Receptor-mediated exopolysaccharide perception controls bacterial infection.

Surface polysaccharides are important for bacterial interactions with multicellular organisms, and some are virulence factors in pathogens. In the legume-rhizobium symbiosis, bacterial exopolysaccharides (EPS) are essential for the development of infected root nodules. We have identified a gene in Lotus japonicus, Epr3, encoding a receptor-like kinase that controls this infection. We show that epr3 mutants are defective in perception of purified EPS, and that EPR3 binds EPS directly and distinguishes compatible and incompatible EPS in bacterial competition studies. Expression of Epr3 in epidermal cells within the susceptible root zone shows that the protein is involved in bacterial entry, while rhizobial and plant mutant studies suggest that Epr3 regulates bacterial passage through the plant's epidermal cell layer. Finally, we show that Epr3 expression is inducible and dependent on host perception of bacterial nodulation (Nod) factors. Plant-bacterial compatibility and bacterial access to legume roots is thus regulated by a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals.

Comparative analyses in Lotus: the cytogenetic map of Lotus uliginosus Schkuhr.

A comparative cytogenetic map was built for Lotusuliginosus (2n = 12), expanding previous analyses that revealed intra- and interspecific chromosomal rearrangements in the model legume L. japonicus, L. filicaulis, and L. burttii. This species is positioned in a sister clade of the previously-mapped species and is proposed as one of the progenitors of L. corniculatus, the main forage crop of the genus. The cytogenetic map allowed the location of 12 genomic regions to be compared between these species. A high macrosynteny was revealed, but it was interrupted by a translocation involving chromosomes 3 and 5, a new rearrangement for the genus. Also, a transposition on chromosome 2 was found in L. japonicus 'Miyakojima'. Furthermore, changes in the number, size, and position of rDNA sites were observed, as well as an intraspecific size heteromorphism of the 5S rDNA site on L. uliginosus chromosome 6. The karyotype differences observed are proportional to the phylogenetic distance among these species.

Exploitation of colinear relationships between the genomes of Lotus japonicus, Pisum sativum and Arabidopsis thaliana, for positional cloning of a legume symbiosis gene.

The Lotus japonicus LjSYM2 gene, and the Pisum sativum orthologue PsSYM19, are required for the formation of nitrogen-fixing root nodules and arbuscular mycorrhiza. Here we describe the map-based cloning procedure leading to the isolation of both genes. Marker information from a classical AFLP marker-screen in Lotus was integrated with a comparative genomics approach, utilizing Arabidopsis genome sequence information and the pea genetic map. A network of gene-based markers linked in all three species was identified, suggesting local colinearity in the region around LjSYM2/PsSYM19. The closest AFLP marker was located just over 200 kb from the LjSYM2 gene, the marker SHMT, which was converted from a marker on the pea map, was only 7.9 kb away. The LjSYM2/PsSYM19 region corresponds to two duplicated segments of the Arabidopsis chromosomes AtII and AtIV. Lotus homologues of Arabidopsis genes within these segments were mapped to three clusters on LjI, LjII and LjVI, suggesting that during evolution the genomic segment surrounding LjSYM2 has been subjected to duplication events. However, one marker, AUX-1, was identified based on colinearity between Lotus and Arabidopsis that mapped in physical proximity of the LjSym2 gene.

Barley disease resistance gene analogs of the NBS-LRR class: identification and mapping.

The majority of verified plant disease resistance genes isolated to date are of the NBS-LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region. We took advantage of the sequence conservation in the NBS motif to clone, by PCR, gene fragments from barley representing putative disease resistance genes of this class. Over 30 different resistance gene analogs (RGAs) were isolated from the barley cultivar Regatta. These were grouped into 13 classes based on DNA sequence similarity. Actively transcribed genes were identified from all classes but one, and cDNA clones were isolated to derive the complete NBS-LRR protein sequences. Some of the NBS-LRR genes exhibited variation with respect to whether and where particular introns were spliced, as well as frequent premature polyadenylation. DNA sequences related to the majority of the barley RGAs were identified in the recently expanded public rice genomic sequence database, indicating that the rice sequence can be used to extract a large proportion of the RGAs from barley and other cereals. Using a combination of RFLP and PCR marker techniques, representatives of all barley RGA gene classes were mapped in the barley genome, to all chromosomes except 4H. A number of the RGA loci map in the vicinity of known disease resistance loci, and the association between RGA S-120 and the nematode resistance locus Ha2 on chromosome 2H was further tested by co-segregation analysis. Most of the RGA sequences reported here have not been described previously, and represent a useful resource as candidates or molecular markers for disease resistance genes in barley and other cereals.

Construction of a genetic linkage map of the model legume Lotus japonicus using an intraspecific F2 population.

