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Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp. michiganensis in transgenic tomato (Solanum lycopersicum)

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Abstract

Two tomato proteins were evaluated by over-expression in transgenic tomato for their ability to confer resistance to Clavibacter michiganensis subsp. michiganensis (Cmm). Snakin-2 (SN2) is a cysteine-rich peptide with broad-spectrum antimicrobial activity in vitro while extensin-like protein (ELP) is a major cell-wall hydroxyproline-rich glycoprotein linked with plant response to pathogen attack and wounding. Tomato plants, cultivar Mountain Fresh, were transformed via Agrobacterium tumefaciens harboring a binary vector for expression of the full-length SN2 gene or ELP cDNA under the regulation of the CaMV 35S promoter. Molecular characterization of PCR-positive putative T0 transgenic plants by Northern analysis revealed constitutive over-expression of SN2 and ELP mRNA. Junction fragment analysis by Southern blot showed that three of the four SN2 over-expressing T0 lines had single copies of complete T-DNAs while the other line had two complete T-DNA copies. All four ELP over-expressing T0 lines had a single copy T-DNA insertion. Semi-quantitative RT-PCR analysis of T1 plants revealed constitutive over-expression of SN2 and ELP. Transgenic lines that accumulated high levels of SN2 or ELP mRNA showed enhanced tolerance to Cmm resulting in a significant delay in the development of wilt symptoms and a reduction in the size of canker lesions compared to non-transformed control plants. Furthermore, in transgenic lines over-expressing SN2 or ELP bacterial populations were significantly lower (100–10,000-fold) than in non-transformed control plants. These results demonstrate that SN2 and ELP over-expression limits Cmm invasiveness suggesting potential in vivo antibacterial activity and possible biotechnological application for these two defense proteins.

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Acknowledgments

We thank Holly Lange and Kevin Conley for maintaining plants in the greenhouse. This research was supported by the New York State Agricultural Experiment Station and the United States-Israel Binational Agricultural Research and Development Fund (award No. IS-4047-07).

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  1. Vasudevan Balaji

    Present address: Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK, 73401, USA

Authors and Affiliations

  1. Department of Plant Pathology and Plant-Microbe Biology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY, 14456, USA

    Vasudevan Balaji & Christine D. Smart

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  1. Vasudevan Balaji
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  2. Christine D. Smart
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Correspondence to Christine D. Smart.

Additional information

The nucleotide sequence of the tomato snakin-2 gene reported in this paper is deposited in GenBank under the accession number HQ008860.

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Fig. S1

Kanamycin sensitivity assay to determine the lowest effective concentration that can inhibit callusing and shoot bud formation in cotyledon explants of tomato. A, Cotyledon explants were excised from aseptically grown 8-day-old seedlings and placed on 2Z medium (Van Eck et al. 2006) supplemented with 25, 50, 75, 100 and 125 mg/l kanamycin. Cotyledon explants placed on 2Z medium without kanamycin were included as a positive control. All treatments contained timentin at a concentration of 300 mg/l and 60 explants were analyzed for each treatment. B, Histogram showing the percentage of cotyledon explants (in a group of 60) that callused and formed shoot buds in the presence of increasing concentration of kanamycin. Data was scored after 18 days of culture (JPG 2.65 MB)

Fig. S2

The T-DNA region of binary vectors pGA482GSN2 (A) and pGA482GELP (B) used for tomato transformation. Diagrams are not to scale. The Cauliflower mosaic virus (CaMV) 35S promoter (P35S)-driven tomato snakin-2 (SN2) gene or extensin-like protein (ELP) encoding cDNA was placed between the right border (RB) and left border (LB) of the T-DNA. The binary vector contains the neomycin phosphotransferase (nptII) gene as the plant selection marker. Pnos, nopaline synthase promoter; Tnos, nopaline synthase terminator; T35S, CaMV 35S polyadenylation signal. Solid arrows below the nptII gene indicate annealing sites of the primer pair used in the polymerase chain reaction. Bold solid lines below the map represent the fragments used as probes in Southern blot analysis. The dotted lines indicate the sizes of internal T-DNA fragments generated by HindIII digestion. The XhoI and BamHI restriction sites used for Southern blot analysis to determine the LB T-DNA junction fragments are marked with an asterisk. The dotted arrows indicate the sizes of junction fragments generated by XhoI (A) or BamHI (B) digestion (JPG 469 KB)

Fig. S3

Screening putative transgenic plants by PCR. The PCR reaction was performed with primer sets specific for the nptII gene (see Table 1). Genomic DNA from putative kanamycin-resistant plants transformed with snakin-2 (SN2) gene (A) or extensin-like protein (ELP) cDNA (B) and non-transgenic plants (NT) were used as template. The 1 kb ladder was used as the size marker (M). S and E denote plants transformed with the SN2 gene or ELP cDNA, respectively. The size of the amplicon was 625 bp (JPG 516 KB)

Fig. S4

Scoring for kanamycin resistance in T1 seeds of transgenic tomato lines over-expressing SN2 and ELP genes. A, Two replicates of a kanamycin sensitivity assay to determine the effective lethal dose that would arrest rooting and growth of tomato seedlings. Seeds of non-transformed tomato plants were surface-sterilized and placed on half-strength MS medium containing 0, 50, 75 or 100 mg/l kanamycin. A total of 30 seedlings were assayed for each treatment. Photographs were taken 10 days after culture. B, Scoring of kanamycin-resistant T1 seedlings in the segregation population of SN2 over-expressing T0 tomato lines S6, S9 and S14. C, Screening for kanamycin resistance in the T1 progeny of ELP over-expressing lines E12, E22 and E24. Kanamycin at a concentration of 100 mg/l was used in the selective screen. Seeds from non-transformed (NT) plant were placed on half-strength MS medium with and without kanamycin and treated as negative and positive controls, respectively (panels B and C, left). The kanamycin based scoring assay was validated by PCR (panels B and C, bottom) with primers specific for the nptII gene (Table S1). Genomic DNA template isolated from representative T1 seedlings sensitive (S) and resistant (R) to kanamycin in the segregating population of SN2 over-expressing T0 lines S6, S9 and S14 (panel B, bottom) and ELP lines E12, E22 and E24 (panel C, bottom). Lane NT indicates non-transformed control. A 1 kb ladder was used the DNA size marker (M) (JPG 3.78 MB)

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Balaji, V., Smart, C.D. Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp. michiganensis in transgenic tomato (Solanum lycopersicum). Transgenic Res 21, 23–37 (2012). https://doi.org/10.1007/s11248-011-9506-x

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