expression and promoting virulence ( 29 , 30 ).
The proto-oncogenic plasmid was suggested
to have originated as a catabolic plasmid. The
largest set of genes inferred to have a shared
evolutionary history includes 52 genes (36% of
those analyzed), of which 10 are implicated in
the uptake of nutrients or metabolism and
another 18 have unknown functions (data S8
and figs. S22 and S23). This set also includes
repABC. We propose that the proto-oncogenic
plasmid led to either the Ti or Ri class of plas-
mid and recombined with a separaterepABC
plasmid to yield the other class.
The next innovation was the acquisition of
oncogenes within T-DNA border sequences.
Every one of the sequenced Ti plasmids has
at least one T-DNA that circumscribestms1
andtms2(aux2/iaaH), and all Ri plasmids
have at least one T-DNA withtms1(Fig. 4B
and data S6). Thetms2gene cooperates withtms1in auxin biosynthesis ( 12 ). Data are con-
sistent with the ancestors of Ti and Ri plasmids
independently acquiringtms1from different
nonagrobacterial sources (fig. S24). Moreover,
even thoughtms1andtms2are a functional
module and are consistently linked in T-DNAs
of Ti plasmids,tms2was potentially acquired
separately fromtms1by an ancestral Ti plas-
mid (fig. S24C). The evidence suggests that in
Ti plasmids,iptwas derived from a duplica-
tion oftzsand the paralog was incorporated
within the T-DNA region (fig. S21).
An unexpectedly small number of events are
sufficient to relate plasmid lineages (Fig. 5 and
figs. S3, S4, and S12 to S24). Single-copy genes
of oncogenic plasmids were grouped into just
18 sets inferred to have similar evolutionary
histories (fig. S23). Five sets were sufficient
to represent 80% of the genes analyzed. In
contrast,traandtrbgenes are distributed
across 50% of the sets. Type IV.c Ti plasmids
are cointegrates of a type I Ti plasmid and a
Ri plasmid. The two sequenced type IV.c Ti
plasmids have edges to every type I.a node,
are nearly twice the size of pTiC58, and have
a secondtralocus located between twovir
loci (Fig. 1A and fig. S10). In a modification
to a previously proposed model, we suggest
that all subtypes of the type IV plasmids are
derived from the same lineage of the co-
integrated ancestor and that type IV.a and
type IV.b Ti plasmids are streamlined variants
( 15 ). The type VI Ti plasmid is also likely a
cointegrate. This plasmid is novel in that it
horizontally acquired, via T-DNA invasion, a
second set oftms1andtms2, which is unlike
the first set found in all types of Ti plasmids
(fig. S24). Type II Ti plasmids are hypothesized
to have recently emerged from a rearrange-
ment event within the type III Ti plasmids.
Type V Ti plasmids are more closely related to
Ri plasmids and are the most genetically and
structurally distinct of the Ti plasmids. The
multiple changes and mosaic structure make
its evolutionary history difficult to interpret.
Its unique structure is predicted to limit op-
portunities for productive recombination events
with other oncogenic plasmids. Although Ti
and Ri classes are very distantly related, they
can co-reside in cells and exchange regions.
Strain Di1411, for example, carries a type I.a
Ti plasmid and a type II Ri plasmid.Modeling disease spread
We next used this evolutionary framework
to identify epidemiological patterns among
strains collected across the world and span-
ning nearly a century of collection. The frame-
work allowed us to analyze strains and plasmids
as independently transmitted entities to ac-
curately model disease spread. An additional
66 genome sequences of hierarchically sam-
pled strains were used to guide grouping of the
agrobacterial strains and oncogenic plasmidsWeisberget al.,Science 368 , eaba5256 (2020) 5 June 2020 5of8
Fig. 4. Diversity of virulence loci of oncogenic plasmids.(A) Compacted de Bruijn graph constructed
from componentk-mers of sequencedvirloci. Traces are colored according to plasmid type and subtype. Key
variations andvirgene regions are indicated; IS, insertion sequence. Large regions that interrupt thevir
loci were not included. Panels at bottom show the traces of pTiC58 and pRi2659, which exemplify the two
main traces. Traces in the inset are colored to differentiate between gene loci. (B) Gene synteny graph of 213
T-DNAs. The main Ti and Ri paths are labeled with dotted arrows. Paths of the three T-DNAs that form the
chimera of the type I.a Ti plasmids are indicated by numbers in red. The two left borders of the chimeric
T-DNA-1 of the type III Ti plasmids are labeled with numbers in green. Nodes are colored according to
category (dark gray, left border; light gray, right border; blue, gene; yellow, insertion sequence). Thetms2*
gene is an independently acquired homolog that failed to cluster withtms2. Edges are colored according
to plasmid type. Line weight is normalized to the proportion of plasmids within a given type. (C)Circosplot
relating oncogenic plasmids to synthesized opine variants (S-A, conjugates of sugars and amino acids;
K-A, conjugates of keto acids and amino acids; S-S, conjugates of sugars). See figs. S11 and S13 for larger
variations of (A) and (B).
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