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bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 genomics of syringae reveals convergent gene gain and loss 2 associated with specialisation onto cherry ( avium) 3 4 Michelle T. Hulin1,4, Andrew D. Armitage1, Joana G. Vicente2, Eric B. Holub2, Laura 5 Baxter2, Helen J. Bates1, John W. Mansfield3, Robert W. Jackson4 and Richard J. Harrison1,4 6 7 1. NIAB EMR, New Road, East Malling, ME19 6BJ 8 2. School of Life Sciences, University of Warwick, Warwick Crop Centre, 9 Wellesbourne, CV35 9EF 10 3. Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, UK 11 4. School of Biological Sciences, University of Reading, Reading, RG6 6AJ, U.K 12 13 Corresponding author – [email protected] 14 15 Running head – Comparative genomics of Pseudomonas from cherry 16 17 Word count: 6500 18 Introduction: 762 19 Methods: 1040 20 Results: 2958 21 Discussion: 1661 22 Acknowledgements: 79 23 24 Figures: 8 (all colour) 25 Tables: 4 26 Supplementary information includes 24 figures, 26 tables and supplementary methods

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27 Summary (200 words) 28 ● Genome-wide analyses of the effector- and toxin-encoding genes were used to 29 examine the phylogenetics and evolution of pathogenicity amongst diverse strains of 30 Pseudomonas syringae causing bacterial canker of cherry (Prunus avium) including 31 pathovars P.s pv. morsprunorum (Psm) races 1 and 2, P.s pv. syringae (Pss) and P.s 32 pv. avii. 33 ● Genome-based phylogenetic analyses revealed Psm races and P.s pv. avii clades were 34 distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were 35 interspersed amongst strains from other host species. 36 ● A maximum likelihood approach was used to predict effectors associated with host 37 specialisation on cherry. Pss possesses a smaller repertoire of III effectors but 38 has more toxin biosynthesis clusters compared with Psm and P.s pv. avii. Evolution 39 of cherry pathogenicity was correlated with gain of genes such as hopAR1 and 40 hopBB1 through putative phage transfer and horizontal transfer, respectively. By 41 contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry- 42 pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the 43 hypersensitive reaction in cherry leaves, confirming computational predictions. 44 ● Cherry canker provides a fascinating example of convergent evolution of 45 pathogenicity that is explained by the mix of effector and toxin repertoires acting on a 46 common host. 47 48 49 Key words- avirulence, bacterial canker, comparative genomics, host specialisation, 50 prediction, Pseudomonas, toxins, type III effectors 51 52 53 54 55 56 57 58 59 60

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61 62 Introduction

63 Pseudomonas syringae is a species complex, associated with plants and the water cycle, 64 comprising several divergent clades that frequently recombine (Young, 2010; Berge et al., 65 2014; Baltrus et al., 2017). Currently, thirteen phylogroups, based on Multi-Locus Sequence 66 Typing (MLST), have been described (Parkinson et al., 2011). As a plant , it is 67 globally important, causing disease on over 180 different host species. P. syringae is 68 responsible for recurring chronic diseases in perennial crops, such as cherry canker 69 (Lamichhane et al., 2014), and also sporadic outbreaks on annual crops, such as bacterial 70 speck of (Şahin, 2001). Individual strains are highly specialised and assigned to over 71 60 host-specific pathovars; some of these are further divided into races which show host 72 genotype specificity (Joardar et al., 2005). This makes P. syringae an important model to 73 study the evolution of host specificity (O’Brien et al., 2011; Mansfield et al., 2012). 74 75 High-throughput sequencing has become a major tool in bacterial studies (Edwards & Holt, 76 2013). With the increasing number of genomes available, population-level studies can now 77 be conducted to ask complex evolutionary questions, such as how disease epidemics emerge 78 and what ecological processes drive the evolution of pathogenicity (Guttman et al., 2014; 79 Monteil et al., 2016). Before genomic methods were available, mutational studies of P. 80 syringae were used to identify functional factors in pathogenesis, such as type III 81 secretion system effector (T3E) repertoires and toxins (Lindgren 1997; Bender et al. 1999). 82 Some T3Es were also shown to act as plant defense elicitors or avirulence (avr) factors when 83 detected by a corresponding pathogen recognition (R) protein in the host (Jones & Dangl, 84 2006). P. syringae has evolved a functionally redundant repertoire of effectors, which allows 85 pathogen populations to lose/modify expendable avr elicitors, with minimal impact on 86 overall virulence (Arnold & Jackson, 2011). It is believed that as pathogen lineages 87 specialise, they fine-tune their effector repertoires to maximize virulence and avoid detection. 88 Host range becomes restricted because the pathogen may lose effectors important for disease 89 on other hosts or gain effectors detected by other plant species (Schulze-Lefert & Panstruga, 90 2011). Many genomics studies have therefore focused on identifying patterns that link 91 effector complements with particular diseases (Baltrus et al. 2011, 2012; O’Brien et al. 92 2012). 93

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94 Much of the understanding of P. syringae – plant interactions has been achieved using 95 herbaceous plant models. Woody, perennial pathosystems provide a greater challenge 96 (Lamichhane et al., 2014). Population genomics of P.s pv. actinidiae, the causal agent of 97 canker, revealed that three pathogen clades, with distinct effector gene sets, have 98 arisen during kiwifruit cultivation (McCann et al., 2013, 2017). A study of the olive 99 pathogen P.s pv. savastanoi revealed that the hopBL effector family is over-represented in 100 wood-infecting pathovars (Matas et al., 2014). Apart from effectors, genes involved in the 101 metabolism of aromatic compounds, phytohormone production and tolerance to reactive 102 oxygen species have been implicated in virulence on olive (Buonaurio et al., 2015). Bartoli et 103 al. (2015a) found that the degradation of the aromatic compound catechol was important for 104 symptom development of P.s pv. actinidiae on kiwifruit. Green et al. (2010), identified 105 differences in metabolism that may dictate the tissue specificity of P.s pv. aesculi 106 strains that infect horse chestnut. Nowell et al. (2016) also identified genes significantly 107 associated with the woody niche. They found candidate effectors, xylose degradation and the 108 α-ketoadipate pathway were associated with this niche. 109 110 This study used genomics to examine the evolution of strains that cause bacterial canker on 111 sweet and wild cherry (both Prunus avium). Clades of P. syringae that constitute the main 112 causal agents of bacterial canker include P.s pv. morsprunorum (Psm) race 1 and race 2 and a 113 P.s pv. syringae (Pss) (Bultreys & Kaluzna, 2010). In addition, P.s pv. avii causes bacterial 114 canker of wild cherry (Ménard et al., 2003). Recently proposed revisions to species names 115 placed Psm R1 in P. amygdali and Psm R2 in P. avellanae (Bull et al., 2010). As they are 116 part of the P. syringae species complex they will be referred to as Psm in this study. The 117 cherry-pathogenic clades of P. syringae are reported to exhibit differences in virulence, host 118 range and lifestyle (Scortichini 2010; Crosse & Garrett 1966), making the P. syringae-cherry 119 pathosystem an intriguing opportunity to study convergent specialisation. The genomic 120 analysis has been coupled with robust pathogenicity testing (Hulin et al., 2018) and 121 functional analysis of potential avirulence (avr) genes. This study provides a proof-of- 122 concept that genomics-based predictions can be used to identify candidate genes involved in 123 disease and will likely become the major tool in disease monitoring, diagnostics and host 124 range prediction. 125 126

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127 Materials and methods 128 129 , plants and pathogenicity tests 130 Methods used for bacterial culture and sources of plants were as described in Hulin et al. 131 (2018) and are detailed in supplementary methods. Bacterial strains are listed in Table 1. 132 Escherichia coli was plated onto Lysogeny Broth (LBA) and grown overnight at 37 °C. 133 Antibiotic concentrations (µg/ml): Kanamycin 50, Gentamycin 10, Spectinomycin 100, 134 Nitrofurantoin 100. X-gal was used at a concentration of 80µg/ml. Tables S1-S3 list the P. 135 syringae mutants, plasmids and primers used in this study. 136 137 Pathogenicity tests, performed on detached cherry leaves and analysed as in Hulin et al. 138 (2018) are described in the supplementary methods. All ANOVA tables are presented in 139 Tables S17-25. 140 141 Genome sequencing, assembly and annotation 142 Genome sequencing using Illumina and genome assembly were performed as in Hulin et al. 143 (2018). For long-read sequencing, PacBio (Pacific Biosystems) and MinION (Oxford 144 Nanopore) were used. High molecular weight DNA was extracted using a CTAB method 145 (Feil et al., 2012). For the PacBio sequencing of Psm R1-5244, R2-leaf and syr9097, DNA 146 samples were sent to the Earlham Institute (Norwich) for PacBio RSII sequencing. 147 148 For MinION sequencing of Psm R1-5300, the DNA library was prepared using the RAD001 149 rapid-prep kit and run on the Oxford Nanopore MinION, flowcell version R9.5 followed by 150 basecalling using Metrichor. MinION reads were extracted from Fast5 files using Poretools 151 (Loman & Quinlan, 2014). The sequencing reads for both long-read technologies were 152 trimmed and assembled using Canu (Berlin et al., 2015) and Circlator was used to circularise 153 contigs (Hunt et al., 2015). The assemblies were polished using error-corrected Illumina 154 reads with bowtie2, samtools and Pilon 1.17 (Li et al., 2009; Langmead & Salzberg, 2013; 155 Walker et al., 2014). 156 157 Plasmid profiling was performed using an alkaline-lysis method (Moulton et al., 1993) and 158 viewed by gel electrophoresis as in Neale et al. (2013). Genomes were submitted to Genbank 159 and accession numbers are listed in Table 1. 160

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161 Orthology analysis 162 OrthoMCL (Li et al., 2003) was used to identify orthologous genes. 108 genomes, including 163 those sequenced and those downloaded from NCBI were re-annotated using RAST (Aziz et 164 al., 2008) to ensure similar annotation quality. For this reason, the Illumina short-read 165 assemblies of the four long-read sequenced genomes (R1-5244, R1-5300, R2-leaf and 166 syr9097) were used in orthology analysis. OrthoMCL was run with default settings and a 50 167 residue cut-off length. 168 169 Phylogenetic and genomic analysis of P. syringae 170 Single-copy genes present in all genomes were aligned using ClustalW (Larkin et al., 2007) 171 and trimmed using Gblocks (Castresana, 2000). Gene alignments were concatenated using 172 Geneious (Kearse et al., 2012). The program jmodeltest 2.1.10 (Posada, 2008) determined the 173 correct evolutionary model for each gene. RAxML-AVX v8.1.15 (Stamatakis, 2014) was 174 used in partitioned mode to build the maximum likelihood phylogeny, with a GTR gamma 175 model and 100 non-parametric bootstrap replicates. To detect core genes that may have 176 undergone recombination, the program GENECONV (Sawyer, 1989) was used as in Yu et al. 177 (2016). Whole genome alignments were performed using progressiveMauve (Darling et al., 178 2010).

179 Virulence and mobility gene identification 180 All T3E encoding sequences were downloaded from pseudomonas-syringae.org, including 181 the recent classification of HopF effectors into four alleles (Lo et al., 2016). tBLASTn 182 (Altschul et al., 1990), was used to search for homologues with a score of ≥70% identity and 183 ≥40% query length to at least one sequence in each effector family. Nucleotide sequences 184 were manually examined for frameshifts or truncations. Disrupted genes were classed as 185 pseudogenes. A heatmap of effector presence was generated using heatmap.2 in gplots 186 (Warnes et al., 2016). Interproscan (Quevillon et al., 2005) identified protein domains and 187 Illustrator for Biological Sequences (IBS) was used for visualisation (Liu et al., 2015). 188 Genomic regions containing effectors were aligned using MAFFT (Katoh et al., 2002). 189 190 A similar analysis was performed for phytotoxin biosynthesis, wood-degradation, ice 191 nucleation and plasmid-associated genes. Protein sequences were obtained from NCBI (Table 192 S4) and blasted against the genome sequence as above. Prophage identification was 193 performed using PHASTER (Arndt et al., 2016).

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194 195 Gain and loss analysis 196 GLOOME (Gain Loss Mapping Engine) was used to plot the gain and loss of genes on the 197 core genome phylogeny (Cohen et al., 2010). Effector genes were considered present even if 198 predicted to be pseudogenes, as these can still be gained and lost. The optimisation level was 199 set to “very high”, a mixture model allowing variable gain and loss distributions was used 200 and the distribution type was GENERAL_GAMMA_PLUS_INV. Highly probable events 201 (probability ≥0.80) on the branches leading to cherry-pathogenic strains were extracted.

202 BayesTraits analysis 203 BayesTraits was used to correlate T3E gene evolution with pathogenicity (Pagel, 2004). A 204 binary matrix was created of effector family presence and pathogenicity of each strain. The 205 effector matrix was collapsed into effector families as the different alleles were predicted to 206 perform similar biological functions in planta (Cunnac et al., 2011). Putative pseudogenes 207 were considered absent as they may be non-functional. A full description of the BayesTraits 208 methodology is described in the supplementary methods. 209 210 Horizontal gene transfer analysis 211 For each effector family, best hit nucleotide sequences were aligned using clustalW (Larkin 212 et al., 2007). RAxML was used to build a phylogenetic tree with a GTR model of evolution 213 and 1000 bootstrap replicates. Incongruence with the core genome tree was examined 214 visually. To further assess horizontal transfer, a species-gene tree reconciliation method 215 RANGER-DTL (Bansal et al., 2012) was used, as in Bruns et al. (2017). Full details of this 216 are described in the supplementary methods. 217 218 Identification of genomic islands 219 Genomic islands (GIs) were identified in the PacBio-sequenced strains using IslandViewer3 220 (Dhillon et al., 2015). Islands were manually delimited as in McCann et al. (2013). BLASTn 221 was utilised to determine if these GIs were present in other P. syringae strains. As most GIs 222 exceeded 10kb, the islands were split into 0.5kb sections prior to analysis. An island was 223 concluded to be fully present if all sections produced hits with a query length >30%. To 224 validate this approach, the Illumina-sequenced genome assemblies of the PacBio-sequenced 225 strains were searched for their own islands. 226

