# Editorial: Population and comparative genomics of plant pathogenic bacteria

Sujan Timilsina, Erica M Goss, Ralf Koebnik, Neha Potnis, Jeffrey Jones

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Sujan Timilsina, Erica M Goss, Ralf Koebnik, Neha Potnis, Jeffrey Jones. Editorial: Population and comparative genomics of plant pathogenic bacteria. Frontiers in Microbiology, 2022, 13, 10.3389/fmicb.2022.1012034. [hal-03813550](https://hal.inrae.fr/hal-03813550)

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OPEN ACCESS

EDITED AND REVIEWED BY
Trevor Carlos Charles,
University of Waterloo, Canada

*CORRESPONDENCE
Sujan Timilsina
sujan.timilsina@ufl.edu
Erica M. Goss
emgoss@ufl.edu
Ralf Koebnik
ralf.koebnik@ird.fr
Neha Potnis
nzp0024@auburn.edu
Jeffrey B. Jones
jbjones@ufl.edu

SPECIALTY SECTION
This article was submitted to
Microbe and Virus Interactions with
Plants,
a section of the journal
Frontiers in Microbiology

RECEIVED 05 August 2022
ACCEPTED 17 August 2022
PUBLISHED 31 August 2022

CITATION
Timilsina S, Goss EM, Koebnik R, Potnis N and Jones JB (2022) Editorial: Population and comparative genomics of plant pathogenic bacteria.
*Front. Microbiol.* 13:1012034.
doi: 10.3389/fmicb.2022.1012034

COPYRIGHT
© 2022 Timilsina, Goss, Koebnik, Potnis and Jones. This is an open-access article distributed under the terms of the [Creative Commons Attribution License (CC BY)](https://creativecommons.org/licenses/by/4.0/). 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.

# Editorial: Population and comparative genomics of plant pathogenic bacteria

Sujan Timilsina<fnref n="1" />*, Erica M. Goss<fnref n="1" />,²*, Ralf Koebnik³*, Neha Potnis<fnref n="4" />* and Jeffrey B. Jones<fnref n="1" />*

KEYWORDS

taxonomy, evolution, ecology, effector, host specificity

Editorial on the Research Topic

Population and comparative genomics of plant pathogenic bacteria

Plant pathogenic bacteria are among the major challenges in crop production, amplified by their continuous and rapid evolution and resulting in the emergence of virulent, competitive, and elusive plant pathogens. To further understand these bacteria, their interactions with host plants, and their responses to disease management, comprehensive methods are necessary. In recent years, phytobacteriology research has used population and comparative studies to quantify the diversity of plant pathogenic bacteria and their virulence mechanisms, including the effectors secreted from different protein secretion systems, phytohormones, or toxins that mediate their interactions with the hosts and/or competing microbes in the phyllosphere. Studies comparing strains over time, within and among geographic regions, and across phylogenetic trees has provided insights into scales of variation and changes in pathogens over time and in response to selection pressures. In this series of articles, population and comparative genomics were used to investigate plant pathogens representing diverse bacterial genera including Gram-negative bacteria composed of facultative anaerobes (i.e., *Dickeya*, *Pantoea*, and *Pectobacterium*) as well as obligate aerobes (i.e., *Pseudomonas*, *Xanthomonas*, and *Xylella*) as well as the Gram-positive genus, *Clavibacter*.

Genomics approaches clarify taxonomy and
provide resources for comparative studies

Since ANI has been introduced as a means to reevaluate taxonomic assignments, genomics approaches have been used for comparative studies at different taxonomic levels. In this Focus Issue, reference pathotype strains of all described *Xanthomonas translucens* pathovars were sequenced using PacBio long-read technology and complete

<footnote n="1">¹Department of Plant Pathology, University of Florida, Gainesville, FL, United States, ²Emerging Pathogens Institute, University of Florida, Gainesville, FL, United States, ³Plant Health Institute of Montpellier, University of Montpellier, CIRAD, INRAE, Institut Agro, IRD, Montpellier, France,</footnote>

<footnote n="4">⁴Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, United States</footnote>
genome sequences were assembled, which gave robust proof for three major clades in this species, each with distinct host preferences (Goettelmann et al.). Moreover, type III effector (T3E) profiling revealed 21 TAL effector classes and 29 additional type III effectors, some of them displaying clade or strain specificity. Further investigation of these genes could help to identify genes that are critically involved in pathogenicity and/or host adaptation, setting the grounds for the development of new resistant cultivars.

Expansion of whole genome sequencing of species within the same genus has offered insight into host specialization and into phylogenetic relationships within and between species within genera. Prior to the identification of the *Xylella* species, *X. taiwanensis*, characterization was restricted to different subspecies of *Xylella fastidiosa*, but lacked analyses at the genus level due to the lack of genome sequences for multiple species (Weng et al.). As a result of having the *X. taiwanensis* genome available as an outgroup in phylogenetic comparisons of *X. fastidiosa* subspecies, there was strong support for *X. fastidiosa* subspecies *pauca* being the basal lineage of *X. fastidiosa* and for *Xylella* being derived from the paraphyletic genus *Xanthomonas*.

