<table><thead><tr><th rowspan="2">TALE</th><th colspan="16">RVDs</th><th colspan="1">PX099A</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th><th>11</th><th>12</th><th>13</th><th>14</th><th>15</th><th>16</th><th>17</th><th>18</th><th>19</th><th>20</th><th>21</th><th>22</th><th>23</th><th>24</th><th>25</th><th>26</th><th>27</th><th>In</th><th>Ortholog</th></tr></thead><tbody><tr><td>Tal1c</td><td>NN</td><td>HD</td><td>NI</td><td>NG</td><td>HD</td><td>NG</td><td>N*</td><td>HD</td><td>HD</td><td>NI</td><td>NG</td><td>NI</td><td>HD</td><td>NG</td><td>NN</td><td>NG</td><td>NI</td><td>NI</td><td>NI</td><td>N*</td><td>NS</td><td>N*</td><td>Both</td><td>PthXo1</td><td></td></tr><tr><td>Tallb</td><td>NI</td><td>HG</td><td>NI</td><td>NN</td><td>NS</td><td>HD</td><td>NN</td><td>HD</td><td>HG</td><td>HD</td><td>NI</td><td>NN</td><td>NI</td><td>HD</td><td>HD</td><td>HG</td><td>NN</td><td>HD</td><td>NS</td><td>NN</td><td>HD</td><td>N*</td><td>NS</td><td>N*</td><td>Both</td><td></td></tr><tr><td>Talla</td><td>NI</td><td>NG</td><td>NN</td><td>NG</td><td>NK</td><td>NG</td><td>NI</td><td>NN</td><td>NI</td><td>NN</td><td>NI</td><td>NN</td><td>NS</td><td>NG</td><td>NS</td><td>NN</td><td>NI</td><td>N*</td><td>NS</td><td>NG</td><td>Both</td><td></td><td></td><td></td><td></td><td>Tal2a</td></tr><tr><td>Tal2b†</td><td>NS</td><td>NG</td><td>NG</td><td>NG</td><td>HD</td><td>HD</td><td>NN</td><td>NG</td><td>H*</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>SK2-3</td><td></td><td></td></tr><tr><td>Tal2b†</td><td>NS</td><td>NG</td><td>NG</td><td>NG</td><td>HD</td><td>HD</td><td>NN</td><td>NG</td><td>HD</td><td>NG</td><td>NG</td><td>HD</td><td>HD</td><td>HD</td><td>H*</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>IX-280</td><td></td></tr><tr><td>Tal2a†</td><td>NS</td><td>HD</td><td>NG</td><td>NG</td><td>HD</td><td>HD</td><td>NN</td><td>HD</td><td>NG</td><td>HD</td><td>NI</td><td>NI</td><td>NI</td><td>N*</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Both</td><td></td></tr><tr><td>Tal3c</td><td>NI</td><td>H*</td><td>NI</td><td>NN</td><td>NN</td><td>NN</td><td>NN</td><td>HD</td><td>NI</td><td>HG</td><td>HD</td><td>NI</td><td>N*</td><td>NS</td><td>NI</td><td>NI</td><td>HG</td><td>HD</td><td>NS</td><td>NS</td><td>NG</td><td></td><td></td><td></td><td>PthXo6</td></tr><tr><td>Tal3b*</td><td>NI</td><td>HG</td><td>ns</td><td>hg</td><td>hg</td><td>hd</td><td>ns</td><td>ng</td><td>hd</td><td>nn</td><td>ng</td><td>hg</td><td>ng</td><td>hd</td><td>hg</td><td>hd</td><td>hd</td><td>ni</td><td>nn</td><td>ng</td><td></td><td></td><td></td><td></td><td>Tal7b</td></tr><tr><td>Tal3a</td><td>NI</td><td>NS</td><td>HD</td><td>HG</td><td>NS</td><td>NN</td><td>HD</td><td>H*</td><td>NG</td><td>NN</td><td>NN</td><td>HD</td><td>NG</td><td>HD</td><td>NG</td><td>NG</td><td>Both</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Tal5a</td></tr><tr><td>Tal4a</td><td>NI</td><td>N*</td><td>NI</td><td>NS</td><td>NN</td><td>NG</td><td>NN</td><td>NS</td><td>N*</td><td>NS</td><td>NN</td><td>NS</td><td>N*</td><td>NI</td><td>HG</td><td>HD</td><td>NI</td><td>HD</td><td>HD</td><td>NG</td><td></td><td></td><td></td><td></td><td>Tal6a</td></tr><tr><td>Tal4b</td><td>NI</td><td>HG</td><td>NI</td><td>HG</td><td>NI</td><td>NI</td><td>NI</td><td>HD</td><td>NN</td><td>HD</td><td>NS</td><td>NG</td><td>SS</td><td>HD</td><td>NI</td><td>NN</td><td>NI</td><td>NN</td><td>NI</td><td>NG</td><td></td><td></td><td></td><td></td><td>Tal6b*</td></tr><tr><td>Tal5a</td><td>NN</td><td>HD</td><td>NS</td><td>NG</td><td>HD</td><td>NN</td><td>N*</td><td>NI</td><td>HD</td><td>NS</td><td>HD</td><td>NN</td><td>HD</td><td>NN</td><td>NN</td><td>NN</td><td>NN</td><td>NN</td><td>NN</td><td>NN</td><td>HD</td><td>NG</td><td></td><td></td><td>Tal9e</td></tr><tr><td>Tal5b</td><td>NI</td><td>HG</td><td>NI</td><td>NI</td><td>NN</td><td>HD</td><td>NS</td><td>NN</td><td>NN</td><td>HD</td><td>NN</td><td>NI</td><td>HD</td><td>NN</td><td>NI</td><td>NG</td><td>HD</td><td>NG</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Tal7a/8a</td></tr><tr><td>Tal5c</td><td>NI</td><td>NN</td><td>N*</td><td>NG</td><td>NS</td><td>NN</td><td>HD</td><td>N*</td><td>NN</td><td>NN</td><td>NI</td><td>NN</td><td>HD</td><td>NG</td><td>HD</td><td>HD</td><td>HD</td><td>NG</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Tal6a</td><td>HD</td><td>HD</td><td>HD</td><td>NG</td><td>N*</td><td>NN</td><td>HD</td><td>HD</td><td>N*</td><td>NI</td><td>NN</td><td>HD</td><td>HI</td><td>ND</td><td>HD</td><td>NI</td><td>HD</td><td>NG</td><td>NG</td><td></td><td></td><td></td><td></td><td></td><td>Tal9a</td></tr><tr><td>Tal6b</td><td>HD</td><td>HD</td><td>NN</td><td>NN</td><td>NI</td><td>NG</td><td>HD</td><td>S*</td><td>HG</td><td>HD</td><td>NG</td><td>N*</td><td>NG</td><td>HD</td><td>HD</td><td>N*</td><td>NI</td><td>NI</td><td>NN</td><td>HD</td><td>HI</td><td>ND</td><td>HD</td><td>NG</td><td>NN</td><td>HG N*</td></tr><tr><td>Tal6c</td><td>NI</td><td>NN</td><td>N*</td><td>NG</td><td>NS</td><td>NN</td><td>NN</td><td>NN</td><td>NI</td><td>NN</td><td>NI</td><td>NG</td><td>HD</td><td>HD</td><td>NI</td><td>HG</td><td>N*</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>AvrXa27</td></tr><tr><td>Tal6d</td><td>NI</td><td>NN</td><td>NI</td><td>HG</td><td>HG</td><td>HD</td><td>NG</td><td>HD</td><td>HG</td><td>HD</td><td>HD</td><td>HD</td><td>NG</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Tal9d</td></tr><tr><td>Tal7</td><td>NI</td><td>NG</td><td>NI</td><td>NI</td><td>N*</td><td>HD</td><td>HD</td><td>HD</td><td>N*</td><td>NI</td><td>NI</td><td>NI</td><td>NG</td><td>HD</td><td>HG</td><td>NN</td><td>NS</td><td>NN</td><td>HD</td><td>HD</td><td>NG</td><td>N*</td><td></td><td></td><td>PthXo7</td></tr></tbody></table>

**FIGURE 4** | RVD sequences of IX-280 and SK2-3 TALEs. An asterisk indicates that the second amino acid in the RVD is absent, resulting in a 33 aa repeat. RVDs in bold are different in PXO99A orthologs. A dagger indicates a truncTALE. The underlined RVD of Tal2a resides in a truncated (28 aa) repeat. Lower case italicized RVDs are untranslated following a frameshift. Blue font highlights TALEs for which EBEs in rice were predicted.

clade III *SWEET* genes. Contrary to our hypothesis, it induced none (Figure 5C). Finally, we tested each of the additional (non-clade III) *SWEET* genes that were identified as candidate targets of the IX-280 and SK2-3 TALEs that differ from TALEs of PXO99A, noted in the previous section: *SWEET2b*, *SWEET1b*, and *SWEET7e*. We also tested *OsSULTR3;3*. None of these was induced either (Supplementary Table S2).

DISCUSSION

This study presents the first completely assembled genome sequence of an Indian Xoo strain and the first genome sequence of a Thai strain. The genome comparisons we carried out (Figure 2A) and comparisons published elsewhere (Salzberg et al., 2008; Quibod et al., 2016) demonstrate the high level of variability in genome structure across different strains of Xoo and a general lack of relationship between genome structure and the geographical location at which a strain was isolated. Like other Xoo strains, both IX-280 and SK2-3 contain hundreds of IS elements and other transposons in their genomes (Table 1) that likely contribute to genome plasticity (Salzberg et al., 2008; Booher et al., 2015). Despite the overall genome structure variability in the species and the geographic separation of IX-280 and SK2-3, strikingly these two strains are part of a young and highly clonal lineage prevalent in India, L-I (Midha et al., 2017), in which no other characterized, non-Indian strains cluster.

This observation and the relative rarity of *xa5* compatibility in Thailand (Figure 1B) suggest introduction of SK2-3 or a recent progenitor in lineage L-I to Thailand directly, or indirectly, from India. Since we cannot rule out L-I having originated outside of India, however, it is alternatively possible that members of the lineage were introduced separately to Thailand and to India.

The strains IX-280 and SK2-3 are of special interest because of their compatibility with multiple single *R* genes (Wonglom et al., 2015; Yugander et al., 2017), in particular *xa5* and *xa13*. Previously, strains compatible with *xa5* and with *xa13* were only found in the genomically and geographically diverse lineage L-III (Midha et al., 2017); IX-280 and SK2-3 represent the first example of strains compatible with *xa5* and with *xa13* in the much more genetically homogeneous lineage L-I. In light of the expanding *R* gene compatibility and geographic spread of strains in L-I, the fact that IX-280 and SK2-3 are incompatible with the *xa5* and *xa13* stacked line IRBB53 underscores the potential benefit of deploying such *R* gene stacks. The results also illustrate the importance of complete genome sequencing in monitoring Xoo populations to develop and deploy varieties with effective disease resistance.

The basis for the compatibility of IX-280 and SK2-3 with *xa5* is almost certainly their ability to sufficiently activate *SWEET11* even under the dampening effect of *xa5* (Figure 5B). Their PthXo1 ortholog, Tal1c, is presumably responsible for
<figure><img src="image_31.png" /></figure>

**FIGURE 5** | Compatibility and ability of IX-280 to induce known or potential bacterial blight S genes in near-isogenic rice lines IR24, IRBB5 (xa5), IRBB13 (xa13), and IRBB53 (xa5 and xa13). (A) Representative lesions at 14 days after clip inoculation. Arrows indicate the distance the lesion progressed. (B) Fold induction of SWEET11, TFX1, and TFIIAγ1 24-27 h after inoculation by syringe infiltration of IX-280 relative to mock (water)-inoculated leaves, measured by qRT-PCR. Each bar represents the mean of three replicates. Error bars represent standard deviation. (C) Fold induction, as in (B), of the other clade III SWEET genes by IX-280 and selected positive control strains. ME2 is a *pthXo1* knockout derivative of PXO99A (Yang and White, 2004) used here to deliver artificial TALEs ArtTAL12-2 and ArtTAL15-1, which are targeted to the SWEET12 and SWEET15 promoters, respectively (Streubel et al., 2013). PXO339 is a Philippines race 9 Xoo strain that induces SWEET13 (Liu et al., 2011). PXO86 is a Philippines race 1 Xoo strain that induces SWEET14 (Bai et al., 2000; Antony et al., 2010).

this; the single difference in RVD sequence between Tal1c and PthXo1 does not affect the base specifying residue (Supplementary Figure S3). Induction of *TFX1* by Tal3c (the PthXo6 ortholog), and of *TFIIAγ1* by Tal7 (ortholog of PthXo7), though reduced by *xa5*, may also contribute. As noted, PthXo6 is a demonstrated virulence factor and *TFX1* is a verified S gene (Sugio et al., 2007). PthXo7 is also a demonstrated virulence factor, and although activation of *TFIIAγ1* was observed only by the *xa5*-compatible strain PXO99A (Sugio et al., 2007), silencing it decreased susceptibility to PXO99A even in an *xa5* background (Yuan et al., 2016). We also observed that despite induction of *TFIIAγ1* by Tal7, activation of *SWEET11*, *TFX1*, and *TFIIAγ1* itself remain dampened in IRBB5 relative to IR24 and IRBB13 (Figure 5B). Thus, activation of *TFIIAγ1* by Tal7 appears to contribute to

susceptibility in some way other than providing a substitute for *TFIIAγ5*.

The basis for the compatibility of IX-280 and SK2-3 with *xa13* is yet to be determined. Despite some of their TALEs being predicted to target clade III *SWEET* genes, no clade III *SWEET* gene was induced by IX-280 in IRBB13 plants. Nor were any of a handful of other candidate targets of interest, including *SWEET* genes of other clades and a paralog of a putative sulfate transporter gene that confers susceptibility to bacterial leaf streak. Possible reasons for such false positive predictions include competing endogenous DNA-binding proteins or DNA methylation at the target, or binding that does not lead to gene activation due to position in the promoter. Compatibility with *xa13*, which harbors a promoter deletion that eliminates the binding site of PthXo1 (and of the IX-280 and SK2-3 ortholog
<table><caption>**TABLE 1** | The IX-280 and SK2-3 genome assemblies.</caption><thead><tr><th></th><th>IX-280</th><th>SK2-3</th></tr></thead><tbody><tr><td>Chromosome</td><td>4,963,593 bp</td><td>4,934,446 bp</td></tr><tr><td>Plasmid</td><td>42,975 bp</td><td>-</td></tr><tr><td>Final coverage</td><td>164.0x</td><td>156.4x</td></tr><tr><td>% Mapped reads</td><td>94.6%</td><td>92.0%</td></tr><tr><td>Annotated genes</td><td>5,041</td><td>4,926</td></tr><tr><td>Annotated IS elements</td><td>411</td><td>407</td></tr><tr><td>Annotated transposases</td><td>730</td><td>698</td></tr><tr><td>tal genes</td><td>17</td><td>17</td></tr></tbody></table>

Tal1c), but not with *xa5* and *xa13* together, thus points to a second major TALE in IX-280 and SK2-3 that activates an alternative, novel S gene. Because of the incompatibility with stacked *xa5* and *xa13*, such as in IRBB53, one would predict that the induction of this alternative S gene by the TALE is not strong enough to remain effective when dampened by *xa5*. Though we predicted targets only for IX-280 (and SK2-3) TALEs most dissimilar to those of the *xa13*-incompatible strain PXO99A, it is possible that one of the IX-280 TALEs more closely related to a PXO99A TALE is responsible: even a single RVD difference could confer the ability to target a new gene. It is also possible that the promoter sequence of the alternative S gene is different in IRBB13 and not represented in our predictions using the Nipponbare reference. Thus, future work should begin with loss- and gain-of-function experiments for each of the IX-280 and SK2-3 TALEs that are not precisely conserved in PXO99A, followed by transcript profiling of the IRBB13 and IR24 responses for any unique TALE revealed to be important for compatibility in IRBB13 plants.

Studies of diverse strains have suggested that induction of a clade III *SWEET* gene is a fundamental requirement for Xoo to cause bacterial blight of rice, but the African Xoo strain BAI3 was recently reported to be compatible on a rice line from which the binding site for its major TALE, TalC, in the promoter of *SWEET14*, was removed by genome editing, and the strain induced no clade III *SWEET* in that line (Blanvillain-Baufume et al., 2017). We have shown for the first time compatibility of an Asian Xoo strain without clade III *SWEET* gene induction. The emerging picture suggests some degree of selection on Xoo populations to evolve to target alternative S genes, perhaps due to the extensive deployment of *R* genes like *xa13* and *xa25* (a recessive allele of the *SWEET13/Xa25* S gene widely used in China; Chen et al., 2002; Liu et al., 2011; Zhou et al., 2015).

It will be of interest to determine whether the evolutionarily divergent IX-280 (and SK2-3) and BAI3 lineages have converged on the same new S gene, or if they target distinct ones. Further, determining the biochemical function(s) of the new S gene(s), which ostensibly can substitute for sucrose export in rendering the plant susceptible, promises to shed light on the mechanism by which clade III *SWEET* genes contribute to disease development.

