Plant Biotechnology Journal (2019) 17, pp. 1069–1080 doi: 10.1111/pbi.13038 Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus Xiaoqing Shi1, Teresa Cordero2, Sandra Garrigues3, Jose F. Marcos3, Jose-Antonio Daro s2,* and Marıa Coca1,* 1 Centre for Research in Agricultural Genomics (CRAG, CSIC-IRTA-UAB-UB), Cerdanyola del Valles, Spain 2 Instituto de Biologıa Molecular y Celular de Plantas (IBMCP, CSIC-Universitat Politecnica de Valencia), Valencia, Spain 3 Instituto de Agroquımica y Tecnologıa de Alimentos (IATA, CSIC), Paterna, Spain Received 30 July 2018; Summary revised 24 October 2018; Fungi that infect plants, animals or humans pose a serious threat to human health and food accepted 8 November 2018. security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules *Correspondence (Tel 34963877893 (JD), that could be used to develop new antifungal therapies in medicine and agriculture. They are and 34935636601 (MC); fax 34963877859 small highly stable proteins with specific potent activity against fungal pathogens. However, their (JD), and 34935636601 (MC); email: exploitation requires efficient, sustainable and safe production systems. Here, we report the jadaros@ibmcp.upv.es (JD), and maria.coca@cragenomica.es (MC)) development of an easy-to-use, open access viral vector based on Tobacco mosaic virus (TMV). Xiaoqing Shi and Teresa Cordero This new system allows the fast and efficient assembly of the open reading frames of interest in contributed equally to this work. small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. Keywords: antifungal proteins, gibson We also demonstrate that this production system renders AFPs fully active against target assembly, Nicotiana benthamiana, pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plant biofactory, tobacco mosaic virus, plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable viral vector. biofactories to bring these antifungal proteins to the market. et al., 2017; Huber et al., 2018; Lo pez-Garcıa et al., 2012; Introduction Meyer, 2008). AFPs are small proteins, usually cationic, that are Disease-causing fungi that infect plants, animals and humans rich in cysteine residues, and are folded in compact structures pose a serious threat to human and animal health, food security supported by disulphide bridges, which make them highly stable and ecosystem resilience (Fisher et al., 2016, 2012). More people and resistant to heat, proteases and extreme pH (Batta et al., die every year from fungal infections than from malaria (Bon- 2009; Campos-Olivas et al., 1995; Garrigues et al., 2017; gomin et al., 2017). Fungal infections can have fatal conse- Hegedu €s and Marx, 2013). They exhibit potent specific antifungal quences for at-risk immunocompromised patients with HIV/AIDS, activity at very low concentrations against important human and anti-cancer chemotherapies, corticosteroid therapies, and organ plant fungal pathogens (Garrigues et al., 2017; Huber et al., transplantation, among others (Campoy and Adrio, 2017). In 2018; Marx et al., 2008; To th et al., 2016; Vila et al., 2001; addition, fungi are a challenge to food security because they Vir agh et al., 2014), and do not have toxic effects on plant or destroy major crops globally and contaminate food and feed with mammalian cells (Moreno et al., 2006; Szappanos et al., 2006). mycotoxins that are detrimental to animal and human health However, the exploitation of AFPs requires efficient, sustainable (Bebber and Gurr, 2015). Only a few classes of antifungal agents and safe production systems. Antifungal proteins have been are available today, and even these are not fully effective due to biotechnologically produced in Pichia pastori at relatively low the development of resistance, host toxicity, and undesirable side yield, and more efficiently in filamentous fungi using a Penicillium effects (Perfect, 2016). There is thus an urgent need to develop chrysogenum-based expression cassette (Garrigues et al., 2018; novel antifungals, whose properties and mechanisms of action Huber et al., 2018; Lo pez-Garcıa et al., 2010; Sonderegger et al., represent improvements on the existing ones, and which can be 2016; To th et al., 2018; Vir agh et al., 2014). Plants represent also applied in diverse fields, including crop and postharvest protec- a good option for producing AFPs that are cysteine-rich proteins tion, preservation in cosmetics, materials and food, and animal that require disulphide bridges formation and proper folding for and human health. activity. Moreover, previous reports indicate that plants can Antifungal proteins (AFPs) produced by filamentous fungi are a sustain AFP production (Coca et al., 2004; Girgi et al., 2006; specific class of antimicrobial peptides (AMPs). Antifungal pro- Moreno et al., 2005; Oldach et al., 2001). These reports show teins are promising biomolecules that could be used to develop that AFPs can be heterologously produced in plants, leading to new antifungal therapies in medicine and agriculture (Garrigues improve resistance to fungal pathogens. ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. 1069 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1070 Xiaoqing Shi et al. Plants are excellent biofactories for producing proteins and important to target these bioactive proteins to the extracellular other metabolites of interest for research, pharma and industry. space, since their toxic effects prevent them from accumulating in They are fueled by sunlight, are free from human pathogens, and intracellular compartments. Moreover, by accumulating the AFPs compared to other systems, production can be scaled up easily. in the extracellular fluids, downstream purification is simpler. We Many biotechnological tools have been developed for molecular show also that the recombinant AFPs produced by the new farming, and those derived from plant viruses are prominent system have exactly the same activity as their native counterparts among them (Hefferon, 2017). Higher plants host a remarkable of fungal origin. Finally, we demonstrate that plant extracellular diversity of viruses with different genomes and strategies for fluids containing the Penicillium digitatum AfpB can protect genome replication and expression. However, they all have small tomato plants from the grey mold disease caused by Botrytis genomes, and thus have limited capacity to harbor genetic cinerea. information. Despite this limitation, plant viruses can complete complex and tightly regulated infectious processes