Disruption of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina.

Plant resistance to necrotrophic fungi is regulated by a complex set of signaling pathways that includes those mediated by the hormones salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and abscisic acid (ABA). The role of ABA in plant resistance remains controversial, as positive and negative regulatory functions have been described depending on the plant-pathogen interaction analyzed. Here, we show that ABA signaling negatively regulates Arabidopsis (Arabidopsis thaliana) resistance to the necrotrophic fungus Plectosphaerella cucumerina. Arabidopsis plants impaired in ABA biosynthesis, such as the aba1-6 mutant, or in ABA signaling, like the quadruple pyr/pyl mutant (pyr1pyl1pyl2pyl4), were more resistant to P. cucumerina than wild-type plants. In contrast, the hab1-1abi1-2abi2-2 mutant impaired in three phosphatases that negatively regulate ABA signaling displayed an enhanced susceptibility phenotype to this fungus. Comparative transcriptomic analyses of aba1-6 and wild-type plants revealed that the ABA pathway negatively regulates defense genes, many of which are controlled by the SA, JA, or ET pathway. In line with these data, we found that aba1-6 resistance to P. cucumerina was partially compromised when the SA, JA, or ET pathway was disrupted in this mutant. Additionally, in the aba1-6 plants, some genes encoding cell wall-related proteins were misregulated. Fourier transform infrared spectroscopy and biochemical analyses of cell walls from aba1-6 and wild-type plants revealed significant differences in their Fourier transform infrared spectratypes and uronic acid and cellulose contents. All these data suggest that ABA signaling has a complex function in Arabidopsis basal resistance, negatively regulating SA/JA/ET-mediated resistance to necrotrophic fungi.

Arabidopsis thaliana DOF6 negatively affects germination in non-after ripened seeds and interacts with TCP14

Seed dormancy prevents seeds from germinating under environmental conditions unfavourable for plant growth and development and constitutes an evolutionary advantage. Dry storage, also known as after-ripening, gradually decreases seed dormancy by mechanisms not well understood. An Arabidopsis thaliana DOF transcription factor gene (DOF6) affecting seed germination has been characterized. The transcript levels of this gene accumulate in dry seeds and decay gradually during after-ripening and also upon seed imbibition. While constitutive over-expression of DOF6 produced aberrant growth and sterility in the plant, its over-expression induced upon seed imbibition triggered delayed germination, abscisic acid (ABA)-hypersensitive phenotypes and increased expression of the ABA biosynthetic gene ABA1 and ABA-related stress genes. Wild-type germination and gene expression were gradually restored during seed after-ripening, despite of DOF6-induced over-expression. DOF6 was found to interact in a yeast two-hybrid system andin planta with TCP14, a previously described positive regulator of seed germination. The expression of ABA1 and ABA-related stress genes was also enhanced in tcp14 knock-out mutants. Taken together, these results indicate that DOF6 negatively affects seed germination and opposes TCP14 function in the regulation of a specific set of ABA-related genes

Arabidopsis heterotrimeric G-protein regulates cell wall defense and resistance to necrotrophic fungi

The Arabidopsis heterotrimeric G-protein controls defense responses to necrotrophic and vascular fungi. The agb1 mutant impaired in the G�� subunit displays enhanced susceptibility to these pathogens. G��/AGB1 forms an obligate dimer with either one of the Arabidopsis G�� subunits (��1/AGG1 and ��2/AGG2). Accordingly, we now demonstrate that the agg1 agg2 double mutant is as susceptible as agb1 plants to the necrotrophic fungus Plectosphaerella cucumerina. To elucidate the molecular basis of heterotrimeric G-protein-mediated resistance, we performed a comparative transcriptomic analysis of agb1-1 mutant and wild-type plants upon inoculation with P. cucumerina. This analysis, together with metabolomic studies, demonstrated that G-protein-mediated resistance was independent of defensive pathways required for resistance to necrotrophic fungi, such as the salicylic acid, jasmonic acid, ethylene, abscisic acid, and tryptophan-derived metabolites signaling, as these pathways were not impaired in agb1 and agg1 agg2 mutants. Notably, many mis-regulated genes in agb1 plants were related with cell wall functions, which was also the case in agg1 agg2 mutant. Biochemical analyses and Fourier Transform InfraRed (FTIR) spectroscopy of cell walls from G-protein mutants revealed that the xylose content was lower in agb1 and agg1 agg2 mutants than in wild-type plants, and that mutant walls had similar FTIR spectratypes, which differed from that of wild-type plants. The data presented here suggest a canonical functionality of the G�� and G��1/��2 subunits in the control of Arabidopsis immune responses and the regulation of cell wall composition.

Identificaci��n y caracterizaci��n de la ruta mediada por la prote��na G heterotrim��rica de Arabidopsis thaliana en la respuesta inmune

