A. Men

Nodulation factor receptor kinase 1α controls nodule organ number in soybean (Glycine max L. Merr)

Two allelic non-nodulating mutants, nod49 and rj1, were characterized using map-based cloning and candidate gene approaches, and genetic complementation. From our results we propose two highly related lipo-oligochitin LysM-type receptor kinase genes (GmNFR1α and GmNFR1β) as putative Nod factor receptor components in soybean. Both mutants contained frameshift mutations in GmNFR1α that would yield protein truncations. Both mutants contained a seemingly functional GmNFR1β homeologue, characterized by a 374-bp deletion in intron 6 and 20-100 times lower transcript levels than GmNFR1α, yet both mutants were unable to form nodules. Mutations in GmNFR1β within other genotypes had no defects in nodulation, showing that GmNFR1β was redundant. Transgenic overexpression of GmNFR1α, but not of GmNFR1β, increased nodule number per plant, plant nitrogen content and the ability to form nodules with restrictive, ultra-low Bradyrhizobium japonicum titres in transgenic roots of both nod49 and rj1. GmNFR1α overexpressing roots also formed nodules in nodulation-restrictive acid soil (pH 4.7). Our results show that: (i) NFR1α expression controls nodule number in soybean, and (ii) acid soil tolerance for nodulation and suppression of nodulation deficiency at low titre can be achieved by overexpression of GmNFR1α.

Inactivation of Duplicated Nod Factor Receptor 5 (NFR5) Genes in Recessive Loss-of-Function Non-Nodulation Mutants of Allotetraploid Soybean (Glycine max L. Merr.)

Chemically induced non-nodulating nod139 and nn5 mutants of soybean (Glycine max) show no visible symptoms in response to rhizobial inoculation. Both exhibit recessive Mendelian inheritance suggesting loss of function. By allele determination and genetic complementation in nod139 and nn5, two highly related lipo-oligochitin LysM-type receptor kinase genes in Glycine max were cloned; they are presumed to be the critical nodulation-inducing (Nod) factor receptor similar to those of Lotus japonicus, pea and Medicago truncatula. These duplicated receptor genes were called GmNFR5alpha and GmNFR5beta. Nonsense mutations in GmNFR5alpha and GmNFR5beta were genetically complemented by both wild-type GmNFR5alpha and GmNFR5beta in transgenic roots, indicating that both genes are functional. Both genes lack introns. In cultivar Williams82 GmNFR5alpha is located in chromosome 11 and in tandem with GmLYK7 (a related LysM receptor kinase gene), while GmNFR5beta is in tandem with GmLYK4 in homologous chromosome 1, suggesting ancient synteny and regional segmental duplication. Both genes are wild type in G. soja CPI100070 and Harosoy63; however, a non-functional NFR5beta allele (NFR5beta*) was discovered in parental lines Bragg and Williams, which harbored an identical 1,407 bp retroelement-type insertion. This retroelement (GmRE-1) and related sequences are located in several soybean genome positions. Paradoxically, putatively unrelated soybean cultivars shared the same insertion, suggesting a smaller than anticipated genetic base in this crop. GmNFR5alpha but not GmNFR5beta* was expressed in inoculated and uninoculated tap and lateral root portions at about 10-25% of GmATS1 (ATP synthase subunit 1), but not in trifoliate leaves and shoot tips.