in their host Results plants. Definitively, evolution has favored small and simple, but A new TMV-based vector system in which ORFs of still powerful, genetic elements in plant virus genomes, a wealthy interest are inserted using the Gibson assembly reaction source of parts for plant biotechnology and synthetic biology. A pioneering example of a plant virus transformed into a As a first step toward a new TMV-derived vector system for plant biotechnological tool is Tobacco mosaic virus (TMV). This is a plus biotechnology that uses the Gibson assembly reaction to insert strand RNA virus of genus Tobamovirus, within the family the ORFs of interest, we constructed a TMV infectious clone that Virgaviridae (Ishibashi and Ishikawa, 2016; Knapp and Lewan- efficiently infects plants when delivered by agroinoculation dowski, 2001; Scholthof, 2004). The genomic RNA is encapsi- (pGTMV; Figures S1 and S2; Addgene plasmid # 118755). We dated by 2130 units of the viral coat protein (CP) into rigid, helical then transferred a fragment of the TMV cDNA containing the rod-shape virions of about 18 nm diameter, and 300-310 nm entire CP ORF (from A5431 to T6278) to a minimal cloning length. TMV RNA encodes four proteins: two 50 -end proximal vector. In the resulting plasmid, the TMV cDNA was flanked by overlapping 126 and 183 kDa replication proteins that are recognition sites for the type-IIS restriction enzyme BsaI. The alternatively produced through a ribosomal readthrough mech- plasmid was manipulated to mutagenize the ATG initiation anism, the 30 kDa movement protein (MP), and the 30 -end codon of the TMV CP into AGA (positions 5712-5714), and to proximal 17.5 kDa CP. The two replication proteins are expressed replace most of the CP ORF (from T5757 to T6176) by a linker from the genomic RNA, while the MP and CP are expressed from consisting of the recognition sites for two unique restriction 30 co-terminal subgenomic RNAs. The TMV genome also contains enzymes (AgeI and XhoI) and the LacZ blue-white selection 50 - and 30 -untranslated regions that are important for virus marker. The resulting minimal intermediate plasmid (pMTMVi-N; translation and replication (Chujo et al., 2015). Addgene plasmid # 118756) is represented in Figure 1 (upper A key property of TMV is the rapid accumulation of large part) and its exact sequence in Figure S2. This small plasmid amounts of the viral CP in infected plant tissues. A combination of (2376 bp) allows us to easily and efficiently insert the ORFs of knowledge and a trial-and-error approach led to the construction interest using the Gibson assembly reaction (Gibson et al., of TMV-derived vectors (Dawson, 2014) such as TB-2 (Donson 2009). The option of assembling two or more DNA fragments in et al., 1991; Kearney et al., 1993), or 30B (Shivprasad et al., a single reaction allows us to fuse any desired peptide tag or 1999), which allow to express a foreign open reading frame (ORF) protein moiety to the recombinant protein of interest. Once the under the control of the viral CP promoter; in these cases, cDNA for the recombinant protein is inserted into pMTMVi-N, encapsidation is achieved by inserting into the recombinant virus the manipulated TMV genome fragment can be transferred into an ORF encoding the CP of a different tobamovirus. Other vectors the TMV infectious clone (pGTMV) using a second Gibson such as the TMV RNA-based overexpression (TRBO) vector, in assembly reaction (Figure 1, upper part). This requires that which most of the viral CP is replaced by the ORF of interest, pGTMV be digested with restriction enzymes NcoI and Pfl23II, produce large amounts of recombinant proteins but are incapable which have unique recognition sites in this plasmid. There are of spreading systemically (Lindbo, 2007). Improvements in TMV- two options for generating the insert from the pMTMVi-N based vectors have also focused on increasing the efficiency of derivatives, the easiest being to digest the plasmid with BsaI. establishing the infection foci and of cloning the genes of However, if the recombinant cDNA contains BsaI sites, the interest. One of the most popular systems uses DNA modules manipulated TMV DNA fragment can be produced by poly- delivered by Agrobacterium tumefaciens and assembled in vivo by merase chain reaction (PCR) using primers PI and PII (Table S1), a site-specific recombinase to produce the TMV-derived vector which flank the recognition sites of NcoI and Pfl23II. (Marillonnet et al., 2004). This de-constructed system was further In pMTMVi-N derivatives, most of the TMV CP is replaced by improved by introducing silent nucleotide substitutions and the ORF for the recombinant protein, such that the recombinant multiple introns into the TMV coding sequences, in order to viruses will be unable to move systemically in infected plants. To increase infectivity (Marillonnet et al., 2005). Another improved obtain recombinant TMV clones that can move within the plant, series of TMV-derived vectors uses Gateway cloning to facilitate we constructed a new minimal intermediate plasmid by transfer- insertion of the recombinant ORFs, which can also be fused to a ring the fragment in the pGTMV from A5431 in TMV cDNA to broad series of epitope tags and fluorescent proteins (Kagale G62 in the delta ribozyme cDNA. We again mutagenized the et al., 2012). TMV CP ATG to AGA and replaced most of the TMV CP with the Here, we report the development of a new TMV-derived vector AgeI-LacZ-XhoI linker, as above. In this case, we replaced the final system that allows quick and easy cloning of the ORFs of interest 30 nt of the TMV genome (from A6366 to the end) with the 30 using the Gibson assembly reaction (Gibson et al., 2009). We end of a Tomato mosaic virus (ToMV) infectious clone (from describe how we have used this system to produce large amounts G5537 to the end). This fragment includes the ToMV CP of AFPs in Nicotiana benthamiana plants. We show that it is promoter, CP ORF and 30 UTR, and provides an alternative ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 1069–1080 A new viral system for efficient AFP production 1071 Figure 1 The TMV vector system, which allows Gibson assembly of the ORF of interest. Schematic representation of intermediate entry plasmids pMTMVi- N and pMTMVi-M, and the Gibson assembly reactions that allow insertion of the gene of interest (GoI) into pGTMV to produce the binary plasmids that will express the recombinant viruses (pGTMVn-GoI and pGTMVm-GoI). Ori pUC and Ori pSa, replication