## DATA AVAILABILITY STATEMENT

The IX-280 genome assembly has been deposited in GenBank under accessions CP019226 (chromosome) and CP019227 (plasmid pXOO43) and the SK2-3 assembly under accession CP019515. Raw data and associated metadata are available from the Sequence Read Archive (SRA) under accessions SRR5989134 (IX-280) and SRR5990719, SRR5990720, and SRR5990721 (SK2-3).

## AUTHOR CONTRIBUTIONS

GL, RO, SP, and RR conceived the study. SC, AB, and RR designed the experiments. SC, PM, CG, PD, LW, SM, WK, JL, and RR performed the experiments and/or generated data. SC, LW, PP, NS, KS, AB, and RR analyzed and interpreted the data. SC, AB, and RR wrote the manuscript with input from RO.

## FUNDING

This work was supported by the Indian Council of Agricultural Research-Networking Project on Transgenic Crops and Department of Biotechnology (award BT/CEIB/12/1/01 to RR) and by the U.S. National Science Foundation (award 1444511 to AB).

## ACKNOWLEDGMENTS

The authors thank R. Sundaram and R. Sonti for helpful comments and discussion. Support from the National Phytotron Facility (NPF) at the Indian Agricultural Research Institute (IARI), New Delhi, India is gratefully acknowledged. A preprint of this article was published (Carpenter et al., 2018).

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018.02703/full#supplementary-material

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Carpenter, Mishra, Ghoshal, Dash, Wang, Midha, Laha, Lore, Kositratana, Singh, Singh, Patil, Oliva, Patarapuwadol, Bogdanove and Rai. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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# Inference of Convergent Gene Acquisition Among *Pseudomonas syringae* Strains Isolated From Watermelon, Cantaloupe, and Squash

Eric A. Newberry¹,², Mohamed Ebrahim³,⁴, Sujan Timilsina³, Nevena Zlatković⁵,
Aleksa Obradović⁵, Carolee T. Bull⁶, Erica M. Goss³,⁷, Jose C. Huguet-Tapia³,
Mathews L. Paret², Jeffrey B. Jones³* and Neha Potnis¹*

¹ Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, United States, ² Department of Plant Pathology, North Florida Research and Education Center, University of Florida, Quincy, FL, United States, ³ Department of Plant Pathology, University of Florida, Gainesville, FL, United States, ⁴ Department of Plant Pathology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt, ⁵ Faculty of Agriculture, University of Belgrade, Belgrade, Serbia, ⁶ Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, State College, PA, United States, ⁷ Emerging Pathogens Institute, University of Florida, Gainesville, FL, United States

**OPEN ACCESS**

**Edited by:**
Dawn Arnold,
University of the West of England,
United Kingdom

**Reviewed by:**
Brian H. Kvitko,
University of Georgia, United States
David John Studholme,
University of Exeter, United Kingdom

*Correspondence:
Jeffrey B. Jones
jbjones@ufl.edu
Neha Potnis
nzp0024@auburn.edu

**Specialty section:**
This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Microbiology

**Received:** 27 November 2018
**Accepted:** 01 February 2019
**Published:** 19 February 2019

**Citation:**
Newberry EA, Ebrahim M, Timilsina S, Zlatković N, Obradović A, Bull CT, Goss EM, Huguet-Tapia JC, Paret ML, Jones JB and Potnis N (2019) Inference of Convergent Gene Acquisition Among Pseudomonas syringae Strains Isolated From Watermelon, Cantaloupe, and Squash. Front. Microbiol. 10:270. doi: 10.3389/fmicb.2019.00270

*Pseudomonas syringae sensu stricto* (phylogroup 2; referred to as *P. syringae*) consists of an environmentally ubiquitous bacterial population associated with diseases of numerous plant species. Recent studies using multilocus sequence analysis have indicated the clonal expansion of several *P. syringae* lineages, located in phylogroups 2a and 2b, in association with outbreaks of bacterial spot disease of watermelon, cantaloupe, and squash in the United States. To investigate the evolutionary processes that led to the emergence of these epidemic lineages, we sequenced the genomes of six *P. syringae* strains that were isolated from cucurbits grown in the United States, Europe, and China over a period of more than a decade, as well as eight strains that were isolated from watermelon and squash grown in six different Florida counties during the 2013 and 2014 seasons. These data were subjected to comparative analyses along with 42 previously sequenced genomes of *P. syringae* stains collected from diverse plant species and environments available from GenBank. Maximum likelihood reconstruction of the *P. syringae* core genome revealed the presence of a hybrid phylogenetic group, comprised of cucurbit strains collected in Florida, Italy, Serbia, and France, which emerged through genome-wide homologous recombination between phylogroups 2a and 2b. Functional analysis of the recombinant core genome showed that pathways involved in the ATP-dependent transport and metabolism of amino acids, bacterial motility, and secretion systems were enriched for recombination. A survey of described virulence factors indicated the convergent acquisition of several accessory type 3 secreted effectors (T3SEs) among phylogenetically distinct lineages through integrative and conjugative element and plasmid loci. Finally, pathogenicity assays on watermelon and squash showed qualitative differences in virulence between strains of the same
clonal lineage, which correlated with T3SEs acquired through various mechanisms of
horizontal gene transfer (HGT). This study provides novel insights into the interplay of
homologous recombination and HGT toward pathogen emergence and highlights the
dynamic nature of *P. syringae sensu lato* genomes.

Keywords: horizontal gene transfer, homologous recombination, pathogen emergence, *Pseudomonas syringae sensu stricto*, cucurbits

INTRODUCTION

The Gram-negative bacterial species, *Pseudomonas syringae sensu lato* (in the largest sense), embodies both a pathogenic and phylogenetic complex of strains, which are responsible for numerous plant diseases of economic importance worldwide. Because many of the phytopathogenic bacteria found within this species complex could not be differentiated using traditional phenotypic and biochemical tests, they were classified into distinct pathogenic populations (i.e., pathovars) as defined by their host specificity (Dye et al., 1980). Currently, over 50 pathovars have been described within the seven named species and one genomospecies in *P. syringae sensu lato* (Gardan et al., 1999). These can be distinguished by multilocus sequence analysis (MLSA; Hwang et al., 2005; Young, 2010; Bull et al., 2011; Berge et al., 2014) and whole genome sequence analysis (Marcelletti and Scortichini, 2014; Nowell et al., 2014; Gomila et al., 2017) into phylogroups which correspond to distinct species.

Aside from its role as a plant pathogen, *P. syringae sensu lato* is common in a variety of habitats outside of the agricultural context, including in precipitation, water, soil, and wild plants as a facultative saprophyte (Hirano and Upper, 2000; Morris et al., 2013). Given the ubiquitous nature of this bacterial species, it is not surprising to note that *P. syringae sensu lato* may exhibit a variety of interactions with plants ranging from commensal leaf inhabitant, to opportunistic, and host-specialized phytopathogen. Similarly, some *P. syringae sensu lato* lineages have evolved differing modes of transmission to plants, including via seed and water, which may be reflected in their ecology, metabolic versatility, and other forms of microbial physiology (Baltrus et al., 2017). Several well characterized plant diseases such as bacterial speck of tomato, bleeding canker of European horse chestnut, or bacterial canker of kiwifruit were each linked to the expansion of a genetically monomorphic pathogen lineage (Green et al., 2010; Cai et al., 2011a; McCann et al., 2013). In some cases, the clonal lineages associated with these diseases were closely related to strains collected from environmental sources that were less virulent and had a broader host range than their host-specialized relatives (Cai et al., 2011b; Monteil et al., 2013). This observation has led to the hypothesis that *P. syringae sensu lato* displays an epidemic population structure, whereby novel pathogen lineages emerge from recombining ancestral populations through the acquisition of genes or alleles that provide an adaptive benefit (Vinatzer et al., 2014). Consistent with this hypothesis, gene

content fluctuation occurs at an over 100-fold greater rate
than amino acid sequence divergence in *P. syringae sensu lato*
genomes (Nowell et al., 2014).

Among the various species found within *P. syringae sensu lato*, *P. syringae sensu stricto* (phylogroup 2; referred to as *P. syringae* in the rest of the manuscript) possesses many traits that are characteristic of the species complex as a whole. The strains described here are commonly recovered from environmental sources, maintain large epiphytic populations, are active ice-nucleators, and cause disease on a wide range of plant species (Canfield et al., 1986; Morris et al., 2008; Berge et al., 2014). A distinguishing feature of this group is the production of the phytotoxins syringomycin, syringopeptin, and syringolin, which are virulence factors that exhibit antimycotic activity and facilitate host colonization (Scholz-Schroeder et al., 2001; Misas-Villamil et al., 2013; Nowell et al., 2016). Although a number of agriculturally relevant pathovars have been described within *P. syringae* (Bull and Koike, 2015), strains are commonly identified as *P. syringae* pv. *syringae* based on the detection of genes associated with the biosynthesis of syringomycin (Little et al., 1998; Sorensen et al., 1998; Bultreys and Gheysen, 1999). *P. syringae* pv. *syringae*, which was named for its original host of isolation (*Syringae vulgaris*), has been recorded as a pathogen of over 40 different plant species and has a host range distinct, but overlapping many of the other pathovars found within the same phylogenetic group (Young, 2010). As a result, it is unclear to what degree many of the plant pathogenic bacteria described here exhibit host-specificity and/or represent ecologically separate populations.

Bacterial leaf spot of watermelon (*Citrullus lanatus*), cantaloupe (*Cucumis melo*), and squash (*Cucurbita pepo*) is a common early spring disease that has a worldwide distribution and can cause significant economic losses under cool, wet environmental conditions (Morris et al., 2000; Riffaud et al., 2003; Newberry et al., 2017). The disease was recently recognized as a seedborne disorder of squash (Manceau et al., 2011); however, its etiology in various cucurbit species is likely to have multiple sources (Monteil et al., 2016). Recently, we characterized the *P. syringae* population responsible for bacterial leaf spot epidemics that occurred in commercial production fields of watermelon and squash throughout Florida. Analysis of the population structure indicated that this newly emerging disease was primarily associated with the expansion of a clonal *P. syringae* lineage throughout the state, that was most closely related to the *P. syringae* pv. *syringae* type/pathotype strain, LMG 1247<sup>PT</sup> (=ICMP 3023<sup>T</sup>), within phylogroup 2b. Additionally,
we identified two other clonal lineages collected from either the same, or previous bacterial leaf spot epidemics in the United States that shared a recent a common ancestor with the aforementioned epidemic clone; however, were located in a separate phylogroup within the same species, namely phylogroup 2a (Newberry et al., 2016, 2018). Although, we were able to precisely classify these pathogens within the phylogenetic structure of *P. syringae sensu lato* using MLSA, we were unable to delineate them from other strains collected from a diverse group of plant species or attribute them to any pathovar previously associated with cucurbit hosts, other than *P. syringae* pv. *syringae*.

Here, we investigated the evolutionary processes that led to the emergence of these similar, yet distinct *P. syringae* lineages as successful pathogens of watermelon, cantaloupe, and squash, as well as the genetic factors that distinguish them from other members of this environmentally ubiquitous bacterial population. We obtained high-quality draft genomes for 11 *P. syringae* strains collected from bacterial leaf spot epidemics in the United States, as well as for three strains isolated from symptomatic squash grown in Italy, Serbia, and China over various years. In order to investigate these strains in the context of the larger diversity of *P. syringae*, we analyzed these data together with the genomes of 42 additional *P. syringae* strains collected from diverse plant species and environments available from public sequence databases. We examined the population structure and analyzed patterns of homologous recombination within *P. syringae*. The distribution of previously described virulence factors including type 3 secreted effectors (T3SEs), phytotoxins, and other biologically relevant features were computationally surveyed. Finally, pan-genome association analysis was carried out to identify orthologous groups potentially involved in niche adaptation of the cucurbit lineages. The combined results of this study demonstrate the presence of a hybrid *P. syringae* clone associated with watermelon, cantaloupe, and squash in the United States and Europe and provide evidence for the

convergent adaptation of two phylogenetically distinct *P. syringae* populations to cucurbit hosts.

## MATERIALS AND METHODS

### Bacterial Strains and Sequencing

We selected 14 *P. syringae* strains that were isolated from the symptomatic tissue of watermelon, cantaloupe, and squash over the period of numerous years for shotgun sequencing, as detailed below. Most of these strains were described previously and altogether, comprised three different multilocus haplotypes located in phylogroups 2a and 2b of *P. syringae* (Table 1). Eight strains collected from bacterial leaf spot epidemics that occurred in Florida between 2013 and 2014 were sequenced. These strains were isolated from watermelon and squash grown in six different Florida counties during these epidemics and comprised haplotypes one and two (Newberry et al., 2018). Three strains included for sequencing comprised haplotype three and were isolated from cantaloupe and squash grown in Florida, Georgia, and California between 2000 and 2006 (Newberry et al., 2016). Finally, three additional strains that were isolated from symptomatic squash grown in Italy, Serbia, and China between 2005 and 2013 were also sequenced because they were found to be identical at four partial housekeeping gene sequences to one of the previously mentioned haplotypes (Hwang et al., 2005).

Bacterial strains were purified from single colonies and cultured overnight in nutrient broth. Genomic DNA was extracted using the CTAB-NaCl method (Ausubel et al., 1994), checked for quality using a NanoDrop 2000 (Thermo Scientific, Waltham, MA, United States) and gel electrophoresis, then quantified using a Qubit 3.0 fluorometer (Thermo Fischer, Waltham, MA, United States). Genomic libraries were prepared using a Nextera library preparation kit (Illumina Inc., San Diego, CA, United States) and the DNA was sequenced using the Illumina MiSeq platform at the Interdisciplinary Center

<table><caption>TABLE 1 | Draft genome sequencing and assembly statistics for *P. syringae* strains isolated from watermelon, cantaloupe, and squash.</caption><thead><tr><th>Strain</th><th>Origin</th><th>Host<sup>a</sup></th><th>Year</th><th>MLST<sup>b</sup></th><th>Phylogroup</th><th>Contigs (N)</th><th>N50 (Kb)</th><th>Genome length (Mb)</th><th>Accession</th></tr></thead><tbody><tr><td>13-C2</td><td>Florida</td><td>WM</td><td>2013</td><td>1</td><td>2b</td><td>83</td><td>178</td><td>5.92</td><td>MUHO00000000</td></tr><tr><td>13-140A</td><td>Florida</td><td>WM</td><td>2013</td><td>1</td><td>2b</td><td>90</td><td>131</td><td>5.91</td><td>MUHL00000000</td></tr><tr><td>13-509A</td><td>Florida</td><td>SQ</td><td>2013</td><td>1</td><td>2b</td><td>107</td><td>111</td><td>5.89</td><td>MUHP00000000</td></tr><tr><td>13-139B</td><td>Florida</td><td>WM</td><td>2013</td><td>2</td><td>2a</td><td>83</td><td>131</td><td>6.37</td><td>MVAT00000000</td></tr><tr><td>13-429</td><td>Florida</td><td>WM</td><td>2013</td><td>2</td><td>2a</td><td>90</td><td>225</td><td>6.25</td><td>MVAY00000000</td></tr><tr><td>14-410</td><td>Florida</td><td>WM</td><td>2014</td><td>1</td><td>2b</td><td>131</td><td>99</td><td>5.91</td><td>MUHQ00000000</td></tr><tr><td>14-32</td><td>Florida</td><td>WM</td><td>2014</td><td>1</td><td>2b</td><td>102</td><td>124</td><td>5.92</td><td>MUHM00000000</td></tr><tr><td>14-Gil</td><td>Florida</td><td>WM</td><td>2014</td><td>1</td><td>2b</td><td>195</td><td>58</td><td>5.91</td><td>MVAU00000000</td></tr><tr><td>03-19A</td><td>Florida</td><td>CL</td><td>2003</td><td>3</td><td>2a</td><td>51</td><td>239</td><td>6.14</td><td>MUHN00000000</td></tr><tr><td>200-1</td><td>Georgia</td><td>SQ</td><td>2000</td><td>3</td><td>2a</td><td>91</td><td>104</td><td>6.14</td><td>MVAZ00000000</td></tr><tr><td>BS2121</td><td>California</td><td>SQ</td><td>2006</td><td>3</td><td>2a</td><td>71</td><td>112</td><td>6.22</td><td>MVAV00000000</td></tr><tr><td>ZUM3584</td><td>Italy</td><td>SQ</td><td>2005</td><td>1</td><td>2b</td><td>129</td><td>108</td><td>5.97</td><td>MVBA00000000</td></tr><tr><td>ZUM3984</td><td>China</td><td>SQ</td><td>2008</td><td>3</td><td>2a</td><td>59</td><td>263</td><td>6.24</td><td>MVAX00000000</td></tr><tr><td>PS711</td><td>Serbia</td><td>SQ</td><td>2013</td><td>1</td><td>2b</td><td>90</td><td>225</td><td>5.89</td><td>RQXZ01000000</td></tr></tbody></table>

