# Complete and Circularized Genome Sequences of 17 Xanthomonas Strains Responsible for Common Bacterial Blight of Bean

Martial Briand, Mylène Ruh, Armelle Darrasse, Marie-Agnès Jacques, Nicolas

W. G. Chen

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## To cite this version:

Martial Briand, Mylène Ruh, Armelle Darrasse, Marie-Agnès Jacques, Nicolas W. G. Chen. Complete and Circularized Genome Sequences of 17 Xanthomonas Strains Responsible for Common Bacterial Blight of Bean. Microbiology Resource Announcements, 2021, 10 (31), pp.e00371. 10.1128/MRA.00371-21. hal-03344454

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Submitted on 30 Aug 2024

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# Complete and Circularized Genome Sequences of 17 *Xanthomonas* Strains Responsible for Common Bacterial Blight of Bean

Martial Briand,ª Mylène Ruh,ª Armelle Darrasse,ª Marie-Agnès Jacques,ª Nicolas W. G. Chenª

ªUniversity of Angers, Institut Agro, INRAE, IRHS, SFR QUASAV, Angers, France

**ABSTRACT** We report the complete and circularized genome sequences of 17 strains of *Xanthomonas citri* pv. fuscans and *Xanthomonas phaseoli* pv. phaseoli, which cause common bacterial blight of bean. These new assemblies combining PacBio and short-read sequencing methods provide high-quality material for studying the evolution of these plant pathogens.

Common bean (*Phaseolus vulgaris* L.) is an important legume crop used for direct human consumption and represents the main source of protein for nearly 500 million people in the world (1). Common bacterial blight of bean (CBB) is a major disease that severely reduces common bean yields worldwide (2). CBB is caused by *Xanthomonas citri* pv. fuscans and *Xanthomonas phaseoli* pv. phaseoli, two phylogenetically distant groups of strains producing indistinguishable symptoms on common bean (3). *X. citri* pv. fuscans is subdivided into three genetic lineages, fuscans, GL2, and GL3, while *X. phaseoli* pv. *phaseoli* is represented by one genetic lineage, GL1 (4).

The first complete genome to be published for CBB agents was that of *X. citri* pv. fuscans strain 4834-R, also known as CFBP 4885 (5). Since then, 69 additional whole genomes have been released, including PacBio assemblies for 17 strains representing the diversity of CBB agents (6). However, these assemblies were noncircularized and contained sequencing errors in important pathogenicity genes encoding transcription activator-like (tal) effectors (6). For 12 of these strains, Illumina assemblies were produced and published independently (7). Moreover, raw reads have not been released so far for both PacBio and short-read sequencing. This announcement aims at clarifying the situation by releasing all of the raw data and producing final circularized versions of these genomes. For this, we used the previous raw data (17 PacBio, 12 Illumina, and a combination of Illumina plus 454 for strain CFBP 4885) to produce *de novo* assemblies, among which 13 combined PacBio and short-read, while the other four corresponded to PacBio only (Table 1).

Strains were originally isolated from bean plants at different dates and places (Table 1) and conserved as lyophilizates at the French Collection for Plant-Associated Bacteria (CIRM-CFBP, Angers, France). Different bacterial cultures and DNA extraction methods were used for PacBio and short-read sequencing.

For short-read sequencing (13 strains), bacteria were grown on Trypticase soy (TS) agar for 2 days at 28°C, and then 5 to 6 clones were scraped, pooled, and cultured overnight in TS broth with shaking. Genomic DNA was extracted and purified using the method of Klotz and Zimm (8). Shotgun and Roche 454 libraries were constructed following the manufacturer's protocols. For strain CFBP 4885, single reads were produced using Illumina Genome Analyzer IIx and Roche 454 GS FLX sequencers, while for the 12 other strains, paired-end sequencing was performed on an Illumina HiSeq 2000 machine. The quality of reads was checked using FastQC v0.11.9.

For PacBio sequencing (17 strains), bacteria were grown on TS agar for 2 days at 28°C, and then 5 to 6 clones were scraped, pooled, and cultured overnight on 10% TS agar to

**Citation** Briand M, Ruh M, Darrasse A, Jacques M-A, Chen NWG. 2021. Complete and circularized genome sequences of 17 *Xanthomonas* strains responsible for common bacterial blight of bean. Microbiol Resour Announc 10:e00371-21. https://doi.org/10.1128/MRA.00371-21.

**Editor** David A. Baltrus, University of Arizona

**Copyright** © 2021 Briand et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Nicolas W. G. Chen, nicolas.chen@agrocampus-ouest.fr.