La resistencia de las plantas a los hongos necr��trofos como Plectosphaerella cucumerina es gen��ticamente compleja y depende de la activaci��n coordinada de distintas rutas de se��alizaci��n (Llorente et al, 2005; Sanchez-Vallet et al, 2010). Entre ��stas se encuentran las mediadas por la prote��na G heterotrim��rica, un complejo formado por tres subunidades (G��, G�� y G��) que regula tanto la respuesta de inmunidad a diferentes pat��genos como distintos procesos de desarrollo (Temple and Jones, 2007). En esta Tesis hemos demostrado que, en Arabidopsis, el mon��mero funcional formado por las subunidades G�� y G��1/G��2 es el responsable de la regulaci��n de la respuesta de defensa, ya que mutantes nulos en estas subunidades (agb1 y agg1 agg2) presentan una alta susceptibilidad al hongo P. cucumerina. Adem��s, hemos identificado varios amino��cidos (Q102, T188 y R235) de la prote��na AGB1 esenciales en la interacci��n con los efectores correspondientes para la regulaci��n de la respuesta inmune (Jiang et al, enviado). Para determinar las bases moleculares de la resistencia mediada por la prote��na G heterotrim��rica, llevamos a cabo un an��lisis transcript��mico comparativo entre los genotipos agb1 y Col-0, el cual revel�� que la resistencia mediada por AGB1 no depende de rutas defensivas implicadas en la resistencia a hongos necrotrofos, como las mediadas por el ��cido salic��lico (SA), etileno (ET), jasm��nico (JA) o ��cido absc��sico (ABA), o la ruta de bios��ntesis de metabolitos derivados del tript��fano. Este estudio mostr�� que un n��mero significativo de los genes desregulados en respuesta a P. cucumerina en el genotipo agb1 respecto a las plantas silvestres codificaban prote��nas con funciones relacionadas con la pared celular. La evaluaci��n de la composici��n y estructura de la pared de los mutantes de las subunidades de la prote��na G heterotrim��rica revel�� que los genotipos agb1 y agg1 agg2 presentaban alteraciones similares diferentes de las observadas en plantas silvestres Col-0, como una reducci��n significativa en el contenido de xilosa en la pared. Estos datos sugieren que la prote��na G heterotrim��rica puede modular la composici��n/estructura de la pared celular y contribuir, de esta manera, en la regulaci��n de la respuesta inmune (Delgado- Cerezo et al, 2011). La caracterizaci��n del interactoma de la prote��na G heterotrim��rica corrobor�� la relevancia funcional que presenta en la regulaci��n de la pared celular, ya que un n��mero significativo de las interacciones identificadas estaban comprendidas por prote��nas relacionadas directa o indirectamente con la biog��nesis y remodelaci��n de la pared celular (Klopffleisch et al, 2011). El papel en inmunidad de algunos de estos potenciales efectores ha sido validado mediante el an��lisis de la resistencia a P. cucumerina de los mutantes de p��rdida de funci��n correspondientes. Con el objetivo de caracterizar las rutas de se��alizaci��n mediadas por AGB1 e identificar efectores implicados en esta se��alizaci��n, llevamos a cabo una b��squeda de mutantes supresores de la susceptibilidad de agb1 a P. cucumerina, identific��ndose varios mutantes sgb (supressor of Gbeta). En esta Tesis hemos caracterizado en detalle el mutante sgb10, que presenta una activaci��n constitutiva de las rutas de se��alizaci��n mediadas por SA y JA+ET y suprime el fenotipo de susceptibilidad de agb1. SGB10 y AGB1 forman parte de rutas independientes en la regulaci��n de la respuesta inmune, mientras que interaccionan de forma compleja en el control de determinados procesos de desarrollo. La mutaci��n sgb10 ha sido cartografiada entre los genes At3g55010 y At3g56408, que incluye una regi��n con 160 genes. ABSTRACT Plant resistance to necrotrophic fungi Plectosphaerella cucumerina is genetically complex and depends on the interplay of different signalling pathways (Llorente et al, 2005; Sanchez-Vallet et al, 2010). Among others, the heterotrimeric G protein complex has a relevant role. The G protein that is formed by three subunits (G��, G�� and G��) is a pleiotropic regulator of immune responses to different types of pathogens and developmental issues (Temple and Jones, 2007). Throughout the Thesis, we have demonstrated that Arabidopsis��� functional monomer formed by the G�� and G��1/G��2 subunits is a key regulator of defense response, as null mutants (agb1 and agg1 agg2) are equally hypersusceptible to P. cucumerina infection. In addition we have identified several AGB1 aminoacids (Q102, T188 y R235) essentials to interact with specific effectors during the regulation of immune response (Jiang et al, sent).To determine the molecular basis of heterotrimeric G protein mediated resistance we have performed a microarray analysis with agb1-1 and wild type Col-0 plants before and after P. cucumerina challenge. A deep and exhaustive comparative transcriptomical analysis of these plants revealed that AGB1 mediated resistance does not rely on salicilic acid (SA), ethylene (ET), jasmonates (JA), abscisic acid (ABA) or triptophan derived metabolites biosynthesis. However the analysis revealed that a significant number of cell wall related genes are misregulated in the agb1 mutant after pathogen challenge when compared to wild-type plants. The analysis of cell wall composition and structure showed similar cell wall alterations between agb1 and agg1 agg2 mutants that are different from those of wild-type plants, so far the mutants present a significant reduction in xylose levels. All these results suggest that heterotrimeric G protein may regulate immune response through modifications in the cell wall composition/structure (Delgado-Cerezo et al, 2011). The characterization of Heterotrimeric G protein interactome revealed highly connected interactions between the G-protein core and proteins involved in cell wall composition or structure (Klopffleisch et al, 2011). To test the role in immunity of several effectors identified above, we have performed resistance analysis of corresponding null mutants against P. cucumerina. In order to characterize AGB1 mediated signalling pathway and identify additional effectors involved in AGB1-mediated immune response against P. cucumerina, we have performed a screening to isolate mutants with suppression of agb1 phenotype. One of the mutants, named sgb10, has been characterized during the Thesis. The mutant shows constitutive expression of SA, JA+ET-mediated defense signaling pathways to suppres agb1 hypersusceptibility. SGB10 and AGB1 proteins seem to be part of independent pathways in immunity, however its function during development remains unclear. At present, we have mapped the sgb10 mutation between At3g55010 and At3g56408 genes. This region contains 160 genes.

Caracterizaci��n funcional de los genes sgb (suppressors of agb1-2 susceptibility to pathogens): Nuevos reguladores de la respuesta de inmunidad innata de Arabidopsis thaliana

