Enhanced resistance to bacterial and fungal pathogens by overexpression of a human cathelicidin antimicrobial peptide (hCAP18/LL-37) in Chinese cabbage
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The human cathelicidin antimicrobial protein hCAP18, which includes the C-terminal peptide LL-37, is a multifunctional protein. As a possible approach to enhancing the resistance to plant disease, a DNA fragment coding for hCAP18/LL-37 was fused at the C-terminal end of the leader sequence of endopolygalacturonase-inhibiting protein under the control of the cauliflower mosaic virus 35S promoter region. The construct was then introduced into Brassica rapa. LL-37 expression was confirmed in transgenic plants by reverse transcription-polymerase chain reaction and western blot analysis. Transgenic plants exhibited varying levels of resistance to bacterial and fungal pathogens. The average size of disease lesions in the transgenic plants was reduced to less than half of that in wild-type plants. Our results suggest that the antimicrobial LL-37 peptide is involved in wide-spectrum resistance to bacterial and fungal pathogen infection.
KeywordsAntimicrobial peptide Cathelicidin Disease resistance Transgenic Chinese cabbage
Bacterial and fungal plant pathogens severely affect crop productivity. For example, Xanthomonas campestris pv. campestris and Pectobacterium carotovorum subsp. carotovorum, which cause black rot and soft rot, respectively, are present worldwide and severely damage plants and reduce their yields, especially in cruciferous plants (Boman 2003). Therefore, the development of cruciferous plants that are resistant to black and soft rot diseases has been a major goal of researchers for several decades. Strategies based on transgenic approaches to enhance plant disease resistance involve the use of genes associated with plant defense pathways (Makandar et al. 2006; Zhang et al. 2007) and genes encoding plant or fungal hydrolytic enzymes (Bieri et al. 2003), defense-related transcription factors (Chen and Chen 2002; Sohn et al. 2006) and antimicrobial peptides (Alan et al. 2004).
A large number of antimicrobial peptides from different organisms have been characterized (Simmaco et al. 1998). The human cathelicidin antimicrobial protein hCAP18 is the only member of the mammalian cathelicidin family of proteins that is present in humans (Gudmundsson et al. 1996). The holoprotein consists of a conserved prodomain, a cathelin domain, and the non-conserved C-terminal peptide LL-37, which is enzymatically cleaved after secretion (Sorensen et al. 2001; Yamasaki et al. 2006). Its precursor molecule, an 18 kDa human cationic antimicrobial protein (hCAP-18), is secreted by activated neutrophil granulocytes. After release, the helical C-terminal end of this precursor comprising 37 amino acids is cleaved off, thereby forming the functional antimicrobial peptide LL-37 (Sorensen et al. 2001). Since LL-37 is the only human antimicrobial peptide that is active at physiological or elevated salt concentration conditions, there is a significant interest in using this peptide for pharmaceutical applications (De Smet and Contreras 2005; Reddy et al. 2004; Travis et al. 2000).
In the present study, we report the transgenic expression of human cathelicidin antimicrobial peptide carrying the substitution Met37Leu in Chinese cabbage. The expression of this peptide in cabbage plants significantly inhibited the growth of Pectobacterium carotovorum subsp. carotovorum on the plant leaves, and it conferred resistance to several fungal pathogens. These results further support the assignment of a defense role to LL-37 and highlight its plant biotechnological potential.
Materials and methods
Expression vector construction
The leader sequence of the gene encoding Phaseolus vulgaris endopolygalacturonase-inhibiting protein (PGIP) (GenBank Accession No. X64769) was fused upstream of an LL-37-coding DNA fragment to cause the extracellular localization of the mature protein. The PGIP signal peptide (87 bp) was amplified by PCR with primers A linked with BamHI site (5′-CCGGATCCATGACTCAATTCAATATCCCA-3′) and B (5′-AGAGAGTGCAGTTCTCAA-3′). The coding region of LL-37 (111 bp) was substituted with Met-LL37-Leu and amplified by PCR from pFALL37 DNA using primers C (5′-ATGCTGCTGGGTGATTTCTTC-3′) and D with SacI site (5′-CGAGAGCTCCTAGGACTCTGTCC TGGG-3′). The two products were ligated into pBlueScript-SK (Stratagene, La Jolla, CA, USA) at the BamHI and SacI restriction sites. The generated LL-37 fragment was further amplified using primers A and D. For Agrobacterium transformation, the PCR product was subcloned into pBI121 binary vector driven by cauliflower mosaic virus 35S (CaMV35S) promoter (Gelvin 1998). The Ti plasmid vector construct pBI–LL37 was confirmed by DNA sequencing (ABI 377 DNA sequencer; Perkin-Elmer, Cypress, CA, USA).
