A szimbiotikus gümő bakteriális inváziójában és a szimbioszóma mükődésében résztvevő növényi gének azonosítása. = Genetic analysis of symbiosome initiation and development in legume nodulation.

A Sinorhizobium meliloti és a Medicago truncatula között kialakuló szimbiotikus nitrogénkötésen hibás növényi mutánsok fenotípusos vizsgálata során megállapítottuk, hogy a 9F és 14S mutánsok a szimbiotikus gümő inváziójában, a többi mutáns a bakteroid átalakulásban vagy pedig a gümő működésében szenvedett hibát. A 9F mutáns mikroszkópos vizsgálata azt is feltárta, hogy a baktériumok az infekciós fonalakból nem voltak képesek lefűződni és a gümő sejtjeibe jutni. A 7Y mutánsban erős autofluoreszcenciát mutató polifenol képződést figyeltünk meg, ami a beinduló patogén válaszreakció eredménye. Megállapítottuk, hogy az 5L és 11S mutánsok egymás alléljai, a 13U a már korábban más kutató csoportok által azonosított dnf5 mutáns, a 6V pedig a dnf7 allélja, többi mutáns pedig különálló komplementációs csoportot képez. Térképezésen alapuló génizolálással klónoztuk a 9F mutánsban hibát szenvedett IPD3 gént. Megállapítottuk, hogy a 9F a mycorrhiza szimbiótikus kapcsolatban is hibát szenvedett, azaz az IPD3 gén a szimbiotikus partnerek befogadásában játszik szerepet. Vizsgálataink kiderítették, hogy a 9F mutánsban a Nod faktor szignálút hibásan működik. Genetikai térképezés és gén chip alapú módszer kombinálásával azonosítottuk az 5L és 11S mutánsokban hibás szulfát transzporter gént. A6V, 12AA és 13U mutánsok esetében is azonosítottuk a fenotípusért felelős deléciókat, genetikai térképezéssel meghatároztuk a 7Y és 14S növényekben hibát szenvedett géneket tartalmazó genomi régiókat. | The phenotypic characterization of the ineffective mutants impaired in the symbiotic interaction between Sinorhizobium meliloti and Medicago truncatula showed that nodules on 9F and 14S mutants were impaired in the invasion of the nodule cells by bacteria. Aberrant infection process was detected in mutant 9F because bacteria were not released from the infection threads. The other mutants showed defects in bacteroid differentiation, displayed disintegration of the symbiotic structures and were impaired in functioning of the symbiotic nodule. Mutant 7Y showed high accumulation of polyphenolic compounds indicating strong defense reaction against rhizobia. We identified allelic relationship between 5L and 11S, 13U and dnf5, 6V and dnf7 fix- mutants. Positional cloning identified that the IPD3 gene is impaired in the 9F mutant. Further characterization showed that the effectiveness of the AM colonization significantly reduced in the 9F mutant indicating that IPD3 functions in the accommodation of the symbiotic partners in the host cells. Expression data suggested that IPD3 acts in the NF signal pathway. Combining genetic mapping and gene chip-based cloning we identified that the SST1 gene was impaired in the 5L and 11S mutants. We also identified the deletions which are probably responsible for the mutant phenotype in the 6V, 12AA and 13U mutants. Map-based cloning experiments also determined the genomic regions containing the mutated Fix genes in 7Y and 14S mutant plants

A lucerna szimbiotikus nitrogénkötés kialakításában kulcsfontosságú szerepet játszó receptor fehérje molekuláris jellemzése = Molecular characterization of a receptor protein playing an essential role in symbiotic nitrogen fixation of alfalfa.

