Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots.

The roots of most higher plants form arbuscular mycorrhiza, an ancient, phosphate-acquiring symbiosis with fungi, whereas only four related plant orders are able to engage in the evolutionary younger nitrogen-fixing root-nodule symbiosis with bacteria. Plant symbioses with bacteria and fungi require a set of common signal transduction components that redirect root cell development. Here we present two highly homologous genes from Lotus japonicus, CASTOR and POLLUX, that are indispensable for microbial admission into plant cells and act upstream of intracellular calcium spiking, one of the earliest plant responses to symbiotic stimulation. Surprisingly, both twin proteins are localized in the plastids of root cells, indicating a previously unrecognized role of this ancient endosymbiont in controlling intracellular symbioses that evolved more recently.

CYCLOPS, a mediator of symbiotic intracellular accommodation.

The initiation of intracellular infection of legume roots by symbiotic rhizobia bacteria and arbuscular mycorrhiza (AM) fungi is preceded by the induction of calcium signatures in and around the nucleus of root epidermal cells. Although a calcium and calmodulin-dependent kinase (CCaMK) is a key mediator of symbiotic root responses, the decoding of the calcium signal and the molecular events downstream are only poorly understood. Here, we characterize Lotus japonicus cyclops mutants on which microbial infection was severely inhibited. In contrast, nodule organogenesis was initiated in response to rhizobia, but arrested prematurely. This arrest was overcome when a deregulated CCaMK mutant version was introduced into cyclops mutants, conferring the development of full-sized, spontaneous nodules. Because cyclops mutants block symbiotic infection but are competent for nodule development, they reveal a bifurcation of signal transduction downstream of CCaMK. We identified CYCLOPS by positional cloning. CYCLOPS carries a functional nuclear localization signal and a predicted coiled-coil domain. We observed colocalization and physical interaction between CCaMK and CYCLOPS in plant and yeast cell nuclei in the absence of symbiotic stimulation. Importantly, CYCLOPS is a phosphorylation substrate of CCaMK in vitro. Cyclops mutants of rice were impaired in AM, and rice CYCLOPS could restore symbiosis in Lotus cyclops mutants, indicating a functional conservation across angiosperms. Our results suggest that CYCLOPS forms an ancient, preassembled signal transduction complex with CCaMK that is specifically required for infection, whereas organogenesis likely requires additional yet-to-be identified CCaMK interactors or substrates.

Gibberellin controls the nodulation signaling pathway in Lotus japonicus.

Root nodule formation is regulated by several plant hormones, but the details of the regulation of the nodulation signaling pathway are largely unknown. In this study, the role of gibberellin (GA) in the control of root nodule symbiosis was investigated at the physiological and genetic levels in Lotus japonicus. Exogenous application of biologically active GA, GA(3), inhibited the formation of infection threads and nodules, which was counteracted by the application of a biosynthesis inhibitor of GA, Uniconazole P. Nod factor-induced root hair deformation was severely blocked in the presence of GA, which was phenocopied by nsp2 mutants. The number of spontaneous nodules triggered by the gain-of-function mutation of calcium/calmodulin-dependent kinase (CCaMK) or the lotus histidine kinase 1 (LHK1) was decreased upon the addition of GA; moreover, the overexpression of the gain-of-function mutation of L. japonicus, SLEEPY1, a positive regulator of GA signaling, resulted in a reduced nodule number, without other aspects of root development being affected. These results indicate that higher GA signaling levels specifically inhibit the nodulation signaling pathway. Nod factor-dependent induction of NSP2 and NIN was inhibited by exogenous GA. Furthermore, the cytokinin-dependent induction of NIN was suppressed by GA. From these results, we conclude that GA inhibits the nodulation signaling pathway downstream of cytokinin, possibly at NSP2, which is required for Nod factor-dependent NIN expression. These results clarify the roles of GA in the nodulation signaling pathway, and in relation to the cytokinin signaling pathway for nodulation in L. japonicus.

Polyubiquitin promoter-based binary vectors for overexpression and gene silencing in Lotus japonicus.

In this study, we compared the transcriptional activities between Cauliflower mosaic virus (CaMV)35S promoter and polyubiquitin (Ljubq1) promoter from Lotus japonicus using beta-glucuronidase (gus) reporter gene in transgenic plants of L. japonicus. The promoter analysis demonstrated that the Ljubq1 promoter possessed higher activity than the CaMV35S promoter in leaves, stems, roots, nodules, and pollen. Finally, we created GATEWAY conversion technology-compatible binary vectors for over-expression and RNA interference under the Ljubq1 promoter. These materials could provide alternative choice for studies in L. japonicus.

