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Investigación » Departamento de Ingeniería Genética » Profesores/Investigadores » Dr. Alvarez Venegas Raul » Laboratorio de Cromatina y Epigenética




Use of natural and synthetic compounds to activate a defense-priming state in the common bean (Phaseolus vulgaris L.).

To survive in adverse conditions, plants have evolved complex mechanisms that “prime” their defense system to respond and adapt to stresses. Their competence to respond to such stresses fundamentally depends on its capacity to modulate the transcriptome rapidly and specifically. Thus, chromatin dynamics is a mechanism linked to transcriptional regulation and enhanced defense in plants. For example, in Arabidopsis, priming of the SA-dependent defense pathway is linked to histone lysine methylation. Such modifications could create a memory of the primary infection that is associated with an amplified gene response upon exposure to a second stress-stimulus. In addition, the priming status of a plant for induced resistance can be inherited to its offspring. However, analyses on the molecular mechanisms of generational and transgenerational priming in the common bean (Phaseolus vulgaris L.), an economically important crop, are absent. 
We hope that an understanding of the molecular changes leading to defense priming and pathogen resistance will provide valuable knowledge for producing disease-resistant crop varieties by exposing parental plants to priming activators, as well as to the development of novel plant protection chemicals that stimulate the plant's inherent disease resistance mechanisms.

The Phaseolus vulgaris histone lysine methyltransferase PvTRX1h is involved in the synthesis of plant hormones in callus from common bean.


Common bean (Phaseolus vulgaris L.) is the most important grain legume in the human diet. Bean improvement efforts have been focused on classical breeding techniques because bean is recalcitrant to both somatic embryogenesis and in vitro regeneration and, therefore, its stable genetic transformation is hard to achieve. This study was undertaken to better understand the process of somatic embryogenesis in the common bean. We focused on the epigenetic mechanisms by which somatic embryogenesis in plants is regulated and the interaction of these mechanisms with plant hormones. Specifically, we examined the role of the gene PvTRX1h, an ortholog of a major known histone lysine methyltransferase in plants. Given the problems with regeneration and transformation, we chose to develop and use regeneration-competent callus that could be successively transformed. This accomplishment should prove a valuable contribution in itself, as the development of regeneration-competent callus and its successful transformation would be a valuable first step towards establishing an efficient plant regeneration system and genetic transformation in this agronomically important species. 
Calli of common bean were generated and transformed with the PvTRX1hRiA construction to down-regulate expression of the histone lysine methyltransferase gene, PvTRX1h. Clones that showed evidence of producing somatic embryos were selected. Plant hormone content was measured with high-performance liquid chromatography and gene expression was assessed with quantitative real-time PCR. Detailed histological analysis was performed on the selected transgenic calli. Down-regulation of the histone lysine methyltransferase gene, PvTRX1h, was accompanied by altered concentrations of plant hormones in the calli. PvTRX1h regulated the expression of genes involved in auxin biosynthesis and embryogenic calli in which PvTRX1h was downregulated were capable of differentiation into somatic embryos. Surprisingly, down-regulation of PvTRX1h revealed an unforeseen effect, the over-expression of a gene coding for a second histone lysine methyltransferase, PvASHH2h.
The PvTRX1h gene is involved in the synthesis of plant hormones in common bean callus. In addition, PvTRX1hand PvASHH2h carry out opposite non-redundant functions in embryogenesis. These results shed light on the crosstalk among histone methyltransferases and plant hormone signaling and on gene regulation during somatic embryo generation.



Molecular cloning and characterization of Trithorax-Group genes from Phaseolus vulgaris nodules.


In eukaryotes, trithorax group proteins play critical roles in the regulation of transcription, cell proliferation, differentiation and development. In this work we report the molecular cloning and characterization of PvuTRX1h and PvuASH1h, two cDNAs from the common bean Phaseolus vulgaris encoding polypeptides homologues of trithorax group members described in animals and yeast. A full length clone of PvTrx1h was isolated from RNA prepared from roots and contained a 3270 bp ORF encoding 1089 amino acids, while the PvAsh1h contained a 1446 bp ORF encoding 481 amino acids. Characterization of the isolated sequences revealed that they contain all the canonical domains present in proteins from the TRX and ASH1 families. A comparison of the common bean SET-domain sequences with homologous proteins from plants, animals and yeast revealed that PvuTRX1h is phylogenetically related to the TRX (trithorax) family of histone lysine methyltransferases while PvuASH1h clusters with members of the ASH1 family. Quantitative RT-PCR analyses of transcript abundance in roots and nodules, at different developmental stages, demonstrated that PvuTRX1h and PvuASH1h are particularly abundant at early stages of nodule development, suggesting that these genes could be involved in the formation of nitrogen fixing nodules in P. vulgaris. This work is the first report of the presence and characterization of Trithorax group homologue genes in P. vulgaris and their expression patterns during nodule development.



Regulation of disease-responsive genes mediated by epigenetic factors: interaction Arabidopsis-Pseudomonas.


Genes in eukaryotic organisms function within the context of chromatin, and the mechanisms that modulate the structure of chromatin are defined as epigenetic. In Arabidopsis, pathogen infection induces the expression of at least one histone deacetylase, suggesting that histone acetylation/deacetylation, has an important role in the pathogenic response in plants. How/whether histone methylation affects gene response to pathogen infection is unknown. To gain a better understanding of epigenetic mechanisms regulating the interaction between Pseudomonas syringae and Arabidopsis thaliana, we analyzed three different Arabidopsis ash1-related (absent, small or homeotic discs 1) mutants. We found that loss of function of ASHH2 and ASHR1 resulted in faster hypersensitive responses (HR) to both the mutant (hrpA) and the pathogenic (DC3000) strains of P. syringae, while control (Col-0) and ashr3 mutants appeared more resistant to the infection after two days. Furthermore, we show that in the ashr3 background the PR1 gene displayed the highest expression levels upon infection with DC3000 correlating with increased resistance against this pathogen. Our results show that in both the ashr1 and ashh2 backgrounds the H3K4me2 levels decreased at the promoter region of PR1 upon infection with the DC3000 strain suggesting that an epigenetically regulated PR1 expression is involved in the plant defense. Our results suggest that histone methylation in response to pathogen infection may be a critical component in the signaling and defense processes occurring between plants and microbes.

Patrones dinámicos y estables de metilación de la histona H3 en Arabidopsis.


Los mecanismos que químicamente modifican los nucleosomas y que conllevan a la activación o represión heredable de genes específicos son definidos como epigenéticos. Metilación de la lisina 4 y de la lisina 27 de la histona H3 (H3K4me3 y H3K27me3) son interpretados como marcas “activadoras” o “represivas”, respectivamente. Recientemente demostramos que aún para genes relacionados, ninguna modificación por si misma puede servir como un indicador del estado de expresión. Por ejemplo, genes pertenecientes a la misma familia génica (FLC y AP1) que son selectivamente activados por ATX1, se encuentran modificados en forma similar, pero sorprendentemente, también de forma diferente. Modificaciones activadoras (H3K4me3) y silenciadoras (H3K27me3) coexisten en la parte 5’ de los nucleosomas del gene FLC, activo transcripcionalmente, mientras que el gene altamente transcrito AP1 no muestra ninguna de las dos marcas. Esto sugiere que distintos mecanismos “leen” y operan en cada locus. Proponemos que H3K4me3 y H3K27me3 generan una marca “bi-modular” en el “código de las histonas”, asignando un distinto significado en genes específicos.