Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection

Abstract

The peptide snakin-2 (StSN2) has been isolated from potato (Solanum tuberosum cv Jaerla) tubers and found to be active (EC(50) = 1-20 microM) against fungal and bacterial plant pathogens. It causes a rapid aggregation of both Gram-positive and Gram-negative bacteria. The corresponding StSN2 cDNA encodes a signal sequence followed by a 15-residue acidic sequence that precedes the mature StSN2 peptide, which is basic (isoelectric point = 9.16) and 66 amino acid residues long (molecular weight of 7,025). The StSN2 gene is developmentally expressed in tubers, stems, flowers, shoot apex, and leaves, but not in roots, or stolons, and is locally up-regulated by wounding and by abscisic acid treatment. Expression of this gene is also up-regulated after infection of potato tubers with the compatible fungus Botritys cinerea and down-regulated by the virulent bacteria Ralstonia solanacearum and Erwinia chrysanthemi. These observations are congruent with the hypothesis that the StSN2 is a component of both constitutive and inducible defense barriers.

Figure 1

Purification and characterization of StSN2. A, reverse phase-HPLC fractionation of the cell wall extract (CWE) from potato tubers. The linear gradient used was water (0.1% [v/v] trifluoroacetic acid)-2-propanol, 0% to 30% for 180 min and 30% to 50% for 15 min. Fraction corresponding to StSN2 is indicated. B, Separation by SDS-PAGE of the purified proteins StSN2 and StSN1 (Segura et al., 1999), and CWE from potato tuber. Molecular mass markers (MW) are indicated. C, Nucleotide sequence of StSN2 cDNA (AJ312904) and amino acid sequence of the corresponding protein. The gray-shaded amino acid sequence was obtained by direct N-terminal Edman degradation of the purified StSN2 and the rest of protein sequence was deduced from the cDNA sequence. Signal peptide (SP) is followed by a black-shaded amino acid sequence corresponding to the acidic sequence that preceded StSN2 mature peptide (MP). Predictions of SP were done by using the SignalP (http://www.cbs.dtu.dk/services/SignalP/) and Psort (http://psort.nibb.ac.jp/) program. Oligonucleotides sequences used for 5′-RACE are indicated by horizontal arrows, and those used for PCR amplification of the StSN2 gene are underlined. The position of the introns (I and II) in the StSN2 gene (AJ312424) are indicated by triangles.

Figure 2

Alignment of snakins/GASAs amino acid sequences. A, Comparison of amino acid sequences of snakins/GASAs of subfamilies II, I, and III, respectively. Amino acids conserved across all the family members are black shaded and indicated in the consensus. Highly conserved residues that are relevant for subfamily classification (conserved in known, non-represented members of each group) are gray shaded. Sequences taken for the alignment are the following: StSN1 (Segura et al., 1999) and StSNIII (BG597515) from potato; AtGASA1 and AtGASA4 (Herzog et al., 1995), AtGASA7 (AC005396.2), and AtGASA8 (AC004218.2) from Arabidopsis; LsGAST1 (Shi et al., 1992), LsGASAI (BG130738), and LsGASAII (AI77478) from tomato; GhGEG from Gerbera hybrida (Kotilainen et al., 1999); FaGAST from Fragraria ananassa (AF039183); Rc153 from Ricinus communis (EMBL T24153); PhGIP1 from Petunia hybrida (Ben-Nissan and Weiss, 1996); and PmGAST1 from Picea marina (AF051227). The putative N-terminal sequences of the proteins have been determined by homology with those of StSN1 and StSN2, and by using the posttranslational predictions programms indicated in Figure 1C. B, Alignment of potato StSN2 sequence with amino acid sequence of Cys-rich domains of proteins from mammals and the hemotoxic snaken venom disintegrin. Identical residues are black shaded and similar, conservative positions (polar, apolar, and charged) are gray shaded. Proteins used in the alignment are the following: HsvWF (X04385), HsMuc (Q9ESP3), HsMDC, and HsMDC2a (Sagane et al., 1998) from human; BtvWF from Bos taurus (P80012); and disintegrin from agkistrodon halys blomhoffii snake (Gloydius blomhoffii; P21858). A stretch of 15 amino acids of HsvWF that has not been included in the alignment is indicated by an asterisk. The RGD motif of disintegrin essential for protein action is underlined. Similarities between StSN2 and the aligned proteins were found using the Jpred program for secondary structure predictions (http://barton.ebi.ac.uk/).

Figure 3

Aggregation of bacteria caused by potato StSN2. A 5-μL suspension (10⁵ colony forming units [cfu] mL⁻¹) of the Gram-positive bacterium C. michiganensis subsp. sepedonicus or the Gram-negative bacterium R. solanacearum was deposited in a microscope slide, then 5 μL of a 20 μm solution of StSN2 or a 5-μL drop of water was added, and a photograph was taken immediately under a microscope. Bar represents 20 μm.

Figure 4

Southern-blot analysis of StSN2 gene. Potato genomic DNA (10 μg) was digested with the EcoRI (E), HindIII (H), BamHI (B), or XhoI (X) restriction endonucleases. The StSN2 cDNA fragment obtained by 5′-RACE (nucleotides 170–616) was [³²P] labeled and used as probe.

Figure 5

Expression of StSN2 gene in potato. Nothern-blot analysis of total RNAs (5 μg) extracted from the indicated parts of the potato plant. Blot was hybridized with the StSN2 probe and an StSN1 probe (Segura et al., 1999). Equal loading was confirmed by rehybridization of the blot with a potato 18S-ribosomal RNA probe.

Figure 6

Expression of StSN2 gene is induced by ABA. Nothern-blot analysis of total RNAs (7.5 μg) extracted from leaves and stems of mock-treated plants (M), or plants treated with 100 μm ABA or 50 μm GA₃. Blot was hybridized with the StSN2 probe, and a potato defensin StPTH1 probe (Moreno et al., 1994). RNA equal loading was confirmed by rehybridization of the blot with a potato 18S-ribosomal RNA probe. This is one of three experiments carried out that gave similar results.

Figure 7

Response of StSN2 gene to wounding and water-stress. A, Northern blot (7.5 μg per sample) of total RNAs extracted from mechanically damaged leaves (WL) and upper systemic non-damaged leaves (WS) from wounded plants, and from same-age leaves of control, non-wounded plants (CL and CS). Blot was hybridized with the StSN2 probe and with a potato StPIN2 probe (Peña-Cortés et al., 1989). B, Northern-blot analysis of total RNAs (7.5 μg) extracted from leaves of excised plants mock treated (M) or incubated with salt (250 mm NaCl; S), intact control plants (C), and plants that were remove from soil and were left to dry (drought treatment; D). Blot was hybridized with the StSN2 probe, and a probe of the potato CI7 gene (Kirch et al., 1997). RNA equal loading was confirmed by rehybridization of the blot with a potato 18S-ribosomal RNA probe. This is one representative experiment of the two carried out that gave similar results.

Figure 8

Response of StSN2 gene to infection of potato tubers with pathogens. Northern blot (7.5 μg per sample) of total RNAs extracted from tubers mock inoculated with 10 mm MgCl₂ (M), infected with bacterial suspensions (50 μL of 10⁷ cfu mL⁻¹) of R. solanacearum (Rs) or E. chrysanthemi (Ec), or inoculated with a spore supension (50 μL of 2 × 10⁵ spores mL⁻¹) of the fungus B. cinerea (Bc). Blot was hybridized with the StSN2 probe and RNA equal loading was confirmed by rehybridization of the blot with a potato 18S-ribosomal RNA probe. This is one of two experiments carried out that gave similar results.