Expression of Human Lactoferrin cDNA Confers Resistance to <i>Ralstonia solanacearum</i> in Transgenic Tobacco Plants

Zhang, Z., Coyne, D. P., Vidaver, A. K., and Mitra, A. 1998. Expression of human lactoferrin cDNA confers resistance to Ralstonia solanacearum in transgenic tobacco plants. Phytopathology 88:730-734. A construct containing a human lactoferrin cDNA was used to transform tobacco (Nicotiana tabacum) using an Agrobacterium-mediated DNA-transfer system to express this human protein in transgenic plants. Transformants were analyzed by Southern, Northern, and Western blots to determine integration of the cDNA into the plant genome and lactoferrin gene expression levels. Most transgenic plants demonstrated significant delays of bacterial wilt symptoms when inoculated with the bacterial pathogen Ralstonia solanacearum. Quantification of the expressed lactoferrin protein by enzyme-linked immunosorbent assay in transgenic plants indicated a significant positive relationship between lactoferrin gene expression levels and levels of disease resistance. Incorporation of the lactoferrin gene into crop plants may enhance resistance to other phytopathogenic bacteria as well. Additional keywords: antibacterial proteins, Burkholderia, lactoferricin, Pseudomonas, shiva-1. Expression of nonplant antibacterial protein genes in engineered plants has great potential for combating bacterial diseases (12,17, 24). Transgenic potato or tobacco plants have been developed using constructs encoding shiva-1 (14), SB37 (10,19), or T4 lysozyme (7) for enhancing resistance of these plants to either bacterial wilt caused by Ralstonia solanacearum or to soft rot incited by Erwinia carotovora pv. atroseptica. Both shiva-1 and SB37 are wellcharacterized bactericidal peptides (about 4 kDa) that are synthetic derivatives of cecropin found in insect guts (6,12–14). T4 lysozyme, on the other hand, is an antibacterial bacteriophage protein (7) that disrupts the bacterial membrane (17). Among numerous nonplant antibacterial proteins isolated to date, mammalian lactoferrin (Lf), an iron-binding glycoprotein, is of special interest to us. This protein has long been reported as a major component of infant defense systems. Its mode of action against bacteria is more bactericidal than bacteriostatic (5,15). A study on human and bovine Lf (both sharing a high degree of identity in their amino acid sequences) revealed that a potent bactericidal domain exists in a short peptide sequence termed lactoferricin near the N-terminal region of the Lf molecule (2). While Lf by itself is at most bacteriostatic, its N-terminal peptide (about 25 to 47 amino acids), which can be released by proteolytic cleavage, is highly bactericidal (23). This peptide, lactoferricin, is the shortest active amino acid sequence that is resistant to further enzymatic cleavage (2). Another prominent property of Lf or lactoferricin is its potent activity against a wide range of microorganisms including both gramnegative and gram-positive bacteria, as well as fungi and viruses. Lethal concentrations of bovine lactoferricin, for instance, ranged from 3 to 150 μg/ml against bacteria (1), 18 to 150 μg/ml against yeast (3,21), and 3 to 60 μg/ml against filamentous fungi (4). Inhibition of a human herpes virus by human and bovine Lf occurs at concentrations ranging from 0.5 to 1 mg/ml (9). Our previous research (18) demonstrated high levels of in vitro inhibition of several phytopathogenic bacteria using extracts from Lf-transformed tobacco calli. These reports suggest that the human Lf gene, once expressed in plants, may enhance plant resistance to some bacterial pathogens. Since the tobacco calli used previously (18) were nonregenerable, the current work was then undertaken to genetically engineer tobacco with this gene and determine the degree of resistance of transgenic plants to R. solanacearum. An in vitro assay was further conducted using synthetic bovine lactoferricin and shiva-1 to compare their antibacterial activity. MATERIALS AND METHODS Binary vectors and Agrobacterium strain. Plasmids employing an Agrobacterium Ti-plasmid binary vector system that contained the human Lf gene (hlf) (pAM1400/hlf) were constructed as previously described (18), except that the neomycin phosphotransferase II selectable marker was retained (Fig. 1). The vector plasmid pAM1400 without an insert was used as a negative control. A. tumefaciens strain LBA 4404 was then transformed with either of these two plasmids as before (18). Plant material and transformation. Leaf disks of Nicotiana tabacum cv. Xanthi nc were transformed as described earlier (11). Kanamycin-resistant primary transformed plants (designated as T0) were obtained as reported previously (24). Stable integration of the Lf gene in the genomes of T0 tobacco plants was confirmed by Southern blots (20) using an hlf cDNA fragment as a probe. Kanamycin-resistant, hlf-cDNA–containing T0 plants were selffertilized to obtain T1 plants. Analysis of Lf gene expression. Young seedlings of T0 progeny were screened on modified Murashige and Skoog medium (24) containing 200 μg of kanamycin per ml, and only kanamycin-resistant seedlings were later challenged with R. solanacearum. For Northern blots, frozen leaf samples were ground in liquid nitrogen, and total RNA was extracted using Trizol reagent (Life Technologies, Inc., Gaithersburg, MD) following the manufacturer’s instructions. Twenty micrograms of total RNA from each sample was electrophoresed on 1% agarose gels in 1× Tris-borate-EDTA buffer (20). The gel was then soaked in 7% formaldehyde and 2× SSPE Corresponding author: A. Mitra; E-mail address: [email protected] Publication no. P-1998-0521-01R © 1998 The American Phytopathological Society