A pillangósvirágú növények egyedülálló szimbiotikus kapcsolatban állnak a rizóbium baktériumokkal, ami egy speciális növényi szerv, a gyökérgümő képződéséhez vezet, ahol a légköri nitrogénkötés történik. A növény-baktérium kapcsolat kölcsönös jelcserét indít be a gümőképződés fejlődési programban. Napjainkban több protein-kinázt azonosítottak, amelyek feltehetőleg a nodulációhoz vezető szignál transzdukcióban vesznek részt. A szignál kaszkád a Nod faktor felismerés és továbbítás (NORK rendszer) során vezet el a nodulációig. A NORK rendszer egyik eleme a NORK (NOdulation Reception Kinase) fehérje, amelyik több más kinázzal együttműködve juttatja el a jelet a Nod-faktor érzékelésétől a gümő képzéséig. Izoláltuk a NORK membrán fehérjét lucerna gyökér extraktumból. Két-dimenziós elektroforézissel elválasztottuk és immunoblotton azonosítottuk. Eredményeink alátámasztására, epitop jelzett NORK fehérje tranziens expresszióját igazoltuk dohánylevélben Agrobacterium tumefaciens infiltrálás segítségével. A NORK fehérje szerepének és funkciójának tisztázására a NORK rendszer más kináz fehérjéivel való kapcsolatát (LYK3, NFP) is vizsgáltuk fehérje-fehérje kapcsolatok kimutatásával. Az LRR1 típusú növényi receptor-szerű kinázok (RLK) filogenetikai analízise alapján arra következthetünk, hogy a NORK/SYMRK fehérje homológjai egy jól elkülönülő monofiletikus csoportot alkotnak az LRR1 RLK családon belül, és pillangósvirágú növényeken kívüli taxonokban is megtalálhatók. | Legume roots engage in a unique symbiotic relationship with rhizobia, in which special plant organs, the nodules, are formed and support nitrogen fixation. The plant-bacteria interactions involve the exchange of signals that trigger specific cellular and developmental programs in both. Recently several protein kinases have identified, which are participating in specialized signal transduction pathways that culminate in the induction of a transcriptional program of nodulation.The signal cascade leading to nodulation is part of the Nod-factor perception/ transduction system (the NORK system) active in legume roots. The identified NOdulation Receptor Kinase, NORK, is an integral component of the signalling pathway, in which the coordinated interaction of the components is needed for Nod-factor signal perception and transduction. Isolation of NORK from root extract of alfalfa, separation by 2D and detection on immunoblot were done. Epitop tagged NORK were transiently expressed in tobacco leaves by Agrobacterium tumefaciens infiltration. To justify interactions with other kinases in the NORK system (LYK3, NFP), we investigated the role and function of NORK by protein-protein interaction studies. Phylogenetic analysis of the LRR1-type plant receptor like kinases (RLK) revealed that homologues of the NORK/SYMRK protein constitute a single monophyletic clade within the LRR1 RLK family and members of this group exist in several non-legume plants

Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant

Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula

The bs5 allele of the susceptibility gene Bs5 of pepper (Capsicum annuum L.) encoding a natural deletion variant of a CYSTM protein conditions resistance to bacterial spot disease caused by Xanthomonas species

The recessive bs5 gene of pepper (Capsicum annuum L.) conditions a non-hypersensitive resistance trait, character- ized by a slightly swollen, pale green, photosynthetically active leaf tissue, following Xanthomonas euvesicatoria infection. The isolation of the bs5 gene by map-based cloning revealed that the bs5 protein was shorter by 2 amino acids as compared to the wild type Bs5 protein. The natural 2 amino acid deletion occurred in the cysteine-rich transmembrane domain of the tail-anchored (TA) protein, Ca_CYSTM1. The protein products of the wild type Bs5 and mutant bs5 genes were shown to be located in the cell membrane, indicating an unknown function in this membrane compartment. Successful infection of the Bs5 pepper lines was abolished by the 6 bp deletion in the TM encoding domain of the Ca_CYSTM1 gene in bs5 homozygotes, suggesting, that the resulting resistance might be explained by the lack of entry of the Xanthomonas specific effector molecules into the plant cells

Amino Acid Polymorphisms in the VHIID Conserved Motif of Nodulation Signaling Pathways 2 Distinctly Modulate Symbiotic Signaling and Nodule Morphogenesis in Medicago truncatula

Legumes establish an endosymbiotic association with nitrogen-fixing soil bacteria. Following the mutual recognition of the symbiotic partner, the infection process is controlled by the induction of the signaling pathway and subsequent activation of symbiosis-related host genes. One of the protein complexes regulating nitrogen-fixing root nodule symbiosis is formed by GRAS domain regulatory proteins Nodulation Signaling Pathways 1 and 2 (NSP1 and NSP2) that control the expression of several early nodulation genes. Here, we report on a novel point mutant allele (nsp2-6) affecting the function of the NSP2 gene and compared the mutant with the formerly identified nsp2-3 mutant. Both mutants carry a single amino acid substitution in the VHIID motif of the NSP2 protein. We found that the two mutant alleles show dissimilar root hair response to bacterial infection. Although the nsp2-3 mutant developed aberrant infection threads, rhizobia were able to colonize nodule cells in this mutant. The encoded NSP2 proteins of the nsp2-3 and the novel nsp2 mutants interact with NSP1 diversely and, as a consequence, the activation of early nodulin genes and nodule organogenesis are arrested in the new nsp2 allele. The novel mutant with amino acid substitution D244H in NSP2 shows similar defects in symbiotic responses as a formerly identified nsp2-2 mutant carrying a deletion in the NSP2 gene. Additionally, we found that rhizobial strains induce delayed nodule formation on the roots of the ns2-3 weak allele. Our study highlights the importance of a conserved Asp residue in the VHIID motif of NSP2 that is required for the formation of a functional NSP1-NSP2 signaling module. Furthermore, our results imply the involvement of NSP2 during differentiation of symbiotic nodule cells

