Agroinoculation and Agroinfiltration: Simple Tools for Complex Gene Function Analyses

  • Zarir Vaghchhipawala
  • Clemencia M. Rojas
  • Muthappa Senthil-Kumar
  • Kirankumar S. Mysore
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 678)

Abstract

Agroinoculation, first developed as a simple tool to study plant–virus interactions, is a popular method of choice for functional gene analysis of viral genomes. With the explosive growth of genomic information and the development of advanced vectors to dissect plant gene function, this reliable method of viral gene delivery in plants, has been recruited and morphed into a technique popularly known as agroinfiltration. This technique was developed to examine the effects of transient gene expression, with applications ranging from studies of plant–pathogen interactions, abiotic stresses, a variety of transient expression assays to study protein localization, and protein–protein interactions. We present a brief overview of literature which document both these applications, and then provide simple agroinoculation and agroinfiltration methods being used in our laboratory for functional gene analysis, as well as for fast-forward and reverse genetic screens using virus-induced gene silencing (VIGS).

Key words

Agroinoculation Agroinfiltration Plant–pathogen interactions Virus-induced gene silencing Abiotic stress Tobacco Rattle Virus vector 

References

  1. 1.
    Grimsley, N., Hohn, B., Hohn, T., and Walden, R. (1986) “Agroinfection,” an alternative route for viral infection of plants by using the Ti plasmid Proc Natl Acad Sci USA 83, 3282–86.PubMedCrossRefGoogle Scholar
  2. 2.
    Zottini, M., Barizza, E., Costa, A., Formentin, E., Ruberti, C., Carimi, F., and Lo Schiavo, F. (2008) Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells Plant Cell Rep 27, 845–53.PubMedCrossRefGoogle Scholar
  3. 3.
    Jefferson, R. A., Kavanagh, T. A., and Bevan, M. W. (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants EMBO J 6, 3901–7.PubMedGoogle Scholar
  4. 4.
    Rossi, L., Escudero, J., Hohn, B., and Tinland, B. (1993) Efficient and sensitive assay for T-DNA-dependent transient gene expression Plant Mol Biol Rep 11, 220–29.CrossRefGoogle Scholar
  5. 5.
    Wroblewski, T., Tomczak, A., and Michelmore, R. (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis Plant Biotechnol J 3, 259–73.PubMedCrossRefGoogle Scholar
  6. 6.
    Johansen, L. K., and Carrington, J. C. (2001) Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system Plant Physiol 126, 930–38.PubMedCrossRefGoogle Scholar
  7. 7.
    Schob, H., Kunz, C., and Meins, F., Jr. (1997) Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing Mol Gen Genet 256, 581–5.PubMedCrossRefGoogle Scholar
  8. 8.
    Silhavy, D. (2005) in “Gene silencing by RNA interference: Technology and application” (Sohail, M., Ed.), pp. 357–63, CRC press, Oxford.Google Scholar
  9. 9.
    Chisholm, S. T., Coaker, G., Day, B., and Staskawicz, B. J. (2006) Host-microbe interactions: shaping the evolution of the plant immune response Cell 124, 803–14.PubMedCrossRefGoogle Scholar
  10. 10.
    Tai, T. H., Dahlbeck, D., Clark, E. T., Gajiwala, P., Pasion, R., Whalen, M. C., Stall, R. E., and Staskawicz, B. J. (1999) Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato Proc Natl Acad Sci USA 96, 14153–8.PubMedCrossRefGoogle Scholar
  11. 11.
    Bendahmane, A., Querci, M., Kanyuka, K., and Baulcombe, D. C. (2000) Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato Plant J 21, 73–81.PubMedCrossRefGoogle Scholar
  12. 12.
    Van der Hoorn, R. A. L., Laurent, F., Roth, R., and De Wit, P. J. G. M. (2000) Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis. Mol Plant-Microbe Interact 13, 439–46.PubMedCrossRefGoogle Scholar
  13. 13.
    Ron, M., and Avni, A. (2004) The receptor for the fungal elicitor ethylene-inducing xylanase is a member of a resistance-like gene family in tomato Plant Cell 16, 1604–15.PubMedCrossRefGoogle Scholar
  14. 14.
    Dinesh-Kumar, S. P., Anandalakshmi, R., Marathe, R., Schiff, M., and Liu, Y. (2003) in “Plant Functional Genomics: Methods and Protocols” (Grotewolk, E., Ed.), Vol. 236, pp. 287–93, Humana Press, Inc, Totowa.Google Scholar