Among leguminous plants, the model legume Lotus japonicus (Regel) Larsen has many biological and genetic advantages. We have developed a genetic linkage map of L. japonicus based on amplified fragment length polymorphism (AFLP), simple sequence repeat polymorphism (SSRP) and derived cleaved amplified polymorphic sequence (dCAPS). The F2 mapping population used was derived from a cross between two L. japonicus accessions Gifu B-129 and Miyakojima MG-20. These parental accessions showed remarkable cytological differences, particularly with respect to size and morphology of chromosomes 1 and 2. Using fluorescence in situ hybridization (FISH) with BAC clones from Gifu B-129 and TAC (Transformation-competent Artificial Chromosome) clones from Miyakojima MG-20, a reciprocal translocation was found to be responsible for the cytological differences between chromosomes 1 and 2. The borders of the translocations were identified by FISH and by alignment toward the L. filicaulis x L. japonicus Gifu B-129 linkage map. The markers from the main translocated region were located on linkage groups 1 and 2 of the two accessions, Gifu B-129 and Miyakojima MG-20, respectively. The framework of the linkage map was constructed based on codominant markers, and then dominant markers were integrated separately in each linkage group of the parents. The resulting linkage groups correspond to the six pairs of chromosomes of L. japonicus and consist of 287 markers with 487.3 cM length in Gifu B-129 and 277 markers with 481.6 cM length in Miyakojima MG-20. The map and marker information is available through the World Wide Web at http://www.kazusa.or.jp/lotus/.

Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus.

Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of approximately 1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction.

Evidence that the plant host synthesizes the heme moiety of leghemoglobin in root nodules

Although it is well established that the plant host encodes and synthesizes the apoprotein for leghemoglobin in root nodules, the source of the heme moiety has been uncertain. We recently found that the transcript for coproporphyrinogen III oxidase, one of the later enzymes of heme synthesis, is highly elevated in soybean (Glycine max L.) nodules compared with roots. In this study we measured enzyme activity and carried out western-blot analysis and in situ hybridization of mRNA to investigate the levels during nodulation of the plant-specific coproporphyrinogen oxidase and four other enzymes of the pathway in both soybean and pea (Pisum sativum L.). We compared them with the activity found in leaves and uninfected roots. Our results demonstrate that all of these enzymes are elevated in the infected cells of nodules. Because these are the same cells that express apoleghemoglobin, the data strongly support a role for the plant in the synthesis of the heme moiety of leghemoglobin.

Positional cloning of a gene for nematode resistance in sugar beet.

The Hs1(pro-1) locus confers resistance to the beet cyst nematode (Heterodera schachtii Schmidt), a major pest in the cultivation of sugar beet (Beta vulgaris L.). The Hs1(pro-1) gene was cloned with the use of genome-specific satellite markers and chromosomal break-point analysis. Expression of the corresponding complementary DNA in a susceptible sugar beet conferred resistance to infection with the beet cyst nematode. The native Hs1(pro-1) gene, expressed in roots, encodes a 282-amino acid protein with imperfect leucine-rich repeats and a putative membrane-spanning segment, features similar to those of disease resistance genes previously cloned from higher plants.

Long-range organization of a satellite DNA family flanking the beet cyst nematode resistance locus (Hs1) on chromosome-1 of B. patellaris and B. procumbens.

New members of a satellite DNA family (Sat 121), specific for wild beets of the section Procumbentes of the genus Beta, were isolated. Sequence analysis showed that the members of Sat-121 fall into two distinct classes. The organization of Sat-121 in the vicinity of a beet cyst nematode (Heterodera schachtii Schm.) resistance locus (Hs1) in B. patellaris and B. procumbens was investigated by pulsed-field gel electrophoresis (PFGE) using DNA from a series of resistant monosomic fragment additions, each containing an extra chromosome fragment of B. patellaris chromosome-1 (pat-1) in B. vulgaris. In this way several clusters of Sat-121 flanking the Hs1 (pat-1) locus were identified. In nematode resistant diploid introgressions (2n=18), which contain small segments of B. procumbens chromosome-1 (pro-1) in B. vulgaris, only two major Sat-121 clusters were detected near the Hs1 (pro-1) locus.

A soybean coproporphyrinogen oxidase gene is highly expressed in root nodules.

In plants the enzyme coproporphyrinogen oxidase catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX in the heme and chlorophyll biosynthesis pathway(s). We have isolated a soybean coproporphyrinogen oxidase cDNA from a cDNA library and determined the primary structure of the corresponding gene. The coproporphyrinogen oxidase gene encodes a polypeptide with a predicted molecular mass of 43 kDa. The derived amino acid sequence shows 50% similarity to the corresponding yeast amino acid sequence. The main difference is an extension of 67 amino acids at the N-terminus of the soybean polypeptide which may function as a transit peptide. A full-length coproporphyrinogen oxidase cDNA clone complements a yeast mutant deleted of the coproporphyrinogen oxidase gene, thus demonstrating the function of the soybean protein. The soybean coproporphyrinogen oxidase gene is highly expressed in nodules at the stage where several late nodulins including leghemoglobin appear. The coproporphyrinogen oxidase mRNA is also detectable in leaves but at a lower level than in nodules while no mRNA is detectable in roots. The high level of coproporphyrinogen oxidase mRNA in soybean nodules implies that the plant increases heme production in the nodules to meet the demand for additional heme required for hemoprotein formation.

Comparative sequence analysis of cis elements present in Glycine max L. leghemoglobin lba and lbc3 genes.

