with diverse developmental stages of the leaves from the same donor plant, which might affect transformation efficiency. Several methods for transient transformation of seedlings have been described. In one of the earliest studies, young tobacco seedlings were used for expression of a marker gene after vacuuminfiltration in Agrobacterium suspension (Rossi et al., 1993). McIntosh et al. (2004) developed an efficient and versatile transient assay system based on vacuum-infiltration of two week old Arabidopsis seedlings. A high level of reporter gene expression was observed five days after 22

ACCEPTED MANUSCRIPT infiltration. This procedure was broadly followed and optimized in another study, where 3 to 4 days old seedlings were used as hosts, allowing the whole assay to be completed within one week (Marion et al., 2008). Vacuum application is not always necessary to achieve accumulation of recombinant products in plant seedlings. Transient transformation has also been performed by simple co-cultivation

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of Arabidopsis seedlings with Agrobacterium rhizogenes (Campanoni et al., 2007). In 2009, a fast

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Agrobacterium-mediated seedling transformation based on incubation of Arabidopsis seedlings with

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A. tumefaciens in the presence of a surfactant was reported (Li et al., 2009). The assay was performed within one week from seed sowing to product analysis. Moreover, it was shown that the procedure was applicable to other plant species including tomato, tobacco, rice and switchgrass with increased

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duration of co-cultivation (Li et al., 2009).

6. Functional assessment of antimicrobial peptides expressed in plants

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Function and antimicrobial activity of AMPs depend on three dimensional amphipathic structure of these peptides and their interaction with microbial membranes (Cruz et al., 2014; Lee et al., 2014). Post-translational modifications such as glycosylation, disulphide bond formation and folding are critical for maintaining AMP structure and biological activity. Although these modifications can be

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performed properly by plant cells, AMPs produced in heterologous plant-based systems might still

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have slight differences compared to their natural counterparts (Obembe et al., 2011; Viana et al., 2013). Similarly, synthetic AMPs might show different level of activity than that predicted in silico,

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after synthesis in a plant host. Furthermore, translational modifications such as fusions to secretion peptides or subcellular targeting signals, employed to boost expression in plant-based systems might alter the structure, function and stability of AMPs. Hence, evaluation of function and stability of

protection.

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heterologously produced AMPs are critical for intended uses in molecular farming and plant

Standard techniques in molecular biology or plant physiology, such as DNA, RNA and protein blotting, conventional or quantitative PCR, immunological assays, microscopy techniques, mass spectrometry, and many others might be employed to demonstrate the presence of transgene, transcript or the heterologous product in an expression host. These techniques should be accompanied by inhibition tests, bioassays or various other methods to show the activity of the AMP produced in a heterologous system. Functional assessment of AMPs can be performed using in vivo or in vitro assays depending on the expression system employed, purpose of peptide production, the AMP synthesized and the target pathogen group. In studies, where improvement of disease resistance in plants was aimed, functional assessment was generally done by challenging transgenic plants with target pathogens. Intact or detached leaves of a transgenic plant expressing an AMP were infiltrated with a suspension of a bacterial pathogen, infected physically with mycelium of a fungus or inoculated mechanically with a viral pathogen 23

ACCEPTED MANUSCRIPT (Donini et al., 2005; Furman et al., 2013; Lee et al., 2011; Muramoto et al., 2012; Yevtushenko and Misra, 2012; Zakharchenko et al., 2013a, 2013b). Whole transgenic tomato plants expressing a cationic peptide (cecropin B) were challenged with pathogens causing bacterial wilt and spot, and were shown to display increased resistance (Jan et al., 2010). Transgenic potato plants over-expressing

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endogenous Snakin-2 were inoculated with a suspension of Pectobacterium atrosepticum at a

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wounded site on the stem. Transgenic potato lines exhibited enhanced resistance to blackleg disease

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caused by P. atrosepticum according to restrained disease symptoms such as chlorosis, necrosis or stem collapse (Mohan et al., 2014). These bioassays, exemplified here and many others, provide valuable information since they present direct evidence for improvement of disease resistance. Proper controls such as untransformed and mock transformed plants or tissues from these plants hold critical

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importance in these bioassays for functional assessment.

