Final Report Summary - RNAI IN PLANTS (Untangling the network of RNA silencing pathways in plants.)
Title of the project: Untangling the network of RNA silencing pathways in plants.
Grant Agreement Number: PIEF-GA-2010-276037
Scientific in Charge: Prof. David Charles Baulcombe dcb40@cam.ac.uk
Name of Researcher: Dr. Adrian Alejandro Valli
Final report.
Introduction:
Because of the fully sequenced haploid genome, the controlled sexual cycle, short generation time and rapidly developing marker systems, the unicellular alga Chlamydomonas reinhardtii is an ideal model for genetic analysis of many biological questions. Moreover, Chlamydomonas is a good model for studying RNA silencing because, like higher plants and other eukaryotes, it produces different types of small RNAs to control gene expression. It is also known that its genome encodes three Dicer-like (DCL) proteins and three Argonaute (Ago) proteins and so it is likely to have RNA silencing systems with different interacting modules. These features, among others, suggest that this organism has the potential to serve as a model for other more complex systems including those in higher plants.
Project summary:
When I jointed Prof. Baulcombe’s laboratory in July 2011, previous attempts to reduce the expression of diverse Ago and DCL proteins in Chlamydomonas had failed. Therefore, the first objective of this project focused on developing an entirely novel forward genetic screen to identify mutants affected in RNA silencing factors, particularly in those affecting components of the micro(mi)RNA-silencing pathway. Firstly, this approach required several techniques and tools to be set up in order to make Chlamydomonas an amenable model system for robust screening. Once established, I took advantage of these procedures and isolated several mutants during the second year of this fellowship. The expression level of different types of small RNAs in the mutant lines was assessed, allowing me to cluster the diverse mutants into several distinct groups based on their molecular phenotypes. Finally, when possible, the specific location of the introduced mutations was mapped in the Chlamydomonas genome. Mutations in several genes that encode proteins expected to belong to RNA silencing pathways highlight the effectiveness of the system I have developed for forward genetics. Furthermore, some novel factors have also been found suggesting that Chlamydomonas reinhardtii will be a simple and exceptional model organism from which we can learn about the complexity of the RNA silencing mechanism and apply these insights to other organisms.
Aims:
- Develop a robust forward genetic screen to isolate mutants affected in the miRNA-mediated RNA silencing pathway in Chlamydomonas reinhardtii.
- Identify the mutated factors and investigate their role in RNA silencing.
- Assess the relevance of miRNA pathway in the life cycle of unicellular organisms.
Results:
The forward genetic screen I developed to find mutants affected in the miRNA-mediated RNA silencing pathway in Chlamydomonas is partially based on the artificial (a)miRNA technology previously published by Prof. Baulcombe laboratory. An amiRNA was designed to silence the expression of the phytoene synthase (PSY), a transferase enzyme involved in the biosynthesis of carotenoids, in an inducible manner. Reporter cells expressing the PSY-amiRNA grow normally without the chemical inducer. However, when the inducer is added to the media the reporter cells die as a consequence of low carotenoid levels due to strong PSY down-regulation. Alternatively, after random insertional mutagenesis, cells carrying a mutation that affect a key factor in the miRNA silencing pathway should also be able to grow. Using this idea, 22 mutated lines were isolated as they were no longer able to silence the PSY gene and hence survive under inducing conditions. Interestingly, small RNA northern blots showed that all of the mutated lines express either no or very low levels of the PSY amiRNA and several endogenous miRNAs, strongly suggesting that the miRNA-mediated silencing pathway has been affected. Additionally, Northern blot analysis allowed me to classify the mutants into several groups based on the accumulation of different small RNA types .
To determine the exact location of the mutation producing inserts, the previously developed RESDA-PCR technique was modified and used with success. Thus, the location of these inserts was mapped, using the latest version of the Chlamydomonas genome (at the time of writing this report it can be found in Phytozome v9.1) in 14 out of 22 mutants.
Mutants from Group I and II share a similar molecular phenotype with regard to their small RNA expression profiles, as determined by Northern blot analysis. The link between these groups was confirmed when the insertional mutagen was located, in most of the cases, in (or very close to) the Ago3 gene. Two different antibodies raised against Ago3 are available in our lab, which allowed the level of Ago3 to be tested in these mutant lines. As expected, Western blot analysis confirmed they do not express the Ago3 protein.
The molecular phenotype observed in mutants belonging to Group III, which displayed no expression of all the tested small RNAs, suggests a key factor that participates in small RNA biogenesis has been knocked out. In agreement with this observation, insertions affecting a particular DCL protein (DCL3), the enzyme that processes pri-miRNA transcripts to produce miRNA, were found in 2 out of 4 mutants (Table 1).
Potential impact:
Chlamydomonas reinhardtii is a unicellular green alga that has proven to be an ideal model for studying several biological processes, including photosynthesis, flagellar functions and cell motility, circadian clock and responses to stress and other stimuli. Moreover, this microalga has the potential to revolutionize biotechnology in a number of areas including nutrition, aquaculture, pharmaceuticals, and biofuels, so that the interest in Chlamydomonas has hugely increased in the last decade.
Following their discovery and characterization in multicellular organisms, it was originally hypothesised that miRNAs might have helped to drive evolution of the multicellular state. However, findings from Prof. Baulcombe’s laboratory in 2007 demonstrated that miRNAs are also present in the unicellular algae Chlamydomonas reinhardtii, casting shadow on all those previous suggestions. Although further studies tried to explain the role of miRNAs in unicellular organisms, the answer to this question is still obscure. Thus, the main future challenge of this project is to understand the biological relevance of the miRNA-mediated silencing pathway in the life of unicellular organisms in general, using Chlamydomonas as an amenable model. I predict my future findings on the role of RNA silencing will impact and revolutionize basic and applied sciences in which Chlamydomonas is being used.