Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system

Abstract

SummaryThe symbiosis island of Mesorhizobium loti strain R7A contains genes with strong similarity to the structural vir genes ( virB1-11 ; virD4 ) of Agrobacterium tumefaciens that encode the type IV secretion system (T4SS) required for T-DNA transfer to plants. In contrast, M. loti strain MAFF303099 lacks these genes but contains genes not present in strain R7A that encode a type III secretion system (T3SS). Here we show by hybridization analysis that most M. loti strains contain the VirB/D4 T4SS and not the T3SS. Strikingly, strain R7A vir gene mutants formed large nodules containing bacteroids on Leucaena leucocephala in contrast to the wild-type strain that formed only uninfected tumour-like structures. A rhcJ T3SS mutant of strain MAFF303099 also nodulated L. leucocephala , unlike the wild type. On Lotus corniculatus , the vir mutants were delayed in nodulation and were less competitive compared with the wild type. Two strain R7A genes, msi059 and msi061 , were identified through their mutant phenotypes as possibly encoding translocated effector proteins. Both Msi059 and Msi061 were translocated through the A. tumefaciens VirB/D4 system into Saccharomyces cerevisiae and Arabidopsis thaliana , as shown using the Cre recombinase Reporter Assay for Translocation (CRAfT). Taken together, these results suggest that the VirB/D4 T4SS of M. loti R7A plays an analogous symbiotic role to that of T3SS found in other rhizobia. The heterologous translocation of rhizobial proteins by the Agrobacterium VirB/D4 T4SS is the first demonstration that rhizobial effector proteins are translocated into plant cells and confirms functional conservation between the M. loti and A. tumefaciens T4SS.

Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72

“…Two other secretion systems are common to plant-associated bacteria, type III and IV secretion systems, which transport a wide variety of effector proteins into the extracellular medium or into the cytoplasm of eukaryotic host cells and affect interaction [26][27][28][29] . Intriguingly, neither system is present in strain BH72, probably preventing the export of toxic proteins to the host.…”

Experimental evolution of nodule intracellular infection in legume symbionts

“…Rhizobia were shown to use T3SS to promote or impair symbiosis upon secretion of type three effectors that often have homologs in pathogenic bacteria (Deakin and Broughton, 2009). Known negative effects of rhizobial T3SS include alteration of root hair infection and nodule formation (Chatterjee et al, 1990), nodule infection (Hubber et al, 2004) or symbiosome differentiation (Yang et al, 2010). Thanks to the dual regulation of HrpG, our work showed that T3SS can block both root hair and intracellular infection on the same host plant.…”

Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium

“…We could not, however, detect transfer of a Cre fusion with the C-terminal 10 aa to the kanamycin reporter line (see below for transfer by using a more sensitive GFP reporter line). Importantly, there was no transfer of Cre::VirF19C from virD4 mutant LBA2587 (27), showing that translocation mediated by this short sequence also still depends on the presence of an intact T4SS.…”

“…Selection conditions of the strains and methods for introduction of plasmids are described in detail elsewhere (26). In some experiments, LBA2587, a derivative of LBA1100 with a precise deletion of virD4 (27), was used.…”