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Ti plasmid pTiChry5 (KX388536; Shao et al. 2018), mannopine Ri plasmid pRi8196 (Weisberg et al. 2020; our own unpublished results), cucumopine Ri plasmid pRi2659 (NZ_CP019703.3; Valdes Franco et al. 2016; Tong et al. 2018), and mikimopine Ri plasmid pRi pRi1724 (NC_002575;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.