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227 General DNA manipulations and bacterial transformations 228 Cloning and other molecular biology techniques including ectopic expression of potential avr 229 genes were as described in earlier works (Staskawicz et al., 1984; Arnold et al., 2001; Kvitko 230 & Collmer, 2011). Details are provided in the supplementary methods. 231 232 Results 233 234 Genome assembly and sequencing statistics 235 Genome information gathered in this study enabled a comprehensive analysis and meaningful 236 comparisons to investigate the evolution of pathogenicity amongst P. syringae of 237 cherry. Eighteen P. syringae strains isolated from cherry and plum were phenotyped for 238 pathogenicity and genome sequenced in a previous study (Hulin et al., 2018). To increase this 239 sample, nine strains isolated from wild cherry and five additional non-Prunus out-groups 240 were genome-sequenced using the Illumina MiSeq. The genomes of eight cherry strains 241 sequenced elsewhere were also downloaded from NCBI (Baltrus et al., 2011; Nowell et al., 242 2016). 243 244 Information on the origin and pathogenicity of each strain is summarised in Table 1. Twenty- 245 eight were considered pathogenic to cherry including all Pss isolated from cherry and plum. 246 In contrast, three Psm race 1 strains from plum (R1-5300, R1-9326 and R1-9629) and one 247 from cherry strain (R1-9657) failed to induce canker on cherry following tree inoculations; 248 and three strains of unknown isolated from plum and cherry (Ps-9643, Ps-7928C 249 and Ps-7969) were non-pathogenic (references in Table 1). The cherry pathogens are 250 referred to as their described pathovar names throughout this study. To simplify figures, the 251 first few letters of the pathovar name were used. “Pss” becomes “syr”, as otherwise Pss could 252 refer to other pathovars beginning with “s”, e.g. savastanoi. 253 254 All cherry/plum isolated strains included in this study were sequenced using Illumina MiSeq. 255 Three representative strains (R1-5244, R2-leaf and syr9097) were sequenced using PacBio 256 and the non-pathogenic Psm R1 strain R1-5300 was sequenced using Oxford Nanopore, to 257 obtain more complete genomes. Table 2 summarises the genome assembly statistics for all 258 strains sequenced in this study and Hulin et al. (2018). Illumina genomes assembled into 23- 259 352 contigs, whilst the long-read sequenced genomes assembled into 1-6 contigs. The 260 numbers of plasmids in each strain was determined by plasmid profiling (Fig. S1). Psm R1

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261 and R2 strains possessed between 2-6 plasmids, P.s. pv. avii 5271 possessed six plasmids, 262 whereas, apart from three strains (syr5275, syr7928A, syr9644) with one plasmid each, most 263 cherry-pathogenic strains of Pss did not possess plasmids. The strain syr9097 that was 264 PacBio-sequenced lacked plasmids. The long-read sequenced genomes all assembled into the 265 correct number of contigs corresponding to chromosome and plasmids, apart from R1-5300. 266 The chromosome of this strain was separated into two contigs (tig0 and tig75) based on a 267 whole genome alignment with Psm R1 strain R1-5244 (Fig. S2). 268 269 The Psm R1 (R1-5244, R1-5300) and Psm R2 (R2-leaf) long-read assemblies contained 270 putatively complete plasmid contigs. These were confirmed to contain plasmid-associated 271 genes (Tables S5-S7). All three strains (R1-5244, R1-5300 and R2-leaf) possessed plasmids 272 with repA homologues, indicating they may belong to common plasmid family pPT23A 273 (Zhao et al., 2005). Several plasmids also contained conjugational machinery so may be 274 conjugative. 275 276 Core-genome phylogenetic analysis 277 To examine the relatedness of strains, an analysis of core genes was carried out. 108 genomes 278 of strains from the well studied phylogroups 1-3 isolated from both plants and aquatic 279 environments were selected. A maximum likelihood phylogeny based on 1035 concatenated 280 core genes was constructed using RAxML (Fig. S3). There was low support for certain P2 281 and P3 clades based on bootstrap analysis. To determine if particular taxa were causing low 282 support, the analysis was systematically repeated for the two phylogroups, with non-cherry 283 strains removed. Support and tree likelihood values were compared (Table S8). Within P3, 284 the removal of P.s pv. eriobotryae or P.s pv. daphniphylli improved support, whilst the 285 removal of P.s pv. syringae 1212 improved support values in P2 (Fig. S4 and S5). The global 286 analysis was then repeated with these taxa removed (Fig. S6-S9). The final phylogeny (Fig. 287 1), with the highest mean branch support (92.8%) lacked P.s. pv. eriobotryae. The 288 phylogeny, built using a 611,888bp alignment, contained 102 taxa due to the removal of 289 identical strains (dendro4219, syr9630, R1-9629, R1-9326 and R1-5269). Most support 290 values exceeded 70%, with good support for branches leading to cherry-pathogenic clades. 291 292 One explanation for the low support within P2 and P3 was that these clades have undergone 293 core genome recombination. The program GENECONV (Sawyer, 1989) showed that 140 294 genes had putatively recombined (127,288bp total length, 20.8% of the alignment). Table S9

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295 lists the number of recombination events per phylogroup. It was observed that the most 296 frequent core gene recombination occurred in P3 (73 genes affected), followed by 31 genes in 297 P2 and only 13 in P1. 298 299 Cherry pathogens were found in all three phylogroups. The two Psm races (R1 in P3, R2 in 300 P1) and P.s pv. avii (P1) formed monophyletic clades. Within Psm R1, cherry pathogenic 301 strains formed a distinct clade from previously classified non-pathogenic strains (Hulin et al., 302 2018). This indicated there has been divergence in their core genomes. By contrast, Prunus- 303 infecting strains of Pss were found across P2, interspersed with strains isolated from other 304 plants and aquatic environments. To ensure that genomic comparisons between P2 strains 305 were based on differential pathogenicity, several closely related non-Prunus strains were 306 pathogenicity tested on detached cherry leaves (Fig. S10). In planta bacterial populations of 307 non-Prunus strains were reduced compared to two cherry and plum Pss strains, indicating 308 that host specificity may exist in P2. 309 310 Search for virulence factors 311 312 The hrp pathogenicity island 313 All sequenced strains were confirmed to contain the hrp pathogenicity island required for 314 conventional Type III secretion. Core effectors genes from the conserved effector locus 315 (CEL, Alfano et al., 2000) such as avrE1, hopM1 and hopAA1, were also present (Fig. S11), 316 However, hopAA1 was truncated in both Psm R1 and R2 due to inversion events. The 317 hopAA1 gene was truncated in Psm R2, whilst in Psm R1 both hopAA1 and hopM1 were 318 truncated (Fig. S12). 319 320 Type III effectors and other virulence genes 321 All 102 genomes used in the phylogenetic analysis were scanned for known T3Es and non- 322 T3 virulence factors. A heatmap of presence, absence and pseudogenisation 323 was constructed (Fig. 2). In terms of T3Es, there was variation both between and within the 324 different cherry-pathogenic clades. Notably, Psm R1 which contained pathogenic and non- 325 pathogenic strains on cherry showed clear differentiation in effector repertoire (Table S10). 326 Psm R1, R2 and P.s pv. avii possessed 24-34 effector genes, whereas Pss strains possessed 9- 327 15. This reduced effector repertoire of Pss was representative of P2 strains as noted by

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328 Dudnik & Dudler (2014). Table 3 lists the effectors in each long-read genome assembly in 329 order of appearance. 330 331 332 Non-T3 virulence factors were also identified. All pathogenic Psm R1 strains possessed the 333 coronatine biosynthesis clusters, which were plasmid-borne in Psm R1-5244. All cherry- 334 pathogenic Pss strains possessed at least one biosynthesis gene cluster for the toxins 335 syringomycin, syringolin and syringopeptin, with several strains possessing all three. Strains 336 within clade P2b possessed the biosynthesis genes for mangotoxin. The non-pathogenic 337 cherry P2b strains Ps7928C and Ps7969 lacked all toxin biosynthesis clusters. 338 339 A cluster of genes named WHOP (woody hosts and Pseudomonas) thought to be involved in 340 aromatic compound (lignin) degradation (Caballo-Ponce et al., 2016) was present in Psm R1 341 and R2, whilst P.s pv. avii and most Pss strains contained no WHOP homologues. Two 342 cherry P2d strains (syr2339 and syr7924) did however possess the catechol catBCA cluster. 343 Finally, the genomes were searched for the ice nucleation gene cluster. Members of Psm R1, 344 Pss and P.s pv. avii strains all possessed genes involved in ice nucleation (Fig. 2). Whilst 345 Psm R2 lacked the complete set of genes for ice nucleation.

346

347 Associating T3E evolution with host specificity 348 T3E evolution was statistically correlated with cherry pathogenicity, using the programs 349 BayesTraits and GLOOME (Pagel, 2004; Cohen et al., 2010). BayesTraits takes a binary 350 matrix of two traits and a phylogeny and determines if changes in the two characters (effector 351 gene and pathogenicity) have evolved independently or dependently. Fig. 3a shows the 352 likelihood ratio of cherry pathogenicity being correlated with each effector family’s 353 evolution, with significantly associated effectors highlighted. 354 355 BayesTraits analysis using the core genome phylogeny predicted the evolution of six T3E 356 families was linked to cherry pathogenicity. These were hopBF, hopAB, hopH, hopAR, 357 avrPto and hopBB. To account for any phylogenetic uncertainty, the program was also run on 358 the full set of 100 bootstrapped trees generated by RAxML. The evolution of T3Es hopBF, 359 hopAR and hopAB was always associated with pathogenicity for all 100 trees, indicating

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360 strong association. Whilst, the T3E genes avrPto, hopBB and hopH were only significantly 361 correlated for 88%, 77% and 62% of trees respectively (Fig. S13). To determine how these 362 genes had been gained or lost across the phylogeny, the program GLOOME was used (Cohen 363 et al., 2010). Fig. 3b illustrates the predicted gain and loss of these T3Es on the branches 364 leading to cherry pathogenic clades (with gene presence/absence profiles shown in Fig. 3c). 365 Those putatively associated with pathogenicity (high probability of gain in cherry-pathogenic 366 clades) included hopAR1, hopBB1, hopBF1 and hopH1. The T3Es hopAB1 and avrPto1 were 367 found to be lost from cherry pathogenic Psm R1, whilst the hopAB1 and hopAB3 alleles were 368 pseudogenised in Psm R2 and P.s pv. avii (Fig. 3c). All effector gain and loss events are 369 presented in Fig. S14 and Table S11. Fig. S15 shows the phylogeny with branch labels used 370 in GLOOME. 371 372 GLOOME predicted that key effectors have been gained in multiple clades. The hopAR1 373 gene has been gained in Psm R1, Psm R2, Pss and P.s pv. avii. The T3E hopBB1 was present 374 in the majority of strains within Psm R1, R2 and P.s pv. avii but was absent in Pss strains. It 375 showed high probability of gain on branches leading to both Psm R2 and P.s pv. avii. 376 However, GLOOME predicted loss in two Psm R1 strains indicating it has experienced 377 dynamic evolution in cherry pathogens. The hopBB1 effector is closely related to members of 378 the hopF family and avrRpm2 (Lo et al., 2016). In addition to the significant acquisition of 379 hopBB1 homologues, the hopF family was expanded in cherry pathogens. Pathogenic strains 380 in Psm R1 and R2 all possessed two hopF alleles each (hopF3 and hopF4/hopF2 and hopF4, 381 see Fig. 2). P.s pv. avii did not possess any hopF homologues, but had gained hopBB1. By 382 contrast, Pss strains lacked all hopF members. 383 384 Origins of key effectors in cherry pathogens 385 To understand the origins of key effectors, gene phylogenies were produced. Incongruence 386 with the core-genome phylogeny indicated that effector sequences had likely experienced 387 HGT between the pathogenic clades, as their sequences clustered together. There has been 388 possible effector exchange between Psm R1, R2 and P.s pv. avii. To predict precisely where 389 transfers had occurred on the phylogeny, the program RANGER-DTL was utilised (Bansal et 390 al., 2012). Table 4 reports T3Es that exhibited evidence of HGT between cherry pathogens 391 (gene trees are presented in Fig. S16-S17). Full transfer events are listed in Table S12 and 392 Fig. S18 shows the phylogeny with branch labels used in RANGER-DTL. The BayesTraits 393 correlated T3Es hopBB and hopBF both showed evidence of HGT. Fig. 4a shows examples

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394 of T3Es putatively undergoing HGT between cherry pathogenic clades highlighted in red. 395 Alignments of the flanking regions (Fig. 4b) showed homology between the cherry pathogens 396 and included mobile elements likely involved in recombination events. For example, the 397 regions surrounding Psm R1 and R2 avrD1 sequences were identical (Fig. 4b), indicating that 398 this effector has probably been gained on the same mobile region in both clades. Putatively 399 transferred effectors were mostly plasmid-encoded in the long-read genomes (Table 3). In 400 R1-5244 several of these genes were encoded on one plasmid (Contig 3), whilst in R2-leaf 401 they were found on two plasmids (Contig 6 and 8). 402 403 The pathogenicity-associated T3E gene hopAR1 was present in 23/28 cherry pathogens and 404 showed probable gain in pathogenic clades. Phylogenetic analysis of this T3E (Fig. 5a) 405 showed that the sequences for the different cherry pathogenic clades did not cluster with each 406 other, indicating convergent acquisition. Prophage-identification (Table S13) did however 407 reveal that this T3E is predicted to have been gained in Psm R1 and R2 within different 408 phage sequences, whilst in Pss it is on a genomic island (Fig. 5b), so has been acquired via 409 distinct mechanisms. The Psm R1 phage is 51.5kb, described as intact and contains both 410 hopAR1 and a truncated version of hopBK1. The Psm R2 phage sequence was 37.1kb and 411 was described as ‘incomplete’, indicating it did not have all the components of an active 412 prophage. Further analysis of this region in Psm R2 and P2 strains revealed a shared adjacent 413 tRNA-Thr gene (Fig. 5c,d). Within P2, although cherry Pss strains lacked the phage, several 414 strains isolated from bean (syr2675, syr2676 and syr2682) possessed the hopAR1 gene within 415 a phage homologous to that in Psm R2. The syr2675 hopAR1 sequence was also the most 416 closely related homologue of Psm R2 hopAR1 (Fig. 5a). This novel evidence suggests that 417 this effector gene may have been transferred via phage between phylogroups. 418 419 Many T3Es are mobilised between bacteria on genomic islands (GI). GIs were identified for 420 the three PacBio-sequenced strains of Psm R1, Psm R2 and Pss (Tables S14-S16). R1-5244 421 GIs contained the coronatine biosynthesis cluster and six T3Es. In R2-leaf, eight T3E genes 422 were located on GIs, whilst in syr9097 three T3Es were found on genomic islands. 423 424 These GIs were then searched for in other P. syringae genomes to identify potential sources 425 of transfer and Fig. 6a shows heatmaps of GI presence. The Psm R1 GIs included several 426 found only in pathogenic Psm R1 strains differentiating them from the non-pathogens. These 427 included the coronatine biosynthesis cluster (GI1), hopF3 (GI6) and hopAT1 (GI14). Most

