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19 páginas.- 9 figuras.- referencias, Tomato varieties resistant to the bacterial wilt pathogen Ralstonia solanacearum have the ability to restrict bacterial movement in the plant. Inducible vascular cell wall reinforcements seem to play a key role in confining R. solanacearum into the xylem vasculature of resistant tomato. However, the type of compounds involved in such vascular physico-chemical barriers remain understudied, while being a key component of resistance. Here we use a combination of histological and live-imaging techniques, together with spectroscopy and gene expression analysis to understand the nature of R. solanacearum-induced formation of vascular coatings in resistant tomato. We describe that resistant tomato specifically responds to infection by assembling a vascular structural barrier formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties, effectively contributing to resistance., Research is funded by MCIN/AEI/10.13039/501100011033 (NSC, MV), MCIN/AEI/PID2019-110330GB-C21 (MF, OS), MCIN/AEI/PID2020-118968RBI00 (JR), through the ‘Severo Ochoa Programme for Centres of Excellence in R&D’ (SEV-2015-0533, CEX2019-000917 and CEX2019-000902-S funded by MCIN/AEI/ 10.13039/501100011033), and by the Spanish National Research Council (CISC) pie-201620E081 (JR, AG) and the Generalitat de Catalunya (2017SGR765 grant). AK is the recipient of a Netaji Subhas – Indian Council of Agricultural Research International Fellowship. SS acknowledges financial support from DOC-FAM, European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement no. 754397. This work was also supported by the CERCA Program/Generalitat de Catalunya.

Trabajo presentado en 5th International Symposium on Plant Apoplastic Diffusion Barriers (PADiBA) celebrado en Dundee (Escocia) del 13 al 15 de septiembre de 2022.

Additional file 6: Figure S3. Temporal dynamics of Fo5176 DEGs during root infection reveal a significant alteration of proteins containing catalytic and carbohydrate-binding modules (CAZY). (A) Clusters of Fo5176 coexpressed DEGs during infection using fuzzy c-means clustering. (B) Number of DEGs encoding proteins with carbohydrate active enzyme-domains (CAZY) and glycosyl hydrolases in the different clusters. DEGs with the entry “no cluster” were not associated to a cluster by the clustering algorithm.

Additional file 14: Table S8. Primers used in this study

Additional file 8: Figure S5. Characterization of cesa3-3. (A) Representative images of 7 day-old WT, cesa3-3 and cev1 seedlings. (B) Primary root length box plots of indicated genotypes. Medians are represented inside the boxes by solid lines, and circles depict individual measurements (n = 38-61). (C) Representative primary root images specifying the initiation of the differentiation zone by the appearance of root hairs (dashed line), and bulging cells in cesa3-3 and cev1 (asterisks). (D) Box plot summary of primary root diameter at the onset of differentiation (n = 31). (E) Lignin deposition visualized by phloroglucinol stain (fuchsia) in primary roots of indicated genotypes. Letters in (B, D) denote statistically significant differences among samples determined by ANOVA followed by Tukey’s HSD test. Scale bar:100 μm (E).