Un porcentaje importante de las p��rdidas de la producci��n agr��cola se deben a las enfermedades que causan en los cultivos los hongos necr��trofos y vasculares. Para mejorar la productividad agr��cola es necesario tener un conocimiento detallado de las bases gen��ticas y moleculares que regulan la resistencia de las plantas a este tipo de pat��genos. En Arabidopsis thaliana la resistencia frente a pat��genos necr��trofos, como el hongo Plectosphaerella cucumerina BMM (PcBMM), es gen��ticamente compleja y depende de la activaci��n coordinada de distintas rutas de se��alizaci��n, como las reguladas por las hormonas ��cido salic��lico (SA), ��cido jasm��nico (JA), etileno (ET) y ��cido absc��sico (ABA), as�� como de la s��ntesis de compuestos antimicrobianos derivados del Tript��fano y de la integridad de la pared celular (Llorente et al., 2005, Hern��ndez-Blanco et al., 2007; Delgado-Cerezo et al., 2012). Uno de los componentes claves en la regulaci��n de la resistencia de las plantas a pat��genos (incluidos hongos necr��trofos y bi��trofos) es la prote��na G heterotrim��rica, un complejo proteico formado por tres subunidades (G��, G�� y G��), que tambi��n regula distintos procesos del desarrollo vegetal. En Arabidopsis hay un gen que codifica para la subunidad �� (GPA1), otro para la �� (AGB1), y tres genes para la subunidad �� (AGG1, AGG2 y AGG3). El complejo GPA1-AGB1-AGG (1-3) se activa y disocia tras la percepci��n de una se��al espec��fica, actuando el d��mero AGB1-AGG1/2 como un mon��mero funcional que regula las respuestas de defensa (Delgado-Cerezo et al., 2012). Estudios transcript��micos y an��lisis bioqu��micos de la pared celular en los que se comparaban los mutantes agb1-2 y agg1 agg2, y plantas silvestres (Col-0) revelaron que la resistencia mediada por G��-G��1/2 no es dependiente de rutas de defensa previamente caracterizadas, y sugieren que la prote��na G podr��a modular la composici��n/estructura (integridad) de la pared celular (Delgado-Cerezo et al., 2012). Recientemente, se ha demostrado que AGB1 es un componente fundamental de la respuesta inmune mediada por Pathogen- Associated Molecular Patterns (PTI), ya que los mutantes agb1-2 son incapaces de activar tras el tratamiento con PAMPs respuestas de inmunidad, como la producci��n de especies reactivas de ox��geno (ROS; Liu et al., 2013). Dada la importancia de la prote��na G heterotrim��rica en la regulaci��n de la respuestas de defensa (incluida la PTI) realizamos un escrutinio de mutantes supresores de la susceptibilidad de agb1-2 al hongo necr��trofo, PcBMM, para identificar componentes adicionales de las rutas de se��alizaci��n reguladas por AGB1. En este escrutinio se aislaron cuatro mutantes sgb (suppressors of agb1-2 susceptibility to pathogens), dos de los cuales, sgb10 y sgb11, se han caracterizado en la presente Tesis Doctoral. El mutante sgb10 es un segundo alelo nulo del gen MKP1 (At3g55270) que codifica la MAP quinasa-fosfatasa 1 (Bartels et al., 2009). Este mutante presenta lesiones espont��neas en plantas adultas y una activaci��n constitutiva de las principales rutas de defensa (SA, JA y ET, y de metabolitos secundarios, como la camalexina), que explicar��a su elevada resistencia a PcBMM y Pseudomonas syringae. Estudios epist��ticos sugieren que la resistencia mediada por SGB10 no es dependiente, si no complementaria a la regulada por AGB1. El mutante sgb10 es capaz de restablecer en agb1-2 la producci��n de ROS y otras respuestas PTI (fosforilaci��n de las MAPK6/3/4/11) tras el tratamiento con PAMPs tan diversos como flg22, elf18 y quitina, lo que demuestra el papel relevante de SGB10/MKP1 y de AGB1 en PTI. El mutante sgb11 se caracteriza por presentar un fenotipo similar a los mutantes irregular xylem (e.g. irx1) afectado en pared celular secundaria: irregularidades en las c��lulas xilem��ticas, reducci��n en el tama��o de la roseta y altura de planta, y hojas con un mayor contenido de clorofila. La resistencia de sgb11 a PcBMM es independiente de agb1-2, ya que la susceptibilidad del doble mutante sgb11 agb1-2 es intermedia entre la de agb1-2 y sgb11. El mutante sgb11 no revierte la deficiente PTI de agb1-2 tras el tratamiento con flg22, lo que indica que est�� alterado en una ruta distinta de la regulada por SGB10. sgb11 presenta una sobreactivaci��n de la ruta del ��cido absc��sico (ABA), lo que podr��a explicar su resistencia a PcBMM. La mutaci��n sgb11 ha sido cartografiada en el cromosoma III de Arabidopsis entre los marcadores AthFUS6 (81,64cM) y nga6 (86,41cM) en un intervalo de aproximadamente 200 kb, que comprende