Targeting microbial pathogens by expression of new recombinant dermaseptin peptides in tobacco.

Abstract Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acid peptide, is produced by Phyllomedusa bicolor. In an attempt to enhance the antimicrobial efficacy of DrsB1, the DrsB1 encoding 93 bp sequence was either fused to the N or C terminus of sequence encoding chitin‐binding domain (CBD) of Avr4 gene from Cladosporium fulvum. Tobacco leaf disk explants were inoculated with Agrobacterium rhizogenes harboring pGSA/CBD‐DrsB1 and pGSA/DrsB1‐CBD expression vectors to produce hairy roots (HRs). Polymerase chain reaction (PCR) was employed to screen putative transgenic tobacco lines. Semi‐quantitative RT‐PCR and western blotting analysis indicated that the expression of recombinant genes were significantly higher, and recombinant proteins were produced in transgenic HRs. The recombinant proteins were extracted from the tobacco HRs and used against Pectobacterium carotovorum, Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas campestris pathogenic bacteria and Alternaria alternata and Pythium sp. fungi. Two recombinant proteins had a statistically significant (p < 0.01) inhibitory effect on the growth and development of plant pathogens. The CBD‐DrsB1 recombinant protein demonstrated a higher antibacterial effect, whereas the DrsB1‐CBD recombinant protein demonstrated greater antifungal activity. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, adhered to each other, and crushed following the antimicrobial activity of the recombinant proteins. Due to the high antimicrobial activity of the recombinant proteins against plant pathogens, this strategy can be used to generate stable transgenic crop plants resistant to devastating plant pathogens.


| INTRODUC TI ON
Plant pests and diseases are among the main factors reducing the production of agricultural products and diminishing their quality and yield as well as threatening food safety (Oerke, 2006). In developing countries, it is estimated that pests and diseases decrease the yield of crop plants by 30%-40% (Flood, 2010). Chemical control is often used to combat devastating plant pathogens. However, given the negative effects of chemical control on human health and the environment, and emergence of resistance by pathogens, it is necessary to employ safer and more sophisticated methods to cope with plant pathogens (Vidaver, 2002).
Fungal cell wall plays a crucial role in fungal pathogenesis since it acts as the major interface with the host immune system (Hopke, Brown, Hall, & Wheeler, 2018), and fungal mutants affected in the biosynthesis of cell wall oligosaccharides are severely affected in their growth and pathogenicity (Bowman & Free, 2006). Chitin is one of the main structural oligosaccharides of the cell wall in various pathogenic fungi, playing a role of an important elicitor of innate defense responses in plants (Sánchez-Vallet, Mesters, & Thomma, 2015). Through their hydrolytic activities, plant chitinases hydrolyze cell wall chitin eventually leading to cell death (Latgé, 2010;Latgé & Beauvais, 2014;Thomma, Nürnberger, & Joosten, 2011). To fight back, fungal pathogens employ various strategies, including changes in cell wall oligosaccharides, secreting effectors, and masking cell wall components (Fujikawa et al., 2012;Latgé & Beauvais, 2014).
For example, Cladosporium fulvum produces and secretes Avr4 effectors at the infection site covering the cell wall chitin inhibiting plant hydrolytic enzymes (van den Burg, Harrison, Joosten, Vervoort, & de Wit, 2006). The Avr4 effector has a CBD binding to the chitin in fungal cell walls, decreasing host chitinases access to fungal cell walls and thus preventing fungal cell wall degradation (van den Burg et al., 2006).
Since correct packaging, disulfide bonds formation, accumulation of multi-subunit proteins, and posttranslational modifications are performed by plant cells accurately, plant systems are used to produce eukaryotic recombinant proteins (Daniell, Streatfield, & Wycoff, 2001;Giddings, Allison, Brooks, & Carter, 2000). However, low production levels, high extraction costs, and limitations in the degree of recombinant protein purification are among major challenges facing recombinant protein production in plants (Borisjuk et al., 1999). The use of Agrobacterium rhizogenes-mediated transformation to produce hairy roots (HRs) is one of the efficient strategies to produce recombinant proteins and secondary metabolites (Borisjuk et al., 1999).
The use of genetic engineering strategies to introduce genes encoding natural and synthetic antimicrobial peptides is a new approach in engineering resistance to a broad spectrum of pathogens (Zasloff, 2002). Various recombinant proteins and peptides have so far been introduced in HRs of different plant species, and their antimicrobial properties have been tested in vitro (Aleinein, Schäfer, & Wink, 2015;Moghadam, Niazi, Afsharifar, & Taghavi, 2016;Pham, Schäfer, & Wink, 2012). For instance, an anti-HIV protein and an anti-tumor protein MAP30, including ribosome-inhibiting proteins were produced in tobacco HRs (Moghadam et al., 2016).
Analysis of total proteins extracted from transgenic HRs indicated a strong antimicrobial activity against both gram-positive and gram-negative bacteria as well as pathogenic fungi (Moghadam et al., 2016). Chahardoli, Fazeli, and Ghabooli (2018) expressed a lactoferricin encoding protein in tobacco HR culture with potent antibacterial activity against Escherichia coli (Chahardoli et al., 2018).
Introduction of ranalexin peptide in tobacco HRs resulted in efficient production of ranalaxin in HRs with strong inhibitory effect on multidrug resistant gram-positive and gram-negative pathogens such as Staphylococcus aureus, Streptococcus pyogenes, and E. coli and on Enterococcus strains resistant to vancomycin, suggesting that tobacco HR culture was a suitable system to produce ranalexin and other recombinant peptides (Aleinein et al., 2015).
In this study, we showed that fusion of dermaseptin B1 (DrsB1) antimicrobial peptide to the chitin-binding domain (CBD) of Avr4 protein from C. fulvum enhanced the antibacterial activity of DrsB1 peptide, suggesting that CBD might facilitate DrsB1 peptide access to the fungal plasma membrane, leading to cell membrane rupture and deformation. promoter. The Arg-Gly-Ser-(His) 6 sequence was engineered after SP to identify the recombinant proteins ( Figure 1).