origins; AmpR and KanR, ampicillin and kanamycin resistance selection markers; LacZ, fragment of E. coli b-galactosidase, which allows blue–white screening in the presence of X-gal; TMV and 30 ToMV, viral cDNAs; LB and RB, left and right borders of the Agrobacterium. tumefaciens transfer DNA; P35S and T35S, Cauliflower mosaic virus (CaMV) 35S promoter and terminator; d, ribozyme. tobamovirus CP to encapsidate the recombinant virus, which antifungal protein, with inhibitory concentrations up to one order allows it to move systemically (Shivprasad et al., 1999). This of magnitude lower than other AFPs (Garrigues et al., 2017). second minimal TMV intermediate plasmid, called pMTMVi-M Therefore, there is a great interest in obtaining large amounts of (Addgene plasmid # 118757), is shown in Figure 1 (lower part, AfpB, so we assessed this possibility using our new TMV vector full sequence in Figure S2). In pMTMVi-M, the M indicates system. We designed two different constructs to target this movement, whereas the N in pMTMVi-N indicates nonmovement. protein to two different subcellular compartments, namely The cDNA corresponding to the recombinant protein can be apoplast and endoplasmic reticulum (ER). Compartmentalizing inserted into the AgeI-XhoI digested plasmid using the Gibson antimicrobial peptides in plant cells is known to favor their assembly reaction, as previously described. The manipulated TMV accumulation by protecting them from host proteases and fragment can also be reintegrated into pGTMV, using the Gibson avoiding their toxic effects on the host cells (Bundo  et al., assembly reaction with a BsaI-digested insert, although in this 2014; Coca et al., 2006; Montesinos et al., 2016). To target AfpB case the destination plasmid (pGTMV) must be digested with NcoI to the apoplast or ER, we added the signal peptide of the tobacco and CpoI. If the cDNA corresponding to the recombinant protein AP24 protein (XP_009782398.1) to the amino terminus of the contains BsaI sites, the insert can also be produced by PCR using mature AfpB protein (Figure 2a). This signal peptide allows the primers PI and PIII (Table S1), which flank the NcoI and CpoI protein to enter the secretory pathway toward the extracellular recognition sites. space. In the second construct, we added an additional KDEL We confirmed that the new TMV vector system performed sequence to the carboxyl terminus of the protein, facilitating correctly by cloning the cDNA of reporter green fluorescent retention inside the ER. We then expressed the corresponding protein (GFP) into both the pMTMVi-N and pMTMVi-M interme- recombinant viruses (TMVDCP-AfpB and TMVDCP-AfpBKDEL; diate plasmids, transferring the manipulated TMV fragments to Figure S4) in N. benthamiana leaves via infiltration with A. tume- pGTMV, and infiltrating N. benthamiana plants (Figure S3, faciens cultures. We observed that leaves agroinfiltrated with sequences in Figure S4). AfpB constructs were damaged, particularly those with the construct designed for AfpB accumulation in ER (Figure 2b). In Production of the antifungal protein AfpB in leaf contrast, leaves agroinfiltrated with a GFP control (TMVDCP-GFP) apoplasts of N. benthamiana plants showed a healthy green appearance, similar to that of the mock- AfpB is an AFP whose gene was identified in the genome of the agroinfiltrated leaves (Figure 2b). This suggests that the negative postharvest phytopathogenic fungus P. digitatum but that, unlike effects might be due to afpB expression rather than to TMVDCP other AFPs, is not naturally produced by the fungus (Garrigues infection. et al., 2016). In a recent study, P. digitatum was genetically To confirm that AfpB was produced in N. benthamiana leaves, modified to produce AfpB and was found to be a highly active we extracted the apoplastic and total protein content, and ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 1069–1080 1072 Xiaoqing Shi et al. Figure 2 Production of AfpB in the apoplast of Nicotiana benthamiana leaves. (a) Schematic representation of the two constructs prepared and designed to accumulate AfpB in the apoplast or endoplasmic reticulum (ER) of plant cells. Both incorporate the signal peptide of tobacco AP24 protein at the N-terminus of the mature AfpB protein, and the one designed for ER accumulation also contains the ER-retention signal KDEL at its C-terminus. RB and LB, right and left border T-DNA; P35S and T35S, CaMV 35S promoter and terminator; d, ribozyme; MP, movement protein; CP, coat protein. (b) Appearance of N. benthamiana leaves 7 days after agroinfiltration with the indicated constructs or with the agrobacterium induction media (mock). (c-f) Analysis of AfpB accumulation in agroinfiltrated leaves from two independent plants (1 and 2). Proteins from extracellular fluids (c, d) or acid extraction (e, f) were separated by tricine-SDS-PAGE and Coomassie-blue stained (c, e) or immunodetected using antibodies anti-PAFB (d, f). Purified AfpB (3.5 or 7 lg) from Penicillium digitatum cultures (AfpB[Pd]) was run in parallel as a control. ECFs from plants accumulating a cysteine-rich protein of unknown function (UP, see Figure 3) was also run in parallel for comparative purposes. Molecular weight markers are shown in kDa on the left. analysed them by SDS-PAGE. Coomassie blue staining showed protein extracts did not detect AfpB in leaves inoculated with the accumulation of a small protein with same apparent TMVDCP-AfpBKDEL, although the protein was detected in molecular weight to that of the P. digitatum AfpB (6.46 kDa) in TMVDCP-AfpB leaves (Figure 2e). These observations indicate the extracellular fluids (ECFs) of TMVDCP-AfpB agroinfiltrated that AfpB did not accumulate in the ER vesicles of N. ben- leaves, but which was absent in leaves that had been agroinfil- thamiana leaf cells, probably due to toxic effects. By comparing trated with TMVDCP-GFP (Figure 2c). This protein was immun- with the known amounts of AfpB produced by P. digitatum, odetected with specific anti-PAFB antibodies by Western blot we estimated that the accumulation of AfpB in the ECFs was analysis (Huber et al., 2018) (Figure 2d), thus demonstrating that ~225  37 lg per gram of N. benthamiana leaves. Thus, the AfpB was produced in N. benthamiana leaves when targeted to recombinant protein accounts for more than 60% of the total the apoplastic space. ECF protein. We observed no differential bands in total acid protein Efficient production of proteins of fungal origin extracts from leaves