Cruciferous weed isolates of Xanthomonas campestris yield insight into pathovar genomic relationships and genetic determinants of host- and tissue-specificity

Zoe Dubrow, Sara Carpenter, Morgan Carter, Ayress Grinage, Carine Gris, E. Lauber, Jules Butchacas, Jonathan Jacobs, Christine Smart, Matthew Tancos, et al.

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To cite this version:

Zoe Dubrow, Sara Carpenter, Morgan Carter, Ayress Grinage, Carine Gris, et al. Cruciferous weed isolates of Xanthomonas campestris yield insight into pathovar genomic relationships and genetic determinants of host- and tissue-specificity. Molecular Plant-Microbe Interactions, 2022, 35 (9), pp.791-802. 10.1094/MPMI-01-22-0024-R. hal-03744707

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# Cruciferous Weed Isolates of *Xanthomonas campestris* Yield Insight into Pathovar Genomic Relationships and Genetic Determinants of Host and Tissue Specificity

Zoë E. Dubrow,<fnref n="1" /> Sara C. D. Carpenter,<fnref n="1" /> Morgan E. Carter,<fnref n="1" />,<fnref n="2" /> ID Ayress Grinage,<fnref n="3" /> Carine Gris,<fnref n="4" />

Emmanuelle Lauber,<fnref n="4" /> Jules Butchachas,<fnref n="5" /> Jonathan M. Jacobs,<fnref n="5" /> Christine D. Smart,<fnref n="1" /> Matthew A. Tancos,<fnref n="6" />

Laurent D. Noël,<fnref n="4" /> and Adam J. Bogdanove<fnref n="1" />,† ID

Accepted for publication 3 May 2022.

Pathovars of *Xanthomonas campestris* cause distinct diseases on different brassicaceous hosts. The genomic relationships among pathovars as well as the genetic determinants of host range and tissue specificity remain poorly understood despite decades of research. Here, leveraging advances in multiplexed long-read technology, we fully sequenced the genomes of a collection of *X. campestris* strains isolated from cruciferous crops and weeds in New York and California as well as strains from global collections, to investigate pathovar relationships and candidate genes for host- and tissue-specificity. Pathogenicity assays and genomic comparisons across this collection and publicly available *X. campestris* genomes revealed a correlation between pathovar and genomic relatedness and provide support for *X. campestris* pv. *barbareae*, the validity of which had been questioned. Linking strain host range with type III effector repertoires identified AvrAC (also 'XopAC') as a candidate host-range determinant, preventing infection of *Matthiola incana*, and this was confirmed experimentally. Furthermore, the presence of a copy of the cellobiosidase gene *cbsA* with coding sequence for a signal peptide

was found to correlate with the ability to infect vascular tissues, in agreement with a previous study of diverse *Xanthomonas* species; however, heterologous expression in strains lacking the gene gave mixed results, indicating that factors in addition to *cbsA* influence tissue specificity of *X. campestris* pathovars.

**Keywords:** AvrAC, black rot, *Brassica*, cabbage, cellobiosidase, pathovar, type III effectors, *Xanthomonas campestris*

*Xanthomonas campestris* is a destructive species of plant-pathogenic bacteria causing disease in brassicaceous plants (e.g., cabbage, cruciferous weeds, and ornamentals). The species comprises multiple pathogenic variants (pathovars) that cause distinct diseases on different plant hosts. Within the genus *Xanthomonas*, multiple changes to species and pathovar nomenclature have been made and other changes have been proposed, reflecting sometimes conflicting views on what pathovars belong in *X. campestris*. Vauterin and colleagues (1995) restricted *Xanthomonas campestris* to only pathovars infecting *Brassica* hosts (pathovars *campestris*, *raphani*, *incanae*, *barbareae*, *armoraciae*, and *abberans*). However, recent phylogenetic data and pathogenicity assays have suggested that this pathovar structure should be revised, with strains belonging to pathovars *abberans*, *armoraciae*, and *barbareae* reassigned as *campestris*, *raphani*, or *incanae*, and any strains not known to be pathogenic designated as "nonpathogenic" (Fargier and Manceau 2007; Fargier et al. 2011). *Xanthomonas campestris* pv. *campestris* is the most agriculturally important of these pathovars. It causes black rot, a globally destructive vascular disease of *Brassica oleracea*, particularly important in cabbage and cauliflower. Black rot is identified by chlorotic and necrotic V-shaped lesions extending from hydathodes and wounds and by blackening of veins. Pathovar *raphani* is a nonvascular pathogen and causes leaf spot on brassicaceous and solanaceous crops. *X. campestris* pv. *incanae* does not cause disease on *Brassica* crop plants but is a vascular pathogen of ornamental crucifers including *Matthiola* and *Erysimum* spp. Some characterized *X. campestris* pv. *incanae* strains (e.g., type strain CFBP 2527) are pathogens of *Matthiola* spp., while others (e.g., CFBP 1606) infect only

Current affiliation for Zoë E. Dubrow: Pairwise Plants, Durham, NC, U.S.A.

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture. USDA is an equal opportunity provider and employer.

**Funding:** This work was supported by a USDA NIFA Pre-doctoral Research Fellowship (2019-67011-29501 to Z. E. Dubrow), the New York Cabbage Research and Development Program (contract number 11404 to C. D. Smart and A. J. Bogdanove), the French Laboratory of Excellence project 'TULIP' (ANR-10-LABX-41 and ANR-11-IDEX-0002-02 to L. D. Noël), and USDA-ARS (Project 8044-22000-047-00D, M. A. Tancos).

**e-Xtra:** Supplementary material is available online.

The author(s) declare no conflict of interest.

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<footnote n="1">¹ Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, U.S.A.</footnote>

<footnote n="2">² School of Plant Sciences, University of Arizona, Tucson, AZ, U.S.A.</footnote>

<footnote n="3">³ Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, U.S.A.</footnote>

<footnote n="4">⁴ LIPME, Université de Toulouse, INRAE, CNRS, Université Paul Sabatier, Castanet-Tolosan, France</footnote>

<footnote n="5">⁵ Department of Plant Pathology, The Ohio State University, Columbus, OH, U.S.A.</footnote>

<footnote n="6">⁶ Foreign Disease-Weed Science Research Unit, United States Department of Agriculture-Agricultural Research Service, Frederick, MD, U.S.A.</footnote>

<footnote>†Corresponding author: A. J. Bogdanove; ajb7@cornell.edu</footnote>
*Erysimum* spp. (Fargier and Manceau 2007; Fargier et al. 2011). The basis for this difference in host specificity is unknown. Many of the *X. campestris* nonpathogenic strains lack a type III secretion system (T3SS), which is essential for pathogenicity (Meline et al. 2019; Lee et al. 2020), while others appear to have a full virulence gene repertoire (Fargier and Manceau 2007; Roux et al. 2015). We recently distinguished the latter as *X. campestris* “unknown pathogenicity” due to their high, whole-genome average nucleotide identity (ANI) to pathogenic strains but lack of a known host (Dubrow and Bogdanove 2021).

