Examinando por Autor "Roeschlin, Roxana Andrea"

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    ÍtemEmbargo
    Caracterización genética y molecular de los efectores TAL PthA de Xanthomonas citri subsp. citri AT, cepa supresora de la cancrosis bacteriana de los cítricos
    (2024) Uviedo, Facundo; Marano, María Rosa; Roeschlin, Roxana Andrea; https://orcid.org/0000-0003-0437-589X
    Resultados preliminares demuestran que los genes pthA de la cepa X. citri AT presentan un patrón polimórfico diferente al de la cepa de referencia X. citri 306, lo que sugiere su posible implicancia en la patogenicidad específica del hospedador cítrico analizado (Siciliano, 2009; Roeschlin, 2015). La enzima de restricción BamHI permite separar los diferentes genes de pthA (Al‐ Saadi y col., 2007; Shiotani y col., 2007) y así obtener un perfil de bandas de ADN correspondiente a los diferentes genes pthA codificados por los plásmidos de las cepas X. citri AT y X. citri 306. Los ensayos de Southern Blot, utilizando una sonda específica para pthA permitieron identificar los diferentes alelos de pthA (Siciliano, 2009). Resultó interesante observar que la cepa de referencia X. citri 306 presenta un fragmento de 3,3 kb que correspondería al gen pthA4 (da Silva y col., 2002; Al‐Saadi y col., 2007; Shiotani y col., 2007), mientras que en la cepa X. citri AT se identificó un fragmento de menor tamaño, aproximadamente 2,3 kb correspondiente a un nuevo alelo presente en esta variante. Además, se detectó un fragmento diferencial de 3,8 kb en X. citri AT. En el presente Capítulo, se propone caracterizar funcionalmente los genes de pthA identificados en la cepa X. citri AT, los cuales difieren de sus homólogos en X. citri 306. Para ello, se plantearon los siguientes objetivos: i) Evaluar la capacidad del efector TAL PthA4 en restaurar la virulencia de X. citri AT en C. limon y C. sinensis; ii) Complementar cepas mutantes de X. citri 306 en los genes pthA con los diferentes plásmidos recombinantes conteniendo los efectores TAL de X. citri AT; iii) Verificar la expresión de los PthA mediante Western Blot utilizando anticuerpos monoclonales anti-3xFLAG; iv) Evaluar el comportamiento de las cepas complementadas con los diferentes efectores TAL de X. citri A T a través de ensayos de patogenicidad en Citrus spp. utilizando dos métodos de inoculación: infiltración e hisopado.
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    ÍtemAcceso Abierto
    Designer TALEs enable discovery of cell death-inducer genes
    (Oxford University Press, 2024-09-09) Roeschlin, Roxana Andrea; Azad, Sepideh M.; Grove, René P.; Chuan, Ana; García, Lucila; Niñoles, Regina; Uviedo, Facundo; Villalobos, Liara; Massimino, Maria E.; Marano, María Rosa; Boch, Jens; Gadea, José; https://orcid.org/0000-0001-6121-7930; https://orcid.org/0009-0001-2033-3430; https://orcid.org/0009-0005-3778-9900; https://orcid.org/0009-0008-0589-9867; https://orcid.org/0000-0002-1625-422X; https://orcid.org/0000-0002-7862-9509; https://orcid.org/0000-0003-0437-589X; https://orcid.org/0000-0001-8399-0107; https://orcid.org/0009-0000-9611-0316; https://orcid.org/0000-0001-6600-8177; https://orcid.org/0000-0001-5883-7506; https://orcid.org/0000-0002-3612-7914
    Transcription activator-like effectors (TALEs) in plant-pathogenic Xanthomonas bacteria activate expression of plant genes and support infection or cause a resistance response. PthA4AT is a TALE with a particularly short DNA-binding domain harboring only 7.5 repeats which triggers cell death in Nicotiana benthamiana; however, the genetic basis for this remains unknown. To identify possible target genes of PthA4AT that mediate cell death in N. benthamiana, we exploited the modularity of TALEs to stepwise enhance their specificity and reduce potential target sites. Substitutions of individual repeats suggested that PthA4AT-dependent cell death is sequence specific. Stepwise addition of repeats to the C-terminal or N-terminal end of the repeat region narrowed the sequence requirements in promoters of target genes. Transcriptome profiling and in silico target prediction allowed the isolation of two cell death inducer genes, which encode a patatin-like protein and a bifunctional monodehydroascorbate reductase/carbonic anhydrase protein. These two proteins are not linked to known TALE-dependent resistance genes. Our results show that the aberrant expression of different endogenous plant genes can cause a cell death reaction, which supports the hypothesis that TALE-dependent executor resistance genes can originate from various plant processes. Our strategy further demonstrates the use of TALEs to scan genomes for genes triggering cell death and other relevant phenotypes.
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    ÍtemAcceso Abierto
    PthA4AT, a 7.5-repeats transcription activator-like (TAL) effector from Xanthomonas citri ssp. citri, triggers citrus canker resistance
    (Wiley Open Access, 2019-10) Roeschlin, Roxana Andrea; Uviedo, Facundo; García, Lucila; Molina, María Celeste; Favaro, María Alejandra; Chiesa, María Amalia; Tasselli, Sabrina; Franco-Zorrilla, José Manuel; Forment, Javier; Gadea, José; Marano, María Rosa
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    ÍtemAcceso Abierto
    Resistance to citrus canker induced by a variant of Xanthomonas citri ssp. citri is associated with a hypersensitive cell death response involving autophagy-associated vacuolar processes
    (Wiley, 2017-11-08) Roeschlin, Roxana Andrea; Favaro, María Alejandra; Chiesa, María Amalia; Alemano, Sergio Gabriel; Vojnov, Adrián Alberto; Castagnaro, Atilio Pedro; Filippone, María Paula; Gmitter, Frederick George; Gadea, José; Marano, María Rosa
    Xanthomonas citri ssp. citri (X. citri) is the causal agent of Asiatic citrus canker, a disease that seriously affects most commercially important Citrus species worldwide. We have identified previously a natural variant, X. citri AT, that triggers a host-specific defence response in Citrus limon. However, the mechanisms involved in this canker disease resistance are unknown. In this work, the defence response induced by X. citri AT was assessed by transcriptomic, physiological and ultrastructural analyses, and the effects on bacterial biofilm formation were monitored in parallel. We show that X. citri AT triggers a hypersensitive response associated with the interference of biofilm development and arrest of bacterial growth in C. limon. This plant response involves an extensive transcriptional reprogramming, setting in motion cell wall reinforcement, the oxidative burst and the accumulation of salicylic acid (SA) and phenolic compounds. Ultrastructural analyses revealed subcellular changes involving the activation of autophagy-associated vacuolar processes. Our findings show the activation of SA-dependent defence in response to X. citri AT and suggest a coordinated regulation between the SA and flavonoid pathways, which is associated with autophagy mechanisms that control pathogen invasion in C. limon. Furthermore, this defence response protects C. limon plants from disease on subsequent challenges by pathogenic X. citri. This knowledge will allow the rational exploitation of the plant immune system as a biotechnological approach for the management of the disease.