Clare Gough

The Medicago truncatula Lysine Motif-Receptor-Like Kinase Gene Family Includes NFP and New Nodule-Expressed Genes

Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysine motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.

A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-specific MtENOD11 expression in roots of Medicago truncatula.

Using dual cultures of arbuscular mycorrhizal (AM) fungi andMedicago truncatula separated by a physical barrier, we demonstrate that hyphae from germinating spores produce a diffusible factor that is perceived by roots in the absence of direct physical contact. This AM factor elicits expression of the Nod factor-inducible gene MtENOD11, visualized using a pMtENOD11-gusA reporter. Transgene induction occurs primarily in the root cortex, with expression stretching from the zone of root hair emergence to the region of mature root hairs. All AM fungi tested (Gigaspora rosea,Gigaspora gigantea, Gigaspora margarita, and Glomus intraradices) elicit a similar response, whereas pathogenic fungi such as Phythophthora medicaginis, Phoma medicaginis var pinodella andFusarium solani f.sp. phaseoli do not, suggesting that the observed root response is specific to AM fungi. Finally, pMtENOD11-gusA induction in response to the diffusible AM fungal factor is also observed with all threeM. truncatulaNod−/Myc− mutants (dmi1,dmi2, and dmi3), whereas the same mutants are blocked in their response to Nod factor. This positive response of the Nod−/Myc− mutants to the diffusible AM fungal factor and the different cellular localization of pMtENOD11-gusA expression in response to Nod factor versus AM factor suggest that signal transduction occurs via different pathways and that expression of MtENOD11 is differently regulated by the two diffusible factors.

The Medicago truncatula LysM-receptor Kinase Gene Family Includes NFP and New Nodule-expressed Genes

Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysine motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.

Expression Profiling in Medicago truncatula Identifies More Than 750 Genes Differentially Expressed during Nodulation, Including Many Potential Regulators of the Symbiotic Program

In this study, we describe a large-scale expression-profiling approach to identify genes differentially regulated during the symbiotic interaction between the model legume Medicago truncatula and the nitrogen-fixing bacterium Sinorhizobium meliloti. Macro- and microarrays containing about 6,000 probes were generated on the basis of three cDNA libraries dedicated to the study of root symbiotic interactions. The experiments performed on wild-type and symbiotic mutant material led us to identify a set of 756 genes either up- or down-regulated at different stages of the nodulation process. Among these, 41 known nodulation marker genes were up-regulated as expected, suggesting that we have identified hundreds of new nodulation marker genes. We discuss the possible involvement of this wide range of genes in various aspects of the symbiotic interaction, such as bacterial infection, nodule formation and functioning, and defense responses. Importantly, we found at least 13 genes that are good candidates to play a role in the regulation of the symbiotic program. This represents substantial progress toward a better understanding of this complex developmental program.

Medicago LYK3, an Entry Receptor in Rhizobial Nodulation Factor Signaling

Rhizobia secrete nodulation (Nod) factors, which set in motion the formation of nitrogen-fixing root nodules on legume host plants. Nod factors induce several cellular responses in root hair cells within minutes, but also are essential for the formation of infection threads by which rhizobia enter the root. Based on studies using bacterial mutants, a two-receptor model was proposed, a signaling receptor that induces early responses with low requirements toward Nod factor structure and an entry receptor that controls infection with more stringent demands. Recently, putative Nod factor receptors were shown to be LysM domain receptor kinases. However, mutants in these receptors, in both Lotus japonicus (nfr1 and nfr5) and Medicago truncatula (Medicago; nfp), do not support the two-receptor model because they lack all Nod factor-induced responses. LYK3, the putative Medicago ortholog of NFR1, has only been studied by RNA interference, showing a role in infection thread formation. Medicago hair curling (hcl) mutants are unable to form curled root hairs, a step preceding infection thread formation. We identified the weak hcl-4 allele that is blocked during infection thread growth. We show that HCL encodes LYK3 and, thus, that this receptor, besides infection, also controls root hair curling. By using rhizobial mutants, we also show that HCL controls infection thread formation in a Nod factor structure-dependent manner. Therefore, LYK3 functions as the proposed entry receptor, specifically controlling infection. Finally, we show that LYK3, which regulates a subset of Nod factor-induced genes, is not required for the induction of NODULE INCEPTION.

The hrp gene locus of Pseudomonas solanacearum, which controls the production of a type III secretion system, encodes eight proteins related to components of the bacterial flagellar biogenesis complex

Five transcription units of the Pseudomonas solanacearum hrp gene cluster are required for the secretion of the HR‐inducing PopA1 protein. The nucleotide sequences of two of these, units 1 and 3, have been reported. Here, we present the nucleotide sequence of the three other transcription units, units 2, 4 and 7, which are together predicted to code for 15 hrp genes. This brings the total number of Hrp proteins encoded by these five transcription units to 20, including HrpB, the positive regulatory protein, and HpaP, which is apparently not required for plant interactions., Among the 18 other proteins, eight belong to protein families regrouping proteins involved in type III secretion pathways in animal and plant bacterial pathogens and in flagellum biogenesis, while two are related solely to proteins involved in secretion systems. For the various proteins found to be related to P. solanacearum Hrp proteins, those in plant‐pathogenic bacteria include proteins encoded by hrp genes. For Hrp‐related proteins of animal pathogens, those encoded by the spa and mxi genes of Shigella flexneri and of Salmonella typhimurium and by the ysc genes of Yersinia are involved in type III secretion pathways. Proteins involved in flagellum biogenesis, which are related to Hrp proteins of P. solanacearum, include proteins encoded by fli and fli genes of S. typhimurium, Bacillus subtils and Escherichia coli and by mop genes of Erwinia carotovora. P. solanacearum Hrp proteins were also found to be related to proteins of Rhizobium fredii involved in nodulation specificity.

