Abstract
We report here the complete genome sequence of the Rhizobium rhizogenes ( formerly Agrobacterium rhizogenes) strain LBA9402 (NCPPB1855rifR), a pathogenic strain causing hairy root disease. In order to assemble a complete genome we obtained short-reads from Illumina sequencing as well as long-reads from Oxford Nanopore Technology sequencing. The genome consists of a 3,958,212 bp chromosome, a 2,005,144 bp chromid (secondary chromosome) and a 252,168 bp Ri plasmid (pRi1855), respectively. The primary chromosome was very similar to that of the avirulent biocontrol strain K84, but the chromid showed a 724 kbp deletion accompanied by a large 1.8 Mbp inversion revealing the dynamic nature of these secondary chromosomes. The sequence of the agropine Ri plasmid was compared to other types of Ri and Ti plasmids. Thus we identified the genes responsible for agropine catabolism, but also a unique segment adjacent to the TL-region that has the signature of a new opine catabolic gene cluster including the three genes that together encode an opine dehydrogenase. Our sequence analysis also revealed a novel gene at the very right end of the TL-DNA, which is unique for the agropine Ri plasmid. The protein encoded by this gene was most related to the succinamopine synthases of chrysopine and agropine Ti plasmids and thus may be involved in synthesis of the unknown opine that can be degraded by the adjacent catabolic cluster. The available sequence will facilitate the use of R. rhizogenes and especially LBA9402 in both the laboratory and for biotechnological purposes.
Key words: Agrobacterium rhizogenes; chromid; opine; ornithine cyclodeaminase; Ri-plasmid; succinamopine synthase
Introduction
Hairy root, a neoplastic plant disease with a wide host range, is characterized by the formation of adventitious roots from infected wound sites (Riker 1930). It was originally encountered as a problem in tree nurseries, but nowadays the disease is also increasingly causing problems in the greenhouse by inducing extensive root mats, thereby reducing the harvest of cucumbers and tomatoes (Weller et al . 2000). The causal agent, a bacterium that was for long calledAgrobacterium rhizogenes, contains a large, about 200 kbp root-inducing (Ri) plasmid, which contains the essential virulence determinants (White and Nester 1980). The molecular mechanism by which hairy root is induced is similar to that which is used by the related bacterium Agrobacterium tumefaciens to induce crown gall tumors in plants. During infection, part of the Ri plasmid (the T-region) is transferred to plant cells and integrated into the plant genome (Bevan and Chilton 1982). Expression of rol -genes located in the T-DNA leads to the transformation of normal cells into tumor cells that develop into roots that can grow in in vitro culture in the absence of added plant growth regulators (Jouanin et al. 1987). In the cells of hairy roots unusual compounds called opines are produced, which are specific condensates of amino acids and keto acids or sugars (Petit et al. 1983). These opines, which are formed by enzymes encoded by the T-DNA, support the growth of the pathogen, which contains the catabolic genes usually in a region adjacent to the T-region on the Ri plasmid (Dessaux et al. 1993). On the basis of the specific opines formed and degraded, agropine, cucumopine, mannopine, and mikimopine Ri plasmids are nowadays distinguished.
Like A. tumefaciens, A. rhizogenes has been disarmed by deleting the T-DNA genes in order to convert it into a vector useful for plant genome engineering (Collier et al . 2018). Also, the bacterium as such is used for biotechnological research and application; in research for instance for gene function and gene expression analysis in roots (Ron et al. 2014), in industry to obtain roots that can be grown in bioreactors for the production of secondary metabolites (Mehrotra et al. 2015).
It has become apparent over the years that in nature various bacteria of the Rhizobiaceae family may cause hairy root or crown gall. On the basis of their physiological properties three different groups (biovars 1-3) were distinguished already long ago (Kerr and Panagopoulos 1977). The species name Rhizobium rhizogenes is now commonly used for the bacteria belonging to biovar 2. R. rhizogenes strains have two megabase DNA circles, a chromosome and a plasmid-derived megacircle, sometimes called a chromid (Jumas-Bilak et al. 1998; Harrison et al. 2010). Draft genome sequences of several strains are available on NCBI, and one draft genome sequence has been published forR. rhizogenes strain ATCC15834 consisting of 43 scaffolds (Kajala et al. 2014). However, up to now only one complete genome sequence is available for R. rhizogenes , that of the avirulent agrocin-producing biocontrol agent Kerr 84 (Slater et al. 2009). Here we present a second complete genomic sequence of R. rhizogenes, that of the hairy root inducing strain LBA9402. This strain is a rifampicin-resistant derivative of wild-type strain NCPPB1855, which is one of the most widely used laboratory strains (Desmet et al. 2020). By comparing the chromosome and chromid of LBA9402 with those of strain K84 we found that the chromosome was very similar, but that the chromid showed large differences due to a large 724 kbb deletion accompanied by a large inversion, underscoring the dynamic nature of the chromid. Analysis of the sequence of the agropine Ri plasmid of LBA9402 revealed that this had a few unique areas including one that we predict encodes a new opine catabolic cluster, including the three genes characteristic for defining an opine dehydrogenase. A candidate gene for a novel opine synthase was identified at the very right end of the TL-DNA.
Materials and methods