2013 Annual Report
1a.Objectives (from AD-416):
With previous funding from the Florida 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 essential for successful colonization of citrus. ARS' U.S. Horticultural Research Lab (USHRL) at Ft. Pierce has developed efficient methods for large scale transformation, regeneration and screening of citrus for resistance to HLB. The objective of this proposal is to transform citrus with genes encoding selected scFv and evaluate the transgenic citrus for resistance to HLB. This proposal relates to NP303, Plant Diseases, goal to 'develop plant disease resistance'. The ARS Cooperator has expertise in plant transformation.
1b.Approach (from AD-416):
ARS/MPPL, Beltsville, will convert scFv genes from phagemid to plasmid inserts, make appropriate modifications, and clone the modified scFv genes into vectors for plant transformation. ARS MPPL, Beltsville, will also lead the molecular characterization of transformed citrus. ARS USHRL Ft. Pierce will use these constructs to transform citrus, identify transformed citrus, produce and propagate transformed trees, and screen them for resistance to ‘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. The goal of the current project is to identify scFv that might inhibit the ability of ‘Ca. Liberibacter asiaticus’ to grow and survive in citrus and thereby prevent disease. These scFv will be engineered into a commercial citrus rootstock and tested to see if they can inhibit the development of citrus greening disease. We have isolated scFv against 3 different proteins produced by ‘Ca. Liberibacter asiaticus’ that we think may be required for survival inside the citrus plant. Our scFv recognize the outer exposed portion of a protein called TolC. This protein is a molecular pump that is used to excrete from the bacterial cell chemicals that are produced by the host in order to defend itself against the pathogen. If the pump is blocked by antibodies, the bacterium is expected to die. The second targeted protein is called InvA. This protein destroys a cellular signal that tells the citrus host cell to die in a planned way when it is infected by the bacterium. If the function of this bacterial protein were impaired by the scFv, the host citrus cell would die upon infection but the plant would survive. We also have scFv that bind to a protein called a pilus, which is required by the bacteria to stick to each other and the host cell in order to form colonies which promote survival. A visiting scientist has recently been hired to develop these ideas, and the next steps are to put the scFv genes into cloning vectors that will be used by a colleague at Fort Pierce, Florida to transform the citrus plants and test for effects.