Implicación del sistema de secreción de tipo 3 de Sinorhizobium (Ensifer) fredii HH103 en la modulación de la respuesta de defensa de Glycine max cv. Williams y estudio de los efectores específicos secretados a través de este sistema

Plants that interact with pathogenic bacteria in their natural environments have developed barriers to block or contain the infection. Phytopathogenic bacteria have evolved mechanisms to subvert these defenses and promote infection. Thus, some Gram-negative phytopathogenic bacteria use the type 3 secretion system (T3SS) to deliver proteins, called effectors, directly into the cytoplasm of the host cells. These effectors suppress plant defense responses to promote infection and growth of the pathogen (Galan and Collmer, 1999). The T3SS has also been found in some symbiotic rhizobial strains and the secreted effectors, collectively known as nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. Sinorhizobium (Ensifer) fredii HH103 is a broad-host range bacterium able to nodulate dozens of legumes including soybean, which is considered its natural host plant. This bacterium secretes at least eight proteins through the T3SS in response to inducer flavonoids (Rodrigues et al., 2007) and the synthesis and secretion of Nops is controlled by the T3SS transcriptional regulator TtsI whose transcription is NodD- and flavonoids-dependent (Deakin and Broughton, 2009). In this thesis we show that the inactivation of the Ensifer (Sinorhizobium) fredii HH103 T3SS negatively affect soybean nodulation very early in the symbiotic process, which is associated with a reduction of the expression of early nodulation genes. This symbiotic phenotype could be the consequence of the bacterial triggering of soybean defense responses associated to the production of salicylic acid (SA) and the impairment of the T3SS mutant to suppress these responses. Interestingly, the early induction of the transcription of GmMPK4, which negatively regulates SA accumulation and defense responses in soybean, in plants inoculated with HH103 could be associated to the differential defense responses induced by the parental and the T3SS mutant strain (Jiménez-Guerrero et al., 2015). S. fredii HH103 secretes at least eight Nops through the T3SS. Some of them cannot be considered real effectors, since they are components of the extracellular appendages of the T3SS machinery. In this thesis, we described for the first time a new Rhizobium-specific effector, which we have called NopI. This effector, like NopL and NopP, was Rhizobium-specific and could be of great importance in the symbiosis with Glycine max (soybean) cv. Williams and Vigna unguiculata. Besides, while inactivation of nopL or nopP was beneficial for symbiosis with these plants, the absence of both NopL and NopP was detrimental, suggesting that these effectors could exert complementary functions in the symbiotic process. We also confirmed that the expression of both nopL and nopI was regulated by inducer flavonoids and by the transcriptional regulators NodD1 and TtsI. In addition, translocation of NopL within soybean root cells was confirmed by the adenylate cyclase assay. Furthermore, we characterized the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in soybean nodules and nodulation assays showed that NopC exerted a positive effect on symbiosis with soybean. Finally, adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103 and not a component of the T3SS machinery. NopL and NopP effectors are phosphorylated by plant kinases, but their exact function in symbiosis is yet unknown (Deakin and Broughton, 2009). However, some results indicate that NopL could be involved in the modulation of the host MAPK signaling and in the suppression of premature senescence of nodules. NopP is also phosphorylated by plant kinases but its exact function in symbiosis is still unknown. However, inactivation of the S. fredii HH103 nopP gene causes an increase in the number of nodules formed in soybean (Deakin and Broughton, 2009). In this thesis, we studied the function of the Rhizobium-specific effectors NopL and NopP secreted by S. fredii HH103 in the symbiosis with soybean, which is considered its natural host plant. Both NopL and NopP were phosphorylated by soybean root kinases and the phosphorylation cascade was Ca2+- and calmodulin-dependent. While the signaling pathway that culminates in the phosphorylation of NopL included ser/thr and MAPKK kinases, in the case of NopP this pathway was composed of ser/thr and tyr kinases but not MAPKK kinases. Transient expression in Nicotiana benthamiana leaves of both nopL and nopP fused to YFP and further confocal imaging indicated that these effectors localized to the nucleus of the host cell and accumulate in nuclear foci, suggesting a possible role in plant gene regulation or responses to DNA stress. In this sense, the use of a yeast based array to determine functions of effectors indicated that NopP could be involved in microtubule-related processes and nuclear localization and migration. Finally, co-immunoprecipitation analyses of N. benthamiana NopL- and NopP-interacting proteins showed that NopL binds to proteins related to the plant immune response and also with calreticulin and NopP interacts with proteins related to nucleic acids (e. g. histone H4) or proteins related to plant immunity (GRAS2 transcription factors or cyclophilin 40).Premio Extraordinario de Doctorado U

The Rhizobial Type 3 Secretion System: The Dr. Jekyll and Mr. Hyde in the Rhizobium–Legume Symbiosis