agroinfiltrated with TMVDCP-AfpBKDEL targeted to different subcellular compartments and mock inoculated leaves (Figure 2e). In contrast, GFP was observed clearly in the total protein extracts from TMVDCP-GFP Since AfpB was not produced when targeted to the ER, we leaves (Figure 2e). Moreover, Western blot analysis of total acid assessed the efficiency of the TMV-based expression system using ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 1069–1080 A new viral system for efficient AFP production 1073 a different fungal protein and different subcellular localizations. The AfpB produced in plants is biologically active and We selected a protein of unknown function (UP, unknown indistinguishable from the produced by P. digitatum protein) encoded by the genome of P. digitatum (PDIG_23520); UP is a small, cysteine-rich secreted protein that has no predicted Having established that AfpB can be produced in plants when antifungal activity (Garrigues and Marcos, unpublished observa- targeted to the extracellular space, we then proceeded to its tions). Preliminary experiments showed that this protein was purification from plant ECFs and to determine its biological readily produced when secreted to the apoplast space (Figure 2c). activity. For purification, we subjected ECFs from agroinfiltrated We prepared three different constructs to target UP to the N. benthamiana leaves to one-step cation-exchange chro- apoplast, the ER, or the vacuole (recombinant viruses TMVDCP- matography. We found that most proteins in the extract, UP, TMVDCP-UPKDEL and TMVDCP-UPVS, respectively; Fig- except for AfpB, were not retained by the cationic column, and ures 3a and S4). The constructs to target UP to the apoplast were found in the flow-through (Figure 4a). We then eluted and ER were prepared as described for AfpB. The vacuolar the retained AfpB from the column as a single peak in construct carries the same N-terminal signal peptide, which fractions 14 and 15 at 0.25 M NaCl concentration, and allows UP to enter the secretory pathway, and also a vacuolar detected a single protein band by SDS-PAGE analysis. Next signal peptide at the carboxyl terminus of the protein (Neuhaus we compared the antifungal activity of the purified recombi- et al., 1991). Nicotiana benthamiana leaves agroinfiltrated with nant AfpB (AfpB[Nb]) to that of the AfpB produced by these recombinant viruses appeared normal with respect to the P. digitatum (AfpB[Pd]). The activity was assayed against the TMVDCP-GFP control (Figure 3b). We observed a protein with the producer fungus P. digitatum, an economically relevant patho- expected mobility of P. digitatum UP in the ECFs obtained from gen of citrus fruits against which the fungal protein showed TMVDCP-UP-infiltrated leaves (Figure 3c, left panel). As judged potent activity (Garrigues et al., 2017). Growth inhibitory assays from the intensity of bands stained with Coomassie blue, this at different protein concentrations showed that the recombi- protein achieved a higher level of accumulation than those nant protein produced in biofactory plants had equivalent reached by AfpB. Moreover, we obtained large amounts of this activity to the fungal version (Figure 4b). Importantly, the crude unknown protein when using TMVDCP-UPKDEL and TMVDCP- ECFs obtained from infiltrated plant tissues showed antifungal UPVS, the constructs designed for intracellular accumulation in ER activity as result of AfpB production, and this activity was vesicles or vacuoles (Figure 3c, right panel). These differences similar to that of the purified AfpB from fungal origin were striking, considering that AfpB was not detected when (Figure 4c). These results indicate that purification to homo- targeted to the ER. We estimated that the recombinant protein geneity is not required in order to obtain a good AfpB activity. accumulated in the ECFs to a concentration of 4.3  1.1 mg per TMV-based production of alternative antifungal gram of leaf fresh weight. These results demonstrate that the proteins in biofactory plants new TMV-based protein production system is highly efficient, and allows one to easily target recombinant proteins to different Finally, we wanted to evaluate the new TMV-based system with subcellular compartments. other antifungal proteins. For this, we chose the well- Figure 3 Production of large amounts of a Penicillium digitatum unknown protein in Nicotiana benthamiana leaves. (a) Schematic representation of the constructs generated to accumulate the protein of unknown function (UP, PDIG_23520) in the apoplast, ER, or vacuole of plant cells. The three constructs include the signal peptide of the tobacco AP24 protein at the N- terminus. A KDEL signal was added at the C- terminus in the construct designed for ER- retention, and the vacuolar signal (VS) was fused at the C-end for vacuolar accumulation. (b) Appearance of N. benthamiana leaves agroinfiltrated with the indicated constructs after 7 days. (c) Analysis of UP accumulation in agroinfiltrated leaves. Proteins from extracellular fluids (ECFs, left panel) or acid extraction (right panel) were separated by tricine-SDS-PAGE and Coomassie-blue stained. Purified UP from P. digitatum cultures was run in parallel as a control. Molecular weight markers are shown in kDa on the left. ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 1069–1080 1074 Xiaoqing Shi et al. characterized AFP from Aspergillus giganteus (Campos-Olivas apoplasts of infiltrated leaves (Figure 5b). By comparison to et al., 1995; Meyer, 2008; Vila et al., 2001). Based on the results known amounts of the fungal protein, we estimated the amount obtained with the AfpB, we decided to directly target AgAFP of protein produced to be around 196  72 lg per gram of leaf accumulation to the apoplastic space. We generated the fresh weight. Moreover, ECFs enriched in AgAFP showed TMVDCP-AgAFP construct containing the sequence encoding antifungal activity against P. digitatum (Figure 5c). This activity the AP24 secretion signal peptide and the mature AgAFP seems to be at least one order of magnitude lower than that of (Figure S4). Nicotiana benthamiana leaves infiltrated with this the ECFs containing AfpB (Figure 4c), in agreement with the high construct had a similar appearance to the mock infiltrated leaves activity reported for AfpB (Garrigues et al., 2017). These results, and leaves inoculated with the TMVDCP-GFP control (Figure 5a). obtained with a different AFP, indicate that our TMV-based We analysed ECFs from N. benthamiana leaves