<footnote><sup>a</sup>WM, watermelon; CL, cantaloupe; SQ, squash. <sup>b</sup>Multilocus sequence type.</footnote>
<table><caption>**TABLE 2** | List of genomes included in comparative analyses including pathovar classification, host of isolation, and phylogenetic classification based on MLSA.</caption><thead><tr><th>Strain</th><th>Pathovar</th><th>Isolation source</th><th>Phylogroup</th><th>Accession</th><th>Reference</th></tr></thead><tbody><tr><td>Alf3</td><td>syringae</td><td>Medicago sativa (Alfalfa)</td><td>2b</td><td>JPNN00000000.1</td><td>Harrison et al., 2016</td></tr><tr><td>BS0292</td><td>aptata</td><td>Beta vulgaris (Sugar beet)</td><td>2b</td><td>FOVV00000000.1</td><td>–</td></tr><tr><td>BS3827</td><td>aptata</td><td>Beta vulgaris (Sugar beet)</td><td>2b</td><td>FOQB00000000.1</td><td>–</td></tr><tr><td>BS3829</td><td>aptata</td><td>Beta vulgaris (Sugar beet)</td><td>2b</td><td>FOPR00000000.1</td><td>–</td></tr><tr><td>CC457</td><td>NA</td><td>Cucumis melo (Cantaloupe)</td><td>2b</td><td>AVEB00000000.2</td><td>Baltrus et al., 2014b</td></tr><tr><td>HS191</td><td>syringae</td><td>Panicum milliaceum (Millet)</td><td>2b</td><td>NZ_CP006256.1</td><td>Ravindran et al., 2015</td></tr><tr><td>ICMP459<sup>PT</sup></td><td>aptata</td><td>Beta vulgaris (Sugar beet)</td><td>2b</td><td>LJRP00000000.1</td><td>Thakur et al., 2016</td></tr><tr><td>PP1</td><td>pisi</td><td>Pisum sativum L. (Pea)</td><td>2b</td><td>AUZR00000000.2</td><td>Baltrus et al., 2014a</td></tr><tr><td>Pav013</td><td>avellanae</td><td>Corylus avellana (European hazelnut)</td><td>2b</td><td>GCA_000302795.1</td><td>O'Brien et al., 2012</td></tr><tr><td>Pav037</td><td>avellanae</td><td>Corylus avellana (European hazelnut)</td><td>2b</td><td>GCA_000302815.1</td><td>O'Brien et al., 2012</td></tr><tr><td>2507</td><td>syringae</td><td>Triticum aestivum (Wheat)</td><td>2b</td><td>LYUO00000000.1</td><td>Sultanov et al., 2016</td></tr><tr><td>41A</td><td>syringae</td><td>Prunus armeniaca (Armenian plum)</td><td>2b</td><td>JYHJ00000000.1</td><td>Bartoli et al., 2015</td></tr><tr><td>1845</td><td>syringae</td><td>Helianthus L. (Sun flower)</td><td>2b</td><td>LYUP00000000.1</td><td>Sultanov et al., 2016</td></tr><tr><td>B64</td><td>syringae</td><td>Triticum aestivum (Wheat)</td><td>2b</td><td>ANZF00000000.1</td><td>Dudnik and Dudler, 2013</td></tr><tr><td>CRAFRU11</td><td>syringae</td><td>Corylus avellana (European hazelnut)</td><td>2b</td><td>ATSU00000000.1</td><td>Scortichini et al., 2013</td></tr><tr><td>CRAFRU12</td><td>syringae</td><td>Corylus avellana (European hazelnut)</td><td>2b</td><td>ATSV00000000.1</td><td>Scortichini et al., 2013</td></tr><tr><td>ICMP11168</td><td>syringae</td><td>A. deliciosa (Kiwifruit)</td><td>2b</td><td>LKGV00000000.1</td><td>Visnovsky et al., 2016</td></tr><tr><td>ICMP3023<sup>T</sup></td><td>syringae</td><td>Syringa vulgaris (Lilac)</td><td>2b</td><td>LJRK00000000.1</td><td>Thakur et al., 2016</td></tr><tr><td>ICMP3947<sup>PT</sup></td><td>lapsa</td><td>Triticum aestivum (Wheat)</td><td>2b</td><td>LJQQ00000000.1</td><td>Thakur et al., 2016</td></tr><tr><td>ICMP4394<sup>PT</sup></td><td>atrofaciens</td><td>Triticum aestivum (Wheat)</td><td>2b</td><td>LJPO00000000.1</td><td>Thakur et al., 2016</td></tr><tr><td>MB03</td><td>NA</td><td>Populus lasiocarpa (Poplar)</td><td>2b</td><td>LAGV00000000.1</td><td>–</td></tr><tr><td>NCPPB4273<sup>T</sup></td><td>coryli</td><td>Corylus avellana (European hazelnut)</td><td>2b</td><td>AWQP00000000.1</td><td>Marcelletti and Scortichini, 2014</td></tr><tr><td>SM</td><td>syringae</td><td>Triticum aestivum (Wheat)</td><td>2b</td><td>APWT00000000.1</td><td>Dudnik and Dudler, 2013</td></tr><tr><td>BRIP34881</td><td>NA</td><td>Hordeum vulgare (Barley)</td><td>2b</td><td>AMXL00000000.1</td><td>Gardiner et al., 2013</td></tr><tr><td>A2</td><td>syringae</td><td>Pyrus calleryana (Pear)</td><td>2a</td><td>LGKU00000000.1</td><td>Mott et al., 2016</td></tr><tr><td>CFBP1754<sup>PT</sup></td><td>papulans</td><td>Malus sylvestris (Apple)</td><td>2a</td><td>JYHI00000000.1</td><td>Bartoli et al., 2015</td></tr><tr><td>UMAF0158</td><td>syringae</td><td>Mangifera indica (Mango)</td><td>2a</td><td>NZ_CP005970.1</td><td>Martínez-García et al., 2015</td></tr><tr><td>31R1</td><td>NA</td><td>Zea mays L. (Corn)</td><td>2a</td><td>GCA_900105295.1</td><td>Lindow, 1987</td></tr><tr><td>BRIP39023</td><td>NA</td><td>Triticum aestivum (Wheat)</td><td>2a</td><td>AMZX00000000.1</td><td>Dudnik and Dudler, 2013</td></tr><tr><td>ICMP11293</td><td>syringae</td><td>A. deliciosa (Kiwifruit)</td><td>2a</td><td>LKEP00000000.1</td><td>Visnovsky et al., 2016</td></tr><tr><td>NFACC10-1</td><td>NA</td><td>Panicum virgatum (Switchgrass)</td><td>2a</td><td>GCF_900119195.1</td><td>–</td></tr><tr><td>Pc58<sup>T</sup></td><td>NA</td><td>Prunus cerasus (Sour cherry)</td><td>2a</td><td>GCA_900074915.1</td><td>Kalužna et al., 2016</td></tr><tr><td>B301D</td><td>syringae</td><td>Pyrus communis (Comice pear)</td><td>2d</td><td>GCA_000988485.1</td><td>Ravindran et al., 2015</td></tr><tr><td>B728a</td><td>syringae</td><td>Phaseolus vulgaris (Bean)</td><td>2d</td><td>NC_007005.1</td><td>Feil et al., 2005</td></tr><tr><td>2339</td><td>syringae</td><td>Prunus avium (Sweet cherry)</td><td>2d</td><td>LIHU00000000.1</td><td>Nowell et al., 2016</td></tr><tr><td>2340</td><td>syringae</td><td>Pyrus sp. (Pear)</td><td>2d</td><td>LIHT00000000.1</td><td>Nowell et al., 2016</td></tr><tr><td>HRI-W7872</td><td>syringae</td><td>Prunus domestica (Plum)</td><td>2d</td><td>LIHS00000000.1</td><td>Nowell et al., 2016</td></tr><tr><td>HRI-W7924</td><td>syringae</td><td>Prunus cerasus (Sour cherry)</td><td>2d</td><td>LIHR00000000.1</td><td>Nowell et al., 2016</td></tr><tr><td>ICMP13102</td><td>syringae</td><td>A. deliciosa (Kiwifruit)</td><td>2d</td><td>LKEO00000000.1</td><td>Visnovsky et al., 2016</td></tr><tr><td>PD2774</td><td>syringae</td><td>A. chinensis (Kiwifruit)</td><td>2d</td><td>LKEL00000000.1</td><td>Visnovsky et al., 2016</td></tr><tr><td>ATCC10853<sup>PT</sup></td><td>aceris</td><td>Acer L. (Maple)</td><td>2d</td><td>LGAR00000000.1</td><td>–</td></tr><tr><td>USA011</td><td>NA</td><td>Freshwater</td><td>2d</td><td>AVDX00000000.2</td><td>Baltrus et al., 2014b</td></tr></tbody></table>

for Biotechnology Research, University of Florida. The raw sequence data were subjected to adapter and quality trimming with Scythe<fnref n="1" /> and SolexaQA, respectively (Cox et al., 2010). The quality-trimmed reads were then *de novo* assembled into contigs using the SPAdes Genome assembler (v3.5.0) with the “careful” option to reduce mismatches in the assembly (Bankevich et al., 2012). The draft genome assemblies were submitted to the Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP)

pipeline and the Joint Genome Institute (IMG-JGI) server for annotation (Markowitz et al., 2012; Tatusova et al., 2016). The sequencing, assembly statistics, and collection information are presented in Table 1.

## Pan-Genome Association Analysis

The strains sequenced in this study were investigated in context of *P. syringae* by including 42 additional genomes that were publicly available from the National Center for Biotechnology Information (NCBI) GenBank for analysis (Table 2). These genomes were selected to represent the diversity

<footnote n="1">¹https://github.com/vsbuffalo/scythe</footnote>
of phytopathogenic bacteria previously described within the species and are distributed among phylogroups 2a, 2b, and 2d (Berge et al., 2014; Bull and Koike, 2015). Representatives of phylogroup 2c, otherwise known as *P. congelans*, were not included in this analysis as they are not known to be phytopathogenic (Mohr et al., 2008). The genome assemblies were re-annotated with Prokka to generate GFF3 files (Seemann, 2014), which were then used as input for Roary pan-genome pipeline (v.3.6.1, Page et al., 2015). Orthologous groups were clustered using the CD-Hit and MCL algorithms with a BLASTp cut-off set to 95% along with the “-s” option to prevent the splitting of orthologous groups containing paralogs. The output of Roary was further analyzed using Scoary to test for associations between the presence/absence of orthologous groups and cucurbit-associated lineages (Brynildsrud et al., 2016). The population structure (as described below) was used to control for spurious associations in the estimation of probabilities and orthologous groups with a Bonferroni $p \le 10^{-5}$ were reported.

## Analysis of Population Structure and Interlineage Recombination

Initial analysis of the population structure was conducted by calculating the average nucleotide identities between genomes using the MUMmer algorithm (Marçais et al., 2018), with the Python package pyani<fnref n="2" />. Subsequently, a core genome alignment was constructed using the program Parsnp (Treangen et al., 2014). Locally collinear blocks (LCBs) of maximal unique matches shared across all genome assemblies were identified and aligned against the gold standard reference genome of *P. syringae* pv. *syringae* strain B728a (NC_007005.1). Single nucleotide polymorphisms (SNPs) located on LCBs < 200 bp or other regions of poor alignment were removed from the data set to generate a concatenated alignment of high-quality core genome SNPs. This concatenated SNP alignment was used to infer a maximum likelihood phylogeny using iqTree (v.1.6.4) with the Jukes-Cantor model of nucleotide substitution (Nguyen et al., 2015). Branch support was assessed using the ultrafast bootstrap method with 1,000 replicates (Minh et al., 2013) and the phylogenetic tree was visualized and annotated using FigTree (v.1.4.2³).

To analyze patterns of homologous recombination within *P. syringae*, the core genome alignment generated with Parsnp was used as input for analysis with fastGEAR using the default settings. The fastGEAR algorithm employs, BAPS, a Bayesian hierarchical clustering method to infer sequence clusters in an alignment (Corander et al., 2003). This was followed by a hidden Markov model (HMM), which was used to collapse clusters that share a common ancestry in at least 50% of the sites into lineages. The program detects “recent” recombination events between lineages using a HMM and the origin of the recombinant sequence is assigned to the lineage with the highest probability at that position. Similarly, “ancestral” recombination events that are shared by all strains which comprise a lineage are identified; however, the origin of these recombination events cannot be

inferred due to their conserved nature (Mostowy et al., 2017). The statistical significance of recombination predictions was tested using a Bayes factor (BF) > 1 for recent recombination events and BF > 10 for ancestral recombination events. This analysis was conducted with two alternative genome alignments using completed reference genomes of *P. syringae* pv. *syringae* strains UMAF0158 (NZ_CP005970.1) and HS191 (NZ_CP006256.1) to validate the lineage prediction and proportion of gene flux between lineages. Finally, to examine the evolutionary history of *P. syringae* in the absence of recombination, the positions in the core genome alignment which corresponded to the predicted recent recombination events were removed and SNPs extracted using the program SNP-sites (Page et al., 2016). This recombination-filtered SNP alignment was then used to construct a maximum likelihood phylogeny as described above.

## Functional Analysis of Recent Recombination Events

To characterize the functional impact of the recombinant genes leading to the emergence of phylogroup 2b-a, BLASTn (E-value $\le 1e^{-50}$) was used to map the recent recombination events predicted by fastGEAR back to the genome assemblies of three strains (13-140A, ZUM3584, HS191), representative of the different recombination profiles in the dataset. The Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology IDs corresponding to recombinant and non-recombinant genes were subsequently extracted from the IMG annotations and a linear regression analysis was conducted to compare the composition of recombinant vs. non-recombinant core genes across the COG functional categories. Additionally, the KEGG orthology IDs were used to compare the distribution of recombinant genes across the KEGG BRITE functional annotations among the three different recombination profiles.

After noting evidence for extensive recombination affecting the *hrp/hrc* pathogenicity island of phylogroup 2b-a cucurbit strains in the fastGEAR output, amino acid sequence alignments for the 27 open reading frames that comprise *hrp/hrc* gene cluster, as described by Alfano et al. (2000), were constructed using a sub-set of 54 *P. syringae* genomes. Individual gene alignments were subjected to a phylogenetic analysis with FastTree 2 using the gamma time reversible model (Price et al., 2010). A gene was considered to be recombinant among the 2b-a lineages if it clustered into a monophyletic group with other of members of phylogroup 2a, rather than 2b, as predicted by the fastGEAR algorithm. To illustrate these relationships, a phylogenetic network was constructed with a concatenated alignment of the entire *hrp/hrc* gene cluster using the NeighborNet method and p-distance in SplitsTree 4 (Huson and Bryant, 2006).

## Analysis of Type 3 Secreted Effectors (T3SEs) and Phytotoxins

Reference sequences were obtained from the public T3SE database<fnref n="4" /> and used to query the genome assemblies using

<footnote n="2">²https://github.com/widdowquinn/pyani</footnote>

<footnote n="3">³http://tree.bio.ed.ac.uk/software/figtree/</footnote>

<footnote n="4">⁴http://www.pseudomonas-syringae.org/</footnote>
BLASTn (E-value ≤ 1e⁻<fnref n="5" />). A given T3SE was considered to be present in a genome if the alignment with the reference sequence displayed at least 80% sequence identity over 80% of the query length. If genomic sequences orthologous to a given reference sequence were split between two contigs, then the sequences were concatenated to determine their presence in the assemblies. For each T3SE, an alignment of the subject sequence with the curated reference sequence was used to record putative frameshift mutations or other disruption of the coding sequence. The distribution of T3SEs was then used to construct a binary matrix, where 1 indicated the presence of a gene, 0.5 indicated a putative pseudogene, and 0 if the gene was absent. Hierarchical clustering of the binary matrix was performed with the Python package, hclust². A similar strategy described by Baltrus et al. (2011) was used to identify genes involved in the biosynthesis of the following phytotoxins: tabtoxin, phaseolotoxin, mangotoxin, syringomycin, syringolin, and syringopeptin.

## Identification of Plasmids and Genomic Islands

The presence of plasmids was predicted for the strains sequenced in this study using plasmidSPAdes (Antipov et al., 2016). Assembled contigs were screened using Microbial Genome BLAST against the complete plasmid database to identify putative plasmid sequences. These contigs were subsequently screened for T3SEs and other virulence factors as described above. Genomic islands and other signatures of horizontal gene transfer (HGT) were detected using the Island Viewer 4 server (Bertelli and Brinkman, 2018). Genomic islands (GIs) were predicted using the reference strains *P. fluorescens* SBW25, *P. protegens* Pf-5, *P. aeruginosa* PAO1, and *P. syringae* pv. *syringae* B728a. The predicted GIs were analyzed for gene content and extracted from the genome assemblies to construct multiple sequence alignments using the Mauve software package (Darling et al., 2004).