Bioremediation of cadmium contaminated soil using symbiosis between leguminous plant and recombinant rhizobia with the MTL4 and the PCS genes.

Cadmium contamination in rice grains is one of the important issues in Asian countries. We have developed a novel bio-remediation system based on the symbiosis between leguminous plant and genetically engineered rhizobia. We designed two types of recombinant rhizobia, carrying two genes, synthetic tetrameric metallothionein (MTL4) and cDNA encoding phytochelatin synthase from Arabidopsis thaliana (AtPCS). The MTL4 and AtPCS genes were transferred to Mesorhizobium huakuii subsp. rengei B3, which can infect and form nodules on Chinese milk vetch, Astragalus sinicus. The two genes were fused to the nolB or nifH promoter, which generated nodule specific expression of these genes in strain B3. The two recombinant strains, B3(pMPnolBMTL4nifHPCS) and B3::nifHMTL4(pMPnifHPCS), showed 25 and 12-fold increase in Cd concentration, in the free-living cells, respectively. When these recombinant strains established the symbiotic relationship with A. sinicus, the symbionts increased Cd accumulation in nodules by two-fold in hydroponic culture. The expression of the both MTL4 and AtPCS genes showed additive effect on cadmium accumulation in nodules. We also applied these recombinant bacteria to rice paddy soil polluted with Cd (1mgkg(-1) dry weight soil). The accumulation of Cd increased not only in nodules but also in the roots of A. sinicus infected by the recombinant rhizobia. The accumulation of Cd in the plant roots infected by B3(pMPnolBMTL4nifHPCS) achieved three-fold than that by the wild-type B3. After two months of cultivation of the symbiont, a maximum of 9% of Cd in paddy soil was removed. Thus, the symbiosis will be useful in phytoremediation for heavy metals.

New nodulation mutants responsible for infection thread development in Lotus japonicus.

Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.

Construction of an additional metal-binding site in human metallothionein-2.

We have constructed a new metal-binding site in the human metallothionein-2 (hMT-2), using the protein as a scaffold to investigate the structure and function of metal-binding. Potential metal-binding sites were designed within hMT-2 on the basis of structures generated by homology modeling. Amino acid residues D11, C13, C26 and S28 in the beta-domain of hMT-2 (hMT-2beta) were found, by computer search, to form a potential tetrahedral Cys4 metal-binding site. Six mutant proteins were constructed with the following amino acid substitutions: D11C, S28C and D11C/S28C in hMT-2 and the same mutations in hMT-2beta, respectively. These single-mutant and double-mutant proteins bound one gram atom of cadmium or zinc ions per gram molecule of protein more than the corresponding wild-type proteins. The circular dichroism spectra suggested that the structures of the single-mutant proteins that bound Cd or Zn were similar to that of the D11C/S28C double-mutant proteins. To evaluate the metal-binding affinity of the mutant proteins, we performed pH titrations of wild-type and mutant proteins. The stability with changes in pH of all the mutant proteins was higher than that of the wild-type proteins, and that of the double-mutant D11C/S28C protein was highest. Consequently, it appears that we were able to create novel proteins that bound metal ions at high density and with high affinity.

Lotus burttii takes a position of the third corner in the lotus molecular genetics triangle.

In order to consolidate molecular genetic system in Lotus japonicus and to further access the biological diversity in Lotea, we introduce here Lotus burttii B-303 derived from West Pakistan as the third crossing partner of the Gifu ecotype (B-129-S9) for a genetic analysis. L. burttii is a relatively small and early flowering plant with non-shattering behavior. The general chromosome morphology is very similar to Gifu, and fluorescence in situ hybridization (FISH) analysis revealed that the short arm of chromosome 1 in L. burttii is comparable to that of Gifu, indicating that the translocation event involving chromosomes 1 and 2, which was observed in L. japonicus Miyakojima MG-20, is not present in L. burttii. In addition L. burttii has a higher level of DNA polymorphism compared to Gifu and MG-20 enabling design of codominant markers such as SSR, CAPS and dCAPS. Using an F2 population from a cross between Gifu and L. burttii, codominant makers that co-segregated at the translocation site could be expanded. In order to normalize the genetic background, L. burttii was inbred for nine generations and the germplasm L. burttii B-303-S9 was established.