Rhizobia are soil bacteria that can establish a symbiotic association with legumes. As a result, plant nodules are formed on the roots of the host plants where rhizobia differentiate to bacteroids capable of fixing atmospheric nitrogen into ammonia. This ammonia is transferred to the plant in exchange of a carbon source and an appropriate environment for bacterial survival. This process is subjected to a tight regulation with several checkpoints to allow the progression of the infection or its restriction. The type 3 secretion system (T3SS) is a secretory system that injects proteins, called effectors (T3E), directly into the cytoplasm of the host cell, altering host pathways or suppressing host defense responses. This secretion system is not present in all rhizobia but its role in symbiosis is crucial for some symbiotic associations, showing two possible faces as Dr. Jekyll and Mr. Hyde: it can be completely necessary for the formation of nodules, or it can block nodulation in different legume species/cultivars. In this review, we compile all the information currently available about the effects of different rhizobial effectors on plant symbiotic phenotypes. These phenotypes are diverse and highlight the importance of the T3SS in certain rhizobium–legume symbioses.Ministerio de Ciencia e Innovación PID2019-107634RB-I00Junta de Andalucía P20_00185Universidad de Sevilla FEDER-US 1259948, FEDER-US 125054

NopC is a rhizobium-specific type 3 secretion system effector secreted by sinorhizobium (ensifer) fredii HH103

Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in hostrange determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1-and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103Junta de Andalucía P11-CVI-7050Ministerio de Economía y Competitividad AGL2012-38831Universidad de Sevill

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

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

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-

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

A transcriptomic analysis of the effect of genistein on Sinorhizobium fredii HH103 reveals novel rhizobial genes putatively involved in symbiosis

Sinorhizobium fredii HH103 is a rhizobial soybean symbiont that exhibits an extremely broad host-range. Flavonoids exuded by legume roots induce the expression of rhizobial symbiotic genes and activate the bacterial protein NodD, which binds to regulatory DNA sequences called nod boxes (NB). NB drive the expression of genes involved in the production of molecular signals (Nod factors) as well as the transcription of ttsI, whose encoded product binds to tts boxes (TB), inducing the secretion of proteins (effectors) through the type 3 secretion system (T3SS). In this work, a S. fredii HH103 global gene expression analysis in the presence of the flavonoid genistein was carried out, revealing a complex regulatory network. Three groups of genes differentially expressed were identified: i) genes controlled by NB, ii) genes regulated by TB, and iii) genes not preceded by a NB or a TB. Interestingly, we have found differentially expressed genes not previously studied in rhizobia, being some of them not related to Nod factors or the T3SS. Future characterization of these putative symbiotic-related genes could shed light on the understanding of the complex molecular dialogue established between rhizobia and legumes.España, Ministerio de Economía y Competitividad BIO2011-30229-C01España, Ministerio de Economía y Competitividad AGL2012-38831Junta de Andalucía, P11-CVI-7050Junta de Andalucía P11-CVI-750

Natural variation in a short region of the Acidovorax citrulli type III‐secreted effector AopW1 is associated with differences in cytotoxicity and host adaptation

Bacterial fruit blotch, caused by Acidovorax citrulli, is a serious disease of melon and watermelon. The strains of the pathogen belong to two major genetic groups: group I strains are strongly associated with melon, while group II strains are more aggressive on watermelon. A. citrulli secretes many protein effectors to the host cell via the type III secretion system. Here we characterized AopW1, an effector that shares similarity to the actin cytoskeleton-disrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, showing 14 amino acid differences between group I and II variants. We show that group I AopW1 is more toxic to yeast and Nicotiana benthamiana cells than group II AopW1, having stronger actin filament disruption activity, and increased ability to induce cell death and reduce callose deposition. We further demonstrated the importance of some amino acid positions within the HVR for AopW1 cytotoxicity. Cellular analyses revealed that AopW1 also localizes to the endoplasmic reticulum, chloroplasts, and plant endosomes. We also show that overexpression of the endosome-associated protein EHD1 attenuates AopW1-induced cell death and increases defense responses. Finally, we show that sequence variation in AopW1 plays a significant role in the adaptation of group I and II strains to their preferred hosts, melon and watermelon, respectively. This study provides new insights into the HopW1 family of bacterial effectors and provides first evidence on the involvement of EHD1 in response to biotic stress.United States-Israel Binational Agriculture Research and Development (BARD) Fund S-5023-17

Rice and bean AHL-mimic quorum-sensing signals specifically interfere with the capacity to form biofilms by plant-associated bacteria

Many bacteria regulate their gene expression in response to changes in their population density in a process called quorum sensing (QS), which involves communication between cells mediated by small diffusible signal molecules termed autoinducers. n-acyl-homoserine-lactones (AHLs) are the most common autoinducers in proteobacteria. QS-regulated genes are involved in complex interactions between bacteria of the same or different species and even with some eukaryotic organisms. Eukaryotes, including plants, can interfere with bacterial QS systems by synthesizing molecules that interfere with bacterial QS systems. In this work, the presence of AHL-mimic QS molecules in diverse Oryza sativa (rice) and Phaseolus vulgaris (bean) plant-samples were detected employing three biosensor strains. A more intensive analysis using biosensors carrying the lactonase enzyme showed that bean and rice seed-extract contain molecules that lack the typical lactone ring of AHLs. Interestingly, these molecules specifically alter the QS-regulated biofilm formation of two plant-associated bacteria, Sinorhizobium fredii SMH12 and Pantoea ananatis AMG501, suggesting that plants are able to enhance or to inhibit the bacterial QS systems depending on the bacterial strain. Further studies would contribute to a better understanding of plant–bacteria relationships at the molecular level