Science - USA (2020-06-05)

RESEARCH ARTICLE

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PLASMID EVOLUTION

Unexpected conservation and global transmission of

agrobacterial virulence plasmids

Alexandra J. Weisberg^1 , Edward W. Davis II1,2, Javier Tabima^1 , Michael S. Belcher^1 , Marilyn Miller^1 ,
Chih-Horng Kuo^3 , Joyce E. Loper1,2,4, Niklaus J. Grünwald^4 , Melodie L. Putnam^1 , Jeff H. Chang1,2,5*

The accelerated evolution and spread of pathogens are threats to host species. Agrobacteria require
an oncogenic Ti or Ri plasmid to transfer genes into plants and cause disease. We developed
a strategy to characterize virulence plasmids and applied it to analyze hundreds of strains collected
between 1927 and 2017, on six continents and from more than 50 host species. In consideration of prior
evidence for prolific recombination, it was surprising that oncogenic plasmids are descended from a
few conserved lineages. Characterization of a hierarchy of features that promote or constrain plasticity
allowed inference of the evolutionary history across the plasmid lineages. We uncovered epidemiological
patterns that highlight the importance of plasmid transmission in pathogen diversification as well as in
long-term persistence and the global spread of disease.

A

gricultural ecosystems promote the rap-
id evolution and diversification of path-
ogens ( 1 ). These ecosystems increase
pathogen population sizes, lower bar-
riers for transmission, and increase
opportunities for horizontal exchange of viru-
lence genes. Understanding the genetic basis
for how pathogens emerge and diversify in
agricultural ecosystems is foundational for
determining their spread and assessing risks.
Such knowledge is critical to policies for im-
proving plant health and preparing against
disease outbreaks to increase global food
security ( 2 ).
Plasmids that confer virulence and antimi-
crobial resistance are evolutionary drivers of
pathogenic bacteria that affect plant, human,
and animal health ( 3 – 6 ). Some plasmids can
mediate conjugation and transmit horizon-
tally within and across species to diversify
pathogen populations. The development of
strategies to infer horizontal transfer of plas-
mids is crucial for accurately assessing dis-
ease outbreaks and instituting measures to
limit risks from disease. However, it is dif-
ficult to trace dissemination of plasmids and
integrate findings with analyses of chromo-
somes, because plasmids can be horizontally
transferred and hence can defy inference of
evolutionary histories ( 7 ). In addition, plas-
mids tend to have few conserved regions, high
numbers of repeated sequences, high rates
of gene exchange, and many structural vari-

ants. Even core genes such as those associated

with replication or mobility are susceptible

to recombination.

Agrobacteria are a diverse, polyphyletic group,

and its members are common in soils and

on many species of plants. These bacteria have

multipartite genomes with two chromosomes

and nonvirulence plasmids, which are extreme-

ly diverse among agrobacteria ( 8 ). Pathogenic

strains additionally carry conjugative onco-

genic tumor-inducing (Ti) or root-inducing

(Ri) plasmids and can infect plants to cause

crown gall or hairy root diseases, respectively

( 9 ). These diseases are incurable and persist

forthedurationofthelivesofinfectedplants,

which continually releaseopines,metabolites

that are central to the ecology and epidemi-

ology of agrobacteria.