by Western blot system can be considered a general platform for producing using the specific antibodies against AgAFP, previously described antifungal proteins in biofactory plants. (Coca et al., 2004), and found that AgAFP accumulated in the Optimization of the AfpB production method for scaling the process Syringe inoculation of plants is a time-consuming and labor intense process, and thus not suitable for large-scale production of AFPs. Figure 4 Equivalence of antifungal activity for the in planta-and in Figure 5 Production of Aspergillus giganteus AFP (AgAFP) in the fungus-produced AfpB. (a) Purification of AfpB from Nicotiana apoplast of Nicotiana benthamiana leaves. (a) Appearance of benthamiana leaves by cationic exchange chromatography. Coomassie- N. benthamiana leaves 7 days after agroinoculation with the indicated blue stained tricine-SDS-PAGE gel loaded with equivalent volumes of constructs. (b) Analysis of AgAFP accumulation in the extracellular fluid extracelular fluid (ECF), flow-through (FT) and different eluted (ECF) from two independent plants (1 and 2) by Western blot chromatographic fractions (1, 5, 13-16), and 3.5 lg of Penicillium immunodetection using anti-AFP-specific antibodies. Purified AgAFP digitatum purified protein (AfpB[Pd]). (b-c) In vitro inhibitory activity of (250 ng) from A. giganteus cultures was run in parallel as a control (AFP AfpB against P. digitatum fungus. Dose–response curves comparing the [Ag]). Molecular weight markers are shown in kDa on the left. (c) fungal growth inhibitory activity of the purified AfpB[Pd] and Antifungal activity of ECFs containing AFP[Nb] against Penicillium N. benthamiana AfpB (AfpB[Nb]) (b), or with ECFs from leaves of digitatum. Dose–response curves of the fungal growth inhibitory activity N. benthamiana producing AfpB (AfpB[Nb] ECF) or GFP (GFP[Nb] ECF) (c). of AFP[Nb] ECF in comparison to GFP[Nb] ECF. Data shown represent the Data shown represent the mean  SD of three biological replicates after mean  SD of three biological replicates after 72 h of incubation with the 72 h of incubation with the indicated amounts of AfpB. indicated amounts of AFP. ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 1069–1080 A new viral system for efficient AFP production 1075 We evaluated simple methods for agroinoculation that could important economic losses (Dean et al., 2012). AfpB produced facilitate and speed up the AFP production process. These methods by P. digitatum has in vitro inhibitory activity against B. cinerea included vacuum infiltration, which can be done on a large scale with a MIC (minimal inhibitory concentration) value of 12.5 lM with robotics, and simpler spray applications, as reported previously (Garrigues et al., 2017). We deposited drops containing fungal (Hahn et al., 2015). To monitor transfection efficiency easily, we conidia on tomato leaves along with N. benthamiana ECF used the construct TMVDCP-GFP to produce GFP that can be containing 10 lM AfpB, as well as, drops with conidia along visualized under UV light. In syringe- or vacuum-agroinfiltrated with the same concentration of pure AfpB produced by leaves, we observed a strong and homogeneous distribution of GFP P. digitatum, and control drops with conidia along with water fluorescence in the leaves at 7 days, while in sprayed leaves we or ECF from GFP producing plants. Where control drops were could distinguish separate GFP foci (Figure 6a, upper panels). deposited, infection symptoms were clearly visible at 4 days However, these GFP foci enlarged over time and merged at 12 days postinoculation (dpi); whereas where drops containing AfpB (Figure 6a, lower panels). These observations indicate that all three were deposited lesions were clearly smaller (Figure 7a). By methods can efficiently produce GFP, although the spray applica- quantifying lesion size using image analysis, we observed a tion method requires a longer timeframe. We also tested these statistically significant decrease in the damaged area in the methods for AfpB production, and found that ECFs from agroinoc- presence of AfpB (Figure 7b). These results demonstrate that ulated leaves showed similar AfpB accumulation at 12 days for all AfpB protects against B. cinerea infection, both for crude ECFs three methods (Figure 6b). These results indicate that the much containing AfpB (AfpB[Nb]ECF), and for AfpB purified from simpler spray application can be implemented for easy and fungal cultures (AfpB[Pd]). Even more interesting, the ECF inexpensive large-scale AfpB production. extracts containing AfpB showed the same protective efficacy. Finally, we evaluated the stability of the in planta accumulated Therefore, depending on the intended use for the AfpB AfpB during storage as dried material, which could allow producers protein, it may not be necessary to include a downstream to uncouple biomass production from processing. Comparing AfpB purification process. This would reduce considerably the costs accumulation in dried leaves with that in immediately frozen leaves, of production and biotechnological application of this antifun- we observed that AfpB remains stable at least for 2 months when gal biomolecule. stored in dried leaves (Figure 6c). Therefore, AfpB can be manu- factured in N. benthamiana without the need for immediate Discussion processing, and can be stored in dried leaves. In this study, we show that N. benthamiana plants are an Protection of tomato plants against Botrytis grey mold excellent biofactory for producing antifungal proteins of fungal by in planta-produced AfpB origin when transiently expressed using a TMV-derived vector. Finally, we assessed the effectiveness of in planta-produced The process we have developed is fast and produces high yields of AfpB in plant protection assays. For that, plant protein extracts antifungal proteins, which, in addition, are bioactive in the crude containing AfpB were evaluated against the broad-spectrum ECF, or can be easily purified from crude extracts. Importantly, fungal pathogen B. cinerea. This fungus causes gray mold in the recombinant proteins produced in plants have exactly the plants and fresh fruits and vegetables, and is responsible for same antifungal activity as their native counterparts purified from Figure 6 Optimization of AfpB production method for scaling the process. (a) Efficacy of different agroinoculation methods using TMVDCP-GFP. Representative Nicotiana benthamiana leaves visualized under UV light at 7 or 12 days postinoculation (dpi), as indicated. (b) Comparative production of AfpB by agroinoculation with TMVDCP-AfpB using the indicated methods. Coomassie-blue staining of ECF proteins from leaves at 12 dpi (c) Stability of AfpB upon dried storage of agroinoculated leaves. Coomassie-blue staining of total protein extracts obtained from leaves that were immediately frozen or dry-stored for 2 months. ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 1069–1080 1076 Xiaoqing Shi et al. Figure 7 Protection against Botrytis cinerea infection on tomato leaves by in planta-produced AfpB produced in Nicotiana benthamiana. Representative leaves at 4 days after drop- inoculation with B. conidia suspensions (0.5 9 106 conidia/mL) along with N. benthamiana ECFs containing AfpB (10 lM AfpB[Nb]), GFP ECFs (GFP[Nb]) or fungal protein (10 lM AfpB[Pd]). (b) Box plot of the percentage of leaf damage area from the indicated treatments. Data represent three independent experiments. Asterisks denote statistical significance (Tukey test, **P < 0.05). fungi. This is a remarkable result because these small cysteine-rich to the scientific community. Here, starting with an infectious proteins have been shown to be difficult to produce in a soluble, clone of a TMV vulgare strain (GenBank accession number correctly folded conformation in bacterial systems (Kiedzierska MK087763) and a small, optimized binary vector (Thole et al., et al., 2008; Rosano and Ceccarelli, 2014). They only had been 2007), we constructed the plasmid pGTMV, which mediates the successfully produced as active proteins in Pichia pastori at fast and efficient infection of N. benthamiana plants after relatively low yields (Garrigues et al., 2017; Lopez-Garcıa et al., infiltration with transformed A. tumefaciens. Marillonnet et al. 2010; Viragh et al., 2014), and at better yields in filamentous (2005) suggested that plant agroinoculation with TMV is an fungi (Garrigues et al., 2017; Huber et al., 2018; Sonderegger inefficient process, attributed to abnormal RNA processing of the et al., 2016; To th et al., 2018). Although plant and fungal viral genome after transcription in the cellular nucleus, a step that systems are similar in terms of yield and activity, the transient is certainly alien to the natural TMV replication cycle. However, production of AFPs in plants represents an attractive alternative as our experimental results demonstrate high infectivity, even at low sustainable bioproduction factories fueled by sunlight and easily densities of A. tumefaciens (Figure S5). The low infectivity scalable. The developments reported here expand the available previously reported in agroinoculation experiments may have alternatives for the exploitation of antifungal proteins and other been due to the use of inefficient genetic elements such as cysteine-rich proteins such as UP, which was produced to promoters and ribozymes, or the use of large, unstable binary extremely high yields. plasmids. To produce these antifungal proteins in plants, we first Several plant-based systems have been used to produce small developed a new TMV-based vector system. The main reason proteins and peptides with antimicrobial activity. While challeng- for developing another TMV vector was to exploit the Gibson ing due to the physicochemical properties and toxicity of many of assembly reaction to insert the ORFs of interest into the viral these peptides, some of these platforms successfully produce genome. The Gibson assembly reaction, which is extremely AMPs. These platforms include leaf chloroplasts (Lee et al., 2011), efficient for inserting DNA fragments into plasmids, provides rice seeds (Bundo  et al., 2014; Montesinos et al., 2016, 2017), unprecedented yields when assembling several DNA fragments barley seeds (Hol askova et al., 2018) and hairy roots (Chahardoli into a plasmid in a single step, provided these fragments contain et al., 2018). However, the yields reported when using these the appropriate overlapping ends (Gibson et al., 2009). This systems were much lower than the AFP yield achieved in reaction facilitates the insertion of tags and the fusion of protein N. benthamiana using the TMV-derived system reported here. moieties. Moreover, we feel that modular TMV-derived systems While our estimated yield of AfpB was approximately 250 lg per that re-assemble in vivo (Marillonnet et al., 2004, 2005) are gram of fresh N. benthamiana leaf tissue, the reported yields for unnecessarily complex, and moreover they are not freely available Retrocyclin-101 and Protegrin-1 were ~5 and 8 lg/g of tobacco ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 1069–1080 A new viral system for efficient AFP production 1077 respectively (Lee et al., 2011), those for Cecropin A were ~40 lg/ conveniently stored before purification. There are clear advantages g of rice seeds (Montesinos et al., 2016), those for LL37 were to decoupling these two processes, such as avoiding immediate ~0.55 lg/g of barley seeds (Hol askov a et al., 2018), and those for processing after harvesting or otherwise freezing the plant mate- Lactoferrin chimera were ~4.8 lg/g of tobacco hairy roots rial, with their associated production costs. These developments (Chahardoli et al., 2018). In addition, all of these plant-based facilitate large-scale production of these proteins in an easy, fast production systems are based on stable transformation, which cost-efficient and safe manner. In our TMV vector, the viral CP is makes the process more complex and time consuming. Other replaced with the AFP genes, so no virus particles are created, and transient expression systems have also been used for AMP therefore there is no risk of viral dissemination in the environment. production, although with lower yields than those we achieved Also, the afp genes are not incorporated into the plant genome so for AFPs (Patin ~o-Rodrıguez et al., 2013; Zeitler et al., 2013). they did not become heritable traits. Nonetheless, industrial Zeitler et al. (2013) used a TMV full-length virus strategy, to production would require contained greenhouse conditions to produce recombinant linear AMP SP1-1 at a yield of ~25 lg/g of avoid the release of the genetically modified A. tumefaciens strains N. benthamiana leaf tissue, while Patin ~o-Rodrıguez et al. (2013) and TMV clones. The green AFP manufacturing process developed used the MagnICON system to produce Protegrin-1 at lower here could contribute to bring these proteins into the market for amounts than for the AFPs, and without providing