RESUMEN
BACKGROUND: Bacteria of the genus Xanthomonas cause economically significant diseases in various crops. Their virulence is dependent on the translocation of type III effectors (T3Es) into plant cells by the type III secretion system (T3SS), a process regulated by the master response regulator HrpG. Although HrpG has been studied for over two decades, its regulon across diverse Xanthomonas species, particularly beyond type III secretion, remains understudied. RESULTS: In this study, we conducted transcriptome sequencing to explore the HrpG regulons of 17 Xanthomonas strains, encompassing six species and nine pathovars, each exhibiting distinct host and tissue specificities. We employed constitutive expression of plasmid-borne hrpG*, which encodes a constitutively active form of HrpG, to induce the regulon. Our findings reveal substantial inter- and intra-specific diversity in the HrpG* regulons across the strains. Besides 21 genes directly involved in the biosynthesis of the T3SS, the core HrpG* regulon is limited to only five additional genes encoding the transcriptional activator HrpX, the two T3E proteins XopR and XopL, a major facility superfamily (MFS) transporter, and the phosphatase PhoC. Interestingly, genes involved in chemotaxis and genes encoding enzymes with carbohydrate-active and proteolytic activities are variably regulated by HrpG*. CONCLUSIONS: The diversity in the HrpG* regulon suggests that HrpG-dependent virulence in Xanthomonas might be achieved through several distinct strain-specific strategies, potentially reflecting adaptation to diverse ecological niches. These findings enhance our understanding of the complex role of HrpG in regulating various virulence and adaptive pathways, extending beyond T3Es and the T3SS.
Asunto(s)
Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Regulón , Xanthomonas , Xanthomonas/patogenicidad , Xanthomonas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Virulencia/genética , Transcriptoma , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Perfilación de la Expresión Génica , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Hereditary, or vertically-transmitted, symbioses affect a large number of animal species and some plants. The precise mechanisms underlying transmission of functions of these associations are often difficult to describe, due to the difficulty in separating the symbiotic partners. This is especially the case for plant-bacteria hereditary symbioses, which lack experimentally tractable model systems. Here, we demonstrate the potential of the leaf symbiosis between the wild yam Dioscorea sansibarensis and the bacterium Orrella dioscoreae (O. dioscoreae) as a model system for hereditary symbiosis. O. dioscoreae is easy to grow and genetically manipulate, which is unusual for hereditary symbionts. These properties allowed us to design an effective antimicrobial treatment to rid plants of bacteria and generate whole aposymbiotic plants, which can later be re-inoculated with bacterial cultures. Aposymbiotic plants did not differ morphologically from symbiotic plants and the leaf forerunner tip containing the symbiotic glands formed normally even in the absence of bacteria, but microscopic differences between symbiotic and aposymbiotic glands highlight the influence of bacteria on the development of trichomes and secretion of mucilage. This is to our knowledge the first leaf symbiosis where both host and symbiont can be grown separately and where the symbiont can be genetically altered and reintroduced to the host.
Asunto(s)
Dioscorea , Hojas de la Planta , Simbiosis , Dioscorea/microbiología , Dioscorea/genética , Hojas de la Planta/microbiologíaRESUMEN
The impact of host diversity on the genotypic and phenotypic evolution of broad-spectrum pathogens is an open issue. Here, we used populations of the plant pathogen Ralstonia pseudosolanacearum that were experimentally evolved on five types of host plants, either belonging to different botanical families or differing in their susceptibility or resistance to the pathogen. We investigated whether changes in transcriptomic profiles, associated with or independent of genetic changes, could occur during the process of host adaptation, and whether transcriptomic reprogramming was dependent on host type. Genomic and transcriptomic variations were established for 31 evolved clones that showed better fitness in their experimental host than the ancestral clone. Few genomic polymorphisms were detected in these clones, but significant transcriptomic variations were observed, with a large number of differentially expressed genes (DEGs). In a very clear way, a group of genes belonging to the network of regulation of the bacterial virulence such as efpR, efpH or hrpB, among others, were deregulated in several independent evolutionary lineages and appeared to play a key role in the transcriptomic rewiring observed in evolved clones. A double hierarchical clustering based on the 400 top DEGs for each clone revealed 2 major patterns of gene deregulation that depend on host genotype, but not on host susceptibility or resistance to the pathogen. This work therefore highlights the existence of two major evolutionary paths that result in a significant reorganization of gene expression during adaptive evolution and underscore clusters of co-regulated genes associated with bacterial adaptation on different host lines.
