Abstract
The tobacco β-1,3-glucanase gene (GLU), alfalfa defensin gene alfAFP, and their bivalent gene GLU-AFP were introduced into tomato cv. Micro-Tom via Agrobacterium-mediated method. Transformants were obtained and confirmed by GUS histochemical staining, PCR, and Southern blotting. Northern blotting analysis with GLU and APF probes revealed a variation in the expression among these transformants at transcription level. One to three copies of the transgene were, respectively, integrated into the tomato nuclear genome. Performance test of resistance to Ralstonia solanacearum with T1 generation transgenic tomato lines showed that the transgenic lines exhibited higher resistance to the infected pathogens than nontransgenic plants, and the resistance levels were related to expression levels of the transgene, showing a dose effect. The transgenic tomato harboring GLU — AFP cassette showed the highest disease resistance, which suggested that the alfAFP and glucanase genes have synergistical effects on the resistance to R. solanacearum. Some independent lines with high disease resistance, low variability, and stable expression of transgenes could be selected for further studies and molecular breeding.
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Abbreviations
- AFP:
-
gene encoding antifungal peptide defensin
- AS:
-
acetosyringone
- CaMV35S:
-
cauliflower mosaic virus 35S promoter
- Cef:
-
cefotaxime
- GLU:
-
gene encoding β-1,3-glucanase
- GUS:
-
β-glucuronidase
- Km:
-
kanamycin
- PCR:
-
polymerase chain reaction
- Pnos :
-
nopaline synthase gene promoter
- Tnos :
-
nopaline synthase terminator
- ZT:
-
zeatin
References
Hayward, A.C., Biology and Epidemiology of Bacterial Wilt Caused by Pseudomonas solanacearum, Annu. Rev. Phytopathol., 1991, vol. 29, pp. 65–87.
Hoda, H.E.H., Mohamed, E.O., and Noha, M.S., Biological Control of Bacterial Spot of Tomato Caused by Xanthomonas campestris pv. vesicatoria by Rahnella aquatilis, Microsc. Res., 2005, vol. 21, pp. 4–10.
Deslandes, L., Olivier, J., Peeters, N., Feng, D.X., Khounlotham, M., Boucher, C., Somssich, I., Genin, S., and Marco, Y., Physical Interaction between RRS1-R, a Protein Conferring Resistance to Bacterial Wilt, and PopP2, a Type III Effector Targeted to the Plant Nucleus, Proc. Natl. Acad. Sci. USA, 2003, vol. 100, pp. 8024–8029.
Tian, C.E., Wang, Z.X., and Chen, T., Transfer of Antibacterial Peptide D Gene into Tomato and Identification of Transgenic Plants, Hereditas, 2000, vol. 22, pp. 86–89.
Sarowar, S., Kim, Y.J., Kim, E.N., Kim, K.D., Hwang, B.K., Islam, R., and Shin, J.S., Overexpression of a Pepper Basic Pathogenesis-Related Protein 1 Gene in Tobacco Plants Enhances Resistance to Heavy Metal and Pathogen Stresses, Plant Cell Rep., 2005, vol. 4, pp. 216–224.
Yanqing, W., Qian, Y., and Yukio, T., Nitric Oxide-Over-producing Transformants of Pseudomonas fluorescens with Enhanced Biocontrol of Tomato Bacterial Wilt, J. Gen. Plant Pathol., 2005, vol. 71, pp. 33–38.
Sujon, S., Eui, N.K., and Young, J.K., Overexpression of a Pepper Ascorbate Peroxidase-Like 1 Gene in Tobacco Plants Enhances Tolerance to Oxidative Stress and Pathogens, Plant Sci., 2005, vol. 169, pp. 55–63.
Krish, K.K., Kandasami, P., and Rangaraj, N., A High Throughput Functional Expression Assay System for a Defense Gene Conferring Transgenic Resistance on Rice against the Sheath Blight Pathogen, Rhizoctonia solani, Plant Sci., 2003, vol. 165, pp. 969–976.
Zhang, X.-H., Guo, D.-J., and Zhang, L.-M., The Research on the Expression of Rabbit Defensin (NP-1) Gene in Transgenic Tomato, Acta Gen. Sinica, 2000, vol. 27, pp. 953–958.
Gao, A.G., Hakimi, S.M., Mittanck, C.A., Wu, Y., Woerner, B.M., Stark, D.M., Shah, D.M., Liang, J., and Rommens, C.M., Fungal Pathogen Protection in Potato by Expression of a Plant Defensin Peptide, Nat. Biotechnol., 2000, vol. 18, pp. 1307–1310.