**Received** 3 May 2021

**Accepted** 9 July 2021

**Published** 5 August 2021
<table><caption>TABLE 1 Accession numbers and statistics<sup>a</sup></caption><thead><tr><th>Pathovar</th><th>Lineage</th><th>Strain<sup>b</sup></th><th>Country (date)</th><th>SRA no. (short reads)<sup>c</sup></th><th>No. of short reads</th><th>Coverage (short reads) (x)</th><th>SRA no. (PacBio reads)</th><th>No. of PacBio reads</th><th>Coverage (PacBio reads) (x)</th><th>N<sup>30</sup> (bp)</th><th>GenBank assembly no.</th><th>Size (bp)</th><th>G+C (%)</th><th>No. of plasmids</th><th>No. of CDSS</th></tr></thead><tbody><tr><td rowspan="10">Xcf</td><td rowspan="10">fuscans</td><td>4885</td><td>France (1998)</td><td>ERP127521</td><td>8,852,907</td><td>78</td><td>SRR13977177</td><td>87,054</td><td>128</td><td>17,839</td><td>GCA_002759355.2</td><td>5,103,871</td><td>64.7</td><td>3</td><td>4,244</td></tr><tr><td>6165</td><td>Canada (1957)</td><td>SRR13997422</td><td>4,510,629</td><td>180</td><td>SRR13975653</td><td>74,194</td><td>100</td><td>10,202</td><td>GCA_002759215.3</td><td>5,064,829</td><td>64.8</td><td>1</td><td>4,214</td></tr><tr><td>6166</td><td>South Africa (1963)</td><td>ERR4913433</td><td>19,858,409</td><td>794</td><td>SRR13976510</td><td>78,691</td><td>91</td><td>8,037</td><td>GCA_002759235.2</td><td>5,158,326</td><td>64.7</td><td>4</td><td>4,326</td></tr><tr><td>6167</td><td>USA (1954)</td><td>NA</td><td>NA</td><td>NA</td><td>SRR13976512</td><td>87,526</td><td>166</td><td>13,753</td><td>GCA_002759415.2</td><td>5,329,619</td><td>64.6</td><td>4</td><td>4,524</td></tr><tr><td>6975</td><td>France (1994)</td><td>NA</td><td>NA</td><td>NA</td><td>SRR13976539</td><td>79,222</td><td>160</td><td>14,492</td><td>GCA_002759255.3</td><td>5,188,573</td><td>64.7</td><td>3</td><td>4,380</td></tr><tr><td>7767R</td><td>Cameroon (2009)</td><td>ERR4913436</td><td>16,368,893</td><td>655</td><td>SRR13976552</td><td>60,016</td><td>146</td><td>22,346</td><td>GCA_002759375.2</td><td>5,297,402</td><td>64.6</td><td>2</td><td>4,461</td></tr><tr><td>6988R</td><td>La Réunion (2000)</td><td>SRR14028005</td><td>36,595,739</td><td>1,464</td><td>SRR13977966</td><td>91,551</td><td>217</td><td>18,042</td><td>GCA_002759275.2</td><td>5,158,358</td><td>64.6</td><td>1</td><td>4,328</td></tr><tr><td>6989</td><td>La Réunion (2000)</td><td>NA</td><td>NA</td><td>NA</td><td>SRR13978504</td><td>82,059</td><td>135</td><td>12,235</td><td>GCA_002759295.2</td><td>5,158,365</td><td>64.6</td><td>1</td><td>4,330</td></tr><tr><td>6990</td><td>La Réunion (2000)</td><td>ERR4913441</td><td>16,805,623</td><td>672</td><td>SRR13977971</td><td>69,567</td><td>137</td><td>14,095</td><td>GCA_002759315.2</td><td>5,159,777</td><td>64.6</td><td>1</td><td>4,330</td></tr><tr><td>6991</td><td>La Réunion (2000)</td><td>ERR4913428</td><td>51,958,687</td><td>2,078</td><td>SRR13978069</td><td>71,661</td><td>116</td><td>11,93</td><td>GCA_002759395.2</td><td>5,269,642</td><td>64.6</td><td>2</td><td>4,477</td></tr><tr><td>6992</td><td>La Réunion (2000)</td><td>ERR4913442</td><td>15,585,602</td><td>623</td><td>SRR13978503</td><td>76,775</td><td>156</td><td>14,339</td><td>GCA_002759335.2</td><td>5,282,885</td><td>64.6</td><td>1</td><td>4,336</td></tr><tr><td>6994R</td><td>Tanzania (1990)</td><td>ERR4913430</td><td>15,635,275</td><td>625</td><td>SRR13978501</td><td>98,141</td><td>259</td><td>19,925</td><td>GCA_002759175.2</td><td>5,167,617</td><td>64.7</td><td>1</td><td>4,246</td></tr><tr><td>6996R</td><td>La Réunion (2000)</td><td>SRR14028037</td><td>11,275,045</td><td>451</td><td>SRR13977968</td><td>85,535</td><td>100</td><td>21,629</td><td>GCA_002759195.2</td><td>5,129,377</td><td>64.7</td><td>1</td><td>4,200</td></tr><tr><td rowspan="4">Xpp</td><td rowspan="4">GL1</td><td>412</td><td>USA (NA)</td><td>ERR4913438</td><td>15,533,646</td><td>621</td><td>SRR13978569</td><td>87,054</td><td>166</td><td>14,063</td><td>GCA_002759095.2</td><td>5,155,153</td><td>64.9</td><td>2</td><td>4,364</td></tr><tr><td>6164</td><td>Romania (1966)</td><td>ERR4913425</td><td>90,543,733</td><td>3,622</td><td>SRR13977151</td><td>79,892</td><td>170</td><td>14,763</td><td>GCA_002759115.2</td><td>5,341,746</td><td>64.6</td><td>2</td><td>4,557</td></tr><tr><td>6546R</td><td>USA (NA)</td><td>SRR14027994</td><td>40,690,726</td><td>1,628</td><td>SRR13978070</td><td>92,581</td><td>100</td><td>18,472</td><td>GCA_002759135.3</td><td>5,169,078</td><td>64.8</td><td>2</td><td>4,398</td></tr><tr><td>6982</td><td>La Réunion (2000)</td><td>NA</td><td>NA</td><td>NA</td><td>SRR13978588</td><td>84,288</td><td>167</td><td>15,913</td><td>GCA_002759155.2</td><td>5,239,376</td><td>64.8</td><td>3</td><td>4,445</td></tr></tbody></table>

