Agroinfiltration

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Agroinfiltration is a method used in plant biology and especially lately in plant biotechnology to induce transient expression of genes in a plant, or isolated leaves from a plant, or even in cultures of plant cells, in order to produce a desired protein. In the method a suspension of Agrobacterium tumefaciens is introduced into a plant leaf by direct injection or by vacuum infiltration, or brought into association with plant cells immobilised on a porous support (plant cell packs), whereafter the bacteria transfer the desired gene into the plant cells via transfer of T-DNA. The main benefit of agroinfiltration when compared to the more traditional plant transformation is speed and convenience, although yields of the recombinant protein are generally also higher and more consistent.

The first step is to introduce a gene of interest to a strain of Agrobacterium tumefaciens. Subsequently the strain is grown in a liquid culture and the resulting bacteria are washed and suspended into a suitable buffer solution. For injection, 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 suspension 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 introduce 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 intercellular spaces within the leaves via the stomata. When the vacuum is released, the pressure difference forces the "Agrobacterium" suspension into the leaves through the stomata into the mesophyll tissue. This can result in nearly all of the cells in any given leaf being in contact with the bacteria.

Once inside the leaf the Agrobacterium remains in the intercellular space and transfers the gene of interest as part of the Ti plasmid-derived T-DNA in high copy numbers into the plant cells. The gene is then transiently expressed through RNA synthesis from appropriate promoter sequences in all transfected cells (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 less often, Nicotiana tabacum.

Transient expression in cultured plant cell packs is a new procedure, recently patented by the Fraunhofer Institute in Aachen, Germany.[1] For this technique, suspension cultured cells of tobacco (eg: NT1 or BY2 cell lines of Nicotiana tabacum) are immobilised by filtration onto a porous support to form a well-aerated cell pack, then incubated with recombinant Agrobacterium for a time to allow T-DNA transfer, before refiltration to remove excess bacteria and liquid. Incubation of the cell pack in a humid environment for time periods up to several days allows transient expression of protein. Secreted proteins can be washed out of the cell pack by application of buffer and further filtration.

Silencing suppressors in agroinfiltration[edit]

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

It’s quite common to coinfiltrate the Agrobacterium carrying the construct of interest together with another Agrobacterium carrying a silencing suppressor protein gene such as the one encoding the p19 protein from the plant pathogenic Tomato bushy stunt virus (TBSV), or the NSs protein [2] from Tomato spotted wilt virus (TSWV). TBSV was first discovered in 1935 in tomatoes and results in plants with stunted growth and deformed fruits. TSWV was discovered in tomatoes in Australia in 1915, and for many years was the only member of what is now known as genus Tospovirus, family Bunyaviridae.

In order to defend itself against viruses and other pathogens that introduce foreign nucleic acids into their cells,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.[3][4] Many plant viruses have developed mechanisms that counter the plants PTGS-systems by evolving proteins, such as p19 and NSs, that interfere with the PTGS-pathway at different levels.[5][6][7]

Although it is not clear exactly how p19 works to suppress RNA silencing, studies have shown that transiently expressed proteins in Nicotiana benthamiana leaves have a up to 50-fold higher yield when coinfiltrated with TBSV p19.[8][9]

TSWV and other tospovirus NSs proteins have been shown to be effective as suppressors of both local and systemic silencing,[10] and may be a useful alternative to p19 where the latter has been shown not to be effective. In other studies, p19 from Artichoke mottled crinckle virus (AMVC) has been shown to have a similar, although weaker, effect to TBSV p19.[11]

See also[edit]

References[edit]

  1. ^ http://www.google.com/patents/EP2623603A1?cl=en
  2. ^ Takeda, A; Sugiyama, K; Nagano, H; Mori, M; Kaido, M; Mise, K; Tsuda, S; Okuno, T (2002). "Identification of a novel RNA silencing suppressor, NSs protein of Tomato spotted wilt virus". FEBS Lett. 532: 75–9. doi:10.1016/s0014-5793(02)03632-3. PMID 12459466. 
  3. ^ Hammond, Scott M.; Caudy, Amy A.; Hannon, Gregory J. (Feb 2001). "Post-transcriptional gene silencing by double-stranded RNA". Nature Reviews Genetics. 2 (2): 110–119. doi:10.1038/35052556. PMID 11253050. 
  4. ^ 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. PMC 1540124Freely accessible. PMID 11457942. 
  5. ^ 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. PMC 23715Freely accessible. PMID 9789044. 
  6. ^ 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. 
  7. ^ 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. 
  8. ^ 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. 
  9. ^ "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. 
  10. ^ "Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing". PLOS ONE. 10: e0134517. doi:10.1371/journal.pone.0134517. 
  11. ^ 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): 96. doi:10.1186/1472-6750-9-96. PMC 2785776Freely accessible. PMID 19930574.