A pH-dependent conformational change in EspA, a component of the Escherichia coli O157:H7 type III secretion system.

pH-Dependent structural changes for Escherichia coli O157:H7 EspA were characterized by CD, 8-anilino-2-naphthyl sulfonic acid (ANS) fluorescence, and sedimentation equilibrium ultracentrifugation. Far- and near-UV CD spectra, recorded between pH 2.0 and 7.0, indicate that the protein has significant amounts of secondary and tertiary structures. An increase in ANS fluorescence intensity (in the presence of EspA) was observed at acidic pH; whereas, no increased ANS fluorescence was observed at pH 7.0. These results suggest the presence of a partially unfolded state. Interestingly, urea-induced unfolding transitions, monitored by far-UV CD spectroscopy, showed that the protein is destabilized at pH 2.0 as compared with EspA at neutral pH. Although increased ANS fluorescence was observed at pH 3.0, the urea-induced unfolding curve is similar to that found at pH 7.0. This result suggests the presence, at pH 3.0, of an ordered, but partially unfolded state, which differs from typical molten globule. The results of analytical ultracentrifugation and infrared spectroscopy indicate that EspA molecules associate at pH 7.0, suggesting the formation of short filamentous oligomers containing alpha-helical structures, whereas the protein tend to form nonspecific aggregates containing intermolecular beta-sheets at pH 2.0. Our experiments indicate that EspA has the potential to spontaneously form filamentous oligomers at neutral pH; whereas the protein is partially unfolded, assuming different conformations, at acidic pH.

EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein.

The structural properties of EspB, a virulence factor of the Escherichia coli O157 type III secretion system, were characterized. Far-UV and near-UV CD spectra, recorded between pH 1.0 and pH 7.0, show that the protein assumes alpha-helical structures and that some tyrosine tertiary contacts may exist. All tyrosine side-chains are exposed to water, as determined by acrylamide fluorescence quenching spectroscopy. An increase in the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate was observed at pH 2.0 in the presence of EspB, whereas no such increase in fluorescence was observed at pH 7.0. These data suggest the formation of a molten globule state at pH 2.0. Destabilization of EspB at low pH was shown by urea-unfolding transitions, monitored by far-UV CD spectroscopy. The result from a sedimentation equilibrium study indicated that EspB assumes a monomeric form at pH 7.0, although its Stokes radius (estimated by multiangle laser light scattering) was twice as large as expected for a monomeric globular structure of EspB. These data suggest that EspB, at pH 7.0, assumes a relatively expanded conformation. The chemical shift patterns of EspB 15N-1H heteronuclear single quantum correlation spectra at pH 2.0 and 7.0 are qualitatively similar to that of urea-unfolded EspB. Taken together, the properties of EspB reported here provide evidence that EspB is a natively partially folded protein, but with less exposed hydrophobic surface than traditional molten globules. This structural feature of EspB may be advantageous when EspB interacts with various biomolecules during the bacterial infection of host cells.

Root hair abundant genes LjRH101 and LjRH102 encode peroxidase and xyloglucan endotransglycosylase in Lotus japonicus.

We have identified root hair abundant genes, LjRH101 and LjRH102, from a model legume Lotus japonicus by cDNA-amplified fragment length polymorphism (AFLP). These two genes will be suitable markers for the molecular biological identification of root hairs.

Expression of a major house dust mite allergen gene from Dermatophagoides farinae in Lotus japonicus accession Miyakojima MG-20.

Transformation of a model legume Lotus japonicus accession Miyakojima MG-20 was examined using Agrobacterium tumefaciens with a binary expression vector. Using the improved transformation method, we introduced a major allergen gene from a house dust mite, Der f 1, into MG-20. Analyses by Southern hybridization, reverse transcription (RT)-PCR, and Western blotting showed that the Der f 1 gene was integrated into the genome of L. japonicus, expressing the gene product in the T1 lines. Our results imply future application of oral allergen-specific immunotherapy using legume plants.

Interactions of arsenic with human metallothionein-2.

Arsenic is a toxic element that is found in the atmosphere, as well as in aquatic and terrestrial environments. We have demonstrated that As(3+) binds to human metallothionein-2 (hMT-2) by UV absorption spectroscopy, inductively coupled plasma-atomic emission spectrometry (ICP-AES), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MALDI-TOF-MS revealed that the structure of the adduct formed by arsenic and hMT-2 (As-hMT-2) was not homogeneous. The maximum molar ratio of arsenic to hMT-2 was found to be more than 6:1 on ICP-AES, UV absorption spectroscopy and MALDI-TOF-MS. The ratio of the number of sulfhydryl groups in hMT-2 that bound arsenic was 3:1, which is the same as the ratios reported previously for arsenic-glutathione and arsenic-phytochelatin complexes.

