Use of the rhizobial type III effector gene nopP to improve Agrobacterium rhizogenes-mediated transformation of Lotus japonicus

Background Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. Results Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti. Conclusion We have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.

Abscisic acid utilizing rhizobacteria disturb nitrogen-fixing symbiosis of pea Pisum sativum L.

Rhizosphere bacteria are capable of utilizing various phytohormones (particularly auxins) as nutrients and thereby affect plant growth, nutrition and interactions with symbiotic microorganisms. Here, for the first time we evaluated the effects of rhizosphere bacteria Novosphingobium sp. P6W and Rhodococcus sp. P1Y capable of utilizing abscisic acid (ABA) on growth and nitrogen-fixing symbiosis of pea (Pisum sativum L.) line SGE and its Cd-insensitive mutant SGECdt using hydroponic culture. The plants were co-inoculated with the ABA-utilizing bacteria and nodule bacterium Rhizobium leguminosarum bv. viciae RCAM1066. Treatment with cadmium (Cd) was applied as an inducer of ABA biosynthesis in plants. In the presence of only nodule bacteria, Cd significantly inhibited the growth of roots and shoots and also decreased the nodule number and nitrogen-fixing activity in SGE peas, but not in the SGECdt mutant. Inoculation with ABA-utilizing bacteria also inhibited biomass production, nodulation and nitrogen-fixation of Cd-untreated SGE plants. This negative effect of bacteria on the SGECdt mutant was less pronounced. Contrary to this, ABA-utilizing bacteria had no effect on SGE plants treated with Cd, but decreased shoot biomass and nitrogen-fixing activity of the SGECdt mutant. Inoculation with ABA-utilizing bacteria had no effect on shoot Cd and nutrient content of both pea genotypes, suggesting that bacterial effects on plants were not associated with the plant nutrient status. We propose that the bacteria counteracted the increased ABA concentrations in SGE roots caused by Cd due to utilization of this phytohormone. However, opposite processes aimed at inhibiting and stimulating growth and legume–rhizobia symbiosis can be caused by the ABA-utilizing bacteria.

Microbial Consortium of PGPR, Rhizobia and Arbuscular Mycorrhizal Fungus Makes Pea Mutant SGECdt Comparable with Indian Mustard in Cadmium Tolerance and Accumulation

Cadmium (Cd) is one of the most widespread and toxic soil pollutants that inhibits plant growth and microbial activity. Polluted soils can be remediated using plants that either accumulate metals (phytoextraction) or convert them to biologically inaccessible forms (phytostabilization). The phytoremediation potential of a symbiotic system comprising the Cd-tolerant pea (Pisum sativum L.) mutant SGECdt and selected Cd-tolerant microorganisms, such as plant growth-promoting rhizobacterium Variovorax paradoxus 5C-2, nodule bacterium Rhizobium leguminosarum bv. viciae RCAM1066, and arbuscular mycorrhizal fungus Glomus sp. 1Fo, was evaluated in comparison with wild-type pea SGE and the Cd-accumulating plant Indian mustard (Brassica juncea L. Czern.) VIR263. Plants were grown in pots in sterilized uncontaminated or Cd-supplemented (15 mg Cd kg−1) soil and inoculated or not with the microbial consortium. Cadmium significantly inhibited growth of uninoculated and particularly inoculated SGE plants, but had no effect on SGECdt and decreased shoot biomass of B. juncea. Inoculation with the microbial consortium more than doubled pea biomass (both genotypes) irrespective of Cd contamination, but had little effect on B. juncea biomass. Cadmium decreased nodule number and acetylene reduction activity of SGE by 5.6 and 10.8 times, whereas this decrease in SGECdt was 2.1 and 2.8 times only, and the frequency of mycorrhizal structures decreased only in SGE roots. Inoculation decreased shoot Cd concentration and increased seed Cd concentration of both pea genotypes, but had little effect on Cd concentration of B. juncea. Inoculation also significantly increased concentration and/or accumulation of nutrients (Ca, Fe, K, Mg, Mn, N, P, S, and Zn) by Cd-treated pea plants, particularly by the SGECdt mutant. Shoot Cd concentration of SGECdt was twice that of SGE, and the inoculated SGECdt had approximately similar Cd accumulation capacity as compared with B. juncea. Thus, plant–microbe systems based on Cd-tolerant micro-symbionts and plant genotypes offer considerable opportunities to increase plant HM tolerance and accumulation.

Two Novel Amyloid Proteins, RopA and RopB, from the Root Nodule Bacterium Rhizobium leguminosarum

Amyloids represent protein fibrils with a highly ordered spatial structure, which not only cause dozens of incurable human and animal diseases but also play vital biological roles in Archaea, Bacteria, and Eukarya. Despite the fact that association of bacterial amyloids with microbial pathogenesis and infectious diseases is well known, there is a lack of information concerning the amyloids of symbiotic bacteria. In this study, using the previously developed proteomic method for screening and identification of amyloids (PSIA), we identified amyloidogenic proteins in the proteome of the root nodule bacterium Rhizobium leguminosarum. Among 54 proteins identified, we selected two proteins, RopA and RopB, which are predicted to have β-barrel structure and are likely to be involved in the control of plant-microbial symbiosis. We demonstrated that the full-length RopA and RopB form bona fide amyloid fibrils in vitro. In particular, these fibrils are β-sheet-rich, bind Thioflavin T (ThT), exhibit green birefringence upon staining with Congo Red (CR), and resist treatment with ionic detergents and proteases. The heterologously expressed RopA and RopB intracellularly aggregate in yeast and assemble into amyloid fibrils at the surface of Escherichia coli. The capsules of the R. leguminosarum cells bind CR, exhibit green birefringence, and contain fibrils of RopA and RopB in vivo.

Complete Genome Sequence of the Symbiotic Strain Bradyrhizobium icense LMTR 13 T , Isolated from Lima Bean ( Phaseolus lunatus ) in Peru

ABSTRACT The complete genome sequence of Bradyrhizobium icense LMTR 13 T , a root nodule bacterium isolated from the legume Phaseolus lunatus , is reported here. The genome consists of a circular 8,322,773-bp chromosome which codes for a large and novel symbiotic island as well as genes putatively involved in soil and root colonization.

THE INFLUENCE OF SYNTHETIC POLYSACCHARIDE ON BOTH EFFICIENCY OF SYMBIOSIS AND PEROXIDASE AND CATALASE ACTIVENESS OF PEA AND SOYA BEANS

The influence of synthetic polysaccharide MOD-19 on the effic¬iency of nitrogen fixation ofpea and soya plants as well as on ferment activity of the antioxidative defense system - peroxidase and catalase was investigated. It has been shown that the metabolic process intensifying is ob¬served in pea and soya plants grown from the seeds, wich had been tilled before sowing by nodule bacterium (pea - Rhizobium leguminosarum bv. viciae 263b and soya beans - Bradyrhizobium japonicum 634b) and by nonspecific for these bean cultures polysaccharide MOD-19. This has been proved by morphological and functional haracteristics of these systems, the increasing of plant biomass and nodules quantiity on the roots. The active functioning period of nodules is elongating at the expense of secondary nodules formation on the side roots and their nitrogen activity increased. Rising of the oxidative ferment activity level for peroxydase and catalase was found in these plants.