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428 Psm R1 GIs produced hits across P. syringae, particularly in P1 and P3. Psm R2 GIs were 429 most commonly found in P1. Several were shared with other cherry-pathogenic clades, 430 including those containing hopAF1 (GI36), hopAT1 (GI3) and hopD1 (GI6). Finally, 431 although most islands identified in syr9097 were commonly found across the species 432 complex, those containing T3Es (GI30, GI23 and GI26) appeared to be P2-specific, 433 indicating that cherry-pathogenic strains likely gained these islands from other members of 434 P2. 435 436 Functional analysis of potential avr genes 437 To validate the predictive power of this analysis, cloning was used to identify avirulence 438 factors active in cherry. The effector genes avrPto and hopAB were absent from cherry 439 pathogens and their evolution was theoretically linked to pathogenicity. Several other 440 candidate avirulence effectors were identified, that were absent from cherry pathogens, but 441 present in close out-groups (Fig. 2). Avirulence-gene identification focused on Psm R1 as 442 any T3E variation within the clade may be due to differences in host specificity rather than 443 phylogenetic distance. Potential avirulence T3E genes included avrA1, avrPto1, hopAA1, 444 hopAB1, hopAO2 and hopG1, which had full-length homologues in non-pathogenic Psm R1 445 strains, but were absent from or truncated in pathogens. These genes were cloned from R1- 446 5300 (except hopAO2, which was cloned from R1-9657). 447 448 The effector avrRps4 was also cloned from P.s pv. avellanae (Psv) BPIC631 a close relative 449 of Psm R2. This effector was absent from most cherry-pathogenic strains. Several pathogens 450 possessed the full-length gene (R2-leaf, R2-9095 and P.s pv. avii), but lacked the KRVY 451 domain that functions in planta (Fig. S19) (Sohn et al., 2009). The hopAW1 gene was cloned 452 from Pph1448A as this T3E has undergone two independent mutations in Pss strains, 453 disrupting the beginning of the gene (Fig. S20). Finally, hopC1 was cloned from the 454 Aquilegia vulgaris pathogen RMA1 which is basal to the Psm R2 clade, as it is absent from 455 all cherry-pathogenic strains. 456 457 Nine effectors were cloned into the expression vector pBBR1MCS-5 and conjugated into 458 three pathogenic strains (R1-5244, R2-leaf and syr9644). Knock-out strains for the T3SS 459 gene hrpA were obtained for R1-5244 and R2-leaf to act as non-pathogenic controls that 460 could not secrete T3Es and failed to cause the HR on tobacco (Fig. S21). 461

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462 Bacterial population counts were conducted in cherry leaves. The transconjugants expressing 463 HopAB1 or HopC1 failed to multiply to the same levels as the pathogenic empty vector (EV) 464 controls or produce disease lesions. The expression of AvrA1, AvrRps4, and HopAW1 also 465 caused significant reductions in population growth, but this reduction was not consistently 466 seen across all three pathogenic strains (Fig. 7a). As the addition of the hopAB1 gene 467 reduced pathogenicity, the hopAB2 and hopAB3 genes were also cloned from PsvBPIC631 468 and RMA1, and were also found to reduce pathogen multiplication (Fig. 7b). 469 470 To further investigate the induction of the HR by the HopAB family and HopC1, inoculations 471 were performed at high concentrations (2x108 CFU/ml) as described in Hulin et al. (2018). 472 In Psm R1 and R2, the addition of these T3Es led to more rapid tissue collapse than observed 473 in EV controls, indicative of HR induction (Fig. 7c,d); HopC1 and HopAB1 were particularly 474 effective. With Pss, however, EV transconjugants themselves caused rapid tissue collapse, 475 making it impossible to recognise an induced HR as symptom development was not 476 significantly different. 477 478 The hopAB1 gene is found in a mobile-element rich ~40kb region in the non-pathogenic Psm 479 R1-5300, missing from the pathogen Psm R1-5244 (Fig. 7d). Meanwhile, Psm R2 and P.s pv. 480 avii possessed putatively pseudogenised hopAB3 alleles (Fig. 7e) and P.s pv. avii possessed a 481 truncated hopAB1 gene (Fig. S22). hopAB3 is truncated in Psm R2 due to a 2bp insertion 482 (GG at position 1404bp) leading to a premature stop codon, whilst in P.s pv. avii a 218bp 483 deletion has disrupted the C-terminus. If expressed, the E3-ubiquitin ligase is completely 484 absent from the Psm R2 protein and disrupted in P.s pv. avii (Fig. 7f). Both HopAB3 alleles 485 were also divergent enough that the Pto-interacting domain (PID) was not identified by 486 Interproscan. To determine if the truncated Psm R2 HopAB3 allele induced any resistance 487 response in cherry leaves, the gene was expressed in Psm R1-5244 and population growth 488 measured. The addition of this gene did not lead to a significant reduction in growth 489 compared to the EV control, unlike other hopAB alleles (Fig. 7g) and the transconjugant was 490 still able to induce disease symptoms 10 dpi (Fig. 7h). 491 492 Discussion 493 494 Core genome phylogenetics

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495 Phylogenetic analysis confirmed that cherry pathogenicity has evolved multiple times within 496 P. syringae. Psm R1, R2 and P.s pv. avii each formed distinct monophyletic clades, whereas 497 cherry-pathogenic Pss strains were distributed across the P2 clade thus indicating that cherry 498 pathogenicity has either evolved multiple times within P2 or that this clade is not highly 499 specialised. To confirm this genomic prediction of pathogenicity, several additional P2 500 strains isolated from bean, and lilac were tested for pathogenicity in cherry. They each 501 produced lower population levels in cherry leaves than cherry pathogens, suggesting that host 502 adaptation has occurred (Fig. S10). Many P2 strains have previously been named Pss on the 503 basis of lilac pathogenicity, despite being pathogenic to other plant species (Young, 1991). A 504 new naming system within this phylogroup would therefore be desirable. 505 506 Search for candidate effectors involved in cherry pathogenicity 507 Gains and losses of T3Es were closely associated with pathogenicity. Virulence-associated 508 effectors hopAR1, hopBB1, hopH1 and hopBF1 had been gained in multiple cherry- 509 pathogenic clades. The hopAR1 effector has been studied in the bean pathogen P.s pv. 510 phaseolicola R3 (1302A), as an GI-located avr gene (avrPphB) whose protein is detected by 511 the corresponding R3 resistance protein in planta (Pitman et al., 2005; Neale et al., 2016). 512 HopAR1 also acts as a virulence factor as a cysteine protease which targets receptor-like 513 kinases to interfere with plant PAMP-triggered immunity (PTI) responses (Zhang et al., 514 2010). This effector could play a similar role in PTI suppression in cherry. 515 516 HopBB1 and other members the HopF family were abundant in cherry pathogens. All HopF 517 members share an N-terminus and myristoylation sites for plant cell membrane localisation 518 (Lo et al., 2016) and interfere with PTI and Effector-triggered immunity (ETI) in model 519 plants (Wang et al., 2010; Wu et al., 2011; Hurley et al. 2014). The presence of multiple 520 hopF homologues in cherry pathogens and specific gain of hopBB1 suggested the importance 521 of their function. In comparison, HopH1 and HopBF1 are under studied. HopH1 is a 522 protease, homologous to the Ralstonia solanacearum Rip36 protein (Nahar et al., 2014). This 523 T3E gene was found on GI37 in Psm R2-leaf and was within 3kb of hopF4 (Fig. S23), 524 indicating that these two T3Es may have been gained together. HopBF1 was first discovered 525 in P.s pvs aptata and oryzae (Baltrus et al., 2011) but its role in planta is undetermined. This 526 study therefore provided candidate T3Es important for cherry pathogenicity which could be 527 the focus of future functional studies.

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528 529 Phytotoxin biosynthesis gene clusters were also identified. Coronatine has been gained on a 530 plasmid in pathogenic Psm R1 and could be one of the factors that differentiate pathogens 531 from non-pathogens in this clade. Coronatine functions in virulence by down-regulating 532 salicylic acid defence signalling (Grant & Jones, 2009). Necrosis-inducing lipodepsipeptide 533 toxins were common in P2. All cherry-pathogenic Pss strains possessed at least one 534 biosynthesis cluster. The ability of Pss strains to cause necrosis on cherry fruits has been 535 linked to toxins (Scholz-Schroeder et al., 2001). Interestingly, two non-pathogenic P2b 536 cherry strains lacked all phytotoxins, a deficiency that could contribute to their lack of 537 pathogenicity. 538 539 All cherry-pathogenic Pss strains had reduced effector repertoires. This observation supports 540 the hypothesis that a phenotypic trade-off exists, with strains retaining few T3Es, whilst 541 relying more on phytotoxins for pathogenicity (Hockett et al., 2014). If this pathogenic 542 strategy has evolved in the P2 clade, this raises the question as to how it affects host 543 specificity and virulence. P2 strains often infect more than one host species (Rezaei & 544 Taghavi, 2014). These strains probably possess fewer ETI-inducing avirulence factors that 545 restrict effector-rich strains to particular hosts, so may be more successful generalists. The 546 reduction in T3E repertoire could however be limiting as strains may be less capable of long- 547 term disease suppression required at the start of a hemi-biotrophic interaction. 548 549 Most cherry-pathogenic clades possessed genes involved in aromatic compound degradation, 550 shown to be important in virulence on olive (Caballo-Ponce et al., 2016) and ice nucleation 551 genes that stimulate damage (Lamichhane et al., 2014). The fact that not all cherry- 552 pathogenic clades possessed these genes suggests they are not crucial for bacterial canker, 553 however they could contribute to niche persistence. For example, Crosse and Garrett (1966), 554 observed that Psm R1 survived in cankers for longer than Pss. Increased persistence could be 555 linked to genes involved in woody-tissue adaptation. 556 557 HGT has been important in the acquisition of key effectors 558 HGT is a key mechanism for effector shuffling within P. syringae (Arnold & Jackson, 2011). 559 Pathogenicity-associated T3Es hopBB1 and hopBF1 were plasmid encoded and showed 560 evidence of HGT between the cherry-pathogenic clades in P1 and P3. Plasmid profiling 561 revealed that cherry pathogens in phylogroups 1 and 3 possessed native plasmids, some of

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562 which were putatively conjugative, indicating the importance of plasmids in gene exchange. 563 By contrast, most cherry-pathogenic Pss strains lacked plasmids. 564 565 The pathogenicity-associated T3E hopAR1 was chromosomal in Psm R1 and R2. This gene 566 was found within distinct prophage sequences in these two clades. To our knowledge this is 567 the first reported example of a plant pathogen T3E located within a prophage sequence. 568 Interestingly, the Psm R2 hopAR1 gene homologue was most similar to hopAR1 from a P2 569 bean strain syr2675, which is a close relative of cherry Pss. This strain possessed the same 570 phage as Psm R2, indicating that horizontal gene transfer of this T3E between phylogroups 571 may have been phage-mediated. The striking example of convergent acquisition of hopAR1 572 in the cherry pathogens, putatively through distinct prophages in Psm R1 and R2, and a GI in 573 Pss indicates that this T3E may have important roles in virulence. The well characterised P.s 574 pv. phaseolicola R3 homologue is not associated with a phage, but has been shown to 575 undergo dynamic evolution on a mobile genomic island in planta in resistant bean cultivars 576 (Neale et al., 2016). It is intriguing that the same T3E may be important in a completely 577 different pathosystem. 578 579 Several T3Es in Psm R1, R2 and Pss were located on GIs. To determine the likely source of 580 GIs in cherry strains, all other P. syringae strains were searched for homologous sequences 581 (Fig. 6). There was evidence of Psm R1 and R2 islands being shared between cherry 582 pathogen clades indicative of HGT events occurring between strains occupying the same 583 ecological niche. 584 585 Functional genomics revealed convergent loss of an avr factor

586 Genes from the hopAB and avrPto families form a redundant effector group (REG) vital for 587 early PTI suppression in herbaceous species (Jackson et al., 1999; Lin & Martin, 2005; 588 Kvitko et al., 2009). Both effectors also trigger ETI by interacting with the serine-threonine 589 kinase R protein Pto in tomato (Kim et al., 2002). 590 591 Across the P. syringae complex, the REG was common (Fig. S24), but cherry pathogens all 592 lacked full-length members of this family. The hopAB1 gene has been lost from Psm R1, 593 whilst the Psm R2 and P.s pv. avii predicted HopAB3 proteins lacked the PID and E3- 594 ubiquitin ligase domains through contrasting mutations. P.s pv. avii also possessed a

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595 truncated hopAB1 gene (Fig. S22), lacking the PID domain. The lack of a PID in the cherry 596 pathogen HopAB proteins suggested that they could have diverged to avoid a Pto-like 597 recognition in cherry. 598 599 Full-length members of this REG were cloned into cherry pathogens to determine their role 600 in planta. The addition of HopAB alleles (HopAB1-3) consistently reduced population 601 growth of pathogenic strains in planta and triggered an HR. HopAB loss or pseudogenisation 602 in cherry pathogens may have been selected for to reduce avirulence activity. The truncated 603 version of HopAB3 in R2-leaf was found not to exhibit avirulence activity as its expression 604 did not reduce the growth of R1-5244 in planta. Although AvrPto is part of the same REG, 605 its expression had no effect on the ability of cherry pathogens to multiply in planta. The 606 absence of AvrPto in cherry pathogens is therefore unlikely to be driven by avirulence, but 607 could be due to the lack of HopAB virulence targets in planta. As this REG is vital for early 608 disease suppression in model strains, cherry pathogens may rely on other T3Es to fulfil this 609 role. 610 611 The variation in hopAB1 presence in Psm R1 is intriguing. Psm R1 strains may be pathogenic 612 on both cherry and plum (ΔhopAB1) or just pathogenic on plum (possessed hopAB1) (Hulin 613 et al., 2018). This suggested that the host proteins in cherry that detect the presence of 614 HopAB are not present/functioning in plum. Future studies may determine how the two host 615 immune responses diverged and could examine hopAB diversity across Prunus pathogens. 616 This study focused on Prunus avium, however strains isolated from additional Prunus spp. 617 were included, such as P.s pv. cerasicola, P.s pv. morsprunorum FTRSU7805, P.s pv. 618 amygdali and P.s pv. persicae (Table 1). All apart from P.s pv. amygdali 3205 and P.s pv. 619 persicae lacked HopAB (Fig. 2), indicating that there may be a conserved resistance 620 mechanism regulating ETI activated by this effector family in Prunus species. 621 622 Linking genomics to host specialisation 623 Cherry pathogenicity has arisen independently within P. syringae, with strains using both 624 shared and distinctive virulence strategies. Cherry-pathogenic clades in P1 and P3 have large 625 effector repertoires. Cherry Pss were found across P2. They possessed reduced T3Es and 626 several phytotoxin gene clusters. Key events in the evolution of cherry pathogenicity (Fig. 8) 627 appear to be the acquisition of virulence-associated effectors, often through horizontal gene 628 transfer, such as hopAR1, members of the hopF family such as hopBB1, hopBF1 and hopH1.

