Abstract
The biolistic transient gene expression assay is a beneficial tool for studying gene function in vivo. However, biolistic transient assay systems have inherent pitfalls that often cause experimental inaccuracies such as poor transformation efficiency, which can be confused with biological phenomena. The double-barreled gene gun device is an inexpensive and highly effective attachment that enables statistically significant data to be obtained with one-tenth the number of experimental replicates compared to conventional biolistic assays. The principle behind the attachment is to perform two simultaneous bombardments with control and test DNA preparations onto the same leaf. The control bombardment measures the efficiency of the transformation while the ratio of the test bombardment to the control bombardment measures the activity of the gene of interest. With care, the ratio between the pair of bombardments can be highly reproducible from bombardment to bombardment. The double-barreled attachment has been used to study plant resistance (R) gene-mediated responses to effectors, induction and suppression of cell death by a wide variety of pathogen and host molecules, and the role of oömycete effector RXLR motifs in cell reentry.
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Klein, T. et al. (1992). Transformation of microbes, plants, and animals by particle bombardment. Nature Biotechnology, 10, 286–291.
Liang, G.H. and Skinner, D.Z. (2004). Genetically modified crops: their development, uses, and risks. Haworth Press, Binghamton, New York.
Barry, M.A. and Johnston, S.A. (1997). Biological features of genetic immunization. Vaccine, 15, 788–791.
Daniel, H. et al. (1990). Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors. Proceedings of the National Academy of Sciences of the United States of America, 87, 88–92.
Remacle, C. et al. (2005). High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes. Proceedings of the National Academy of Sciences of the United States of America, 103, 4771–4776.
Qutob, D., Kamoun, S., and Gijzen, M. (2002). Expression of a Phytophthora sojae necrosis-inducing protein occurs during transition from biotrophy to necrotrophy. Plant Journal, 32, 361–373.
Dou, D., Kale, S.D. et al. (2008). Conserved C-Terminal motifs required for avirulence and suppression of cell death by Phytophthora sojae effector Avr1b. The Plant Cell, 20, 1118–1133.
Dou, D., Kale, S.D. et al. (2008). RXLR-mediated entry of Phytophthora sojae effector Avr1b into soybean cells does not require pathogen-encoded machinery. The Plant Cell, 20, 1930–1947.
Jefferson, R.A. (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Molecular Biology Reporter 5, 387–405.
Sanford, J.C. et al. (1991). An improved, helium-driven biolistic device. Technique, 3, 3–16.
Sokal, R.R. and Rohlf, F.J. (1969) Biometry. W.H. Freeman and Company, San Francisco.
Sokal, R.R. and Rohlf, F.J. (1969) Statistical tables. W.H. Freeman and Company, San Francisco.
Benjamini, Y. and Hochberg, Y. (1995). Controlling the false discovery rate – a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society. Series B, 57, 289–300.
Benjamini, Y. and Yekutieli, D. (2001). The control of the false discovery rate in multiple testing under dependency. Annals of Statistics, 29, 1165–1188.
Gijzen, M. et al. (1996) Temperature induced susceptibility to Phytophthora sojae in soybean isolines carrying different Rps genes. Physiological and Molecular Plant Pathology, 48, 209–215.
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Kale, S.D., Tyler, B.M. (2011). Assaying Effector Function in Planta Using Double-Barreled Particle Bombardment. In: McDowell, J. (eds) Plant Immunity. Methods in Molecular Biology, vol 712. Humana Press. https://doi.org/10.1007/978-1-61737-998-7_13
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DOI: https://doi.org/10.1007/978-1-61737-998-7_13
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