Transgenic citrus expressing the antimicrobial gene Attacin E (attE) reduces the susceptibility of ‘Duncan’ grapefruit to the citrus scab caused by Elsinoë fawcettii
- 353 Downloads
Citrus scab, caused by Elsinoë fawcettii (anamorph Sphaceloma fawcettii), is a common foliar fungal disease affecting many citrus cultivars, including grapefruit. No commercial grapefruit cultivar is resistant to scab, and the disease results in severely blemished fruit which reduces its marketability. Transgenic ‘Duncan’ grapefruit trees expressing the antimicrobial attE gene were produced via Agrobacterium-mediated transformation. In in vitro leaf and greenhouse assays, several transgenic-lines had significantly lower susceptibility to E. fawcettii compared to the non-transformed control (P < 0.0001). In the greenhouse studies, sporulation on all transgenic lines except 1 was significantly reduced (P < 0.0001) but the level of sporulation over time did not correspond to disease severity ratings. Lesion size was also significantly reduced on transgenic lines compared to the non-transformed control (P < 0.0001) and the least susceptible line A-23 had the smallest lesions, but in general there was no correlation between lesion size and disease susceptibility. The level of attE mRNA was inversely related to the number of copies detected by Southern blot. The least susceptible line had a single inserted copy of the attE transgene whereas more susceptible lines had multiple copies. Since the attacin mode of action was thought to be specific to Gram-negative bacteria, it was unexpected to find that there was a significant activity against E. fawcettii.
KeywordsCitrus paradisi Agrobacterium-mediated transformation Antimicrobial peptide
The authors would like to thank Dr. Herb Aldwinckle, NYSAES Cornell University, USA for providing us with the pCa2Att/121 clone and Drs. Dennis Gray and Zhijian Li, MREC, University of Florida, USA for providing us with a binary vector containing the bifunctional nptII/egfp fusion gene.
- Bitancourt, A. A., & Jenkins, A. E. (1936). Elsinoë fawcettii, the perfect stage of citrus scab fungus. Phytopathology, 26, 393–396.Google Scholar
- Broekaert, W. F., Cammue, B. P. A., De Bolle, M. F. C., Thevissen, K., De Samblanx, G. W., & Osborn, R. W. (1997). Antimicrobial peptides from plants. Critical Review of Plant Science, 16, 297–323.Google Scholar
- Brunner, E., Domhof, S., & Langer, F. (2002). Nonparametric analysis of longitudinal data in factorial experiments. New York: John Wiley & Sons.Google Scholar
- Cardoso, S. C., Mendes, J. M. B., Camargo, R. L. B., Christiano, R. S. C., Filho, A. B., Vieira, M. L. C., et al. (2010). Transgenic sweet orange (Citrus sinensis L. Osbeck) expressing the attacin A gene for resistance to Xanthomonas citri subsp. citri. Plant Molecular and Biological Report, 28, 185–192.CrossRefGoogle Scholar
- Carlsson, A., Nystrom, T., de Cock, H., & Bennich, H. (1998). Attacin - an insect immune protein - binds LPS and triggers the specific inhibition of bacterial outer-membrane protein synthesis. Microbiology-Society for General Microbiology, 144, 2179–2188.Google Scholar
- Carlsson, A., Engstrom, P., Palva, E. T., & Bennich, H. (1991). Attacin, an antibacterial protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins in Escherichia coli by interfering with omp gene transcription. Infection and Immunity, 59, 3040–3045.Google Scholar
- DeLucca, A. J., Bland, J. M., Jacks, T. J., Grimm, C., Cleveland, T. E., & Walsh, T. J. (1997). Fungicidal activity of cecropin A. Antimicrobial Agents and Chemotherapy, 4, 481–483.Google Scholar
- Dewdney, M. M., & Timmer, L. W. (2010) Citrus scab. In M. E. Rogers, M. M. Dewdney, & T. M. Spann (Eds), 2011 Florida Citrus Pest Management Guide: University of Florida, IFAS. pp. 3. http://edis.ifas.ufl.edu/cg020).
- Dutt, M., Orbovic, V., & Grosser, J. W. (2009). Cultivar dependent gene transfer into citrus using Agrobacterium. Proceedings of the Florida State Horticultural Society, 122, 85–89.Google Scholar
- Ieki, H. (1981). Resistance of Citrus to scab. International Citrus Congress (4th : 1981 : Tokyo, Japan) International Society of Citriculture, 1, 340–344.Google Scholar
- Ko, K., Norelli, J. L., Reynoird, J.-P., Boresjza-Wysocka, E., Brown, S. K., & Aldwinckle, H. S. (2000). Effect of untranslated leader sequence of AMV RNA4 and signal peptide of pathogenesis-related protein 1b on attacin gene expression, and resistance to fire blight in transgenic apple. Biotechnology Letters, 22, 373–381.CrossRefGoogle Scholar
- Norelli, J. L., Borejsza-Wysocka, E., Reynoird, J.-P., & Aldwinckle, H. S. (2000). Transgenic ‘Royal Gala’ apple expressing Attacin E has increased field resistance to Erwinia amylovora (fire blight). Acta Hortculturae, 538, 631–633.Google Scholar
- Reddy, M. S., Dinkins, R. D., & Collins, G. B. (2003). Gene silencing in transgenic soybean plants transformed via particle bombardment. Plant Cell Report, 21, 676–683.Google Scholar
- Timmer, L. W. (2000). Scab diseases. In L. W. Timmer, S. M. Garnsey, & J. H. Graham (Eds.), Compendium of citrus diseases (2nd ed., pp. 31–32). St. Paul: American Phytopathological Society Press.Google Scholar
- Timmer, L. W., & Zitko, S. E. (1993) Techniques for greenhouse evaluation of fungicides for control of citrus scab. In E. Rabe (Ed.), Proceedings of the IV Congress of the International Society of Citrus Nurserymen (pp. 125–129). Johannesburg: The South African Citrus Nurserymen’s Association.Google Scholar
- Timmer, L. W., Roberts, P. D., Chung, K. R., & Bhatia, A. (2001). Citrus scab (PP153): University of Florida Institute of Food and Agricultural Sciences. http://edis.ifas.ufl.edu/ch014.
- Timmer, L. W., Mondal, S. N., Peres, N. A. R., & Bhatia, A. (2004). Fungal diseases of fruit and foliage of citrus trees. In S. A. M. H. Naqvi (Ed.), Diseases of Fruits and Vegetables – Diagnosis and Management, vol. 1, (pp. 191–227). Dordrecht: Kluwer Academic Publishers.Google Scholar