One of the most widely employed bioassays is inhibition tests performed in vitro. Total or crude

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protein extracts from transgenic plants expressing AMPs are used to inhibit microbial growth in diffusion assays on solid media or in liquid bacterial suspension cultures (Jan et al., 2010; Zakharchenko et al., 2013a, 2013b). Number of viable microbial cells, concentration of AMP in the

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protein extract, culture conditions, duration of incubation, and various other parameters might affect

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the results obtained from these inhibition assays. Additionally, antimicrobial activity of contaminating endogenous proteins, peptides or metabolites from the host organism might also deviate the results.

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Hence, cautiously selected control reactions should be included alongside protein extracts from transgenic plants or cells. Extracts from transgenic tomato expressing cecropin B were added to bacterial liquid suspension cultures containing 1 × 104 colony forming units (CFU) of either Escherichia coli, Salmonella enteritidis, or Erwinia carotovora to evaluate the inhibitory effect of

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heterologous cecropin B on bacterial growth. According to optical density recordings after an incubation for 17 h, growth inhibition of 16 – 35% was determined in bacteria treated with extracts of transgenic tomato compared to wild type plants (Jan et al., 2010). Since there is no single type of peptides, peptide structures or mode of actions, there is no universal bioassay for functional evaluation of AMPs. Instead, various in vitro and in vivo tests might be developed depending on the nature of a specific AMP and its target and the properties of the expression host. 7. Considerations on commercial production of antimicrobial peptides in plants Although AMP production in plants presents promising strategies for novel applications in medicine and agriculture, commercial production is a challenging path. Technical challenges of plant-based AMP manufacturing are comparable to those of any other biological manufacturing systems. The challenges might arise at different stages of development or manufacturing process including, but not limited to, the design of expression strategy, transformation and validation of transgenic organisms, multiplication of clones, and development of extraction and purification techniques (Broz et al., 2013). 24

ACCEPTED MANUSCRIPT Further challenges reside in attaining product yield, function and stability that should meet the demand in market. Additionally, plant-derived drugs should meet the same standards of safety, quality and effectiveness offered by counterparts produced using other heterologous expression systems.

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7.1. Selection of a production platform for molecular farming of antimicrobial peptides and

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regulations on genetically modified plants

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A key issue for commercial production of AMPs is the selection of a proper plant production platform. Most processes in plant-based AMP production should largely follow classical regulatory requirements already established for non-plant produced recombinant pharmaceuticals as well as regulatory requirements related with GM plants. There are many factors that must be considered

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including compatibility of the product with the expression system, plant-specific post-translational modifications, product yield, storage conditions, purification strategy and many others (Broz et al.,

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2013). There are also several unique challenges regarding AMP production in plants, which arise from the simple fact that certain plant production systems might include use of food crops and involve production of pharmaceutical peptides in open field.

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Related with GM plant risk assessment and regulatory framework, there are certain differences

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between Europe and the United States (Sparrow et al., 2013). For example in the US, there are three coordinated agencies responsible for regulation of all biotechnological products, namely the Food and

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Drug Administration (FDA), the Environmental Protection Agency (EPA) and the US Department of Agriculture (USDA). Each of these institutional bodies have their own regulatory scope (Shama and Peterson, 2004). Overall, FDA evaluates safety (toxicity and allergenicity) of food produced from GM plant material, and is also responsible for regulation of pharmaceuticals. EPA is mainly responsible for