genes, entre los que no se encuentra ninguno previamente descrito como IRX. El aislamiento y caracterizaci��n de SGB11 apoya la relevancia de la prote��na G heterotrim��rica en la regulaci��n de la interconexi��n entre integridad de la pared celular e inmunidad. ABSTRACT A significant percentage of agricultural losses are due to diseases caused by necrotrophic and vascular fungi. To enhance crop yields is necessary to have a detailed knowledge of the genetic and molecular bases regulating plant resistance to these pathogens. Arabidopsis thaliana resistance to necrotrophic pathogens, such as Plectosphaerella cucumerina BMM (PcBMM) fungus, is genetically complex and depends on the coordinated activation of various signaling pathways. These include those regulated by salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) hormones and the synthesis of tryptophan-derived antimicrobial compounds and cell wall integrity (Llorente et al., 2005, Hern��ndez-Blanco et al., 2007; Delgado-Cerezo et al., 2012). One key component in the regulation of plant resistance to pathogens (including biotrophic and necrotrophic fungi) is the heterotrimeric G-protein. This protein complex is formed by three subunits (G��, G�� and G��), which also regulates various plant developmental processes. In Arabidopsis only one gene encodes for subunits �� (GPA1) and �� (AGB1), and three genes for subunit �� (AGG1, AGG2 y AGG3). The complex GPA1- AGB1-AGG(1-3) is activated and dissociates after perception of an specific signal, AGB1- AGG1/2 acts as a functional monomer regulating defense responses (Delgado-Cerezo et al., 2012). Comparative transcriptomic studies and biochemical analyses of the cell wall of agb1-2 and agg1agg2 mutant and wild plants (Col-0), showed that G��-G��1/2-mediated resistance is not dependent on previously characterized defense pathways. In addition, it suggests that G protein may modulate the composition/structure (integrity) of the plant cell wall (Delgado-Cerezo et al., 2012). Recently, it has been shown that AGB1 is a critical component of the immune response mediated by Pathogen-Associated Molecular Patterns (PTI), as agb1-2 mutants are unable to activate immune responses such as oxygen reactive species (ROS) production after PAMPs treatment (Liu et al., 2013). Considering the importance of the heterotrimeric G protein in regulation of defense responses (including PTI), we performed a screening for suppressors of agb1-2 susceptibility to the necrotrophic fungus PcBMM. This would allow the identification of additional components of the signaling pathways regulated by AGB1. In this search four sgb mutants (suppressors of agb1-2 susceptibility to pathogens) were isolated, two of which, sgb10 and sgb11, have been characterized in this PhD thesis. sgb10 mutant is a second null allele of MKP1 gene (At3g55270), which encodes the MAP kinase-phosphatase 1 (Bartels et al., 2009). This mutant exhibits spontaneous lesions in adult plants and a constitutive activation of the main defense pathways (SA, JA and ET, and secondary metabolites, such as camalexin), which explains its high resistance to Pseudomonas syringae and PcBMM. Epistatic studies suggest that SGB10- mediated resistance is not dependent, but complementary to the regulated by AGB1. The sgb10 mutant is able to restore agb1-2 ROS production and other PTI responses (MAPK6/3/4/11 phosphorylation) upon treatment with PAMPs as diverse as, flg22, elf18 and chitin, demonstrating the relevant role of SGB10/MKP1 and AGB1 in PTI. sgb11 mutant is characterized by showing a similar phenotype to irregular xylem mutants (e.g. irx1), affected in secondary cell wall: irregular xylems cells, rosette size reduction and plant height, and higher chlorophyll content on leaves. The resistance of sgb11 to PcBMM is independent of agb1-2, as susceptibility of the double mutant agb1-2sgb11 is intermediate between agb1-2 and sgb11. The sgb11 mutant does not revert the deficient PTI response in agb1-2 after flg22 treatment, indicating that is altered in a pathway different to the one regulated by SGB10. sgb11 presents an over-activation of the abscisic acid pathway (ABA), which could explain its resistance to PcBMM. The sgb11 mutation has been mapped on chromosome III of Arabidopsis, between AthFUS6 (81.64 cM) and nga6 (86.41 cM) markers, in 200 kb interval, which does not include previously known IRX genes. The isolation and characterization of SGB11 supports the importance of heterotrimeric G protein in the regulation of the interconnection between the cell wall integrity and immunity.