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629 Significantly, the loss/pseudogenisation of HopAB effectors has also occurred in multiple 630 clades. Within P2, the different cherry-infecting Pss clades have slight differences in their 631 virulence factor repertoires which may reflect their convergent host adaptation. Clades 632 differed in T3E content, phytotoxin genes and possession of genes for catechol degradation. 633 This study demonstrated that populations genomics can be used to examine a complex 634 disease of a perennial plant species. A huge dataset was narrowed down to several candidate 635 host-specificity controlling genes, two of which (hopAB and hopC1) encode proteins that had 636 putative avirulence functions in planta. 637 638 Acknowledgements 639 We acknowledge the East Malling Trust, University of Reading and BBSRC for funding 640 (BB/P006272/1). Library preparation for PacBio sequencing was performed at the Earlham 641 Institute, whilst for wild cherry strains Illumina MiSeq sequencing, libraries was prepared at 642 the Genomics Facility, University of Warwick. We thank Steve Roberts, Helen Neale, Mateo 643 San José and David Guttman for providing bacterial strains. We thank the East Malling Farm 644 and Glass staff for plant maintenance. The authors declare no conflict of interest. 645 646 Author contributions 647 MTH, JWM, RWJ and RJH conceived and designed the study as well as writing the 648 manuscript. MTH performed bioinformatics, statistical analysis and laboratory work. JV 649 isolated some of the strains used in this study, JV and EH selected representative strains and 650 prepared DNA for MiSeq sequencing of nine strains and LB assembled these nine sequences. 651 HJB performed the MinION library preparation and sequencing. ADA assisted in 652 bioinformatics pipeline development. All authors read and reviewed the final manuscript. 653 654 655 656

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32 bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, 2018. 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.

1059 Figure legends 1060 1061 Fig. 1: Core genome phylogenetic tree. Multi-locus phylogeny based on 1035 genes which 1062 represent the core genome of P. syringae. Strains from cherry and plum are highlighted in 1063 pink and blue respectively. Strains pathogenic to cherry (assessed in Hulin et al., 2018 and 1064 Vicente et al., 2004) are labelled with red circles. * indicates non-pathogenic to cherry in 1065 controlled pathogenicity tests. Phylogroups are also labelled for reference. Percentage 1066 bootstrap support values below 99% are shown for each node. The scale is nucleotide 1067 substitutions per site. 1068 1069 Fig. 2: Virulence gene identification. a: Heatmap of virulence gene presence and absence 1070 across P. syringae. The dark green squares indicate presence of a full-length T3E homologue 1071 whereas light green squares indicate that the gene is disrupted or truncated in some way. 1072 Other non-T3 secreted virulence factors are coloured in dark and light blue. Strains infecting 1073 cherry and plum are highlighted in pink and blue respectively. * indicates non-pathogenic to 1074 cherry in controlled pathogenicity tests. The cherry-pathogenic clades are illustrated via 1075 horizontal shading of cells with Psm R1 in blue, Psm R2 in light green, Pss in light red and 1076 P.s pv. avii in orange. Strains are ordered based on the core genome phylogenetic tree which 1077 is represented by the dendrogram. b: The total number of full-length and pseudogenised T3E 1078 genes plotted for each strain. 1079 1080 Fig. 3: Association of T3E evolution with cherry pathogenicity. a: Barplot showing the 1081 likelihood ratio for the correlation of each effector gene family with cherry pathogenicity 1082 based on BayesTraits analysis using the core genome phylogeny. The values are obtained 1083 from means of 100 independent runs of the program with error bars showing standard error. 1084 Those effectors that were not significantly associated with pathogenicity are coloured in grey. 1085 Coloured bars were associated with pathogenicity. GLOOME analysis revealed those 1086 significant effector genes where presence of the gene was associated with pathogenicity 1087 (p≤0.05). These are coloured in shades of blue, whilst where the significant gene was absent 1088 in cherry pathogenic clades the bar is coloured in shades of red. b: Gain and loss of 1089 BayesTraits-associated T3Es in cherry-pathogenic clades on the core genome phylogeny 1090 predicted using GLOOME (probability ≥0.8). For visualisation, clades within the 1091 phylogenetic tree have been collapsed with cherry pathogenic clades in pink (Psm R1 plum 1092 strains in blue). Psa: P.s pv. avii. Effectors are colour-coded based on the bar colours in A.

33 bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, 2018. 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.

1093 *: The probability of this effector being gained/lost was slightly lower than 0.8 (see Table 1094 S11 for details). c: Heatmap of presence and absence of associated effectors across P. 1095 syringae. This was constructed as in Fig. 2. Cherry-pathogenic strains are highlighted by pink 1096 vertical shading of columns. 1097 1098 Fig. 4: Horizontal gene transfer has played a key role in the evolution of cherry 1099 pathogenicity. a: Transfer of avrD1, hopBB1 and hopBF1 between different cherry- 1100 pathogenic clades based on the P. syringae core genome phylogeny. Strains that possess the 1101 T3E are coloured in blue, and those that are cherry-pathogenic are highlighted in red. The 1102 transfer events predicted by RANGER-DTL are shown by purple arrows. The scale-bar 1103 shows substitutions per site. b: DNA alignments of genomic regions containing these 1104 effectors. Alignments are colour-coded based on similarity where identical residues are in 1105 grey, whereas dissimilar residues appear in black. The effector gene is coloured in red, 1106 mobile element genes are in green and other CDS are in blue. Cherry-pathogenic strains are 1107 named in pink. Gene name abbreviations: ME, mobile element, Res, resolvase, Hypo, 1108 hypothetical protein gene; ISPsy4, insertion sequence, PHP, polymerase and histidinol 1109 phosphatase. 1110 1111 Fig. 5: Evolution of hopAR1 in different clades of P. syringae containing cherry 1112 pathogens. a: Maximum-likelihood phylogenetic tree built using the nucleotide sequences of 1113 the hopAR1 gene. Cherry and plum isolated strains are highlighted in pink and blue 1114 respectively. R1-9657* is classed as a non-pathogen of cherry. Bootstrap supports less than 1115 100% are shown. The scale is nucleotide substitutions per site. * points to the clustering of 1116 Psm R2 sequences with syr2675. b: Genomic locations of the hopAR1 gene in the three 1117 PacBio-sequenced cherry pathogens. The gene is located within prophage sequences in Psm 1118 R1 and R2 (see Table S13 for details), whereas in syr9097 it is on a genomic island adjacent 1119 to a tRNA gene. Effector genes are coloured in red, other CDS in blue, phage genes predicted 1120 by PHASTER and mobile element genes are in green, tRNA genes in pink and genomic 1121 islands predicted (GI14 in Psm R2 and GI23 in Pss) in light blue. # Indicates that hopBK1 is 1122 a pseudogene in this strain. The end of predicted prophage sequences is denoted with a 1123 dashed green line. c: Alignment of the region surrounding the hopAR1 gene in several strains. 1124 The blue box indicates the region in d. All sequences share an upstream tRNA-Thr gene in 1125 pink. The phage region in the bean pathogens syr2676 and syr2675 (shortened due to it being 1126 at the end of a contig) and Psm R2-leaf are highlighted in a green box. Other mobile elements

34 bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, 2018. 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.

1127 are also coloured in green. The hopAR1 gene is coloured in red and outlined with a red box. 1128 All strains with the phage sequences and syr9097 also contain attL and attR repeats which are 1129 putative insertion sites. Additional closely related phylogroup 2 (P2) strains (USA011, 1130 syr1212, syrB728A) which lack the hopAR1 gene are included for comparison. Homologous 1131 regions are highlighted in yellow to show regions of similarity between P2 strains. d: Close- 1132 up alignment of the genomic regions surrounding the hopAR1 gene in strains of P2, Psm R2, 1133 P.s pv. amygdali and P.s pv. avii that share homology in the surrounding regions. tRNA 1134 genes are in pink, phage/mobile element genes are in green, CDS in blue and the hopAR1 1135 T3E gene is coloured red and highlighted with a red box. Alignments are colour-coded based 1136 on similarity where identical residues are in grey, whereas dissimilar residues appear in 1137 black. * The syr2675 hopAR1 gene is similar to Psm R2 sequences and also has homologous 1138 phage sequences upstream. 1139 1140 Fig. 6: Genomic islands characteristic of cherry pathogens are found across P. syringae. 1141 The heatmap shows the presence and absence of the genomic islands identified in the PacBio 1142 sequenced pathogenic strains of (a) Psm R1, (b) Psm R2 and (c) Pss across the P. syringae 1143 complex. Dark green squares indicate that the full-length GI is putatively present. Light green 1144 squares are where the GI was predicted to be partially present. The names of strains that 1145 infect cherry and plum are coloured in pink and blue, respectively. Strains that were not 1146 pathogenic but were isolated from cherry are marked with an asterisk. The different 1147 phylogroups 1,2 and 3 are delimited by black borders. Pink arrows point to GIs that contain 1148 T3E or phytotoxin genes. PsmR1 GIs - 1: coronatine biosynthesis genes, 2: hopF3, 3: hopA1, 1149 4: hopAT1, 5: hopBL2, 6: hopAO2, 7: hopAY1, Psm R2 GIs - 8: hopAT1, 9: hopD1, 10: 1150 hopX1, 11: hopAR1, 12: hopE1, 13: hopAE1, 14: avrB2, 15: hopAZ1, 16: hopAF1, 17: 1151 hopH1, Pss GIs – 18: hopAR1, 19: avrRpm1, 20: hopBE1. Full details are in Tables S14-S16. 1152 1153 Fig. 7: Identification of avirulence factors activating ETI in cherry. a: Boxplot of an 1154 initial ten-day population count analysis of cherry pathogens (R1-5244, R2-leaf and syr9644) 1155 transconjugants expressing candidate avirulence genes. The data presented are based on one 1156 experiment, with three leaf replicates and three nested technical replicates (n=9). Controls 1157 included the wildtype strain, a strain containing the empty pBBR1MCS-5 vector and a ∆hrpA 1158 deletion mutant (for R1-5244 and R2-leaf). A separate ANOVA was performed for each 1159 cherry pathogen (R1-5244, R2-leaf and syr9644) and the Tukey-HSD significance groups 1160 (p=0.05, confidence level: 0.95) for each strain are presented above each boxplot. b: Boxplot

35 bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, 2018. 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.

1161 of ten-day population counts of cherry pathogens (R1-5244, R2-leaf and syr9644) expressing 1162 different HopAB alleles and HopC1. The data presented are based on three independent 1163 experiments (n=27). Tukey-HSD significance groups (p=0.05, confidence level: 0.95) are 1164 presented above each boxplot. c: Symptom development of R1-5244, R2-leaf, syr9644 1165 transconjugants. Mean symptom score values are presented and represent two independent 1166 experiments (n=6). Error bars show standard error above and below the mean. Symptoms 1167 assessed as degree of browning of the infiltration site, 1: some browning, 2:< 50%, 3:>50%, 1168 4: 100% of the infiltrated area brown. Analysis was based on Area Under Disease Progress 1169 Curves values (0-48 h), see Tables S25-S26. An ANOVA was performed on AUDPC values, 1170 with * indicating significantly different from the empty vector (EV) control. d: Symptom 1171 development over time on a leaf inoculated with R1-5244 transconjugants. HPI: Hours post 1172 inoculation. The order of strains: 1: EV, 2: hopAB1, 3: hopAB2, 4: hopAB3, 5: hopC1. 1173 Arrows indicate the first appearance of symptoms associated with each strain and are 1174 coloured based on the graph in c. e: Alignment of the DNA region surrounding hopAB1 in 1175 Psm R1 strains. Grey indicates sequence identity whereas black indicates divergence. The 1176 effector genes are coloured in red, whereas other CDS are in blue and putative mobile 1177 element genes are in green. * indicates the location of hopAB1 in R1-5300, whilst the 1178 upstream effectors are hopQ1, hopD1 and hopR1. The second alignment shows the hopAB3 1179 gene of Psm R2 and close out-groups. * The hopAB3 gene has been truncated due to a GG 1180 insertion leading to a frameshift in Psm R2, whilst in P.s pv. avii (avii3846) there is a 1181 deletion at the end of the gene. f: Diagrams showing the location of key domains in the 1182 HopAB3 protein including the Pto-interaction domain (PID), BAK1-interacting domain 1183 (BAK1) and E3 ubiquitin ligase (E3). The E3 domain is lost completely from the Psm R2 1184 protein whereas in avii3846 the beginning of this domain is lost. The PID domain was not 1185 detected in the cherry pathogen sequences. g: Boxplot of ten-day population counts of R1-

1186 5244 trans-conjugants expressing three different hopAB alleles, including hopAB3R2-leaf and 1187 hopC1. The data presented are based on two independent experiments (n=18). Tukey-HSD 1188 significance groups (p=0.05, confidence level: 0.95) are presented above each boxplot. h: 1189 Representative image of symptoms 10 dpi with the different R1-5244 transconjugants when 1190 inoculated at a low level to observe pathogenicity. Arrows point to pathogenic symptoms in

1191 the strain expressing hopAB3R2-leaf and the EV strain, colour-coded as in g. ANOVA tables 1192 for all statistical analyses are presented in Tables S17-S25. 1193

36 bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, 2018. 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.

1194 Fig. 8: Model highlighting genomic events that have led to the evolution of pathogenicity 1195 towards cherry. a: The core genome phylogeny is presented. Scale bar shows substitutions 1196 per site. For visualisation, clades within the phylogenetic tree have been collapsed with 1197 clades containing cherry pathogens in pink (Psm R1 plum strains in blue). Examples of 1198 cherry pathogens within each clade of phylogroup 2 are named. HGT events predicted using 1199 phylogenetic analysis and RANGER-DTL are shown. b: The key gains and losses of 1200 associated virulence genes in strains pathogenic to cherry are described. Where a gene is 1201 present but not necessarily predicted to be gained in this clade, it is shown with a plus sign, 1202 whilst the use of the words gain or loss specifically denotes results based on GLOOME 1203 analysis. *: The probability of this effector being gained/lost predicted using GLOOME was 1204 slightly lower than 0.8 (see Table S11 for details). For Pss strains within P2, present toxin 1205 biosynthesis gene clusters are shown as dots for comparison. Orange: mangotoxin, blue: 1206 syringopeptin, green: syringolin, pink: syringomycin. 1207 1208

37 certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder bioRxiv preprint

1209 Table 1: List of bacterial strains used in this study. Pathovar designation, phylogroup, isolation information, cherry pathogenicity (reference doi: 1210 for when tested and Nt for not tested) and reference for publication of genome sequence are included. NCBI accession numbers are also listed. https://doi.org/10.1101/244715 1211 Strains in bold were considered pathogenic in cherry. # The pathogenic status of MAFF302280 on cherry is debated. This strain is reported to be 1212 the pathotype strain of P.s pv. morsprunorum (Sawada et al., 1999), so is assumed to be equivalent to CFBP 2351, NCPPB2995, ICMP5795 and 1213 LMG5075. The strain NCPPB2995 was reported to be potentially non-pathogenic (Gardan et al., 1999). In comparison, the ‘same’ strain 1214 LMG5075 tested positive for pathogenicity in a recent publication (Gilbert et al., 2009). There is no definite link showing that MAFF302280 is 1215 the same strain as the others listed as it is not linked to them in online databases (http://www.straininfo.net/) or taxonomy-focused publications a CC-BY-NC-ND 4.0Internationallicense ;

1216 (Bull et al., 2010). It is assumed to be putatively pathogenic in this study due to its close relatedness to other Psm R2 strains, however further this versionpostedJanuary8,2018. 1217 pathogenicity tests would be required to fully confirm this.

