Diversification of plasmids in a genus of pathogenic and nitrogen-fixing bacteria.

Members of the agrobacteria-rhizobia complex (ARC) have multiple and diverse plasmids. The extent to which these plasmids are shared and the consequences of their interactions are not well understood. We extracted over 4000 plasmid sequences from 1251 genome sequences and constructed a network to reveal interactions that have shaped the evolutionary histories of oncogenic virulence plasmids. One newly discovered type of oncogenic plasmid is a mosaic with three incomplete, but complementary and partially redundant virulence loci. Some types of oncogenic plasmids recombined with accessory plasmids or acquired large regions not known to be associated with pathogenicity. We also identified two classes of partial virulence plasmids. One class is potentially capable of transforming plants, but not inciting disease symptoms. Another class is inferred to be incomplete and non-functional but can be found as coresidents of the same strain and together are predicted to confer pathogenicity. The modularity and capacity for some plasmids to be transmitted broadly allow them to diversify, convergently evolve adaptive plasmids and shape the evolution of genomes across much of the ARC. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

The Agrobacterium rhizogenes GALLS gene encodes two secreted proteins required for genetic transformation of plants.

Agrobacterium tumefaciens and Agrobacterium rhizogenes are related pathogens that cause crown gall and hairy root diseases, which result from integration and expression of bacterial genes in the plant genome. Single-stranded DNA (T strands) and virulence proteins are translocated into plant cells by a type IV secretion system. VirD2 nicks a specific DNA sequence, attaches to the 5' end, and pilots the DNA into plant cells. A. tumefaciens translocates single-stranded DNA-binding protein VirE2 into plant cells where it likely binds T strands and may aid in targeting them into the nucleus. Although some A. rhizogenes strains lack VirE2, they transfer T strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant for tumor formation. Unlike VirE2, full-length GALLS (GALLS-FL) contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. GALLS-FL and VirE2 contain nuclear localization signals (NLS) and secretion signals. Mutations in any of these domains abolish the ability of the GALLS gene to substitute for virE2. Here, we show that the GALLS gene encodes two proteins from one open reading frame: GALLS-FL and a protein comprised of the C-terminal domain, which initiates at an internal in-frame start codon. On some hosts, both GALLS proteins were required to substitute for VirE2. GALLS-FL tagged with yellow fluorescent protein localized to the nucleus of tobacco cells in an NLS-dependent manner. In plant cells, the GALLS proteins interacted with themselves, VirD2, and each other. VirD2 interacted with GALLS-FL and localized inside the nucleus, where its predicted helicase activity may pull T strands into the nucleus.

VirE1-Mediated Resistance to Crown Gall in Transgenic Arabidopsis thaliana.

ABSTRACT Crown gall disease, caused by Agrobacterium tumefaciens, remains a serious agricultural problem despite current biocontrol methods. Agrobacterium tumefaciens transfers single-stranded DNA (T-strands) into plant cells along with several virulence proteins, including a single-stranded DNA-binding protein (VirE2). In plant cells, T-strands are protected from nucleases and targeted to the nucleus by VirE2, which is essential for efficient transmission (transfer and integration) of T-strands. VirE1 is the secretory chaperone for VirE2; it prevents VirE2 from forming aggregates and from binding the T-strands in bacterial cells. Therefore, we hypothesized that sufficient quantities of VirE1 expressed in plant cells might block T-DNA transmission by preventing VirE2 from binding T-strands. Here we show that root explants from Arabidopsis thaliana plants that expressed virE1 formed 3.5-fold fewer tumors than roots from plants without virE1. Also, this resistance was specific for VirE2-mediated Agrobacterium transformation. Plants that have been genetically altered to resist crown gall may prove more effective than biological control.

Draft Genome Sequence of Erwinia billingiae OSU19-1, Isolated from a Pear Tree Canker.

Plant-associated Erwinia include pathogenic and nonpathogenic species. We report the 5.6-Mb genome sequence of Erwinia billingiae OSU19-1, isolated from a canker on a pear tree inoculated with Erwinia amylovora. OSU19-1 and a closely related European isolate, E. billingiae Eb661(T), share many similarities including 40 kb of plasmid sequence.

Agrobacterium tumefaciens and A. rhizogenes use different proteins to transport bacterial DNA into the plant cell nucleus.

Agrobacterium tumefaciens and A. rhizogenes transport single-stranded DNA (ssDNA; T-strands) and virulence proteins into plant cells through a type IV secretion system. DNA transfer initiates when VirD2 nicks border sequences in the tumour-inducing plasmid, attaches to the 5' end, and pilots T-strands into plant cells. Agrobacterium tumefaciens translocates ssDNA-binding protein VirE2 into plant cells where it targets T-strands into the nucleus. Some A. rhizogenes strains lack VirE2 but transfer T-strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant. VirE2 and full-length GALLS (GALLS-FL) contain nuclear localization sequences that target these proteins to the plant cell nucleus. VirE2 binds cooperatively to T-strands allowing it to move ssDNA without ATP hydrolysis. Unlike VirE2, GALLS-FL contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. VirE2 may accumulate in the nucleus and pull T-strands into the nucleus using the force generated by cooperative DNA binding. GALLS-FL accumulates inside the nucleus where its predicted ATP-dependent strand transferase may pull T-strands into the nucleus. These different mechanisms for nuclear import of T-strands may affect the efficiency and quality of transgenic events in plant biotechnology applications.

Agrobacterium rhizogenes GALLS protein contains domains for ATP binding, nuclear localization, and type IV secretion.

Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2.

Agrobacterium rhizogenes GALLS protein substitutes for Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.

Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4. Bacteria also secrete certain Vir proteins into plant cells via this pore. One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration. VirE2 binds incoming ssT-DNA and helps target it into the nucleus. Some strains of A. rhizogenes lack VirE2, but they still transfer T-DNA efficiently. We isolated a novel gene from A. rhizogenes that restored pathogenicity to virE2 mutant A. tumefaciens. The GALLS gene was essential for pathogenicity of A. rhizogenes. Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein. Despite their lack of similarity, GALLS substituted for VirE2.