## Pathogenicity Assays

The pathogenicity of most of the bacterial strains sequenced in this study was examined previously (Newberry et al., 2016, 2018). However, to make direct comparisons with strains collected from independent disease outbreaks, a sub-set of 10 *P. syringae* strains representative of the genetic diversity and geographic source of isolation in the collection were selected for further pathogenicity testing. A strain that was originally isolated from diseased proso millet, *P. syringae* pv. *syringae* HS191 (Gross and DeVay, 1976; Ravindran et al., 2015), was also included in these experiments due to its genetic similarity to the cucurbit strains examined here. Bacterial strains were cultured overnight on King's medium B agar, suspended in a sterile MgSO₄*7H₂O solution (10 mM), and adjusted to ~1 × 10⁸ colony forming units (CFU ml⁻¹) spectrophotometrically (OD₆₀₀ = 0.1). Two-week-old seedlings of watermelon cv. Troubadour (Harris Seeds, Rochester, NY, United States) and squash cv. Conqueror III (Seminis Vegetable Seeds) were sprayed with the bacterial suspension until run-off, incubated in a humidity chamber at 100% RH for 48 h,

then placed under growth room conditions at 21°C and ~70% RH with a 12 h photoperiod. Plants sprayed with the sterile MgSO₄*7H₂O solution served as a negative control. Two weeks after inoculation, the total proportion of necrotic/symptomatic leaf tissue was rated visually from 0 to 8 using a modified version of the Horsfall-Barratt scale (Horsfall and Barratt, 1945): 0 = no symptoms, 1 = 1–3%, 2 = 3–6%, 3 = 6–12%, 4 = 12–25%, 5 = 25–50%, 6 = 50–75%, 7 = 75–87%, 8 = 87–100%. Five replicates were included for each strain/host combination, and the experiment was conducted twice. A non-parametric analysis of variance (ANOVA) was used to test for differences among the treatments and all statistical analyses were conducted using JMP Pro 13 (SAS Institute, Cary, NC, United States).

# RESULTS

## Population Structure of *P. syringae* sensu stricto

Previous studies using MLSA described three phylogroups of phytopathogenic bacteria within *P. syringae* sensu stricto (Berge et al., 2014; Bull and Koike, 2015). Maximum likelihood reconstruction of the *P. syringae* core genome based on 246,510 polymorphic sites extracted from an alignment of 2.43 Mb revealed the presence of two well-supported phylogenetic groups in addition to those delineated through MLSA (2a, 2b, and 2d), designated here as 2b-a and Pav (Figure 1). The between-phylogroup average nucleotide identities (ANI) were primarily equal and ranged from 94.61 to 95.54% ANI. However, phylogroup 2b-a was intermediate of 2b (97.43% ANI), Pav (96.15% ANI), and 2a (96.00% ANI). Similar levels of sequence identity (between 98.16 and 98.98% ANI) were recorded within all *P. syringae* phylogroups (Table 3). Phylogroup 2b contained a number of strains collected from diverse monocot and herbaceous dicot plant species including the type strain, *P. syringae* pv. *syringae* ICMP3023T, as well as other previously described pathovars such as *P. syringae* pvs. *aptata* ICMP459PT, *atrofaciens* ICMP4394PT, *pisi* PP1, and *lapsa* ICMP3947PT. Phylogroup 2b-a branched as a sister group to 2b and contained many cucurbit strains that were isolated from plants grown in Florida, Italy, France, and Serbia between 2003 and 2014. All of these strains were of the same clonal lineage, except for ZUM3584, and shared a recent common ancestor with that of *P. syringae* pv. *syringae* HS191, which was isolated in Australia from diseased proso millet in 1969 and was more distantly related. The other novel *P. syringae* phylogroup inferred in this analysis, phylogroup Pav, consisted of strains associated exclusively with hazelnut decline that were classified primarily as *P. syringae* pv. *avellanae* (O'Brien et al., 2012) and branched between phylogroups 2b and 2d. The cucurbit strains located in phylogroup 2a branched from each other into two distinct lineages that were nested among other strains isolated from diverse plants and environments. A well supported split was observed between the clones collected in California and China (BS2121 and ZUM3984) and those in Florida and Georgia (03-19A and 200-1), which differed by less than 300 SNPs in the

<footnote n="5">⁵https://bitbucket.org/nsegata/hclust2</footnote>
<figure><img src="image_33.png" /><figcaption>**FIGURE 1** | Population structure of *P. syringae* sensu stricto. Mid-point rooted, maximum likelihood phylogeny based on 246,510 SNPs extracted from a core genome alignment of 2.43 Mb. Sequence clusters identified by BAPS analysis are color coded and phylogroup designations are labeled at the nodes. Bootstrap support was ≥98% except where otherwise indicated and the scale bar shows the number of substitutions per site.</figcaption></figure>

<table><caption>**TABLE 3** | Average percent identity within and between *P. syringae* phylogroups<sup>a</sup>.</caption><thead><tr><th></th><th>2a</th><th>2b</th><th>Pav</th><th>2b-a</th><th>2d</th></tr></thead><tbody><tr><td>2a</td><td>98.95</td><td></td><td></td><td></td><td></td></tr><tr><td>2b</td><td>94.80</td><td>98.35</td><td></td><td></td><td></td></tr><tr><td>Pav</td><td>95.20</td><td>96.55</td><td>98.16</td><td></td><td></td></tr><tr><td>2b-a</td><td>96.00</td><td>97.43</td><td>96.15</td><td>98.68</td><td></td></tr><tr><td>2d</td><td>94.61</td><td>95.20</td><td>95.54</td><td>95.05</td><td>98.75</td></tr></tbody></table>

core genome alignment and represented distinct haplotypes within the lineage (Figure 1).

## Patterns of Homologous Recombination Between *P. syringae* sensu stricto Phylogroups

The sequence clusters identified by the Bayesian statistical clustering method were congruent to the phylogroups supported

by the maximum likelihood phylogeny. However, fastGEAR collapsed phylogroups 2b and 2b-a into a single lineage, while the others remained distinct. The average proportion of recent recombination in the core genome of *P. syringae* phylogroups (± the standard deviation) was estimated to be 0.71 ± 1.13% for 2a, 1.44 ± 1.16% for 2b, 2.03 ± 0.06% for Pav, and 0.81 ± 0.20% for 2d. In contrast, the proportion of recent recombination in the core genome of phylogroup 2b-a ranged from 27.98 to 30.54%, with ≥98.69% of the recombinant sequences predicted

<footnote><sup>a</sup>Percent identity was calculated after correcting for clones in the dataset.</footnote>
<figure><img src="image_34.png" /></figure>

**FIGURE 2** | Inference of recent homologous recombination between *P. syringae* sensu stricto phylogroups. Distribution, origin, and proportion of recent recombination events across the *P. syringae* core genome as predicted by fastGEAR. The lineage predictions are color coded and phylogroups labeled accordingly. Strains isolated from cucurbits hosts are shown in bold (A). Unrooted, maximum likelihood phylogeny based on 246,510 core genome SNPs (B) and unrooted, maximum likelihood phylogeny based on 81,421 recombination free SNPs (C). Branches corresponding to strains isolated from cucurbit hosts and the millet strain, HS191, are labeled in red. The scale bar indicates the number of substitutions per site.

to have originated from phylogroup 2a. Between 178 and 213 recent recombination events were dispersed across the core genome of 2b-a lineages, where ZUM3584 and HS191 displayed variation in distribution of recent recombination events in relation to the strains collected in Florida, France, and Serbia (Figure 2A). Although evidence of ancestral recombination was minimal or undetected among the 2a (0.49%), 2b (0.00%), and 2d (1.32%) lineages, 40.09% of the phylogroup Pav core genome was acquired through ancestral recombination events (Supplementary Figure S1). After removing 1,592,717 positions affected by recent recombination from the core genome alignment (64% of the original alignment), a second phylogenetic analysis was conducted using 81,421 recombination-free SNPs. Overall, the topology of the recombination filtered phylogeny was congruent to that of the unfiltered tree. However, phylogroup 2b-a appeared as a branch within phylogroup 2b, rather than as a distinct group and with considerably shorter branch lengths as compared to the unfiltered phylogeny (Figures 2B,C).

## Functional Analysis of Recombinant Genes Leading to the Emergence of Phylogroup 2b-a

The recombinant regions predicted by fastGEAR corresponded to 3,094 coding sequences among strains 13-140A, ZUM3584,

and HS191, of which 2,812 were assigned to a COG functional category. Likewise, 6,875 of 7,654 coding sequences were assigned to COG categories corresponding to regions of the core genome where no signal of recombination was detected. No significant difference in the effect of COG category on recombination was observed between strains (P = 0.15) and a strong linear relationship between recombinant and recombination-free core genes was noted across the general functional groups (R² = 0.886; Figure 3A). Although, a Grubb's test for outliers indicated that no COG category was significantly over- or underrepresented with recombinant genes (data not shown), the top three categories skewed by recombination included amino acid transport and metabolism (E), cell motility (N), and extracellular structures (W). Functional categorization of recombinant core genes utilizing the KEGG BRITE database supported these observations. Approximately 25% of the classifiable genes impacted by recombination were ATP-dependent transporters associated with numerous metabolic pathways, followed by bacterial motility proteins and secretions systems, which each comprised approximately 7–8% of the recombinant genes in each strain (Figure 3B).

Examination of the specific secretion systems impacted by recombination indicated that many of the genes associated with the biosynthesis of type III secretion system (i.e., *hrp/hrc* gene cluster) were recombinant among strains 13-140A and
<figure><img src="image_35.png" /></figure>

**FIGURE 3** | Functional impact of recombination on phylogroup 2b-a. Comparison of COG category compositions corresponding to the recombination-free and recombinant core genome of phylogroup 2b-a. (A). Stacked bar-chart showing the proportion of KEGG BRITE functional categories assigned to the recombinant core genome of three strains representative of phylogroup 2b-a recombination profiles (B).

ZUM3584, whereas no signals of recombination were detected in the *hrp/hrc* cluster of strain HS191. Phylogenetic analyses showed that 17 *hrp/hrc* genes clustered the cucurbit strains isolated in Florida, France, and Serbia into a monophyletic group with other members of phylogroup 2a and were therefore considered recombinant, while strain ZUM3584 carried 11 recombinant alleles (data not shown). A phylogenetic network constructed with a concatenated alignment of the entire *hrp/hrc* cluster (7,509 aa) was largely concordant with that of the core genome phylogeny. This analysis showed the phylogroup 2b-a cucurbit strains branching from other 2a lineages, while ZUM3584 was

placed in a hybrid position in the phylogenetic network and HS191 clustered among members of phylogroup 2b (Figure 4).

## Distribution of Type 3 Secreted Effectors (T3SEs) and Phytotoxins

The T3SE repertoires of the 56 genomes analyzed in this study are presented in Figure 5. The cucurbit strains carried between 17 and 21 potentially functional or disrupted effector genes, while an average of 13 T3SEs were present among other *P. syringae* lineages. Hierarchical clustering based on the presence/absence of effector genes revealed a loose correlation between the core
<figure><img src="image_36.png" /><figcaption>**FIGURE 4** | Evolutionary history of the *P. syringae* hrp/hrc pathogenicity island. Phylogenetic network based on a concatenated amino acid alignment of the 27 open reading frames that comprise the hrp/hrc gene cluster (7,509 aa) for a sub-set of 54 *P. syringae* genomes. The network was generated using the NeighborNet method and p-distance in Splitstree 4 software. The phylogroup classifications of lineages based on the core-genome (Figure 1) are color coded.</figcaption></figure>

genome evolution and effector repertoires, with most members of the primary *P. syringae* phylogroups clustering into distinct groups. The phylogroup 2b-a and 2a cucurbit strains carried similar effector profiles and clustered together along with other

2a lineages. The T3SE *hopA1* was exclusive to most of the strains within this group and was noted to be the only gene present in the exchangeable effector locus of phylogroup 2b-a strains (except for HS191 which carried *hopA2*), along with its corresponding chaperone, *shcA*. The effector *hopZ5* was present exclusively in the genomes of all but two cucurbit strains (13-509 and PS711) within *P. syringae sensu stricto* and displayed 98.27% amino acid sequence identity to the *hopZ5* allele present in the *P. syringae* pv. *actinidiae* biovar 3 of phylogroup 1. This effector was linked to *hopH1*, present with a point deletion, resulting a frameshift mutation in the gene. Several effector genes found to be sparsely distributed across *P. syringae* were also identified among the cucurbit-associated lineages. Most of these genes were present on an integrative and conjugative element (ICE) or plasmid loci (see results below) and included *avrRpt2* in strains 13-509A and PS711; *hopAR1* in strains 200-1 and 03-19A; *avrPto1*, *hopAU1*, and *hopX2* in strains 13-139B and 13-429; and *hopAW1*, which was common to all phylogroup 2a-cucurbit strains except 200-1 and 03-19A.

We also investigated the presence of phytotoxin biosynthetic gene clusters in the genome assemblies, which displayed a simpler distribution. The mangotoxin gene cluster was conserved across *P. syringae*. Evidence for the complete syringomycin, syringolin, and syringopeptin biosynthetic pathways was also present in most *P. syringae* genomes including the cucurbit strains examined here, although was notably absent from strains associated with diseases of woody hosts including all members of phylogroup Pav, *P. cerasi* 58<sup>T</sup>, and *P. syringae* pv. *papulans* CFBP1754<sup>PT</sup>. Additionally, phylogroup 2a cucurbit

<figure><img src="image_37.png" /><figcaption>**FIGURE 5** | Type 3 secreted effector profiles of *P. syringae sensu stricto*. Black squares indicate the presence of an intact coding sequence and gray squares indicates the presence of a putative pseudogene. The dendrogram was constructed based on the presence/absence of T3SEs using Pearson's correlation coefficient and the average linkage method with Hclust2. The effector profiles for strains isolated from cucurbit hosts and the millet strain, HS191, are shown inside the cyan box. Phylogroup classifications of lineages based on the core-genome are color coded.</figcaption></figure>
strains 03-19A, 200-1, BS2121, and ZUM3984 carried *ccd2*, which involved in phaseolotoxin biosynthesis, but lacked other components of this biosynthetic pathway (Supplementary Figure S2). Ravindran et al. (2015) previously noted that phylogroup 2b-a strain HS191 was negative for ice-nucleation activity and carried an ~2 Kb truncation in the center of the ice-nucleation protein, *inaZ*. As the 2b-a cucurbit strains were previously found to be negative for ice-nucleation activity (Newberry et al., 2018), we investigated the presence of *inaZ* in the genome assemblies of these strains and found the same truncation (data not shown).

## Pan-Genome Association Analysis

We investigated the pan-genome of *P. syringae* to identify orthologous groups (OGs) associated the cucurbit strains and therefore, potentially involved in niche adaptation. Among the 19,613 OGs that comprised the *P. syringae* pan-genome, only seven were significantly associated with both phylogroup 2a and 2b-a cucurbit strains (Bonferroni *p* ≤ 10⁻⁵). Among these, two displayed 100% specificity, indicating that these OGs were completely absent from the genomes of related *P. syringae* strains and included the T3SE *hopZ5* and a hypothetical protein also present in the ICE locus. Several other OGs with known functions in virulence displayed significant associations and included the T3SE *hopA1*, its corresponding chaperone *shcA*, and the type VI secreted effector *vgrG*. The gene that displayed the strongest association with the cucurbit lineages was a hypothetical protein of 63 aa in length, adjacent to a putative iron-sulfur binding gene cluster (Table 4).

## Convergent Acquisition of T3SEs and Other Putative Virulence Factors Through Integrative and Conjugative Elements and Plasmids

Analysis with Island Viewer 4 revealed that all cucurbit strains carried a predicted genomic island between approximately 90 and 125 Kb in size that was similar in structure to an ICE described in *P. syringae* pvs. *actinidiae* and *phaseolicola* of phylogroups 1 and 3, respectively (Pitman et al., 2005; McCann et al., 2013). The boundaries of the island were delineated by *parA* and *xerC* at opposite ends, each flanking a repeat region

overlapping *tRNA*lys, which serve as the attachment sites for the island (Pitman et al., 2005). The contigs corresponding to predicted ICE loci were subsequently extracted from the genomes of representative cucurbit strains (divided between one and five contigs per genome) and used to construct a multiple sequence alignment along with the complete ICE sequences of *P. syringae* pv. *actinidiae* (Psa) SR121 (Accession no. KX009066) and *P. syringae* pv. *syringae* HS191 (Figure 6). We were unable to confirm whether the predicted ICE carried by strains 13-429 and 13-139B was intact due to the presence of the pyoverdine biosynthetic gene cluster (~60 Kb) in the center of the ICE region of these two strains and therefore, were not included in the alignment.

This analysis revealed that the putative ICEs displayed a mosaic structure characterized by differing mobile genetic elements and gene cassettes. It also revealed the overall synteny of the ICEs was conserved and that the core ICE genes displayed a high degree of homology (ranging from 96 to 99% average pairwise identity) with the ICE of Psa strain SR121. Similar to the ICE described in *P. syringae* pv. *phaseolicola* strain 1302A (Pitman et al., 2005), *hopAR1* was carried in the ICE of strain 200-1 between a predicted helicase and *pilL*, while *avrRpt2* was found at the same locus in strain 13-509A and HS191 carried a predicted type VI secretion protein (*hcp1*). Similarly, *hopZ5* and a disrupted copy of *hopH1* was present in the ICEs of all cucurbit strains except 13-509A, which carried *hopC1* and an intact copy of *hopH1* at the same locus. Both of these effector pairs were flanked by a predicted phage integrase and site-specific recombinase (*xerD*) that shared 97.05 and 93.13 aa identity, respectively.