  15. 15.
    Mysore, K. S., and Ryu, C. M. (2004) Nonhost resistance: how much do we know? Trends Plant Sci 9, 97–104.PubMedCrossRefGoogle Scholar
  16. 16.
    Burch-Smith, T. M., Anderson, J. C., Martin, G. B., and Dinesh-Kumar, S. P. (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants Plant J 39, 734–46.PubMedCrossRefGoogle Scholar
  17. 17.
    Grimsley, N., Hohn, T., Davies, J. W., and Hohn, B. (1987) Agrobacterium-mediated delivery of infectious maize streak virus into maize plants Nature 325, 177–79.CrossRefGoogle Scholar
  18. 18.
    Elmer, J. S., Sunter, G., Gardiner, W. E., Brand, L., Browning, C. K., Bisaro, D. M., and Rogers, S. G. (1988) Agrobacterium-mediated inoculation of plants with tomato golden mosaic virus DNAs Plant Mol Biol 10, 225–34.CrossRefGoogle Scholar
  19. 19.
    Sung, Y., and Coutts, R. (1995) Mutational analysis of potato yellow mosaic geminivirus J Gen Virol 76, 1773–80.PubMedCrossRefGoogle Scholar
  20. 20.
    Boulton, M. I., King, D. I., Markham, P. G., Pinner, M. S., and Davies, J. W. (1991) Host range and symptoms are determined by specific domains of the maize streak virus genome Virology 181, 312–18.PubMedCrossRefGoogle Scholar
  21. 21.
    Kheyr-Pour, A., Gronenborn, B., and Czosnek, H. (1994) Agroinoculation of tomato yellow leaf curl virus (TYLCV) overcomes the virus resistance of wild Lycopersicon species Plant Breed 112, 228–33.CrossRefGoogle Scholar
  22. 22.
    Ding, X. S., Liu, J., Chen, N. -H., Folimonov, A., Hou, Y. -M., Bao, Y., Katagi, C., Carter, S. A., and Nelson, R. S. (2004) The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing Mol Plant Microbe Interact 17, 583–92.PubMedCrossRefGoogle Scholar
  23. 23.
    Chen, S., Vaghchhipawala, Z., Li, W., Asard, H., and Dickman, M. B. (2004) Tomato phospholipid hydroperoxide glutathione peroxidase inhibits cell death induced by Bax and oxidative stresses in yeast and plants Plant Physiol 135, 1630–41.PubMedCrossRefGoogle Scholar
  24. 24.
    Yang, Y., Li, R., and Qi, M. (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves Plant J 22, 543–51.PubMedCrossRefGoogle Scholar
  25. 25.
    Senthil-Kumar, M., Rame Gowda, H. V., Hema, R., Mysore, K. S., and Udayakumar, M. (2008) Virus-induced gene silencing and its application in characterizing genes involved in water-deficit-stress tolerance J Plant Physiol 165, 1404–21.PubMedCrossRefGoogle Scholar
  26. 26.
    Anand, A., Vaghchhipawala, Z., Ryu, C. M., Kang, L., Wang, K., del-Pozo, O., Martin, G. B., and Mysore, K. S. (2007) Identification and characterization of plant genes involved in Agrobacterium-mediated plant transformation by virus-induced gene silencing, Mol Plant Microbe Interact 20, 41–52.PubMedCrossRefGoogle Scholar
  27. 27.
    Sheludko, Y. V. (2008) Agrobacterium-mediated transient expression as an approach to production of recombinant proteins in plants Recent Pat Biotechnol 2, 198–208.PubMedCrossRefGoogle Scholar
  28. 28.
    Jia, H., Pang, Y., Chen, X., and Fang, R. (2006) Removal of the selectable marker gene from transgenic tobacco plants by expression of Cre recombinase from a tobacco mosaic virus vector through agroinfection Transgenic Res 15, 375–84.PubMedCrossRefGoogle Scholar
  29. 29.
    Anand, A., Krichevsky, A., Schornack, S., Lahaye, T., Tzfira, T., Tang, Y., Citovsky, V., and Mysore, K. S. (2007) Arabidopsis VIRE2 INTERACTING PROTEIN2 is required for Agrobacterium T-DNA integration in plants Plant Cell 19, 1695–708.PubMedCrossRefGoogle Scholar
  30. 30.
    Frederick, R. D., Thilmony, R. L., Sessa, G., and Martin, G. B. (1998) Recognition specificity for the bacterial avirulence protein AvrPto is determined by Thr-204 in the activation loop of the tomato Pto kinase Mol Cell 2, 241–5.PubMedCrossRefGoogle Scholar
  31. 31.
    Ryu, C. M., Anand, A., Kang, L., and Mysore, K. S. (2004) Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species Plant J 40, 322–31.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Zarir Vaghchhipawala
    • 1
  • Clemencia M. Rojas
    • 1
  • Muthappa Senthil-Kumar
    • 1
  • Kirankumar S. Mysore
    • 1
  1. 1.Plant Biology DivisionThe Samuel Roberts Noble FoundationArdmoreUSA

Personalised recommendations