<sup>a</sup>NA, not available.

<sup>b</sup>Strains ending with “R” indicate Rif-resistant variants of the original CFBP isolate.

<sup>c</sup>Illumina paired reads (2 × 100 bp) except for strain 4885, which is a combination of Illumina single reads (36 bp) and 454 GS-FLX (247 bp).
<figure><img src="image_4.png" /></figure>

obtain fresh cultures. A loop (~5 µl) of cells was suspended in sterile distilled H₂O and collected by centrifugation. Genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega) according to the manufacturer's recommendations. DNA was mechanically sheared using g-TUBE columns (Covaris). PacBio SMRTbell libraries were prepared from ~10 µg of genomic DNA and size-selected to 15 to 20 kb using BluePippin cassettes (Sage Scientific). Single-molecule real-time (SMRT) cell sequencing was performed on a PacBio RS II machine using P5-C3 chemistry (one SMRT cell per strain).

PacBio reads were filtered using PreAssembler Filter v1 of the SMRT Portal version 2.3 (Pacific Biosciences, Inc., CA) and then assembled using Canu v1.5 (9) with the setting genomeSize = 5m. Circularization was done using Berokka v0.2.3 (https://github.com/tseemann/berokka). For some assemblies where molecules could not be circularized, assembly was performed again on a subset of reads selected with Filtlong (https://github.com/rrwick/Filtlong), using the Illumina reads as external reference if available. The sequence start was fixed using the fixstart option of Circlator v1.5.1 (10). Polishing was performed using variantCaller v2.2.2 (https://github.com/PacificBiosciences/GenomicConsensus) with the setting --algorithm best. For the 13 strains with short reads, correction of PacBio assemblies was done using Pilon v1.23 (11) with the setting --mindepth 0.5. Coding sequence (CDS) predictions were retrieved from the NCBI Prokaryotic Genome Annotation Pipeline (12). Default parameters were used for all software unless otherwise specified.

The genomes consisted of 5,064,829- to 5,341,746-bp sequences comprising a chromosome plus one to four plasmids, with an average G+C content of 64.7%, and 4,200 to 4,557 predicted CDSs (Table 1). Genomes were estimated to be >99.6% complete and <0.4% contaminated using CheckM v1.0.7 (13). This apparent incompleteness corresponded to the absence of CheckM marker PF13603 (leucyl-tRNA synthetase) in all strains from lineages GL1, GL3, and fuscans, suggesting that this absence reflected a specificity of these lineages rather than incomplete genomes. On the other hand, apparent contamination corresponded to the duplication of one CheckM marker in CFBP strains 6166 and 6982. Assembly quality, estimated by homogeneity of the coverage of short reads, showed lower relative standard deviations for the new genomes than those of previous PacBio assemblies (6), indicating that the overall quality has been improved. New assemblies led to the elimination of six plasmids from previous PacBio genomes of CFBP strains 4885, 6165, 6975, 6164, and 6546R, corresponding to redundant sequences with poor coverage. All *tal* gene sequences were correct according to the previous sequence checks (6). Finally, circularization resulted in the merging of overlapping ends for each molecule, which led to the elimination of dozens of artifactual genes, including *tal18H2_CFBP6164* and *tal18H*-CFBP6546R (6). In all, this release provides enhanced versions of 17 CBB agent genomes, which constitute an important basis for further studies of these plant pathogens.