Plant transformation and regeneration
The prepared construct was transformed into Chinese cabbage using the protocol described in Min et al. (2007). A total of 168 hypocotyls from in vitro grown seedlings of Chinese cabbage (Brassica rapa cv. Osome) were inoculated with Agrobacterium tumefaciens strain LBA4404 carrying either pBI-LL37 or pBI121. Green shoots that developed in the selective medium were transferred to a rooting medium containing 100 mg L−1 kanamycin and 500 mg L−1 carbenicillin. Rooted shoots were screened by PCR for the presence of the transgene before transfer to plastic pots.
Estimation of transformants and generation of homozygous lines
Self-pollinated seeds obtained from T0 plants were sown into plastic pots in the greenhouse. Two weeks after germination, seedlings were sprayed with 400 mg L−1 kanamycin in water; they were sprayed again 2 days later. Three days after the second spray, the ratios of green seedlings to bleached seedlings were determined, and the results were analyzed by a Chi-square test for goodness of fit to the ratios 3:1, 15:1, or 63:1. In order to obtain transformants homozygous for the LL-37 gene, kanamycin-resistant T1 progenies were grown to produce selfed T2 seeds. T2 lines that had no bleached segregants after kanamycin sprays were assumed to be homozygous for the LL-37 and nptII genes.
Molecular analysis of transformants
PCR analyses were conducted to detect the presence of LL-37- or nptII-specific fragments. Primers 35SF (5′-TCCACTGACGTAAGGGATGA-3′) and LL-37R (5′-CGAGAGCTCCTAGGACTCTGTCCTGGG-3′), which amplified a fragment of size approximately 750 bp, including sequences from the 3′ end of the 35S promoter, signal peptide (SP), and part of the LL-37 gene, were used to screen for putative LL-37 transformants. Putative transformants were screened using primers nptIIF (5′-TCGGCTATGACTGGGCACAACAGC-3′) and nptIIR (5′-AAGA AGGCGATAGAAGGCGATGCG-3′), which amplified a 722-bp nptII-specific fragment. Genomic DNA was isolated from young leaves of Chinese cabbage plants using a DNeasy Plant kit (Qiagen, Germantown, MD, USA.) as per the manufacturer’s instructions. PCR was performed and the reaction conditions were followed (1 cycle of 94°C for 1 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and 1 cycle of 72°C for 10 min). The reaction products were electrophoresed on a 2% (w/v) agarose gel with 1× TAE buffer and visualized by staining with ethidium bromide.
In order to analyze gene expression in transgenic plants by RT-PCR, total RNA from wild-type and transgenic plants was reverse transcribed using AMV reverse transcriptase (Roche, USA) with oligo (dT) primers for 1 h at 42°C. The expression level of actin mRNA was used as a quantitative control.
Western blot analysis was performed by following standard molecular techniques (Sambrook et al. 1998). Briefly, for western blotting, 200 ng of purified protein or synthetic peptide was electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Blots were probed with 1:1,000 dilution of polyclonal Hbt Ab antiserum raised in rabbit and then with 1:10,000 dilution of goat anti-rabbit immunoglobulin-peroxidase conjugate (Vector, Burlingame, CA, USA). The blots were then developed using the enhanced chemiluminescence (ECL) developing system (GE Health Care, USA). The Low Range BioRad 161-0304 markers (BioRad, Hercules, CA, USA) were used as the molecular size markers.
P. carotovorum subsp. carotovorum KACC 10057 obtained from the Korean Agricultural Culture Collection (http://kacc.rda.go.kr) was grown in Luria–Bertani (LB) medium until the absorbance at 600 nm (A 600) was 0.2, which corresponds to a concentration of approximately 2 × 108 CFU/mL. Three different concentrations of the culture (104, 106, and 108 CFU/mL) with 10 mM MgCl2 were inoculated by syringe infiltration. The inoculated plants were transferred to a growth chamber and incubated at 28°C under continuous light. They were examined for 12–96 h after inoculation. Lesion length (cm) and disease index (DI) were recorded for each individual plant; the disease index ranged from 0 to 6 on the basis of the development of the disease lesions: 0, no lesion; 1, lesion size 0.1–0.5 cm; 2, 0.5–1.5 cm; 3, 1.5–3.5 cm; 4, 3.5–5.5 cm; 5, 5.5–8.5 cm; and 6, over 8.5 cm or plant dead.
The fungi Fusarium oxysporum f. sp. Lycopersici (KACC 40032), Colletotrichum higginsianum (KACC 40807), and Rhizoctonia solani (KACC 40107) were inoculated on plant leaves by placing 10 μl of an aqueous suspension containing 106 spores/mL on the leaves. Plants were maintained in highly humidified conditions (100% RH) at 25°C with 16 h of light in a growth chamber.
Evaluation of in vitro inhibition assays
Total and extracellular fluids were extracted by the methods described in Alan et al. (2004). Protein concentrations in the leaflet fluids were determined by the Bradford assay (Bradford 1976). The in vitro inhibition assays were evaluated by the protocol from Alan et al. (2004). Briefly, the experiments tested whether total fluid (TF) and extracellular fluid (EF) from three homozygous lines possessed antimicrobial activity. Assays were also performed using TF and EF from wild-type plant and LB medium as controls. A volume of 248 μL of TF, EF, and LB was mixed with 2.5 μL of 108 CFU/mL P. carotovorum subsp. carotovorum in Eppendorf tubes and incubated on a shaker for 4 h. The samples remaining in the tubes were mixed in the ratio 1:9 with LB, returned to the shaker, and incubated overnight at 37°C. The bacterial growth in these tubes was determined by a spectrophotometer at 600 nm.