Despite causing different diseases (or no disease) and ex-
hibiting different host ranges, *X. campestris* pvs. *campestris*,
*raphani*, *incanae*, and *X. campestris* nonpathogenic or unknown
pathogenicity strains have greater than 96% ANI (Dubrow
and Bogdanove 2021). Apart from the lack of the T3SS in
*X. campestris* nonpathogenic strains, the molecular mechanisms
underlying these differences in pathogenicity are unknown. It
has been hypothesized that divergence in host range is due to
differences in virulence gene content, such as presence or ab-
sence of type III-secreted effectors (T3Es) (Roux et al. 2015).
A comparison of the T3E repertoires of eight *X. campestris* pv.
*campestris*, two *X. campestris* pv. *raphani*, two *X. campestris*
pv. *incanae*, and an *X. campestris* unknown pathogenicity strain
revealed multiple, though often unconserved, differences in T3E
content across these groups (Bolot et al. 2013b). Also, whole-
genome sequences of 10 *Xanthomonas* strains representing vas-
cular and nonvascular pathogens of dicot or monocot hosts, in-
cluding one *X. campestris* pv. *raphani* and three *X. campestris*
pv. *campestris* strains, were compared to gain insight into tissue
specificity as well as host specificity (Bogdanove et al. 2011), but
no specific genes were identified as determinants. Recently how-
ever, a comparison of 54 *Xanthomonas* and five *Xylella* genomes
(Gluck-Thaler et al. 2020) uncovered significant association of
the *cbsA* gene, encoding a predicted extracellular cellobiosidase,
with vascular pathogenesis, and experimental results support a
contributing role. Transfer of the gene from a vascular patho-
var to a nonvascular pathovar of *X. translucens* enabled vas-
cular pathogenesis by the latter, a knockout of the gene in the
vascular *X. translucens* pathovar expanded symptom develop-
ment to the adjacent nonvascular tissue, and knockouts of the
gene in the vascular pathogens *X. oryzae* pv. *oryzae*, *Ralstonia
solanacearum*, and *Xylella fastidiosa* impaired pathogenicity
(Gluck-Thaler et al. 2020; Jha et al. 2007; Liu et al. 2005).

*X. campestris* pv. *campestris* does not appear to have a strongly
geographically based population structure, and strains isolated
in a particular region, for example, in the northeastern United
States state of New York, have been observed not to persist from
year to year, together indicating the prevalence of dissemination
on seed (Denance et al. 2018; Fargier et al. 2011; Lange et al.
2016). Cultural practices such as seed saving, insufficient sani-
tation of transplant production facilities, or lack of field rotation
may result in endemic *X. campestris* populations, but this is not
common (Bella et al. 2019).

In addition to infecting crops, *X. campestris* is often as-
sociated with cruciferous weeds. However, studies on collec-
tions of strains isolated from cruciferous weeds in Germany
(Krauthausen et al. 2018), New York (Lange et al. 2022), and
California (Ignatov et al. 2007) support the conclusion that
weeds are not a significant source of *X. campestris* pv. *campestris*
inoculum to crops. While certain weed isolates were observed to
be pathogenic in cabbage (particularly those from the California
collection), in each study, the weed isolates grouped separately
from *X. campestris* pv. *campestris* crop strains by multilocus
sequence analysis and most were not pathogenic to crop plants.

Though apparently not a significant cause of crop disease out-
breaks, weed isolates nonetheless are a potential resource for
probing pathovar relationships and genetic determinants of host

range and tissue specificity, a better understanding of which
could lead to the discovery of new mechanisms of resistance
to be bred into economically important cruciferous species. To
this end, we first selected 24 strains from the California weed
isolate collection (Ignatov et al. 2007), nine strains from New
York weed isolate collections (Burkholder 1941; Lange et al.
2022), 20 crop isolates of *X. campestris* pv. *campestris* from the
eastern United States (18 from New York and two from Michi-
gan) (Lange et al. 2016), and two *X. campestris* pv. *campestris*
isolates, two *X. campestris* pv. *incanae* isolates, and one
*X. campestris* pv. *raphani* isolate originating from various loca-
tions from public collections (Supplementary Table S1). We then
completed whole-genome sequencing using single molecule
long-read technology (SMRT) and determined relationships
among these and 49 publicly available *X. campestris* genomes,
including one of a weed isolate from the eastern United States
state of Maryland we recently reported (Tancos et al. 2022).
Next, we assayed pathogenicity and tissue-specificity on rep-
resentative subsets of the California weed isolates and Eastern
United States crop isolates that we sequenced, all but one of
the weed isolates from the eastern United States that we se-
quenced (strain CFBP 5824) (Burkholder 1941), plus the weed
isolate from Maryland, and several reference strains, on a panel
of plant species, to determine pathovar and to identify associa-
tions of pathovar and genetic relatedness. Finally, we analyzed
T3E gene content to determine any relationships to host or tis-
sue specificity and the presence or absence of the *cbsA* gene to
determine its relationship to tissue specificity in *X. campestris*.

RESULTS AND DISCUSSION

Fifty-nine new complete *X. campestris* genome assemblies.

SMRT sequencing was completed for 59 *X. campestris* strains in three separate runs. Coverage ranged from 176 to 1,030×. Each genome was circular and complete after assembly, consisting of an approximately 5-Mb chromosome and up to three plasmids of varying sizes, but plasmids were not well-conserved among strains. Average GC content of each was approximately 60 to 65%. The assemblies and raw sequences for all strains have been deposited in GenBank under BioProject PRJNA689092 (Supplementary Table S1 lists accession numbers for individual strains).

Isolate pathovar designations and support
for *X. campestris* pv. *barbareae*.

To determine pathovar designations for the newly sequenced isolates, we performed pathogenicity assays for representative strains on an array of hosts, including the sequenced kale-like *Brassica oleracea* var. *alboglabra* TO1000, to identify *X. campestris* pv. *campestris* strains, the ornamentals *Erysimum cheiri* and *Matthiola incana*, to identify *X. campestris* pv. *incanae* strains, *Solanum lycopersicum*, to identify *X. campestris* pv. *raphani* strains (tomato is a host of *X. campestris* pv. *raphani* but no other *X. campestris* pathovar), as well as two cruciferous weed species found in New York state, *Barbarea vulgaris* and *Sinapis arvensis* (Fig. 1; Supplementary Table S2). We assayed 48 strains total, including 13 of the California weed isolates selected to represent the genetic diversity of that group (Fig. 2), 12 of the New York crop isolates plus two more that were not sequenced, all nine of the weed isolates from New York plus the Maryland weed isolate, as well as three *X. campestris* pv. *campestris*, four *X. campestris* pv. *raphani*, and four *X. campestris* pv. *incanae* strains that were previously sequenced. To inoculate leaves, we used a clip-and-dip technique (discussed below) that allows for invasion by vascular or nonvascular colonizers.
As expected, previously characterized agronomic *X. campestris* pv. *campestris* strains caused vascular symptoms (expanding chlorotic and necrotic lesions) on *Brassica oleracea* and *Sinapis arvensis*. *X. campestris* pv. *raphani* strains caused spots on leaves of *Brassica oleracea*, *Solanum lycopersicum*, and *Sinapis arvensis*, though certain strains caused symptoms on the ornamental or other weed hosts (Supplementary Table S2). *X. campestris* pv. *incanae* strains caused vascular symptoms on either *M. incana*, *E. cheiri*, or both, but no symptoms on *Brassica oleracea* or *Barbarea vulgaris*. *X. campestris* pv. *incanae* CFPB1606 was observed only to cause symptoms on *E. cheiri* and not *M. incana* (Supplementary Table S2), confirming previously reported results (Fargier et al. 2011).