The effector *hopZ1* was also present in the ICE of all but two cucurbit strains (ZUM3584, 200-1) and was located approximately 10 Kb downstream of *hopZ5/hopC1*, near the boundary of the island. Interestingly, *hopZ1* was also carried by strain HS191 and was linked to a similar integrase/recombinase gene cassette. However, in HS191, *hopZ1* was present in the exchangeable effector locus, as indicated by the presence of *queA* and *tRNA*leu, which delineate the boundary of this pathogenicity island (Supplementary Figure S3). Several other accessory genes and putative virulence factors were identified in ICEs. Strain ZUM3985 carried *hopAW1* at a locus unique to the ICE found in this strain. It also carried a predicted calcium binding protein (1,691 aa) of the RTX toxin superfamily, which is a protein family

<table><caption>TABLE 4 | Top orthologous groups associated with phylogroup 2b-a and 2a cucurbit lineages.</caption><thead><tr><th>Locus tag<sup>a</sup></th><th>Gene/Annotation<sup>b</sup></th><th>Sensitivity<sup>c</sup></th><th>Specificity<sup>c</sup></th><th>Bonferroni (p)</th></tr></thead><tbody><tr><td>106332</td><td>Hypothetical protein</td><td>100</td><td>95.12</td><td>1.36E-07</td></tr><tr><td>1061120</td><td>Hypothetical protein (ICE)</td><td>86.67</td><td>100</td><td>9.03E-07</td></tr><tr><td>106173</td><td>hopZ5 (ICE)</td><td>86.67</td><td>100</td><td>9.03E-07</td></tr><tr><td>1099230</td><td>shcA</td><td>100</td><td>87.80</td><td>1.55E-05</td></tr><tr><td>1056171</td><td>Hypothetical protein</td><td>93.33</td><td>92.68</td><td>2.67E-05</td></tr><tr><td>107291</td><td>vgrG</td><td>100</td><td>86.37</td><td>5.43E-05</td></tr><tr><td>1099229</td><td>hopA1</td><td>100</td><td>86.37</td><td>5.43E-05</td></tr></tbody></table>

<footnote><sup>a</sup>IMG locus tags reported for strain 13-140A, the identifier Ga0170668_precedes each locus tag number. <sup>b</sup>ICE shown in parentheses indicates that a gene was present in the integrative and conjugative element locus. <sup>c</sup>Sensitivity indicates the proportion of the target population (cucurbit lineages) in which an orthologous group was present, and specificity shows the proportion of the non-target population in which an orthologous group was absent.</footnote>
<figure><img src="image_38.png" /></figure>

commonly secreted via the type I secretion system (Linhartová et al., 2010). Likewise, a cassette of small peptides between 62 and 83 aa in length with a predicted *papB* domain, which is involved in the regulation of adhesin biosynthesis (Xia et al., 2000), was conserved in ICE of several strains including 13-140A, ZUM3584, 200-1, HS191, and SR121 (Figure 6).

Analysis with plasmidSPAdes provided no evidence of plasmid
sequences in phylogroup 2b-a cucurbit strains, whereas several
were assembled among strains within phylogroup 2a. These
results were consistent with the size of genome assemblies,
which averaged 5.92 and 6.23 Mb for the 2b-a and 2a cucurbit
strains, respectively (Table 1). A 52.42 Kb contig was assembled
in strains 200-1 and 03-19A which displayed 93% nucleotide
identity with 66% query coverage to the complete plasmid
sequence from strain HS191 (NZ_CP006257.1). This plasmid
carried the effectors *hopC1* and *hopH1*, which marked the
second copy of *hopH1* for these two strains, in addition to
the disrupted allele present in the ICE. A putative virulence
plasmid of 16.51 Kb in size was assembled in strains 13-
429 and 13-139B which harbored four accessory effector genes
including *hopAU1*, *hopAW1*, *hopAF1*, and *avrPto*. The top
hit for this contig in the NCBI complete plasmid database
was plasmid PP3 (NZ_LT963405.1) of *P. syringae* pv. *avii*
strain CFBP3846 and displayed only distant homology (95%
nucleotide identity and 39% query coverage). Strains BS2121
and ZUM3984 also carried two putative plasmids, for which
the top BLAST hits included the complete plasmids pCC1557
(NZ_CP007015.1) and pB13-200A (NZ_CP019872.1). However,

no apparent virulence factors were identified in these contigs.
A summary of the origin of T3SEs among representative cucurbit
strains is presented in **Figure 7C** and a compiled list of
putative plasmid sequences and BLAST results are available in
**Supplementary Table S1**.

Correlation Between T3SE Repertoires
and Pathogenicity

The results from two independent pathogenicity experiments
produced similar results and showed significant differences
in disease severity between the treatments (*P* < 0.0001).
Phylogroup 2b-a strains 13-140A, 13-C2, and ZUM3854 induced
expanding lesions and foliar blighting on watermelon and
squash, with mean severity ratings ranging from 5.60 to 7.50
among the three strains. In contrast, the squash strains 13-
509A and PS711 produced only superficial lesions on both
hosts and mean severity ratings that did not significantly differ
from that of the millet strain, HS191 (mean severity ratings
ranging from 0.70 to 2.40). Two effector pairs differentiated
the virulent from weakly virulent strains within this group
and included *hopZ5* and the disrupted copy of *hopH1* among
the virulent strains, while the weakly virulent strains carried
*hopC1* and an intact copy of *hopH1*. Within phylogroup 2a,
no significant differences in disease severity were observed
among strains BS2121, ZUM3984, and 13-139B, which produced
moderate to high levels of disease severity on both hosts.
While strains 03-19A and 200-1 also induced expanding
lesions and foliar blighting on squash (between 5.00 and
<figure><img src="image_39.png" /><figcaption>**FIGURE 7** | Correlation between T3SE profiles and pathogenicity of *P. syringae* strains to watermelon and squash. Pathogenicity analysis for representative cucurbit strains and the millet strain, HS191, from phylogroups 2a and 2b-a. Mean severity rating from 0 to 8, taken 14 days after inoculation from two independent growth room experiments (n = 10) (A). Representative bacterial spot symptoms on squash and watermelon, respectively (B). Comparison of effector repertoires among strains used in pathogenicity testing for phylogroups 2b-a (left) and 2a (right). An asterisk indicates a predicted disruption in the coding sequence and the superscript indicates the genomic regions of the effector gene in the genome assemblies: ¹conserved effector locus, ²exchangeable effector locus, ³integrative and conjugative element locus, ⁴other genomic island, ⁵phage region, ⁶plasmid. An effector gene with multiple superscripts indicates a T3SE was acquired through different sources among strains (C).</figcaption></figure>

7.00 mean severity), they were weakly virulent on watermelon (between 1.40 and 2.00 mean severity). Four T3SEs were unique to the squash limited strains of phylogroup 2a and included *hopC1*, an intact copy of *hopH1*, *hopAR1*, and

*hopM1* disrupted by a frameshift mutation. Likewise, *hopAW1*, *hopZ1*, and an intact *hopM1* allele were unique effectors common among the strains virulent to both watermelon and squash (Figure 7).
DISCUSSION

Bacterial leaf spot of watermelon, cantaloupe, and squash, caused by *P. syringae* (referring to sensu stricto unless otherwise stated), is a sporadic plant disease and often attributed to contaminated seed. Here, we provide the first genomic insights into the *P. syringae* strains associated with these three cucurbit hosts, with a focus on three apparently clonal lineages located in phylogroups 2a and 2b. We sequenced the genomes of six strains that were isolated from diseased cucurbits grown in the United States, Europe, and China over the period of a more than a decade, as well as eight strains that were isolated from watermelon and squash grown in six different Florida counties during the 2013 and 2014 seasons (Table 1). Reconstruction of the *P. syringae* core genome revealed that the cucurbit-associated lineages of phylogroup 2b formed a novel phylogenetic group, designated here as phylogroup 2b-a (Figure 1), which emerged through genome-wide homologous recombination between phylogroups 2a and 2b (Figure 2). While the majority of this group consisted of a single clonal lineage isolated from plants grown in the United States, France, and Serbia, strains ZUM3584 and HS191 were more distantly related and displayed variation in the distribution of recombinant loci that were dispersed across the core genome (Figures 1, 2). As the overall proportion of recombinant sequences was similar among all 2b-a lineages (ranging from 27.98 to 30.54% of the core genome), the distribution of recent recombination events, rather than the quantity, was the primary factor driving diversification within this group. These observations were supported by the recombination filtered core-genome phylogeny, which showed phylogroup 2b-a as a branch within phylogroup 2b, rather than as a distinct group, and more recently diverged as compared to the unfiltered core-genome phylogeny (Figures 2B,C). In contrast to the 2b-a strains, the *P. syringae* strains isolated from watermelon, cantaloupe, and squash in phylogroup 2a were nested among the lineages of other strains isolated from corn, wheat, switchgrass roots, and various tree species (Figure 1).

Unexpectedly, another example of genome-wide homologous
recombination was inferred in our analysis among the *P. syringae*
pv. *avellanae* (*Pav*) lineages of phylogroup Pav, whereby
an estimated 40.09% of the core genome was acquired
through ancestral recombination events (Supplementary
Figure S1). These results were particularly interesting as the
four *Pav* genomes analyzed were not clonal and displayed
levels of sequence divergence equivalent to that of other
*P. syringae* phylogroups (Table 3), suggesting that the fixation
of recombinant alleles across phylogroup Pav (i.e., inference of
ancestral recombination) was not an artifact of small sample
size. These results were striking given the minimal impact of
recombination on the evolution of the primary *P. syringae*
phylogroups, which displayed an average of 95% pairwise
identity between groups and were at the edge of the proposed
species delimitation boundary for prokaryotes (Table 3). Both
the recent and ancestral recombination profiles suggested
admixture between phylogroups Pav and 2b (Figure 2A and
Supplementary Figure S1). However, because phylogroup

Pav did not display levels of pairwise identity intermediate of
two different phylogroups, as was observed for 2b-a (Table 3),
this could indicate that recombination has occurred between
a currently unsampled *P. syringae* population. Taken together,
these observations indicate that *P. syringae* phylogroups may
inhabit overlapping environmental niches and are consistent
with that of an epidemic population structure.

The link between macro-scale recombination events and the emergence of hybrid, epidemic-clones has been well documented among human and animal bacterial pathogens (Spoor et al., 2015). As such, genome-wide recombination events are associated with niche adaptation and remodeling of the host-pathogen interaction. Functional analysis of the recent recombination events that led to the emergence of phylogroup 2b-a painted a similar picture. Although a strong linear relationship between the COG category compositions suggested an overall proportional change among the general functional groups (Figure 3A), analysis utilizing the KEGG BRITE database revealed that pathways involved in the ATP-dependent transport of amino acids and other organic compounds, bacterial motility, and secretion systems were enriched for recombinant genes (Figure 3B). Interestingly, most of the specific pathways found within these functional groups were similarly affected by recombination in the millet pathogen, *P. syringae* pv. *syringae* HS191, except for those genes encoding for the type 3 secretion system, otherwise known as the *hrp/hrc* pathogenicity island. A phylogenetic analysis confirmed that the recombinant *hrp/hrc* genes shared a common evolutionary history with that of phylogroup 2a (Figure 4) and therefore, may be a significant factor contributing to the convergent pathogenicity of 2a and 2b-a lineages to cucurbit hosts. This finding was reminiscent of bacterial etiolation and decline of creeping bentgrass, for which the host-specificity of two phylogenetically distinct *Acidovorax avenae* populations was associated with three ancestral recombination events affecting the *hrp/hrc* gene cluster (Zeng et al., 2017).

In addition to genome-wide recombination, we found
evidence for the remodeling of T3SE repertoires of phylogroup
2b-a. This remodeling was marked by the acquisition of *hopA1*,
which was carried exclusively by most phylogroup 2a lineages
within *P. syringae*, including those isolated from cucurbit hosts.
The *hopA1* and *shcA* genes, which form an effector-chaperone
complex, were the only two genes occupying the exchangeable
effector locus of phylogroup 2b-a lineages (except for the millet
strain HS191 which carried *hopA2*), suggesting that this effector
was recently acquired. Previous studies have demonstrated that
strains of *P. syringae sensu stricto* carry fewer T3SEs than
other *Pseudomonas* spp. and this was associated with the
production of broad host-range toxins such as syringomycin
(Baltrus et al., 2011; Hulin et al., 2018). Although, we found
evidence for the syringomycin, syringopeptin, and syringolin
biosynthetic clusters in the genome assemblies (Supplementary
Figure S2), on average, phylogroup 2b-a and 2a cucurbit strains
carried more T3SEs (between 17 and 21 potentially functional
or disrupted effector genes) than other *P. syringae* lineages
(Figure 5). Most of these accessory T3SEs were acquired through
independent mechanisms of HGT. These included an ICE,
present in both 2a and 2b-a lineages (Figure 6), as well as two different plasmids harboring effector genes among phylogroup 2a lineages (Supplementary Table S1). Interestingly, a positive correlation between the proportion of recent recombination in the core genome and the number of T3SEs was observed in other *P. syringae* strains, including *P. cerasi* 58T, *P. syringae* pv. *papulans* CFBP1754PT, and *P. syringae* pv. *pisi* PP1 and highlights the interplay of homologous recombination and HGT in pathogen emergence (Figures 2, 5).

Despite the open pan-genome exhibited by *P. syringae* (Nowell et al., 2014), pan-genome association analysis identified only a handful of orthologous groups that were significantly associated (Bonferroni $p \le 10^{-5}$) with both the 2a and 2b-a cucurbit strains (Table 4). Four of the seven significantly associated genes included previously described virulence factors such as the T3SE *hopZ5*, the *hopA1/shcA* effector-chaperone complex, and the type VI secreted effector *vgrG*, suggesting a role suggesting a role in pathoadaptation. The effector *hopZ5* was among several accessory T3SEs acquired through the ICE (Figure 6) and was the only virulence factor which distinguished the cucurbit-associated lineages from related pathogens of various plant species (Table 4). Interestingly, *hopZ5* was also among the few virulence factors acquired by the pandemic strains of *P. syringae* pv. *actinidiae* (*Psa*) biovar 3 (which may be considered a species distinct of *P. syringae sensu stricto*) and to date, has only been recorded in the genomes of *Pseudomonas* spp. associated with diseases of woody hosts (McCann et al., 2013; Nowell et al., 2016). Although *hopZ5* was not linked to the ICE described in *Psa* biovar 3, it was curious to find that many of the core ICE genes associated with the horizontal transfer of this effector among the cucurbit strains shared a recent evolutionary history with the ICE carried by *Psa* strain SR121 (Figure 6).

The acquisition of novel virulence factors through HGT is commonly attributed to changes in bacterial phenotypes. Hence, it was striking to note the contrasting pathogenicity among the cucurbit strains (Figure 7). As these differences in pathogenicity were observed among strains of the same clonal lineage, this indicated that components of the accessory genome, rather than the underlying genetic background was likely the key factor influencing these phenotypes. We found that two phylogroup 2b-a strains collected in Florida and Serbia (13-509A and PS711, respectively) were both weak pathogens of watermelon and squash and produced superficial lesions like that of the millet strain HS191 (Figures 7A,B). Interestingly, 13-509A and PS711 also carried effector repertoires more like that of the millet strain than other virulent 2b-a strains isolated from cucurbits (Figure 5). This difference was primarily accounted for by the presence of *hopC1*, in place of *hopZ5* in the ICE locus, while *hopC1* was carried in the exchangeable effector locus of strain HS191 (Figure 7C). Both *hopZ5* and *hopC1* were adjacent to a second T3SE, *hopH1* (Figure 6). However, the *hopH1* allele present in the virulent cucurbit strains carried a point deletion, rendering a frameshift mutation in the gene and was therefore not predicted to be translocated or expressed.

A similar negative association between the *hopC1* and *hopH1* effector pair and virulence was observed among phylogroup 2a

strains 03-19A and 200-1. Both strains were weakly pathogenic to watermelon and carried *hopC1* and an intact copy of *hopH1* on a putative ~52 Kb plasmid (Figure 7C). While the disruption of *hopH1* among highly virulent strains within phylogroups 2a and 2b-a may serve to avoid host recognition, we cannot discount the possibility that the disruption of this effector was an artifact due its horizontal transfer and linkage to *hopZ5*, rather than selection pressure. Furthermore, we observed that strains 03-19A and 200-1 induced severe foliar blighting on squash, rather than being weakly pathogenic in general (Figures 7A,B). This indicates a difference in the nature of the host-pathogen interaction and perhaps suggests induction of an effector triggered immunity in watermelon. These strains also carried *hopAR1* in the ICE locus (Figure 7C), which is an avirulence gene that has been well described in *P. syringae* pv. *phaseolicola* (Tsiamis et al., 2000) and is another candidate potentially limiting the host-range of these two stains to squash. Further analysis is required to determine whether the expression of any of the gene candidates identified here, including *hopC1*, *hopH1*, and *hopAR1* may serve as negative pathogenicity factors in cucurbits, and conversely, whether *hopZ5* promotes the virulence of these pathogens.