Asunto(s)
Ralstonia solanacearum , Humanos , Virulencia/genética , Ralstonia solanacearum/genética , Ralstonia/genética , Perfilación de la Expresión GénicaRESUMEN
Symbiotic nitrogen fixation (SNF) can play a key role in agroecosystems to reduce the negative impact of nitrogen fertilizers. Its efficiency is strongly affected by the combination of bacterial and plant genotypes, but the mechanisms responsible for the differences in the efficiency of rhizobium strains are not well documented. In Medicago truncatula, SNF has been mostly studied using model systems, such as M. truncatula A17 in interaction with Sinorhizobium meliloti Sm2011. Here we analyzed both the wild-type (wt) A17 and the Mtefd-1 mutant in interaction with five S. meliloti and two Sinorhizobium medicae strains. ETHYLENE RESPONSE FACTOR REQUIRED FOR NODULE DIFFERENTIATION (MtEFD) encodes a transcription factor, which contributes to the control of nodule number and differentiation in M. truncatula. We found that, in contrast to Sm2011, four strains induce functional (Fix+) nodules in Mtefd-1, although less efficient for SNF than in wt A17. In contrast, the Mtefd-1 hypernodulation phenotype is not strain-dependent. We compared the plant nodule transcriptomes in response to SmBL225C, a highly efficient strain with A17, versus Sm2011, in wt and Mtefd-1 backgrounds. This revealed faster nodule development with SmBL225C and early nodule senescence with Sm2011. These RNA sequencing analyses allowed us to identify candidate plant factors that could drive the differential nodule phenotype. In conclusion, this work shows the value of having a set of rhizobium strains to fully evaluate the biological importance of a plant symbiotic gene.
Asunto(s)
Medicago truncatula , Sinorhizobium meliloti , Sinorhizobium , Sinorhizobium/genética , Sinorhizobium meliloti/genética , Fijación del Nitrógeno/genética , Factores de Transcripción/genética , Simbiosis/genética , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/microbiologíaRESUMEN
Orobanche cumana Wall., sunflower broomrape, is one of the major pests for the sunflower crop. Breeding for resistant varieties in sunflower has been the most efficient method to control this parasitic weed. However, more virulent broomrape populations continuously emerge by overcoming genetic resistance. It is thus essential to identify new broomrape resistances acting at various stages of the interaction and combine them to improve resistance durability. In this study, 71 wild sunflowers and wild relatives accessions from 16 Helianthus species were screened in pots for their resistance to broomrape at the late emergence stage. From this initial screen, 18 accessions from 9 species showing resistance, were phenotyped at early stages of the interaction: the induction of broomrape seed germination by sunflower root exudates, the attachment to the host root and the development of tubercles in rhizotron assays. We showed that wild Helianthus accessions are an important source of resistance to the most virulent broomrape races, affecting various stages of the interaction: the inability to induce broomrape seed germination, the development of incompatible attachments or necrotic tubercles, and the arrest of emerged structure growth. Cytological studies of incompatible attachments showed that several cellular mechanisms were shared among resistant Helianthus species.
RESUMEN
Senescence determines plant organ lifespan depending on aging and environmental cues. During the endosymbiotic interaction with rhizobia, legume plants develop a specific organ, the root nodule, which houses nitrogen (N)-fixing bacteria. Unlike earlier processes of the legume-rhizobium interaction (nodule formation, N fixation), mechanisms controlling nodule senescence remain poorly understood. To identify nodule senescence-associated genes, we performed a dual plant-bacteria RNA sequencing approach on Medicago truncatula-Sinorhizobium meliloti nodules having initiated senescence either naturally (aging) or following an environmental trigger (nitrate treatment or salt stress). The resulting data allowed the identification of hundreds of plant and bacterial genes differentially regulated during nodule senescence, thus providing an unprecedented comprehensive resource of new candidate genes associated with this process. Remarkably, several plant and bacterial genes related to the cell cycle and stress responses were regulated in senescent nodules, including the rhizobial RpoE2-dependent general stress response. Analysis of selected core nodule senescence plant genes allowed showing that MtNAC969 and MtS40, both homologous to leaf senescence-associated genes, negatively regulate the transition between N fixation and senescence. In contrast, overexpression of a gene involved in the biosynthesis of cytokinins, well-known negative regulators of leaf senescence, may promote the transition from N fixation to senescence in nodules.