quantitative practical use as antifungals. To support these applications, we also data. In any case, even our yield of 250 lg/g for AfpB is far from demonstrated the effectiveness of plant ECF containing AfpB in the 4 mg/g achieved for other proteins, such as GFP or the protecting tomato plants against Botrytis grey mold. Botrytis P. digitatum unknown protein UP, which has no predicted cinerea is one of the top ten fungal phytopathogens, causing antimicrobial activity. This highlights the difficulties associated important economic losses due to the broad range of hosts, and with producing bioactive peptides in biofactory plants. that damage that can occur during the production and posthar- Importantly, our results with AfpB show that the subcellular vesting of vegetables, fruits and flowers (Dean et al., 2012). localization is relevant to achieve high accumulations of bioactive Fungicides are the commonly used method for controlling peptides in plant tissues. AfpB accumulates at high levels when B. cinerea, and botryticides represent more than 10% of the targeted to the extracellular space, whereas intracellular accu- world’s fungicide market. This work supports the use of green AfpB mulation is hindered, probably by toxic effects. This observation as an environment friendly and sustainable alternative to chemical was somehow unexpected because AFP has not previously been fungicides. found to be toxic in plant cells (Coca et al., 2004; Girgi et al., 2006; Moreno et al., 2006, 2005; Oldach et al., 2001; Vila et al., Experimental procedures 2001). However, these previous studies used external application Plasmid construction or extracellular localization of AFPs in plant tissues, so it is possible that AFPs interfere with plant cell functions once inside the cells. To build pGTMV, pMTMVi-N and pMTMVi-M (Addgene plasmids In contrast, the other fungal protein produced in our experiments, # 118755, 118756 and 118757, respectively), PCR amplification UP, which has no predicted antifungal activity, accumulated to reactions were performed using the Phusion high-fidelity DNA similarly high intracellular and extracellular levels. These observa- polymerase (Thermo Scientific) in HF buffer. The Gibson assembly tions reveal the importance of targeting protein accumulation reactions were performed using the NEBuilder HiFi DNA assembly depending on the characteristics and activity of the recombinant master mix (New England Biolabs). The NEBuilder assembly tool protein. (https://nebuilder.neb.com/; New England Biolabs) was used to Additionally, apoplastic targeting of AFPs in plant tissues design the primers. Escherichia coli DH5a were electroporated greatly facilitates their purification. As observed in our work, an with the products of the Gibson assembly reaction. The full extremely simple purification scheme is required to purify AFPs sequence of these plasmids, which was confirmed experimentally from N. benthamiana tissue. ECFs can be obtained easily by by nucleotide sequencing, is shown in Figure S2. To build vacuum infiltration of harvested leaves and sieving, yielding highly plasmids expressing the various recombinant TMV clones used in AFP-rich solutions, in which more than half of the protein content this work, the intermediate plasmids pMTMVi-N or pMTMVi-M is AFPs. Starting with these ECFs, a single chromatographic step is were digested with AgeI and XhoI (Thermo Scientific), and the required to purify these small proteins to homogeneity. Even DNA fragments corresponding to the ORFs of interest were better, our results show that ECFs can be used directly as obtained by PCR and assembled using the Gibson reaction. Next, antifungals with no further purification. Thus, the downstream the manipulated fragment of the TMV cDNA was excised from processing of recombinant AFPs can be greatly reduced and, the resulting plasmids by digestion with BsaI (New England consequently, the overall manufacturing costs. Biolabs), and assembled with the large fragment of pGTMV In addition to leaf infiltration, we have also shown that the TMV- digested with NcoI-Pfl23II (pMTMVi-N derivatives) or NcoI-CpoI derived system can be used to produce AFPs by spraying N. ben- (pMTMVi-M derivatives) (Thermo Scientific). The full sequence of thamiana tissues with A. tumefaciens suspensions. This simplifica- all recombinant viruses is shown in Figure S4. tion of the inoculation process was previously reported for the Agroinoculation of N. benthamiana leaves MagnICON system (Hahn et al., 2015), and we have successfully implemented it for our new TMV-derived system. We demonstrate Agrobacterium tumefaciens GV3101 carrying the helper plas- that the AFP yields achieved after 12 days by spraying leaves are mid pSoup (Hellens et al., 2000) was transformed with similar to those achieved after 7 days by vacuum or syringe plasmids to express the different TMV recombinant clones. infiltration, although spraying is a much simpler inoculation For plant agroinoculation, overnight cultures of A. tumefaciens strategy. Moreover, we observed that AFPs remain stable in stored were diluted in induction medium (10 mM MES, 10 mM MgCl2, N. benthamiana leaves for at least 2 months at room temperature. 200 lM acetosyringone) at the appropriate optical density at This observation suggests that the AFP production process can be 600 nm (OD600), incubated for 3 h at room temperature, and decoupled from the purification process, and leaves can be infiltrated using a needle-less syringe (0.5 OD600) or sprayed on ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 1069–1080 1078 Xiaoqing Shi et al. the underside of leaves using an aerograph at 2 atmospheres apoplastic fluids were dialysed against water and used at the of pressure (1.0 OD600). Importantly, the surfactant Silwet L-77 concentrations indicated in antifungal assays. (0.1% v/v) needs to be added to the induction medium in the spray inoculation experiments for the effective agroinoculation. Plant protection assays N. benthamiana plants were grown in the greenhouse at 24 °C Botrytis cinerea was kindly provided by Prof. A. Molina (CBGP with a 14 h light-10 h dark photoperiod. For the inoculation collection, Madrid). Tomato plants (Solanum lycopersicum cultivar experiments, we used plants at the 4- to 5-leaf stage, without Marmande, known as the Mediterranean tomato) were cultivated visible flower buds. Unless indicated in the results, leaves were in growth chambers at 22 °C with a 16 h light-8 h dark harvested at 7 dpi or 12 dpi