bioRxiv preprint doi: https://doi.org/10.1101/731166; this version posted August 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 CRISPR-Cas systems in the plant pathogen Xanthomonas spp. and their impact on 2 genome plasticity 3 Paula Maria Moreira Martins a*; Andre da Silva Xavier c*; Marco Aurelio Takita a 4 Poliane Alfemas-Zerbini b; Alessandra Alves de Souza a#. 5 *These authors contributed equally to this work 6 aCitrus Biotechnology Lab, Centro de Citricultura Sylvio Moreira, Instituto Agronômico 7 de Campinas, Cordeirópolis-SP, Brazil 8 bDepartament of Microbiology, Instituto de Biotecnologia Aplicada à Agropecuária 9 (BIOAGRO), Universidade Federal de Viçosa, Viçosa-MG, Brazil 10 cDepartament of Agronomy/NUDEMAFI, Universidade Federal do Espírito Santo, 11 Brazil. 12 13 Key words: Phage, plasmids, Xanthomonadaceae, Xylella. 14 Running title: CRISPR-Cas systems in Xanthomonas spp. 15 Abstract 16 Xanthomonas is one of the most important bacterial genera of plant pathogens 17 causing economic losses in crop production worldwide. Despite its importance, many 18 aspects of basic Xanthomonas biology remain unknown or understudied. Here, we 19 present the first genus-wide analysis of CRISPR-Cas in Xanthomonas and describe 20 specific aspects of its occurrence. Our results show that Xanthomonas genomes harbour 21 subtype I-C and I-F CRISPR-Cas systems and that species belonging to distantly 22 Xanthomonas-related genera in Xanthomonadaceae exhibit the same configuration of bioRxiv preprint doi: https://doi.org/10.1101/731166; this version posted August 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. 23 coexistence of the I-C and I-F CRISPR subtypes. Additionally, phylogenetic analysis 24 using Cas proteins indicated that the CRISPR systems present in Xanthomonas spp. are 25 the result of an ancient acquisition. Despite the close phylogeny of these systems, they 26 present significant variation in both the number and targets of spacers. An interesting 27 characteristic observed in this study was that the identified plasmid-targeting spacers 28 were always driven toward plasmids found in other Xanthomonas strains, indicating that 29 CRISPR-Cas systems could be very effective in coping with plasmidial infections. 30 Since many effectors are plasmid encoded, CRISPR-Cas might be driving specific 31 characteristics of plant-pathogen interactions. 32 33 Introduction 34 Phytopathogenic bacteria are a global threat to crop production worldwide. 35 Xanthomonas spp. is one of the most important genera of phytopathogens since these 36 species can infect at least 120 monocotyledonous and 260 dicotyledonous species of 37 economic importance (1,2). These pathogens are able to live both inside and outside of 38 plant hosts. Regardless of their lifestyle, bacteria are constantly exposed to many 39 different threats, such as the constant pressure in the form of exogenous DNA invasions 40 from both viruses and invading plasmids from other bacteria (3,4). Many basic aspects 41 of how these phytopathogens react and protect themselves from such threats remain 42 understudied. 43 Bacteriophages (or simply “phages”) are one of the most abundant entities 44 across the biosphere and one of the most potent pathogens of bacteria (5). Many aspects 45 of both bacterial and phage genomes have been shaped by this never-ending war, in 46 which both groups have had to develop defence and attack systems (6,7). In addition to bioRxiv preprint doi: https://doi.org/10.1101/731166; this version posted August 9, 2019. 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. 47 virus attack, plasmid invasions can also be deleterious to bacteria. The most urgent topic 48 concerning the negative effects of plasmidial invasions can be linked to the so-called 49 “metabolic burden” (4,8), consisting of physiological disturbance due to the presence of 50 exotic genetic material and its associated metabolism that drains important energetic 51 resources of the host cell, negatively impacting its fitness. 52 For every horizontal genetic transfer that takes place in a prokaryotic cell, 53 specific intra-cellular protection systems may come into play. Despite the fact that 54 genomic rearrangements can lead to positive outcomes, there must be a balance between 55 stability and tolerance of these events (3). Many biological systems have evolved to 56 protect the integrity of the genetic information of prokaryotes. One of the first types of 57 system ever discovered that eradicates exogenous DNA infections at their onset was 58 restriction-modification systems, which recognize self-DNA by its methylation pattern 59 and enzymatically destroy the invader DNA, thereby “restricting” its occurrence (9). 