Transformation of tobacco plants for single-chain antibody expression via apoplastic and symplasmic routes, and analysis of their susceptibility to stolbur phytoplasma infection

Phytoplasmas are sieve tube restricted, wall-less phytopathogenic bacteria belonging to the class Mollicutes that have resisted cultivation. In previous studies we have shown that single-chain Fv antibodies (scFv) directed against an immunodominant membrane protein of the culturable plant pathogenic mollicute Spiroplasma citri inhibit its growth in vitro. Based on these observations, we considered a plantibody-based strategy for controlling phytoplasmas in plants. Different genetic constructs, in which the anti-stolbur phytoplasma scFv 2A10 gene was placed either under the control of the constitutive CaMV 35S or the phloem-specific rice sucrose synthase (RSs1) promoters, were engineered for expressing antibodies in tobacco plants via the apoplastic (signal peptide) or the symplasmic (no signal peptide) route. Transformed plants were analyzed for scFv 2A10 protein expression and for their resistance to the stolbur disease both in greenhouse and field experiments. This is the first time that a plantibody strategy for pathogen resistance has been evaluated in field experiments. In the transformed plants, the apoplast-targeted scFv was functional and was produced at an average of 0.04% of soluble proteins. When the stolbur phytoplasma was graft-transmitted to tobacco plants producing secreted scFv, a short delay in symptom appearance and phytoplasma multiplication (2-weeks at maximum) was observed, together with a slight reduction in symptom severity. However, no significant resistance was observed when the phytoplasma was transmitted by its insect vector. When the single-chain molecule was targeted to the cytoplasm, the transcripts were detected with both promoters but the scFv 2A10 protein could not be detected, and the plants did not show any resistance to the disease.