| Agrobacterium rhizogenes-mediated transformation
Tobacco (Nicotiana tabacum) Xanthi cultivar seeds were disinfected in a detergent solution (5% sodium hypochlorite and Triton X-100) for 10 min and then washed three times with distilled water to remove detergent residues. The seeds were then germinated on MS culture medium under 16 hr light/8 hr dark photoperiod at 24 ± 2°C.
Two A. rhizogenes (ATCC15834) clones harboring the pGSA1285/ CBD-DrsB1 and pGSA1285/DrsB1-CBD expression vectors were used to inoculate tobacco leaf disks. Briefly, sterilized 3-week-old tobacco leaf disks (1 cm 2 ) were cut and put in an A. rhizogenes twoday inoculation suspension for 10 min. The inoculated leaves were dried using a sterile filter paper and then placed on the hormone and antibiotic-free MS culture medium. The inoculated leaf disks were incubated at 24 ± 2°C for 2-3 days in dark and were eventually transferred to the selective medium containing kanamycin (50 mg/L) and cefotaxime (200 mg/L) for HR induction. The explants were regularly subcultured once every 2 weeks until root formation (Tempe & Casse-Delbart, 2012).

| Expression analysis of the recombinant genes
Total RNA was extracted following lithium chloride method (Li, Wang, Sun, & Li, 2011) followed by DNase treatment to remove genomic DNA contamination. cDNA was synthesized using a cDNA synthesis kit (Thermo Fisher Scientific Inc., #k1622).

| Total protein extraction from HRs
One-gram HR tissue from transgenic and control lines were ground in liquid nitrogen and homogenized in a potassium phosphate buffer (50 mM at pH7, 1 mM phenylmethylsulfonyl fluoride). The homogenized samples were vigorously vortexed for 2-5 min and centrifuged at 13,000 rpm for 30 min at 4°C, and the supernatants were filtered using 0.45 µm membranes (Stone & Gifford, 1997). Furthermore, total protein was also extracted from a DrsB1-expressing (DrsB1-04) transgenic HR with no CBD fused as a control (Alibakhshi, 2017).
The concentration of extracted proteins was measured using the Bradford method (Bradford, 1976), and proteins were stored at −20°C.

| Purification of the recombinant proteins and Western blot analysis
To purify the expressed recombinant proteins, total protein isolated from transgenic and controls was transferred to a chromatography column containing the PrepEase Ni-IDA resin. The column was prewashed with 300 mM NaCl and then with 50 mM NaH 2 O 4 . The purified recombinant proteins were removed from the column by rinsing the column, using 250 mM imidazole. Purified proteins were then loaded on 14% acrylamide gel and electrophoresed at 150 V.
The recombinant proteins were electroblotted by using a Mini-Protean II Multiscreen Apparatus (Bio-Rad). The nitrocellulose blot was blocked for 1 hr, using tris buffered saline (TBS) containing 5% powdered milk. The blots were washed three times with TBS buffer and then exposed to 1:2000 dilution of mouse monoclonal anti-His antibody at 37°C for 1 hr followed by 3,3′-diaminobenzidine tetrahydrochloride (DAB) detection according to the manufacturer's instructions (Bollag, Edelstein, & Rozycki, 1996).