Strain Pathovar Race PG Isolation source Isolator Prunus cv. Sequenced Pathogenicity BioProject/accession tested on cherry (Prunus avium) avii5271 avii 1 Prunus avium Garrett, Wild This study Vicente et al., 2004 NBAO00000000 1990, UK cherry

R1-5270 morsprunorum 1 3 Prunus avium Garrett, Wild This study Vicente et al., 2004 NBAN00000000 1990, UK cherry

The copyrightholderforthispreprint(whichwasnot R2-7968A morsprunorum 2 1 Prunus avium Vicente, Wild This study Vicente et al., 2004 NBAI00000000 . 2000, UK cherry R2-9095 morsprunorum 2 1 Prunus avium Roberts, Wild This study M. Hulin, pers. obs. MLED00000000 2010, UK cherry syr5264 syringae 2 Prunus avium Garrett, 1990 Wild This study Vicente et al., 2004 NBAQ00000000 UK cherry syr5275 syringae 2 Prunus avium Garrett, 1990 Wild This study Vicente et al., 2004 NBAP00000000 UK cherry syr7928A syringae 2 Prunus avium Vicente, Wild This study Vicente et al., 2004 NBAL00000000 2000, UK cherry syr8094A syringae 2 Prunus avium Vicente, Wild This study Vicente et al., 2004 NBAK00000000 2001, UK cherry Ps-7928C unknown 2 Prunus avium Vicente, 2000, Wild cherry This study Vicente et al., 2004 NBAM00000000 UK

1 certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder bioRxiv preprint

Ps-7969 unknown 2 Prunus avium Vicente, 2000, Wild cherry This study Vicente et al., 2004 NBAJ00000000

UK doi: syr2675 syringae 2 vulgaris 1965, Kenya This study This study MLEX00000000 https://doi.org/10.1101/244715 syr2676 syringae 2 Phaseolus vulgaris 1990, Lesotho This study This study MLEY00000000 syr2682 syringae 2 Phaseolus vulgaris 1990, Lesotho This study This study MLFA00000000 syr3023 syringae 2 vulgaris 1950, UK This study This study MLFD00000000 syr100 syringae 2 Phaseolus lunatus 1962, Kenya This study This study MLEV00000000

R1-5244 morsprunorum 1 3 Prunus avium Cross, 1960, Unknown Hulin et al., 2018 Hulin et al., 2018 MLEB00000000

UK a CC-BY-NC-ND 4.0Internationallicense ;

R1-5300 morsprunorum 1 3 Prunus domestica Prunier, UK Victoria Hulin et al., 2018 Hulin et al., 2018 MLEN00000000 this versionpostedJanuary8,2018. R1-9326 morsprunorum 1 3 Prunus domestica Roberts, 2011, Victoria Hulin et al., 2018 Hulin et al., 2018 MLEO00000000 UK R1-9629 morsprunorum 1 3 Prunus domestica Roberts, 2012, Victoria Hulin et al., 2018 Hulin et al., 2018 MLEP00000000 UK R1-9646 morsprunorum 1 3 Prunus avium Roberts, Stella Hulin et al., 2018 Hulin et al., 2018 MLEE00000000 2012, UK R1-9657 morsprunorum 1 3 Prunus avium Roberts, 2012, Kiku- Hulin et al., 2018 Hulin et al., 2018 MLEF00000000 UK Shidare R2-5255 morsprunorum 2 1 Prunus avium Prunier, UK Napoleon Hulin et al., 2018 Hulin et al., 2018 MLEC00000000 R2-5260 morsprunorum 2 1 Prunus avium Garrett, UK Roundel Hulin et al., 2018 Hulin et al., 2018 MLEG00000000 R2-leaf morsprunorum 2 1 Prunus avium Hulin, 2014, Napoleon Hulin et al., 2018 Hulin et al., 2018 MLEH00000000

UK The copyrightholderforthispreprint(whichwasnot R2-SC214 morsprunorum 2 1 Prunus avium Roberts, Wild Hulin et al., 2018 Hulin et al., 2018 MLEI00000000 . 1983, UK cherry syr9097 syringae 2 Prunus avium Roberts, Unknown Hulin et al., 2018 Hulin et al., 2018 MLEJ00000000 2010, UK syr9293 syringae 2 Prunus domestica Roberts, Victoria Hulin et al., 2018 Hulin et al., 2018 MLEQ00000000 2011, UK syr9630 syringae 2 Prunus domestica Roberts, Victoria Hulin et al., 2018 Hulin et al., 2018 MLER00000000 2012, UK syr9644 syringae 2 Prunus avium Roberts, Stella Hulin et al., 2018 Hulin et al., 2018 MLEK00000000 2012, UK syr9654 syringae 2 Prunus domestica Roberts, Victoria Hulin et al., 2018 Hulin et al., 2018 MLES00000000 2012, UK syr9656 syringae 2 Prunus avium Roberts, Kiku- Hulin et al., 2018 Hulin et al., 2018 MLEM00000000 2012, UK Shidare

2 certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder bioRxiv preprint

syr9659 syringae 2 Prunus avium Roberts, Kiku- Hulin et al., 2018 Hulin et al., 2018 MLEL00000000

2012, UK Shidare doi: Ps-9643 unknown 1 Prunus domestica Roberts, 2012, Victoria Hulin et al., 2018 Hulin et al., 2018 MLET00000000 UK https://doi.org/10.1101/244715 avii3846 avii 1 Prunus avium 1991, France Wild Nowell et al., 2016 Ménard et al., 2003 LIIJ00000000 cherry R1-2341 morsprunorum 1 3 Prunus cerasus 1988, Unknown Nowell et al., 2016 Nowell et al., 2016 LIIB00000000 Hungary R1-5269 morsprunorum 1 3 Prunus cerasus Garrett, Wild Nowell et al., 2016 Vicente et al., 2004 LIHZ00000000 1990, UK cherry R2-5261 morsprunorum 2 1 Prunus avium Garrett, UK Roundel Nowell et al., 2016 Vicente et al., 2004 LIIA00000000 R2-302280 morsprunorum 1 Prunus domestica USA Unknown Baltrus et al., 2011 Gilbert et al., 2009# AEAE00000000

syr2339 syringae 2 Prunus avium 1984, Unknown Nowell et al., 2016 Nowell et al., 2016 LIHU00000000 a CC-BY-NC-ND 4.0Internationallicense ;

Hungary this versionpostedJanuary8,2018. syr7872 syringae 2 Prunus domestica Lewis, 2000, Opal Nowell et al., 2016 Vicente et al., 2004 LIHS00000000 UK syr7924 syringae 2 Prunus avium Vicente, Wild Nowell et al., 2016 Vicente et al., 2004 LIHR00000000 2000, UK cherry acer302273 aceris 2 Acer sp. USA Baltrus et al., 2011 Nt AEAO00000000 acti18884 actinidiae 1 2010, NZ McCann et al., 2013 Nt AOKO00000000 acti19073 actinidiae 1 Actinidia deliciosa 1998, Korea McCann et al., 2013 Nt AOJR00000000 acti212056 actinidiae 1 Actinidia deliciosa 2012, Japan Sawada et al., 2014 Nt BBWG00000000 acti302091 actinidiae 1 Actinidia deliciosa 1984, Japan Baltrus et al., 2011 Nt AEAL00000000 actiCRAFRU actinidiae 1 Actinidia deliciosa 2010, Italy Butler et al., 2013 Nt ANGD00000000 The copyrightholderforthispreprint(whichwasnot actiNCPPB3871 actinidiae 1 Actinidia deliciosa 1992, Italy Marceletti et al., 2011 Nt ANGD00000000 . aes089323 aesculi 3 Aesculus India, 1980 Baltrus et al., 2011 Nt AEAD00000000 hippocastanum aes2250 aesculi 3 Aesculus 2008, UK Green et al., 2010 Nt ACXT00000000 hippocastanum aes3681 aesculi 3 Aesculus 1969, India Green et al., 2010 Nt ACXS00000000 hippocastanum amy3205 amygdali 3 Prunus dulcis 1967, Greece Bartoli et al., 2015b Nt JYHB00000000 amyICMP3918 amygdali 3 Prunus dulcis Panagopoulos, Thakur et al., 2016 Nt LJPQ00000000 1967, Greece avelBP631 avellanae 1 Corylus avellana 1976, Greece O'Brien et al., 2012 Hulin et al., 2018 AKBS00000000 avelVe037 avellanae 2 Corylus avellana 1990, Italy O'Brien et al., 2012 Nt AKCJ00000000

3 certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder bioRxiv preprint

BRIP34876 unknown 2 Hordeum vulgar 1971, Gardiner et al., 2013 Nt AMXK00000000

Australia doi: castCFBP4217 castaneae 3 Castanea crenata 1977, Japan Nowell et al., 2016 Nt LIIH00000000 https://doi.org/10.1101/244715 CC1416 unknown 1 Epilithon USA Baltrus et al., 2014 Nt AVEP00000000 CC1544 unknown 1 Lake water France Baltrus et al., 2014 Nt AVEI00000000 CC1559 unknown 1 France Baltrus et al., 2014 Nt AVEG00000000 CC94 unknown 2 Cantaloupe France Baltrus et al., 2014 Nt AVEA00000000 cera6109 cerasicola 3 Prunus yedoensis 1995, Japan Nowell et al., 2016 Nt LIIG00000000 ceraICMP17524 cerasicola 3 Prunus yedoensis Japan Thakur et al., 2016 Nt LJQA00000000 ciccICMP5710 ciccaronei 3 Ceratonia siliqua Italy Thakur et al., 2016 Nt LJPY00000000 a CC-BY-NC-ND 4.0Internationallicense ; cunnICMP11894 cunninghamiae 3 Cunninghamia China Thakur et al., 2016 Nt LJQE00000000 this versionpostedJanuary8,2018. lanceolata daphICMP9757 daphniphylli 3 Daphniphyllum Japan Thakur et al., 2016 Nt LJQF00000000 teijsmannii delphi569 delphinii 1 Delphinium sp. NZ Thakur et al., 2016 Nt LJQH00000000 dendro3226 dendropanacis 3 Dendropanax 1979, Japan Bartoli et al., 2015b Nt JYHG00000000 trifidus dendro4219 dendropanacis 3 Dendropanax 1981, Japan Bartoli et al., 2015b Nt JYHD00000000 trifidus dendro9150 dendropanacis 3 Dendropanax Japan Thakur et al., 2016 Nt LJQG00000000 trifidus erio4455 eriobotryae 3 Eriobotrya japonica USA Thakur et al., 2016 Nt LJQI00000000

The copyrightholderforthispreprint(whichwasnot

glyR4 glycinea 3 Glycine max Cross, 1960, Qi et al., 2011 Nt AEGH00000000 . USA ICMP19498 unknown 3 Actinidia deliciosa 2010, NZ Visnovsky et al., 2016 Nt LKCH00000000 lach301315 lachrymans 3 Cucumis sativus Japan Baltrus et al., 2011 Nt AEAF00000000 lach302278 lachrymans 1 Cucumis sativus USA Baltrus et al., 2011 Nt AEAM00000000 lapsaICMP3947 lapsa 2 Zea sp. Unknown Thakur et al., 2016 Nt LJQQ00000000 meli6289 meliae 3 Melia azedarach Japan Thakur et al., 2016 Nt LJQT00000000 morsU7805 morsprunorum 3 Prunus mume Unknown Mott et al., 2016 Nt LGLQ00000000 myriAZ8448 myricae 3 Myrica rubra Japan Thakur et al., 2016 Nt LGLA00000000 neriiICMP16943 savastanoi 3 Olea europea Spain Thakur et al., 2016 Nt LJQW00000000

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paniLMG2367 panici 2 miliaceum Unknown Liu et al., 2012 Nt ALAC00000000 papu1754 papulans 2 1973, Canada Nowell et al., 2016 Nt JYHI00000000 doi: persNCPPB2254 persicae 1 Prunus persica 1972, France Zhao et al., 2015 Nt LAZV00000000 https://doi.org/10.1101/244715 photICMP7840 photiniae 3 Photinia glabra Japan Thakur et al., 2016 Nt LJQO00000000 pisiPP1 pisi 2 Pisum sativum Japan Baltrus et al., 2014b Nt AUZR00000000 phas1448a phaseolicola 3 Phaseolus vulgaris Teverson, Joardar et al., 2005 Hulin et al., 2018 CP000058 1965, Ethiopia rhapCFBP4220 rhaphiolepidis 3 Rhaphiolepis 1980, Japan Nowell et al., 2016 Nt LIHV00000000 umbellata RMA1 unknown 1 Aquilegia vulgaris Jackson, Hulin et al., 2018 Hulin et al., 2018 MLEU00000000 a CC-BY-NC-ND 4.0Internationallicense 2012, UK ; sava3335 savastanoi 3 Olea europea Stead, France Rodriguez-Palenzuela Nt ADMI00000000 this versionpostedJanuary8,2018. et al., 2010 sava4352 savastanoi 3 Olea europea Yugoslavia Thakur et al., 2016 Nt LGKR00000000 savaDAPP- savastanoi 3 Olea europea Italy Moretti et al., 2014 Nt JOJV00000000 PG722 savaPseNe107 savastanoi 3 Olea europea Balestra, bartoli et al., 2015b Nt JYHF00000000 Nepal soliICMP16925 solidagae 2 Solidago altissima Japan Thakur et al., 2016 Nt JYHF00000000 syr1212 syringae 2 Pisum sativum UK Baltrus et al., 2014 This study AVCR00000000 syr2340 syringae 2 Pyrus sp. 1985, Nowell et al., 2016 Nt LIHT00000000 Hungary syr41a syringae 2 Prunus armeniaca 2011, France Bartoli et al., 2015b Nt JYHJ00000000 The copyrightholderforthispreprint(whichwasnot . syrB301D syringae 2 Pyrus communis Garrett, 1959, Ravindran et al., 2015 Nt CP005969 UK syrB64 syringae 2 Triticum aestivum Wilcoxson, Dudnik and Dudler Nt ANZF00000000 USA 2013 syrB728a syringae 2 Phaseolus vulgaris 1987, USA Feil et al., 2005 This study CP000075 syrHS191 syringae 2 Panicum miliaceum Hayward, Ravindran et al., 2015 Nt CP006256 Australia, 1969 syrUMAF0158 syringae 2 Mangifera indica Cazorla, 2010, Martínez-García et al., Nt CP005970 Spain 2015 thea3923 theae 1 Camelia sinensis 1974, Japan Mazzaglia et al., 2012 Nt AGNN00000000 tomaDC3000 tomato 1 Solanum 1960, UK Buell et al., 2003 Nt AE016853

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lycopersicum tomaT1 tomato 1 Solanum 1986, Canada Almeida et al., 2009 Nt ABSM00000000 doi:

lycopersicum https://doi.org/10.1101/244715 UB303 unknown 2 Lake water France Baltrus et al., 2014 Nt AVDZ00000000 ulmiICMP3962 ulmi 3 Ulmus sp. Yugoslavia Thakur et al., 2016 Nt LJRQ00000000 USA007 unknown 1 Stream water USA Baltrus et al., 2014 Nt AVDY00000000 USA011 unknown 1 Stream water USA Baltrus et al., 2014 Nt AVDX00000000

a CC-BY-NC-ND 4.0Internationallicense ; this versionpostedJanuary8,2018. The copyrightholderforthispreprint(whichwasnot .

