Caracterización de genes de Rhizobium tropici CIAT 899 implicados en la biosíntesis de los factores de nodulación independiente de la activación por flavonoides e inducidos por estrés osmótico

En el establecimiento de la simbiosis rizobio-leguminosa, los flavonoides secretados por las raíces activan un regulador transcripcional (proteína NodD) que por unión a unas secuencias promotoras conservadas en los rizobios (nod-box) activan la transcripción de los genes conocidos como genes de nodulación (o genes nod). Estos genes codifican para unas proteínas que están implicadas en la biosíntesis y secreción de una molécula de naturaleza lipoquitooligosacarídica conocida como factor de nodulación (o factor Nod). Este factor inicia una cascada regulatoria en la leguminosa que culmina con la formación de unas estructuras conocidas como nódulos en las raíces donde el rizobio se establecerá y fijará nitrógeno atmosférico hasta una forma utilizable por la planta. Rhizobium tropici CIAT 899 es una estirpe de rizobio que, además de tolerar diferentes estreses abióticos, sintetiza factores Nod bajo estrés salino. Así, en la presente Tesis Doctoral se ha estudiado la regulación de la síntesis de factores Nod en condiciones estresantes y no estresantes, y su implicación en el establecimiento de la simbiosis con diferentes leguminosas como Phaseolus vulgaris, Leucaena leucocephala, Lotus japonicus y Lotus burttii. Para ello, se llevaron a cabo estudios transcriptómicos donde se observó que los genes de nodulación de CIAT 899 se inducen tanto en presencia del flavonoide apigenina como en condiciones de estrés osmótico, ya sea estrés iónico salino o estrés no iónico en presencia de manitol. A continuación, se buscó qué genes están implicados en las vías de síntesis de factores Nod bajo estrés osmótico. Así, por mutagénesis dirigida se determinó que el regulador NodD2 y un regulador perteneciente a la familia AraC son esenciales en la activación de los genes de nodulación y en la síntesis de factores Nod bajo estrés osmótico. Por otro lado, también se identificó a un regulador transcripcional NrcR que, aunque promovió la activación de los genes nod en presencia de apigenina y estrés salino, también reprimió el número de decoraciones que presentaron los factores Nod, además de afectar a otros fenotipos como movilidad de superficie y producción de exopolisacáridos. También se estudió el papel de las tres copias del gen nodA presentes en el genoma de este rizobio. Así, por mutagénesis dirigida por deleción no polar se observó que NodA1 y NodA3 (este a pesar de que nodA3 carece de nod-box) son esenciales en la síntesis de factores Nod en presencia de apigenina y estrés salino, mientras que NodA2 por sí solo no es capaz de sintetizar estas moléculas en presencia de apigenina y solo lo hace ligeramente en presencia de estrés salino. Por último, en cuanto a los fenotipos simbióticos de los mutantes obtenidos anteriormente, se observó que NodD1 es esencial para establecer simbiosis con L. leucocephala y L. japonicus, mientras que ambos NodD1 y NodD2 lo son en las otras dos leguminosas de estudio: P. vulgaris y L. burttii. Por otro lado, se observó que NodA1 o NodA3 por sí solos son suficientes para establecer simbiosis con todas las leguminosas ensayadas, mientras que NodA2 por sí solo no puede establecer simbiosis con L. leucocephala y L. japonicus. Todos estos datos apuntan a que la activación de la síntesis de los factores Nod bajo estrés osmótico juega un papel en el establecimiento de la simbiosis con determinadas leguminosas. Futuros trabajos aportaran más datos acerca de este hecho.In the establishment of the rhizobia-legume symbiosis, the flavonoids secreted by the plant roots activate a transcriptional regulator (NodD protein) that activates the transcription of the nodulation genes (known as nod genes) by binding to the rhizobia-conserved promoter sequences (nod-boxes). The nod genes encode for proteins that are involved in the biosynthesis and secretion of the Nodulation factors (also known as Nod factors). These Nod factors start a regulatory pathway in the legume that culminates in the formation of structures on the roots called nodules, where the established rhizobia will fix atmospheric nitrogen into a reduced form appropriate for the plant metabolism. Rhizobium tropici CIAT 899 is a rhizobial strain which tolerates different abiotic stresses and it is able to synthesizes Nod factors under salt stress. Due to this fact, in this Thesis it has been studied the regulation of the synthesis of Nod factors under stressful and non-stressful conditions, and their involvement in the establishment of the symbiosis with different legumes including Phaseolus vulgaris, Leucaena leucocephala, Lotus japonicus and Lotus burttii. To achieve this goal, ARNseq studies were carried out. Here, it was observed that the CIAT 899 nod genes are induced both in the presence flavonoid apigenin and under osmotic stress, either salt stress or non-ionic stress produced by mannitol. Then, it has been studied which genes are involved in the activation of the Nod factor synthesis pathway under osmotic stress. Thus, directed mutagenesis determined that NodD2 and a new regulator belonging to the AraC family are essentials in the activation of the nod genes and therefore in the synthesis of Nod factors under osmotic stress. On the other hand, a new transcriptional regulator NrcR was also identified. This protein promoted the activation of the nod genes in the presence of apigenin and salt stress but at the same time, it repressed the number of decorations of the Nod factors. Moreover, NrcR regulates other phenotypes such as surface motility and exopolysaccharides production. In addition, it has been also studied the role of the three copies of the nodA gene which are located in the genome of CIAT 899. Thus, non-polar deletion mutagenesis demonstrated that NodA1 and NodA3 (nodA3 gene lacks nod-box upstream) were both required for the synthesis of Nod factors in the presence of apigenin and salt stress, while NodA2 is not able to synthesize these molecules in the presence of apigenin but it does slightly under salt stress. Finally, the symbiotic phenotypes of the mutants obtained before were studied. Here, it was observed that NodD1 is essential to establish symbiosis with L. leucocephala and L. japonicus, while both NodD1 and NodD2 are necessary for the establishment of the symbiosis in the other two legumes of this study: P. vulgaris and L. burttii. In addition, the presence of NodA1 or NodA3 is sufficient to establish symbiosis with all the legumes tested, while NodA2 by itself does not nodulate L. leucocephala and L. japonicus. All these data indicate that the activation of the synthesis of the Nod factors under osmotic stress plays a role in the establishment of the symbiosis with certain legumes. Future jobs will provide more information about this fact.Premio Extraordinario de Doctorado U