The weed isolates from California, like the agronomic *X. campestris* pv. *campestris* strains, elicited vascular symptoms on *Brassica oleracea*, as observed by Ignatov and colleagues (2007), and *Sinapis arvensis* and were therefore designated as *X. campestris* pv. *campestris*. In contrast to previously characterized agronomic *X. campestris* pv. *campestris* strains, of which a small percentage caused disease on *M. incana* (three out of

<table><thead><tr><th>Disease Type</th><th>Vascular</th><th>Vascular</th><th>Vascular</th><th>Nonvascular</th><th>Nonvascular</th><th>None</th></tr></thead><tbody><tr><td><strong>Pathovar</strong></td><td><strong>campestris</strong></td><td><strong>campestris</strong><br>(CA weed)</td><td><strong>incanae</strong></td><td><strong>raphani</strong></td><td><strong>barbareae</strong></td><td><strong>nonpathogenic</strong></td></tr><tr><td><strong>Brassica oleracea</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Sinapis arvensis</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Matthiola incana</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Erysimum cheiri</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Solanum lycopersicum</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Barbarea vulgaris</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

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<figure><img src="image_39.png" /><figcaption>**Fig. 1.** Symptoms and host specificity of infection by representative strains of *Xanthomonas campestris* pathovars and subgroups examined in this study. Leaves of *Brassica oleracea*, selected brassicaceous weed and ornamental species, and tomato 14 days after clip-and-dip inoculation with a representative strain of each pathovar (or subgroup). Red borders highlight interactions that resulted in disease symptoms.</figcaption></figure>
<figure><img src="image_40.png" /><figcaption>**Fig. 2.** Grouping of *Xanthomonas campestris* strains based on average nucleotide identity (ANI) is congruent with grouping by pathovar. ANI matrix of 109 *X. campestris* genomes constructed using the Enveomics Collection Toolbox genome-based distance matrix calculator. Red boxes in the matrix indicate compact clusters of closely related genomes calculated using Ward's method as part of the Enveomics Toolbox. Colored circles depict geographic origin and host species from which the strain was isolated. Xcc = *X. campestris* pv. *campestris*; Xci = *X. campestris* pv. *incanae*; Xcb = *X. campestris* pv. *barbareae*; Xcnp = *X. campestris* nonpathogenic strain; Xcr = *X. campestris* pv. *raphani*; Xcc-CA denotes strains isolated in California (Ignatov et al. 2007) and Xcc-CN denotes strains isolated in China (He et al. 2007). Asterisks indicate New York weed isolates.</figcaption></figure>
17 strains), *E. cheiri* (seven out of 17 strains), or both ornamentals (three out of 17 strains), all but one California weed isolate caused disease on both ornamental crucifers. That isolate, 40-2, was pathogenic on *M. incana* but not *E. cheiri* (Supplementary Table S2).

Among the ten weed isolates from the eastern United States that were tested, four from New York and the single Maryland isolate caused vascular symptoms and four New York isolates caused spots. One New York weed isolate, 5053, caused no symptoms. Three of the vascular strains caused disease on *M. incana*, all five were pathogenic on *E. cheiri*, and none caused symptoms on *Brassica oleracea*. We therefore designated these strains as *X. campestris* pv. *incanae*. One of the spot-causing isolates, 5055, did so on *Brassica oleracea* and *Sinapis arvensis* but not on any other plant species and was therefore designated as *X. campestris* pv. *raphani*. These results were consistent with observed phenotypes in cabbage for these strains in a previous study, except for 16_8, which we did not observe to cause V-shaped lesions on cabbage, while Lange and colleagues (2022) did, perhaps due to the use of a different *Brassica oleracea* cultivar or inoculation method. The remaining three strains, 10_16, 11_19, and 3054, caused symptoms only on *Barbarea vulgaris* (Fig. 1). These symptoms, severe black and water-soaked spots, were strikingly similar to those of “black rot of *Barbarea vulgaris*,” also caused by *X. campestris* isolates from New York weeds (Burkholder 1941). Two of those original isolates, now CFBP 5825 and CFBP 5826 (originally accessioned as ICPB XB2 and XB1, respectively), were designated as nonpathogenic *Xanthomonas campestris* (Fargier et al. 2011; Dubrow and Bogdanove 2021). Sequencing of a rifampicin-resistant derivative of CFBP 5825 (“CFBP 5825R”) showed it has a T3SS and effector genes (Roux et al. 2015), so we previously considered it an *X. campestris* unknown pathogenicity strain (Dubrow and Bogdanove 2021). Fargier and colleagues 2011 did not find CFBP 5825 to be pathogenic on *Barbarea vulgaris* and they proposed that *X. campestris* pv. *barbareae*, as described by Vauterin et al. (1995), be abandoned, as it had not been confirmed in any other published study since its discovery (Fargier and Manceau 2007; Fargier et al. 2011). We did not have strains CFBP 5825 or CFBP 5826 to test on the New York cultivar of *Barbarea vulgaris*. However, because the sequence of CFBP 5825R is available and we had included in our sequencing another of the isolates from New York weeds that was reported to possibly cause weak spotting on *Barbarea vulgaris* (CFBP 5824 [Burkholder 1941]), we were able to determine, as detailed further below, that these strains are genetically closely related to the more recent New York isolates causing disease on *Barbarea vulgaris* (Fig. 2; Supplementary Fig. S1). Our results with this new group of strains isolated in New York and causing spot symptoms on *Barbarea vulgaris* but no other tested species support the *X. campestris* pv. *barbareae* pathovar designation.

**Associations of pathovar and genetic relatedness.**

To determine whether strains of the same pathovar are genetically more closely related to each other than to other strains, the 59 new assemblies and 49 published *X. campestris* genome sequences were first used to build an ANI matrix (Fig. 2). ANI was greater than 96% in all pairwise comparisons, surpassing the 95% ANI threshold for species-level identification. The strains nonetheless group into five clusters, containing i) all California weed isolates (*X. campestris* pv. *campestris*) and some *X. campestris* pv. *campestris* strains from China, ii) all New York *X. campestris* pv. *campestris* crop isolates and other *X. campestris* pv. *campestris* strains from China and elsewhere around the globe, iii) all *X. campestris* pv. *incanae* and *X. campestris* unknown pathogenicity (or rather *X. campestris* pv. *barbareae*) strains, including those isolated from weeds in New York and

Maryland as well as CFBP 5824 and CFBP 5825R representing the isolates described in 1940 (Burkholder 1941), iv) all *X. campestris* pv. *raphani* strains, including those isolated from weeds and crops in New York, and v) all *X. campestris* non-pathogenic strains, including the New York weed isolate 5053. This result reveals a strong association of pathovar with genetic similarity and supports the use of genetic relatedness to predict host range and tissue specificity of new *X. campestris* isolates.

