TITLE:
“Immunomodulation of Plum pox virus infection by stable and transient expression of recombinant antibody against viral Nib replicase in Nicotiana benthamiana plants”

SUMMARY:
Plum pox virus (PPV) is member of Potyvirus genus, and causes sharka disease. Sharka had a serious agronomic and economical impact on stone fruit crops. The use of resistant varieties is the most long-term and definitive strategy in the fight against viral diseases in plants. Although, great efforts are spend looking for resistant cultivars or at least agronomically tolerant against viral infections, by classical methods or biotechnological technology.
In this context, the main objective of this PhD work would be to evaluate if the expression of recombinant antibodies specific of PPV proteins in Nicotiana benthamiana plants can interfere or immunomodulate viral infection, and if it could be use as a strategy to obtain resistance.
In this work, it has been use the recombinant antibody fragment scFv2A, that consist of variable regions from the light and heavy chains of the monoclonal antibody 2A- IVIA (mAb 2A-IVIA) sequentially cloned and joined by a linker peptide, so that they are expressed like an unique protein or single chain Fv fragment (scFv). ScFv2A as well as mAb 2A-IVIA recognize specifically the NIb replicase protein of PPV, that has RNA-dependent RNA-polymerase activity (RdRp). The NIb protein localise mainly in nucleus of infected plant cells, thought his replication activity takes place in membrane structures derived from the endoplasmic reticulum (RE) in the cytosol.
Nicotiana benthamiana plants were transformed with three versions of the scFv2A recombinant fragment addressed to different cell compartments. A cytosolic version (scFv2A), a nuclear version (NLSscFv2A) and a third one addressed to RE membranes but cytosol facing version (6K2scFv2A). Several transgenic lines were obtained: lines A, lines N and lines K, transformed respectively with the constructions scFv2A, NLSscFv2A and 6K2scFv2A. To assess the level of resistance of different transgenic lines independent challenge experiments, by mechanical inoculation or by inoculation with a recombinant PPV clones fused to GFP (PPV-GFP), were performed. The transgenic plants that expressed the scFv2A, 6K2scFv2A o NLSscFv2A fragments showed different levels of protection against PPV infection. In some of them partial protection was observed, the symptom appearance was delayed and the virus accumulation was lower that in nontransgenic plants. In some of them the infection was totally blocked, they didn’t shoe symptoms or virus accumulation during the period  assayed. Although none of transgenic lines assayed showed protection against PPV in all their plants, at least with the inoculum doses and inoculation methods used, it could be state that expression of diferent versions of scFv2A fragment in plants is able to interfer or immunomodulate the infection process blocking its normal development. In any case, the low level of accumulation of scFv2A fragments would restrict the protection effect.
Moreover, combining the transient expression of scFv2A fragments by agroinfiltration with the agroinoculation with the recombinant viral clone PPV-GFP, it has been established a quicker alternative methodology to evaluate the effectiveness of scFv fragments to interfere with viral infection. This methodology has been applied to quantitatively evaluate the effectiveness of the different scFv2A versions. This methodology could be used to select new scFv2A versions with improved stability or scFv fragments with new specificities that show the better effectiveness for the stable transformation, and in this way assure the result of immunomodulation strategy for the abstention of PPV resistance.