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environmental risk assessment and will help to protect environment. Role of USDA is evaluation of GM plants in terms of their impacts on agriculture and environment (Shama and Peterson, 2004). In Europe, risk assessment related with GM plants is carried out by the European Food Safety Authority (EFSA). All pharmaceutical AMPs produced in plants should be assessed favorably on their safety as well as efficacy by the legislative agencies. The assessment is based on investigation of pharmaceutical efficacy in clinical trials, chemistry control, good manufacturing practices, pharmacology information, and adequate and sufficient inspection of the manufacturing facility (Elbehri, 2005). Open field in planta molecular pharming is associated with potential risk of pollen escape and contamination of the food supply (Shama and Peterson, 2004). Hence, there are strict regulations on physical isolation of GM crops from non-transgenic plants (Elbehri, 2005). Since the isolation requirement largely influences the cost in PMF, self-pollinating cereal crops (e.g., barley, rice and wheat) with their heavy pollen are considered attractive host plants. These cereal crops minimize the risk of gene flow due to their physiological properties. The isolation distances of these crops are 25

ACCEPTED MANUSCRIPT maximally several tens of meters, which is in contrast to at least one mile isolation distance for maize with its wind-borne, relatively light pollen (Elbehri, 2005). For instance, Ventria Bioscience (http://www.ventria.com) grows transgenic rice in fields for heterologous production of recombinant

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human enzymes with a wide breadth of applications in medicine.

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On the other hand, regulations on agricultural activities employing GM plants in open field are pushing most of the biotechnology companies to move indoors, which increases production cost due to

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energy requirements. Nevertheless, in Iceland, for example, most of the primary energy supply is derived from renewable or cheap sources such as geothermal energy, hence price of energy in Iceland is among the cheapest in the world. This has most probably attracted attention of ORF Genetics Ltd.

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(http://www.orfgenetics.com) to build their large greenhouses in Iceland. The company uses endosperm of barley, a self-pollinating plant, for manufacturing of recombinant growth factors and

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other proteins.

The pharmaceutical company Synthon (http://www.synthon.com/corporate) employs yet another solution to overcome the issues related with profitable production of biopharmaceuticals indoors. In 2012, Synthon acquired the LEX System, a proprietary of Biolex Therapeutics. The patented LEX

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System is a manufacturing platform where Lemna minor (a duckweed), a fast-growing aquatic plant

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which doubles its biomass every 36 h, is cultivated in disposable bags with a simple growth media. This clinically validated technology significantly reduces manufacturing time and costs. The

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Greenovation GmbH (http://www.greenovation.com) makes use of Physcomitrella patens (a moss) for recombinant protein production. The technology is based on culturing of the moss in photobioreactors with a low-cost mineral media. Moreover, the genome of Physcomitrella patens is completely sequenced (Rensing et al., 2008), and the organism offers a unique possibility in precise gene targeting

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via highly efficient homologous recombination in vegetative cells. Glycosylation pathways used by Physcomitrella patens have also been engineered towards human-like glycans (Decker and Reski, 2012), which is favorable, as plant glycans bear specific sugar residues that might cause immune reactions in human (Gomord et al., 2010). 7.2. Considerations on target antimicrobial peptides for commercial production Economic feasibility of AMP production in plants is mainly dependent on governmental legislation processes. It is essential to develop a proper business strategy to match the anticipated benefit with biopharming; i.e., reduced cost of goods. One of the basic and most difficult practical considerations is the choice of final pharmaceutical product itself. There are many factors influencing the product selection. These factors include structural properties of AMP, pharmaceutical efficacy during various phases of preclinical and clinical trials, patent protection on AMP or pharmaceutical product, anticipated sales volume, pricing, lifetime and stability, profit potential, regulatory oversight and few others (Davies, 2010). 26

ACCEPTED MANUSCRIPT There are currently several AMPs which were developed from natural peptides and went through various phases of clinical trial pipeline. These AMPs might be promising candidates for plant-based biomanufacturing. Examples for candidate peptides include a derivative of bovine indolicine called omiganan, which is currently in phase III clinical trials for the treatment of papulopustular rosacea