Asunto(s)
Medicago truncatula , Rhizobium , Regulación de la Expresión Génica de las Plantas , Medicago truncatula/metabolismo , Fijación del Nitrógeno/fisiología , Proteínas de Plantas/metabolismo , ARN de Planta/metabolismo , Rhizobium/genética , Nódulos de las Raíces de las Plantas/metabolismo , Simbiosis/genética , Transcriptoma/genéticaRESUMEN
Plant diseases are an important threat to food production. While major pathogenicity determinants required for disease have been extensively studied, less is known on how pathogens thrive during host colonization, especially at early infection stages. Here, we used randomly barcoded-transposon insertion site sequencing (RB-TnSeq) to perform a genome-wide screen and identify key bacterial fitness determinants of the vascular pathogen Xanthomonas campestris pv campestris (Xcc) during infection of the cauliflower host plant (Brassica oleracea). This high-throughput analysis was conducted in hydathodes, the natural entry site of Xcc, in xylem sap and in synthetic media. Xcc did not face a strong bottleneck during hydathode infection. In total, 181 genes important for fitness were identified in plant-associated environments with functional enrichment in genes involved in metabolism but only few genes previously known to be involved in virulence. The biological relevance of 12 genes was independently confirmed by phenotyping single mutants. Notably, we show that XC_3388, a protein with no known function (DUF1631), plays a key role in the adaptation and virulence of Xcc possibly through c-di-GMP-mediated regulation. This study revealed yet unsuspected social behaviors adopted by Xcc individuals when confined inside hydathodes at early infection stages.
Asunto(s)
Brassica , Xanthomonas campestris , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Brassica/microbiología , Enfermedades de las Plantas/microbiología , Virulencia/genética , Xilema/metabolismoRESUMEN
Rhizobium-legume nitrogen-fixing symbiosis involves the formation of a specific organ, the root nodule, which provides bacteria with the proper cellular environment for atmospheric nitrogen fixation. Coordinated differentiation of plant and bacterial cells is an essential step of nodule development, for which few transcriptional regulators have been characterized. Medicago truncatula ETHYLENE RESPONSE FACTOR REQUIRED FOR NODULE DIFFERENTIATION (MtEFD) encodes an APETALA2/ETHYLENE RESPONSIVE FACTOR (ERF) transcription factor, the mutation of which leads to both hypernodulation and severe defects in nodule development. MtEFD positively controls a negative regulator of cytokinin signaling, the RESPONSE REGULATOR 4 (MtRR4) gene. Here we showed that that the Mtefd-1 mutation affects both plant and bacterial endoreduplication in nodules, as well as the expression of hundreds of genes in young and mature nodules, upstream of known regulators of symbiotic differentiation. MtRR4 expressed with the MtEFD promoter complemented Mtefd-1 hypernodulation but not the nodule differentiation phenotype. Unexpectedly, a nonlegume homolog of MtEFD, AtERF003 in Arabidopsis (Arabidopsis thaliana), could efficiently complement both phenotypes of Mtefd-1, in contrast to the MtEFD paralog MtEFD2 expressed in the root and nodule meristematic zone. A domain swap experiment showed that MtEFD2 differs from MtEFD by its C-terminal fraction outside the DNA binding domain. Furthermore, clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) mutagenesis of MtEFD2 led to a reduction in the number of nodules formed in Mtefd-1, with downregulation of a set of genes, including notably NUCLEAR FACTOR-YA1 (MtNF-YA1) and MtNF-YB16, which are essential for nodule meristem establishment. We, therefore, conclude that nitrogen-fixing symbiosis recruited two proteins originally expressed in roots, MtEFD and MtEFD2, with distinct functions and neofunctionalization processes for each of them.