Medicago LINC complexes function in nuclear morphology, nuclear movement, and root nodule symbiosis 1[OPEN]

Nuclear movement is involved in cellular and developmental processes across eukaryotic life, often driven by Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, which bridge the nuclear envelope (NE) via the interaction of Klarsicht/ ANC-1/Syne-1 Homology (KASH) and Sad1/UNC-84 (SUN) proteins. Arabidopsis (Arabidopsis thaliana) LINC complexes are involved in nuclear movement and positioning in several cell types. Observations since the 1950s have described targeted nuclear movement and positioning during symbiosis initiation between legumes and rhizobia, but it has not been established whether these movements are functional or incidental. Here, we identify and characterize LINC complexes in the model legume Medicago truncatula. We show that LINC complex characteristics such as NE localization, dependence of KASH proteins on SUN protein binding for NE enrichment, and direct SUN-KASH binding are conserved between plant species. Using a SUN dominant-negative strategy, we demonstrate that LINC complexes are necessary for proper nuclear shaping and movement in Medicago root hairs, and are important for infection thread initiation and nodulation.National Science Foundation NSF-1440019, NSF-1613501Biotechnology and Biological Sciences Research Council BB/P007112/1European Molecular Biology Organization 699

OnfD, an AraC-Type Transcriptional Regulator Encoded by Rhizobium tropici CIAT 899 and Involved in Nod Factor Synthesis and Symbiosis

Rhizobium tropici CIAT 899 is a broad-host-range rhizobial strain that establishes symbiotic interactions with legumes and tolerates different environmental stresses such as heat, acidity, or salinity. This rhizobial strain produces a wide variety of symbiotically active nodulation factors (NF) induced not only by the presence of plant-released flavonoids but also under osmotic stress conditions through the LysR-type transcriptional regulators NodD1 (flavonoids) and NodD2 (osmotic stress). However, the activation of NodD2 under high-osmotic-stress conditions remains elusive. Here, we have studied the role of a new AraC-type regulator (named as OnfD) in the symbiotic interaction of R. tropici CIAT 899 with Phaseolus vulgaris and Lotus plants. We determined that OnfD is required under salt stress conditions for the transcriptional activation of the nodulation genes and therefore the synthesis and export of NF, which are required for a successful symbiosis with P. vulgaris. Moreover, using bacterial two-hybrid analysis, we demonstrated that the OnfD and NodD2 proteins form homodimers and OnfD/NodD2 form heterodimers, which could be involved in the production of NF in the presence of osmotic stress conditions since both regulators are required for NF synthesis in the presence of salt. A structural model of OnfD is presented and discussed. IMPORTANCE The synthesis and export of rhizobial NF are mediated by a conserved group of LysR-type regulators, the NodD proteins. Here, we have demonstrated that a non-LysR-type regulator, an AraC-type protein, is required for the transcriptional activation of symbiotic genes and for the synthesis of symbiotically active NF under salt stress conditions.España Ministerio de Economía y Competitividad (project AGL2016-77163-R

RNA-seq analysis of the Rhizobium tropici CIAT 899 transcriptome shows similarities in the activation patterns of symbiotic genes in the presence of apigenin and salt

