Edición de genes de tomate que codifican potenciales factores provirales para el virus del mosaico del pepino dulce

Resumen

Pepino mosaic virus (PepMV; species Pepino mosaic virus, genus Potexvirus; family Alphaflexiviridae) is a positive-sense single-stranded RNA virus that causes major diseases affecting tomato crops worldwide, resulting in significant economic losses. Currently, no resistant tomato varieties are commercially available and the described sources of resistance are not suitable for their use in breeding programs. Thus, the identification of new genetic targets to obtain resistance to PepMV is very interesting from an applied point of view. On the other hand, the experimental system PepMV/tomato is acquiring an increasing importance, so deepening the knowledge of the molecular aspects of this interaction has on itself a great importance from the fundamental point of view. In the first chapter of this thesis, I describe the identification of 21 tomato proteins that interacted with PepMV proteins in a yeast-two hybrid screening using a normalized cDNA library from healthy and infected tomato plants as bait. In most cases, the virus-induced silencing of the genes encoding these proteins did not result in any different phenotype compared with the wild type (WT) plants. Interestingly, the plants silenced for the genes encoding the RdRp-IP5, TGB1-IP12 and TGB2-IP18 proteins showed a decrease in PepMV accumulation in addition to preserving a phenotype similar to the WT plants. TGB2-IP12 was functionally annotated as a serine/threonine kinase, but the other two proteins were not annotated in public databases. The bioinformatic analysis of RdRp-IP5 suggested that it belongs to the NTF2 protein superfamily, with a possible role in the response to biotic stresses. For TGB2-IP18, I could not find a clear indication of a function, although it has an orthologous gene in Arabidopsis thaliana annotated as part of the cytochrome b6f complex. I also studied the subcellular localization of these two proteins fused to fluorescent proteins by confocal laser scanning microscopy in healthy and infected Nicotiana benthamiana plants. I found colocalization of RdRp-IP5 and TGB1-IP12 in structures similar to the viral replication complexes (VRCs) described previously. However, the TGB2-IP18 protein kept the same chloroplastic localization in healthy or in infected cells. Finally, I generated knockout homozygous mutants of the genes encoding RdRp-IP5 and TGB1-IP12 respectively, and a heterozygous mutant with a single edited allele of the gene encoding TGB2-IP18 using the CRISPR/Cas9 system. PepMV accumulation was not altered in rdrp-ip5 and tgb2-ip18 infected mutants. However, the tgb1-ip12 infected mutant showed an increased accumulation of the virus, suggesting that TGB1-IP12 may have a role during PepMV infection in tomato. In the work described in the second chapter, I generated seven mutants of tomato cv. Micro-Tom on genes encoding TGB2 interacting proteins (TGB2-IP14-21) by the CRISPR/Cas9 technology. To test the role of these proteins during viral infection I challenged these mutants with PepMV; tgb2-ip19 and tgb2-ip21 infected mutants showed significantly higher viral title compared with WT plants. TGB2-IP19 was annotated as an S-adenosylmethionine-dependent methyltransferase type 11, while TGB2-IP21 was annotated as a protein of unknown function. An in silico study suggested nuclear and cytoplasmic localization of TGB2-IP19 and its possible implication in the response to biotic stresses. The predicted localization of the TGB2-IP21 protein was anchored to the membranes of the endoplasmic reticulum (ER) or mitochondria and it was possibly involved in processes related to the response to biotic stress, although a specific function could not be predicted. In the work described in the third chapter, I expressed TGB2 of PepMV fused to fluorescent proteins by agroinfiltration in N. benthamiana plants. The confocal laser scanning microscopy analysis of TGB2 showed a fluorescent signal that colocalized with an ER-specific marker protein. The expression of TGB2 in plants infected with PepMV resulted in the relocation of the fluorescent signal of TGB2 into the VRCs. The co-expression of TGB2 with its interactor TGB2-IP19 showed a colocalization of both fluorescent signals at nuclei and cytoplasm in uninfected plants, and a relocation of the fluorescent signal of both proteins to the VRCs in infected plants.