[CA]: Ralstonia solanacearum és un dels patògens bacterians de plantes més devastadors del món. Aquest bacteri és l’agent causant del marciment bacterià en més de 450 espècies de plantes i causa una devastació severa en cultius de solanàcies com la tomaquera. R. solanacearum accedeix a sistema radicular a través de ferides i llocs d’emergència d’arrels laterals i després es desplaça fins a la vasculatura radicular, on es multiplica al xilema i després es dissemina verticalment a través del xilema i horitzontalment entre els vasos i els teixits circumdants. Fins ara, l’estratègia de maneig més fiable per controlar R. solanacearum ha estat l’ús de resistència genètica. En tomàquet, entre les millors fonts de resistència es troba el conrear resistent Hawaii 7996 (H7996), que s’usa comunament com a patró en el cultiu comercial de tomàquet. Aquesta varietat resistent ha desenvolupat mecanismes de defensa efectius per prevenir la colonització de vasos o el moviment entre vasos un cop que ha passat la colonització vascular. Publicacions recents mostren que el cilindre vascular del xilema de l’arrel actua com a teixit predominant per muntar una resposta de defensa eficaç contra els invasors vasculars, restringint l’avanç del patogen cap al teixit aeri. No obstant això, els mecanismes que regulen aquesta forma de resistència segueixen sent desconeguts en gran manera. Desentranyar els mecanismes a través dels quals les plantes bloquegen o alenteixen la progressió de patògens en la vasculatura del xilema de l’arrel pot ser vital en el desenvolupament de varietats resistents mitjançant intervencions biotecnològiques. Per tant, en aquesta tesi intentem aprofundir en les respostes de defensa que actuen en la vasculatura del xilema de l’arrel que restringeixen efectivament la colonització pel patogen bacterià vascular R. solanacerum en tomàquet. Els estudis histopatològics comparatius en línies de tomàquet resistents i susceptibles indiquen que un reforç vascular compost de ferulats, que culmina en la formació d’una zona ferulo-suberínica que pot actuar com una forta barrera fisico-química contra la invasió de R. solanacearum (Capítol 1). Aquesta barrera fenòlic-alifàtica reforça les parets del teixit vascular del xilema en H7996 i pot restringir el moviment del bacteri des de l’interior del vas del xilema cap al xilema circumdant, cèl·lules de l’parènquima, vasos propers i espais intercel·lulars. Els canvis de composició en les parets es van estudiar mitjançant espectroscòpia, la qual cosa va mostrar una intensa acumulació de metabòlits compatibles amb la suberina específicament en les arrels de H7996 resistent, després de la infecció de R. solanacearum (Capítol 2). Es van observar diferències en l’estat de polimerització de la lignina en les arrels després de la infecció entre tomàquet resistent i susceptible. L’H7996 contenia una major proporció de lignina de tipus G, que és més resistent a la degradació, mentre que Marmande susceptible contenia més lignina de tipus S, que és més propensa a degradar-se. L’expressió de gens de la via biosintètica d’àcids grassos de la suberina es va incrementar significativament en el teixit vascular de l’xilema de l’arrel primària de les plantes infectades amb H7996 en comparació amb els controls o el tomàquet susceptible (Capítol 3). Això indica que la regulació positiva d’aquests gens és una resposta específica de plantes H7996 resistents que té lloc a la vasculatura suberizada després de la infecció per R. solanacearum. A més, es va observar inducció de ProSlFHT::GUS en la vasculatura del xilema de l’arrel principal de les plantes H7996 infectades, així com en teixits que se sap que dipositen suberina com ara epidermis, exodermis i teixits que experimenten cicatrització de ferides., [ES]: Ralstonia solanacearum es uno de los patógenos bacterianos de plantas más devastadores del mundo. Esta bacteria del suelo es el agente causante de la marchitez bacteriana en más de 450 especies de plantas y causa una devastación severa en cultivos de solanáceas como el tomate. R. solanacearum accede al sistema radicular a través de heridas y sitios de emergencia de raíces laterales y luego se desplaza hasta la vasculatura radicular, donde se multiplica en el xilema y luego se disemina verticalmente a través del xilema y horizontalmente entre los vasos y los tejidos circundantes. Hasta la fecha, la estrategia de manejo más fiable para controlar R. solanacearum ha sido el uso de resistencia genética. En tomate, entre las mejores fuentes de resistencia se encuentra el cultivar resistente Hawaii 7996 (H7996), que se usa comúnmente como patrón en el cultivo comercial de tomate. Este cultivar resistente ha desarrollado mecanismos de defensa efectivos para prevenir la colonización de vasos o el movimiento entre vasos una vez que ha ocurrido la colonización vascular. Publicaciones recientes muestran que el cilindro vascular del xilema de la raíz actúa como tejido predominante para montar una respuesta de defensa eficaz contra los invasores vasculares, lo que restringe el avance del patógeno hacia el tejido aéreo. Sin embargo, los mecanismos que regulan esta forma de resistencia siguen siendo desconocidos en gran manera. Desentrañar los mecanismos a través de los cuales las plantas bloquean o ralentizan la progresión de patógenos en la vasculatura del xilema de la raíz puede ser vital en el desarrollo de cultivares resistentes mediante intervenciones biotecnológicas. Por lo tanto, en esta tesis intentamos arrojar luz sobre las respuestas de defensa que actúan en la vasculatura del xilema de la raíz que restringen efectivamente la colonización por el patógeno bacteriano vascular R. solanacerum en tomate. Los estudios histopatológicos comparativos en líneas de tomate resistentes y susceptibles indican que un refuerzo vascular compuesto de ferulatos, que culmina en la formación de una zona ferulo-suberínica, puede