Attila Kereszt

A receptor kinase gene regulating symbiotic nodule development

Leguminous plants are able to establish a nitrogen-fixing symbiosis with soil bacteria generally known as rhizobia. Metabolites exuded by the plant root activate the production of a rhizobial signal molecule, the Nod factor, which is essential for symbiotic nodule development. This lipo-chitooligosaccharide signal is active at femtomolar concentrations, and its structure is correlated with host specificity of symbiosis, suggesting the involvement of a cognate perception system in the plant host. Here we describe the cloning of a gene from Medicago sativa that is essential for Nod-factor perception in alfalfa, and by genetic analogy, in the related legumes Medicago truncatula and Pisum sativum. The identified ‘nodulation receptor kinase’, NORK, is predicted to function in the Nod-factor perception/transduction system (the NORK system) that initiates a signal cascade leading to nodulation. The family of ‘NORK extracellular-sequence-like’ (NSL) genes is broadly distributed in the plant kingdom, although their biological function has not been previously ascribed. We suggest that during the evolution of symbiosis an ancestral NSL system was co-opted for transduction of an external ligand, the rhizobial Nod factor, leading to development of the symbiotic root nodule.

Plant Peptides Govern Terminal Differentiation of Bacteria in Symbiosis

Legume Symbiosome Leguminous plants (peas and beans) are major players in global nitrogen cycling by virtue of their symbioses with nitrogen-fixing bacteria that are harbored in specialized structures, called nodules, on the plants roots. Van de Velde et al. (p. 1122) show that the host plant, Medicago truncatula produces nodule-specific cysteine-rich peptides, resembling natural plant defense peptides. The peptides enter the bacterial cells and promote its development into the mature symbiont. In a complementary study, D. Wang et al. (p. 1126), have identified the signal peptidase, also encoded by the plant, that is required for processing these specialized peptides into their active form. Products encoded by the leguminous plant Medicago direct the differentiation of the bacterial partner in symbiosis. Legume plants host nitrogen-fixing endosymbiotic Rhizobium bacteria in root nodules. In Medicago truncatula, the bacteria undergo an irreversible (terminal) differentiation mediated by hitherto unidentified plant factors. We demonstrated that these factors are nodule-specific cysteine-rich (NCR) peptides that are targeted to the bacteria and enter the bacterial membrane and cytosol. Obstruction of NCR transport in the dnf1-1 signal peptidase mutant correlated with the absence of terminal bacterial differentiation. On the contrary, ectopic expression of NCRs in legumes devoid of NCRs or challenge of cultured rhizobia with peptides provoked symptoms of terminal differentiation. Because NCRs resemble antimicrobial peptides, our findings reveal a previously unknown innovation of the host plant, which adopts effectors of the innate immune system for symbiosis to manipulate the cell fate of endosymbiotic bacteria.

An ERF Transcription Factor in Medicago truncatula That Is Essential for Nod Factor Signal Transduction

Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor–induced gene expression and for spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.

Comparative mapping between Medicago sativa and Pisum sativum

Comparative genome analysis has been performed between alfalfa ( Medicago sativa) and pea ( Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.

Agrobacterium rhizogenes-mediated transformation of soybean to study root biology.

This protocol is used to induce transgenic roots on soybean to study the function of genes required in biological processes of the root. Young seedlings with unfolded cotyledons are infected at the cotyledonary node and/or hypocotyl with Agrobacterium rhizogenes carrying the gene construct to be tested and the infection sites are kept in an environment of high humidity. When the emerged hairy roots can support the plants, the main roots are removed and the transgenic roots can be tested. Using this method, almost 100% of the infected plants form hairy roots within 1 month from the start of the experiments.

Natural roles of antimicrobial peptides in microbes, plants and animals.

Antimicrobial peptides (AMPs) are ribosomally synthesized natural antibiotics that are crucial effectors of innate immune systems in all living organisms. AMPs are diverse peptides, differing in their amino acid composition and structure, that generally display rapid killing and broad-spectrum antimicrobial activities. Therefore, AMPs have high potential for therapeutic use in healthcare and agriculture. This review focuses on in vivo studies relating how organisms - bacteria, plants, insects and mammals - employ AMPs in their interactions with microbial competitors, pathogens and symbionts.

The pha gene cluster of Rhizobium meliloti involved in pH adaptation and symbiosis encodes a novel type of K+ efflux system

The fix‐2 mutant of Rhizobium meliloti affected in the invasion of alfalfa root nodules (Inf−/Fix−) is K+ sensitive and unable to adapt to alkaline pH in the presence of K+. Using directed Tn5 mutagenesis, we delimited a 6 kb genomic region in which mutations resulted in both Inf−/Fix− and K+‐sensitive phenotypes. In this DNA region, seven open reading frames (ORFs) were identified and the corresponding genes were designated phaA, B, C, D, E, F and G. The putative PhaABC proteins exhibit homology to the subunits of a Na+/H+ antiporter from an alkalophilic Bacillus strain. Moreover, PhaA and PhaD also show similarity to the ND5 and ND4 subunits of the proton‐pumping NADH:ubiquinone oxidoreductase respectively. Computer analysis suggests that all seven proteins are highly hydrophobic with several possible transmembrane domains. Some of these domains were confirmed by generating active alkaline phosphatase fusions. Ion transport studies on phaA mutant cells revealed a defect in K+ efflux at alkaline pH after the addition of a membrane‐permeable amine. These results suggest that the pha genes of R. meliloti encode for a novel type of K+ efflux system that is involved in pH adaptation and is required for the adaptation to the altered environment inside the plant.

A Paradigm for Endosymbiotic Life: Cell Differentiation of Rhizobium Bacteria Provoked by Host Plant Factors

Symbiosis between Rhizobium bacteria and legumes leads to the formation of the root nodule. The endosymbiotic bacteria reside in polyploid host cells as membrane-surrounded vesicles where they reduce atmospheric nitrogen to support plant growth by supplying ammonia in exchange for carbon sources and energy. The morphology and physiology of endosymbionts, despite their common function, are highly divergent in different hosts. In galegoid plants, the endosymbionts are terminally differentiated, uncultivable polyploid cells, with remarkably elongated and even branched Y-shaped cells. Bacteroid differentiation is controlled by host peptides, many of which have antibacterial activity and require the bacterial function of BacA. Although the precise and combined action of several hundred host peptides and BacA has yet to be discovered, similarities, especially to certain insect-bacterium symbioses involving likewise host peptides for manipulation of endosymbionts, suggest convergent evolution. Rhizobium-legume symbiosis provides a rich source of information for understanding host-controlled endosymbiotic life in eukaryotic cells.

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.