Viroids are exclusively plant pathogens with the lowest biological complexity.
In the last years they have been reported as infectious agents on a broad number of
species (herbaceous and woody) of agricultural interest. Their study, apart from the
agro-economic interest that it could report, is interesting by the fact that it is the
simpliest model to study the interactions between RNA pathogens and their hosts. As
viroids genome does not codify for proteins, all the steps of their life cycle are strictly
dependents on the interaction with host factors. The classical point of view of the viroid
pathogenic process asumes that the alterations induced in the plant are consequence of
the competence host-pathogen for the cellular factors that have a role in the
transcription, cell to cell movement or the systemic movement of the viroid.
Nevertheless some new results suggest that the host RNA silencing activation against
these pathogens is the responsible of the pathogenic process asociated with viroid
infection. On this thesis we have studied the relationship between the RNA silencing
mechanism and the viroid pathogenesis (concretely the induced by HSVd).
The first step (chapter 1) was the characterization of the developmental
alterations induced by the HSVd on transgenic Nicotiana benthamiana plants, that were
transformed with a construction that carried a dimerico form of this viroid. In a general
view, the analysis of the phenotipic alterations on N.benthamiana-HSVd2x transgenic
plants, confirmed their similarity with the symptoms asociated on natural hosts with
viroid infection, what lead us to justify our system (N.benthamiana-HSVd2x transgenic
plants) as a model to study the pathogenic process induced by nuclear viroids.
Facing a series of contradictory results in the literature about the involvement of
RNA silencing on viroid pathogesis, we considered that it was necesary to carry a direct
approach in order to study the interaction between main factors of the RNA silencing
machinery and viroids.
To carry this analysis (chapter 2) we studied 2 variables that were previously
reported to affect RNA silencing: a biochemical variable, RDR6, and an enviromental
variable, temperature. The results shown on this chapter point to RNA silencing
mediated by RDR6 as the main contributor to symptom expression on the interaction
HSVd-N.benthamiana, that, moreover, is independent of the pathogen accumulation
levels. This is the first direct experimental evidence about the existence of a close
relationship between viroid pathogenesis and viroid specific RNA silencing, that was
previously suggested by several authors.
One of the questions that arrise from chapter 2 is which could be the RNA
silencing RDR6 dependent pathway responsible for the pathogenic process of nuclear
viroids. Several observations lead us to propose that the pathogenic process induced by
viroids may be asociated with the trans-acting siRNAs (tasiRNAs) biogenesis pathway.
With the aim of getting deeper on vd-sRNAs biogénesis, which will offer a
much exact portrait about which components of the silencing machinery and which
viroid form could be implied in the production of vd-sRNAs, we carried a vd-sRNAs
masive sequencing on two different Cucumis sativus (a HSVd natural host) tissues, leaf
and phloem exudate. This data, shown on chapter 3, indicate that vd-sRNAs are
originated as the result of the jerarquic action of different DCL proteins agains a dsRNA
generated from a linear precursor and not from the mature form. Moreover, the phloem
library vd-sRNA data showed that there is a differential distribution in terms of size and
sequence on this tissue.
Although previous reports about how diverse pathogens change the miRNA
levels in plants, there were not deep studies about the effect of viroid infection over this
kind of RNA regulators in their hosts. Keeping in mind that, moreover, miRNAs hasn’t
been descrived in Cucumis sativus, our first step was the description of the endogenous
sRNA repertoire and the characterization of the miRNAs and their precursors (chapter
4).
Once determined the characteristics of Cucumis sativus endogenous sRNAs, we
had a reference to analyze their alterations asociated with the infectious process induced
by HSVd (chapter 5). Our results lead us to identify 4 different kinds of alterations
induced by HSVd infection: a) sRNA general profile alteration, being the 21 nts size
class the most accumulated on infected plant, b) repression and over-expression of some
miRNAs, c) differential expression of specie-specific miRNAs on infected plants and d)
alteration of the fidelity of procesing precission of miRNAs on infected plants.
Finally we conclude the thesis with a bioinformatic analysis of one of the main
features of the viroid pathogenesis RNA silencing related hypothesis, the potentiallity of
vd-sRNAs to regulate endogenous gene expression on plants. Potential targets are
mainly related with developmental processes, protein metabolism, stress response,
transcription regulation, cellular biogenesis etc. This allow us to speculate about the
existence of a direct relationship of the negative regulation of some of these genes
mediated by vd-sRNAs and the sympthoms observed on infected plants.