Asunto(s)
Medicago truncatula , Simbiosis , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas , Medicago truncatula/metabolismo , Nitrógeno/metabolismo , Fijación del Nitrógeno/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nódulos de las Raíces de las Plantas/microbiología , Simbiosis/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Xanthomonas campestris pv. campestris (Xcc) is a seed-transmitted vascular pathogen causing black rot disease on cultivated and wild Brassicaceae. Xcc enters the plant tissues preferentially via hydathodes, which are organs localized at leaf margins. To decipher both physiological and virulence strategies deployed by Xcc during early stages of infection, the transcriptomic profile of Xcc was analysed 3 days after entry into cauliflower hydathodes. Despite the absence of visible plant tissue alterations and despite a biotrophic lifestyle, 18% of Xcc genes were differentially expressed, including a striking repression of chemotaxis and motility functions. The Xcc full repertoire of virulence factors had not yet been activated but the expression of the HrpG regulon composed of 95 genes, including genes coding for the type III secretion machinery important for suppression of plant immunity, was induced. The expression of genes involved in metabolic adaptations such as catabolism of plant compounds, transport functions, sulphur and phosphate metabolism was upregulated while limited stress responses were observed 3 days postinfection. We confirmed experimentally that high-affinity phosphate transport is needed for bacterial fitness inside hydathodes. This analysis provides information about the nutritional and stress status of bacteria during the early biotrophic infection stages and helps to decipher the adaptive strategy of Xcc to the hydathode environment.
Asunto(s)
Brassica , Xanthomonas campestris , Xanthomonas , Adaptación Fisiológica/genética , Proteínas Bacterianas/metabolismo , Brassica/genética , Regulación Bacteriana de la Expresión Génica , Enfermedades de las Plantas/genética , Transcriptoma/genética , Virulencia/genética , Xanthomonas/metabolismo , Xanthomonas campestris/genéticaRESUMEN
Leghemoglobins enable the endosymbiotic fixation of molecular nitrogen (N2) in legume nodules by channeling O2 for bacterial respiration while maintaining a micro-oxic environment to protect O2-sensitive nitrogenase. We found that the NIN-like protein (NLP) transcription factors NLP2 and NIN directly activate the expression of leghemoglobins through a promoter motif, resembling a "double" version of the nitrate-responsive elements (NREs) targeted by other NLPs, that has conserved orientation and position across legumes. CRISPR knockout of the NRE-like element resulted in strongly decreased expression of the associated leghemoglobin. Our findings indicate that the origins of the NLP-leghemoglobin module for O2 buffering in nodules can be traced to an ancient pairing of NLPs with nonsymbiotic hemoglobins that function in hypoxia.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Leghemoglobina/genética , Medicago truncatula/genética , Nódulos de las Raíces de las Plantas/metabolismo , Factores de Transcripción/metabolismo , Fabaceae/genética , Fabaceae/metabolismo , Leghemoglobina/química , Medicago truncatula/metabolismo , Fijación del Nitrógeno , Oxígeno/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nodulación de la Raíz de la Planta , Regiones Promotoras Genéticas , Simbiosis , Factores de Transcripción/genéticaRESUMEN
In legumes interacting with rhizobia, the formation of symbiotic organs involved in the acquisition of atmospheric nitrogen gas (N2) is dependent on the plant nitrogen (N) demand. We used Medicago truncatula plants cultivated in split-root systems to discriminate between responses to local and systemic N signaling. We evidenced a strong control of nodule formation by systemic N signaling but obtained no clear evidence of a local control by mineral nitrogen. Systemic signaling of the plant N demand controls numerous transcripts involved in root transcriptome reprogramming associated with early rhizobia interaction and nodule formation. SUPER NUMERIC NODULES (SUNN) has an important role in this control, but we found that major systemic N signaling responses remained active in the sunn mutant. Genes involved in the activation of nitrogen fixation are regulated by systemic N signaling in the mutant, explaining why its hypernodulation phenotype is not associated with higher nitrogen fixation of the whole plant. We show that the control of transcriptome reprogramming of nodule formation by systemic N signaling requires other pathway(s) that parallel the SUNN/CLE (CLAVATA3/EMBRYO SURROUNDING REGION-LIKE PEPTIDES) pathway.