Deciphering the genetic basis for polyketide variation among mycobacteria producing mycolactones

BackgroundMycolactones are immunosuppressive and cytotoxic polyketides, comprising five naturally occurring structural variants (named A/B, C, D, E and F), produced by different species of very closely related mycobacteria including the human pathogen, Mycobacterium ulcerans. In M. ulcerans strain Agy99, mycolactone A/B is produced by three highly homologous type I polyketide megasynthases (PKS), whose genes (mlsA1: 51 kb, mlsA2: 7.2 kb and mlsB: 42 kb) are found on a 174 kb plasmid, known as pMUM001.ResultsWe report here comparative genomic analysis of pMUM001, the complete DNA sequence of a 190 kb megaplasmid (pMUM002) from Mycobacterium liflandii 128FXT and partial sequence of two additional pMUM replicons, combined with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. These data reveal how PKS module and domain differences affecting MlsB correlate with the production of mycolactones E and F. For mycolactone E these differences from MlsB in M. ulcerans Agy99 include replacement of the AT domain of the loading module (acetate to propionate) and the absence of an entire extension module. For mycolactone F there is also a reduction of one extension module but also a swap of ketoreductase domains that explains the characteristic stereochemistry of the two terminal side-chain hydroxyls, an arrangement unique to mycolactone FConclusionThe mycolactone PKS locus on pMUM002 revealed the same large, three-gene structure and extraordinary pattern of near-identical PKS domain sequence repetition as observed in pMUM001 with greater than 98.5% nucleotide identity among domains of the same function. Intra- and inter-strain comparisons suggest that the extreme sequence homogeneity seen among the mls PKS genes is caused by frequent recombination-mediated domain replacement. This work has shed light on the evolution of mycolactone biosynthesis among an unusual group of mycobacteria and highlights the potential of the mls locus to become a toolbox for combinatorial PKS biochemistry.

A bacterial artificial chromosome library of Lotus japonicus constructed in an Agrobacterium tumefaciens-transformable vector.

We constructed a BAC library of the model legume Lotus japonicus with a 6-to 7-fold genome coverage. We used vector PCLD04541, which allows direct plant transformation by BACs. The average insert size is 94 kb. Clones were stable in Escherichia coli and Agrobacterium tumefaciens.

An integrated functional genomics and genetics approach for the plant's function in symbiotic nodulation.

Recent advances of high throughput DNA sequencing, bioinformatics robotics, BAC libraries, microarrays, insertional mutagenesis as well as promoter trapping open the opportunity for an integrated function and structure analysis of the genomes of soybean (Glycine max) and the model legume Lotus japonicus. We are specifically interested in the plant’s role during the establishment of nodule morphogenesis, and the genes shared during seemingly related developmental programs leading either to nodule or lateral root formation. Additionally our research seeks to elucidate plant genetic controls over plant-microbe interactions and symbiotic signaling of both Rhizobium and mycorrhizal symbioses. Nodulation in legumes involves the complex interaction of bacterial genes and their products with plant developmental processes governing mitogenic signal perception, signal transduction and morphogenesis. The challenge is to understand the plant’s genetic contribution to this symbiosis with the aim to improve natural associations of benefit for agriculture and the environment. Plant mutations were induced using EMS, fast neutron deletion as well as insertion mutagenesis. Single recessive loci were mapped using molecular markers, which were used to isolate soybean BAC clones to generate contigs spanning mutant deletions. Special emphasis was given to the Nts-1 locus of soybean that governs autoregulation of nodulation. If mutated, this locus leads to abundant nodulation (supernodulation) as well as nitrate tolerance in nodulation. Expression analysis of nodulation events using 4,200 micro-arrayed root ESTs was initiated to detect gene products temporarily expressed during early nodulation. Lotus japonicus as compared to soybean facilitates high throughput insertional mutagenesis and promoter trapping. Insertion of a promoter-less gus-reporter gene allowed the isolation of activated plant lines that showed development specific gus-gene expression. Isolation of flanking DNA sequences provided information of potential promoters and gene function as well as providing a link between structural and functional elements of nodulation-related genes. Evidence suggests that many nodule initiation functions evolved or are shared with lateral root related processes. The possibility exists that several non-legumes share such genes.