Evidence that the hrpB gene encodes a positive regulator of pathogenicity genes from Pseudomonas solanacearum

The hrp gene cluster of Pseudomonas solanacearum GMI1000 strain encodes functions that are essential for pathogenicity on tomato and for the elicitation of the hypersensitive response on tobacco. In this study, we present the nucleotide sequence of one of the hrp genes (hrpB) located at the left‐hand end of the cluster and we show that hrpB encodes a positive regulator controlling the expression of hrp genes. hrpB has a coding capacity for a 477‐amino‐acid polypeptide, which shows significant similarity to several prokaryotic transcriptional activators including the AraC protein of Escherichia coli, the XylS protein of Pseudomonas putida and the VirF protein of Yersinia enterocolitica. The predicted hrpB gene product belongs to a family of bacterial regulators different from the previously described HrpS protein of the hrp gene cluster of Pseudomonas syringae pv. phaseolicola. Genetic evidence demonstrates that the hrpB gene product acts as a positive regulator of the expression in minimal medium of all but one of the putative transcription units of the hrp gene cluster and also controls the expression of genes located outside this cluster. We also show in this paper that the transcription of hrpB is induced in minimal medium and is partly autoregulated.

Lipo-chitooligosaccharide signaling in endosymbiotic plant-microbe interactions.

The arbuscular mycorrhizal (AM) and the rhizobia-legume (RL) root endosymbioses are established as a result of signal exchange in which there is mutual recognition of diffusible signals produced by plant and microbial partners. It was discovered 20 years ago that the key symbiotic signals produced by rhizobial bacteria are lipo-chitooligosaccharides (LCO), called Nod factors. These LCO are perceived via lysin-motif (LysM) receptors and activate a signaling pathway called the common symbiotic pathway (CSP), which controls both the RL and the AM symbioses. Recent work has established that an AM fungus, Glomus intraradices, also produces LCO that activate the CSP, leading to induction of gene expression and root branching in Medicago truncatula. These Myc-LCO also stimulate mycorrhization in diverse plants. In addition, work on the nonlegume Parasponia andersonii has shown that a LysM receptor is required for both successful mycorrhization and nodulation. Together these studies show that structurally related signals and the LysM receptor family are key components of both nodulation and mycorrhization. LysM receptors are also involved in the perception of chitooligosaccharides (CO), which are derived from fungal cell walls and elicit defense responses and resistance to pathogens in diverse plants. The discovery of Myc-LCO and a LysM receptor required for the AM symbiosis, therefore, not only raises questions of how legume plants discriminate fungal and bacterial endosymbionts but also, more generally, of how plants discriminate endosymbionts from pathogenic microorganisms using structurally related LCO and CO signals and of how these perception mechanisms have evolved.

hrp genes of Pseudomonas solanacearum are homologous to pathogenicity determinants of animal pathogenic bacteria and are conserved among plant pathogenic bacteria.

The majority of bacterial plant diseases are caused by members of three bacterial genera, Pseudomonas, Xanthomonas, and Erwinia. The identification and characterization of mutants that have lost the abilities to provoke disease symptoms on a compatible host and to induce a defensive hypersensitive reaction (HR) on an incompatible host have led to the discovery of clusters of hrp genes (hypersensitive reaction and pathogenicity) in phytopathogenic bacteria from each of these genera. Here, we report that predicted protein sequences of three hrp genes from Pseudomonas solanacearum show remarkable sequence similarity to key virulence determinants of animal pathogenic bacteria of the genus Yersinia. We also demonstrate DNA homologies between P. solanacearum hrp genes and hrp gene clusters of P. syringae pv. phaseolicola, Xanthomonas campestris pv. campestris, and Erwinia amylovora. By comparing the role of the Yersinia determinants in the control of the extracellular production of proteins required for pathogenicity, we propose that hrp genes code for an export system that might be conserved among many diverse bacterial pathogens of plants and animals but that is distinct from the general export pathway.

Transcriptional organization and expression of the large hrp gene cluster of Pseudomonas solanacearum.

Cloning and localized mutagenesis of the larger cluster of hrp genes of Pseudomonas solanacearum strain GMI1000 allowed the definition of the borders of this cluster, which now extends about 2 kb to the left of the insert of the previously described plasmid pVir2 (Boucher et al. 1987, J. Bacteriol. 169:5626-5632). The size of the cluster has also been expanded 3 kb to the right to include a region previously described as dsp; our present data demonstrate that insertions occurring in these 3 kb lead to leaky mutations affecting both pathogenicity on tomato and ability to induce the hypersensitive response (HR) on tobacco. Therefore, the size of the entire hrp gene cluster is estimated to be about 22 kb. The use of transposon Tn5-B20, which promotes transcriptional gene fusions, allowed us to demonstrate that the hrp gene cluster is organized in a minimum of six transcriptional units, which are transcribed when the culture is grown in minimal medium but are repressed during growth in rich medium or in the presence of peptone or Casamino Acids. The level of expression in minimal medium is modulated by the carbon source provided; pyruvate is the best inducer. Under these conditions the level of expression observed in vitro appears to be representative of the actual expression observed in planta.