60 Other mechanisms include the extreme abortive infection system, which kills the 61 infected cell, preventing the phage from spreading throughout the bacterial population 62 (10). Curiously, systems that were designed to aid in the maintenance of infective 63 DNAs within cells have been co-opted for other functions. That is the case for the toxin- 64 antitoxin operons (TA), which were originally described as a postsegregational killing 65 system present in plasmids; infected cells that lose these invasive molecules will die, 66 which increases plasmid prevalence among a given bacterial population (11). Few TA 67 systems, such as the mazEF (12), hok/soc (13) and especially toxIN (14,15) systems, 68 have been reported to exclude phages, mainly through the induction of premature cell 69 death after phage invasion. 70 In the last decade, another bacterial defence system that has been in the spotlight 71 is the CRISPR-Cas system (16). There are three types of CRISPR-Cas systems (I, II and bioRxiv preprint doi: https://doi.org/10.1101/731166; this version posted August 9, 2019. 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. 72 III), each with many subtypes (17). These systems are basically characterized by the 73 genomic presence of a module of repetitive DNA interpolated by “spacer” sequences 74 consisting of previous invasive DNAs. During the occurrence of another invasion, these 75 spacers are used to positively identify exogenous DNA and oppose the infective 76 molecules (18). With the recent discovery of CRISPR-Cas as a defence mechanism in 77 bacteria, its presence and abundance have been the focus of studies in the genomes of 78 many prokaryotes, especially those of human-associated genera (19–22). However, in- 79 depth analyses are lacking for phytopathogens, even in economically important genera 80 such as the closely related taxa Xanthomonas and Xylella fastidiosa (23,24). In this 81 work, we performed a genome-wide investigation of CRISPR-Cas in both of these 82 phytopathogens, which cause diseases in different plant species, and showed that these 83 systems may be a driving force for genetic diversity, impacting pathogenicity and host- 84 range distribution. 85 86 Materials and Methods 87 Genome analysis 88 An in-depth analysis of both prophage and CRISPR arrays (and the 89 identification of putative protospacer targets when CRISPR was present) was carried 90 out in 10 Xanthomonas genomes that we previously selected (25). The complete list is 91 shown in Table 1. The Xylella fastidiosa strains analysed for CRISPR arrays are also 92 shown in Table 1, and subspecies were selected as phylogenetically representative 93 members of these species (26). We expanded the number of genomes analysed only for 94 the cas operon search to strengthen our conclusions about what subtypes of CRISPR- 95 Cas systems are present in the Xanthomonas and Xylella genera. Therefore, the total bioRxiv preprint doi: https://doi.org/10.1101/731166; this version posted August 9, 2019. 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. 96 numbers of genomes in this analysis were as follows: 121 Xanthomonas strains 97 spanning 27 different species/pathovars (Supplemental File S1), 20 Xylella strains of 98 four subspecies (Supplemental File 2S), and 7 other Xanthomonadaceae isolates 99 (Supplemental File S3). 100 101 Table 1: Selection of genomes used for CRISPR array searches in both Xanthomonas spp. and 102 Xylella fastidiosa ssp. genomes. (*) also used for prophage analysis. Xylella fastidiosa genomes Accession number X. f. subsp. fastidiosa Temecula NC_004556.1 X. f. subsp. pauca 9a5c NC_002488.3 X. f. subsp. fastidiosa M23 NC_010577.1 X. f. subsp. multiplex M12 NC_010513.1 X. f. subsp. fastidiosa GB514 NC_017562.1 X. f. subsp. fastidiosa MUL0034 NZ_CP006740.1 X. f. subsp. sandyi Ann-1 AAAM04000275.1 Xanthomonas genomes * X. axonopodis pv. citri 306 NC_003919.1 * X. axonopodis Xac29-1 NC_020800.1 * X. citri subsp. citri Aw12879 NC_020815.1 * X. campestris pv. vesicatoria 85-10 AM039952.1 * X. campestris pv. raphani 756C NC_017271.1 * X. campestris pv. campestris ATCC33913 NC_003902.1 * X. campestris pv. campestris 8004 NC_007086.1 * X. albilineans GPE PC73 NC_013722.1 * X. oryzae pv. oryzicola BLS256 NC_017267.2 * X. oryzae pv. oryzae PXO99A NC_010717.2 103 104 CRISPR arrays and putative protospacer target identification bioRxiv preprint doi: https://doi.org/10.1101/731166; this version posted August 9, 2019.