and examined for protein photoperiod. AfpB protection assays against Botrytis cinerea production. In storage experiments, leaves were dried in an infection were performed on detached tomato leaves of 3-week- incubator at 37 °C for 2 months. Plants accumulating the GFP old plants on 1% (w/v) agar in water containing 2 lg/mL kinetin. were photographed at different time points with a Nikon Leaves were locally infected at two points with conidial suspen- D7000 digital camera under illumination with a hand-held UV sions (106 conidia/mL) by applying 20 lL drops containing 10 lM lamp. AfpB of purified protein or extracellular fluids. The progression of Protein analysis and purification symptoms was followed visually. Lesion area was measured by image analysis using the Fiji ImageJ2 package. We analysed two Protein samples were prepared by freezing agroinfiltrated leaves infection points on three leaves from three independent plants in in liquid nitrogen and then grinding to a fine power with a mortar three independent experiments. and pestle. Total soluble proteins were obtained in two volumes of 50 mM sodium phosphate buffer pH 7.2, 10 mM EDTA, 10 mM DTT, 0.1% (w/v) SDS and 0.1% (v/v) Triton X-100. Extracts were Acknowledgements clarified by centrifugation at 16000 g for 15 min at 4 °C. Acidic This work was supported by grants BIO2017-83184-R, BIO2015- extraction was used with AFP samples, as these proteins are pH- 68790-C2-1-R and BIO2015-68790-C2-2-R and through the stable, and can be selectively extracted at low pH. The acidic ‘Severo Ochoa Programme for Centres of Excellence in R&D’ buffer (pH 2.8) contained 84 mM citric acid, 30 mM Na2HPO4, n (SEV-2015-0533) from Spanish Ministerio de Ciencia, Innovacio 6 mM ascorbic acid, 0.1% (v/v) 2-mercaptoethanol. For dried y Universidades (co-financed FEDER funds) and by the CERCA leaves, total proteins were extracted in 50 mM sodium acetate Programme/Generalitat de Catalunya. We thank Laia Castillo for (pH 5.5), 100 mM NaCl, and 10% (v/v) glycerol. Apoplastic fluids technical support. were extracted from fresh leaves by vacuum infiltration using phosphate buffered saline (PBS) buffer supplemented with 0.02% (v/v) Silwet L-77. Protein concentrations were determined Conflict of interest using the Bio-Rad protein assay and bovine serum albumin (BSA) The authors declare no conflict of interest. as standard. Protein preparations were separated in tricine-SDS-PAGE (16.5%). Gels were stained with Coomassie blue to detect References proteins, or transferred to nitrocellulose membranes (Protran Batta, G., Barna, T., Gaspari, Z., Sandor, S., Ko €ver, K.E., Binder, U., Sarg, B., et al. 0.2 lm) to immunodetect proteins, as described previously (Coca (2009) Functional aspects of the solution structure and dynamics of PAF - A et al., 2004; Garrigues et al., 2017). To detect AfpB, we used highly-stable antifungal protein from Penicillium chrysogenum. FEBS J. 276, antiserum against P. chrysogenum PAFB, which was kindly 2875–2890. provided by Dr. Florentine Marx (Medical University of Innsbruck, Bebber, D.P. and Gurr, S.J. (2015) Crop-destroying fungal and oomycete Austria) (Huber et al., 2018). AFPs were purified to homogeneity pathogens challenge food security. Fungal Genet. Biol. 74, 62–64. by cationic chromatography using an AKTA Purifier system Bongomin, F., Gago, S., Oladele, R. and Denning, D. (2017) Global and multi- equipped with a 5 mL HiTrap SP HP column (GE Healthcare), as national prevalence of fungal diseases—estimate precision. J. Fungi 3, 57. described previously (Garrigues et al., 2017). AfpB from P. dig- Bundo , M., Montesinos, L., Izquierdo, E., Campo, S., Mieulet, D., Guiderdoni, itatum (AfpB[Pd]) was obtained from the genetically modified E., Rossignol, M., et al. (2014) Production of cecropin A antimicrobial peptide in rice seed endosperm. BMC Plant Biol. 14, 102. strain PDSG3543 and purified from culture supernatants using Campos-Olivas, R., Bruix, M., Santoro, J., Lacadena, J., del Pozo, A.M., the same cationic chromatography (Garrigues et al., 2017). Gavilanes, J.G. and Rico, M. 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(2006) The antifungal protein AFP secreted by Aspergillus giganteus antimicrobial peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to does not cause detrimental effects on certain mammalian cells. Peptides 27, control viral and bacterial infections. Plant Biotechnol. J. 9, 100–115. 1717–1725. ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 1069–1080 1080 Xiaoqing Shi et al. Thole, V., Worland, B., Snape, J.W. and Vain, P. (2007) The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation. Plant Supporting information Physiol. 145, 1211–1219. Additional supporting information may be found online in the th, L., Kele, Z., Borics, A., Nagy, L.G., Varadi, G., Viragh, M., et al. (2016) To Supporting Information section at the end of the article. NFAP2, a novel cysteine-rich anti-yeast protein from Neosartorya fischeri NRRL 181: isolation and characterization. AMB Express 6, 75. Figure S1 TMV infectious clone. th, L., V To  et al. (2018) aradi, G., Borics, A., Batta, G., Kele, Z., Vendrinszky, A., Figure S2 Full sequences of pGTMV, pMTMVi-N and pMTMVi-M Anti-candidal activity and functional mapping of recombinant and synthetic plasmids. Neosartorya fischeri antifungal protein 2 (NFAP2). Front. Microbiol. 9, 1–12. Figure S3 Analysis of GFP produced in Nicotiana benthamiana Vila, L., Lacadena, V., Fontanet, P., Martinez del Pozo, A. and San Segundo, B. plants using the TMV-derived system. (2001) A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens. Mol. Plant Microbe Interact. 14, 1327–1331. Figure S4 Sequence of TMV-derived recombinant viruses Viragh, M., Vo € ro  Lakatos, G., et al. (2014) €s, D., Kele, Z., Kovacs, L., Fizil, A., TMVDCP-GFP, TMV(ToCP)-fGFP, TMVDCP-AfpB, TMVDCP- Production of a defensin-like antifungal protein NFAP from Neosartorya AfpBKDEL, TMVDCP-UP, TMVDCP-UPKDEL, TMVDCP-UPVS, fischeri in Pichia pastoris and its antifungal activity against filamentous fungal and TMVDCP-AgAFP. isolates from human infections. Protein Expr. Purif. 94, 79–84. Figure S5 Dilution analysis of TMVΔCP-GFP infectivity in Nico- Zeitler, B., Bernhard, A., Meyer, H., Sattler, M., Koop, H.U. and Lindermayr, C. tiana benthamiana. (2013) Production of a de-novo designed antimicrobial peptide in Nicotiana Table S1 Primers used in this work. benthamiana. Plant Mol. Biol. 81, 259–272. ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 1069–1080