Comparing strain relatedness with the original plant host and geographic origin provides potentially useful insight. For example, one of the *X. campestris* unknown pathogenicity/*X. campestris* pv. *barbareae* strains was found on *Capsella bursa-pastoris*. Thus, these strains, all of which were isolated in New York and all of which infect *Barbarea vulgaris*, may have a broader host range than we observed, as pathogens or as endo- or epiphytes. As another example, the relationship of the California weed isolates to strains from China may be informative. In the previous study of the California isolates, it was noted that all were pathogens on both the weeds they were isolated from and *Brassica oleracea* but that they grouped separately from crop isolates tested (Ignatov et al. 2007). The expanded analysis here, including all available *X. campestris* genomes, reveals that the California weed isolates are related specifically to strains from China isolated from *Brassica rapa*, *Brassica juncea*, and *Brassica napus* rather than from *Brassica oleracea* as most other represented *X. campestris* pv. *campestris* strains were. This observation suggests that, in the strains isolated in China and in the California weed isolates, there are i) adaptations that make them better able to infect the other crop brassicas and possibly weed species or ii) avirulence or other limiting factors that make them less fit on *Brassica oleracea*. The latter is consistent with the conclusion of the authors of the original study that weeds are not a major source of inoculum for disease on *Brassica oleracea* crops in California, though it remains possible that such weed strains might spread to such crops under certain environmental conditions (Ignatov et al. 2007).

The California weed isolate collection contrasts with the New York weed isolate collection and the one from Germany in that the isolates from California were pathogenic on *Brassica oleracea* while none of the New York weed isolates tested or the Maryland weed isolate were, and only a few of the isolates in the German collection were observed to cause disease on a crop species (Ignatov et al. 2007; Krauthausen et al. 2018; our results). These differences may be the result of sampling bias, however. Drawing firm conclusions about the origin of the weed-associated *X. campestris* pv. *campestris* strains and the overall *X. campestris* population on weeds in California would benefit from further sampling.

Next, to examine phylogeny, the whole-genome sequences were used to construct a maximum likelihood (ML) tree (Fig. 3; Supplementary Fig. S1). The tree confirms the groups from ANI analysis and provides additional insight into relationships within those groups. For example, the California weed isolates and the related *X. campestris* pv. *campestris* strains from China did not separate out from the rest of the *X. campestris* pv. *campestris* crop isolates monophyletically. However, a large subgroup of those strains does form a single clade. Also, the one Maryland and four New York weed isolates designated as *X. campestris* pv. *incanae* and the three New York weed isolates designated as *X. campestris* pv. *barbareae* form a clade with all other *X. campestris* pv. *incanae* and *X. campestris* unknown pathogenicity or *X. campestris* pv. *barbareae* strains, with the *X. campestris* unknown pathogenicity and *X. campestris* pv. *barbareae* strains forming a subclade within it. As noted earlier, CFBP 5824 and CFBP 5825R, representing the early New York weed isolates (Burkholder 1941), also reside in this *X. campestris* pv. *barbareae* clade. Burkholder (1941) classified CFBP 5824 as
pathovar *armoracae*, causing symptoms on horseradish and “no
or very slight” symptoms on *Barbarea vulgaris*. Also, although
CFBP 5825 was described as causing a spot disease on *Barbarea
vulgaris* (Burkholder 1941), Fargier and Manceau (2007) did not
observe symptoms following inoculation of CFBP 5825 to *Bar-
barea vulgaris*. We surmise that *Barbarea vulgaris* accessions
differ in their susceptibility to different strains in this group.
Specifically, the accession used by Fargier and Manceau (2007)
from a Swiss collection may not be susceptible to CFBP 5825,
while the accession we used, which originated in New York and
is likely more genetically similar to the plants initially used by
Burkholder (1941), clearly is. It would be of interest to assay
CFBP 5824, CFBP 5825R, and the other strains in this group
on a collection of *Barbarea vulgaris* accessions to determine
whether there is indeed variation across different strain and ac-
cession combinations. With respect to CFBP 5825, alternative
explanations for the difference between results of Burkholder
(1941) and those of Fargier and Manceau (2007) include at-
tenuation of the strain during subculturing and differences in
assay conditions. Though re-examination of the pathogenicity
of CFBP 5824 and CFBP 5825 on *Barbarea vulgaris* is war-
ranted, overall, the information available and our results strongly
suggest a shared genetic basis for pathovar *barbareae*, as a de-
scendant of *X. campestris* pv. *incanae*. Finally, the single New
York isolate designated as *X. campestris* pv. *raphani* grouped in
a clade with all other *X. campestris* pv. *raphani* strains as well as
all *X. campestris* nonpathogenic strains, with the latter forming a
single subclade. Included in the latter is the sole New York weed
isolate found to be nonpathogenic, 5053. A BLAST search of the
5053 genome confirmed the absence of a T3SS and T3Es. These
relationships suggest that loss of the T3SS and associated T3E

<figure><img src="image_41.png" /><figcaption>**Fig. 3.** Maximum likelihood tree of *Xanthomonas campestris* genomes sequenced in this study and publicly available genomes reflects pathovar relationships. Representing 108 total strains, the tree was constructed using the reference sequence alignment-based phylogeny builder (REALPHY) (Bertels et al. 2014). Orthologous single nucleotide polymorphisms were called within REALPHY using the *X. campestris* pv. *campestris* (*Xcc*) B100, ATCC33913, 8004, and Xca5 genomes as references. The resulting alignments were then merged as a part of the REALPHY pipeline. This merged alignment was used to reconstruct the phylogeny using RAxML HPC Black-box on the CIPRES portal. Branch colors are based on pathovar classification. Gray branches indicate strains for which pathotype was not tested in this or other studies but was inferred to be nonpathogenic due to lack of a type III secretion system.</figcaption></figure>

genes from an *X. campestris* pv. *raphani* progenitor may have
given rise to the *X. campestris* nonpathogenic strains or, though
seemingly less likely, that lack of a T3SS and T3Es could be the
ancestral state and *X. campestris* nonpathogenic strains never
gained these genes.

T3E repertoires.

Due to distinct differences in pathovar tissue specificities
and host ranges despite their high genetic percent identity, we
hypothesized that there may be pathovar-specific differences
in T3E repertoires. Using TBLASTN and a cut-off of 60%
identity and 60% coverage, we queried the whole-genome se-
quences with a list of known *Xanthomonas* T3Es (available on-
line from The *Xanthomonas* Resource) (Supplementary Table
S3). The genomes were found to harbor between zero and 19
T3E genes, with *X. campestris* nonpathogenic strains contain-
ing none, *X. campestris* pv. *raphani* strains averaging about six,
and *X. campestris* pv. *incanae*, *X. campestris* pv. *barbareae*,
and *X. campestris* pv. *campestris* strains averaging 14 to 16.
While overall patterns of T3E gene content reflect phylogeny
at the pathovar level, there are no conserved pathovar-specific
T3E genes or individual T3E genes that distinguish vascular and
nonvascular pathovars. However, for each pathovar, there are
T3E genes that are present in all strains, and T3E genes that are
absent from all strains, as has been observed by others (Roux
et al. 2015). Most striking in this regard are the *X. campestris*
pv. *raphani* strains, all of which have the same six T3E genes
and few or no others (Supplementary Table S3).