The widespread distribution of two genetically monomorphic *P. syringae* populations in association with multiple cucurbit hosts suggests a role for natural selection in maintaining these populations. Furthermore, the convergent acquisition of alleles through homologous recombination and multiple virulence factors through HGT among the cucurbit strains examined here may be interpreted as genomic signatures of host-adaption (Sheppard et al., 2018). Although the evolutionary processes inferred here mirrored those of other plant-pathogenic bacteria responsible for an array of emerging diseases, we do not know if these recombination events occurred upon colonization of cucurbit hosts or if these events happened prior to exposure to this ecological niche. Clues as to the answer of this question may found in the recombinant genome of the millet pathogen, *P. syringae* pv. *syringae* HS191, which lacked similar signatures in ecologically significant loci such as the *hrp/hrc* pathogenicity island and ICE locus. The significance of other numerous functional pathways affected by recent recombination events remains to be explored and provides further evidence for the emerging paradigm that plant-pathogen compatibility is not defined solely by T3SE repertoires but is likely a multifactorial process involving the acquisition/metabolism of plant derived nutrients, bacterial chemotaxis, and evasion of plant-innate immunity, among other processes (Jacques et al., 2016). Ultimately, this hypothesis will need to be tested through more extensive sampling of *P. syringae* strains from multiple environments coupled with functional analysis.

## AUTHOR CONTRIBUTIONS

EN, ME, JJ, EG, and MP conceived the project. MP, CB, NZ, EN, and AO provided *P. syringae* strains. EN conducted the pathogenicity assays. ME prepared the genomic DNA. ME, ST,
and JJ oversaw the sequencing experiments. ST and NP assembled the draft genomes. ME conducted the toxin analysis and EN performed all other computational analyses with support from JH-T and NP. ME submitted the genome sequences to NCBI-GenBank and JGI-IMG. EN wrote the manuscript and all authors provided a critical review of the paper.

## FUNDING

This research was supported in part by the Southern IPM Center, Florida Watermelon Association, the National Watermelon Association, and USDA-NIFA. NZ and AO were supported by the national project III46008. NP and EN were supported by funding from NIFA-Hatch and Alabama Agriculture Experiment Station. Publication of this manuscript

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was supported by the University of Florida Open Access Publishing Fund.

## ACKNOWLEDGMENTS

Thanks to Dr. Bert Woudt for providing *P. syringae* strains collected in Italy and China. The authors declare that all standard biosecurity and institutional safety procedures were adhered to during this work.

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The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2019.00270/full#supplementary-material

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Newberry, Ebrahim, Timilsina, Zlatković, Obradović, Bull, Goss, Huguet-Tapia, Paret, Jones and Potnis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
# Corrigendum: Inference of Convergent Gene Acquisition Among *Pseudomonas syringae* Strains Isolated From Watermelon, Cantaloupe, and Squash

Eric A. Newberry¹,², Mohamed Ebrahim³,⁴, Sujan Timilsina³, Nevena Zlatković⁵, Aleksa Obradović⁵, Carolee T. Bull⁶, Erica M. Goss³,⁷, Jose C. Huguet-Tapia³, Mathews L. Paret², Jeffrey B. Jones³* and Neha Potnis¹*

OPEN ACCESS

**Approved by:**
Frontiers Editorial Office,
Frontiers Media SA, Switzerland

*Correspondence:
Jeffrey B. Jones
jbjones@ufl.edu
Neha Potnis
nzp0024@auburn.edu

**Specialty section:**
This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Microbiology

**Received:** 04 April 2019
**Accepted:** 16 April 2019
**Published:** 03 May 2019

**Citation:**
Newberry EA, Ebrahim M, Timilsina S, Zlatković N, Obradović A, Bull CT, Goss EM, Huguet-Tapia JC, Paret ML, Jones JB and Potnis N (2019) *Corrigendum: Inference of Convergent Gene Acquisition Among Pseudomonas syringae Strains Isolated From Watermelon, Cantaloupe, and Squash*. Front. Microbiol. 10:963. doi: 10.3389/fmicb.2019.00963

¹Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, United States. ²Department of Plant Pathology, North Florida Research and Education Center, University of Florida, Quincy, FL, United States. ³Department of Plant Pathology, University of Florida, Gainesville, FL, United States. ⁴Department of Plant Pathology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. ⁵Faculty of Agriculture, University of Belgrade, Belgrade, Serbia. ⁶Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, State College, PA, United States. ⁷Emerging Pathogens Institute, University of Florida, Gainesville, FL, United States

**Keywords:** horizontal gene transfer, homologous recombination, pathogen emergence, *Pseudomonas syringae sensu stricto*, cucurbits

## A Corrigendum on

### Inference of Convergent Gene Acquisition Among *Pseudomonas syringae* Strains Isolated From Watermelon, Cantaloupe, and Squash

by Newberry, E. A., Ebrahim, M., Timilsina, S., Zlatković, N., Obradović, A., Bull, C. T., et al. (2019). Front. Microbiol. 10:270. doi: 10.3389/fmicb.2019.00270

In the original article, we neglected to acknowledge the University of Florida Open Access Publishing Fund in supporting the publication of this manuscript. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Copyright © 2019 Newberry, Ebrahim, Timilsina, Zlatković, Obradović, Bull, Goss, Huguet-Tapia, Paret, Jones and Potnis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
<figure><img src="image_40.png" /></figure>

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# Multiple Recombination Events Drive the Current Genetic Structure of *Xanthomonas perforans* in Florida

Sujan Timilsina¹, Juliana A. Pereira-Martin², Gerald V. Minsavage¹, Fernanda Iruegas-Bocardo¹, Peter Abrahamian², Neha Potnis³, Bryan Kolaczkowski⁴, Gary E. Vallad²*, Erica M. Goss¹,⁵* and Jeffrey B. Jones¹*

¹ Department of Plant Pathology, University of Florida, Gainesville, FL, United States, ² Gulf Coast Research and Education Center, University of Florida, Gainesville, FL, United States, ³ Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, United States, ⁴ Microbiology and Cell Science, University of Florida, Gainesville, FL, United States, ⁵ Emerging Pathogens Institute, University of Florida, Gainesville, FL, United States

Prior to the identification of *Xanthomonas perforans* associated with bacterial spot of tomato in 1991, *X. euvesicatoria* was the only known species in Florida. Currently, *X. perforans* is the *Xanthomonas* sp. associated with tomato in Florida. Changes in pathogenic race and sequence alleles over time signify shifts in the dominant *X. perforans* genotype in Florida. We previously reported recombination of *X. perforans* strains with closely related *Xanthomonas* species as a potential driving factor for *X. perforans* evolution. However, the extent of recombination across the *X. perforans* genomes was unknown. We used a core genome multilocus sequence analysis approach to identify conserved genes and evaluated recombination-associated evolution of these genes in *X. perforans*. A total of 1,356 genes were determined to be “core” genes conserved among the 58 *X. perforans* genomes used in the study. Our approach identified three genetic groups of *X. perforans* in Florida based on the principal component analysis (PCA) using core genes. Nucleotide variation in 241 genes defined these groups, that are referred as Phylogenetic-group Defining (PgD) genes. Furthermore, alleles of many of these PgD genes showed 100% sequence identity with *X. euvesicatoria*, suggesting that variation likely has been introduced by recombination at multiple locations throughout the bacterial chromosome. Site-specific recombinase genes along with plasmid mobilization and phage associated genes were observed at different frequencies in the three phylogenetic groups and were associated with clusters of recombinant genes. Our analysis of core genes revealed the extent, source, and mechanisms of recombination events that shaped the current population and genomic structure of *X. perforans* in Florida.

**Keywords:** core genome multilocus sequence typing, bacterial evolution, recombination, horizontal gene transfer (HGT), *Xanthomonas perforans*, bacterial spot

## INTRODUCTION

Bacterial pathogens challenge the sustainability and economics of agricultural production. The most damaging bacterial plant pathogens combine rapid evolution with a tendency for emerging strains to spread quickly over long-distances (Carroll et al., 2014). Characterizing bacterial strains associated with disease outbreaks advances our understanding of changes in pathogen

**OPEN ACCESS**

**Edited by:**
Dawn Arnold,
University of the West of England,
United Kingdom

**Reviewed by:**
Prabhu B. Patil,
Institute of Microbial Technology
(CSIR), India
Marcus Michael Dillon,
University of Toronto, Canada

**\*Correspondence:**

Gary E. Vallad
gvallad@ufl.edu
Erica M. Goss
emgoss@ufl.edu
Jeffrey B. Jones
jbjones@ufl.edu

**Specialty section:**
This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Microbiology

**Received:** 30 November 2018

**Accepted:** 20 February 2019

**Published:** 13 March 2019

**Citation:**
Timilsina S, Pereira-Martin JA, Minsavage GV, Iruegas-Bocardo F, Abrahamian P, Potnis N, Kolaczkowski B, Vallad GE, Goss EM and Jones JB (2019) Multiple Recombination Events Drive the Current Genetic Structure of Xanthomonas perforans in Florida. *Front. Microbiol.* 10:448. doi: 10.3389/fmicb.2019.00448
populations and geographic distribution of genetic variation as well as the potential to trace the source of outbreaks. Technological advancements in both sequencing and computational tools have facilitated translational research for bacterial disease management via epidemiological and resistance-based approaches in hosts ranging from humans to plants (Köser et al., 2012; Gétaz et al., 2018).

Evolutionary and epidemiological studies of bacterial populations use core genomes, pan-genomes, and intergenic regions to uncover patterns and processes of strain emergence and spread (Biek et al., 2015; McNally et al., 2016; Jibrin et al., 2018). The process of whole genome sequencing followed by gene-by-gene comparisons to identify core genes, which are present in all sampled genomes, expands MLSA (Multilocus Sequence Analysis) from a half-dozen to several hundred or even a thousand genes (Bialek-Davenet et al., 2014). This reproducible approach for phylogenetic comparisons is termed core genome MLSA (cgMLSA) (Maiden and Harrison, 2016; Ghanem et al., 2017; Moura et al., 2017).

The genus *Xanthomonas* is comprised of plant pathogenic bacteria affecting multiple plant hosts. Fresh market tomato production in Florida is severely affected by bacterial spot disease of tomato caused by *Xanthomonas perforans* (Jones et al., 2004; Horvath et al., 2012). Previous studies on *X. perforans* strains isolated from Florida have shown shifts in the bacterial population with regards to species, races, bactericide resistance, bacteriocin production, effector profiles, and phylogenetic groups (Timilsina et al., 2014; Schwartz et al., 2015; Abrahamian et al., 2018). Prior to the initial identification of *X. perforans* in 1991, only tomato race 1 (T1) strains of *Xanthomonas euvesicatoria* were reported on tomato in Florida (Horvath et al., 2012; Timilsina et al., 2016). The first *X. perforans* strains from Florida were identified as tomato race 3 (T3) strains (Jones, 2004; Timilsina et al., 2016). T3 strains carry the functional XopAF (*avrXv3*) and XopJ4 (*avrXv4*) effectors. In 1998, a tomato race 4 (T4) *X. perforans* strain was identified (Minsavage et al., 2003) that lacked a functional XopAF effector. Various surveys and independent isolations over the last two decades determined that T4 *X. perforans* has become the dominant pathogen causing bacterial spot on tomato in Florida (Horvath et al., 2012; Vallad et al., 2013). While selection for widespread copper tolerance in bacteria is expected due to the historical reliance on copper-based bactericides for the management of bacterial spot disease (Vallad et al., 2010), the drivers of tomato race change (in the absence of host resistance), host expansion, and introduction of novel effector genes are less obvious.

We previously identified at least two phylogenetic groups of *X. perforans* in strains isolated from Florida in 2006 and 2012 using MLSA of six housekeeping genes (Timilsina et al., 2014). Among the two groups, group 2 strains appeared recombinant based on the sequences of two housekeeping genes that were identical to *X. euvesicatoria* strain Xe85-10, isolated from pepper (Timilsina et al., 2014). Although *X. perforans* strains are regarded as tomato specific, a group 2 *X. perforans* strain, Xp2010, was isolated from pepper, and other group 2 strains from tomato were shown to cause disease on pepper (Timilsina et al., 2014; Schwartz et al., 2015). The phenotypic and genotypic

changes in group 2 strains suggests that the genomic impact of recombination likely extends beyond the few genes we have previously reported (Jibrin et al., 2018).

Phylogenetic methods are commonly applied to the study of bacterial strain ancestry and diversification (Didelot and Falush, 2007). However, most phylogenetic analysis methods assume recombination is absent, and the presence of recombination in the history of a sample can cause incorrect phylogenies. For multilocus sequence analysis of bacterial populations, the tendency has been to remove recombination in order to correctly interpret ancestral relationships for the unrecombined portion of the genome, the “clonal frame” (Wicker et al., 2012; Croucher et al., 2014; Lu et al., 2016). However, considering the ubiquity and impact of recombination on bacterial genetic diversity and evolution, the effect of recombination on phylogenetic relationships should be considered (Didelot and Wilson, 2015; Mostowy et al., 2017). Horizontal gene transfer can expedite evolution and may influence host-specificity in bacteria (Ochman et al., 2000; Yan et al., 2008). Genetic transfer may result in trait convergence due to shared genes acquired by horizontal gene transfer, or lead to the formation of distinct lineages or phylogroups (McNally et al., 2016). Transduction via virus, transformation by donor DNA, and conjugation with the donor are the three mechanisms by which bacteria acquire genetic material (Ochman et al., 2000). The acquisition of genomic DNA can leave specific signals surrounding the introduced genes at the integration sites (Ochman et al., 2000). For example, genomic movement between bacterial species by transduction is limited by phage-host specificity and the events are mediated by mobile DNA vectors observed along with the translocated genomic DNA (Popa et al., 2017).

Our objectives were to determine the extent of recombination in *X. perforans* genomes from Florida strains, identify recombined genes that contribute to the observed population structure in Florida, and evaluate putative mechanisms of genetic transfer of recombined regions. Using a cgMLSA approach, our study provides insights into the extent of recombination and mechanisms of horizontal gene transfer affecting the core genes that constitute the majority of the genomic background of phylogenetically divergent *X. perforans* genomes. The presence of multiple recombination mechanism signals throughout the genome, affecting both core and pathogenicity associated genes, is consistent with high genome plasticity in *X. perforans*. We provide empirical evidence that recombination of core genes has defined the existing phylogenetic groups of *X. perforans* in Florida. The observed genomic patterns appear to be correlated with traits like host-specificity and overall pathogen fitness and indicate that recombination has an extraordinary impact on evolutionary processes in *X. perforans*.

# MATERIALS AND METHODS

## Bacterial Strains, Genome Assembly, and Genome Similarity

The genomes of 58 *X. perforans* strains isolated from Florida in 1991, 2006, 2012/13, and 2015 were used in this study
<table><caption>**TABLE 1** | List of strains used in this study.</caption><thead><tr><th>Phylogenetic group</th><th>Year</th><th>Strains</th><th>Source</th></tr></thead><tbody><tr><td rowspan="3">Group 1</td><td>1991</td><td>Xp91-118</td><td>Potnis et al., 2011</td></tr><tr><td>2006</td><td>Xp4B, Xp4-20, Xp5-6, Xp11-2, Xp15-11, Xp18-15</td><td>Schwartz et al., 2015</td></tr><tr><td>2012</td><td>GEV872, GEV893, GEV904, GEV909, GEV915, GEV917, GEV936, GEV940, GEV968, GEV993, GEV1026</td><td>Schwartz et al., 2015</td></tr><tr><td rowspan="7">Group 2</td><td>2006</td><td>Xp3-15, Xp7-12, Xp8-16, Xp9-5, Xp10-13</td><td>Schwartz et al., 2015</td></tr><tr><td>2010</td><td>Xp2010</td><td></td></tr><tr><td>2012</td><td>GEV839, GEV1001, GEV1044, GEV1054, GEV1063</td><td></td></tr><tr><td>2013</td><td>TB6, TB9, TB15</td><td></td></tr><tr><td>2015/16</td><td>GEV1921, GEV1989, GEV2004, GEV2009, GEV2015, GEV2049, GEV2063, GEV2098, GEV2115, GEV2116, GEV2117, GEV2120, GEV2129, GEV2132, GEV2135</td><td>This study</td></tr><tr><td>2006</td><td>Xp17-12</td><td>Schwartz et al., 2015</td></tr><tr><td>2015/16</td><td>GEV2010, GEV2097, GEV2112, GEV2121, GEV2122, GEV2124, GEV2125, GEV2127, GEV2130, GEV2134</td><td>This study</td></tr></tbody></table>

(Table 1). Draft whole genome sequences of 33 strains, including reference strain Xp91-118, were previously published (Potnis et al., 2011; Schwartz et al., 2015). The remaining 25 X. perforans strains collected in 2015 are also publicly available (Supplementary Table 1). The raw Illumina MiSeq 2x250 basepair reads were reassembled using Spades v.3.11 with read error correction and “--careful” switch (Bankevich et al., 2012). The assembled sequences were validated using filter-spades.py<fnref n="1" /> and Bowtie2 was used to align the assembled reads to identify inconsistencies (Langmead and Salzberg, 2012). Pilon (Walker et al., 2014) was used to remove the inconsistencies identified by Bowtie2. The assembled sequences were filtered to remove sections with coverage less than 2 and contig size less than 500 nucleotides. CheckM identified more than 99% genome completeness with less than 0.6% contamination per genome (Parks et al., 2015; Supplementary Table 1). The genomes were annotated using the IMG/JGI platform (Markowitz et al., 2013). Following assembly, pairwise Average Nucleotide Identity (ANI) based on blast was calculated using jSpecies v 1.2.1 (Richter and Rosselló-Móra, 2009).