Caracterizaci��n de la necrosis sist��mica inducida por la interacci��n sin��rgica entre el Virus X de la patata y potyvirus en Nicotiana benthamiana

Los virus de plantas pueden causar enfermedades severas que conllevan serias p��rdidas econ��micas a nivel mundial. Adem��s, en la naturaleza son comunes las infecciones simult��neas con distintos virus que conducen a la exacerbaci��n de los s��ntomas de enfermedad, fen��meno al que se conoce como sinergismo viral. Una de las sintomatolog��as m��s severas causadas por los virus en plantas susceptibles es la necrosis sist��mica (NS), que incluso puede conducir a la muerte del hu��sped. Este fenotipo ha sido comparado en ocasiones con la respuesta de resistencia de tipo HR, permitiendo establecer una serie de paralelismos entre ambos tipos de respuesta que sugieren que la NS producida en interacciones compatibles ser��a el resultado de una respuesta hipersensible sist��mica (SHR). Sin embargo, los mecanismos moleculares implicados en el desarrollo de la NS, su relaci��n con procesos de defensa antiviral o su relevancia biol��gica a��n no son bien entendidos, al igual que tampoco han sido estudiados los cambios producidos en la planta a escala gen��mica en infecciones m��ltiples que muestran sinergismo en patolog��a. En esta tesis doctoral se han empleado distintas aproximaciones de an��lisis de expresi��n g��nica, junto con otras t��cnicas gen��ticas y bioqu��micas, en el sistema modelo de Nicotiana benthamiana para estudiar la NS producida por la infecci��n sin��rgica entre el Virus X de la patata (PVX) y diversos potyvirus. Se han comparado los cambios producidos en el hu��sped a nivel gen��mico y fisiol��gico entre la infecci��n doble con PVX y el Virus Y de la patata (PVY), y las infecciones simples con PVX o PVY. Adem��s, los cambios transcript��micos y hormonales asociados a la infecci��n con la quimera viral PVX/HC���Pro, que reproduce los s��ntomas del sinergismo entre PVX���potyvirus, se han comparado con aquellos producidos por otros dos tipos de muerte celular, la PCD ligada a una interacci��n incompatible y la PCD producida por la disfunci��n del proteasoma. Por ��ltimo, t��cnicas de gen��tica reversa han permitido conocer la implicaci��n de factores del hu��sped, como las oxilipinas, en el desarrollo de la NS asociada al sinergismo entre PVXpotyvirus. Los resultados revelan que, respecto a las infecciones con solo uno de los virus, la infecci��n doble con PVX���PVY produce en el hu��sped diferencias cualitativas adem��s de cuantitativas en el perfil transcript��mico relacionado con el metabolismo primario. Otros cambios en la expresi��n g��nica, que reflejan la activaci��n de mecanismos de defensa, correlacionan con un fuerte estr��s oxidativo en las plantas doblemente infectadas que no se detecta en las infecciones simples. Adem��s, medidas en la acumulaci��n de determinados miRNAs implicados en diversos procesos celulares muestran como la infecci��n doble altera de manera diferencial tanto la acumulaci��n de estos miRNAs como su funcionalidad, lo cual podr��a estar relacionado con los cambios en el transcriptoma, as�� como con la sintomatolog��a de la infecci��n. La comparaci��n a nivel transcript��mico y hormonal entre la NS producida por PVX/HC���Pro y la interacci��n incompatible del Virus del mosaico del tabaco en plantas que expresan el gen N de resistencia (SHR), muestra que la respuesta en la interacci��n compatible es similar a la que se produce durante la SHR, si bien se presenta de manera retardada en el tiempo. Sin embargo, los perfiles de expresi��n de genes de defensa y de respuesta a hormonas, as�� como la acumulaci��n relativa de ��cido salic��lico (SA), ��cido jasmonico (JA) y ��cido absc��sico, en la interacci��n compatible son m��s semejantes a la respuesta PCD producida por la disfunci��n del proteasoma que a la interacci��n incompatible. Estos datos sugieren una contribuci��n de la interferencia sobre la funcionalidad del proteasoma en el incremento de la patogenicidad, observado en el sinergismo PVX���potyvirus. Por ��ltimo, los resultados obtenidos al disminuir la expresi��n de 9���LOX, �����DOX1 y COI1, relacionados con la s��ntesis o con la se��alizaci��n de oxilipinas, y mediante la aplicaci��n ex��gena de JA y SA, muestran la implicaci��n del metabolismo de las oxilipinas en el desarrollo de la NS producida por la infecci��n sin��rgica entre PVXpotyvirus en N. benthamiana. Adem��s, estos resultados indican que la PCD asociada a esta infecci��n, al igual que ocurre en interacciones incompatibles, no contiene necesariamente la acumulaci��n viral, lo cual indica que necrosis e inhibici��n de la multiplicaci��n viral son procesos independientes. ABSTRACT Plant viruses cause severe diseases that lead to serious economic losses worldwide. Moreover, simultaneous infections with several viruses are common in nature leading to exacerbation of the disease symptoms. This phenomenon is known as viral synergism. Systemic necrosis (SN) is one of the most severe symptoms caused by plant viruses in susceptible plants, even leading to death of the host. This phenotype has been compared with the hypersensitive response (HR) displayed by resistant plants, and some parallelisms have been found between both responses, which suggest that SN induced by compatible interactions could be the result of a systemic hypersensitive response (SHR). However, the molecular mechanisms involved in the development of SN, its relationship with antiviral defence processes and its biological relevance are still unknown. Furthermore, the changes produced in plants by mixed infections that cause synergistic pathological effects have not been studied in a genome���wide scale. In this doctoral thesis different approaches have been used to analyse gene expression, together with other genetic and biochemical techniques, in the model plant Nicotiana benthamiana, in order to study the SN produced by the synergistic infection of Potato virus X (PVX) with several potyviruses. Genomic and physiological changes produced in the host by double infection with PVX and Potato virus Y (PVY), and by single infection with PVX or PVY have been compared. In addition, transcriptional and hormonal changes associated with infection by the chimeric virus PVX/HC���Pro, which produces synergistic symptoms similar to those caused by PVX���potyvirus, have been compared with those produced by other types of cell death. These types of cell death are: PCD associated with an incompatible interaction, and PCD produced by proteasome disruption. Finally, reverse genetic techniques have revealed the involvement of host factors, such as oxylipins, in the development of SN associated with PVX���potyvirus synergism. The results revealed that compared with single infections, double infection with PVX���PVY produced qualitative and quantitative differences in the transcriptome profile, mainly related to primary metabolism. Other changes in gene expression, which reflected the activation of defence mechanisms, correlated with a severe oxidative stress in doubly infected plants that was undetected in single infections. Additionally, accumulation levels of several miRNAs involved in different cellular processes were measured, and the results showed that double infection not only produced the greatest variations in miRNA accumulation levels but also in miRNA functionality. These variations could be related with transcriptomic changes and the symptomatology of the infection. Transcriptome and hormone level comparisons between SN induced by PVX/HCPro and the incompatible interaction produced by Tobacco mosaic virus in plants expressing the N resistance gene (SHR), showed some similarities between both responses, even though the compatible interaction appeared retarded in time. Nevertheless, the expression profiles of both defence���related genes and hormoneresponsive genes, as well as the relative accumulation of salicylic acid (SA), jasmonic acid (JA) and abscisic acid in the compatible interaction are more similar to the PCD response produced by proteasome disruption. These data suggest that interference with proteasome functionality contributes to the increase in pathogenicity associated with PVX���potyvirus synergism. Finally, the results obtained by reducing the expression of 9���LOX, �����DOX1 and COI1, related with synthesis or signalling of oxylipins, and by applying exogenously JA and SA, revealed that oxylipin metabolism is involved in the development of SN induced by PVX���potyvirus synergistic infections in N. benthamiana. Moreover, these results also indicated that PVX���potyvirus associated PCD does not necessarily restrict viral accumulation, as is also the case in incompatible interactions. This indicates that both necrosis and inhibition of viral multiplication are independent processes.