Sequences of strains CFBP 6690 and WHRI8481, which were
accessioned as *X. campestris* pv. *raphani* and *X. campestris*,
respectively, and which genetically group with *X. campestris*
pv. *raphani* via ANI (though WHRI8481 grouped with
*X. campestris* pv. *incanae* strains in the ML tree [Fig. 3]), lack
T3E genes entirely. Further investigation revealed that both also
lack a T3SS and are therefore likely to be *X. campestris* non-
pathogenic strains, though we did not test this prediction with
pathogenicity assays. Since CFBP 6690 and WHRI8481 group
distinctly from the *X. campestris* nonpathogenic strain clade,
evolution toward an endo- or epiphytic lifestyle via loss of the
T3SS and associated T3E genes may have occurred in multiple
lineages.

Transcription activator-like effector (TALE) variation.

Some *Xanthomonas campestris* strains encode TALEs, a class
of DNA-binding T3E that directly activate specific host genes,
some of which are 'susceptibility' genes that contribute to dis-
ease. TALE structure is well-conserved among homologs, and
target specificity depends on pairs of hypervariable amino acids
in a central repeat region, termed repeat variable di-residues,
that specify individual DNA bases (Boch et al. 2009; Moscou
and Bogdanove 2009). The central repeats of TALEs are usually
each 33 to 35 amino acids long. Thus, *tal* genes, in which the
repeats are 99 to 105 bp, are often missed or misassembled in
short read-derived genomes. The published genomes included
in our analysis that were assembled from short-read data, there-
fore, may not accurately reflect *tal* gene content. Furthermore,
as TALEs are of particular interest to our research groups, in this
and previous studies (e.g., Denance et al. 2018), toward capturing
the diversity of *tal* genes present in *X. campestris* populations,
strains were selected for sequencing based, in part, on prescreen-
ing for *tal* genes by PCR or Western blot analysis; therefore, the
proportion of strains in the sequenced collection that harbor *tal*
genes, particularly *X. campestris* pv. *campestris* strains, may not
represent that in nature.

With these two caveats, analysis of TALE gene content (Sup-
plementary Table S3) revealed the following. The *X. campestris*
pv. *campestris* and *X. campestris* pv. *incanae* strains contain
up to four TALE genes, though some strains contain none. Indeed, no TALE genes were found in any California *X. campestris* pv. *campestris* weed isolate from the sequenced collection. TALE genes are present in all the *X. campestris* pv. *barbareae* genome assemblies that were generated using long-read technology. Yet, none of the *X. campestris* pv. *raphani* genome assemblies contains TALE sequences, including the long-read assemblies. This was unexpected since *X. campestris* pv. *raphani* and *X. campestris* pv. *campestris* are often found in the same field or even on the same plant, and the TALE genes in *X. campestris* pv. *campestris* are found either on plasmids, in association with mobile elements, or both, which suggests that they could be horizontally transferred.

While TALE function has been investigated more thoroughly in other *Xanthomonas* species, TALEs are a recent discovery in *X. campestris* pv. *campestris* (Denance et al. 2018). Three TALEs (Hax2, Hax3, and Hax4) from *X. campestris* pv. *campestris* Xca5 (previously classified as pathovar *armoraciae*) were found to collectively contribute to virulence (Bolot et al. 2013a; Kay et al. 2005), but susceptibility gene targets of these or other *X. campestris* pv. *campestris* TALEs have not yet been identified. More research is needed to dissect the significance of the unusual TALE distribution in *X. campestris* and any virulence functions of TALEs in strains of different pathovars.

**AvrAC restricts host range on M. incana.**

AvrAC (also called XopAC) is a T3E found so far only in *X. campestris* (Xu et al. 2008). *X. campestris* pv. *campestris* strains expressing AvrAC are unable to cause disease on *Ara-bidopsis thaliana* accession Col-0 due to recognition of the effector

by the ZAR1 resistance protein complex and subsequent activation of plant defense, characterized by the plant hypersensitive reaction (HR), a rapid, localized, cell death (Adachi et al. 2019; Feng and Tang 2019; Guy et al. 2013b). AvrAC is a uridylyl transferase, and its avirulence function depends on this activity (Feng et al. 2012; Wang et al. 2015). The *avrAC* gene is present in the majority of the *X. campestris* pv. *campestris* strains isolated from *Brassica oleracea* worldwide but missing from all but one of the *X. campestris* pv. *campestris* isolates from California weeds (Supplementary Table S3), 40_2. As noted above, isolate 40_2 is the only California weed isolate that did not cause disease on *M. incana* (Supplementary Table S2). The *avrAC* gene is present in all the *X. campestris* pv. *barbareae* and *X. campestris* pv. *raphani* strains, but only in the subset of *X. campestris* pv. *incanae* strains that did not cause disease on *M. incana* (e.g., CFBP 1606). The *avrAC* gene is in fact absent from every *X. campestris* strain that did cause symptoms on *M. incana* (Supplementary Table S2). These observations suggest that AvrAC acts as an avirulence protein when delivered by the pathogen into *M. incana* cells.

To explore this hypothesis further, we inoculated *M. incana* leaves, using syringe infiltration, with a selection of *X. campestris* pv. *campestris* strains that contain or lack the *avrAC* gene, including the one California weed isolate that contains it, a pair of *X. campestris* pv. *incanae* strains with and without the gene, and an *X. campestris* pv. *barbareae* strain (which has it) (Fig. 4A). By 28 h after inoculation, HR was apparent in all leaves inoculated with any *avrAC*-containing strain but not in those inoculated with a strain lacking *avrAC*. As a more conclusive test, we assayed, alongside the *avrAC*-containing

<figure><img src="image_42.png" /><figcaption>**Fig. 4.** The *avrAC* gene causes avirulence on *Matthiola incana*. A, Results in *M. incana* leaves 28 h after syringe infiltration with strains of *Xanthomonas campestris* pv. *campestris* (*Xcc*), *X. campestris* pv. *incanae* (*Xci*), and *X. campestris* pv. *barbareae* (*Xcb*) naturally containing or lacking *avrAC* or mock inoculum and B, 48 h after syringe infiltration with *X. campestris* pv. *campestris* 8004, 8004Δ*avrAC*(pCZ917), 8004Δ*avrAC*(pCZ917:*avrAC*), or 8004*avrAC*-H469A. Ten replicate inoculations were carried out, one per leaf, for each. Representative leaves are shown, with the tally of leaves showing HR indicated below. The experiments were repeated once (A) or twice (B) with equivalent results.</figcaption></figure>
*X. campestris* pv. *campestris* strain 8004, the following strains generated in an earlier study (Guy et al. 2013a): an *avrAC* knockout derivative of 8004 carrying the empty plasmid vector pCZ917, the derivative carrying *avrAC* on the plasmid, and an 8004 derivative with the endogenous *avrAC* gene replaced by *avrAC-H469A*, which encodes an enzymatically inactive protein. This experiment was scored at 48 h. The results (Fig. 4B) confirmed that AvrAC causes avirulence on *M. incanae* and demonstrated that this function depends on its enzymatic activity, together suggesting the presence of ZAR1 or a close functional analog in *M. incanae*.

