Tesis doctoral de Marta Barcala Rodriguez
Summary meloidogyne spp. Is a worldwide spread nematode genus that belongs to the plant sedentary endoparasites. These nematodes induce the development of special feeding cells, called giant cells (gcs) by still unclear mechanisms that allow them to establish and reproduce successfully within their host roots. During this differentiation process, the gcs enlarge, become multinucleate, and acquire a dense cytoplasm and a highly active metabolism. All these alterations are accompanied by extensive gene expression changes which are still poorly understood. As a result of the efforts of several laboratories to bring some light to these gene reprogrammation events, the induction and/or repression of several genes in either gcs or galls has been reported. However, we are still far from understanding how the nematode impinges on the developmental program of the selected root pro-vascular cells and redirect them to become gcs. It is generally accepted that many of the events leading to gene expression changes are caused directly or indirectly by molecular components of the nematode secretions. Furthermore, the mechanisms underlying the plant root response to nematodes that lead to the development of a new organ, the gall, are also unidentified. in this context, based on previous studies that reported the induction by nematodes of genes differentially regulated during embryogenesis (abi3 and genes encoding lea-like proteins) in galls and gcs, we aimed to study the expression of other genes similarly regulated. Thus, we analysed the induction of a class i small heat shock protein gene from sunflower (hahsp17.7g4; g4) in nicotiana tabacum plants after m. Incognita infection by using promoter-gus fusions. We found that g4::gus, described to be differentially regulated during both heat shock and embryogenesis, was active within gcs at mid-infection times, and to a lesser extend in gc younger than 7 days post infection (dpin). Additionally, 5¿deletions of the g4 promoter were tested and a minimal nematode-responsive promoter could be defined. When we transformed arabidopsis thaliana plants with the shortest g4::gus carrying only one heat-shock element (hse) arrangement, gus expression patterns were faithfully reproduced during heat-shock and after nematode infection. Moreover, the activation of two different promoter versions mutated in nucleotides crucial for heat-shock factors (hsfs) binding was analyzed in gcs. A mutant version of the g4 promoter not responsive to heat-shock (g4 mute) was also inactive in gcs, whereas another mutant construct showing a decrease in both the embryogenesis and heat-hock response (g4 muta) showed a similar induction as the wt (g4) in gcs. All these results suggested the involvement of hses in nematode-responsiveness of the g4 promoter, probably through hsfs. Presumably, this finding indicated differences between the regulatory mechanisms acting during heat-shock or embryogenesis from those operating in gc induction. In order to confirm this hypothesis, we analyzed the regulation of a third mutant version of g4 promoter (g4 mutp) and two additional shsp promoters (hahsp17.6 (g1) and hashp18.6 (g2)). Tobacco g4 mutp::gus transgenic plants carried point mutations in its promoter that improved hsf binding and showed a reduced late-embryogenesis response, but maintained its activity in gcs. G1 promoter was silent in tobacco plants both during heat-shock and after nematode infection, but not during late-embryogenesis. On the contrary, g2, a heat-shock responsive gene that is not expressed during late-embryogenesis was positively regulated in gc. Hence, this analysis further supported a role of hsf in shsp promoter induction in developing gc and indicated similarities (but not identity) in the regulatory mechanisms taking part in gcs as compared to those involved in the heat-shock response. It also confirmed a role of specific hses arrangements in shsp promoter activation in gcs. although no functional assays were performed to establish a role of the shsp in gc development, immunolocalization experiments indicated that class i shsp accumulated specifically in the cytoplasm of gcs, suggesting that they must have a relevant role possibly related to gc formation, maintenance and/or nematode feeding habits. This finding also indicates that not only a heterologous promoter was induced in these cells, but also the endogenous gene(s) coding for at least one class i shsps was active. in order to get new insights into the global reprogrammation events taking place during gc differentiation, a transcriptome analysis was carried out with rna from the first stages of gcs development (3 dpin) in a. Thaliana plants. To this end, we combined the microarray hybridization technology with laser capture microdissection, what allowed us to specifically analyze the 3 dpin gcs transcriptome. Remarkably, most of the differentially expressed genes observed (1160), were repressed. This may indicate the importance of the reprogrammation events based in gene silencing to change the developmental program of the vascular root stem cells in order to differentiate into gcs; particularly genes coding for enzymes related to plant defence, like peroxidases, glutathione-s transferases or pathogenesis related proteins, showed an overall down-regulation. In contrast, at this very early infection stages, genes coding for proteins involved in cell wall modifications, cell cycle changes or cytoskeleton rearrangements were mostly induced, in agreement with the dramatic morphological changes observed in the gcs. moreover, the relevance of analysing the transcriptome of specific cells instead of tissues or organs, which can be facilitated by microdissection, was highlighted in the comparison between microarray analyses carried out with rna from microdissected gcs and hand-dissected whole galls in arabidopsis. Although the results must be taken cautiously, since neither the infection time nor the nematode species were fully equivalent, few similarities were encountered in the set of differentially expressed genes in both experiments. This finding reinforces the importance of the dilution effect caused by the presence of several different tissues and cell types within a gall, where only 4-8 gcs contribute to the total mrna extracted. finally, comparisons with other transcriptome data downloaded from the public genevestigator database revealed unexpected similarities with the gcs differential transcriptome. One of the most notably similar situations in terms of specifically regulated genes was that of agrobacterium tumefaciens-induced crown-galls. The other one was obtained with arabidopsis cell suspension cultures undergoing differentiation towards xylem cells, providing the first molecular evidence that cells from pro-vascular tissues could be the stem or initial cells for the nematode-induced differentiation of gcs.
Datos académicos de la tesis doctoral «Análisis de los cambios en la expresión génica producidos por los nematodos endoparásitos en los sitios de alimentación«
- Título de la tesis: Análisis de los cambios en la expresión génica producidos por los nematodos endoparásitos en los sitios de alimentación
- Autor: Marta Barcala Rodriguez
- Universidad: Castilla-la mancha
- Fecha de lectura de la tesis: 23/05/2008
Dirección y tribunal
- Director de la tesis
- Carolina Escobar Lucas
- Tribunal
- Presidente del tribunal: francisca Fernández del campo
- godelieve Gheysen (vocal)
- keith Lindsey (vocal)
- blanca San segundo de los mozos (vocal)