Agroinfiltration

Agroinfiltration is a method in plant biology to induce transient expression of genes in a plant or to produce a desired protein. In the method a suspension of Agrobacterium tumefaciens is injected into a plant leaf, where it transfers the desired gene to plant cells. The benefit of agroinfiltration when compared to traditional plant transformation is speed and convenience.

First step of the protocol is to introduce a gene of interest to a strain of Agrobacterium. Subsequently the strain is grown in a liquid culture and the resulting bacteria are washed and suspended into a suitable buffer solution. This solution is then placed in a syringe (without a needle). The tip of the syringe is pressed against the underside of a leaf while simultaneously applying gentle counterpressure to the other side of the leaf. The Agrobacterium solution is then injected into the airspaces inside the leaf through stomata, or sometimes through a tiny incision made to the underside of the leaf.

Vacuum infiltration is another way to penetrate Agrobacterium deep into plant tissue. In this procedure, leaf disks, leaves, or whole plants are submerged in a beaker containing the solution, and the beaker is placed in a vacuum chamber. The vacuum is then applied, forcing air out of the stomata. When the vacuum is released, the pressure difference forces solution through the stomata and into the mesophyll.

Once inside the leaf the Agrobacterium remains in the intercellular space and transfers the gene of interest in high copy numbers into the plant cells. The gene is then transiently expressed (no selection for stable integration is performed). The plant can be monitored for a possible effect in the phenotype, subjected to experimental conditions or harvested and used for purification of the protein of interest. Many plant species can be processed using this method, but the most common ones are Nicotiana benthamiana and Nicotiana tabacum.

p19 in agroinfiltration

Agroinfiltration using a promoter::GUS construct in Nicothiana benthamiana with TBSV p19 (right leaf disc) and without TBSV p19 (left leaf disc).

It’s common to coinfiltrate the Agrobacterium carrying the construct of interest together with Agrobacterium carrying the p19 protein from the plant pathogen Tomato bushy stunt virus (TBSV). TBSV is a virus that was first discovered in 1935 in tomatoes and results in plants with stunted growth and deformed fruits. In order to defend itself against viruses (and other exogenous DNA and RNA) plants have developed a system of post-transcriptional gene silencing (PTGS) where small interfering RNAs are produced from double-stranded RNA in order to create a sequence specific degradation pathway that efficiently silence non-native genes.[1][2] Many plant viruses has developed mechanisms that counter the plants PTGS-systems by evolving proteins, such as TBSVs p19, that interfere with the PTGS-pathway at different levels.[3][4][5]

Although it is not clear exactly through which mechanism p19 works studies has shown that transiently expressed proteins in Nicotiana benthamiana leaves has a up to 50-fold higher yield when coinfiltrated with TBSV p19.[6][7] In other studies, p19 from Artichoke mottled crinckle virus (AMVC) has been shown to have a similar, although smaller, effect as TBSV p19.[8]

See also

References

    1. ↑ Hammond, Scott M.; Caudy, Amy A.; Hannon, Gregory J. "Post-transcriptional gene silencing by double-stranded RNA". Nature Reviews Genetics 2 (2): 110–119. doi:10.1038/35052556.
    2. ↑ Johansen, Lisa K.; Carrington, James C. (2001-07-01). "Silencing on the Spot. Induction and Suppression of RNA Silencing in the Agrobacterium-Mediated Transient Expression System". Plant Physiology 126 (3): 930–938. doi:10.1104/pp.126.3.930. ISSN 1532-2548. PMID 11457942.
    3. ↑ Anandalakshmi, Radhamani; Pruss, Gail J.; Ge, Xin; Marathe, Rajendra; Mallory, Allison C.; Smith, Trenton H.; Vance, Vicki B. (1998-10-27). "A viral suppressor of gene silencing in plants". Proceedings of the National Academy of Sciences 95 (22): 13079–13084. doi:10.1073/pnas.95.22.13079. ISSN 0027-8424. PMID 9789044.
    4. ↑ Kasschau, Kristin D.; Carrington, James C. (1998-11-13). "A Counterdefensive Strategy of Plant Viruses". Cell 95 (4): 461–470. doi:10.1016/S0092-8674(00)81614-1. ISSN 0092-8674. PMID 9827799.
    5. ↑ Voinnet, Olivier (2001-08-01). "RNA silencing as a plant immune system against viruses". Trends in Genetics 17 (8): 449–459. doi:10.1016/S0168-9525(01)02367-8. ISSN 0168-9525. PMID 11485817.
    6. ↑ Voinnet, Olivier; Rivas, Susana; Mestre, Pere; Baulcombe, David (2003-03-01). "Retracted: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus". The Plant Journal 33 (5): 949–956. doi:10.1046/j.1365-313X.2003.01676.x. ISSN 1365-313X.
    7. ↑ "Retraction: ‘An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus’". The Plant Journal 84 (4): 846–846. 2015-11-01. doi:10.1111/tpj.13066. ISSN 1365-313X.
    8. ↑ Lombardi, Raffaele; Circelli, Patrizia; Villani, Maria; Buriani, Giampaolo; Nardi, Luca; Coppola, Valentina; Bianco, Linda; Benvenuto, Eugenio; Donini, Marcello (2009-11-20). "High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus". BMC Biotechnology 9 (1). doi:10.1186/1472-6750-9-96. PMC 2785776. PMID 19930574.
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