Background Rhizobium tropici strain CIAT 899 establishes effective symbioses with several legume species, including Phaseolus vulgaris and Leucaena leucocephala. This bacterium synthesizes a large variety of nodulation factors in response to nod-gene inducing flavonoids and, surprisingly, also under salt stress conditions. The aim of this study was to identify differentially expressed genes in the presence of both inducer molecules, and analyze the promoter regions located upstream of these genes. Results Results obtained by RNA-seq analyses of CIAT 899 induced with apigenin, a nod gene-inducing flavonoid for this strain, or salt allowed the identification of 19 and 790 differentially expressed genes, respectively. Fifteen of these genes were up-regulated in both conditions and were involved in the synthesis of both Nod factors and indole-3-acetic acid. Transcription of these genes was presumably activated through binding of at least one of the five NodD proteins present in this strain to specific nod box promoter sequences when the bacterium was induced by both apigenin and salt. Finally, under saline conditions, many other transcriptional responses were detected, including an increase in the transcription of genes involved in trehalose catabolism, chemotaxis and protein secretion, as well as ribosomal genes, and a decrease in the transcription of genes involved in transmembrane transport. Conclusions To our knowledge this is the first time that a transcriptomic study shows that salt stress induces the expression of nodulation genes in the absence of flavonoids. Thus, in the presence of both nodulation inducer molecules, apigenin and salt, R. tropici CIAT 899 up-regulated the same set of symbiotic genes. It could be possible that the increases in the transcription levels of several genes related to nodulation under saline conditions could represent a strategy to establish symbiosis under abiotic stressing conditions.España, Ministerio de Economía y Competitividad AGL2012-1España, Junta de Andalucía P11-CVI-705

Osmotic stress activates nif and fix genes and induces the Rhizobium tropici CIAT 899 Nod factor production via NodD2 by up-regulation of the nodA2 operon and the nodA3 gene

The symbiosis between rhizobia and legumes is characterized by a complex molecular dialogue in which the bacterial NodD protein plays a major role due to its capacity to activate the expression of the nodulation genes in the presence of appropiate flavonoids. These genes are involved in the synthesis of molecules, the nodulation factors (NF), responsible for launching the nodulation process. Rhizobium tropici CIAT 899, a rhizobial strain that nodulates Phaseolus vulgaris, is characterized by its tolerance to multiple environmental stresses such as high temperatures, acidity or elevated osmolarity. This strain produces nodulation factors under saline stress and the same set of CIAT 899 nodulation genes activated by inducing flavonoids are also up-regulated in a process controlled by the NodD2 protein. In this paper, we have studied the effect of osmotic stress (high mannitol concentrations) on the R. tropici CIAT 899 transcriptomic response. In the same manner as with saline stress, the osmotic stress mediated NF production and export was controlled directly by NodD2. In contrast to previous reports, the nodA2FE operon and the nodA3 and nodD1 genes were up-regulated with mannitol, which correlated with an increase in the production of biologically active NF. Interestingly, in these conditions, this regulatory protein controlled not only the expression of nodulation genes but also the expression of other genes involved in protein folding and synthesis, motility, synthesis of polysaccharides and, surprinsingly, nitrogen fixation. Moreover, the non-metabolizable sugar dulcitol was also able to induce the NF production and the activation of nod genes in CIAT 899.España, MINECO AGL2016-77163-

The Rhizobium tropici CIAT 899 NodD2 protein promotes symbiosis and extends rhizobial nodulation range by constitutive nodulation factor synthesis

In the symbiotic associations between rhizobia and legumes, the NodD regulators orchestrate the transcription of the specifc nodulation genes. This set of genes is involved in the synthesis of nodulation factors, which are responsible for initiating the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. Among the fve NodD regulators present in this rhizobium, only NodD1 and NodD2 seem to have a role in the symbiotic process. However, the individual role of each NodD in the absence of the other proteins has remained elusive. In this work, we show that the CIAT 899 NodD2 does not require activation by inducers to promote the synthesis of nodulation factors. A CIAT 899 strain overexpressing nodD2, but lacking all additional nodD genes, can nodulate three different legumes as effciently as the wild type. Interestingly, CIAT 899 NodD2- mediated gain of nodulation can be extended to another rhizobial species, since its overproduction in Sinorhizobium fredii HH103 not only increases the number of nitrogen-fxing nodules in two host legumes but also results in nodule development in incompatible legumes. These fndings potentially open exciting opportunities to develop rhizobial inoculants and increase legume crop production.Spanish Ministry of Science and Innovation funded by MCIN/AEI/10.13039/501100011033 AGL2016-77163-R and PID2019- 107634RB-I00Ministerio de Economía y Competitividad FPU18/0624

Plant growth promotion in cereal and leguminous agricultural important plants: From microorganism capacities to crop production

