2013 Annual Report
1a.Objectives (from AD-416):
With previous funding from the FL Citrus Research and Development Foundation (CRDF), ARS' Molecular Plant Pathology Laboratory (MPPL) has developed a library of scFv antibody fragments that bind specific targets on the HLB pathogen that are believed to be exposed on the surface of the pathogen and therefore useful for novel ultrasensitive detection assays. The University of Florida has developed expertise in the use of antibodies to enhance visualization of bacteria for detection and characterization of host/pathogen interactions. The objectives of this proposal are to transfer recently developed scFv antibodies to commercial partners for the development of novel diagnostic tests, as well as to use them to study host/pathogen interactions at the ultrastructural level.
1b.Approach (from AD-416):
ARS' MPPL, Beltsville, will convert scFv genes from phagemid to plasmid inserts, ensure the sequence is free of unwanted stop codons and send both purified scFv and the genes encoding them to commercial partners. ARS MPPL, Beltsville, will also purify and transfer scFv to the University of Florida to develop serologically enhanced methods to detect and visualize ‘Ca. Liberibacter asiaticus’ in infected plants and insects. The University of Florida and ARS will both maintain HLB infected plants, and the University of Florida will maintain colonies of infected citrus psyllids. Agdia will carry out serological characterization of scFv received from ARS and test them in serological assays for detection of ‘Ca. Liberibacter asiaticus’.
The goal of the parent project ’Invasive Citrus Pathogens’ is to prevent the introduction or spread within the citrus industry of any of a number of graft-transmissable and invasive pathogens of citrus. In this project, funded by the Citrus Research and Development Foundation, we are working with special antibodies that bind to the pathogen that causes citrus greening disease, ‘Ca. Liberibacter asiaticus’. These antibodies have been developed and initially characterized with funding from a previous grant of CRDF and the assistance of a commercial partner. ‘Ca. Liberibacter asiaticus’ can’t be grown in laboratory culture, and so conventional antibodies, which are prepared by injecting a rabbit with relatively large amounts of purified bacteria grown in the laboratory, are not available. We have therefore used recombinant DNA technology to prepare a series of antibodies against ‘Ca. Liberibacter asiaticus’. Our antibodies are termed ‘single chain fragment variable’ (scFv) antibodies and are produced in a laboratory strain of a bacterial virus. This allows production of the scFv in the laboratory and the availability of the full genome sequence of the pathogen allows us to screen the scFv to find those that bind to interesting targets in the pathogen. We have in hand a ‘library’ of some 20 million scFv and have identified scFv with useful properties. In the past year we have developed a much improved protocol for the production of the recombinant scFv antibodies. We have improved the yield and reproducibility of the production system by about 4 fold, which will improve the efficiency of experimental work moving forward. We have also developed a detection assay for the pathogen in plant samples. This assay uses extracts of plants suspected of being infected with the pathogen in what is called a ‘dot blot’ format. We have also developed a technology in which the pathogen can be visualized directly in plant tissue, a technique called a ‘tissue print’. The ‘tissue prints’ are made by taking a section of a leaf or stem of a plant and pressing it on a special type of paper. The sap of the plant containing the pathogen is bound to the paper but is invisible. However the anatomical structure of the sample is preserved on the paper, just as a finger print preserves the anatomical structure of the surface of a person’s finger. A second commercial antibody is used to bind to our scFv, and the antibodies produce a red colored reaction product when the appropriate chemicals are added. When the tissue print is viewed with a microscope, the pathogen can be seen in relation to the anatomy of the plant. This visualization provides both direct detection of the pathogen and scientific information about the distribution within the plant. We continue to isolate more scFv that bind to different targets on or within ‘Ca. Liberibacter asiaticus’ and to resolve technical problems that remain in production system for the scFv.