Tesis doctoral de Mary Esperanza Portillo Bocanegra
Root-knot nematodes (rkn; meloidogyne spp.) Are sedentary parasites that affect a high variety of plants with a negative impact in the production of crops worldwide among those tomato, important vegetable in the human nutrition and health. The infective rkn in the root induces 4-7 highly specialized feeding cells called giant cells (gcs), nutrient sinks that nematode uses to complete its life cycle. Drastic alterations in gene expression during gcs differentiation are correlated with cell wall expansion, nuclear division without cytokinesis, endoreduplication and the development of a metabolically active cell as well as the hypertrophy of the cells surrounding gcs shaping a visible gall in the root. Multiple studies have identified different gene expression changes both in galls and gcs, as well as different roles of nematode secretions in the migration and in the re-differentiation process of root cells; although the specific molecular mechanisms that lead the complex reprogramming events of vascular cells to nematode feeding cells induction and development are unknown yet . Further understanding of these events may offer new strategies for the improvement of nematode control in crops. In order to advance in this objective, we performed a comprehensive examination of the tomato (solanum lycopersicum) transcriptome during the course of meloidogyne javanica infection using tom1 microarray independently in whole galls and isolated gcs. to ensure the high quality of differential gene expression study vigorous quality control measures were required at each individual step. Initially, a strict monitorization of the infection time was designed and performed in vitro to guaranty minimal sample age variation since root-knot nematodes penetration and establishment are not synchronous. Additionally, the reliability of the output data is strongly related to the high quality and integrity of the rna isolated from the tissues. Therefore, three different commercial reagents and three homogenization procedures, commonly used in most laboratories, were assessed to avoid partial degradation and loss during rna isolation from root and gall tissue. The best combination with the highest yields and quality-integrity was used in the galls transcriptomic study. on the other hand, in plant-nematode interaction the study of the gcs specific transcriptome has not been possible to date by the difficult to precise isolation of gcs without contamination from other cells in the heterogeneous gall tissue. To gain access to gc-specific mrnas was developed a simple, quick and efficient method based in cryopreservation, suitable for laser capture microdissection (lcm) of pure gcs content. Using this approach, it was possible to specifically identify and capture gcs as early as 3 days post-infection (dpi), and the isolation of sufficient rna from a few quantities of cells with a high quality/integrity, which after two rounds of amplification was successfully used for microarray hybridization. following these procedures, a differential gene expression study was carried out with rna from hand-dissected galls at different infection stages (1, 3, 7 and 14 dpi) compared with equivalent uninfected root segments taken as controls. Independently, other study to determine the transcriptional changes occurred specifically within gcs at 3 and 7dpi was developed. In addition, from these studies a comparison among whole galls and gcs at 3 and 7 dpi was performed, in order to identify key genes and/or pathways that may play important roles in the gcs induction, differentiation and function. in galls among the approximately 8500 tomato genes or unigenes monitored in the tom1 microarray were identified 1839 unigenes displaying significant differences in mrna levels between hand-dissected galls and controls with strong differences between gene number and their expression over time of infection. One of the most striking observations in our investigation was the high proportion of genes down regulated at early stages of infection mainly at 1 dpi, especially associated with regulation of transcription, stress and defense. This suggests that for a successful initial establishment, a generalized repression of genes may be necessary. At 7 and 14 dpi, a greater impact of transcriptional changes was evident, as the number of differentially regulated transcripts increased by two orders of magnitude in comparison to the earliest stages. Notable changes in the protein synthesis, regulation of transcription and metabolism were observed. Interestingly, genes encoding chromatin remodelling proteins, histone deacetylases and methyltransferases were induced mainly at 14 dpi. in gcs, our approach led to the identification of 1051 unigenes with significant changes in the expression level at 3 and 7 dpi, as compared to cells of the vascular cylinder from non infected roots. At 3 dpi, a large proportion of down regulated genes was observed. This result confirms the importance of a cellular reprogramming, based in down regulation of gene expression, to shift the stem cells differentiation program towards a gc differentiation program. However, among the few up regulated genes, some genes encoding chromatin remodeling protein and a methyltransferase could be found at this early stage. At 7dpi in gcs the number of differentially expressed genes was drastically increased, with a clear change in the expression level and an equilibrate proportion between down and up regulated genes. The transcriptional changes reflected alterations in all cellular processes associated with the cellular morphology transformation. Genes related to protein synthesis, metabolism and regulation of transcription were the most affected. Remarkably, genes related with ribosomal protein were up regulated, suggesting that the level of protein synthesis is substantially altered; a process likely associated to the cell developmental status and/or the nematode nutritional needs. Differences in the regulation of transcription also were observed in gcs at 3 and 7dpi, as they could be key regulators of different pathways putatively involved in gcs differentiation and/or maintenance. additionally, a high number of genes encoding histones and ribosomal proteins, as well as a gen involved in the nucleosome assembly and two histone deacetylases, were exclusively up regulated at 7 dpi. Dynamic changes in chromatin structure are associated with epigenetic processes and transcriptional regulation. Our finding may be interpreted as a widespread form of transcriptional control induced by nematode throughout development of feeding cells. These data suggest that most transcriptional changes related to chromatin remodelling and protein synthesis are different across giant cells stages, perhaps reflecting distinct cell morphogenesis processes. the comparison between the differential transcriptomes data of whole galls and gcs at the same developmental stages for 3 and 7 dpi, allowed us to obtain exclusive genes for both gcs and galls, as well as co-regulated genes. A functional classification of exclusive genes established clear differences in gene expression pattern between gcs and galls, as in the secondary metabolism related with the plant defense. Interestingly, when the genes co-regulated in gcs-galls were compared, the gall log 2 values were lower in galls as compared to gcs. This reinforces the importance of the dilution effect of gcs-specific transcripts on hand-dissected galls and explains that small changes in gene expression within gcs may be undetectable in whole gall transcriptome analysis. In the present work, we made the first direct analysis that, by using comparable biological samples analyzed in the same microarray platform, demonstrates the sensitivity of the lcm approach to study transcriptional changes during gcs development. finally, the results were confirmed by in situ reverse transcription-pcr localization and by real time rt-pcr (qpcr). In situ reverse transcription-pcr demonstrated the induction of specific genes with specific localization only in gcs or gcs plus surrounding cells, on 3 and 7 dpi tomato gall sections. The qpcr revealed for 15 genes similar transcriptional patterns to those found in the microarray hybridizations; these results indicate a high reliability of the data obtained from our approach. in conclusion this study has identified a large panel of tomato genes expressed in gcs, revealing pathways that appear to play important roles in their induction, formation, and function as nutrients sinks to support nematode development and reproduction. Thus, this analysis provides a broad basis for future research to understand rkn nematode parasitism and to develop effective management tools against this parasite.
Datos académicos de la tesis doctoral «Reprogramacion global de la transcripcion durante el desarrollo de las celulas gigantes y agallas inducidas en solanum licopersicon (tomate) por meloidogyne javanica.«
- Título de la tesis: Reprogramacion global de la transcripcion durante el desarrollo de las celulas gigantes y agallas inducidas en solanum licopersicon (tomate) por meloidogyne javanica.
- Autor: Mary Esperanza Portillo Bocanegra
- Universidad: Castilla-la mancha
- Fecha de lectura de la tesis: 25/05/2009
Dirección y tribunal
- Director de la tesis
- Carolina Escobar Lucas
- Tribunal
- Presidente del tribunal: francisca Fernández del campo
- vivian Blok (vocal)
- alfonso g. Navas sanchez (vocal)
- María mercedes Lucas sanchez (vocal)