Assessment of Candidatus Liberibacter asiaticus in transgenic citrus trees generated with prophage genes

Abstract

HLB is the most destructive citrus disease worldwide, since there are no resistant commercial varieties, the disease causes a significant reduction in production and there are no curative methods. In Brazil, the disease is associated with Candidatus Liberibacter americanus (Lam) and Ca. L. asiaticus (Las), being Las cosmopolitan and Lam restricted to Brazil. The absence of commercial citrus varieties resistant to HLB, nor curative measures for the management of this disease, show the necessity of the search for new strategies for its management. A feasible approach to the disease control is the use of biotechnology to generate genetically modified plants resistant to the bacteria. For that, the production and validation of GM plants with resistance to these bacteria become a feasible alternative as a tactic to avoid the disease negative impacts and the maintenance of the citrus production worldwide. Bacteriocins are anti-bacterial proteins that specifically binding to receptors from the bacteria external membranes and consequently, kill the cells that lack proteins responsible for the immunity. In Las genome, two prophages are described: SC1 and SC2. In SC1 prophage is notorious the presence of a putative bacteriocin, similar to 'colicin la' (SC1_gp060). In the SC2 prophage occurs a gene (SC2_gp255) that codify a protein that express immunity to colicin. Our hypothesis is that citrus plants overexpressing colicin could reduce both bacteria infection in commercial citrus plants. Based on this information, the aim of this work is to evaluate the presence and Las and Lam titer for three Pera sweet orange events focused on prophage genes, specifically a Las colicin gene. These are events will be compared to non-transformed plants (same variety), allowing to determine if the transgene effectively can induce resistance against one or both bacteria. For that, the Sweet Orange GM events generated during the project (2016/01993-1) were propagated and challenged by Las and Lam, through grafting buds from infected source plants. in the present project will be assessed the presence of these bacterium on these events, and, with the confirmation of the presence of bacteria, the bacterial titer will be quantified. The presence and titer quantification will be performed by qPCR, based on its high specificity and quickness when compared to conventional PCR. Moreover, statistical analyses will be performed aiming to verify the reduction or absence of bacterial titer of one or both targets in the GM events related to control (same variety - non transformed). (AU)