| Antimicrobial activity of the recombinant proteins
Antimicrobial activities of the recombinant proteins were determined by studying the activity of the total protein extracted from Briefly, 100 µl of 21 hr old culture of the bacterial suspensions of half-MacFarland (1.5 × 10 8 cfu/ml) was poured onto the surface of the nutrient agar culture medium and spread by using a sterilized cotton swab. Next, 50 µl of the filter sterilized extracted fusion proteins (60 μg/ml) were added to each of the sterile 6-mm disks. The disks containing the recombinant proteins were then placed on the bacterial culture medium. The Petri dishes were maintained at 4°C for 30 min to fix the proteins in the disks and then incubated at 28°C or 30°C (depending on the bacterium type) for 16 hr. In order to determine the antimicrobial activity of fusion proteins, the diameter of inhibition zones surrounding the disks was measured in triplicates in millimeter (r zone − r disk ). It must be mentioned that the gentamicin disk (10 μg/disk) was used as the positive control and the proteins extracted from the non-transgenic HRs as the negative control.
Statistical analysis of the treatments was conducted using MSTATC and SAS 9.1 softwares in a factorial experiment using a completely randomized design with three replications. If there were significant differences between the treatments, Duncan's multiple range test at the relevant significant level was used to compare the means.

| Minimum inhibitory concentrations of the recombinant proteins
The microdilution method was used to determine the minimum in-

| Scanning electron microscopy analysis
Alternaria alternata (PTCC 5,224) and Pythium sp. fungi were cultured on the PDA medium for 4 days. Fungal plugs (1 cm 2 ) were injected with each of the recombinant proteins (50 μg/ml) and incubated at 25°C for 24 hr followed by freezing at −80°C for 24 hr. The frozen plugs were then put in a freeze-drying machine for 4 hr. The fixed samples were coated with gold nanoparticles using a Desk Sputter Coater-DSRI, and finally scanned with an electron microscope (FE-SEM, Tescan Mira3 LMU, at HV = 20 kV).

| Molecular analysis of transgenic plants
Transgenic HRs were produced and designated as CBD-DrsB1-XX and DrsB1-CBD-XX based on the type of the construct employed to express the recombinant proteins, XX represents the transgenic line number.
Polymerase chain reaction analysis of putative transgenic HRs using rolC and DrsB1 peptide specific primers resulted in amplification of 600 bp and 100 bp PCR products, respectively, suggesting that HRs are transgenic, whereas no PCR product was amplified in non-transgenic HRs. Moreover, PCR analysis using the VirG specific primers did not lead to any amplification, ruling out possible A. rhizogenes contamination (Appendix Figure A1).

| Antimicrobial activity of the recombinant proteins
Sixty micrograms per milliliter (60 μg/ml) total protein from the CBD-DrsB1-8, CBD-DB1-14, and DrsB1-04 lines (Alibakhshi, 2017) were used to compare the activity of recombinant proteins against four pathogenic bacteria. Total protein from transgenic HRs lines had a significant (p < 0.01) higher antibacterial activity than total protein extracted from non-transgenic HRs. Interestingly, the total protein from transgenic HRs lines exhibited a higher activity than DrsB1-04 transgenic line (Table 1).
The antibacterial activity of the recombinant proteins was assayed for bactericidal activity against four gram-negative bacteria.
The recombinant proteins showed a stronger activity against P. The inhibition zone diameter is presented as the mean ± standard deviation of three replicates