A novel bioremediation system for heavy metals using the symbiosis between leguminous plant and genetically engineered rhizobia.

A novel plant-bacterial remediation system for heavy metals (HM) was developed by expression of tetrameric human metallothionein (MTL4) in Mesorhizobium huakuii subsp. rengei B3, a strain which infects and forms nodules on a green manure, Astragalus sinicus. The MTL4 gene was fused to the nifH and nolB promoters, which generated nodule- specific expression of the MTL4 gene. The expression analysis of the MTL4 gene was demonstrated in free-living cells in the presence of Cd(2+) and Cu(2+), under the low oxygen condition. The MTL4 under the nifH and nolB promoters was expressed and increased the accumulation of Cd(2+), but not Cu(2+) in free-living cells. The expression of the integrated nifH-MTL4 gene in the chromosome of strain B3 was also expressed stably and accumulated Cd(2+) in the bacterial cells. The MTL4 transcripts were detected by in situ hybridization in bacteroids of mature nodules of A. sinicus containing nifH-MTL4 and nolB-MTL4 fusion gene. Moreover the MTL4 protein was detected by immunostaining. By infection of the recombinant B3, A. sinicus established symbiosis with the recombinant B3 that was grown in Cd(2+) and Cu(2+)-polluted soils. The symbionts increased Cd(2+) accumulation in nodules 1.7-2.0-fold, whereas, no significantly increase in Cu(2+) accumulation was noted.

Genetic manipulation system in propionibacteria.

Members of the genus Propionibacterium are widely used in the production of vitamin B12, tetrapyrrole compounds, and propionic acid as well as in probiotic and cheese industries. Shuttle vectors were developed in propionibacteria using replicons from endogenous plasmids in Propionibacterium and Escherichia coli and an appropriate selection marker. The efficient transformation was achieved using the shuttle vector prepared from Propionibacterium freudenreichii to overcome the high restriction modification system in propionibacteria. Expression vectors with native promoters for use in propionibacteria were also developed. Using this system, cholesterol oxidase, which is used as a diagnostic enzyme, was produced in P. freudenreichii. Genes involved in 5-aminolevulinic acid (ALA) and vitamin B12 biosynthesis in propionibacteria were isolated. ALA in propionibacteria could be synthesized via both the C4 pathway (condensation of glycine and succinyl CoA) and the C5 pathway (from glutamate). The hemA gene encoding ALA synthase from Rhodobacter spheroides, was overexpressed and ALA accumulated in P. freudenreichii. Thus, the genetic manipulation systems in propionibacteria will facilitate genetic studies of probiotics and the vitamin B12 biosynthetic pathway.

Variation of the amino acid content of Arabidopsis seeds by expressing soybean aspartate aminotransferase gene.

We have increased the contents of several amino acids in the seeds of Arabidopsis thaliana by introduction of aspartate aminotransferase (AAT), an enzyme of the aspartate biosynthetic pathway. mRNA was prepared from one-week-old seedlings of Glycine max cv. enrei and the cDNA encoding AAT5 was isolated and linked to the CaMV35S promoter in the plant vector pBI121. The AAT5 gene encodes a protein of 462 amino acid residues that shows 51% amino acid sequence similarity to A. thaliana chloroplast Asp3. The soybean AAT5 also contains a chloroplast transit peptide and is able to functionally complement a Saccharomyces cerevisiae mutant lacking the Asp5 gene. A. thaliana was transformed with the AAT5 gene from Agrobacterium tumefaciens by the vacuum infiltration method. The AAT5 gene was detected in the transcript and genomic DNA from the transgenic T2 plants. The T3 progeny showed a 3:1 segregation ratio indicating the presence of a single integration. Expression of G. max AAT5 in A. thaliana transformants caused 3-, 4-, 23-, and 50-fold increases in the contents of free glycine, alanine, asparagine, and glutamine, respectively, in the T3 seeds. A decrease in the contents of valine, tyrosine, isoleucine, leucine, and phenylalanine by several folds was also observed. Thus, it is of interest that a key gene expression resulted in marked changes of metabolites in plant seeds.

Molecular analysis of promoter elements from Propionibacterium freudenreichii.

