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Title:   Author  item
                                            Hao, Guixiaitem
                                            BOYLE, MICHAEL - Smithsonian Tropical Researchitem
                                            ZHOU, LIJUAN - University Of Floridaitem
                                            Duan, Ping
  Submitted to: PLOS ONE
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 6/4/2013
                                    Publication  Date: 7/11/2013
                                    Citation: Hao, G., Boyle, M., Zhou, L., Duan, Y. 2013. The intracellular citrus huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ encodes two novel autotransporters. PLoS ONE 8(7):e68921. doi:10.1371/journal/pone.0068921.Interpretive Summary: Citrus huanglongbing (HLB), or citrus greening has severely affected citrus industry worldwide. Although citrus HLB is known to be caused by three species of ‘Candidatus Liberibacter’, including ‘Ca. L. asiaticus’ (Las), little is known about molecular mechanism of the host-pathogen interactions. In this study, we discovered two novel Las autotransporter proteins that belong to type V secretion system. We proved that these proteins localized at the surface of the bacteria when expressed in Escherichia coli and targeted to mitochondria in plant cells. The characterization of these autotransporters will lead to better understanding how the bacterial pathogen cause citrus HLB and how can we control citrus HLB by blocking the functions of these autotransporter proteins.
                                Technical Abstract: Proteins secreted by the type V secretion system (T5SS), known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. In this study, we characterized two novel autotransporter proteins of ‘Candidatus Liberibacter asiaticus’ (Las), and designated them as LasAI and LasAII in replacement of the previous name HyvI and HyvII.  As a phloem-limited, intracellular bacterial pathogen, Las has a significantly reduced genome but causes Huanglongbing, one of the most devastating diseases of citrus worldwide. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a C-terminal translocator domain. When fused to the GFP gene and expressed in Escherichia coli, the LasAI C-terminus and full length LasAII were localized to the bacterial poles, similar to members of autotransporter family from other bacteria. Despite the absence of signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by removal of the LasAI antigen using protease treatment of the intact bacterial cells. When transiently expressed in tobacco leaves, along with a co-inoculation of a P19 gene-silencing-suppressor, the GFP-LasAI translocator targeted to mitochondria. This is the first report that Las encodes unique autotransporters that target to mitochondria. These findings may lead to a better understanding of the pathogenesis of this intracellular “energy parasitic” bacterium.


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