## Pan-Genome Size

For evaluation of the pan-genome of the 58 X. perforans strains, all genes were extracted from the 58 X. perforans strains using

roary (Page et al., 2015) following gene annotation from prokka (Seemann, 2014). The method yielded a total of 7,245 genes. The pan-genome matrix of gene presence/absence in each genome was used as input for a rarefaction analysis to calculate the average number of genes added with each additional genome (Méric et al., 2014). The calculation was randomized by resampling 100 times. The Heaps law function was fitted to the data using the micropan package in R to the rarefaction curve (Tettelin et al., 2008; Snipen and Liliand, 2015). The Heaps law model estimates the parameter alpha. When alpha > 1, this suggests a closed pan genome and saturated sampling of the gene pool, while alpha < 1 suggests an open pan-genome.

## Core Gene Identification and Alignment

The IMG/JGI annotated sequences were used to identify core genes among the 58 genomes. Nucleotide and amino acid sequences of annotated genes were used as input for core gene identification using get_homeologues v.2.0.1.9 (Contreras-Moreira and Vinuesa, 2013). Genes present in at least 95% of the genomes and with 75% pairwise alignment coverage were retained. The genes were parsed using python scripts to strictly define core genes as genes present in 100% of the genomes with intact start and stop codons. This approach was taken to limit the core genes to those most likely to be functional, based on genome annotation, in all strains. Genes with multiple copies were also removed. A total of 1,356 genes met the above criteria. Nine genes annotated as functional by the get_homeologues built-in annotation algorithm were not annotated by NCBI nor IMG/JGI, but were included in the analysis. The resulting nucleotide sequences of single copy core genes were individually aligned by MAFFT (Katoh and Standley, 2013) using a biopython script (Cock et al., 2009). Individual gene alignments were concatenated using sequence matrix software (Vaidya et al., 2011) to create a circa 1.09 megabases long sequence for each strain.

## Sequence Typing and Gene Mapping

Individual core genes were sequence typed based on nucleotide sequence identity using a python script. Genes with identical sequences were assigned the same number, representing the sequence type. The process was repeated in a loop for all core genes and an output sequence type matrix was generated. This allowed quick comparison of core genes based on allelic variation. Invariable genes were stripped from the matrix to generate a heat map of allelic profiles using the ggplot2 package (Wickham, 2010) in R (R Core Team, 2013). The heat map was color coded to illustrate the allelic patterns for variable core genes, thus providing a genetic fingerprint.

The relative positions of the core genes were mapped based on the complete genome of X. perforans Xp91-118 (NCBI accession number: GCA_000192045.3). We used the collated nucleotide sequences of the core genes of Xp91-118 as queries to BLAST (Zhang et al., 2000) against the complete genome. The output was configured to list the start and end positions in the complete genome for all core genes, which were sorted by position using a python script. BRIG (BLAST Ring Image Generator) software v. 0.95 (Alikhan et al., 2011) was used to visualize the positions of individual core genes in Xp91-118.

<footnote n="1">¹https://github.com/drpowell/utils/blob/master/filter-spades.py</footnote>
## Phylogenetic Analysis

Single gene evolution may be different from the evolution of the organism as a whole, particularly when there is horizontal gene transfer (Gogarten and Townsend, 2005). PhyML v.3.1 (Guindon et al., 2010) was used to construct maximum likelihood phylogenetic trees for single gene and concatenated core gene sequences. Nucleotide substitution models were estimated independently for individual genes and selected based on the log likelihood Akaike Information Criterion result calculated using `jModelTest2` (Darriba et al., 2012). General time reversible model with gamma distributed rates and invariant sites (GTR+G+I) was identified as the best nucleotide substitution model for the concatenated sequence. Maximum likelihood trees were constructed with 500 bootstrap samples for both concatenated and single genes using the suggested substitution model. ClonalFrameML (Didelot and Wilson, 2015) was used to reconstruct maximum likelihood trees while accounting for recombination. ClonalFrameML calculates R/theta, nu, and 1/delta, which represent the relative rate of recombination to mutation, new polymorphisms introduced from recombination, and the inverse of average tract length of recombination (Didelot and Falush, 2007; Didelot and Wilson, 2015). The three parameters were calculated for all single gene trees and for the concatenated sequence tree (hereafter referred to as core genome tree). Additionally, genomic clustering observed in the phylogenetic trees was confirmed by principal component analysis (PCA). The sequence types of the core genes were used as input to conduct PCA using micropan package in R (Snipen and Liliand, 2015).

## Detecting Genes Driving Phylogenetic Relationships

Phylogenetic distances between the unrooted single gene trees and core genome tree, along with the sequence type matrix, were used to determine the genes influencing the core genome tree topology of the 58 strains of *X. perforans*. Congruency of single gene trees to the core genome tree was assessed using Robinson-Foulds (RF) symmetry. This index represents the distance between two phylogenetic trees by evaluating the number of nodes in a tree that are shared with a reference tree (Robinson and Foulds, 1981). We used the core genome tree as the reference tree. The RF symmetry values range between 0 and 1, such that 0 indicates identical tree topology and 1 indicates completely different tree topologies. For example, phylogenetic trees for genes that were identical in nucleotide sequence among all the 58 *X. perforans* strains did not share any nodes with the core genome tree and the RF value was 1. Alternatively, if any nodes in a single gene tree supported a node in the core genome tree, the resulting RF value was less than 1. RF symmetry was calculated using ETE3 Toolkit (Huerta-Cepas et al., 2016) for each single gene tree against the core genome tree to determine the genes that supported some part of the topology of the core genome tree. The sequence types of genes with RF symmetry < 1 were extracted. The variable genes that exhibited RF values < 1 and supported the phylogenetic grouping in the core genome topology are hereafter referred to as Phylogenetic

group-Defining (PgD) genes. Maximum likelihood phylogenetic trees were constructed using concatenated sequences of 241 PgD and 1,115 non-PgD genes separately. The total tree length of the two phylogenetic trees were computed using Analysis of Phylogenetics and Evolution (ape) package in R (Paradis and Schliep, 2018) to confirm the role of PgD genes in phylogenetic grouping of *X. perforans* in the core genome tree.

## Identifying Recombination Sources and Recombination Mechanisms

We used two methods to determine if PgD genes may have been horizontally transferred. The sequences of the PgD genes were compared to the NCBI sequence database to determine if alleles were shared with other closely related *Xanthomonas* species. We also calculated the relative impact of recombination to mutation on nucleotide substitution using ClonalFrameML. Clusters of genes identified as variable or PgD, particularly those with high recombination values, were identified and their gene neighborhoods and flanking regions were examined. Gene neighborhood regions from representatives of each phylogenetic group were aligned and examined for the presence of genes suggestive of prior transfer events, including features of plasmids, phages, and transposable elements (Chiu and Thomas, 2004). In addition to clusters of core genes, we confirmed the presence of these recombination associated signatures in neighborhood regions of effector genes that were previously suspected to be horizontal transferred (Timilsina et al., 2016).

## RESULTS

### Xanthomonas perforans Pan-Genome

A total of 7,245 genes was identified in the pan-genome of 58 *X. perforans* strains and 2,866 genes were considered as core genes by roary (Supplementary Figure 1A). The Heaps law estimate, on the rarefaction curve of the number of new genes identified after randomly adding a genome, suggested an open pan-genome (Supplementary Figure 1B). The estimate for alpha was 0.813, suggesting that additional genes will be found upon sampling more *X. perforans* genomes.

### Core Genome Phylogeny

The get_homeologues pipeline identified 2,031 genes as core genes present in at least 95% of the sampled genomes. The variation in the core genes identified from roary and get_homeologues is likely due to the two different annotation pipelines used to generate inputs for these programs. Roary used the annotation output from prokka whereas annotation based on Clusters of Orthologous Groups of proteins (COG) downloaded from IMG/JGI were used for the get_homeologues platform for core gene extraction. We manually curated these to 1,356 core genes that were present and intact in all 58 *X. perforans* genomes (Supplementary Table 2). Nucleotide sequence comparison revealed that 783 genes were identical among all 58 genomes. At least two allele types were found in the remaining 573 genes (Supplementary Table 3).
The core genome phylogenetic analysis identified a third phylogenetic group in addition to the two previously described groups of *X. perforans* in Florida (**Figure 1A**). PCA of sequence types of the 1,356 core genes confirmed the three phylogenetic groups (**Figure 2**). Strain Xp17-12, which was

previously considered to be within group 1 (Schwartz et al., 2015), clustered with 10 strains isolated in 2015/16 to form a separate phylogenetic clade that we refer as group 3. Among the 58 strains, 18 strains were designated group 1, 29 strains were designated group 2, and 11 strains were

A

<figure><img src="image_41.png" /></figure>

B

<figure><img src="image_42.png" /><figcaption>**FIGURE 1** | The core genome of Florida *X. perforans* strains shows three groups of strains. (A) Maximum likelihood phylogeny based on concatenated sequences of 1,356 core genes (~1.3 Mb) and the branch topology were maintained regardless of the bootstrap support. (B) Heat map based on the allelic profile (sequence type) of variable genes (573) found within the core genes.</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>**FIGURE 2** | Principal component analysis showing three distinct clusters of *X. perforans* strains from Florida based on the sequence types of 1,356 conserved genes.</figcaption></figure>
designated group 3. Group 1 is a heterogeneous group that includes 6 strains from 2006 and 11 from 2012 along with the reference tomato race 3 (T3) strain Xp91-118 from 1991. Strains from the 2015/16 season were in group 2 (15 strains) or group 3 (10 strains).

Within groups, the majority of strains shared more than 99.8% pairwise nucleotide sequence identity in the core genome (Supplementary Table 4). Between the groups, identity was reduced to 99.5%. The group 1 strains had relatively lower sequence identities of ~99.5% in pairwise comparisons, and some strains had group 2 sequence types for several genes as observed in the heatmap and sequence type table (Figure 1B and Supplementary Table 3). Group 2 strains formed a monophyletic group with sequence identity above 99.7% among core genomes except for comparisons with Xp8-16 and Xp2010 (Figure 1A and Supplementary Table 4). Group 3 showed relatively low polymorphism with the majority of strains sharing core genome sequence identity above 99.9%. Average nucleotide identity based on BLAST using the whole genomes of these strains showed similar pairwise sequence identities to core genome comparisons (Supplementary Table 5).

## Variable Core Genes by Phylogenetic Group

Core genes were distributed throughout the Xp91-118 genome (Figure 3). Among the 573 genes that had at least two allele types, referred to as variable genes, allelic variation was often between phylogenetic groups (Figure 1B). Sequences were generally monomorphic or had a single SNP at low frequency within phylogenetic groups. While only 783 genes were monomorphic across the 58 genomes, the number of genes with identical nucleotide sequences within groups were 1124, 1195, and 1239 for groups 1, 2, and 3, respectively.

## Phylogenetic Group Defining Genes

We identified 241 core genes that supported one or more branches of the core genome tree topology that defined the phylogenetic groups (RF < 1). These genes are collectively distinguished as the phylogenetic group defining (PgD) genes as they drive the observed phylogenetic grouping of the 58 strains (Figure 1A and Supplementary Figure 2A). In particular, we observed that these PgD genes carried allele types that were often specific to phylogenetic groups (Figure 1B, Supplementary Table 3, and Supplementary Figure 2A). The annotations of genes identified as PgD are listed in Supplementary Table 2, among which 59 were annotated as hypothetical proteins and 2 as genes with domains of unknown functions. A total of 1,115 single gene trees did not support the core genome tree topology (RF = 1). These included 783 genes that were identical among all the *X. perforans* strains, plus 332 genes that had a variant allele type for at least one strain. The total length of tree based on 241 PgD genes was six times the length of the tree based on the remaining 1,115 core genes, signifying the larger contribution of PgD genes to strain variation (Supplementary Figure 2B). The non-PgD genes contribute to variation shared among a small numbers of strains.

Allele types distinguishing different groups were found in the PgD genes. Among the 241 PgD genes, 96 genes carried an allele specific to group 2 strains (different from the allele in group 1 and 3 strains), and 78 (81%) of those group 2 alleles were identical to *X. euvesicatoria* reference strain Xe85-10 (NCBI Accession no GCA_000009165.1). We found 142 genes with group 3-specific alleles, out of which 64 genes (45%) had alleles identical to *X. euvesicatoria* Xe85-10. An additional five PgD genes with group 3-specific alleles were identical to those of *X. axonopodis* pv. *citrumelo* strain F1 (NCBI Accession no. GCA_000225915.1), which included two hypothetical proteins, a protease modulator HflC, an anti-anti-sigma factor, and a type VI secretion system associated gene. Finally, group 3 alleles of two PgD genes were identical to those of *X. perforans* strain LH3 (NCBI Accession no. GCA_001908855.1), which was isolated from Mauritius in 2010 (Richard et al., 2017). These genes were N-acetyl-gamma-glutamyl-phosphate reductase (AQS75037.1) and aminoglycoside phosphotransferase (AQS78190.1). Therefore, LH3 is the only group 1 strain to contain these group 3 allele types. BLAST searches did not produce exact sequence matches to group 3 alleles for 71 genes. Unique allele types of PgD genes were distributed among group 1 strains. Group 1 strains isolated in 2006 carried specific allele types for 13 PgD genes that were identical to Xe85-10. Xp4-20 and Xp5-6 carried an additional 51 and 19 unique allele types, respectively. The remaining four group 1 strains isolated in 2006 (Xp4B, Xp15-11, Xp11-2, and Xp18-15) had specific allele types for 15 additional genes. Group 1 strains isolated in 2012 were homogenous with 15 genes among the PgD genes identical to Xe85-10. Some of the allele types carried by group 1 strains collected in 2006 were identical to group 2 but different from the reference strain Xp91-118. Among all 241 PgD genes, we found three genes that each had three alleles that were specific to group: endopeptidase (AQS77891.1), TonB-dependent siderophore receptor (AQS78913.1), and septum formation protein Maf (AQS76051.1).

Mapping PgD genes to the complete genome of Xp91-118 identified the positions and proximity of these genes (Figure 3). For example, a ~22 kb region between tryptophan-tRNA ligase (AQS77329.1) and catalase (AQS77307.1), encompassing 16 core genes (14 designated as PgD genes), exhibited diverged haplotypes specific to group 2 strains compared to group 1 and 3 strains. Similarly, an ~8 kb region, between co-chaperone YbbN (AQS78328.1) and peptidyl-prolyl *cis-trans* isomerase (AQS78967.1) genes, exhibited a distinct haplotype in group 3 strains compared to the other two groups. The overall ratio of changes introduced by recombination relative to mutation in the concatenated core genome tree was estimated to be 16.75 by ClonalFrameML. These values ranged between 0.063 (AQS77927.1) and 184.159 (AQS77019.1) among the individual PgD gene trees (Supplementary Table 2).

## Recombining Genes and Mechanism of Horizontal Gene Transfer

Genomic regions acquired via horizontal gene transfer may have signatures of integration associated with different modes of horizontal gene transfer (Ochman et al., 2000). We examined
<figure><img src="image_44.png" /><figcaption>**FIGURE 3** | A circular representation of complete genome of Xp91-118 indicating relative positions of core, variable, and phylogenetic group defining (PgD) genes. Clusters of PgD suggest potential recombination hotspots.</figcaption></figure>

genomes representing each phylogenetic group for signals of recombination flanking clusters of PgD genes. We used two criteria to select genomic regions. First, we were interested in alleles of closely clustered PgD genes that were identical to Xe85-10 indicating gene transfer from *X. euvesicatoria*. Second, we focused on gene trees that exhibited higher ratios of recombination to mutation than the concatenated gene tree. Gene neighborhood comparisons around PgD genes showed the presence of multiple tRNAs, phage-associated and plasmid mobilization genes, along with site specific two-component

system *XerC* and *XerD*, which were previously described to be associated with horizontal gene transfer (Aussel *et al.*, 2002). For instance, group 1 strains isolated in 2012 have ~9.6 kb of phage-associated genes between the tRNA-Leucine and tRNA-Glycine adjacent to the PgD genes AQS75151.1-AQS78555.1 (distinct alleles in group 2 and group 1 strains isolated in 2006). This unique ~9.6 kb region found only in group 1 strains isolated in 2012 include AAA-domain containing protein, phage major capsid protein, phage portal protein, phage terminase-like protein with HTH domain, and hypothetical proteins. Nucleotide
BLAST search in NCBI revealed 98% sequence similarity with only LH3 strain. The site-specific recombinase (*XerD*) gene was observed in all the group 1 strains between the tRNA-Leucine and phage associated genes, suggesting the integration of the unique

genomic region was facilitated by bacteriophages (Figure 4A). In general, multiple copies of *XerD*, ranging from four to eight, were observed in all *X. perforans* genomes except for the 1991 reference strain, Xp91-118, which carries only one copy.