Genome sequencing is also revealing previously unrecognized diversity, as is illustrated by the characterization of *Xanthomonas* causing bacterial leaf canker on water spinach (*Ipomoea aquatic*) by Hu et al. a survey of the disease in Dongguan City, China, where water spinach is grown in small plots, found the *Xanthomonas* causal agent along with *Pantoea*, which had a synergistic effect on disease. Whole genome sequencing using short and long-read technologies of a representative *Xanthomonas* strain showed >97% ANI to *X. perforans*, which is a classic pathogen of tomato, but was more recently also identified on pepper and eucalyptus. The ANI below 98% indicates that this is an atypical *X. perforans*. The catalog of effectors in this strain will advance efforts to understand the relationships between effector repertoires and host range in *Xanthomonas*.

Soft rot pectobacteria are devastating plant pathogens with a global distribution and a broad host range. To close the shortage for genome data of *Pectobacterium aroidearum*, the causal agent of soft rot on the popular houseplant *Syngonium podophyllum*. Xu et al. present the complete genome sequence of strain L6. About 10% of the predicted genes were potentially related to pathogenesis using the Virulence Factors of Pathogenic Bacteria database, including genes related to toxins, plant cell-wall degrading enzymes, and bacterial secretion systems. This study provides novel information for the discovery of potential pathogenicity factors and the development of more effective strategies against this pathogen.

Historically, less work has been done on Gram-positive plant pathogens. Among them, *Clavibacter michiganensis*, a member of the Actinobacteria, is a causal agent of bacterial canker of tomato. Oh et al. provide the complete genome sequence of the type strain of *C. michiganensis*. When compared

with other strains, it turned out that the chromosomal DNA sequences were almost identical, whereas its plasmids were found to carry distinct gene content among *C. michiganensis* strains. The genome information of the type strain LMG 7333T will help in understanding the genetic diversity of *Clavibacter*'s plasmid complements and how they may relate to virulence on host plants.

## Using genomics to investigate variation and evolution of type III effector repertoires

Traditionally, T3Es have been thought to be important host-range determinants. Such effectors have been characterized as avirulence factors that define race structure in plant pathogens. Understanding the race structure in pathogen population and factors associated with race specificity are of importance when assessing efficacy and durability of host resistance. Rosenthal et al. used a comparative genomics approach on genome sequences of different races of *Xanthomonas hortorum* pv. *vitians* and identified two T3Es, XopAQ and XopAF2, as potential mediators of gene-for-gene interactions between race 1 and 3 strains and wild lettuce. The close examination of the neighborhood regions of these effectors indicated proximity of *xopAF2* to prophage sequences, thus, likelihood of gain or loss of this effector in the pathogen population via phage-mediated transfer, in response to host selection pressure.

Shah et al. analyzed T3Es of *X. translucens* strains that cause bacterial leaf streak in small grain cereals, which include three pathovars: *cerealis*, *translucens*, and *undulosa*. Four effectors, XopAJ, XopAL1, XopE3, and XopM were found in all analyzed *X. translucens* pv. *translucens* strains, but not in members of the other two pathovars, *cerealis* and *undulosa*. Whether these effectors act as host specificity factors, as hypothesized by the authors, remains to be experimentally proven. Interesting differences were also reported in the TAL effectors, which activate the expression of host genes, including two novel dipeptides (called RVDs for Repeat Variable Diresidues) that contact the bound DNA and provide sequence specificity to these transcription activators (Shah et al.).

The *Pseudomonas syringae* species complex represents a classic system to address convergence of diverse strains belonging to distantly related phylogroups onto a common host. Ruinelli et al. conducted association analyses on genomes of *P. syringae* strains associated with diseases on *Prunus* as well as those from other hosts and from non-agricultural environments. They identified horizontal gene transfer (HGT) as a predominant evolutionary force that has mediated gain/loss of genes such as the T3E, *hopAY*, among strains belonging to diverse phylogroups in a relatively short evolutionary timescale. Such independent acquisition events of genes important for successful pathogen colonization, but also loss of specific genes
or their inactivation, have shaped what we define as host ranges
in plant pathogenic bacteria.

Heritable changes in bacteria, including effectors, often
occur during conjugation or horizontal gene transfer of mobile
genetic elements. Integrative Conjugative Elements (ICEs) are
among the mobile replicons that combine the characteristics
of plasmids with an ability to carry genes. ICEs are also
known to carry cargo regions that can drastically change
bacterial phenotypes, including their interactions with hosts.
Baltrus et al. demonstrated that a T3E ICE from P. syringae
pv. maculicola—PmaICE-DQ—can transfer to other strains
via conjugation, resulting in new phenotypes of the recipient.
Moreover, another ICE carrying multiple effector genes—
PmaICE-AOAB—was found to be adjacent to the PmaICE-DQ.
The study demonstrated the transfer of ICEs regions to naive
strains. The presence of effectors within ICEs enables quick loss
and gain of effectors, thus providing additional flexibility to the
pathogen under selection pressure.