**Data availability.** The reads and assemblies were all deposited at GenBank under the accession numbers listed in Table 1. The novel assemblies were deposited to replace the previous PacBio assemblies (6).

## ACKNOWLEDGMENTS

The research leading to these results received grants from Genoscope (3X 154/AP2006-2007 and XANTHOMICS 18/AP2009-2010) and the French National Research Agency (XANTHOMIX ANR-2010-GENM-013-02 and CROpTAL ANR-14-CE19-0002-04). This work was supported by France Génomique National infrastructure, funded as part of the "Investissement d'Avenir" program managed by the French National Research Agency (ANR-10-INBS-0009).

We thank the CIRM-CFBP (Angers, France) for strain preservation and supplies. We thank L. Legrand for fruitful discussions and CATI BARIC for bioinformatics facilities.

## REFERENCES

1. Bitocchi E, Rau D, Bellucci E, Rodriguez M, Murgia ML, Gioia T, Santo D, Nanni L, Attene G, Papa R. 2017. Beans (*Phaseolus* ssp.) as a model for

understanding crop evolution. Front Plant Sci 8:1–21. https://doi.org/10.3389/fpls.2017.00722.
<figure><img src="image_5.png" /></figure>

2. EPPO/CABI. 1997. *Xanthomonas axonopodis* pv. phaseoli. EPPO Quar Pest 1997:1-6.

3. Chen NWG, Ruh M, Darrasse A, Foucher J, Briand M, Costa J, Studholme DJ, Jacques M-A. 2021. Common bacterial blight of bean: a model of seed transmission and pathological convergence. Mol Plant Pathol https://doi.org/10.1111/mpp.13067.

4. Mhedbi-Hajri N, Hajri A, Boureau T, Darrasse A, Durand K, Brin C, Saux MF-L, Manceau C, Poussier S, Pruvost O, Lemaire C, Jacques M-A. 2013. Evolutionary history of the plant pathogenic bacterium *Xanthomonas axonopodis*. PLoS One 8:e58474. https://doi.org/10.1371/journal.pone.0058474.

5. Darrasse A, Carrère S, Barbe V, Boureau T, Arrieta-Ortiz ML, Bonneau S, Briand M, Brin C, Cociancich S, Durand K, Fouteau S, Gagnevin L, Guérin F, Guy E, Indiana A, Koebnik R, Lauber E, Munoz A, Noël LD, Pieretti I, Poussier S, Pruvost O, Robène-Soustrade I, Rott P, Royer M, Serres-Giardi L, Szurek B, van Sluys M-A, Verdier V, Vernière C, Arlat M, Manceau C, Jacques M-A. 2013. Genome sequence of *Xanthomonas fuscans* subsp. *Fuscans* strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads. BMC Genomics 14:761. https://doi.org/10.1186/1471-2164-14-761.

6. Ruh M, Briand M, Bonneau S, Jacques M-A, Chen NWG. 2017. *Xanthomonas* adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors. BMC Genomics 18:670. https://doi.org/10.1186/s12864-017-4087-6.

7. Chen NWG, Serres-Giardi L, Ruh M, Briand M, Bonneau S, Darrasse A, Barbe V, Gagnevin L, Koebnik R, Jacques M-A. 2018. Horizontal gene transfer plays a major role in the pathological convergence of *Xanthomonas* lineages on common bean. BMC Genomics 19:606. https://doi.org/10.1186/s12864-018-4975-4.

8. Klotz LC, Zimm BH. 1972. Size of DNA determined by viscoelastic measurements: results on bacteriophages, *Bacillus subtilis* and *Escherichia coli*. J Mol Biol 72:779-800. https://doi.org/10.1016/0022-2836(72)90191-X.

9. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 27:722-736. https://doi.org/10.1101/gr.215087.116.

10. Hunt M, De Silva N, Otto TD, Parkhill J, Keane JA, Harris SR. 2015. Circulator: automated circularization of genome assemblies using long sequencing reads. Genome Biol 16:1-10. https://doi.org/10.1186/s13059-015-0849-0.

11. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963. https://doi.org/10.1371/journal.pone.0112963.

12. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI Prokaryotic Genome Annotation Pipeline. Nucleic Acids Res 44:6614-6624. https://doi.org/10.1093/nar/gkw569.

13. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043-1055. https://doi.org/10.1101/gr.186072.114.