The soybean leghemoglobin lba gene promoter sequence was determined and aligned with the promoter sequence of the soybean lbc3 gene from the same gene family. Five highly conserved regions were found. There are two large conserved regions, one of which overlaps the basic promoter while the other defines a minimal enhancer in the upstream positive elements. Within the minimal enhancer, an inverted repeat with similarity to the binding site of a yeast transcription factor, GCN4, was found. This particular repeat is conserved in the promoters of all functional soybean lb genes as well as in lb gene promoters from other legumes. This suggests that the inverted repeat is important for leghemoglobin gene expression.

Isolation of DNA markers linked to a beet cyst nematode resistance locus in Beta patellaris and Beta procumbens.

In cultivated beet no useful level of resistance of the beet cyst nematode (BCN) Heterodera schachtii Schm. has been found, unlike the situation in wild species of the section Procumbentes. Stable introgression of resistance genes from the wild species into Beta vulgaris has not been achieved, but resistant monosomic additions (2n = 18 + 1), diploids of B. vulgaris with an extra alien chromosome carrying the resistance locus, have been obtained. Here we describe a new series of resistant monosomic fragment addition material of B. patellaris chromosome 1 (pat-1). We further describe the cloning of a single-copy DNA marker that specifically hybridizes with a monosomic addition fragment of approximately 8 Mb (AN5-90) carrying the BCN resistance locus. This marker and another fragment-specific, single-copy DNA marker probably flank the BCN locus on the addition fragment present in the AN5-203 material, which is approximately 19 Mb in size. Furthermore, several specific repetitive DNA markers have been isolated, one of which hybridizes to AN5-90 and also to DNA from a smaller DNA segment of Beta procumbens, present in line B883, carrying a BCN resistance locus introgressed into the B. vulgaris genome. This suggests that the specific repetitive marker is closely linked to the BCN locus.

Characterization and genomic organization of a highly expressed late nodulin gene subfamily in soybeans.

A soybean nodulin cDNA clone (E41) hybrid-selected mRNA for three in vitro translation products with apparent molecular weights of 26 kDa, 25 kDa and 24 kDa. Based on Southern analysis of soybean genomic DNA, combined with mapping and sequencing of genomic clones, we identified four genes that are related to E41, one of which was identified to be the previously characterized N-20 gene. Our data indicate the linkage of three of the genes, of which one is a truncated version and suggest that they originated by gene duplication combined with deletion and conversion. The genes are highly expressed and we postulate that the sequence conservation in the 5' and 3' flanking regions of all four genes, has a functional role in their expression. Hybrid-selected translation products of E41 are not immunoprecipitable with antibody to the soluble fraction of nodules suggesting that they are membrane associated. The N-20 gene, which is a member of this gene subfamily, showed sequence similarity to four previously characterized nodulin genes and a phylogenetic tree is proposed based on the extent of sequence similarity.

Evolution of the MIP family of integral membrane transport proteins.

Six integral membrane proteins of bacterial, animal, and plant origin, which are believed to function in solute transport, share sequence identity and are grouped together as members of the MIP family. These include the Escherichia coli glycerol facilitator, the major intrinsic protein from bovine lens fibre junction membranes, a plant tonoplast membrane protein, a soybean protein from the peribacteroid membrane, and a Drosophila neurogenic protein. These proteins, each of which appears to consist of six transmembrane helical segments per subunit, apparently arose by internal duplication of a three-transmembrane segment. Phylogenetic 'trees' interrelating these proteins and segments are presented.

5' Analysis of the soybean leghaemoglobin lbc(3) gene: regulatory elements required for promoter activity and organ specificity.

The soybean leghaemoglobin lbc(3) gene promoter was analysed in transgenic Lotus corniculatus plants. Hybrid-promoter constructions and 5' deletions were studied using chimeric genes composed of the various promoters, the chloramphenicol acetyltransferase (CAT) coding sequence and the lbc(3) 3' flanking region. A 5' Bal31 deletion series mapped a strong positive regulatory element between -1100 and -950. A weaker element located between -230 and -170 defined the minimum 5' region required for detectable promoter activity. Reactivation of inactive promoters with deletion endpoints between -230 and the transcription initiation site was obtained employing the constitutive cauliflower mosaic virus (CaMV) 35S enhancer. The position of cis regulatory element(s) required for nodule-specific expression was defined to 37 bp between -139 and -102. This region contains sequences conserved in other leghaemoglobin and nodulin genes. No indispensable control elements were found on the lbc(3) 3' flanking region.

A small family of nodule specific genes from soybean.

The primary structure of two nodule specific soybean genes are presented. The two genes code for primary products of 20.0 (nodulin 20) and 22.7 (nodulin 22) kdaltons, respectively. Both genes are related to the nodulin 23 and 44 genes. Alignment of the deduced amino acid sequences of all four genes revealed three domains of high homology interrupted by highly diverged regions due to numerous duplication and insertion events. The first conserved domain codes for a putative signal peptide, while the two others each contain four Cys residues that can be arranged in a way reminiscent of the metal binding domains present in some enzymes and in several DNA binding proteins.