Whole genome sequencing can capture shifts in effector
content in pathogens over time. Bernal et al. screened hundreds
of X. perforans strains from tomato fields in the Midwestern
USA over a period of 4 years, representing multiple varieties,
seed producers, and growers. They found that the X. perforans
population shifted from race T3 to race T4, mostly caused by
a SNP in *avrXv3* that inactivated this effector gene by an early
stop codon. While host selection is a likely explanation for the
loss of effector function, few tomato varieties with resistance to
X. perforans T3 made up <7% of the acreage in this geographic
region. Analysis of whole genome sequences showed that most
strains were nearly identical to each other. The authors discuss
the possible mechanisms of persistence of this clonal population
from year to year, which is critical knowledge needed to manage
bacterial spot of tomato.

Beyond type III effectors

A recent paradigm to explain genetic basis of host adaptation
or specificity has suggested involvement of multiple genetic
determinants (not limited to T3Es), each contributing small or
large effects toward adaptation of pathogen to a specific host
or tissue type. Liyanapathiranage et al. conducted large-scale
comparative and evolutionary analyses to test the hypothesis
that specific gain or loss events of multiple type VI secretion
systems are not random, but occurred as independent events
during the adaptation of Xanthomonas species to specific hosts.
They observed that specific cases of loss or gain events happened
in certain clades. Whether these secretion systems and their
effectors contribute to an adaptation to vascular or non-vascular
lifestyles or provide preference toward dicots or monocots
remains to be addressed experimentally.

Exhaustive genomics resources stimulate comparative
studies aiming at cataloging known and discovering new traits,

thus better understanding how they are linked to the ecology of
the pathogen. Bacterial toxin-antitoxin (TA) systems, consisting
of two or more adjacent genes, are one of these traits of interest,
which are implicated in genome maintenance, antibiotics
persistence, phage defense, and virulence. Kandel et al. used
bioinformatics tools to screen the genomes of hundreds of P.
syringae strains representing the genetic and lifestyle diversity
of the P. syringae species complex for TA systems. They show
that P. syringae strains encode on average 15 TA systems
per genome, which belong to 26 different families and are
thought to target diverse cellular functions. Further functional
characterization of the predicted TA systems could reveal how
these widely prevalent gene modules may affect P. syringae
ecology, virulence, and disease management practices.

Another approach to identify previously unknown virulence
factors is using genome-wide association studies (GWAS).
Agarwal et al. applied a GWAS approach on Pantoea ananatis
that causes center rot of onion. This bacterium does not
utilize the type II or type III secretion system, but possesses
a biosynthetic gene cluster—HiVir/PASVIL—for production
of a phosphonate secondary metabolite and also another
gene cluster—alt—that confers tolerance to thiosulfinates.
The pangenome association analyses indicated that although
HGT events, including transfer of PASVIL cluster, may have
contributed toward diversification and niche adaptation of P.
ananatis, other factors (e.g. tyrosine kinase, N-acetylmuramoyl-
L-alanine amidase, and HTH-type transcriptional regulator) are
likely also involved in the pathogenicity of P. ananatis on onion.

High-throughput sequencing offers incredible opportunities
for identifying genes involved in various microbial processes
including fitness, virulence, and antibiotic resistance.
Transposon mutagenesis was useful in identifying essential
genes for growth on media as well as in planta. Random
barcode transposon-site sequencing (RB-TnSeq) was used
in comparing three *Dickeya* strains from two species to
identify genes involved in fitness as measured by bacterial
growth in potato tubers and growth in minimal and complex
media (Helmann et al.). Notably, many of the metabolic
traits that were required for growth in minimal medium were
also important for efficient growth in potato tubers, such
as amino acid, carbohydrate, and nucleotide biosynthesis.
Moreover, growth of all three *Dickeya* species required the
pectate degradation gene, *kduD*, whereas disruption in three
putative DNA-binding proteins had different effects at the
strain level.

The last two decades have witnessed adoption of
comparative and population genomics methods being
integrated into addressing several questions related to
pathogen biology, epidemiology as well as translational research
to guide disease management practices based on changes in
pathogen population structure. The articles published in this
Research Focus show such translational aspects, informing
meaningful hypotheses that demand further functional
genomics studies. Thanks to the plant pathologists who have taken initiative to sequence several hundreds, if not thousands, of genomes of their favorite bacterial genus, we have rich databases with genotypic and phenotypic data. The articles published in this issue also address this important aspect of the need to explore already available data in genome databases, rather than focusing efforts on sequencing more isolates.

## Author contributions

ST, EG, RK, NP, and JJ contributed to the manuscript preparation and finalizing. All authors contributed to the article and approved the submitted version.

## Conflict of interest

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.

## Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.