3.3 General properties of the Ri plasmids
The pRi1855 plasmid comprises 252,168 bp. It has an approximately 4%
lower GC content than the rest of the genome. In total, 236 protein
coding sequences were identified with an average size of 898 bp
(supplementary figure S5). We compared the agropine Ri plasmid pRi1855
sequence to publicly available Ri and Ti plasmid sequences: octopine Ti
plasmid pTiAch5 (CP007228; Henkel et al. 2014; Huang et al. 2015),
nopaline Ti plasmid pTiC58 (AE007871; Goodner et al. 2001; Wood et al.
2001), succinamopine Ti plasmid pTiEU6 (KX388535; Shao et al. 2019),
agropine Ti plasmid pTiBo542 (DQ058764; Oger et al. 2001), chrysopine Ti
plasmid pTiChry5 (KX388536; Shao et al. 2018), mannopine Ri plasmid
pRi8196 (Weisberg et al. 2020; our own unpublished results), cucumopine
Ri plasmid pRi2659
(NZ_CP019703.3; Valdes Franco et
al. 2016; Tong et al. 2018), and mikimopine Ri plasmid pRi pRi1724
(NC_002575; Moriguchi et al.
2001). The conservation of the different areas in these plasmids is
visualized in figure 2 and we shall discuss these in the following
parts.
In previous studies dealing with the closely related agropine Ri plasmid
pRiA4, the replication (repABC ) and conjugative transfer
(tra, trb ) genes in the agropine Ri plasmid were already
described (Nishiguchi et al. 1987; Wetzel et al. 2015) and are very
similar to those in other Ti and Ri plasmids.
The agropine Ri plasmid has two T-regions, one of which, the TL-region
contains the rol -genes that are necessary and sufficient for the
formation of hairy roots. Other Ri plasmids have only one T-region with
very similar genes (fig. 3; Otten 2018). In the agropine Ri plasmid,
however, a copy of IS630 has inserted between orf3 andorf8 . At the very left end in the agropine TL-region and the
mannopine T-region a gene for agrocinopine synthase is present, but only
remnants of these genes are still present in the cucumopine and
mikimopine Ri plasmids. At the very right end of the T-region one (in
the cucumopine and mikimopine Ri plasmids) or two (in the mannopine Ri
plasmids) non-conserved genes are present. These encode the cucumopine
(Valdes Franco et al. 2016) and mikimopine synthase (Moriguchi et al.
2001), respectively, and the two genes necessary for mannopine synthesis
in the mannopine Ri plasmid (fig. 3 ). The TL-region of the agropine Ri
plasmid pRiA4 was previously sequenced (Slightom et al. 1986) and this
revealed at the right end the presence of orf15 /rolD and
three smaller orfs . In our pRi1855 sequence we find besidesorf15/rolD only one larger orf , hereinafter calledorf16 . The function of orf16 is unknown, but may encode an
unknown opine synthase as will be discussed in a next paragraph. The
agropine Ri plasmid has besides the conserved T-region (TL-region) an
additional T-region (TR-region) containing aux -genes involved in
biosynthesis of the auxin indole acetic acid (Offringa et al .
1986) and the genes mas1 , mas2 and ags for agropine
biosynthesis (Bouchez and Tourneur 1991).
The virulence region of pRi1855 responsible for transfer of the T-DNA
into plant cells contains the essential virulence genesvirA-virD5 , but although virE3 is present the virE1and virE2 genes are missing and replaced by a new orf with
some similarity to nopaline pTi virF . The sequence of the
cucumopine and mikimopine Ri plasmids in this area is almost identical
suggesting that the deletion of virE2 occurred once before the
divergence of these Ri plasmids. The virE2 gene is functionally
replaced by a gene called GALLS as reported before for pRiA4
(Hodges et al . 2004). TheGALLS gene was previously also identified in the
cucumopine and mikimopine Ri
plasmids by Southern analysis, but is not present in the mannopine Ri
plasmid, which still carries the virE2 gene (Hodges et al .
2004). Together with a tzs gene, which encodes an enzyme that
catalyzes the synthesis of the cytokinin zeatin riboside 5’-phosphate
(Krall et al . 2002), GALLS is located outside thevir -region, about 65 kbp clockwise from the virE3 gene,
near opine catabolic genes in the
cucumopine and mikimopine Ri plasmids. We could now identify and locate
the GALLS gene in the pRi1855 sequence at a completely different
location next to the traG gene almost 90 kbp counterclockwise
from the virE3 gene. Besides the core set of vir genes
mentioned above, the vir region of the agropine Ri plasmid
contains next to the virA gene the tzs gene, virK ,
a second nopaline Ti-like virF gene, and finally virH1 ,virH2 . It resembles in this respect the vir -region of the
nopaline Ti plasmid and like in the nopaline Ti plasmids virJ is
absent. Other types of Ri plasmids have similar vir -regions, but
a virH2 gene is absent from the cucumopine and mikimopine Ri
plasmids, and as mentioned above a tzs gene is present, but
located in an entirely different area of the plasmid.
3.4 Agropine and
agrocinopine catabolism genes
Hairy roots formed by agropine
strains contain agropine, agropinic acid, mannopinic acid and mannopine
(Petit et al. 1983). The agropine
Ri plasmid enables host strains to degrade agropine. R.
rhizogenes strains such as A4 , but not 1855 or HRI contain a second,
catabolic plasmid with genes for catabolism of the other three mannityl
opines (Petit et al. 1983). We have now identified the genes for
agropine transport and catabolism in pRi1855, which are located in a
segment of the plasmid adjacent to the TR-region (fig. 2). This region
contains genes with high similarity to the genes described by Kim and
Farrand (1996) on the octopine Ti plasmid involved in agropine uptake
and degradation. These genes encode an agropine permease and also
comprize agcA for the delactonase converting agropine into
mannopine, mocC for oxidizing mannopine into deoxyfructosyl
glutamine and mocDE determining the deconjugase liberating an
amino acid and a phosphorylated sugar. In the octopine Ti plasmid the
genes mocA and mocB encode enzymes with weak homology to
glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydratase
(Kim and Farrand 1996). These are probably involved in further
catabolism of the release phosphorylated sugar. However, while an intact
homolog of mocA was present in pRi1855, as well as a homolog ofmocC , in between only a truncated remnant of a gene homologous tomocB was present due to a deletion of more than 1 kbp. Regulators
closely related to mocS and mocR are present in an
identical position in front of mocA and between mocC andmocD . Our detection of amocD gene in pRi1855 was remarkable as such gene was thought to
be absent from the agropine Ri plasmid (Baek et al. 2005). The agropine
Ri plasmid has adjacent to the left end of the TL-region in pRi1855 a
set of acc genes for agrocinopine catabolism (fig. 2), which
matches the presence of an acs gene for the biosynthesis of
agrocinopine in the TL-region. These genes are also present in the
mannopine Ri plasmid, but absent from the cucumopine and mikimopine Ri
plasmids.