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.