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(Moual et al., 2013). Pexiganan, an analogue of magainin II from Xenopus laevis, is another promising

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AMP, and it is currently being tested in phase III clinical trial program as a topical antibiotic peptide

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for the treatment of mild infections of diabetic foot ulcers (www.dipexiumpharmaceuticals.com). Few other promising peptide agents, which are currently being evaluated in preclinical and clinical trials for development of various potent antimicrobial drugs, include human lactoferrin 1-11 (hLF1-11), histatin 5 derivative P-113, and a synthetic peptide (CKPV)2 designed based on α-melanocyte stimulating

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hormone (Seo et al., 2012). Besides their potential for use against antimicrobial diseases, AMPs intended for production using plant-based molecular farming should be evaluated for potential toxicity

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and allergenicity when consumed in food made from transgenic plant material. To sum up, future commercial applications of in planta AMP production will mainly depend on the compatibility of AMP production strategy with the regulatory standards, agricultural value of the host

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8. Concluding remarks

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plant as well as end-user needs and concerns.

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Diverse natural and synthetic peptides with antimicrobial activities hold great promises for innovative approaches in medical and agricultural biotechnology. They present novel alternatives or substitutes for both antibiotics in treatment or control of microbial infections in human and agrochemicals in plant protection in agriculture. Unique properties of AMPs allow use of different peptides from various

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sources against diverse human and plant pathogens. Disease resistance in crop plants conferred by AMPs might reduce losses in yield, quality and safety of agricultural products due to phytopathogens. On the other hand, heterologous expression of AMPs in plant-based systems is regarded as a key to the bottleneck for large-scale cost-efficient production of therapeutic AMPs for medical use. Although AMP production in plants is considered a prospective tool for innovative applications in medicine and agriculture, certain technical limitations still remain. Improvement of plant expression systems and related biotechnological tools will allow us to overcome these limitations and respond accurately to increasing demand for recombinant peptides. On the other hand, plant-based AMP production faces limitations in authorization processes by local legislatives and concerns raised by public. Field trials of novel GM plant varieties are often smashed by activists opposed to GM crops in Europe (Kuntz, 2012). The doubts of opponents are linked not only to ethical deliberations, but also to a risk that modified genetic material might accidentally end up in the food chain due to cross-pollination or horizontal gene transfer (McHughen and Wager, 2010). Additionally, food crops such as maize employed for production of pharmaceuticals might simply mix 27

ACCEPTED MANUSCRIPT with counterparts and contaminate food supply, as in the case of ProdiGene incident. Privately owned ProdiGene Inc. produces diverse therapeutic or industrial compounds including trypsin, laccase, aprotinin and others in grains of GM corn. In 2001, officials from the USDA working with FDA realized that field grown transgenic corn developed by ProdiGene had contaminated nearby soybean in

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a test plot in Nebraska. Furthermore, in a second test plot in Iowa USDA required a local producer to

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destroy more than 60 hectares of corn that was believed to be cross-pollinated by ProdiGene‘s

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transgenic corn (Fox, 2003).

Field-grown plants on agricultural scale are advantageous in terms of production capacity and cost. However, pharmaceutical companies using plant biotechnology are struggling with strict regulations

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on field-grown GM plants. Utilization of self-pollinating plants and geographic separation of these transgenic crops in tightly isolated fields away from other agricultural practices might provide feasible solutions to comply with regulations. Although a wide range of platforms for AMP molecular farming

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in plants is available, considerable effort needs to be invested in choice and improvement of optimal production strategy to fulfill the requirements for economic feasibility and comply with the legislative

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regulations for handling GM plant material.

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Acknowledgements

The writing for this manuscript was supported by grant LO1204 from the National Program of

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Figure legends

Fig. 1. Comparison of heterologous expression systems for production of biological molecules.

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Coloring is used to rank expression systems from least to most desirable according to various parameters and properties. Adapted from Spök and Karner (2008), Daniell et al. (2001), Fischer et al.