Asunto(s)
Medicago truncatula , Rhizobium , Homeostasis , Medicago truncatula/genética , Medicago truncatula/metabolismo , Nitrógeno , Fijación del Nitrógeno , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nodulación de la Raíz de la Planta , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/metabolismo , SimbiosisRESUMEN
The evolutionary and adaptive potential of a pathogen is a key determinant for successful host colonization and proliferation but remains poorly known for most of the pathogens. Here, we used experimental evolution combined with phenotyping, genomics, and transcriptomics to estimate the adaptive potential of the bacterial plant pathogen Ralstonia solanacearum to overcome the quantitative resistance of the tomato cultivar Hawaii 7996. After serial passaging over 300 generations, we observed pathogen adaptation to within-plant environment of the resistant cultivar but no plant resistance breakdown. Genomic sequence analysis of the adapted clones revealed few genetic alterations, but we provide evidence that all but one were gain of function mutations. Transcriptomic analyses revealed that even if different adaptive events occurred in independently evolved clones, there is convergence toward a global rewiring of the virulence regulatory network as evidenced by largely overlapping gene expression profiles. A subset of four transcription regulators, including HrpB, the activator of the type 3 secretion system regulon and EfpR, a global regulator of virulence and metabolic functions, emerged as key nodes of this regulatory network that are frequently targeted to redirect the pathogen's physiology and improve its fitness in adverse conditions. Significant transcriptomic variations were also detected in evolved clones showing no genomic polymorphism, suggesting that epigenetic modifications regulate expression of some of the virulence network components and play a major role in adaptation as well.
Asunto(s)
Adaptación Biológica/genética , Ralstonia solanacearum/genética , Regulón , Evolución Biológica , Mutación con Ganancia de Función , Aptitud Genética , Solanum lycopersicum/microbiología , Ralstonia solanacearum/patogenicidad , TranscriptomaRESUMEN
Legumes have the capacity to develop root nodules hosting nitrogen-fixing bacteria, called rhizobia. For the plant, the benefit of the symbiosis is important in nitrogen-deprived conditions, but it requires hosting and feeding massive numbers of rhizobia. Recent studies suggest that innate immunity is reduced or suppressed within nodules [1-10]; this likely maintains viable rhizobial populations. To evaluate the potential consequences and risks associated with an altered immuni`ty in the symbiotic organ, we developed a tripartite system with the model legume Medicago truncatula [11, 12], its nodulating symbiont of the genus Sinorhizobium (syn. Ensifer) [13, 14], and the pathogenic soil-borne bacterium Ralstonia solanacearum [15-18]. We show that nodules are frequent infection sites where pathogen multiplication is comparable to that in the root tips and independent of nodule ability to fix nitrogen. Transcriptomic analyses indicate that, despite the presence of the hosted rhizobia, nodules are able to develop weak defense reactions against pathogenic R. solanacearum. Nodule defense response displays specificity compared to that activated in roots. In agreement with nodule innate immunity, optimal R. solanacearum growth requires pathogen virulence factors. Finally, our data indicate that the high susceptibility of nodules is counterbalanced by the existence of a diffusion barrier preventing pathogen spreading from nodules to the rest of the plant.
Asunto(s)
Medicago truncatula/microbiología , Enfermedades de las Plantas/microbiología , Ralstonia solanacearum/fisiología , Nódulos de las Raíces de las Plantas/microbiología , Sinorhizobium meliloti/fisiología , Sinorhizobium/fisiología , Medicago truncatula/inmunología , Inmunidad de la Planta , Nódulos de las Raíces de las Plantas/inmunologíaRESUMEN
Advances in deciphering the functional architecture of eukaryotic genomes have been facilitated by recent breakthroughs in sequencing technologies, enabling a more comprehensive representation of genes and repeat elements in genome sequence assemblies, as well as more sensitive and tissue-specific analyses of gene expression. Here we show that PacBio sequencing has led to a substantially improved genome assembly of Medicago truncatula A17, a legume model species notable for endosymbiosis studies1, and has enabled the identification of genome rearrangements between genotypes at a near-base-pair resolution. Annotation of the new M. truncatula genome sequence has allowed for a thorough analysis of transposable elements and their dynamics, as well as the identification of new players involved in symbiotic nodule development, in particular 1,037 upregulated long non-coding RNAs (lncRNAs). We have also discovered that a substantial proportion (~35% and 38%, respectively) of the genes upregulated in nodules or expressed in the nodule differentiation zone colocalize in genomic clusters (270 and 211, respectively), here termed symbiotic islands. These islands contain numerous expressed lncRNA genes and display differentially both DNA methylation and histone marks. Epigenetic regulations and lncRNAs are therefore attractive candidate elements for the orchestration of symbiotic gene expression in the M. truncatula genome.