actuar como una fuerte barrera físico-química contra la invasión de R. solanacearum (Capítulo 1). Esta barrera fenólico-alifática refuerza las paredes del tejido vascular del xilema en H7996 y puede restringir el movimiento de la bacteria desde la luz del vaso del xilema hacia el xilema circundante, células del parénquima, vasos cercanos y espacios intercelulares. Los cambios de composición en las paredes se estudiaron mediante espectroscopia, lo cual mostró una intensa acumulación de metabolitos compatibles con la suberina específicamente en las raíces de H7996 resistente, tras la infección de R. solanacearum (Capítulo 2). Se observaron diferencias en el estado de polimerización de la lignina en las raíces después de la infección entre tomate resistente y susceptible. El H7996 contenía una mayor proporción de lignina de tipo G, que es más resistente a la degradación, mientras que Marmande susceptible contenía más lignina de tipo S, que es más propensa a degradarse. La expresión de genes de la vía biosintética de ácidos grasos de la suberina se incrementó significativamente en el tejido vascular del xilema de la raíz primaria de las plantas infectadas con H7996 en comparación con los controles o el tomate susceptible (Capítulo 3). Esto indica que la regulación positiva de estos genes es una respuesta específica de plantas H7996 resistentes que tiene lugar en la vasculatura suberizada tras la infección por R. solanacearum. Además, se observó inducción de ProSlFHT::GUS en la vasculatura del xilema de la raíz principal de las plantas H7996 infectadas, así como en tejidos que se sabe que depositan suberina tales como epidermis, exodermis y tejidos que experimentan cicatrización de heridas., [EN]: Ralstonia solanacearum is one of the world's most devastating bacterial pathogens of plants. This soil-borne bacterium is the causative agent of bacterial wilt on more than 450 plant species, and it causes severe devastation on Solanaceous crops like tomato. R. solanacearum gains access to the root system through wounds and lateral root emerging sites and then moves to the root vasculature, where it multiplies in the xylem and later spreads vertically within the xylem sap and horizontally between vessels and to the surrounding tissues. To date, the most reliable management strategy to control R. solanacearum has been the use of genetic resistance. In tomato, among the best resistance sources is the resistant cultivar Hawaii 7996 (H7996), which is commonly used as a rootstock in commercial tomato cultivation. This resistant cultivar has evolved effective defense mechanisms to prevent vessel colonization or movement between vessels once vascular colonization has occurred. Recent reports show that the root xylem vascular cylinder acts as a predominant tissue for mounting an efficient defense response against vascular invaders, restricting onward movement of the pathogen to the aerial tissue. However, the mechanisms regulating this form of resistance remain elusive. Unravelling the mechanisms through which the plants block or slow down pathogen progression at the root xylem vasculature can be vital in the development of resistant cultivars by biotechnological interventions. Hence, in this thesis we attempted to shed light on the defense responses acting at the root xylem vasculature that effectively restrict colonization by the vascular bacterial pathogen R. solanacerum in tomato. Comparative histopathological studies in resistant and susceptible tomato lines indicated that a ferulate vascular reinforcement, culminating into a ferulo-suberin zone, may act as a strong physico-chemical barrier against R. solanacearum invasion (Chapter 1). This phenolic- aliphatic barrier reinforces the walls of xylem vascular tissue in H7996 and may restrict movement of the bacterium from the xylem vessel lumen to the surrounding xylem parenchyma cells and nearby vessels and inter-cellular spaces. Compositional changes in walls were studied using spectroscopy, which showed strong accumulation of suberin- compatible metabolites specifically in roots of resistant H7996, upon infection of R. solanacearum (Chapter 2). Differences in the polymerization state of lignin in roots were observed after infection between resistant and susceptible tomato. H7996 contained a higher proportion of G-type lignin, which is more resistant to degradation, whereas susceptible Marmande contained more S-type lignin, which is more prone to degradation. Expression of genes from the suberin fatty acid biosynthetic pathway was significantly upregulated in the taproot xylem vascular tissue of H7996 infected plants compared to the mock controls or susceptible tomato (Chapter 3). This indicates that upregulation of these genes is a specific response of resistant H7996 plants that takes place in suberizing vasculature upon R. solanacearum infection. Further, induction of ProSlFHT::GUS was observed in taproot xylem vasculature of infected H7996 plants, as well as in tissues known to deposit suberin such as epidermis, exodermis and tissues undergoing wound healing. Implications of overexpressing genes from pathway of suberin and the associated soluble phenolics synthesis, were evaluated in susceptible tomato background. Overexpression of suberin feruloyl transferase (FHT), which catalyzes the formation feruloyl esters showed limited restriction against R. solanacearum. In contrast, overexpression of tyramine N-hydroxycinnamoyltransferase (THT 1-3), responsible for synthesis of hydroxycinnamic acid amides (HCAA) such as feruloyl tyramine resulted in an increase of resistance against R. solanacearum with disease progressing remarkably slower in this line compared to wild type plants. Accumulation of such aminated phenolics may act as a chemical barrier acting as phytoalexin but could also act as physical barrier if cross-linked to the poly-aromatic domain of suberin.

