Comparative phylogenomic analysis reveals evolutionary genomic changes and novel toxin families in endophyticLiberibacterpathogens
Authors: Tan Yongjun , Wang Cindy , Schneider Theresa , Li Huan , de Souza Robson Francisco , Tang Xueming , Hsieh Tzung-Fu , Wang Xu , Li Xu , Zhang Dapeng 
Published: June 2021

Liberibacterpathogens are the causative agents of several severe crop diseases worldwide, including citrus Huanglongbing and potato Zebra Chip. These bacteria are endophytic and non-culturable, which makes experimental approaches challenging and highlights the need for bioinformatic analysis in advancing our understanding aboutLiberibacterpathogenesis. Here, we performed an in-depth comparative phylogenomic analysis of theLiberibacterpathogens and their free-living, nonpathogenic, ancestral species, aiming to identify the major genomic changes and determinants associated with their evolutionary transitions in living habitats and pathogenicity. We found that prophage loci represent the most variable regions amongLiberibactergenomes. Using gene neighborhood analysis and phylogenetic classification, we systematically recovered, annotated, and classified all prophage loci into four types, including one previously unrecognized group. We showed that these prophages originated through independent gene transfers at different evolutionary stages ofLiberibacterand only the SC-type prophage was associated with the emergence of the pathogens. Using ortholog clustering, we vigorously identified two additional sets of genomic genes, which were either lost or gained in the ancestor of the pathogens. Consistent with the habitat change, the lost genes were enriched for biosynthesis of cellular building blocks. Importantly, among the gained genes, we uncovered several previously unrecognized toxins, including a novel class of polymorphic toxins, a YdjM phospholipase toxin, and a secreted EEP protein. Our results substantially extend the knowledge on the evolutionary events and potential determinants leading to the emergence of endophytic, pathogenicLiberibacterspecies and will facilitate the design of functional experiments and the development of new detection and blockage methods of these pathogens.

Importance

Liberibacterpathogens are associated with several severe crop diseases, including citrus Huanglongbing, the most destructive disease to the citrus industry. Currently, no effective cure or treatments are available, and no resistant citrus variety has been found. The fact that these obligate endophytic pathogens are not culturable has made it extremely challenging to experimentally uncover from the whole genome the genes/proteins important toLiberibacterpathogenesis. Further, earlier bioinformatics studies failed to identify the key genomic determinants, such as toxins and effector proteins, that underlie the pathogenicity of the bacteria. In this study, an in-depth comparative genomic analysis ofLiberibacterpathogens together with their ancestral non-pathogenic species identified the prophage loci and several novel toxins that are evolutionarily associated with the emergence of the pathogens. These results shed new lights on the disease mechanism ofLiberibacterpathogens and will facilitate the development of new detection and blockage methods targeting the toxins.