bioRxiv preprint doi: https://doi.org/10.1101/2021.06.02.446850; this version posted June 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Comparative phylogenomic analysis reveals evolutionary genomic 2 changes and novel toxin families in endophytic Liberibacter 3 pathogens 4 5 Yongjun Tan1, Cindy Wang1, Theresa Schneider1, Huan Li1, Robson Francisco de Souza2, 6 Xueming Tang3, Tzung-Fu Hsieh4,5, Xu Wang6,7,8, Xu Li4,5, and Dapeng Zhang1,9,* 7 8 1Department of Biology, College of Arts & Sciences, Saint Louis University, St. Louis, MO 63103 9 2Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 10 São Paulo 05508-900, Brazil 11 3School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China 12 4Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695 13 5Plants for Human Health Institute, North Carolina State University, Kannapolis, NC 28081 14 6Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 15 36849 16 7Alabama Agricultural Experiment Station, Auburn University, Auburn, AL 36849 17 8HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806 18 9Bioinformatics and Computational Biology Program, College of Arts & Sciences, Saint Louis 19 University, St. Louis, MO 63103 20 21 Correspondence and requests for materials should be addressed to D.Z. (email: 22 [email protected]) 23 24 Running title: Comparative Phylogenomics of Liberibacter Pathogens 25 Keywords: Liberibacter pathogens, huanglongbing, zebra chip, toxins, prophages, 26 pathogenesis, evolution, comparative genomics 27 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.02.446850; this version posted June 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 28 Abstract 29 Liberibacter pathogens are the causative agents of several severe crop diseases worldwide, 30 including citrus Huanglongbing and potato Zebra Chip. These bacteria are endophytic and non- 31 culturable, which makes experimental approaches challenging and highlights the need for 32 bioinformatic analysis in advancing our understanding about Liberibacter pathogenesis. Here, 33 we performed an in-depth comparative phylogenomic analysis of the Liberibacter pathogens 34 and their free-living, nonpathogenic, ancestral species, aiming to identify the major genomic 35 changes and determinants associated with their evolutionary transitions in living habitats and 36 pathogenicity. We found that prophage loci represent the most variable regions among 37 Liberibacter genomes. Using gene neighborhood analysis and phylogenetic classification, we 38 systematically recovered, annotated, and classified all prophage loci into four types, including 39 one previously unrecognized group. We showed that these prophages originated through 40 independent gene transfers at different evolutionary stages of Liberibacter and only the SC-type 41 prophage was associated with the emergence of the pathogens. Using ortholog clustering, we 42 vigorously identified two additional sets of genomic genes, which were either lost or gained in 43 the ancestor of the pathogens. Consistent with the habitat change, the lost genes were 44 enriched for biosynthesis of cellular building blocks. Importantly, among the gained genes, we 45 uncovered several previously unrecognized toxins, including a novel class of polymorphic toxins, 46 a YdjM phospholipase toxin, and a secreted EEP protein. Our results substantially extend the 47 knowledge on the evolutionary events and potential determinants leading to the emergence of 48 endophytic, pathogenic Liberibacter species and will facilitate the design of functional 49 experiments and the development of new detection and blockage methods of these pathogens. 50 51 Importance 52 Liberibacter pathogens are associated with several severe crop diseases, including citrus 53 Huanglongbing, the most destructive disease to the citrus industry. Currently, no effective cure 54 or treatments are available, and no resistant citrus variety has been found. The fact that these 55 obligate endophytic pathogens are not culturable has made it extremely challenging to 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.02.446850; this version posted June 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 56 experimentally uncover from the whole genome the genes/proteins important to Liberibacter 57 pathogenesis. Further, earlier bioinformatics studies failed to identify the key genomic 58 determinants, such as toxins and effector proteins, that underlie the pathogenicity of the 59 bacteria. In this study, an in-depth comparative genomic analysis of Liberibacter pathogens 60 together with their ancestral non-pathogenic species identified the prophage loci and several 61 novel toxins that are evolutionarily associated with the emergence of the pathogens. These 62 results shed new lights on the disease mechanism of Liberibacter pathogens and will facilitate 63 the development of new detection and blockage methods targeting the toxins. 64 65 66 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.02.446850; this version posted June 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 67 Introduction 68 Candidatus Liberibacter, a genus of Gram-negative bacteria in the order of Rhizobiales, 69 has recently received increasing attention due to the fact that several of its species are 70 associated with severe diseases on multiple crop plants, such as citrus, potato, and tomato. 71 These include three species, Candidatus Liberibacter asiaticus found in Asia and North America, 72 Candidatus Liberibacter africanus found in Africa 1, and Candidatus Liberibacter americanus 73 found in Brazil 2, that cause the citrus Huanglongbing (HLB) disease, and Candidatus 74 Liberibacter solanacearum, that causes similar diseases in tomato and potato, referred to as 75 Psyllid yellows and Zebra Chip (ZC), respectively 3,4. The HLB disease, also known as citrus 76 greening, is the most destructive, worldwide disease of citrus 5. It is characterized by yellowing 77 of citrus tree leaves, premature defoliation, decay of feeder rootlets and lateral roots, production 78 of small bitter fruit, and eventually death of the citrus tree 6. Thus, HLB has led to a substantial 79 loss of citrus production and severe damage to the economy and job market 7. Unfortunately, 80 there is currently no effective cure or treatments available, and no resistant citrus variety has 81 been found so far. In a similar vein, ZC is a new disease of potato that was first identified in 82 Mexico in 1994 8 and has quickly spread to many countries in recent years. It negatively affects 83 growth, yield, propagation potential, and qualities of tubers and thus impacts international trade 84 and the economy significantly 9. 85 Given the severity and rapid spread of these diseases in commercial crops, extensive 86 research efforts have been made to better understand the basic biology of these pathogens and 87 the pathogenesis mechanisms of the diseases, especially on HLB. However, the progress has 88 been slow due to two main obstacles. First, the Liberibacter pathogens of these diseases are 89 endophytic bacteria, whose transmissions are naturally vectored by several psyllids, such as 90 Asian citrus psyllid (Diaphorina citri) 10, African citrus psyllid (Trioza erytreae) 11, and Bactericera 91 cockerelli 9. Therefore, they are not culturable which makes it difficult for wet-lab researchers to 92 conduct functional studies. Second, secretion of toxins or effectors represents one of the most 93 important mechanisms of bacterial pathogenesis and virulence. These toxins either manipulate 94 host/vector immunity and physiology or damage the host cells 12. However, such molecules 95 involved in the HLB disease have remained elusive for years. Previous research has focused 96 primarily on secreted proteins in HLB-associated pathogens which carry T2SS specific signal 97 peptides 13. Thus far, the only proteins claimed to be the potential toxins behind the HLB 98 disease have been the short proteins carrying these signal peptides 14-17. However, these 99 proteins are mainly present in one strain of the C.L. asiaticus species, and not found in other 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.02.446850; this version posted June 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 100 C.L. asiaticus strains and other Liberibacter pathogens associated with HLB (C.L. americanus 101 and C.L. africanus)14,16-21, suggesting that other types of toxins