Cycling Dof Factors: Molecular and functional characterization of Arabidopsis thaliana AtCDF3 and tomato (Solanum lycopersicum L.) SlCDF3 in response to abiotic stress

El tomate (Solanum lycopersicum L.) es considerado uno de los cultivos hort��colas de mayor importancia econ��mica en el territorio Espa��ol. Sin embargo, su producci��n est�� seriamente afectada por condiciones ambientales adversas como, salinidad, sequ��a y temperaturas extremas. Para resolver los problemas que se presentan en condiciones de estr��s, se han empleado una serie de t��cnicas culturales que disminuyen sus efectos negativos, siendo de gran inter��s el desarrollo de variedades tolerantes. En este sentido la obtenci��n y an��lisis de plantas transg��nicas, ha supuesto un avance tecnol��gico, que ha facilitado el estudio y la evaluaci��n de genes seleccionados en relaci��n con la tolerancia al estr��s. Estudios recientes han mostrado que el uso de genes reguladores como factores de transcripci��n (FTs) es una gran herramienta para obtener nuevas variedades de tomate con mayor tolerancia a estreses abi��ticos. Las prote��nas DOF (DNA binding with One Finger) son una familia de FTs espec��fica de plantas (Yangisawa, 2002), que est��n involucrados en procesos fisiol��gicos exclusivos de plantas como: asimilaci��n del nitr��geno y fijaci��n del carbono fotosint��tico, germinaci��n de semilla, metabolismo secundario y respuesta al fotoperiodo pero su preciso rol en la tolerancia a estr��s abi��tico se desconoce en gran parte. El trabajo descrito en esta tesis tiene como objetivo estudiar genes reguladores tipo DOF para incrementar la tolerancia a estr��s abiotico tanto en especies modelo como en tomate. En el primer cap��tulo de esta tesis se muestra la caracterizaci��n funcional del gen CDF3 de Arabidopsis, as�� como su papel en la respuesta a estr��s abi��tico y otros procesos del desarrollo. La expresi��n del gen AtCDF3 es altamente inducido por sequ��a, temperaturas extremas, salinidad y tratamientos con ��cido absc��sico (ABA). La l��nea de inserci��n T-DNA cdf3-1 es m��s sensible al estr��s por sequ��a y bajas temperaturas, mientras que l��neas transg��nicas de Arabidopsis 35S::AtCDF3 aumentan la tolerancia al estr��s por sequ��a, osm��tico y bajas temperaturas en comparaci��n con plantas wild-type (WT). Adem��s, estas plantas presentan un incremento en la tasa fotosint��tica y apertura estom��tica. El gen AtCDF3 se localiza en el n��cleo y que muestran una uni��n espec��fica al ADN con diferente afinidad a secuencias diana y presentan diversas capacidades de activaci��n transcripcional en ensayos de protoplastos de Arabidopsis. El dominio C-terminal de AtCDF3 es esencial para esta localizaci��n y su capacidad activaci��n, la delecci��n de este dominio reduce la tolerancia a sequ��a en plantas transg��nicas 35S::AtCDF3. An��lisis por microarray revelan que el AtCDF3 regula un set de genes involucrados en el metabolismo del carbono y nitr��geno. Nuestros resultados demuestran que el gen AtCDF3 juega un doble papel en la regulaci��n de la respuesta a estr��s por sequ��a y bajas temperaturas y en el control del tiempo de floraci��n. En el segundo cap��tulo de este trabajo se lleva a cabo la identificaci��n de 34 genes Dof en tomate que se pueden clasificar en base a homolog��a de secuencia en cuatro grupos A-D, similares a los descritos en Arabidopsis. Dentro del grupo D se han identificado cinco genes DOF que presentan caracter��sticas similares a los Cycling Dof Factors (CDFs) de Arabidopsis. Estos genes son considerados ort��logos de Arabidopsis CDF1-5, y han sido nombrados como Solanum lycopersicum CDFs o SlCDFs. Los SlCDF1-5 son prote��nas nucleares que muestran una uni��n espec��fica al ADN con diferente afinidad a secuencias diana y presentan diversas capacidades de activaci��n transcripcional in vivo. An��lisis de expresi��n de los genes SlCDF1-5 muestran diferentes patrones de expresi��n durante el d��a y son inducidos de forma diferente en respuesta a estr��s osm��tico, salino, y de altas y bajas temperaturas. Plantas de Arabidopsis que sobre-expresan SlCDF1 y SlCDF3 muestran un incremento de la tolerancia a la sequ��a y salinidad. Adem��s, de la expresi��n de varios genes de respuesta estr��s como AtCOR15, AtRD29A y AtERD10, son expresados de forma diferente en estas l��neas. La sobre-expresi��n de SlCDF3 en Arabidopsis promueve un retardo en el tiempo de floraci��n a trav��s de la modulaci��n de la expresi��n de genes que controlan la floraci��n como CONSTANS (CO) y FLOWERING LOCUS T (FT). En general, nuestros datos demuestran que los SlCDFs est��n asociados a funciones aun no descritas, relacionadas con la tolerancia a estr��s abi��tico y el control del tiempo de floraci��n a trav��s de la regulaci��n de genes espec��ficos y a un aumento de metabolitos particulares. ABSTRACT Tomato (Solanum lycopersicum L.) is one of the horticultural crops of major economic importance in the Spanish territory. However, its production is being affected by adverse environmental conditions such as salinity, drought and extreme temperatures. To resolve the problems triggered by stress conditions, a number of agricultural techniques that reduce the negative effects of stress are being frequently applied. However, the development of stress tolerant varieties is of a great interest. In this direction, the technological progress in obtaining and analysis of transgenic plants facilitated the study and evaluation of selected genes in relation to stress tolerance. Recent studies have shown that a use of regulatory genes such as transcription factors (TFs) is a great tool to obtain new tomato varieties with greater tolerance to abiotic stresses. The DOF (DNA binding with One Finger) proteins form a family of plant-specific TFs (Yangisawa, 2002) that are involved in the regulation of particular plant processes such as nitrogen assimilation, photosynthetic carbon fixation, seed germination, secondary metabolism and flowering time bur their precise roles in abiotic stress tolerance are largely unknown. The work described in this thesis aims at the study of the DOF type regulatory genes to increase tolerance to abiotic stress in both model species and the tomato. In the first chapter of this thesis, we present molecular characterization of the Arabidopsis CDF3 gene as well as its role in the response to abiotic stress and in other developmental processes. AtCDF3 is highly induced by drought, extreme temperatures, salt and abscisic acid (ABA) treatments. The cdf3-1 T-DNA insertion mutant was more sensitive to drought and low temperature stresses, whereas the AtCDF3 overexpression enhanced the tolerance of transgenic plants to drought, cold and osmotic stress comparing to the wild-type (WT) plants. In addition, these plants exhibit increased photosynthesis rates and stomatal aperture. AtCDF3 is localized in the nuclear region, displays specific binding to the canonical DNA target sequences and has a transcriptional activation activity in Arabidopsis protoplast assays. In addition, the C-terminal domain of AtCDF3 is essential for its localization and activation capabilities and the deletion of this domain significantly reduces the tolerance to drought in transgenic 35S::AtCDF3 overexpressing plants. Microarray analysis revealed that AtCDF3 regulated a set of genes involved in nitrogen and carbon metabolism. Our results demonstrate that AtCDF3 plays dual roles in regulating plant responses to drought and low temperature stress and in control of flowering time in vegetative tissues. In the second chapter this work, we carried out to identification of 34 tomato DOF genes that were classified by sequence similarity into four groups A-D, similar to the situation in Arabidopsis. In the D group we have identified five DOF genes that show similar characteristics to the Cycling Dof Factors (CDFs) of Arabidopsis. These genes were considered orthologous to the Arabidopsis CDF1 - 5 and were named Solanum lycopersicum CDFs or SlCDFs. SlCDF1-5 are nuclear proteins that display specific binding to canonical DNA target sequences and have transcriptional activation capacities in vivo. Expression analysis of SlCDF1-5 genes showed distinct diurnal expression patterns and were differentially induced in response to osmotic, salt and low and high temperature stresses. Arabidopsis plants overexpressing SlCDF1 and SlCDF3 showed increased drought and salt tolerance. In addition, various stress-responsive genes, such as AtCOR15, AtRD29A and AtERD10, were expressed differently in these lines. The overexpression of SlCDF3 in Arabidopsis also results in the late flowering phenotype through the modulation of the expression of flowering control genes such CONSTANS (CO) and FLOWERING LOCUS T (FT). Overall, our data connet SlCDFs to undescribed functions related to abiotic stress tolerance and flowering time through the regulation of specific target genes and an increase in particular metabolites.