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1218 Table 2: Assembly statistics for all strains in this study and by Hulin et al. (2018). Cherry pathogens are in bold. N50: The weighted median doi: 1219 contig size in the assembly. Features: The number of protein encoding and RNA sequences in the annotated genome. https://doi.org/10.1101/244715

Assembly Sequencing Reference No. contigs Plasmids Total length GC N50 Average coverage Features % R1-5270 Illumina This study 185 3 6258313 58.10 202152 134 5770 R1-5244 Illumina Hulin et al., 2018 198 4 6302385 58.08 227422 265 5810 R1-5300 Illumina Hulin et al., 2018 201 4 6342586 57.88 142021 180 5844 R1-9326 Illumina Hulin et al., 2018 268 4 6353636 57.91 142021 81 5874 a CC-BY-NC-ND 4.0Internationallicense ; R1-9629 Illumina Hulin et al., 2018 216 3 6341664 57.94 142021 172 5856 this versionpostedJanuary8,2018. R1-9646 Illumina Hulin et al., 2018 171 3 6302776 58.03 235429 180 5801 R1-9657 Illumina Hulin et al., 2018 191 4 6317852 57.91 145272 158 5848 R2-5255 Illumina Hulin et al., 2018 206 2 6448834 58.38 102760 112 5966 R2-7968A Illumina This study 278 6 6498711 58.42 91262 134 6016 R2-5260 Illumina Hulin et al., 2018 223 3 6495620 58.41 101794 458 5995 R2-9095 Illumina This study 304 2 6418849 58.48 92453 100 5887 R2-leaf Illumina Hulin et al., 2018 203 5 6366714 58.48 100658 180 5846 R2-SC214 Illumina Hulin et al., 2018 203 3 6253818 58.56 108341 180 5747 The copyrightholderforthispreprint(whichwasnot avii5271 Illumina This study 352 6 6243644 58.56 56064 127 5809 . Ps-9643 Illumina Hulin et al., 2018 58 1 5937102 58.78 243355 212 5386 syr9097 Illumina Hulin et al., 2018 66 0 5892389 59.35 316078 158 5117 syr9293 Illumina Hulin et al., 2018 73 0 6135031 58.84 557853 196 5302 syr9630 Illumina Hulin et al., 2018 57 0 5940819 59.33 347701 206 5175 syr9644 Illumina Hulin et al., 2018 75 1 6173193 59.13 251053 208 5334 syr9654 Illumina Hulin et al., 2018 49 0 5941610 59.37 245023 147 5148 syr9656 Illumina Hulin et al., 2018 39 0 5980728 59.10 1007808 205 5184 syr9659 Illumina Hulin et al., 2018 51 0 5943090 59.37 235830 116 5148

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syr5264 Illumina This study 59 0 6029896 59.08 380149 114 5314 syr5275 Illumina This study 64 1 5994091 59.30 371492 145 5207 doi: syr7928A Illumina This study 59 1 6129363 59.26 371492 141 5338 https://doi.org/10.1101/244715 syr8094A Illumina This study 71 0 5942438 59.33 265238 106 5184 syr7928C Illumina This study 49 1 5994455 59.17 325175 124 5318 syr7969 Illumina This study 92 0 6185932 59.01 164374 151 5476 RMA1 Illumina Hulin et al., 2018 95 1 6306889 58.73 187448 320 5825 syr100 Illumina This study 23 0 5872916 59.33 893822 83 5140 syr2675 Illumina This study 65 0 5994384 59.34 227612 83 5177 a CC-BY-NC-ND 4.0Internationallicense ; syr2676 Illumina This study 90 1 6158476 59.30 259660 78 5387 this versionpostedJanuary8,2018. syr2682 Illumina This study 185 1 6259099 59.21 242212 84 5405 syr3023 Illumina This study 228 0 6203212 58.90 456738 88 5365 R1-5244 PacBio This study 5 4 6445963 58.05 6109228 82 6024 R2-leaf PacBio This study 6 5 6576340 58.41 6242845 141 6093 syr9097 PacBio This study 1 0 5929959 59.30 5929959 100 5147 R1-5300 MinION This study 6 4 6645601 57.87 5688034 100 6449

The copyrightholderforthispreprint(whichwasnot .

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Table 3: List of effectors in genomes sequenced using PacBio/Minion methods. Effectors are listed in order of appearance on each assembly doi: contig (labelled as chromosomal or plasmid). Where effectors could be considered as linked (within 10kb of each other) they are underlined. * https://doi.org/10.1101/244715 indicates effectors within the conserved effector locus. #: Effector gene is disrupted and is labelled as a pseudogene.

R1-5244 Contig Length Effectors Chromosome tig0 6109228 hopAZ1, hopA2#, avrE1, hopM1#, hopAA1# *, hopZ4, hopAT1, hopQ1, hopD1, hopR1, hopF4, hopBL2, hopAV1, hopAO2#, hopAY1, #

hopF3, hopAS1, hopI1, hopAE1, hopAF1-2, hopAU1, hopAH1, hopV1, hopAR1, hopBK1 a CC-BY-NC-ND 4.0Internationallicense ; Plasmid tig3 168854 hopAF1-1, hopBF1, avrD1, avrRpm2, hopBD1 this versionpostedJanuary8,2018. Plasmid tig4 81536 hopA1 Plasmid tig5 45535 - Plasmid tig6 40810 - R1-5300 Contig Length Effectors Chromosome tig0 5688034 hopV1, hopAZ1, avrA1, hopQ1-2, hopA2#, avrE1, hopM1#, hopAA1*, hopAB1, hopQ1, hopD1, hopR1, hopAO2#, avrRpm2, avrPto1, hopAS1, hopAT1#, hopBL2#, hopI1, hopAE1, hopAF1-2, hopF3, hopAY1#, hopAU1, hopAH1 Chromosome tig75 697453 hopW1, hopBK1#, hopAR1 Plasmid tig46 52059 - The copyrightholderforthispreprint(whichwasnot Plasmid tig65 47809 hopX1, hopBB1, hopG1 . Plasmid tig84 57689 hopAO1# Plasmid tig113 102557 avrD1 R2-leaf Contig Length Effectors Chromosome tig0 6242845 hopY1, hopAS1, hopAT1, hopH1, hopF4, hopW1, hopR1, hopAG1#, hopAH1-2, hopAI1, hopN1, hopAA1#, hopM1, avrE1*, hopF2, hopE1, hopA2, hopAH1-1, hopAH1-1, hopAB3#, avrRps4, hopS2, hopI1, hopAR1 Plasmid tig5 102862 hopAO1#, hopAZ1, hopAY1 Plasmid tig4 97840 hopD1#, hopAU1 Plasmid tig6 69519 hopAF1-1, hopBF1

Plasmid tig8 42783 hopBB1, hopBD1 Plasmid tig9 20491 avrB2, hopX1 9 syr9097 Contig Length Effectors certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder bioRxiv preprint

Chromosome tig0 5929959 hopAG1, hopAH1, hopA1I, avrRpm1, hopAR1, hopI1, hopAE1, hopBE1, hopAF1, hopAH1, hopAW1#, hopH1, hopA2, avrE1, hopM1,

hopAA1* doi:

https://doi.org/10.1101/244715 a CC-BY-NC-ND 4.0Internationallicense ; this versionpostedJanuary8,2018. The copyrightholderforthispreprint(whichwasnot .

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doi:

https://doi.org/10.1101/244715 Table 4: List of putative horizontal gene transfer events that have occurred between Prunus-infecting clades within P. syringae. Where the effector gene is present in the PacBio- or Minion-sequenced strains its chromosomal or plasmid location is indicated. #: Effector gene is disrupted and is labelled as a pseudogene.

Predicted in

Effector Putative transfers Region Plasmid location RangerDTL a CC-BY-NC-ND 4.0Internationallicense ; avrD1 R1/R2/P.s pv. avii Plasmid tig3 (R1-5244) Y this versionpostedJanuary8,2018. avrRps4# R2/P.s pv. avii Next to cluster of mobile elements - Y hopAF1 R1/R2/P.s pv avii Plasmid tig3 (R1-5244), tig6 (R2) Y tig5(R2), tig84 (R1- N hopAO1 R2/P.s pv avii/R1-5300 Plasmid 5300) hopAT1 R1/R2 Genomic island - N hopAU1 R2/P.s pv. avii Plasmid tig4 (R2) Y hopAY1 R2/P.s pv. avii Plasmid tig5 (R2) Y tig8 (R2), tig65 (R1- Y The copyrightholderforthispreprint(whichwasnot

hopBB1 R1/R2/P.s pv. avii Plasmid 5300) . hopBD1 R2/P.s pv. avii Plasmid tig3 (R1-5244), tig8 (R2) Y hopBF1 R1/R2/P.s pv. avii Plasmid tig3 (R1-5244), tig6 (R2) Y hopD1 R2/P.s pv. avii Plasmid tig4(R2) N hopO1 R2/P.s pv. avii Next to cluster of mobile elements (next to hopT1) - Y hopT1 R2/P.s pv. avii Next to cluster of mobile elements (next to hopO1) - Y

11 bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, 2018. 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.

● syr9097 ● syr7872 96 ● syr8094A syrB728a 64 ● syr9654 ● syr9659 76 ● syr5275 syr2682 83 syr2676 79 syr1212 syr2675 USA011 76 acer302273 soli16925 syr2340 2d CC94 ● syr2339 53 ● syr7924 51 UB303 syrB301D ● syr9644 ● syr9656 96 ●syr9293 syr41a 38 laps3947 ● syr5264 syr3023 syrB64 BRIP34876 pani2367 Phylogroup 2 pisiPP1 2b syrHS191 avelVe037 Ps7969 Ps7928C ● syr7928A syr100 syrUMAF0158 papu1754 savaPseNe107 sava4352 savaPG722 nerii16943 sava3335 79 cicc5710 cunn11894 daph9757 79 amyg3205 ● R1-2341 73 ● R1−9646 ● R1−5270 ● R1−5244

72 87 R1−9657 R1-5300 cast4217 myricaeAZ8448 76 rhap4220 dendro3226 dendro9150 phot7840 aes3681 aes089323 aesc2250 ulmi3962 56 meliae6289 cera6109 cera17524 morsU7805 phas1448A glycR4 Phylogroup 3 lach301315 amyg3918 ICMP19498 ● R2-302280 ● R2-9095 ● R2−7968A ● R2−leaf ● R2−sc214 ● R2−5255 ● R2-5261 ● R2-5260 act18884 actCRAFRU 99 act3871 act302291 act212056 80 act19073 thea3923 avelBP631 CC1559 1b delphi569 RMA1 CC1544 USA007 Phylogroup 1 CC1416 tomaDC3000 pers2254 lach302278 1a 96 tomaT1 Ps9643 0.01 ● avii3846 ● avii5271

● Pathogenic on cherry certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder bioRxiv preprint doi: https://doi.org/10.1101/244715 a

avrB3avrA1 avrB4avrB2 avrD1avrE1 avrPto1avrB3 avrPto2avrB4 avrPto3avrD1 avrPto4avrE1 avrRpm1avrPto5avrPto1 avrRpm2avrRps4avrPto2 avrRpt2 a avrPto3hopA1 CC-BY-NC-ND 4.0Internationallicense hopAA1avrPto4hopA2 ; this versionpostedJanuary8,2018. hopAB1avrPto5 avrRpm1hopAB2 avrRpm2hopAD1hopAB3 avrRps4hopAE1hopAF1 hopAG1hopAH1 avrRpt2hopAL1hopAI1 hopAM1hopAO1hopA1 hopAO2hopA2 hopAQ1hopAR1hopAA1 hopAS1hhopAT1opAB1 hopAU1hopAV1 hopAW1hopAX1hopAB2 hopAZ1hopAY1 hopBA1hopABhopB1 3 hopBB1hopAD1 hopBC1hopAE1 hopBD1hopAF1 hopBD2 hopBE1hopBF1hopAG1 hopBG1hopBH1 hopAH1hopBJ1hopBI1 hopBK1hopBL1hopAI1 hopBL2hopAL1hopC1 hWHOP INA hopD1opAM1hopE1IAA−H EFE hopAO1hopF1IAAIAA−M−L R2−5260 R2−5261 R2−5255 R2−leaf R2−7968A R2−9095 R2−302280 R2−sc214 avii5271 avii3846 Ps9643 Ps7969 Ps7928C syr7928A syr9293 syr9656 syr5264 syr9644 syr7924 syr2339 syr8094A syr7872 syr9097 syr5275 syr9659 syr9654 R1−5244 R1−5270 R1−9646 R1−2341 R1−5300 R1−9657* hopAO2hopF2 act19073 act212056 acti302091 acti3871 actiCRAFRU act18884 thea3923 avelBP631 delphi569 CC1559 CC1544 RMA1 CC1416 USA007 pers2254 tomaDC3000 lach302278 tomaT1 syrUMAF0158 syr100 papu1754 avelVe037 syrHS191 pisiPP1 pani2367 BRIP34876 syrB64 syr3023 syr41a laps3947 syrB301D UB303 CC94 syr2340 soli16925 acer302273 USA011 syrB728a syr2675 syr1212 syr2676 syr2682 ICMP19498 amyg3918 lach301315 glycR4 phas1448A meliae6289 ulmi3962 aes2250 aes089323 aes3681 morsU7805 cera17524 cera6109 phot7840 dendro9150 dendro3226 rhap4220 myricaeAZ8448 cast4217 cicc5710 sava3335 savaPG722 sava4352 savaPseNe107 nerii16943 cunn11894 amyg3205 daph9757 * * hopAQ1hopF3 * hopAR1hopF4 hopG1hopH1 hopAS1 hopH3hopI1 hopAT1hopM1hopK1 hopAU1hopO1hopN1 hopAV1hopQ1hopR1 hopAW1hopS1 The copyrightholderforthispreprint(whichwasnot hopS2hopT1

hopAX1 . hopAY1hopT2 hopU1hopV1 hopW1hopAZ1hopX1 hopX2hopB1 hopBA1hopY1hopZ1 hopBB1hopZ2 hopBC1hopZ3 hopBD1hopZ4 hopBD2 Syringomycin hopZ5 SyringopeptinSyringolinhopBE1 MangotoxinCoronatine hopBF1 Phaseolotoxin Tabtoxin hopBG1 hopBH1 hopBI1 hopBJ1 hopBK1 hopBL1 hopBL2 hopC1 hopD1 hopE1 hopF1 hopF2 hopF3 hopF4 hopG1 hopH1 hopH3 hopI1 hopK1 hopM1 hopN1 hopO1 hopQ1 hopR1 hopS1 hopS2 hopT1 hopT2 hopU1 hopV1 hopW1 hopX1 hopX2 hopY1 hopZ1 hopZ2 hopZ3 hopZ4 hopZ5 Syringomycin Syringolin Syringopeptin Coronatine Mangotoxin Tabtoxin Phaseolotoxin WHOP INA EFE IAA−H IAA−L IAA−M myriAZ8448 act19073 act212056 acti302091 acti3871 actiCRAFRU act18884 thea3923 R2−5260 R2−5261 R2−5255 R2−leaf R2−7968A R2−9095 R2−302280 R2−sc214 avelBP631 delphi569 CC1559 CC1544 RMA1 CC1416 USA007 avii5271 avii3846 pers2254 tomaDC3000 lach302278 Ps9643 tomaT1 syrUMAF0158 syr100 papu1754 Ps7969 avelVe037 Ps7928C syr7928A syrHS191 pisiPP1 pani2367 BRIP34876 syrB64 syr3023 syr9293 syr9656 syr41a syr5264 laps3947 syr9644 syrB301D UB303 syr7924 syr2339 CC94 syr2340 soli16925 acer302273 USA011 syr8094A syr7872 syr9097 syrB728a syr5275 syr9659 syr9654 syr2675 syr1212 syr2676 syr2682 ICMP19498 amyg3918 lach301315 glycR4 phas1448A meliae6289 ulmi3962 aes2250 aes089323 aes3681 morsU7805 cera17524 cera6109 phot7840 dendro9150 dendro3226 rhap4220 cast4217 R1−5244 R1−5270 R1−9646 R1−2341 R1−5300 R1−9657 cicc5710 sava3335 savaPG722 sava4352 savaPseNe107 nerii16943 cunn11894 amyg3205 daph9757 * * * *

b strain

Number ofeffectorgenes ●

10

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●● 40 ● bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, 2018. 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.