Quyang, B., Li, H.-Y., and Ye, Z.-B., Increased Resistance to Fusarium Wilt in Transgenic Tomato Expressing Bivalent Hydrolytic Enzymes, J. Plant Physiol. Mol. Biol., 2003, vol. 29, pp. 179–184.
Yuexia, W., Albert, P.K., and Joel, M.C., Co-Transfer and Expression of Chitinase, Glucanase and Bar Genes in Creeping Bent Grass for Conferring Fungal Disease Resistance, Plant Sci., 2003, vol. 165, pp. 497–506.
Mao, B.-Z., Li, D.-B., Li, Q., and He, Z.-H., Transgenic Rice with Double Defense Genes Exhibiting Resistance to Sheath Blight (Rhizoctonia solani Kuhn.), J. Plant Physiol. Mol. Biol., 2003, vol. 29, pp. 322–326.
Hideki, T., Ayano, S., Tsutomu, A., Syofi, R., Sumire, F., Mari, K., and Yasufumi, H., Catalog of Micro-Tom Tomato Responses to Common Fungal, Bacterial, and Viral Pathogens, J. Gen. Plant Pathol., 2005, vol. 71, pp. 8–22.
Naoki, Y., Taneaki, T., and Manabu, W., Expressed Sequence Tags from the Laboratory-Grown Miniature Tomato (Lycopersicon esculentum) Cultivar Microtom and Mining for Single Nucleotide Polymorphisms and Insertions/Deletions in Tomato Cultivars, Gene, 2005, vol. 4, pp. 1–8.
Tzfira, T., Jensen, C.S., Wang, W.X., Zuker, A., Vinocur, B., Altman, A., and Vainstein, A., Transgenic Populus tremul: A Step-by-Step Protocol for Its Agrobacterium Meditated Transformation, Plant Mol. Biol. Rep., 1997, vol. 15, pp. 219–235.
Chomczynski, N. and Sacchi, N., Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate Phenol-Chloroform Extraction, Anal. Biochem., 1987, vol. 161, pp. 156–159.
Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Springer Harbor: Cold Spring Harbor Lab., 1989.
Deslandes, L., Pileur, F., Liaubet, L., Camut, S., Can, C., Williams, K., Holub, E., Beynon, J., Arlat, M.L., and Marcol, Y., Genetic Characterization of RRS1, a Recessive Locus in Arabidopsis thaliana That Confers Resistance to the Bacterial Soilborne Pathogen Ralstonia solanacearum, Mol. Plant-Microbe Interact., 1998, vol. 11, pp. 659–667.
Nariyoshi, K., Hitoshi, K., and Takayuki, A.B.E., Control of Tomato Bacterial Wilt without Disinfections Using a New Functional Polymer That Captures Microbial Cells Alive on the Surface and Is Highly Biodegradable, BioSci. Biotechnol. Biochem., 2005, vol. 69, pp. 326–333.
Gelvin, S.B., Agrobacterium-Mediated Plant Transformation: The Biology behind the “Gene-Jocking” Tool, Microbiol. Mol. Biol. Rev., 2003, vol. 67, pp. 16–37.
Wen, Y.C., and Tim, M.I., Molecular Mechanism for Silencing Virally Transduced Genes Involves Histone Deacetylation and Chromatin Condensation, Proc. Natl. Acad. Sci. USA, 2000, vol. 97, pp. 377–382.
Covey, S.N. and Al-Kaff, N.S., Plant DNA Viruses and Gene Silencing, Plant Mol. Biol., 2000, vol. 43, pp. 307–322.
Sang, M.P., Jung, S., and Sung, J., Transgenic Watermelon Rootstock Resistant to CGMMV (Cucumber Green Mottle Mosaic Virus) Infection, Plant Cell Rep., 2005, vol. 24, pp. 350–356.
Allen, G., Spiker, S., and Thompson, W., Use of Matrix Attachment Regions (MARs) to Minimize Transgene Silencing, Plant Mol. Biol., 2000, vol. 43, pp. 241–256.
Fagard, M. and Vaucheret, H., Systemic Silencing Signal, Plant Mol. Biol., 2000, vol. 43, pp. 285–293.
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Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 5, pp. 756–763.
The text was submitted by the authors in English.
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Chen, S.C., Liu, A.R. & Zou, Z.R. Overexpression of glucanase gene and defensin gene in transgenic tomato enhances resistance to Ralstonia solanacearum . Russ J Plant Physiol 53, 671–677 (2006). https://doi.org/10.1134/S1021443706050116
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DOI: https://doi.org/10.1134/S1021443706050116