The Intracellular Citrus Huanglongbing Bacterium, 'Candidatus Liberibacter asiaticus' Encodes Two Novel Autotransporters

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 redesignated them as LasA(I) and LasA(II) in lieu of the previous names Hyv(I) and Hyv(II). As a phloem-limited, intracellular bacterial pathogen, Las has a significantly reduced genome and causes huanglongbing (HLB), a devastating disease of citrus worldwide. Bioinformatic analyses revealed that LasA(I) and LasA(II) share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasA(I) C-terminus and the full length LasA(II) were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of a typical signal peptide, LasA(I) was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasA(I) protein. Its surface localization was also confirmed by the removal of the LasA(I) antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasA(I) translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria when expressed in the plants. These findings may lead to a better understanding of the pathogenesis of this intracellular bacterium.