Propionibacterium freudenreichii is a commercially important microorganism that is used in the production of cheeses, cobalamin (vitamin B(12)), and propionic acid. Although a host-vector system in propionibacteria has been developed, there is little information available on the genetic background of the bacteria. To obtain genetic information to facilitate genetic engineering in propionibacteria, we cloned promoter regions from P. freudenreichii using Escherichia coli as a host at the first screening and a promoter-probe vector, pCVE1, which consists of the cholesterol oxidase (choA) gene from Streptomyces sp. as a reporter gene. Finally, nine clones with strong promoter activities in P. freudenreichii were screened by monitoring the choA gene expression and determining if the nucleotide sequences of the cloned DNA fragment were aligned. The initiation sites of these transcripts were determined by primer extension analysis. The putative consensus sequences corresponding to a -35 and -10 hexamer were found to be specific for P. freudenreichii, but not E. coli or other bacteria. Moreover, a new consensus heptamerous sequence between the -35 and -10 regions, termed the -16 region, was also found. It is possible that the putative consensus heptamer is functional and essential to promoter activity in P. freudenreichii. These results should provide new opportunities for controlled gene expression in P. freudenreichii.

Production of vitamin B12 in genetically engineered Propionibacterium freudenreichii.

Since the chemical synthesis of vitamin B12 requires more than 70 steps, the production of vitamin B12 has been achieved by microorganism fermentation with additional brief chemical modifications. In an effort to increase the productivity of vitamin B12, we tried to express 10 genes belonging to the hem, cob and cbi gene families involved in the synthesis of vitamin B12 in Propionibacterium freudenreichii, which is a known producer of vitamin B12. In a recombinant P. freudenreichii clone that harbored the expression vector containing a cobA, cbiLF, or cbiEGH, we obtained an increase in vitamin B12 production of 1.7-, 1.9-, and 1.5-fold higher, respectively, than that in the microorganism without any cloned genes in the expression vector pPK705. The cobU and cobS genes caused a slight increase in the production of vitamin B12. Furthermore, we achieved multigene expression in P. freudenreichii. In a recombinant P. freudenreichii clone that harbored an exogenous gene, hemA, from Rhodobacter sphaeroides and endogenous hemB and cobA genes, we successfully achieved the production of about 1.7 mg/l vitamin B12, 2.2-fold higher than that produced by P. freudenreichii harboring pPK705.

Toward elucidating the full spectrum of mite allergens--state of the art.

Our research has focused on the molecular design of immunotherapeutic vaccines and the advancement of mite-allergy diagnosis. Here, we describe the research history of the major group 1 and group 2 allergens, immunoelectrophoretic analyses covering the complete spectrum of mite allergens, our results on allergens with distinctive characteristics (a conjunctival congestion-eliciting antigen [LM2], an immunotherapeutic antigen [HM2] with high efficacy and without definite adverse reactions, and a potent T-cell stimulatory antigen [HM1] with secretion of IFN-gamma), the full spectrum and immunochemical properties of the major and other important mite allergens (including our newly described allergens: a pan-allergen [tropomyosin, group 10], a potent T-cell stimulatory allergen [M-177, apolipophorin, group 14] and its peptide fragments Mag1 and Mag3, a moderate IgE-binding allergen [gelsolin/villin, group 16], an EF-hand Ca2+-binding allergen [group 17], and a less IgE-binding allergen [heat shock protein 70]), and prospects for the development of immunotherapeutic and diagnostic agents.

Prophylactic effect of Lactobacillus oral vaccine expressing a Japanese cedar pollen allergen.

Lactic acid bacteria (LAB) represent an attractive delivery vehicle for oral allergy vaccine because of their safety as a food microorganism as well as their potent adjuvant activity triggering anti-allergic immune response. Here, we report the generation of recombinant LAB expressing a major Japanese cedar pollen allergen Cry j 1 (Cry j 1-LAB), and their prophylactic effect in vivo. To facilitate heterologous expression, the codon usage in the Cry j 1 gene was optimized for the host LAB strain Lactobacillus plantarum by the recursive PCR-based exhaustive site-directed mutagenesis. Use of the codon-optimized Cry j 1 cDNA and a lactate dehydrogenase gene fusion system led to a successful production of recombinant Cry j 1 in L. plantarum NCL21. We also found that oral vaccination with the Cry j 1-LAB suppressed allergen-specific IgE response and nasal symptoms in a murine model of cedar pollinosis.