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(2004), Ma et al. (2003), and Parachin et al. (2012).

Fig. 2. Evaluation of plant-based heterologous expression of human cathelicidin LL-37. Agroinfiltration of Nicotiana benthamiana leaves with a syringe (A) and western blot detection of LL-

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37 (B) are displayed. Agroinfiltration was performed according to a previously described method

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(Sparkes et al., 2006). Western blot analysis of protein extracts from infiltrated tobacco leaves was performed against anti-LL-37 primary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX). The

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lanes in western blot analysis were loaded with (1) 90 ng of synthetic LL-37, (2) 35 ng of synthetic LL-37, (4) protein extracts of tobacco leaves infiltrated with a gene construct to express LL-37 fused with a secretion signal at N-terminus and a KDEL endoplasmic reticulum retention signal at Cterminus, and (6) protein extracts of tobacco leaves infiltrated with a gene construct to express LL-37

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fused with a secretion signal at N-terminus. Lanes 3 and 5 were left empty. Protein extracts of uninfiltrated tobacco leaves and leaves infiltrated with various other constructs to express LL-37 without a secretion signal at N-terminus did not produce signals in western blot analyses (data not shown).

46

ACCEPTED MANUSCRIPT Table 1 Selected list of web-based databases and resources on antimicrobial peptides (AMPs). Information and tools available AMPer http://marray.cmdr.ubc.ca/cgi-bin/amp.pl Gene-encoded AMPs, hidden Markov models for recognition of AMP classes, novel AMP discovery AntiBP2 http://www.imtech.res.in/raghava/antibp2 Server for antibacterial peptide prediction in a protein sequence APD http://aps.unmc.edu/ap Database of AMPs from various organisms, AMP prediction, novel peptide design BACTIBASE http://bactibase.pfba-lab-tun.org Natural bacteriocin database, tools for structural prediction and characterization BAGEL http://bagel.molgenrug.nl AMPs from prokaryotes, bacteriocin mining tool for identification of candidate AMP genes CAMP http://www.camp.bicnirrh.res.in Experimentally validated and predicted AMPs, analysis tools on sequence and structure of AMPs DAMPD http://apps.sanbi.ac.za/dampd Manually curated database of AMPs, various analysis tools Defensins http://defensins.bii.a-star.edu.sg A database of knowledgebase defensin family of AMPs LAMP http://biotechlab.fudan.edu.cn/database/lamp A database linking AMPs, various search and analysis tools Norine http://bioinfo.lifl.fr/norine Database of nonribosomally synthesized bioactive peptides PepBank http://pepbank.mgh.harvard.edu Database of peptides that are 20 amino acids-long or shorter PhytAMP http://phytamp.pfba-lab-tun.org Database of plant

References Fjell et al., 2007, 2011

CE P

TE

D

MA

NU

SC R

IP

T

Web site

AC

Database

Lata et al., 2010

Wang et al., 2009

Hammami et al., 2010

van Heel et al., 2013

Thomas et al., 2010; Waghu et al., 2014

Seshadri Sundararajan et al., 2012 Seebah et al., 2007 Zhao et al., 2013 Caboche et al., 2008

Shtatland et al., 2007 Hammami et 47

ACCEPTED MANUSCRIPT

Li and Chen, 2008

CE P

TE

D

MA

NU

SC R

IP

T

http://faculty.ist.unomaha.edu/chen/rapd

al., 2009

AC

RAPD

AMPs, information on taxonomic, microbiological and physicochemical data Database of recombinantlyproduced AMPs and expression strategies

48

ACCEPTED MANUSCRIPT Table 2 Selected list of antimicrobial peptides expressed in plants for plant protection (PP). ND: not determined. Promote r