a Likelihood of orthogroup presence/absence being correlated with cherry pathogenicity b

30 Psa + hopBF1* + hopAR1 + hopBB1

Gained Lost

P1 + hopH1 + hopBF1 20 + hopAR1 + hopBB1 Psm R2

P3 Likelihood ratio Likelihood

10 + hopBF1* + hopAR1 Psm R1 - hopAB1 - avrPto1* - hopBB1 in two strains

P2a 0 avrD hopI avrA avrB hopT hopZ hopF hopY hopS hopK hopE hopX hopB hopV hopU hopN hopC hopD hopR hopH hopO hopG hopQ hopM hopA avrE1 hopBI hopW hopAI + hopBF1* hopAT hopBJ avrPto hopBL hopAL hopAY hopAV hopAF hopAZ hopBF hopBK hopAX hopBA hopAS hopAA hopBE hopAE hopBB hopAB hopAU hopAD hopBH hopBC hopAH hopBD hopAR hopAQ hopAG hopAO avrRpt hopBG hopAM Pss P2 hopAW avrRps avrRpm c Effector family avrPto1 + hopAR1* avrPto2 avrPto3 avrPto4 P2d avrPto5 hopAB1 hopAB2 hopAB3 hopAR1 hopBB1 hopBF1 hopH1

hopH3 act19073 act212056 acti302091 acti3871 actiCRAFRU act18884 thea3923 R2 − 5260 R2 − 5261 R2 − 5255 R2 − leaf R2 − 7968A R2 − 9095 R2 − 302280 R2 − sc214 avelBP631 delphi569 CC1559 CC1544 RMA1 CC1416 USA007 avii5271 avii3846 pers2254 tomaDC3000 lach302278 Ps9643 tomaT1 syrUMAF0158 syr100 papu1754 Ps7969 avelVe037 Ps7928C syr7928A syrHS191 pisiPP1 pani2367 BRIP34876 syrB64 syr3023 syr9293 syr9656 syr41a syr5264 laps3947 syr9644 syrB301D UB303 syr7924 syr2339 CC94 syr2340 soli16925 acer302273 USA011 syr8094A syr7872 syr9097 syrB728a syr5275 syr9659 syr9654 syr2675 syr1212 syr2676 syr2682 ICMP19498 amyg3918 lach301315 glycR4 phas1448A meliae6289 ulmi3962 aes2250 aes089323 aes3681 morsU7805 cera17524 cera6109 phot7840 dendro9150 dendro3226 rhap4220 myricaeAZ8448 cast4217 R1 − 5244 R1 − 5270 R1 − 9646 R1 − 2341 R1 − 5300 R1 − 9657 cicc5710 sava3335 savaPG722 sava4352 savaPseNe107 nerii16943 cunn11894 amyg3205 daph9757 * * * *

0.03 bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, 2018. 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.

aA

syr9097 syr7872 syr9097 syr8094A syr9097 syr7872 syrB728a syr7872 syr8094A syr9654syr9097 syr8094A syrB728a syr7872 syrB728a syr9097syr9654 syr9659 syr9097syr9654 syr7872syr9659 syr5275syr8094A syr7872 syr8094A syr2682syrB728a syr9659 syr5275 syr9654 syr8094Asyr5275 syrB728asyr2682 A syr2676 syrB728asyr2682 syr9654syr2676 syr9659syr1212 syr5275 syr9654syr2676 syr9659syr1212 syr2675 syr9659syr1212 syr5275syr2675 syr2682USA011 syr5275syr2675 syr2682USA011 syr2676acer302273 syr2682USA011 syr2676acer302273 avrD1 syr1212soli16925 hopBB1 syr2676acer302273 hopBF1 syr1212soli16925 syr2340syr2675 syr1212soli16925 syr2675syr2340 CC94USA011 syr2675 USA011 acer302273 syr2340 CC94 syr2339syr9097 CC94USA011 syr2339syr9097acer302273 avrD1 syr7924syr7872soli16925 hopBB1 syr2339acer302273 hopBF1 syr7924soli16925 syr2340 syr9097 syr7872 A UB303syr8094A syr7924syr7872soli16925 syr2340UB303syr8094A syrB301DCC94syrB728a UB303syr2340syr8094A CC94syrB301DsyrB728a syr9644syr2339syr9654 CC94syrB301DsyrB728a syr9644syr2339syr9654 syr7924syr9659syr9656 syr9644syr2339syr9654 syr7924syr9659syr9656 UB303syr5275syr9293 syr7924syr9659syr9656 UB303syr5275syr9293 syrB301Dsyr2682syr41a UB303syr5275syr9293 syrB301Dsyr2682syr41a syr9644syr2676 syrB301Dsyr41a syr9644 laps3947 syr9644syr2682 syr2676laps3947 syr1212syr9097syr5264syr9656 syr2676laps3947 syr1212syr9656syr5264 syr2675syr3023syr7872syr9293 syr1212syr9656syr5264 syr2675syr9097syr3023syr9293 syrB64USA011syr8094Asyr41a syr2675syr9097syr3023syr9293 syr7872USA011syr41asyrB64 syrB728aBRIP34876acer302273laps3947 USA011syr7872syrB64syr41a syr8094Aacer302273laps3947BRIP34876 avrD1 syr9654syr5264soli16925 hopBB1 syr8094Aacer302273laps3947BRIP34876 hopBF1 syrB728apani2367syr5264soli16925 syr2340pani2367 syrB728asyr5264 syr9654syr3023 pisiPP1syr3023syr9659 syr9654pani2367soli16925 syr2340pisiPP1syr9659 CC94syrB64syr5275 syr2340pisiPP1syr3023 syrHS191CC94syrB64 syrHS191syr2339syr2682BRIP34876 syrHS191CC94syrB64syr9659 syr2339syr5275BRIP34876 avelVe037 syr5275BRIP34876 avelVe037syr2682 syr7924pani2367syr2676 avelVe037syr2339syr2682 Ps7969syr7924pani2367syr2676 Ps7969UB303syr1212 syr7924pani2367 UB303pisiPP1 Ps7928CpisiPP1 Ps7969pisiPP1syr2676 Ps7928Csyr1212 syrHS191syrB301Dsyr2675 Ps7928CUB303syr1212 syr7928AsyrHS191syrB301Dsyr2675 syr7928Asyr9644USA011 syrHS191syrB301D avelVe037syr9644 syr100avelVe037 syr7928AavelVe037syr2675 syr100 USA011 Ps7969acer302273syr9656 syr100syr9644USA011 syrUMAF0158Ps7969acer302273syr9656 avrD1 syrUMAF0158Ps7928Csoli16925syr9293 hopBB1 syrUMAF0158Ps7969acer302273syr9656 hopBF1 Ps7928Csoli16925syr9293 papu1754 papu1754syr2340syr41a papu1754Ps7928Csoli16925syr9293 syr7928Asyr2340syr41a syr7928ACC94 syr7928Asyr41a savaPseNe107 savaPseNe107syr100 laps3947 savaPseNe107syr2340 sava4352syr100 CC94laps3947 sava4352 syr2339syr5264 sava4352syr100 CC94laps3947 syrUMAF0158syr2339syr5264 syrUMAF0158syr7924 syrUMAF0158syr2339syr5264 savaPG722syr7924 savaPG722papu1754 syr3023 savaPG722 nerii16943papu1754 syr3023 nerii16943 UB303syrB64 nerii16943papu1754syr7924syr3023 savaPseNe107UB303syrB64 sava3335savaPseNe107syrB301DBRIP34876 savaPseNe107UB303syrB64 sava4352sava3335 syrB301DBRIP34876 sava4352syr9644 sava4352sava3335 syrB301DBRIP34876 cicc5710syr9644 savaPG722cicc5710 pani2367syr9656 cicc5710syr9644 savaPG722 pani2367 cunn11894 pisiPP1 savaPG722cunn11894 pani2367 nerii16943cunn11894pisiPP1syr9656 nerii16943 syr9293 nerii16943 pisiPP1syr9656 daph9757 syrHS191syr9293 daph9757sava3335 syrHS191syr41a sava3335daph9757 syrHS191syr9293 sava3335amyg3205 syr41a amyg3205avelVe037laps3947 amyg3205 syr41a cicc5710avelVe037laps3947 R12341cicc5710 Ps7969 R12341cicc5710avelVe037laps3947 cunn11894R12341 Ps7969 cunn11894 syr5264 cunn11894Ps7969 R19646Ps7928Csyr5264 R19646 Ps7928Csyr3023 R19646 Ps7928Csyr5264 daph9757 syr3023 daph9757syr7928AsyrB64 R15270daph9757 syr3023 R15270amyg3205syr7928AsyrB64 R15270amyg3205 amyg3205syr7928AsyrB64 R15244syr100 R12341R15244syr100 BRIP34876 R12341R15244syr100 R12341R19657 BRIP34876 R19657syrUMAF0158pani2367 R19657 BRIP34876 R19646syrUMAF0158pani2367 R19646 pisiPP1 R19646syrUMAF0158pani2367 R15270R15300papu1754pisiPP1 R15270R15300papu1754 R15270R15300papu1754pisiPP1 savaPseNe107cast4217 savaPseNe107cast4217 syrHS191 savaPseNe107cast4217 R15244 syrHS191 R15244sava4352avelVe037 R15244 syrHS191 R19657sava4352myriAZ8448avelVe037 R19657myriAZ8448 R19657myriAZ8448sava4352avelVe037 savaPG722rhap4220Ps7969 rhap4220savaPG722Ps7969 rhap4220savaPG722Ps7969 R15300 R15300nerii16943Ps7928C R15300dendro3226 dendro3226nerii16943cast4217Ps7928C dendro3226syr7928A nerii16943cast4217Ps7928C dendro9150sava3335syr7928A sava3335cast4217 dendro9150sava3335syr7928A myriAZ8448syr100 dendro9150myriAZ8448cicc5710syr100 phot7840myriAZ8448syr100 rhap4220phot7840cicc5710 phot7840rhap4220syrUMAF0158 rhap4220cicc5710 aes3681cunn11894syrUMAF0158 cunn11894 aes3681dendro3226cunn11894syrUMAF0158 dendro3226papu1754 aes3681dendro3226daph9757papu1754 aes089323daph9757papu1754 aes089323dendro9150daph9757savaPseNe107 aes089323savaPseNe107 dendro9150savaPseNe107 aes2250amyg3205 dendro9150sava4352amyg3205 aes2250phot7840amyg3205 phot7840sava4352 aes2250phot7840R12341 ulmi3962R12341sava4352 aes3681R12341ulmi3962savaPG722 aes3681ulmi3962savaPG722 aes3681savaPG722 aes089323R19646meliae6289nerii16943 meliae6289R19646nerii16943 aes089323R19646meliae6289nerii16943 R15270cera6109 aes089323R15270sava3335 cera6109R15270 aes2250sava3335 aes2250cera6109 aes2250cera17524R15244sava3335 ulmi3962R15244cera17524cicc5710 cera17524R15244cicc5710 ulmi3962cicc5710 R19657cunn11894morsU7805 ulmi3962R19657cunn11894 meliae6289R19657morsU7805cunn11894 meliae6289R15300 meliae6289morsU7805R15300daph9757 phas1448AR15300daph9757 phas1448Acera6109daph9757 phas1448A cera6109cast4217 glycR4cera17524amyg3205cast4217 cera6109amyg3205cast4217 glycR4cera17524amyg3205 lach301315R12341myriAZ8448 glycR4cera17524R12341myriAZ8448 lach301315R12341morsU7805myriAZ8448 morsU7805rhap4220 lach301315R19646 amyg3918R19646rhap4220 amyg3918phas1448AR19646 morsU7805rhap4220 phas1448A ICMP19498R15270dendro3226 amyg3918phas1448AR15270dendro3226 ICMP19498glycR4R15270dendro3226 glycR4R15244dendro9150 ICMP19498R15244 R2302280 R15244dendro9150 R2302280 lach301315 glycR4dendro9150 lach301315R19657 R29095 phot7840R19657 R2302280 lach301315phot7840R19657 R29095 amyg3918phot7840 amyg3918aes3681R15300 R29095 R15300 R27968A aes3681R15300 R27968A ICMP19498 cast4217 amyg3918aes3681cast4217 ICMP19498aes089323cast4217 R2leaf aes089323 R27968A ICMP19498aes089323 R2302280R2leaf myriAZ8448 R2302280 aes2250myriAZ8448 R2leaf aes2250myriAZ8448 R2sc214 aes2250 R29095R2sc214 rhap4220 R2302280 rhap4220 R29095 ulmi3962rhap4220 R25255 ulmi3962dendro3226 R29095R2sc214 ulmi3962dendro3226 R27968AR25255 meliae6289dendro3226 R27968A meliae6289 R27968AR25255 meliae6289 R2leafR25261 dendro9150 R2leafR25261 cera6109dendro9150 dendro9150 R25260 cera6109 R2sc214R25260 phot7840 R2leafR25261 cera6109phot7840 R2sc214 cera17524phot7840 act18884 aes3681cera17524 R2sc214R25260 aes3681cera17524 R25255act18884 aes3681 R25255 morsU7805 actiCRAFRU aes089323morsU7805 R25261actiCRAFRU phas1448Aaes089323 R25255act18884 aes089323morsU7805 R25261 phas1448A acti3871 aes2250 R25261actiCRAFRU phas1448Aaes2250 R25260acti3871 aes2250 R25260 glycR4ulmi3962 acti302091 glycR4ulmi3962 act18884acti302091 lach301315 R25260acti3871 glycR4ulmi3962 act18884 lach301315meliae6289 meliae6289 acti302091 lach301315meliae6289 actiCRAFRUact212056 amyg3918 actiCRAFRUact212056 amyg3918cera6109 act18884 act19073 cera6109 acti3871act19073 ICMP19498 actiCRAFRUact212056 amyg3918cera6109 acti3871 ICMP19498cera17524 thea3923 cera17524 act19073 ICMP19498cera17524 R2302280thea3923acti302091 morsU7805 R2302280acti302091 morsU7805 acti3871 morsU7805 avelBP631 R29095avelBP631act212056 phas1448A R2302280thea3923acti302091 R29095act212056 phas1448A CC1559 avelBP631R29095act212056 phas1448A CC1559R27968Aact19073 glycR4 R27968Aact19073 glycR4 CC1559R27968A glycR4 delphi569R2leafthea3923 lach301315 delphi569R2leafthea3923 lach301315 act19073 lach301315 RMA1avelBP631 amyg3918 RMA1avelBP631R2sc214 amyg3918 delphi569R2leafthea3923 R2sc214 CC1544 ICMP19498 RMA1R2sc214 amyg3918 CC1544CC1559R25255 ICMP19498 CC1559R25255 avelBP631 ICMP19498 USA007delphi569R2302280R25261 USA007delphi569R2302280R25261 CC1544CC1559R2302280R25255 R29095 CC1416R29095R25260 USA007R25261 CC1416RMA1R25260 RMA1R27968A delphi569R29095R25260 tomaDC3000CC1544R27968Aact18884 CC1544tomaDC3000act18884 CC1416RMA1R27968A pers2254R2leaf USA007pers2254R2leafactiCRAFRU R2leafact18884 USA007R2sc214actiCRAFRU R2sc214 tomaDC3000CC1544actiCRAFRU CC1416lach302278acti3871 CC1416lach302278R25255acti3871 pers2254USA007R2sc214 tomaT1R25255acti302091 tomaDC3000tomaT1acti302091 lach302278R25255acti3871 tomaDC3000R25261 R25261 CC1416acti302091 pers2254Ps9643R25260act212056 pers2254Ps9643R25260act212056 tomaDC3000tomaT1R25261 lach302278avii3846act19073 lach302278avii3846act18884act19073 R25260act212056 0.02 act18884 0.02 thea3923 pers2254Ps9643act18884act19073 avii5271tomaT1thea3923actiCRAFRU tomaT1avii5271actiCRAFRU avii3846lach302278 Ps9643avelBP631acti3871 Ps9643avelBP631acti3871 0.02 tomaT1thea3923actiCRAFRU CC1559 CC1559acti302091 avii5271avelBP631acti3871 0.02 avii3846acti302091 0.02 avii3846 Ps9643acti302091 avii5271delphi569act212056 avii5271delphi569act212056 avii3846CC1559 RMA1act19073 RMA1act19073 0.02 delphi569act212056 CC1544thea3923 avii5271RMA1act19073 CC1544thea3923 thea3923 USA007avelBP631 USA007avelBP631 CC1544 CC1416CC1559 CC1416CC1559 USA007avelBP631 delphi569 CC1559 tomaDC3000delphi569 tomaDC3000 CC1416 pers2254RMA1 pers2254RMA1 tomaDC3000delphi569 CC1544 CC1544 RMA1 lach302278 USA007lach302278 pers2254CC1544 USA007tomaT1 tomaT1 lach302278 CC1416Ps9643 CC1416Ps9643 USA007tomaT1 tomaDC3000 tomaDC3000 CC1416 0.02 avii3846pers2254 0.02 pers2254avii3846 tomaDC3000Ps9643 avii5271lach302278 avii5271lach302278 0.02 avii3846pers2254 tomaT1 tomaT1 avii5271lach302278 Ps9643 Ps9643 tomaT1 avii3846 avii3846 Ps9643 0.02 avii5271 0.02 avii5271 avii3846 bB 0.02 avii5271