Asunto(s)
Epigénesis Genética , Genoma de Planta/genética , Medicago truncatula/genética , ARN no Traducido/genética , Simbiosis/genética , Metilación de ADN , Regulación de la Expresión Génica de las Plantas , Genómica , Familia de Multigenes , Proteínas de Plantas/genética , ARN de Planta/genética , Nódulos de las Raíces de las Plantas/genéticaRESUMEN
Understanding the development of multicellular organisms requires the identification of regulators, notably transcription factors, and specific transcript populations associated with tissue differentiation. Laser capture microdissection (LCM) is one of the techniques that enable the analysis of distinct tissues or cells within an organ. Coupling this technique with RNA sequencing (RNAseq) makes it extremely powerful to obtain a genome-wide and dynamic view of gene expression. Moreover, RNA sequencing allows two or potentially more interacting organisms to be analyzed simultaneously. In this chapter, a LCM-RNAseq protocol optimized for root and symbiotic root nodule analysis is presented, using the model legume Medicago truncatula (in interaction with Sinorhizobium meliloti in the nodule samples). This includes the description of procedures for plant material fixation, embedding, and micro-dissection; it is followed by a presentation of techniques for RNA extraction and amplification, adapted for the simultaneous analysis of plant and bacterial cells in interaction or, more generally, polyadenylated and non-polyadenylated RNAs. Finally, step-by-step statistical analyses of RNAseq data are described. Those are critical for quality assessment of the whole procedure and for the identification of differentially expressed genes.
Asunto(s)
Captura por Microdisección con Láser/métodos , Medicago truncatula/genética , Medicago truncatula/microbiología , Modelos Biológicos , Análisis de Secuencia de ARN/métodos , Sinorhizobium meliloti/fisiología , Adhesión en Parafina , ARN de Planta/genética , ARN de Planta/aislamiento & purificación , ARN Ribosómico/genética , Fijación del TejidoRESUMEN
Substantial progress has been made in the understanding of early stages of the symbiotic interaction between legume plants and rhizobium bacteria. Those include the specific recognition of symbiotic partners, the initiation of bacterial infection in root hair cells, and the inception of a specific organ in the root cortex, the nodule. Increasingly complex regulatory networks have been uncovered in which cytokinin (CK) phytohormones play essential roles in different aspects of early symbiotic stages. Intriguingly, these roles can be either positive or negative, cell autonomous or non-cell autonomous, and vary, depending on time, root tissues, and possibly legume species. Recent developments on CK symbiotic functions and interconnections with other signaling pathways during nodule initiation are the focus of this review.
Asunto(s)
Citocininas/fisiología , Nodulación de la Raíz de la Planta/fisiología , Citocininas/genética , Fabaceae/microbiología , Fabaceae/fisiología , Fijación del Nitrógeno/fisiología , Nodulación de la Raíz de la Planta/genética , Raíces de Plantas/microbiología , Raíces de Plantas/fisiología , Rhizobium/fisiología , Simbiosis/fisiologíaRESUMEN
Plant NF-Y transcription factors control a wide array of biological functions enabling appropriate reproductive and developmental processes as well as adaptation to various abiotic and biotic environments. In Medicago truncatula, MtNF-YA1 was previously identified as a key determinant for nodule development and establishment of rhizobial symbiosis. Here, we highlight a new role for this protein in compatibility to Aphanomyces euteiches, a root pathogenic oomycete. The Mtnf-ya1-1 mutant plants showed better survival rate, reduced symptoms, and increased development of their root apparatus as compared to their wild-type (WT) background A17. MtNF-YA-1 was specifically up-regulated by A. euteiches in F83005.5, a highly susceptible natural accession of M. truncatula while transcript level remained stable in A17, which is partially resistant. The role of MtNF-YA1 in F83005.5 susceptibility was further documented by reducing MtNF-YA1 expression either by overexpression of the miR169q, a microRNA targeting MtNF-YA1, or by RNAi approaches leading to a strong enhancement in the resistance of this susceptible line. Comparative analysis of the transcriptome of WT and Mtnf-ya1-1 led to the identification of 1509 differentially expressed genes. Among those, almost 36 defense-related genes were constitutively expressed in Mtnf-ya1-1, while 20 genes linked to hormonal pathways were repressed. In summary, we revealed an unexpected dual role for this symbiotic transcription factor as a key player in the compatibility mechanisms to a pathogen.