Plant growth-promoting rhizobacteria (PGPR) are free-living bacteria which actively colonize plant roots, exerting beneficial effects on plant development. The PGPR may (i) promote the plant growth either by using their own metabolism (solubilizing phosphates, producing hormones or fixing nitrogen) or directly affecting the plant metabolism (increasing the uptake of water and minerals), enhancing root development, increasing the enzymatic activity of the plant or “helping” other beneficial microorganisms to enhance their action on the plants; (ii) or may promote the plant growth by suppressing plant pathogens. These abilities are of great agriculture importance in terms of improving soil fertility and crop yield, thus reducing the negative impact of chemical fertilizers on the environment. The progress in the last decade in using PGPR in a variety of plants (maize, rice, wheat, soybean and bean) along with their mechanism of action are summarized and discussed here

The Rhizobium tropici CIAT 899 NodD2 protein regulates the production of Nod factors under salt stress in a flavonoidindependent manner

In the symbiotic associations between rhizobia and legumes, NodD promotes the expression of the nodulation genes in the presence of appropriate flavonoids. This set of genes is implied in the synthesis of Nodulation factors, which are responsible for launching the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. This strain produces Nodulation factors under abiotic stress such as acidity or high concentration of salt. Genome sequencing of CIAT 899 allowed the identification of five nodD genes. Whereas NodD1 is essential to nodulate Leucaena leucocephala, Lotus japonicus and Macroptilium atropurpureum, symbiosis with P. vulgaris and Lotus burtii decreased the nodule number but did not abolish the symbiotic process when NodD1 is absent. Nodulation factor synthesis under salt stress is not regulated by NodD1. Here we confirmed that NodD2 is responsible for the activation of the CIAT 899 symbiotic genes under salt stress. We have demonstrated that NodD1 and NodD2 control the synthesis of the Nod factor necessary for a successful symbiosis with P. vulgaris and L. burtii. This is the first time that NodD is directly implied in the activation of the symbiotic genes under an abiotic stress

Transcriptomic studies of the effect of nod gene-inducing molecules in rhizobia: Different weapons, one purpose

Simultaneous quantification of transcripts of the whole bacterial genome allows the analysis of the global transcriptional response under changing conditions. RNA-seq and microarrays are the most used techniques to measure these transcriptomic changes, and both complement each other in transcriptome profiling. In this review, we exhaustively compiled the symbiosis-related transcriptomic reports (microarrays and RNA sequencing) carried out hitherto in rhizobia. This review is specially focused on transcriptomic changes that takes place when five rhizobial species, Bradyrhizobium japonicum (=diazoefficiens) USDA 110, Rhizobium leguminosarum biovar viciae 3841, Rhizobium tropici CIAT 899, Sinorhizobium (=Ensifer) meliloti 1021 and S. fredii HH103, recognize inducing flavonoids, plant-exuded phenolic compounds that activate the biosynthesis and export of Nod factors (NF) in all analysed rhizobia. Interestingly, our global transcriptomic comparison also indicates that each rhizobial species possesses its own arsenal of molecular weapons accompanying the set of NF in order to establish a successful interaction with host legumes.Ministerio de Economía y Competitividad BIO2016-78409-R, AGL2016-77163-

The symbiotic biofilm of Sinorhizobium fredii SMH12, necessary for successful colonization and symbiosis of glycine max cv osumi, is regulated by quorum sensing systems and inducing Flavonoids via NodD1

Bacterial surface components, especially exopolysaccharides, in combination with bacterial Quorum Sensing signals are crucial for the formation of biofilms in most species studied so far. Biofilm formation allows soil bacteria to colonize their surrounding habitat and survive common environmental stresses such as desiccation and nutrient limitation. This mode of life is often essential for survival in bacteria of the genera Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Rhizobium. The role of biofilm formation in symbiosis has been investigated in detail for Sinorhizobium meliloti and Bradyrhizobium japonicum. However, for S. fredii this process has not been studied. In this work we have demonstrated that biofilm formation is crucial for an optimal root colonization and symbiosis between S. fredii SMH12 and Glycine max cv Osumi. In this bacterium, nod-gene inducing flavonoids and the NodD1 protein are required for the transition of the biofilm structure from monolayer to microcolony. Quorum Sensing systems are also required for the full development of both types of biofilms. In fact, both the nodD1 mutant and the lactonase strain (the lactonase enzyme prevents AHL accumulation) are defective in soybean root colonization. The impairment of the lactonase strain in its colonization ability leads to a decrease in the symbiotic parameters. Interestingly, NodD1 together with flavonoids activates certain quorum sensing systems implicit in the development of the symbiotic biofilm. Thus, S. fredii SMH12 by means of a unique key molecule, the flavonoid, efficiently forms biofilm, colonizes the legume roots and activates the synthesis of Nod factors, required for successfully symbiosis