BR1-5244 BR1-5244 R2-5255 R1R2-52445255 R1-5244 avii3846 R2avii3846-5255 R2-5255

avii3846

R1-9646 R1-9646

R1-9646

R2-leaf R2-leaf R2-leaf R2-leaf avii3846 avii3846 avii3846 avii3846

R1-5244 R1-5244 R1-5244 R1-5244 R2-leaf R2--lleafeaf R2-leaf avii3846 syr9293syr9293 syr9293 R1-9646

R1-5270 A myriAZ8448 R1-9657*

R1-5300

R1-5244

R1-2341

morsU7805

avelBP631

avii5271

avii3846

lach301315

amyg3918

syr2675 50 R2-7968A * R2-sc214

47 R2-302280 bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, R2-leaf2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to displayR2-9095 the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 InternationalR2-5255 license. R2-5261

R2-5260 R1-5244 syr2682 syr7872 syr8094A# hopAR1 hopBK1syr9097 syr9659

syr5275

soli16925

syr2676 syr9644

syr9654

CC94 R2-leaf acti302091 hopAR1acti3871 0.04

R1-9646

R1-5270 A myriAZ8448 R1-9657*

R1-5300

R1-5244

R1-2341

morsU7805 avelBP631 R1-9646 avii5271

avii3846 R1-5270 syr9097 lach301315 myriAZ8448 A amyg3918 B syr2675 R1-9657* 50 R2-7968A * R1-5300 R2-sc214 hopAR1 47 R2-302280 R1-5244 R2-leaf

R2-9095 R1-2341 R2-5255 morsU7805 R2-5261

R2-5260 avelBP631 syr2682 avii5271 syr7872

syr8094A avii3846

syr9097 lach301315 Bacterial CDS Phage T3Es tRNA Genomic islandsyr9659 syr5275 amyg3918 soli16925 syr2675 syr2676 50 syr9644 R2-7968A syr9654 *

CC94 R2-sc214

acti302091 47 R2-302280 acti3871 a c R2-leaf 0.04 R2-9095 tRNA-Thr/attL R2-5255 attR R2-5261 R1-9646 1. syr9097 R2-5260 R1-5270 A myriAZ8448 B 2. USA011 syr2682 R1-9657* 3. syr1212 syr7872 R1-5300 syr8094A R1-5244 4. syrB728A syr9097 R1-2341 morsU7805 5. syr2676 syr9659 avelBP631 6. syr2675 syr5275 avii5271 soli16925 * avii3846 7. R2-leaf syr2676 lach301315 amyg3918 syr9644 50 syr2675 * syr9654 R2-7968A CC94 R2-sc214 acti302091 47 R2-302280 R2-leaf acti3871 R2-9095 R2-5255 * 0.04 R2-5261 R2-5260 syr2682 syr7872 d syr8094A syr9097 syr9659 B syr5275 soli16925 1. syr9097 syr2676 syr9644 2. syr7872 syr9654 CC94 3. syr8094A acti302091 acti3871 4. syr9654 0.04 5. syr9659 tRNA-Thr 6. syr5275 C 7. syr9644 B b 8. soli16925 tRNA-Thr attL 9. syr2676 attR 10. syr2682 1. R1-5244 * 11. syr2675 12. R2-302280 hopAR1 hopBK1# 12. R2-302280 13. R2-5255 14. R2-5260 attL 15. R2-5261 attR 2. R2-leaf hopAR1 16. R2-7968A * 17. R2-9095 18. R2-leaf 19. R2-SC214 3. syr9097 20. CC94 hopAR1 21. amyg3918 22. avii3846 Bacterial CDS Phage/ME T3Es tRNA Genomic island C C tRNA-Thr tRNA-Thr 1. syr9097 2. syrUMAF0158 3. syr2675 4. syr2686 5. R2-SC214

attL attR attL attR bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, 2018. 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.

a

1 1 2 3 4 5 2 6 7 8 Plum Psm R1 3 9 10 11 12 13 4 14 15 16 5 17 18 19 20 21 22 6 23 24 25 26 27 28 29 30 31 7 32 33 34 35 36 act19073 act212056 acti302091 acti3871 actiCRAFRU act18884 thea3923 R2−5260 R2−5261 R2−5255 R2−leaf R2−7968A R2−9095 R2−302280 R2−sc214 avelBP631 delphi569 CC1559 CC1544 RMA1 CC1416 USA007 avii5271 avii3846 pers2254 tomaDC3000 lach302278 Ps9643* tomaT1 syrUMAF0158 syr100 papu1754 Ps7969* avelVe037 Ps7928C* syr7928A syrHS191 pisiPP1 pani2367 BRIP34876 syrB64 syr3023 syr9293 syr9656 syr41a syr5264 laps3947 syr9644 syrB301D UB303 syr7924 syr2339 CC94 syr2340 soli16925 acer302273 USA011 syr8094A syr7872 syr9097 syrB728a syr5275 syr9659 syr9654 syr2675 syr1212 syr2676 syr2682 ICMP19498 amyg3918 lach301315 glycR4 phas1448A meliae6289 ulmi3962 aes2250 aes089323 aes3681 morsU7805 cera17524 cera6109 phot7840 dendro9150 dendro3226 rhap4220 myriAZ8448 cast4217 R1−5244 R1−5270 R1−9646 R1−2341 cicc5710 sava3335 savaPG722 sava4352 savaPseNe107 nerii16943 cunn11894 amyg3205 daph9757 R1-5300 R1-9657*

b

1 2 3 8 4 5 6 9 7 8 9 10 11 10 12 13 14 11 15 16 17 18 12 19 20 21 22 23 24 13 25 26 27 28 29 14 30 31 32 15 33 34 35 16 36 37 38 39 17 40 R1-5300 R1-9657* act19073 act212056 acti302091 acti3871 actiCRAFRU act18884 thea3923 R2−5260 R2−5261 R2−5255 R2−leaf R2−7968A R2−9095 R2−302280 R2−sc214 avelBP631 delphi569 CC1559 CC1544 RMA1 CC1416 USA007 avii5271 avii3846 pers2254 tomaDC3000 lach302278 Ps9643* tomaT1 syrUMAF0158 syr100 papu1754 Ps7969* avelVe037 Ps7928C* syr7928A syrHS191 pisiPP1 pani2367 BRIP34876 syrB64 syr3023 syr9293 syr9656 syr41a syr5264 laps3947 syr9644 syrB301D UB303 syr7924 syr2339 CC94 syr2340 soli16925 acer302273 USA011 syr8094A syr7872 syr9097 syrB728a syr5275 syr9659 syr9654 syr2675 syr1212 syr2676 syr2682 ICMP19498 amyg3918 lach301315 glycR4 phas1448A meliae6289 ulmi3962 aes2250 aes089323 aes3681 morsU7805 cera17524 cera6109 phot7840 dendro9150 dendro3226 rhap4220 myriAZ8448 cast4217 R1−5244 R1−5270 R1−9646 R1−2341 cicc5710 sava3335 savaPG722 sava4352 savaPseNe107 nerii16943 cunn11894 amyg3205 daph9757

c

1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 18 24 24 25 25 26 26 19 27 27 28 28 29 29 30 30 20 31 31 32 32 33 33 R1-2341 act19073 act212056 acti302091 acti3871 actiCRAFRU act18884 thea3923 R2−5260 R2−5261 R2−5255 R2−leaf R2−7968A R2−9095 R2−302280 R2−sc214 avelBP631 delphi569 CC1559 CC1544 RMA1 CC1416 USA007 avii5271 avii3846 pers2254 tomaDC3000 lach302278 Ps9643* tomaT1 syrUMAF0158 syr100 papu1754 Ps7969* avelVe037 Ps7928C* syr7928A syrHS191 pisiPP1 pani2367 BRIP34876 syrB64 syr3023 syr9293 syr9656 syr41a syr5264 laps3947 syr9644 syrB301D UB303 syr7924 syr2339 CC94 syr2340 soli16925 acer302273 USA011 syr8094A syr7872 syr9097 syrB728a syr5275 syr9659 syr9654 syr2675 syr1212 syr2676 syr2682 ICMP19498 amyg3918 lach301315 glycR4 phas1448A meliae6289 ulmi3962 aes2250 aes089323 aes3681 morsU7805 cera17524 cera6109 phot7840 dendro9150 dendro3226 rhap4220 myriAZ8448 cast4217 R1−5244 R1−5270 R1−9646 cicc5710 sava3335 savaPG722 sava4352 savaPseNe107 nerii16943 cunn11894 amyg3205 daph9757 R1-5300 R1-9657* act19073 act212056 acti302091 acti3871 actiCRAFRU act18884 thea3923 R2 − 5260 R2 − 5261 R2 − 5255 R2 − leaf R2 − 7968A R2 − 9095 R2 − 302280 R2 − sc214 avelBP631 delphi569 CC1559 CC1544 RMA1 CC1416 USA007 avii5271 avii3846 pers2254 tomaDC3000 lach302278 Ps9643 tomaT1 syrUMAF0158 syr100 papu1754 Ps7969 avelVe037 Ps7928C syr7928A syrHS191 pisiPP1 pani2367 BRIP34876 syrB64 syr3023 syr9293 syr9656 syr41a syr5264 laps3947 syr9644 syrB301D UB303 syr7924 syr2339 CC94 syr2340 soli16925 acer302273 USA011 syr8094A syr7872 syr9097 syrB728a syr5275 syr9659 syr9654 syr2675 syr1212 syr2676 syr2682 ICMP19498 amyg3918 lach301315 glycR4 phas1448A meliae6289 ulmi3962 aes2250 aes089323 aes3681 morsU7805 cera17524 cera6109 phot7840 dendro9150 dendro3226 rhap4220 myricaeAZ8448 cast4217 R1 − 5244 R1 − 5270 R1 − 9646 R1 − 2341 R1 − 5300 R1 − 9657 cicc5710 sava3335 savaPG722 sava4352 savaPseNe107 nerii16943 cunn11894 amyg3205 daph9757 Ă bioRxiv preprint doi: https://doi.org/10.1101/244715; this version posted January 8, 2018. 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-ND5OHDI 4.0 International license.        D      D  DE  D                                                 DE                            D          DEFG         D        D  D     DE  DE DE      D  D                 DE                        EF       F D      EFG          DEF  DEF        DEF                            DEF                                      FG                                                              G                                                             G                             G                                      E                                                                                                                                                                                                                                                                      H                                                                                                                                                                                                                                                                                                                                                                          F       F                                                                  I                                                                       F                                               I                                                                                                                                                                                                                                                                                                               

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a b avrRps4 hopAU1 P1 (P.s pv. avii) Gain of hopBF1*, hopAR1, hopBB1 hopAY1 hopBB1 Truncation of hopAB1, hopAB3 hopBD1 hopBF1 hopO1 hopT1 avrD1 P1 (Psm R2) Gain hopH1, hopBF1, hopAR1, hopBB1 hopAF1 Truncation of hopAB3 hopAY1 hopBF1 avrD1 hopAF1 hopBB1 Gain hopAR1, hopBF1*, cor+ hopBF1 P3 (Psm R1) Loss hopAB1

P2b (syr9656) Gain of hopBF1* P2b (syr7928A) hopBF1+ P2d (syr9644) Gain of hopAR1*, hopH1+ P2d (syr2339) hopH1+, catABC+ P2d (syr9097) hopAR1+, hopH1+

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