Oncogenic plasmids are exceptionally well

characterized because of their applications

in plant biotechnology and hence are models

for understanding the impact of plasmids on

disease ecology ( 10 , 11 ). Core to oncogenic

plasmids arerepABCreplication genes,tra

andtrbinterbacterial conjugation genes, and

virgenes. Vir proteins are necessary to process

and escort a region, the transfer DNA (T-DNA),

of oncogenic plasmids into host cells, where

it recombines into the genome to genetically

transform host plants. T-DNAs include on-

cogenes that reprogram transformed cells to

proliferate. One oncogene present on T-DNAs

in all oncogenic plasmids istms1(aux1/iaaM),

which is involved in the synthesis of auxins, a

class of plant growth–promoting hormones

( 12 ). T-DNAs also include genes responsible

for the synthesis of opines. More than 20 chem-

ically diverse opines have been identified among

oncogenic plasmids ( 13 ). Opines are nutrients

for the pathogen and act as signals that trigger

replication and interbacterial conjugation of

oncogenic plasmids. Genes necessary for uptake

and catabolism of opines are spatially sepa-

rated from cognate synthesis genes and are

located outside of T-DNAs. This arrangement

led to the hypothesis that instigating pathogens

are privileged in accessing the opines ( 14 ).

Beyond the core set of genes, oncogenic plas-

mids vary extensively in composition and struc-

ture ( 15 ). This diversity is a major challenge,

best expressed by the sentiment that it is

practically impossible to reconstruct the evo-

lution of agrobacteria and oncogenic plasmids,

even with the availability of an extraordinarily

large genomic dataset ( 16 ). Here, we overcame

this challenge and analyzed genomic data from

hundreds of strains, and applied the results to

infer the evolutionary history and transmis-

sion of oncogenic plasmids.

Chromosomal ancestry

A robust phylogeny of bacteria is an essential

foundation for understanding the evolution

and transmission of the plasmids that they

host. Hence, it was critical to reconcile contro-

versies over the complex evolutionary rela-

tionships among agrobacteria (see data S1

for synonymous terms) ( 17 , 18 ). Sequenced

strains were reclassified in the context of a

dataset of nearly 1500 additional strains that

represent families within the order Rhizobiales

(data S1 and S2). Combined phylogenetic and

genomic analyses indicated that agrobacte-

ria,Rhizobium,Allorhizobium,Neorhizobium,

Ensifer/Sinorhizobium,Pararhizobium, and

Shinellaare related at a level of a genus, re-

ferred to as the agrobacteria-rhizobia complex

(ARC; fig. S1 and data S3). Within the ARC,

groups traditionally known asAgrobacterium

tumefaciens,Agrobacterium rhizogenes, and

Agrobacterium vitisare called biovar (BV) 1,

BV2, and BV3, respectively ( 19 ). BV1 was prev-

iously subclassified into genomospecies ( 20 ).

Whole-genome analysis suggested that most

genomospecies (excepting G7 and G8, which

each represent three groups) are commensu-

rate with species-level groups (data S4). BV2 is

homogeneous and represents a single species-

level group. BV2 is sister to a newly defined

clade of pathogenic agrobacteria (BV2-like).

BV3 forms multiple species-level groups.

A single lineage of agrobacteria was previ-

ously estimated to have diverged from rhizobia

between ~250 million and ~150 million years

ago ( 21 , 22 ). Considering the new interpreta-

tion of their phylogeny, we reevaluated esti-

mates of divergence times (Fig. 1, fig. S2, and

data S5). Lineages of agrobacteria emerged

independently, at different times in the his-

tory of the ARC, and are more closely related

to lineages of rhizobia than to each other. BV1

emerged 48 ± 10 million years ago, and its

clade has substructure with long branch lengths,

which suggests that species-level groups di-

verged early in its history. The BV2 lineage is

RESEARCH

Weisberget al.,Science 368 , eaba5256 (2020) 5 June 2020 1of8

(^1) Department of Botany and Plant Pathology, Oregon State
University, Corvallis, OR 97331, USA.^2 Molecular and Cellular
Biology Program, Oregon State University, Corvallis, OR 97331,
USA.^3 Institute of Plant and Microbial Biology, Academia Sinica,
Taipei 11529, Taiwan.^4 Horticultural Crops Research Laboratory,
USDA Agricultural Research Service, Corvallis, OR 97331, USA.
(^5) Center for Genome Research and Biocomputing (CGRB),
Oregon State University, Corvallis, OR 97331, USA.
*Corresponding author. Email: [email protected]