Epigenetic changes in galls induced by the nematode meloidogyne javanica in arabidopsis
- Autores: Ana Cláudia Pereira da Silva
- Directores de la Tesis: Carolina Escobar de Lucas (dir. tes.), Carmen Fenoll (codir. tes.)
- Lectura: En la Universidad de Castilla-La Mancha ( España ) en 2020
- Idioma: español
- Tribunal Calificador de la Tesis: Lee Robertson (presid.), Virginia Ruíz Ferrer (secret.), Sara López Gomollón (voc.)
- Programa de doctorado: Programa de Doctorado en Ciencias Agrarias y Ambientales por la Universidad de Castilla-La Mancha
- Materias:
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- Resumen
Root-knot nematodes (RKN), Meloidogyne spp., are obligate plant-parasites that can parasite a large number of host plant species and threaten the agricultural production. They cause existing root vascular tissue to re-differentiate into a pseudo-organ, called a gall, where several cells are transformed into highly metabolically active giant cells (GCs), their feeding sites. In the last decade, several studies have provided a better understanding of the mechanisms underlying GC formation. Different studies in two hosts, Arabidopsis and tomato, have reported a massive repression of gene expression during the formation of these structures. Although the mechanisms behind this transcriptomic pattern are not well understood, several studies proposed that this gene repression could be mediated by epigenetic mechanisms, including small RNAs (sRNAs). More recent results showed massive and differential accumulation of 24-nucleotides (nt) sRNAs, which can be involved in epigenetic mechanisms such as RNA-directed DNA methylation (RdDM), in early galls. Moreover, repeat-associated small interfering RNAs (rasiRNAs) enriched in early-developing galls targeted primarily retrotransposons, some of whose major members were repressed, suggesting an implication of other epigenetic mechanisms, as RdDM, during gall formation. Considering this, we have decided to study in depth the DNA methylation changes occurring in the galls formed during the interaction between Meloidogyne javanica and Arabidopsis thaliana, as the host plant.
Firstly, we have developed and optimized two protocols for an easiest study of the interaction. In order to correlate the epigenetic changes at the DNA methylation level and changes on the transcriptome in the same biological independent replicate, we have developed a method that allows the simultaneous extraction and purification of genomic DNA and total RNA from the same sample with enough quality for posterior library construction and sequencing. As loss of function mutants or overexpressing lines are routinely used in nematology and other research areas to infer the role of specific genes in different processes, in this case in gall formation, we have also developed a detailed methodology to assess differences in nematode infectivity with minimal interference of altered root architecture phenotypes in the results.
Using these methods, we have studied the changes occurring in the methylome and transcriptome during gall formation by M. javanica in A. thaliana. A generalized hypermethylation was observed in early developing (3 days post infection, dpi) galls when compared to the respective non-infected control root tissue in A. thaliana and in tomato. However, at later stages (14 dpi), no significant changes in the methylome were observed. Hypermethylated GCs within the gall seemed to be contributing for the generalized gall hypermethylation observed. Several Differentially Methylated Regions (DMRs) were identified at 3 and 14 dpi. In 3 dpi galls, most of them overlapped transposable elements (TEs), mainly located at the pericentromeric regions of the chromosomes. sRNA libraries from previous studies were also used to examine how they distribute along the different chromosome regions and assess if they match with DMRs. Predominantly gall-exclusive (eGall) 24-nt siRNAs accumulated at DMRs overlapping TEs, genes and promoters, followed by 22-nt siRNAs. Moreover, eGall-rasiRNAs, previously shown to accumulate mainly at pericentromeric regions of the Arabidopsis chromosomes, accumulated preferentially at DMRs overlapping TEs, while eGall-non-rasiRNAs mainly matched promoters and gene sequences. The gall transcriptome also revealed some correlation with genes and gene promoters overlapping DMRs, in which members of transcription factors families and proteins involved in epigenetic processes were present. Mutants impaired in different proteins involved in (de)methylation revealed the involvement of different methylation regulatory pathways during gall formation. Our results show that DNA methylation mediated by RNA is contributing to the epigenetic changes occurring during gall formation, which might be essential for TE stability and therefore maintenance of genome integrity during the cell differentiation processes occurring in galls and giant cells.
