Functional disruption of the NodT outer membrane protein of �Candidatus Liberibacter asiaticus� for rootstock-mediated resistance to citrus greening using a phloem-directed, single-chain antibody

Functional disruption of the NodT outer membrane protein of �Candidatus Liberibacter asiaticus� for rootstock-mediated resistance to citrus greening using a phloem-directed, single-chain antibody

Report Date: 08/03/2018
Project: 11-125-424   Year: 2018
Category: Horticultural & Management
Author: Tim McNellis
Sponsor: Citrus Research and Development Foundation

This project was based on the idea that blocking the function of the NodT outer membrane transporter of ‘Candidatus Liberibacter asiaticus’ (CLas) would block pathogenicity or survival of the bacterium within citrus plants. Single-chain, mini-antibodies (scFvs) recognizing a peptide corresponding to the major, predicted extracellular loop of CLas were isolated. The scFv with the strongest binding in a qualitative assay was selected and fused to the C-terminal end of the citrus Flowering Locus T (FT) protein as a gene fusion, encoding an FT-scFv protein. The antibody was fused to FT in order to promoter stability, mobility, and expression of the protein in the phloem. The FT-scFv coding region was placed under the control of the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter and introduced into ‘Duncan’ grapefruit (Citrus paradisi) using Agrobacterium-mediated transformation. Fifteen (15) independent transgenic lines were obtained, most of them expressing high levels of the FT-scFv protein, as determined by protein gel immunoblot analysis. Eight lines are maintained in Florida at the United States Horticultural Laboratory (USHRL) and seven lines are maintained at Penn State. Many of the FT-scFv lines have a precocious blooming phenotype, which could be useful for accelerated citrus breeding purposes. Prior attempts to overproduce FT in citrus have encountered problems with lack of plant survival, while FT-scFv plants survive and can produce fruit. All lines have been propagated vegetatively, and they continue to express FT-scFv after propagation. The HLB resistance or tolerance phenotype of the FT-scFv lines has not yet been tested, however. Graft-transmission of the FT-scFv protein has also not yet been tested. However, the materials need to accomplish these last two goals have been produced and this represents an opportunity for future analysis.


Your browser does not support pdfs, click here to download the file.