While we only tested pathogenicity on one cultivar of *M. incana*, other researchers reported that *X. campestris* pv. *incanae* CFBP 1606 expressing AvrAC is avirulent on a different *M. incana* cultivar (Fargier and Manceau 2007). Thus, AvrAC-specific resistance may be characteristic of *M. incana* and may be present in other, closely related species. No *M. incana* genome sequence is currently available, so it is unknown whether *ZAR1* or some other gene is responsible for the resistance. *Arabidopsis thaliana* and *M. incana* are more genetically similar to *Brassica* crop species that lack AvrAC-specific resistance than to each other (Fig. 5), so the resistance may have convergently evolved. Alternatively, *ZAR1*, which is an ancestral nucleotide-binding domain and leucine-rich repeat containing (NLR) gene present in many plant species, or another gene required for its function may have been lost in the lineage that gave rise to the crop species.

The T3E analysis we carried out suggests that the majority of *X. campestris* pv. *campestris* strains isolated from *Brassica oleracea* around the world contain *avrAC* (Supplementary Table S3). Thus, introduction of the gene or genes needed for ZAR1-mediated resistance into cabbage by breeding or genetic transformation could provide broadly effective disease control. If pathogen loss of the effector to evade detection were to reduce virulence, the resistance would also likely be relatively durable. In *Arabidopsis*, AvrAC has been shown to inhibit pattern-triggered plant immunity (PTI) by uridylating Bik1 (Feng et al. 2012; Meng and Zhang 2013) but its contribution to virulence, under controlled conditions, depends on the

strain (Guy et al. 2013b). Whether AvrAC is broadly important to *X. campestris* pv. *campestris* virulence on cabbage under field conditions is yet to be tested. Even so, because *X. campestris* pv. *campestris* has not been found to persist in fields in crop production areas such as New York (Lange et al. 2016), the likelihood of resistance-breaking strains evolving from local populations is low. However, if F1 seed is repeatedly generated in an area where the pathogen does persist year-round, resistance may break down through pathogen loss of *avrAC*.

Importantly, though, through interactions with distinct receptor-like cytoplasmic kinases (RLCKs), ZAR1 mediates recognition of several *Pseudomonas* effectors, including HopBA1, HopF1/HopF2, HopO1, HopX1, and HopZ1 (Laflamme et al. 2020). Further, ZAR1 of *Nicotiana benthiana* mediates recognition of the *X. populans* effector XopJ4, a member of the HopZ/YopJ superfamily (Schultink et al. 2019). Homologs of HopX1 and three members of the HopZ/YopJ superfamily, XopE and XopJ1, Xop-J3, and Xop-J5, respectively, are encoded among the *X. campestris* genomes examined here (Supplementary Table S3). The xopE gene is found in most of the *X. campestris* pv. *campestris* genomes, many of the *X. campestris* pv. *incanae* genomes, and one of the *X. campestris* pv. *barbareae* genomes. The xopJ1 and xopJ3 genes are present in one or two of the *X. campestris* pv. *barbareae* genomes, and xopJ5 is found in many *X. campestris* pv. *campestris*, several *X. campestris* pv. *incanae*, and one of the *X. campestris* pv. *barbareae* genomes. Thus, ZAR1 may be effective even against strains that lack AvrAC. Notably however, *X. campestris* pv. *campestris* 8004 contains both xopE and xopJ5, yet the 8004 *avrAC* knockout mutant grew in population and caused symptoms in *M. incana* leaves following infiltration and pinprick inoculation, respectively (Supplementary Fig. S2). Though we did not repeat the experiment, this result suggests that those Xops are not detected in *M. incanae*, and it underscores the likelihood that the breadth of ZAR1 activity will depend on host genetic background, specifically on the presence of cognate RLCK genes.

### Involvement of *cbsA* in *X. campestris* tissue specificity.

To determine whether the presence of *cbsA* correlates with differences in tissue specificity in *X. campestris*, we searched all available *X. campestris* genomes for *cbsA*. Two copies of *cbsA* were identified. One copy, with a 1,701-bp coding sequence ("*cbsA*1701"), was found in all *X. campestris* pv. *campestris* and *X. campestris* pv. *incanae* strains (i.e., all vascular disease-causing strains) and was absent from all *X. campestris* pv. *barbareae*, *X. campestris* nonpathogenic, and *X. campestris* pv. *raphani* strains (i.e., all nonvascular or nonpathogenic strains) (Supplementary Table S4). This copy shows >99% nucleotide identity across the strains that have it. A second copy of *cbsA*, lacking 210 bp coding for the N-terminal signal peptide found in *cbsA*1701, was found in all *X. campestris* strains, with slightly less sequence conservation, at >95% nucleotide identity.

To test whether *cbsA*1701 is, indeed, a determinant of vascular pathogenicity in *X. campestris*, we expressed a clone of the gene from *X. campestris* pv. *campestris* 8004 under its native promoter in *X. campestris* pv. *barbareae* strains 3054 and 11_19 and in *X. campestris* pv. *raphani* 756c and compared these transformants to transformants carrying the empty vector. We hypothesized that the gene would convert the *X. campestris* pv. *incanae* and *X. campestris* pv. *raphani* strains to vascular pathogens on their hosts. Because *X. campestris* pv. *barbareae* is closely related to *X. campestris* pv. *incanae*, which is a vascular pathogen of *E. cheiri*, we further hypothesized that the addition of *cbsA*1701 might expand the host range of *X. campestris* pv. *barbareae* to include *E. cheiri*. Syringe infiltration of *E. cheiri* leaves with wild-type *X. campestris* pv. *barbareae* 11_19 resulted in a HR, while *X. campestris* pv. *barbareae* 3054 did not cause HR,

<figure><img src="image_43.png" /><figcaption>**Fig. 5.** Relationships among the host species of origin for the *Xanthomonas campestris* strains examined in this study. The hierarchical tree was generated using the NCBI taxonomy browser.</figcaption></figure>
suggesting the presence of an avirulence factor in 11_19 but not 3054, recognized by *E. cheiri* (Supplementary Fig. S3A and B). Based on this result, we inoculated the *X. campestris* pv. *barbareae* 11_19 and 3054 transformants to *Barbarea vulgaris* and the 3054 transformants to *E. cheiri*. We inoculated the *X. campestris* pv. *raphani* transformants to *Brassica oleracea* TO1000. For all inoculations, we used the clip-and-dip method.

On *Barbarea vulgaris*, *cbsA*<sub>1701</sub> made no difference to symptoms caused by *X. campestris* pv. *barbareae* 3054 or *X. campestris* pv. *barbareae* 11_19, each causing only spots (Supplementary Fig. S3C to F). On *E. cheiri*, however, *X. campestris* pv. *barbareae* 3054 carrying the *cbsA*<sub>1701</sub> plasmid (pBBR1::*cbsA*<sub>1701</sub>) caused chlorotic and necrotic lesions extending from the clipped end of the leaf (Fig. 6), similar in appearance to symptoms caused by *X. campestris* pv. *incanae* (Fig. 1), though slower to develop. In *X. campestris* pv. *raphani* 756c inoculated to *Brassica oleracea* TO1000, as in the *X. campestris* pv. *barbareae* strains inoculated to *Barbarea vulgaris*, *cbsA*<sub>1701</sub> had no effect on symptom development, with both the *cbsA*<sub>1701</sub> transformant and the empty vector control causing spots only (Fig. 6).

