Transient plant production of Salmonella Typhimurium diagnostic antibodies

Kopertekh, Lilya; Meyer, Torsten; Freyer, Cornelia; Hust, Michael

The transient expression exploiting Agrobacterium-mediated delivery of expression vectors has become the preferred plantbased platform due to its advantage in speed, yield of recombinant proteins and the reduced concern for transgene escape. Transient recombinant protein production depends on both the expression vector and the plant host. Currently two types of expression vectors, non-viral or plant virus-based, can be used for transient protein production. Expression vectors utilizing a plastocyanin promoter [37] and the 5 0 -and 3 0 -translated region of CPMV [38] are the most efficient non-viral systems described in the literature. The latest development in plant virus vectors is the vector deconstruction strategy. Deconstructed viral vectors based on TMV [18] , CPMV [39] , PVX [21] and BeYDV [40] provide high protein yield through a reduction to components essential for recombinant protein production. In comparison to a deleted PVX vector developed by Giritch et al. [21] , the PVX vector described in this work uses the fused TGB1 and coat protein subgenomic promoter for foreign gene expression and is completely deficient in cell-tocell and long distance movement. We have taken advantage of Agrobacterium-mediated delivery of an expression cassette controlled by the 35S promoter. Infiltration of N. benthamiana leaves with pLH-PVX-m and pLH-gb-PVX-m vectors carrying scFv and scFv-Fc formats of a TM43-E10 antibody resulted in accumulation of mRNA and proteins as was evidenced by qPCR, Western blot analysis and ELISA. One of the factors reducing the efficiency of transient expression is mRNA degradation by RNA silencing. The gene silencing is involved in regulating expression of endogenous genes as well as reducing or eliminating the effects of invading pathogens such as viruses [41, 42] . To neutralize this silencing response and enhance the level of foreign gene expression, co-expression of proteins that are capable to interfere with components of this resistance is used routinely. These factors are known as "suppressors" of RNA silencing. So far, several suppressors of RNA silencing have been evaluated for their ability to counteract gene silencing and enhance foreign gene expression in transient assay, including p19 from either Artichoke mottled crinkle virus (AMCV) [43] or Tomato bushy stunt virus (TBSV) [44] , HcPro from Potato virus Y (PVY) [37] , p25 from PVX [45] and gb from Barley stripe mosaic virus (BSMV) [46] . We investigated the influence of the gb protein from Poa semilatent virus (PSLV) on foreign protein accumulation in a transient assay. PSLV is a plus-strand RNA virus of the genus Hordeivirus with a tripartite genome consisting of RNAα, RNAβ and RNAg. RNAα encodes a component of viral replicase. RNA β encodes coat protein and three movement proteins. RNAg is bicistronic and encode the other component of viral replicase and a non-structural gb protein [47, 48] . gb contributes to symptom severity, systemic viral movement and RNA silencing suppression [33, 49] . Although the anti-silencing activity of PSLV gb protein has been shown a decade ago [33] , the mechanism of gb action has not been investigated. Several lines of evidence indicate that silencing suppressor properties of gb may be mediated by its RNA binding activities. The gb protein likely binds in a sequence unspecific manner to ssRNA via a coiled-coil domain with zinc binding sites, to prevent RNA degradation by RNA-induced silencing complex [33, 50] . In thi