A

<figure><img src="image_45.png" /></figure>

B

<figure><img src="image_46.png" /></figure>

**FIGURE 4** | Genomic regions with genes exhibiting group specific alleles and recombination signals. (A) A genomic region present in group 1 strains, which contains phage associated genes and is flanked with tRNAs and XerD, is absent in Group 2 and 3 strains. Phylogenetic group defining (PgD) genes are indicated by pink boxes between the alignments with recombinant allele types in group 2 and group 1 2006 strains. The coordinates of this region in Xp91-118 are between 323182 and 325825 (B) Group 1 and 2 strains carry XerD, the site specific recombination gene, along with plasmid mobilization and Tra genes (green and blue boxes). This region is missing in Group 3 strains. PgD genes are indicated by pink and purple boxes with recombinant alleles in group 3. The coordinates of this region in Xp91-118 are 1604360-1620009.
<figure><img src="image_47.png" /><figcaption>**FIGURE 5** | Recombination signals observed surrounding the gene for XopJ4 effector. The XopJ4 gene is flanked by phage associated genes in most strains, including T3 strains Xp91-118. In strain Xp2010, isolated from pepper, the effector gene and phage associated genes were replaced by a putative translocase.</figcaption></figure>

Group 3 strains carried 71 PgD genes with unique alleles specific to the group. For example, group 3 alleles of six PgD genes between locus tags AQS76085.1-AQS76101.1 were distinct from the other two groups and were unique among sequences available in NCBI. In group 1 and 2, this genomic region is adjacent to *XerD*, mobile element protein (*MobA*), conjugal transfer protein (*traD*) and phage integrase family protein, suggesting integration via plasmid mobilization and site-specific recombination mechanisms (**Figure 4B**). The genomic region between the multiple tRNA sites and *XerD* present in group 1 and 2 included several unique hypothetical genes and DNA methyltransferase gene. Furthermore, the genetic variation in group 3 strains indicate the acquisition of novel genomic traits via recombination from multiple donors in addition to *X. euvesicatoria*.

## Recombination Affecting Type III Effectors

Following the observation of at least two different mechanisms of genetic exchange that were associated with phage transfer and plasmid mobilization in core genes, we examined these signals throughout representative genomes. Interestingly, signatures of horizontal gene transfer were found surrounding effectors that show presence/absence polymorphism among strains and previously predicted to be acquired via recombination. Among the effectors found in bacterial spot causing *X. perforans*, *avrXv4* (*xopJ4*) is found in all tomato pathogenic strains but not found in the strain, Xp2010, isolated from pepper. We found phage associated genes flanking XopJ4 (**Figure 5**). In Xp2010, both phage associated genes and the *xopJ4* effector are absent. Similarly, the gene coding another XopJ family

effector, *avrBsT* (XopJ2), found in the majority of tomato race 4 *X. perforans*, is flanked by genes for type IV secretion system proteins, conjugative transfer, chromosome partitioning protein, and hypothetical proteins (**Supplementary Figure 3**).

## DISCUSSION

Using a cgMLSA approach, we determined that the *X. perforans* strains isolated in Florida at different times is defined by three groups of strains that are differentiated by hundreds of variable genes, the majority of which appear to be recombinant. All of the strains we analyzed were tomato race 4 *X. perforans* that were collected in the past two decades, except for the 1991 T3 reference strain, Xp91-118, and a pepper strain, Xp2010 (Schwartz et al., 2015). A high-resolution phylogeny of *X. perforans* strains was constructed as well as a core genome fingerprint, which shows allelic variation affecting genes throughout the chromosome. Core genome multilocus sequence analysis allows a holistic comparison of phylogenetic groups and their evolution while minimizing the individual differences between the strains (Maiden et al., 2013; Maiden and Harrison, 2016). Recombination was inferred to be widespread in core genes showing allelic variation with *X. euvesicatoria* strains as a major donor. Furthermore, we observed plasmid and phage associated site-specific recombination mechanisms surrounding clusters of putatively recombinant genes as well as genes that influence host-specificity and pathogen fitness. The open pan-genome further suggests high genomic plasticity in *X. perforans*. We hypothesize that these recombinant strains are epidemic lineages that have emerged in Florida tomato production from a highly recombinogenic source population.
We previously showed two major phylogenetic groups of *X. perforans* in Florida (Timilsina et al., 2014, 2016; Schwartz et al., 2015), but strains collected in the 2015/16 growing season revealed a third monophyletic group of 11 strains. Consistent with recent emergence, it was the most homogenous of the three groups as shown by pairwise nucleotide identity and PCA of sequence types. One strain from this group, Xp17-12, was isolated in 2006 and was previously described as an outlier within group 1 (Schwartz et al., 2015). The relative homogeneity among group 3 strains over time suggests that the 2015/16 group 3 strains were from the same source population. Group 1, which includes strains isolated in 1991, 2006 and 2012, is more heterogeneous than other groups (Figures 1, 2). Group 2 strains have been dominant in Florida since at least 2006 (Timilsina et al., 2016) but appear to be largely clonal (Figure 2). Three genes with alleles specific to each phylogenetic group were observed that could be used for assigning strains to groups to monitor their prevalence in Florida populations going forward. In general, we identified only a quarter of the total genes being shared among all *X. perforans* genomes, which suggests high genome plasticity in the species and is consistent with our finding of an open genome and is in agreement with another study of *X. perforans* (Jibrin et al., 2018).

We inferred recombination to have been the major source of genetic variation in the core genome. Horizontal inheritance of genes from multiple transfer events can obscure ancestral relationships among bacterial strains (Gogarten and Townsend, 2005). Consequently, recombinant loci are often removed from alignments prior to phylogenetic analysis. However, these loci also define the evolution of the recombinant strains (Didelot and Wilson, 2015). Phylogenetic reconstruction without the presence of these potentially recombined PgD genes significantly altered the observed phylogenetic relationships among the *X. perforans* groups (Supplementary Figure 2A). This variation, likely due to recombination from multiple donors, reinforces the necessity to include recombination in bacterial population studies. For group 2, most of the recombinant sequences appeared to have been acquired from *X. euvesicatoria*, which was displaced in Florida by *X. perforans* producing antagonistic bacteriocins against *X. euvesicatoria* (Hert et al., 2005; Timilsina et al., 2016). For group 3, *X. euvesicatoria* was one of multiple donors. The earliest strains of *X. perforans* isolated in Florida belonged to group 1, which had the least recombination signatures relative to the other two groups, as reflected by the copy numbers of site-specific recombinase XerCD genes. Correspondingly, the frequency of recombination observed with *X. euvesicatoria* and other closely related *Xanthomonas* was relatively low in these strains. Although, horizontal gene transfer is largely associated with acquisition of new traits, the tracts of homologous genes potentially acquired from closely related species shows the impact of recombination on the tempo and direction of *X. perforans* genome evolution. Recombination affecting the genetic background of a pathogen can affect niche adaptation, fitness, and microbial competition, and thus the establishment of recombinant strains (Alizon et al., 2009).

Our observations suggest horizontal gene transfer of long fragments of shared recombinant alleles through different genomic vectors. The horizontally introduced genomic fragments

can be regulated by the carrying capacity of plasmid or phage vectors (Ochman et al., 2000; Bobay and Ochman, 2017). These two mechanisms of vector associated horizontal gene transfer were clearly visible in our *X. perforans* genomes. Phage and plasmid associated genes were present in the flanking regions of clusters of core genes showing evidence of recombination. Among the three modes of horizontal gene transfer, we were not able to directly identify genomic fragment acquisition via transformation. Several variable genes had variation attributed to recombination, but without any evidence of phage or plasmid associated genes in the flanking regions. These genes may have been transferred via transformation.

Phage mediated gene transfer appears to play a major role in influencing genomic diversity and evolution of *X. perforans*. We found phage genes throughout the genome in regions with potentially recombined core and effector genes. One such example is the XopJ family effector, *avrXv4* (*xopJ4*). XopJ4 is a member of the XopJ effector family, which is similar to the YopJ, serine/threonine acetyltransferase superfamily (Szczesny et al., 2010). A previous study reported that *xopJ4* was conserved in all *X. perforans* strains except for the Xp2010 strain that was isolated from pepper (Schwartz et al., 2015). The *xopJ4* gene was located between phage associated genes. The whole genomic region including phage associated and *XopJ4* genes is missing in the Xp2010 pepper strain. A similar XopJ family effector (98% amino acid identity) in *X. citri* pv. *vignicola* strain CFBP 7112 (Ruh et al., 2017) is also located between phage associated genes. Effector *avrBsT* is another XopJ family effector found in *X. perforans* that is generally associated with plasmids (Ciesiolka et al., 1999; Kay and Bonas, 2009; White et al., 2009). The *avrBsT* effector was not found in *X. perforans* until 1998 (Timilsina et al., 2016). The nucleotide sequence of *avrBsT* in *X. perforans* is identical to that in the more distantly related bacterial spot species, *X. vesicatoria* (Timilsina et al., 2016). An identical allele type of the plasmid-borne *avrBsT* gene in different *Xanthomonas* species, including *X. perforans*, suggests the gene is horizontally transferred across the genus (Timilsina et al., 2014, 2016; Jibrin et al., 2018). The majority of T4 *X. perforans* strains carry *avrBsT* and the gene has been found to provide a competitive advantage to bacterial strains in field conditions (Abrahamian et al., 2018). The variation created by mobile genetic elements in the core and accessory genes could influence host preference and pathogenicity of *X. perforans* strains.

Several PgD genes also showed evidence of plasmid associated horizontal gene transfer. An intriguing pattern that was evident in the genomes was the density of tRNAs and site-specific tyrosine recombinase genes flanking these recombined regions. The two-component site-specific tyrosine recombinase, XerC and XerD, catalyzes crossover and recombination at specific sites (Midonet and Barre, 2015). Site-specific recombination is characterized by cleavage of both DNA strands at two recombination sites that are later joined to new DNA partners without DNA degradation and phosphodiesters hydrolysis (Subramanya et al., 1997). The XerD recombinase works together with XerC, both of which belong to the λ-integrase family. XerD is reported to initiate recombination by strain exchange to form the Holliday junctions and that is reconstructed by XerC (Aussel et al., 2002;
Crozat et al., 2015). In *X. perforans* genomes, the site-specific XerD gene was co-located with a plasmid mobilization and transfer (*tra*) gene where a ~9.6-kb genomic region was present in group 1 genomes but absent in groups 2 and 3. Sequence comparison showed this region is specific to group 1 *X. perforans* strains. Along with XerCD genes, this genomic island was flanked by tRNAs (**Figure 4A**). The tRNAs serve as a gateway for integration of foreign DNA (Ochman et al., 2000; Williams, 2002). Boyd et al. (2009) reported that tRNA-Arg, -Leu, -Thr, and -Ser were commonly observed insertion sites. The genomic islands introduced by phage or plasmid in *X. perforans* seem to have specific attachment sites, facilitated by tRNAs and site-specific recombinase genes, that altered the core genomes and ultimately shaped the population of *X. perforans* in Florida. These specific sites serve as recombination hotspots in the bacterial genome.

Bacterial spot disease of tomato has posed a series of management challenges in Florida, including the introduction of *X. perforans*. In this study, we have begun to tease apart the genetic mechanisms driving population changes in *X. perforans* since its emergence in 1991, which appear primarily due to phage and plasmid-mediated horizontal gene transfer followed by integration into the chromosome. Our findings indicate rapid genomic evolution in the *X. perforans* population in Florida, which together with our previous findings of extensive recombination in strains from Nigeria and Italy (Jibrin et al., 2018), suggest a pathogen with a high probability of overcoming management practices, i.e., *X. perforans* poses a high “evolutionary risk” (McDonald and Linde, 2002). However, the clonal structure of the Florida population also indicates that a limited number of recombinant genotypes have been introduced to or have successfully established in Florida tomato production. If we could determine the population or populations that are highly recombinogenic, these populations could be specifically managed or movement out of these populations curtailed. For example, recombination events could be occurring in seed sources or other production regions

that are not closely connected to Florida, thus exchanging few migrants. Efforts are also needed to understand why *X. perforans* readily recombines when other bacterial phytopathogens, including the bacterial spot pathogen *X. gardneri*, appear highly clonal (Schwartz et al., 2015; Timilsina et al., 2014).

## DATA AVAILABILITY

The datasets generated for this study can be found in NCBI, PRJNA436012.

## AUTHOR CONTRIBUTIONS

ST, NP, GV, JJ, and EG conceived the project. PA collected additional bacterial strains and provided their genomes. JP-M and FI-B oversaw the genome assembly. GM and ST conducted the sequencing experiments. ST conducted all computational analyses and interpreted with the help of BK, EG, GV, and JJ. ST, EG, JJ, and GV wrote the final manuscript. All authors approved the final manuscript.

## FUNDING

This research was supported in part by the United States Department of Agriculture, National Institute of Food and Agriculture under award number 2015-51181-24312. Publication of this article was funded in part by the University of Florida Open Access Publishing Fund.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2019.00448/full#supplementary-material

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Timilsina, Pereira-Martin, Minsavage, Iruegas-Bocardo, Abrahamian, Potnis, Kolaczkowski, Vallad, Goss and Jones. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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# Molecular Evolution of *Pseudomonas syringae* Type III Secreted Effector Proteins

Marcus M. Dillon¹, Renan N.D. Almeida¹, Bradley Laflamme¹, Alexandre Martel¹, Bevan S. Weir², Darrell Desveaux¹,³ and David S. Guttman¹,³*

¹ Department of Cell & Systems Biology, University of Toronto, Toronto, ON, Canada, ² Landcare Research, Auckland, New Zealand, ³ Centre for the Analysis of Genome Evolution & Function, University of Toronto, Toronto, ON, Canada

Diverse Gram-negative pathogens like *Pseudomonas syringae* employ type III secreted effector (T3SE) proteins as primary virulence factors that combat host immunity and promote disease. T3SEs can also be recognized by plant hosts and activate an effector triggered immune (ETI) response that shifts the interaction back toward plant immunity. Consequently, T3SEs are pivotal in determining the virulence potential of individual *P. syringae* strains, and ultimately help to restrict *P. syringae* pathogens to a subset of potential hosts that are unable to recognize their repertoires of T3SEs. While a number of effector families are known to be present in the *P. syringae* species complex, one of the most persistent challenges has been documenting the complex variation in T3SE contents across a diverse collection of strains. Using the entire pan-genome of 494 *P. syringae* strains isolated from more than 100 hosts, we conducted a global analysis of all known and putative T3SEs. We identified a total of 14,613 putative T3SEs, 4,636 of which were unique at the amino acid level, and show that T3SE repertoires of different *P. syringae* strains vary dramatically, even among strains isolated from the same hosts. We also find substantial diversification within many T3SE families, and in many cases find strong signatures of positive selection. Furthermore, we identify multiple gene gain and loss events for several families, demonstrating an important role of horizontal gene transfer (HGT) in the evolution of *P. syringae* T3SEs. These analyses provide insight into the evolutionary history of *P. syringae* T3SEs as they co-evolve with the host immune system, and dramatically expand the database of *P. syringae* T3SEs alleles.

## OPEN ACCESS

**Edited by:**
Dawn Arnold,
University of the West of England,
United Kingdom

**Reviewed by:**
Brian H. Kvitko,
University of Georgia, United States
David Baltrus,
The University of Arizona,
United States

*Correspondence:
David S. Guttman
david.guttman@utoronto.ca

**Specialty section:**
This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science

Received: 21 December 2018

Accepted: 19 March 2019

Published: 05 April 2019

**Citation:**
Dillon MM, Almeida RND, Laflamme B, Martel A, Weir BS, Desveaux D and Guttman DS (2019) Molecular Evolution of Pseudomonas syringae Type III Secreted Effector Proteins. Front. Plant Sci. 10:418. doi: 10.3389/fpls.2019.00418

**Keywords:** *Pseudomonas syringae*, type III secreted effectors, type III secretion system, plant-pathogen, host-microbe interactions, virulence, immunity

## INTRODUCTION

Over the past three decades, type III secreted effectors (T3SEs) have been recognized as primary mediators of many host-microbe interactions (Michiels and Cornelis, 1991; Salmond and Reeves, 1993; Hueck, 1998; Coburn et al., 2007; Deng et al., 2017; Hu et al., 2017; Rapisarda and Fronzes, 2018). These proteins are translocated directly from the pathogen cell into the host cytoplasm by the type III secretion system (T3SS), where they perform a variety of functions that generally promote virulence and suppress host immunity (Coburn et al., 2007; Zhou and Chai, 2008; Cunnac et al., 2009; Oh et al., 2010; Buttner, 2016; Khan et al., 2018). However, T3SEs can also be recognized by the host immune system, which allows the host to challenge the invading microbe. In plants, this immune response is called effector triggered immunity (ETI) (Jones and Dangl, 2006;