DAF yields a cloned marker linked to the soybean (Glycine max) supernodulation nts-1 locus

We report a further characterization of the genomic region containing the soybean supernodulation gene NTS-1. We performed a search for new markers linked to NTS-1 by combining DNA amplification fingerprinting (DAF) and bulked segregant analysis (BSA). The search resulted in one cloned polymorphism (B44-456) linked in trans, 8.5cM from the locus. Southern hybridization showed duplication of the B44-456 sequence in the soybean genome. Additionally, a DNA database search revealed one Arabidopsis thaliana genomic clone from chromosome I possessing 62% homology to the B44-456 marker. A relatively low number of polymorphisms were identified by several PCR marker technologies for this soybean genomic region, providing an additional support for its highly conserved and/or duplicated organization.

Metagenomics and beyond: new toolboxes for microbial systematics

An extraordinary DNA sequencing revolution has taken place over the past decade, which has seen exciting, yet challenging times for microbial genomics and systematics. Numerous metagenomics and metatranscriptomics projects have provided us with an unprecedented glimpse at the vast biological diversity that exists in minute amounts of samples obtained from environments such as ocean water, soil or human distal gut. One of the key challenges is how we catalogue and classify this vast diversity of microbial life (much of which represents unculturable mixtures) discovered in the last few years alone. Of even greater challenge is the fact that biological mechanisms that rule bacterial plasticity and ecological fitness are far more complex than previously thought, resulting in new concepts of ‘pan’ or ‘supra-genomes’ that appear to be much larger than any individual bacterial genome.

Nodulation Control in Legumes

Nodulation and concomitant symbiotic nitrogen fixation are critical for the productivity of the legume, yielding food, feed and fuel. The nodule number in legumes is regulated by numerous factors including the number and efficiency of the interacting Rhizobium bacteria and abiotic stresses as well as endogenous processes involving phytohormones, nodulation reception systems and autoregulation of nodulation (AON; Kinkema et al., 2006). The original discovery of the AON-controlling LRR receptor kinases, GmNARK/ LjHAR1/MtSUNN, which is active in leaf tissue of several legu-mes, now has led to an analysis of the mechanism underlying the signal transduction.

Functional Genomics of the Regulation of Nodule Number in Legumes

Peter M. Gresshoff, Gustavo Gualtieri, Titeki Laniya, Arief Indrasumunar, Akira Miyahara, Sureeporn Nontachaiyapoom, Tim Wells, Bandana Biswas, Pick Kuen Chan, Paul Scott, M. Kinkema, M. Djordjevic, Dana Hoffmann, Lisette Pregelj, Diana M. Buzas, Dong Xi Li, Artem Men, Qunyi Jiang, Cheol-Ho Hwang and Bernard J. Carroll ARC Centre of Excellence for Integrative Legume Research; School of Life Sciences, and School of Molecular and Microbial Sciences and LAFS, The University of Queensland, St. Lucia, Brisbane QLD 4072, AGRF; Genome Interaction Group, RSBS, ANU, Canberra, ACT, Australia.

Functional Genomics and Genetic Analysis of Nodulation of Soybean and Lotus japonicus

Plant genes control nodulation and nitrogen fixation in all legume symbioses. The need discover the function and structure of these genes, their interplay, their “side-effects” on other morphogenetic steps, and their relationship to other genes involved in the metabolism, and signal transduction and cell division is becoming progressively and feasible in light of recent advances, commonly referred to as functional and structural genomics. Here we demonstrate how map-based cloning as well as promoter trapping have been applied to two legumes, namely soybean and model legume Lotus japonicus to discover genes involved in early nodulation responses. The results suggest that the developmental program underlying nodule initiation and pattern control “borrowed” many genes involved in lateral root formation and control. We focused initially on the supernodulation locus nts-1 (Kolchinsky et al, 1997) to develop strategies of map-based cloning. In parallel we initiated a large scale, Y-DNA-based transformation of the model legume Lotus japonicus (Jiang and Gresshoff, 1997; Handberg and Stougaard, 1992) to trap plant promoters and to possibly obtain flanking sequence as well as knock-out mutants (Stiller et al, 1997; Martirani et al, 1999).