While genomic data show a perfect correlation between the presence of the *cbsA*<sub>1701</sub> gene and vascular pathogenicity and heterologous expression of *cbsA*<sub>1701</sub> conferred apparent weak vascular pathogenicity to one *X. campestris* pv. *barbareae* strain on one of two *Brassica* species tested, additional factors must be involved in vascular versus nonvascular disease development. These may be species-specific avirulence factors, as suggested by the avirulence of *X. campestris* pv. *barbareae* 11_19 on *E. cheiri* (Supplementary Fig. S3A and B). Alternatively and not mutually exclusive, they may be positive-acting factors present in some strains and not others, as suggested for other *Xanthomonas* species (Gluck-Thaler et al. 2020). This seems particularly plausible for *X. campestris* pv. *raphani*, given that *X. campestris* pv. *raphani* strains have a highly reduced effector repertoire in comparison to *X. campestris* pv. *campestris* (Supplementary Table S3). Host factors also could play a role.

<figure><img src="image_44.png" /><figcaption>**Fig. 6.** Heterologous expression of *cbsA*<sub>1701</sub> confers apparent vascular pathogenicity to *Xanthomonas campestris* pv. *barbareae* 3054 on *Erysimum cheiri* but not to *X. campestris* pv. *raphani* 756c on *Brassica oleracea*. *E. cheiri* and *Brassica oleracea* TO1000 leaves are shown 21 days after clip-and-dip inoculation with the *X. campestris* pv. *barbareae* and *X. campestris* pv. *raphani* strains carrying one or both pBBR1::*cbsA*<sub>1701</sub> and the empty vector or with the wild-type strains.</figcaption></figure>

## Summary and conclusions.

Genome sequencing of 59 additional *X. campestris* genomes expanded the existing collection to include a more diverse array of strains isolated from weed and crop species. Pathogenicity assays on selected *Brassica* species and tomato were used to determine pathovar. Strain groupings by pathovar were reflected in whole genome sequence comparisons. Pathogenicity profiles and genetic relationships of strains isolated from weeds in New York that formed a subclade in the larger group of *X. campestris* pv. *incanae* strains provided support for pathovar *barbareae*, rejection of which had been proposed. Mining of the genomic data in the context of the pathovar information provided new insight also into virulence factors and determinants of host and tissue specificity.

Three conclusions emerged regarding tissue-specificity and host range of *X. campestris*. First, nonpathogenic isolates do not have a T3SS, while pathogenic isolates do (Arlat et al. 1991). We determined that a group of *X. campestris* strains isolated in New York cause spot symptoms on *Barbarea vulgaris*. These strains are closely related to ones isolated from New York weeds in the early 1940s, including CFBP 5825, which was originally reported to be the type strain for *X. campestris* pv. *barbareae*. While CFBP 5825 had most recently been considered nonpathogenic and pv. *barbareae* removed from the pathovar naming scheme, our phenotyping provides evidence that *barbareae* is a true pathovar represented by CFBP 5825 and additional recently isolated New York weed strains, which all contain a T3SS and T3E repertoires. It remains to be seen if strains CFBP 6690 and WHR18481, which genetically group with *X. campestris* pv. *raphani* and *X. campestris* pv. *incanae*, respectively, but lack a T3SS, are pathogenic, though we would expect not. Based on our data, within *X. campestris*, in contrast to some other *Xanthomonas* species (Jacobs et al. 2015; Pieretti et al. 2009), presence of a T3SS and T3Es is tied to pathogenicity and absence to a nonpathogenic endo- or epiphytic lifestyle.

Second, while no clear associations of T3E content and tissue specificity were apparent, the T3E AvrAC appears to act as a host-range determinant, preventing infection of the ornamental species *M. incana*. *Arabidopsis thaliana* Col-0 recognizes AvrAC by virtue of the NLR protein ZAR1 and associated proteins, but *Arabidopsis* is less closely related to *M. incana* than it is to *Brassica oleracea* (Fig. 5). A recent phylogenetic analysis of NLR proteins from diverse plant species suggests that ZAR1 may be the most conserved coiled-coil NLR in angiosperms (Adachi et al. 2020). ZAR1 may be present in additional *Brassica* species and could be leveraged for breeding resistance to black rot. By expanding the pool of sequenced *X. campestris* pv. *campestris* genomes, our study suggests that the majority of *X. campestris* pv. *campestris* crop isolates have AvrAC, as well as other potential elicitors of ZAR1-mediated resistance (i.e., XopE and XopJ5) and that such resistance could be broadly effective.

Last, the observed presence of *cbsA*<sub>1701</sub> in vascular but not nonvascular *Xanthomonas* pathogens (Gluck-Thaler et al. 2020) extends to *X. campestris* pathovars, including all of the newly sequenced strains in this study. All strains of *X. campestris* that cause vascular disease, i.e., strains of *X. campestris* pv. *campestris* and *X. campestris* pv. *incanae*, have *cbsA*<sub>1701</sub>, while all nonvascular or nonpathogenic strains, *X. campestris* pv. *raphani*, *X. campestris* pv. *barbareae*, and *X. campestris* nonpathogenic strains, lack the gene. Experimentally, transfer of *cbsA*<sub>1701</sub> to one *X. campestris* pv. *barbareae* strain led to symptoms similar in appearance to those caused by the vascular pathogen *X. campestris* pv. *incanae* in *E. cheiri*. However, a similar result was not observed for a second *X. campestris* pv. *barbareae* strain, and introduction of *cbsA*<sub>1701</sub> did not preclude *X. campestris* pv. *barbareae* strains from causing nonvascu-
lar symptoms in *Barbarea vulgaris*. Additionally, transfer of *cbsA*1701 to *X. campestris* pv. *raphani* did not alter tissue specificity. These data bolster conclusions from previous studies that *cbsA*1701 may contribute but that there are additional factors involved in vascular versus nonvascular disease development (Cerutti et al. 2017; Gluck-Thaler et al. 2020; Jha et al. 2007).

In addition to the insight gained from our study, we expect the new genome sequences and phenotypic data we have presented to facilitate future studies to further improve understanding of *X. campestris* pathogenicity. Investigations of host range determinants and novel factors involved in tissue specificity will be especially valuable in developing strategies for improved resistance in the field.

## MATERIALS AND METHODS

### Bacterial strains and plant varieties used.

Wild-type *X. campestris* strains used or referenced are listed in Supplementary Table S1. Plants used include *Barbarea vulgaris* and *Sinapis arvensis* (Cornell University Weed Garden), *Brassica oleracea* var. oleracea TO1000 (Vilmorin, Salinas, CA, U.S.A.), *Solanum lycopersicum* cv. Moneymaker (Eden Brothers, Arden, NC, U.S.A.), *Matthiola incana* cv. Iron Cherry (Johnny's Selected Seed, Fairfield, ME, U.S.A.), and *Erysimum cheiri* cv. English Wallflower (Eden Brothers, Arden, NC, U.S.A.).