New insights into the regulation of NADPH oxidase dependent reactive oxygen species signaling during the plant immune response

Las NADPH oxidasas de plantas, denominadas ���respiratory burst oxidase homologues��� (RBOHs), producen especies reactivas del ox��geno (ROS) que median un amplio rango de funciones. En la c��lula vegetal, el ajuste preciso de la producci��n de ROS aporta la especificidad de se��al para generar una respuesta apropiada ante las amenazas ambientales. RbohD y RbohF, dos de los diez genes Rboh de Arabidopsis, son pleiotr��picos y median diversos procesos fisiol��gicos en respuesta a pat��genos. El control espacio-temporal de la expresi��n de los genes RbohD y RbohF podr��a ser un aspecto cr��tico para determinar la multiplicidad de funciones de estas oxidasas. Por ello, generamos l��neas transg��nicas de Arabidopsis con fusiones de los promoters de RbohD y RbohF a los genes delatores de la B-glucuronidasa y la luciferasa. Estas l��neas fueron empleadas para revelar el patr��n de expresi��n diferencial de RbohD y RbohF durante la respuesta inmune de Arabidopsis a la bacteria pat��gena Pseudomonas syringae pv. tomato DC3000, el hongo necr��trofo Plectosphaerella cucumerina y en respuesta a se��ales relacionadas con la respuesta inmune. Nuestros experimentos revelan un patr��n de expresi��n diferencial de los promotores de RbohD y RbohF durante el desarrollo de la planta y en la respuesta inmune de Arabidopsis. Adem��s hemos puesto de manifiesto que existe una correlaci��n entre el nivel de actividad de los promotores de RbohD y RbohF con la acumulaci��n de ROS y el nivel de muerte celular en respuesta a pat��genos. La expression de RbohD y RbohF tambi��n es modulada de manera diferencial en respuesta a patrones moleculares asociados a pat��genos (PAMPs) y por ��cido absc��sico (ABA). Cabe destacar que, mediante una estrategia de intercambio de promotores, hemos revelado que la regi��n promotora de RbohD, es necesaria para dirigir la producci��n de ROS en respuesta a P. cucumerina. Adicionalmente, la activaci��n del promotor de RbohD en respuesta al aislado de P. cucumerina no adaptado a Arabidopsis 2127, nos llev�� a realizar ensayos de susceptibilidad con el doble mutante rbohD rbohF que han revelado un papel desconocido de estas oxidasas en resistencia no-huesped. La interacci��n entre la se��alizaci��n dependiente de las RBOHs y otros componentes de la respuesta inmune de plantas podr��a explicar tambi��n las distintas funciones que median estas oxidasas en relaci��n con la respuesta inmune. Entre la gran cantidad de se��ales coordinadas con la actividad de las RBOHs, existen evidencias gen��ticas y farmacol��gicas que indican que las prote��nas G heterotrim��ricas est��n implicadas en algunas de las rutas de se��alizaci��n mediadas por ROS derivadas de los RBOHs en respuesta a se��ales ambientales. Por ello hemos estudiado la relaci��n entre estas RBOH-NADPH oxidasas y AGB1, la subunidad �� de las prote��nas G heterotrim��ricas en la respuesta inmune de Arabidopsis. An��lisis de epistasis indican que las prote��nas G heterotrim��ricas est��n implicadas en distintas rutas de se��alizaci��n en defensa mediadas por las RBOHs. Nuestros resultados ilustran la relaci��n compleja entre la se��alizaci��n mediada por las RBOHs y las prote��nas G heterotrim��ricas, que var��a en funci��n de la interacci��n planta-pat��geno analizada. Adem��s, hemos explorado la posible asociaci��n entre AGB1 con RBOHD y RBOHF en eventos tempranos de la respuesta immune. Cabe se��alar que experimentos de co��mmunoprecipitaci��n apuntan a una posible asociaci��n entre AGB1 y la kinasa citoplasm��tica reguladora de RBOHD, BIK1. Esto indica un posible mecanismo de control de la funci��n de esta NADPH oxidase por AGB1. En conjunto, estos datos aportan nuevas perspectivas sobre c��mo, a trav��s del control transcripcional o mediante la interacci��n con las prote��nas G heterotrim��ricas, las NADPH oxidases de plantas median la producci��n de ROS y la se��alizaci��n por ROS en la respuesta inmune. Nuestro trabajo ejemplifica c��mo la regulaci��n diferencial de dos miembros de una familia multig��nica, les permite realizar distintas funciones fisiol��gicas especializadas usando un mismo mecanismo enzim��tico. ABSTRACT The plant NADPH oxidases, termed respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) which mediate a wide range of functions. Fine tuning this ROS production provides the signaling specificity to the plant cell to produce the appropriate response to environmental threats. RbohD and RbohF, two of the ten Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes in response to pathogens. One aspect that may prove critical to determine the multiplicity of functions of RbohD and RbohF is the spatio-temporal control of their gene expression. Thus, we generated Arabidopsis transgenic lines with RbohD- and RbohF-promoter fusions to the ��-glucuronidase and the luciferase reporter genes. These transgenics were employed to reveal RbohD and RbohF promoter activity during Arabidopsis immune response to the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, the necrotrophic fungus Plectosphaerella cucumerina and in response to immunity-related cues. Our experiments revealed a differential expression pattern of RbohD and RbohF throughout plant development and during Arabidopsis immune response. Moreover, we observed a correlation between the level of RbohD and RbohF promoter activity, the accumulation of ROS and the amount of cell death in response to pathogens. RbohD and RbohF gene expression was also differentially modulated by pathogen associated molecular patterns and abscisic acid. Interestingly, a promoter-swap strategy revealed the requirement for the promoter region of RbohD to drive the production of ROS in response to P. cucumerina. Additionally, since the RbohD promoter was activated during Arabidopsis interaction with a non-adapted P. cucumerina isolate 2127, we performed susceptibility tests to this fungal isolate that uncovered a new role of these oxidases on non-host resistance. The interplay between RBOH-dependent signaling with other components of the plant immune response might also explain the different immunity-related functions mediated by these oxidases. Among the plethora of signals coordinated with RBOH activity, pharmacological and genetic evidence indicates that heterotrimeric G proteins are involved in some of the signaling pathways mediated by RBOH���derived ROS in response to environmental cues. Therefore, we analysed the interplay between these RBOH-NADPH oxidases and AGB1, the Arabidopsis ��-subunit of heterotrimeric G proteins during Arabidopsis immune response. We carried out epistasis studies that allowed us to test the implication of AGB1 in different RBOH-mediated defense signaling pathways. Our results illustrate the complex relationship between RBOH and heterotrimeric G proteins signaling, that varies depending on the type of plant-pathogen interaction. Furthermore, we tested the potential association between AGB1 with RBOHD and RBOHF during early immunity. Interestingly, our co-immunoprecipitation experiments point towards an association of AGB1 and the RBOHD regulatory kinase BIK1, thus providing a putative mechanism in the control of the NADPH oxidase function by AGB1. Taken all together, these studies provide further insights into the role that transcriptional control or the interaction with heterotrimeric G-proteins have on RBOH-NADPH oxidase-dependent ROS production and signaling in immunity. Our work exemplifies how, through a differential regulation, two members of a multigenic family achieve specialized physiological functions using a common enzymatic mechanism.

Functional characterization of YODA, a mitogen-activated protein kinase kinase kinase (MAP3K) that regulates a novel innate immunity pathway in Arabidopsis thaliana

