New molecular mechanisms of Arabidopsis thaliana resistance to necrotrophic pathogens = New molecular mechanisms of Arabidopsis thaliana resistance to necrotrophic pathogens

Resumen

The resistance of plants to necrotrophic fungi is genetically complex and depends on the interplay of different signalling pathways. To get further insights on the mechanisms controlling plant defence to this type of pathogens, we have explored the relevance of additional, uncharacterized molecular components of Arabidopsis thaliana. Thus, we have demonstrated that A. thaliana secondary cell wall is not just a structural defensive barrier, but is also an important component of specific signalling in defence responses. The broad spectrum resistance of irx1 and irx5 mutants, impaired in the Cellulose Synthases (CESAs) required for the synthesis of cellulose of secondary cell wall (Hernandez-Blanco et al., 2007), was found to depend on the constitutive accumulation of antimicrobial peptides, such as LTP3, LTP4 and thionins, and tryptophan-derived metabolites, as demonstrated by genetic, transcriptomic and metabolomic analyses of these mutants. The in vitro antimicrobial activity of some tryptophan-derived compounds (indol-3-ylmethylamine and 4-methoxy indol-3-ylmethylamine) was demonstrated, further supporting their role in defence against necrotrophic pathogens. Moreover, analysis of the susceptibility to adapted P. cucumerina isolates of Arabidopsis mutants impaired in the synthesis (cyp79B2 cyp79B3), activation (pen2, cyp81F2 and pad2) or delivery (pen3) of indole-derived compounds corroborated that these metabolites are essential for basal resistance. Interestingly, we also found that the accumulation/delivery of indolederived metabolites, which is blocked or impaired in cyp79B2 cyp79B3, pad2, or pen3 mutant lines, was required for full Arabidopsis resistance to nonadapted P. cucumerina isolates. These data corroborated the relevant contributions of antimicrobials in Arabidopsis resistance to both adapted and nonadapted necrotrophic pathogens. The irx-mediated resistance was found to be independent of the ethylene (ET), salicylic acid (SA) and jasmonic acid (JA) mediated pathways, but seemed to depend on abscisic acid (ABA) signalling, based on the constitutive, enhanced accumulation of this homone in irx mutants. We have explored, using genetic and transcriptomic tools, the function of ABA signalling on plant resistance to necrotroph. ABA biosynthetic (aba1, aba2 and aba3) and signalling (abi1) mutants were found to be more resistant to P. cucumerina. Transcriptomic analysis of aba1 revealed that its enhanced resistance was probably due to a constitutive, up-regulation of defence-related genes, since it was observed a high overlap between the constitutive gene expression pattern of aba1 mutant and that of wild-type plants upon P. cucumerina inoculation. The transcriptomic analysis also revealed a complex crosstalk between ABA and other defence signalling pathways, and allowed the identification of gene clusters relevant for resistance as evidenced by genetic analysis. RESUMEN La resistencia de las plantas a hongos necrotrofos es geneticamente compleja y depende de la activacion de distintas rutas de senalizacion. Para conocer con mas detalle los mecanismos de defensa para el control de este tipo de agentes patogenos, se ha estudiado la importancia de otros componentes moleculares de Arabidopsis thaliana aun por caracterizar. Se ha demostrado que la pared celular secundaria de A. thaliana no es solo una barrera defensiva estructural, sino que tambien es un componente importante de la senalizacion en las respuestas de defensa. Asi, la resistencia de amplio espectro de los mutantes de las celulosa sintasas (CESAs) de la pared celular secundaria irx1 e irx5 (irx, irregular xylem; Hernandez-Blanco et al., 2007), se debe parcialmente a un alto contenido de peptidos antimicrobianos, como LTP3, LTP4 y tioninas, y de derivados del triptofano, tal y como se demostro mediante los analisis genetico, transcriptomico y metabolomico de dicho mutante. Algunos de estos compuestos (3-metilaminoindol, 4-metoxi-3-metilaminoindol) presentan actividad antimicrobiana in vitro frente a patogenos necrotrofos lo que manifiesta su papel en defensa. Ademas, el analisis de la susceptibilidad de mutantes de sintesis (cyp79B2 cyp79B3), de activacion (pen2, cyp81F2, pad2) y de transporte (pen3) de los compuestos derivados del triptofano a los aislados de P. cucumerina incapaces de infectar al ecotipo silvestre demostro la relevancia de los compuestos derivados del triptofano en la respuesta no-huesped de A. thaliana a hongos necrotrofos. Por otro lado, la resistencia mediada por IRX es independiente de las rutas del etileno (ET), acido salicilico (SA) y acido jasmonic (JA); sin embargo, parece depender de la senalizacion mediada por el acido abscisico (ABA), debido a un mayor contenido constitutivo de dicha hormona en los mutantes irx. Por ello, se analizo la funcion del ABA en la resistencia de la planta a necrotrofos, utilizando herramientas geneticas y transcriptomicas. Los mutantes de sintesis (aba1, aba2 y aba3) y de senalizacion (abi1) del ABA presentan una mayor resistencia a P. cucumerina que se explica por una expresion constitutiva de genes de defensa, puesto que existe un alto solapamiento entre el patron de expresion del mutante aba1 y el de la infeccion con el patogeno en plantas silvestres. El analisis transcriptomico tambien evidencio la existencia de interacciones entre la ruta mediada por ABA y otras rutas de senalizacion de defensa, y permitio la identificacion de los grupos de genes esenciales para la resistencia como lo demuestra el analisis genetico.