Application

CaMV35 S

CaMV35 S

Arabidops is thaliana

Transient expression via agroinfiltra tion Constitutiv e

Oryza sativa

Stable constitutive

Maize ubiquitin1 Maize ubiquitin1

Stable constitutive

CaMV35 S

Cathelicidi n

CecA

AC

CE P

TE

O. sativa

LL-37, derivative of hCAP18 from human Cecropin A from Hyalophor a cecropia

Reference s

PP against Xanthomona s axonopodis pv. citri Analysis of subcellular localization

ND

Boscariol et al., 2006

ND

Company et al., 2014

Transgenic plants failed to develop into maturity PP against bacterial pathogens1 PP against Dickeya chrysanthem i and Fusarium verticillioide s, Improved tolerance to oxidative stress1 PP against bacterial and fungal diseases

ND

Company et al., 2014

0.5% TSP

Company et al., 20141 Nadal et al., 20121

ND

Jung et al., 2012

ND

Coca et al., 2006

IP

SC R

Citrus sinensis (sweet orange) Nicotiana benthamia na

Yield

T

Expressio n system and strategy Constitutiv e

NU

BP100 and derivatives

Plant host

MA

AttA

Descriptio n and peptide origin Attacin A from Tricloplusi a ni Artificially designed

D

Peptide name

ND

Brassica rapa

Constitutiv e

CaMV35 S

O. sativa

Stable constitutive

Maize ubiquitin1

PP against Magnaporth e grisea

O. sativa

Endosperm specific

Rice glutelin B1, Rice glutelin B4

PP against F. 1–4 verticillioide μg g1 s and Dickeya seeds dadantii1

Bundó et al., 20141

49

ACCEPTED MANUSCRIPT Stable constitutive

CaMV35 S

CecP1

Cecropin P1 from Sus scrofa

Camelina sativa, B. napus

Stable constitutive

CaMV35 S

S. tuberosum

Stable constitutive

CaMV35 S

N. tabacum

Pathogen responsive

Poplar win3.12T

PP against Ralstonia solanacearu m and X. campestris pv. vesicatoria PP against Erwinia carotovora and F. sporotrichioi des, Improved tolerance to salinity and oxidative stress PP against Phytophthor a infestans, Sclerotinia sclerotiorum PP against F. solani

∼0.0 Jan et al., 01 μg 2010 mg-1 leave s

PP against fungal pathogens PP against various bacterial and fungal pathogens PP against Alternaria solani and F. oxysporum

T

Solanum lycopersic um

IP

Cecropin B from Hyalophor a cecropia

ND

Zakharche nko et al., 2013a,b

ND

Zakharche nko et al., 2007

ND

Yevtushen ko et al., 2005

ND

Rajasekara n et al., 2005 Portieles et al., 2010

Defensins

Defensin 02 from N. megalosip hon

Wasabi defensin from Wasabia japonica Defensin 1 from Medicago sativa Defensin 1 from B. rapa

MA

D

TE

Gossypiu m hirsutum N. tabacum, S. tuberosum

Constitutiv e

CaMV35 S

Constitutiv e

CaMV35 S

Colocynth is citrullus (Egusi melon)

Constitutiv e

CaMV35 S

Lycobersi cum esculentu m Mill (tomato) O. sativa

Constitutiv e

Stable constitutive

CE P

D4E1

Cecropin A-melittin hybrid peptide, artifical Artificially designed

AC

CEMA

NU

SC R

CecB

ND

ND

Ntui et al., 2010

CaMV35 S

PP against F. ND oxysporum

Abdallah et al., 2010

Rice cytochro me C

PP against a sap-sucking insect, Nilaparvata lugens

Choi et al., 2009

ND

50

ACCEPTED MANUSCRIPT CaMV35 S

S. tuberosum

Stable constitutive

CaMV35 S

Ipomoea batatas (sweet potato)

Constitutiv e, Organspecific, Sugarinducible

Metchniko win

Furman et al., 2013

ND

Rivero et al., 2012

PP against Ceratocystis fimbriata

ND

Muramoto et al., 2012

PP against apple scab caused by Venturia inaequalis PP against Agrobacteri um vitis and Uncinula necator