Las cascadas de se��alizaci��n mediadas por prote��na quinasas activadas por mit��geno (MAP quinasas) son capaces de integrar y transducir se��ales ambientales en respuestas celulares. Entre estas se��ales se encuentran los PAMPs/MAMPs (Pathogen/Microbe-Associated Molecular Patterns), que son mol��culas de pat��genos o microorganismos, o los DAMPs (Damaged-Associated Molecular Patterns), que son mol��culas derivadas de las plantas producidas en respuesta a da��o celular. Tras el reconocimiento de los PAMPs/DAMPs por receptores de membrana denominados PRRs (Pattern Recognition Receptors), como los receptores con dominio quinasa (RLKs) o los receptores sin dominio quinasa (RLPs), se activan respuestas moleculares, incluidas cascadas de MAP quinasas, que regulan la puesta en marcha de la inmunidad activada por PAMPs (PTI). Esta Tesis describe la caracterizaci��n funcional de la MAP quinasa quinasa quinasa (MAP3K) YODA (YDA), que act��a como un regulador clave de la PTI en Arabidopsis. Se ha descrito previamente que YDA controla varios procesos de desarrollo, como la regulaci��n del patr��n estom��tico, la elongaci��n del zigoto y la arquitectura floral. Hemos caracterizado un alelo mutante hipom��rfico de YDA (elk2 o yda11) que presenta una elevada susceptibilidad a pat��genos bi��trofos y necr��trofos. Notablemente, plantas que expresan una forma constitutivamente activa de YDA (CA-YDA), con una deleci��n en el dominio N-terminal, presentan una resistencia de amplio espectro frente a diferentes tipos de pat��genos, incluyendo hongos, oomicetos y bacterias, lo que indica que YDA juega un papel importante en la regulaci��n de la resistencia de las plantas a pat��genos. Nuestros datos indican que esta funci��n es independiente de las respuestas inmunes mediadas por los receptores previamente caracterizados FLS2 y CERK1, que reconocen los PAMPs flg22 y quitina, respectivamente, y que est��n implicados en la resistencia de Arabidopsis frente a bacterias y hongos. Hemos demostrado que YDA controla la resistencia frente al hongo necr��trofo Plectosphaerella cucumerina y el patr��n estom��tico mediante su interacci��n gen��tica con la RLK ERECTA (ER), un PRR implicado en la regulaci��n de estos procesos. Por el contrario, la interacci��n gen��tica entre ER y YDA en la regulaci��n de otros procesos de desarrollo es aditiva en lugar de epist��tica. An��lisis gen��ticos indicaron que MPK3, una MAP quinasa que funciona aguas abajo de YDA en el desarrollo estom��tico, es un componente de la ruta de se��alizaci��n mediada por YDA para la resistencia frente a P. cucumerina, lo que sugiere que el desarrollo de las plantas y la PTI comparten el m��dulo de transducci��n de MAP quinasas asociado a YDA. Nuestros experimentos han revelado que la resistencia mediada por YDA es independiente de las rutas de se��alizaci��n reguladas por las hormonas de defensa ��cido salic��lico, ��cido jasm��nico, ��cido absc��sico o etileno, y tambi��n es independiente de la ruta de metabolitos secundarios derivados del tript��fano, que est��n implicados en inmunidad vegetal. Adem��s, hemos demostrado que respuestas asociadas a PTI, como el aumento en la concentraci��n de calcio citopl��smico, la producci��n de especies reactivas de ox��geno, la fosforilaci��n de MAP quinasas y la expresi��n de genes de defensa, no est��n afectadas en el mutante yda11. La expresi��n constitutiva de la prote��na CA-YDA en plantas de Arabidopsis no provoca un aumento de las respuestas PTI, lo que sugiere la existencia de mecanismos de resistencia adicionales regulados por YDA que son diferentes de los regulados por FLS2 y CERK1. En l��nea con estos resultados, nuestros datos transcript��micos revelan una sobre-representaci��n en plantas CA-YDA de genes de defensa que codifican, por ejemplo, p��ptidos antimicrobianos o reguladores de muerte celular, o prote��nas implicadas en la biog��nesis de la pared celular, lo que sugiere una conexi��n potencial entre la composici��n e integridad de la pared celular y la resistencia de amplio espectro mediada por YDA. Adem��s, an��lisis de fosfoprote��mica indican la fosforilaci��n diferencial de prote��nas relacionadas con la pared celular en plantas CA-YDA en comparaci��n con plantas silvestres. El posible papel de la ruta ER-YDA en la regulaci��n de la integridad de la pared celular est�� apoyado por an��lisis bioqu��micos y glic��micos de las paredes celulares de plantas er, yda11 y CA-YDA, que revelaron cambios significativos en la composici��n de la pared celular de estos genotipos en comparaci��n con la de plantas silvestres. En resumen, nuestros datos indican que ER y YDA forman parte de una nueva ruta de inmunidad que regula la integridad de la pared celular y respuestas defensivas, confiriendo una resistencia de amplio espectro frente a pat��genos. ABSTRACT Plant mitogen-activated protein kinase (MAPK) cascades transduce environmental signals and developmental cues into cellular responses. Among these signals are the pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) and the damage-associated molecular patterns (DAMPs). These PAMPs/DAMPs, upon recognition by plant pattern recognition receptors (PRRs), such as Receptor-Like Kinases (RLKs) and Receptor-Like Proteins (RLPs), activate molecular responses, including MAPK cascades, which regulate the onset of PAMP-triggered immunity (PTI). This Thesis describes the functional characterization of the MAPK kinase kinase (MAP3K) YODA (YDA) as a key regulator of Arabidopsis PTI. YDA has been previously described to control several developmental processes, such as stomatal patterning, zygote elongation and inflorescence architecture. We characterized a hypomorphic, non-embryo lethal mutant allele of YDA (elk2 or yda11) that was found to be highly susceptible to biotrophic and necrotrophic pathogens. Remarkably, plants expressing a constitutive active form of YDA (CA-YDA), with a deletion in the N-terminal domain, showed broad-spectrum resistance to different types of pathogens, including fungi, oomycetes and bacteria, indicating that YDA plays a relevant function in plant resistance to pathogens. Our data indicated that this function is independent of the immune responses regulated by the well characterized FLS2 and CERK1 RLKs, which are the PRRs recognizing flg22 and chitin PAMPs, respectively, and are required for Arabidopsis resistance to bacteria and fungi. We demonstrate that YDA controls resistance to the necrotrophic fungus Plectosphaerella cucumerina and stomatal patterning by genetically interacting with ERECTA (ER) RLK, a PRR involved in regulating these processes. In contrast, the genetic interaction between ER and YDA in the regulation of other ER-associated developmental processes was additive, rather than epistatic. Genetic analyses indicated that MPK3, a MAP kinase that functions downstream of YDA in stomatal development, also regulates plant resistance to P. cucumerina in a YDA-dependent manner, suggesting that the YDA-associated MAPK transduction module is shared in plant development and PTI. Our experiments revealed that YDA-mediated resistance was independent of signalling pathways regulated by defensive hormones like salicylic acid, jasmonic acid, abscisic acid or ethylene, and of the tryptophan-derived metabolites pathway, which are involved in plant immunity. In addition, we showed that PAMP-mediated PTI responses, such as the increase of cytoplasmic Ca2+ concentration, reactive oxygen species (ROS) burst, MAPK phosphorylation, and expression of defense-related genes are not impaired in the yda11 mutant. Furthermore, the expression of CA-YDA protein does not result in enhanced PTI responses, further suggesting the existence of additional mechanisms of resistance regulated by YDA that differ from those regulated by the PTI receptors FLS2 and CERK1. In line with these observations, our transcriptomic data revealed the over-representation in CA-YDA plants of defensive genes, such as those encoding antimicrobial peptides and cell death regulators, and genes encoding cell wall-related proteins, suggesting a potential link between plant cell wall composition and integrity and broad spectrum resistance mediated by YDA. In addition, phosphoproteomic data revealed an over-representation of genes encoding wall-related proteins in CA-YDA plants in comparison with wild-type plants. The putative role of the ER-YDA pathway in regulating cell wall integrity was further supported by biochemical and glycomics analyses of er, yda11 and CA-YDA cell walls, which revealed significant changes in the cell wall composition of these genotypes compared with that of wild-type plants. In summary, our data indicate that ER and YDA are components of a novel immune pathway that regulates cell wall integrity and defensive responses, which confer broad-spectrum resistance to pathogens.