Generation and characterization of LL-37 transgenic plants
Expression of LL-37 in homozygous lines
Increased resistance of the transgenic plants to soft rot
Increased resistance of the transgenic plants to fungal pathogens
Bacterial inhibition assays in transgenic leaflets
In response to a microbial attack, plants activate a complex series of responses that lead to the local and systemic induction of a broad spectrum of antimicrobial defenses (Kim and Martin 2004; Kunkel 2002). When induced defense responses are rapidly and coordinately triggered during a given plant–pathogen interaction, plants become broadly resistant to diseases. These defense responses include the strengthening of mechanical barriers, oxidative burst, and production of antimicrobial compounds (Hammond-Kosack and Parker 2003; Park 2005). Some research has been performed to bolster plant defenses against bacteria and fungi by genetically engineering plants to express antimicrobial peptides (Lee et al. 2008; Prasad et al. 2008).
In this study, we have chosen a variant of the antimicrobial peptide LL-37 as an interesting candidate for transgenic plant expression; this variant is designed to target the peptide into extracellular spaces. Extracellular targeting was originally intended to prevent possible deleterious effects of the peptide in plant cells. Moreover, secretion into extracellular spaces allows the plant-produced peptide to come into direct contact with pathogens growing and multiplying extracellularly before attacking the cells (Ponti et al. 2003). We developed homozygous Chinese cabbage lines stably expressing LL-37, which did not cause adverse effects on the plant phenotypes. These transgenic homozygous lines were tested with four important pathogens of this crop and found to inhibit the growth of the bacterial pathogen causing Chinese cabbage rot and also that of three fungal pathogens.
The results of our pathogenicity assays suggest that the expression of LL-37 provides a moderate level of resistance against a bacterial pathogen (P. carotovorum subsp. carotovorum) at the inoculum concentration of 104 CFU/mL. However, the extent of disease suppression provided by LL-37 expression was reduced as the inoculum concentrations were increased to 106 and 108 CFU/mL. Moreover, we observed reduction in the survival of P. carotovorum subsp. carotovorum cells incubated with EF from LL-37-expressing Chinese cabbage cell lines. However, bacteria grew normally when they were incubated in LB medium only and in TF or EF obtained from a non-transformed cell line. Therefore, leaves appeared to express LL-37 at sufficient levels in the extracellular spaces to retard bacterial multiplication and hence decrease disease severity. The functionality of this transgene and the presence of antimicrobial activity in the EF indicates that the LL-37 peptide was properly targeted to the extracellular space even with a foreign plant signal peptide. Furthermore, the mammalian peptide was not subjected to a processing step in the foreign plant cell environment that rendered it inactive.
Until now, studies involving the enhancement of resistance to various bacterial, fungal, and oomycete pathogens by the expression of antimicrobial peptides have been reported for rice, tobacco, poinsettia, banana, and more host species (Chakrabarti et al. 2003; Liang et al. 2002; Smith et al. 1998). However, progress on identifying the defense mechanisms in Chinese cabbage (B. rapa), an important vegetable crop in Asia, has been very slow. Previously, we reported an enhancement in the resistance to bacterial soft rot by the expression of the bromelain gene in Chinese cabbage (Jung et al. 2008). Here, we demonstrate that expression of the human LL-37 peptide has antimicrobial activity toward both bacterial and fungal pathogens of Chinese cabbage. Since pathogens have the ability to overcome gene-for-gene host defense mechanisms in the field by undergoing mutations in the cognate avirulence genes, any transgenic model based on such resistance-conferring genes may easily be evaded by pathogens. On the other hand, this problem is less likely to occur in transgenic plants overexpressing genes with more general antimicrobial activity. Therefore, the expression of the human LL-37 peptide is expected to confer durable resistance (i.e., field resistance) to a wide variety of pathogens infecting Chinese cabbage plants.
In order to obtain a high level of resistance as observed in the case of R gene-mediated resistance, the overexpression of multiple antifungal proteins with different functions may be necessary. In a R. solani infection assay, tobacco plants coexpressing the barley transgenes (a class II chitinase, a class II β-1,3-glucanase, and a type I ribosome-inactivating protein) were reported to elicit significantly enhanced protection against fungal attack as compared to that of the corresponding isogenic lines expressing a single barley transgene at a similar level (Brogue et al. 1991). Thus, other antimicrobial genes such as the bromelain gene (Jung et al. 2008) could be stacked with the human LL-37 gene by crossing different transgenic lines and this may be able to strongly and durably inhibit the growth of pathogens.
We thank to Dr. Philip Becraft for critical reviews and comments. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008085), Rural Development Administration, Republic of Korea.
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