RESUMEN
The legume-Rhizobium symbiosis leads to the formation of a new organ, the root nodule, involving coordinated and massive induction of specific genes. Several genes controlling DNA methylation are spatially regulated within the Medicago truncatula nodule, notably the demethylase gene, DEMETER (DME), which is mostly expressed in the differentiation zone. Here, we show that MtDME is essential for nodule development and regulates the expression of 1,425 genes, some of which are critical for plant and bacterial cell differentiation. Bisulphite sequencing coupled to genomic capture enabled the identification of 474 regions that are differentially methylated during nodule development, including nodule-specific cysteine-rich peptide genes. Decreasing DME expression by RNA interference led to hypermethylation and concomitant downregulation of 400 genes, most of them associated with nodule differentiation. Massive reprogramming of gene expression through DNA demethylation is a new epigenetic mechanism controlling a key stage of indeterminate nodule organogenesis during symbiotic interactions.
Asunto(s)
Metilación de ADN , Medicago truncatula/crecimiento & desarrollo , Medicago truncatula/genética , Proteínas de Plantas/genética , Nódulos de las Raíces de las Plantas/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Rhizobium/fisiología , Nódulos de las Raíces de las Plantas/genética , SimbiosisRESUMEN
Nod factors (NFs) are lipochitooligosaccharidic signal molecules produced by rhizobia, which play a key role in the rhizobium-legume symbiotic interaction. In this study, we analyzed the gene expression reprogramming induced by purified NF (4 and 24 h of treatment) in the root epidermis of the model legume Medicago truncatula Tissue-specific transcriptome analysis was achieved by laser-capture microdissection coupled to high-depth RNA sequencing. The expression of 17,191 genes was detected in the epidermis, among which 1,070 were found to be regulated by NF addition, including previously characterized NF-induced marker genes. Many genes exhibited strong levels of transcriptional activation, sometimes only transiently at 4 h, indicating highly dynamic regulation. Expression reprogramming affected a variety of cellular processes, including perception, signaling, regulation of gene expression, as well as cell wall, cytoskeleton, transport, metabolism, and defense, with numerous NF-induced genes never identified before. Strikingly, early epidermal activation of cytokinin (CK) pathways was indicated, based on the induction of CK metabolic and signaling genes, including the CRE1 receptor essential to promote nodulation. These transcriptional activations were independently validated using promoter:ß-glucuronidase fusions with the MtCRE1 CK receptor gene and a CK response reporter (TWO COMPONENT SIGNALING SENSOR NEW). A CK pretreatment reduced the NF induction of the EARLY NODULIN11 (ENOD11) symbiotic marker, while a CK-degrading enzyme (CYTOKININ OXIDASE/DEHYDROGENASE3) ectopically expressed in the root epidermis led to increased NF induction of ENOD11 and nodulation. Therefore, CK may play both positive and negative roles in M. truncatula nodulation.
Asunto(s)
Citocininas/metabolismo , Lipopolisacáridos/metabolismo , Medicago truncatula/metabolismo , Epidermis de la Planta/metabolismo , Raíces de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Rayos Láser , Lipopolisacáridos/farmacología , Medicago truncatula/genética , Epidermis de la Planta/efectos de los fármacos , Epidermis de la Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Plantas Modificadas Genéticamente , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/metabolismo , Análisis de Secuencia de ARN/métodos , Transducción de SeñalRESUMEN
BACKGROUND: The bacterial species Xanthomonas campestris infects a wide range of Brassicaceae. Specific pathovars of this species cause black rot (pv. campestris), bacterial blight of stock (pv. incanae) or bacterial leaf spot (pv. raphani). RESULTS: In this study, we extended the genomic coverage of the species by sequencing and annotating the genomes of strains from pathovar incanae (CFBP 1606R and CFBP 2527R), pathovar raphani (CFBP 5828R) and a pathovar formerly named barbareae (CFBP 5825R). While comparative analyses identified a large core ORFeome at the species level, the core type III effectome was limited to only three putative type III effectors (XopP, XopF1 and XopAL1). In Xanthomonas, these effector proteins are injected inside the plant cells by the type III secretion system and contribute collectively to virulence. A deep and strand-specific RNA sequencing strategy was adopted in order to experimentally refine genome annotation for strain CFBP 5828R. This approach also allowed the experimental definition of novel ORFs and non-coding RNA transcripts. Using a constitutively active allele of hrpG, a master regulator of the type III secretion system, a HrpG-dependent regulon of 141 genes co-regulated with the type III secretion system was identified. Importantly, all these genes but seven are positively regulated by HrpG and 56 of those encode components of the Hrp type III secretion system and putative effector proteins. CONCLUSIONS: This dataset is an important resource to mine for novel type III effector proteins as well as for bacterial genes which could contribute to pathogenicity of X. campestris.