| D ISCUSS I ON
Over the past few decades, various approaches have been employed in molecular biology to improve and increase plant resistance to a broad spectrum of plant pathogens (Cao, Li, & Dong, 1998). For instance, expression of fungal and bacterial cell wall-degrading enzymes (chitinases), expression of pathogenesis-related proteins, increase in production of host proteins and metabolites involved in plant defense pathways, and expression of plant antimicrobial proteins and peptides have been reported (Punja, 2001).
All living organisms produce different classes of antimicrobial peptides as a part of their innate immune system to combat pathogens (Hancock & Scott, 2000;Holaskova, Galuszka, Frebort, & Oz, 2015). To enhance the antibacterial activity of natural peptides, researchers design and synthesize new variants or recombinant peptides for pharmaceutical and agricultural industries (Melo, Ferre, & Castanho, 2009;Yevtushenko & Misra, 2012;Yevtushenko et al., 2005). In this study, the DrsB1 peptide was fused to the CBD of the Avr4 gene from C. fulvum so that the recombinant protein could bind to the fungal cell wall as well as the peptidoglycans of bacterial cell walls perturbing the integrity of cell wall components. Although the DrsB1 peptide exhibited strong in vitro antimicrobial activity against bacteria, fungi, protozoans, and yeasts, the antibacterial activity was significantly increased when the N-terminal region of the DrsB1 peptide and MsrA2 analog was manipulated to bind to the negatively charged lipids (Osusky et al., 2005). The expression of modified peptides in potato and tobacco plants led to the production of transgenic lines with enhanced resistance to a number of devastating plant pathogens. Interestingly, the recombinant proteins extracted from transgenic HRs in this study, exhibited a significant (p < 0.01) antibacterial activity against plant pathogenic gram-negative bacteria. Similarly, Badrhadad, Nazarian-Firouzabadi, and Ismaili (2018) provided strong evidence that fusion of an alfalfa antibacterial peptide to rice chitinase CBD inhibited the growth and development of plant pathogens (Badrhadad et al., 2018).
The inhibitory concentrations of the recombination proteins varied significantly for different bacteria and fungi. It was found that the fungi are more sensitive to recombinant proteins than bacteria.
The susceptibility of bacteria and fungi to recombinant proteins may be attributed to the variation of different cell components present in bacteria and fungi (Marcos, Muñoz, Pérez-Payá, Misra, & López-García, 2008).
Although the mechanism by which antimicrobial peptides attack pathogens is not fully understood, the mode of action may involve interaction of charged components (Nguyen et al., 2011;Toke, 2005). A relatively higher positive charge of the recombinant peptides and the binding affinity of the CBD toward cell wall building blocks may accumulate more DrsB1 peptide on the surface of the pathogen leading to effective interaction between positively charged recombinant peptides and the negatively charged membrane surface of the pathogens. It is documented that the positive charge in the hydrophobic part of cationic peptides is essential for their antimicrobial activity (Yin, Edwards, Li, Yip, & Deber, 2012). In other words, fusion of the CBD from Avr4 along with addition of histidine residues increases the antimicrobial activity of the recombinant proteins against pathogenic microbes. There seems to be a relationship between the chitin-binding capability and antimicrobial activity of the CY-AMP peptide against the gram-positive bacteria Lactococcus lactis, Streptococcus mutans, and Clavibacter michiganensis, the gram-negative Erwinia carotovora and Enterobacter cloacae, and the fungi Fusarium oxysporum and Geotrichum candidum (Yokoyama et al., 2009). Mutations in the CBD of the CY-AMP peptide led to a decrease in chitin-binding ability and hence antifungal activity, whereas the antibacterial activity of CY-AMP against grampositive and gram-negative bacteria did not change in comparison with that of the wild-type peptide (Yokoyama et al., 2009). It is noteworthy that the antifungal activity of a barley chitinase mutated at the important catalytic domain of acidic residues declined by 75% in comparison with that of wild-type chitinase (Andersen, Jensen, Robertus, Robert, & Skriver, 1997). Overall, it can be concluded that the antifungal activity of the recombinant proteins increases as CBD shows intrinsic affinity for chitin. Therefore, the recombinant proteins in this study had a relatively higher inhibitory effect against A. alternata in comparison to Pythium sp. Furthermore, the presence of chitin in the cell wall seems to be crucial for antifungal activity of the recombinant proteins (Yan et al., 2008). The results of a similar study indicated that a protein bound to a chitin-binding protein with antifungal activity from Moringa oleifera seeds (MO-CBP3) had a strong antifungal activity against Fusarium solani, F. oxysporum, but did not inhibit the growth and germination of Pythium oligandrum, suggesting that adhesion to the chitin is vital for antibacterial activity (Gifoni et al., 2012).
In conclusion, results of the present study indicated that the cell wall chitin can be targeted to control plant pathogenic fungi containing chitin (Yan et al., 2008). Accumulation of DrsB1 peptide on the surface of fungal pathogens in a carpet-like manner (Pouny, Rapaport, Mor, Nicolas, & Shai, 1992;Shai, 1999) may result in instability of the fungal cell, eventually leading to the fungal cell death ( Figure 5). The DrsB1-CBD recombinant protein had a higher inhibitory effect than CBD-DrsB1 recombinant protein, suggesting that DrsB1 peptide may interfere with CBD affinity for cell wall chitin, leading to a lower concentration of the CBD-DrsB1 recombinant protein at the cell wall surface. It is noteworthy that no plant chitinase has so far been identified with the CBD at the C-terminal part of the main catalytic domains (Beintema, 1994;Iseli, Boller, & Neuhaus, 1993). Due to the high antimicrobial activity of the recombinant proteins of this study, it would be interesting to introduce the recombinant genes to crop plants and generate resistant lines to devastating plant pathogens.

ACK N OWLED G EM ENTS
This study was supported by the Lorestan University research grant.
We appreciate Laboratory of plant pathology at Lorestan and Gillan universities for their excellent assistance. We are also grateful to Dr. Dhananjay Dhokane from Reliance Industries for reading our manuscript.

CO N FLI C T O F I NTE R E S T S
None declared.

E TH I C S S TATEM ENT
None required.

DATA ACCE SS I B I LIT Y
All data used in this study are presented in the manuscript.