The Medicago truncatula Vacuolar iron Transporter-Like proteins VTL4 and VTL8 deliver iron to symbiotic bacteria at different stages of the infection process

The symbiotic relationship between legumes and rhizobium bacteria in root nodules has a high demand for iron, and questions remain regarding which transporters are involved. Here, we characterize two nodule-specific Vacuolar iron Transporter-Like (VTL) proteins in Medicago truncatula. Localization of fluorescent fusion proteins and mutant studies were carried out to correlate with existing RNA-seq data showing differential expression of VTL4 and VTL8 during early and late infection, respectively. The vtl4 insertion lines showed decreased nitrogen fixation capacity associated with more immature nodules and less elongated bacteroids. A mutant line lacking the tandemly-arranged VTL4-VTL8 genes, named 13U, was unable to develop functional nodules and failed to fix nitrogen, which was almost fully restored by expression of VTL8 alone. Using a newly developed lux reporter to monitor iron status of the bacteroids, a moderate decrease in luminescence signal was observed invtl4mutant nodules and a strong decrease in 13U nodules. Iron transport capability of VTL4 and VTL8 was shown by yeast complementation. These data indicate that VTL8, the closest homologue of SEN1 in Lotus japonicus, is the main route for delivering iron to symbiotic rhizobia. We propose that a failure in iron protein maturation leads to early senescence of the bacteroids

The Medicago truncatula Vacuolar iron Transporter-Like proteins VTL4 and VTL8 deliver iron to rhizobium bacteria at different stages of the infection process

The symbiotic relationship between legumes and rhizobium bacteria in root nodules has a high demand for iron, and questions remain regarding which transporters are involved. Here, we characterize two nodule-specific Vacuolar iron Transporter-Like (VTL) proteins in Medicago truncatula. Localization of fluorescent fusion proteins and mutant studies were carried out to correlate with existing RNA-seq data showing differential expression of VTL4 and VTL8 during early and late infection, respectively. The vtl4 insertion lines showed decreased nitrogen fixation capacity associated with more immature nodules and less elongated bacteroids. A mutant line lacking the tandemly-arranged VTL4–VTL8 genes, named 13U, was unable to develop functional nodules and failed to fix nitrogen, which was almost fully restored by expression of VTL8 alone. Using a newly developed lux reporter to monitor iron status of the bacteroids, a moderate decrease in luminescence signal was observed in vtl4 mutant nodules and a strong decrease in 13U nodules. Iron transport capability of VTL4 and VTL8 was shown by yeast complementation. These data indicate that VTL8, the closest homologue of SEN1 in Lotus japonicus, is the main route for delivering iron to symbiotic rhizobia. We propose that a failure in iron protein maturation leads to early senescence of the bacteroids

Host-secreted antimicrobial peptide enforces symbiotic selectivity in Medicago truncatula

Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula-Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix-). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host-strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation

The Medicago truncatula nodule‐specific cysteine‐rich peptides, NCR343 and NCR‐new35 are required for the maintenance of rhizobia in nitrogen‐fixing nodules

In the nodules of IRLC legumes, including Medicago truncatula, nitrogen-fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialized for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule-specific cysteine-rich (NCR) peptides, of which c. 700 are encoded in the M. truncatula genome but only few of them have been proved to be essential for nitrogen fixation. We carried out the characterization of the nodulation phenotype of three ineffective nitrogen-fixing M. truncatula mutants using confocal and electron microscopy, monitored the expression of defence and senescence-related marker genes, and analysed the bacteroid differentiation with flow cytometry. Genetic mapping combined with microarray- or transcriptome-based cloning was used to identify the impaired genes. Mtsym19 and Mtsym20 mutants are defective in the same peptide NCR-new35 and the lack of NCR343 is responsible for the ineffective symbiosis of NF-FN9363. We found that the expression of NCR-new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCRs. The fluorescent protein-tagged version of NCR343 and NCR-new35 localized to the symbiotic compartment. Our discovery added two additional members to the group of NCR genes essential for nitrogen-fixing symbiosis in M. truncatula