Additional file 7: Figure S4. ctl1-2 is impaired in crystalline cellulose deposition in the root. Cellulose content of roots from 10 day-old light-grown plants represented as ug of D-glucose derived from crystalline cellulose per mg of dried alcohol-insoluble residue (AIR) [102]. N = 2 biological replicates +/- standard deviation; 3 technical replicates per biological replicate. Welch’s unpaired t-test; ***P-value = 0.0004.

Additional file 5: Figure S2. RNAseq validation by qRT-PCR. (A) and (B) The expression of 6 randomly picked DEGs from Arabidopsis (A) or Fo5176 (B) expressed at different levels based on the RNA-sequencing (left panels) was confirmed by qRT-PCR (right panels) using de novo generated RNA samples. The qRT-PCR-based expression of each gene was determined relative to the corresponding reference gene; i.e. At GAPDH in (A) and Fo5176 β-Tub in (B). (C) and (D) Correlation (log10 fold changes) between RNAseq and qPCR derived expression data from Arabidopsis (A and C) and Fo5176 (B and D). Very good Pearson correlations of r = 0.79 for Arabidopsis and r = 0.97 for the fungus were obtained (p-value

Additional file 9: Table S4 A-F. Statistical analysis of root vascular penetrations upon Fo5176 pSIX1::GFP infection. Repeated measures two-way ANOVA with post-hoc Tukey test for multiple comparisons corresponding to root vascular penetration events (p-value 0.05) are not included in the tables.

Additional file 2: Figure S1. Complete MDS-Analysis of Arabidopsis transcriptional profiles. Multidimensional scaling analysis (MDS) of transcriptional profiles of Arabidopsis. Samples clearly deviating from the rest of the samples at the same time point and condition (surrounded by a red square) were not used for further analysis.

Trabajo presentado al Seminario del CRAG (Internal Webinar), celebrado Online el 25 de septiembre de 2020.

Ralstonia solanacearum is a devastating bacterial vascular pathogen causing bacterial wilt. In the field, resistance against this disease is quantitative and only available for breeders in tomato and eggplant. To understand the basis of resistance in tomato, we have investigated the spatio-temporal bacterial colonization dynamics using non-invasive live monitoring techniques coupled to grafting of susceptible and resistant varieties. We revealed four different restrictions to the bacterium in resistant tomato: root colonization, vertical movement from roots to shoots, circular vascular bundle invasion and radial apoplastic spread in the cortex. We also show that the radial invasion of cortical extracellular spaces occurs mostly at late disease stages but is observed throughout plant infection. This work shows that resistance is expressed both in root and shoot tissues and highlights the importance of structural constraints to bacterial spread as a resistance mechanism. It also shows that R. solanacearum is not only a vascular pathogen but spreads "out of the xylem", occupying the plant apoplast niche. Our work will help elucidate the complex genetic determinants of resistance, setting the foundations to decipher the molecular mechanisms that limit pathogen colonization, which may provide new potential precision tools to fight bacterial wilt in the field.