ND

Krens et al., 2011

ND

Vidal et al., 2006

IP

Constitutiv e

Chimeric E12Ω (variant of CaMV35 S), Potato βamylase CaMV35 S

Vitis vinifera

Stable constitutive

Arabidop sis ubiquitin3

Musa spp. (banana), N. tabacum

Stable constitutive

Arabidop sis ubiquitin3

PP against bacterial and fungal diseases

ND

Chakrabart i et al., 2003

H. vulgare

Pathogeninducible

A. tumefacie ns mannopin e synthase

PP against Blumeria graminis and F. graminearu m

ND

Rahnamae ian et al., 2009

AC

Magainin2 from Xenopus laevis and a derivative of magainin-2 (MSI99) Derivative of magainin-2 (MSI99), artificial From Drosophila melanogas ter

ND

SC R TE

CE P

Apple

Mag2 and derivatives

PP against citrus canker caused by X. axonopodis PP against bacterial and fungal diseases, Gene stacking

T

Constitutiv e

D

HT

C. sinensis

NU

Dermasept in and/or AP24 and/or lysozyme

From Phyllomed usa sauvagii Various combinatio ns of dermasepti n from P. sauvagii, AP24 osmotin from N. tabacum and lysozyme from Gallus gallus Hordothio nin from Hordeum vulgare

MA

Dermasept in

51

ACCEPTED MANUSCRIPT Pathogen responsive

Poplar win3.12T

PP against fungal diseases

S. tuberosum

Organspecific

Douglasfir BiP Pro1-1

CaMV35 S

Post-harvest PP against Pectobacteri um carotovorum PP against E. carotovora, Phytophthor a infestans and Phytophthor a erythroseptic a PP against Sclerotinia homoecarpa and Rhizoctonia solani PP against E. carotovora

Derivative of temporin A from Rana temporaria , artificial

S. tuberosum

Constitutiv e

Pen4-1

Penaeidin4 -1 from Litopenaeu s setiferus

Agrostis stolonifera (creeping bentgrass)

Stable constitutive

PG1

Protegrin-1 from porcine leukocytes

N. tabacum

Stable chloroplast

Tobacco psbA

Rev4

Reverse peptide of indolicidin isolated from bovine neutrophils , artificial Rev4 and a derivative of magainin-2 (Myp30)

N. tabacum, A. thaliana

Stable constitutive

Peanut chlorotic streak caulimovi rus (PClSV)

A. thaliana

Stable constitutive

CaMV35 S

D

TE

CE P

AC

MA

NU

MsrA3

Rev4 and Myp30

ND

T

N. tabacum, S. tuberosum , Poplar

IP

Derivative of dermasepti n B1 from P. sauvagii and P. bicolor, artificial

SC R

MsrA2

Maize ubiquitin1

8 µg g-1 fresh tuber tissue ND

ND

17% – 26% of TSP PP against E. ND carotovora, Peronospora tabacina and Pseudomona s syringae, Enhanced yield PP against ND Pseudomona s syringae and Peronospora parasitica var Noco2 Gene stacking

Yevtushen ko and Misra, 2007; Yevtushen ko and Misra, 2012 Yevtushen ko and Misra, 2012 Osusky et al., 2004

Zhou et al., 2011

Lee et al., 2011

Xing et al., 2006

Xing et al., 2006

52

ACCEPTED MANUSCRIPT

Heveinlike AMPs from seeds of Stellaria media

Tobacco psbA

PP against E. carotovora and tobacco mosaic virus

Triticum aestivum

Stable constitutive

Maize ubiquitin1

O. sativa

Stable constitutive

Maize ubiquitin1

A. thaliana, N. tabacum

Constitutiv e

PP against F. graminearu m and Rhizoctonia cerealis PP against Magnaporth e oryzae and Rhizoctonia solani PP against Bipolaris sorokiniana and Thielaviopsi s basicola PP against fungal diseases