### Genome sequencing.

Bacteria were cultured at 28°C with shaking (225 rpm) for 24 h in nutrient broth (BD Biosciences, San Jose, CA, U.S.A.), and genomic DNA was isolated using the MasterPure gram-positive DNA purification kit (Lucigen, Middleton, WI, U.S.A.), according to manufacturer instructions. Ten-kilobase genomic libraries were prepared and size-selected as previously described (Booher et al. 2015). The libraries were multiplexed on a SMRT cell (10, 24, or 48 libraries per cell) on a SEQUEL I or SEQUEL II machine (Pacific Biosciences, Menlo Park, CA, U.S.A.) at the Icahn School of Medicine at Mt. Sinai (New York). The genomes were assembled using the HGAP assembler version 4.0 or the PacBio Microbial Assembler and were annotated via the National Center for Biotechnology Information (NCBI) with the Prokaryotic Genome Annotation Pipeline (Chin et al. 2013; Li et al. 2021).

### Pathogenicity assays.

Strains were grown on glucose yeast extract (GYE) (5 g of glucose and 10 g of yeast extract per liter) agar plates for 24 h before being collected with a spatula and resuspended in 10 mM MgCl₂. The cell suspension was adjusted to an optical density at 600 nm (OD₆₀₀) of 0.1 (about 10⁸ CFU/mL) in 50 ml, and Silwet (Phytotech Labs, Lenexa, KS) was added to 0.0125%. Plants were grown in 4-inch pots in a chamber at 90% relative humidity for 4 weeks with 12-h days at 25°C and 12-h nights at 21°C. Plants were inoculated by clipping two leaves and dipping the entire leaves in the prepared inoculum for 10 s. Plants were then returned to the growth chamber for 14 days, after which leaves were observed for the presence of vascular disease or bacterial spot symptoms. This clip-and-dip inoculation technique was used to allow invasion by vascular pathogens through hydathodes and cut veins and by nonvascular pathogens through stomates. A minimum of three technical replicates were completed for each inoculation and all experiments were repeated at least twice. A negative control of 10 mM MgCl₂ was included for inoculation in each round of experiments. For the assays in *M. incana* presented in Supplementary Figure S2 only, to assay disease development, fully expanded leaves of 6-week-old plants grown in a greenhouse were inoculated by piercing the middle

of the central vein with a bacterial inoculum at 10⁸ CFU/ml (OD₆₀₀ = 0.1) in 1 mM MgCl₂. After inoculation, plants were moved to a growth chamber at 22°C with 70% humidity and an 8-h light period. Disease symptoms were observed 10 days after inoculation. For determination of bacterial populations in *M. incana*, leaves were infiltrated using a 1-ml needleless syringe with a bacterial inoculum at 10⁶ CFU/ml (OD₆₀₀ = 0.001) in 1 mM MgCl₂ and were then moved to a growth chamber as above. After 3 days, leaf discs were sampled using a 0.65 cm diameter cork borer (surface area 0.33 cm²) and were individually ground using a TissueLyser MM 400 grinder (Retsch, Hann, Germany), two times for 30 s at a frequency of 30 per second with two glass beads (diameter 4.5 mm) in 200 µl of sterile water. The homogenates were serially diluted in sterile water and 5-µl drops were spotted three times on MOKA (Blanvillain et al. 2007) agar supplemented with rifampicin (50 µg/ml) and pimaricin (30 µg/ml). Plates were incubated at 28°C for 48 h, and colonies were enumerated in spots containing 1 to 30 colonies. Bacterial densities in leaves were calculated as log CFU per square centimeter.

### Avirulence assays.

Bacteria were either grown on GYE agar plates and harvested as described above (Fig. 4A) or were grown in liquid GYE to late log phase (Fig. 4B) and were harvested by centrifugation, then were resuspended to OD₆₀₀ = 0.2 in 10 mM MgCl₂. Leaves at approximately the middle of the stem of 4- to 6-week-old *Matthiola incana* plants, four to six leaves per plant, were pricked with a pin on the abaxial side and were infiltrated through the pin-hole with about 0.1 ml of bacterial suspension or 10 mM MgCl₂, using a needleless syringe. For each inoculum, 10 leaves from 10 different plants were used. Inoculated plants were incubated in a growth chamber at 25°C and ambient humidity under constant light and were monitored for up to 48 h for development of the HR.

### Phylogeny and ANI analysis.

ML trees were created from whole-genome sequences using REALPHY (Bertels et al. 2014). Orthologous single nucleotide polymorphisms were called within REALPHY, using *X. campestris* pv. *campestris* reference genomes B100, ATCC33913, 8004, and Xca5, and the resulting alignments were merged as a part of the REALPHY pipeline. This merged alignment was used to reconstruct the phylogeny using RAxML HPC Blackbox on the CIPRES Portal (Miller et al. 2010). For the rooted tree (Supplementary Fig. S1) *X. oryzae* PX099A (GenBank CP000967.2) was used as the outgroup. Support was assessed using 1,000 bootstrap pseudoreplicates. The trees were visualized in FigTree and coloring was added in FigTree or Adobe Illustrator (Adobe Inc., San Jose, CA, U.S.A.). ANI matrices were made with the Enveomics Toolkit ANI Matrix calculator (Rodriguez-R and Konstantinidis 2016).

### Effector comparisons.

Presence or absence of T3E genes and *cbsA* was determined by a TBLASTN search using the genomes as a BLAST database in Geneious Prime (Biomatters, San Diego, CA, U.S.A.) queried with amino acid sequences of known *Xanthomonas* T3E proteins (The *Xanthomonas* Resource) and those encoded by the two *cbsA* genes in *X. campestris* pv. *campestris* (Genbank Protein accession numbers QCX69893.1 and QCX65459.1). Hits with greater than 60% query coverage and 60% identity were listed as present. TALE sequences were annotated manually by searching for the TALE repeat motifs in chromosome and plasmid sequences. Some effectors within the same family (e.g., XopE1
and XopE2) were grouped together under the family name (i.e., XopE) due to high sequence similarity.

**Cloning of *cbsA* and *X. campestris* transformation.**

The 1,701-bp open reading frame of *cbsA*1701 and 1,000 bp upstream of the gene were amplified from *X. campestris* pv. *campestris* 8004 and were cloned into a mini-Tn7 vector, were subcloned into pCZ1013, and were then transferred into pBBR1-MCS2 (Kovach et al. 1995), using EcoRI and HindIII, yielding pBBR1::cbsA1701. *X. campestris* competent cells were transformed by electroporation as described (White and González 1991). Transformants were selected on GYE amended with 15 µg of kanamycin per milliliter.

**Data availability.**

Genome data generated in the current study are available through GenBank (project number PRJNA689092). Individual strain accession numbers are listed in Supplementary Table S1.

**ACKNOWLEDGMENTS**

The authors thank L.-M. Rodríguez, H. Lange, and M. Arlat for advice on design and execution of the study. We also thank K. Howard, director of the Cornell Weed Garden, for providing rare seed for cruciferous weeds found in New York, and M. Paauw for providing various *X. campestris* pv. *campestris* genomic DNA samples for sequencing. Finally, we thank A. Read, S. Baruah, and T. Bauer for critical feedback on the manuscript and assistance with inoculations.

**AUTHOR-RECOMMENDED INTERNET RESOURCES**

CIPRES portal: https://www.phylo.org/index.php

Enveomics Collection Toolbox genome based distance matrix calculator:
http://enve-omics.ce.gatech.edu/g-matrix

NCBI taxonomy browser:
https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi

The *Xanthomonas* Resource:
http://www.biopred.net/xanthomonas/t3e.html

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