Lee et al., 2011

ND

Li et al., 2011

ND

Jha et al., 2010

IP

SC R

CaMV35 S

ND

Shukurov et al., 2012

From skin N. Pathogen Poplar ND Yevtushen secretion tabacum responsive win3.12T ko and of Rana Misra, temporaria 2007 Thanatin From O. sativa Constitutiv CaMV35 PP against ND Imamura Podisus e S Magnaporth et al., 2010 maculivent e oryzae ris Thionins From B. S. Constitutiv CaMV35 PP against ND Hoshikaw oleracea, tuberosum e S Botrytis a et al., Nasturtium cinerea 2012 officinale and Barbarea vulgaris 1 Potential use of the plant host and/or the expression system for molecular farming of therapeutic antimicrobial peptides has been indicated in the study.

AC

CE P

TE

Temporin A

32% – 38% of TSP

T

Stable chloroplast

NU

SmAMPs

N. tabacum

MA

RsAFPs

Retrocycli n-101 based on Macaca mulatta minidefens ins, artificial Antifungal proteins from Raphanus sativus

D

RC101

53

ACCEPTED MANUSCRIPT Table 3 Selected list of antimicrobial peptides expressed in plants for plant molecular farming (PMF). ND: not determined.

Cathelici din

SMAP-29 from sheep myeloid cells Glucagonlike peptide 1 from human

Nicotian a tabacum Oryza sativa

Interferon-α1 from Salmo salar (SasaIFN-α1)

Solanum tuberosu m, O. sativa

Constitutiv e

Interferon-α from chicken (ChIFN-α)

Lactuca sativa

Transient expression via agroinfiltra tion

O. sativa

MsrA2

rC4V3

AC

CE P

Lactostat Derived from in βlactoglobulin in cow's milk

Derivative of dermaseptin B1 from Phyllomedus a sauvagii and P bicolor, artificial V3 loop fused to C4 domain from gp120 of human immunodefici ency virus (HIV), artificial

Application

CaMV 35S

PMF

NU

Endosperm Rice -specific glutelin B1

MA

CaMV 35S

CaMV 35S

D

Interfero ns

Promo ter

TE

GLP-1

Expressio n system and strategy Constitutiv e

Endosperm Rice -specific 10-kDa prolam in, Rice glutelin B4

Poplar

Constitutiv e

CaMV 35S

N. tabacum

Stable chloroplast

Prrn

Yield

Referenc es

ND

Morassut ti et al., 2002 Yasuda et al., 2005

T

Plant host

IP

Description and peptide origin

SC R

Peptide name

PMF, Potential use as a therapeutic agent for type II diabetes PMF, Disease prevention in fish against viral infections PMF, Viral disease prevention in chicken

PMF using cereal seeds, Potential clinical use as an antihypercholestero lemic peptidebased drug PMF

PMF, Induction of mammalian immune response against HIV, Potential HIV vaccine

ND

ND

Fukuzaw a et al., 2010

0.393 μg kg-1 tissue (0.000 4% TSP) 2 mg g-1 dry seeds

Song et al., 2008

2–6 µg g-1 fresh mass

Yevtushe nko and Misra, 2012

ND

RubioInfante et al., 2012

Cabanos et al., 2013

54

ACCEPTED MANUSCRIPT N. benthami ana

Transient

SP6

PMF, Potential use against both plant and human pathogens

0.025 mg g-1 leaf mass

Zeitler et al., 2013

CE P

TE

D

MA

NU

SC R

IP

T

de novo designed, artificial

AC

SP1-1

55

AC

Figure 1

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

56

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

Figure 2

57

Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology.

Antimicrobial peptides (AMPs) are vital components of the innate immune system of nearly all living organisms. They generally act in the first line of...
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