Department of Plant Sciences

Professor David Baulcombe

Find out more about David Baulcombe and his group or email David Baulcombe@plantsci.cam.ac.uk

Project titles:

  1. Genetic analysis of RNA silencing mechanisms
  2. RNA silencing, natural variation and hybrid incompatability in plants
  3. RNA silencing and heritable epigenetic changes
  4. Artificial evolution of disease resistance

We work on two main topics that are linked by a long standing interest in the interactions between viruses and their hosts. The first area involves protein-based innate immunity mechanisms in plants against viruses and we are focusing on aspects of the molecular recognition process whereby plants recognize the presence of the virus. Our aim in this area is to develop approaches to breeding or engineering of disease resistance in plants.

The second topic involves family of RNA-based processes collectively known as RNA silencing. The defining feature of these processes is the involvement of short RNA molecules that guide an Argonaute/Piwi protein to their target. We developed an interest in this process because it explains immunity and cross protection phenomena that have been known about in the virological literature for decades. The observation was that plants infected with a virus become resistant to secondary infection by the same or related viruses but not to distant or non relatives. We now understand that the immunity results from presence in the infected plant of short silencing RNAs that are derived from the viral genome. These RNAs then guide Argonaute so that it can destroy the RNA of secondarily inoculated viruses.

We continue to address the role of RNA silencing in virus-host plant interactions. However we have also broadened our interest because short silencing RNAs have general roles in genetic and epigenetic regulation in plants. We are exploring the various mechanisms of RNAs silencing and are particularly interested in the extent to which it influences natural variation and responses to stresses. Until now most of our work has been with Arabidopsis but we have now adopted the use of other model plants including tomato and the unicellular alga Chlamydomonas reinhardtii. Our approaches include genetic and biochemical methods. We have recently adopted the use of novel high throughput sequencing technologies that lead us into genomics and systems biology. To allow us to take full advantage of these approaches we are setting up a computational biology team to be linked with our group.

Please refer to our website for more complete details of our work and downloads of our publications: http://www.plantsci.cam.ac.uk/research/davidbaulcombe.html.

 

Project Title: Genetic analysis of RNA silencing mechanisms.

Supervisor: Professor D.Baulcombe

Project outline:

The basic mechanisms of RNA silencing are now well understood (Baulcombe, 2004). The main players are RNA dependent RNA polymerases, dsRNA, siRNAs or miRNAs, Dicer and Argonaute nucleases. However there are still many aspects of the mechanism that are unclear. It is not known for example how the surveillance mechanism operates so that an siRNA or miRNA complex with Argonaute is able to find its target RNA: do the potential targets and the Argonaute complexes get channeled through the same subcellular location to increase the chance of interaction or is the complex able to move rapidly through the cell so that target RNAs can be found? What determines whether an siRNA mediates transcriptional or posttranscriptional silencing? In transcriptional silencing how does an siRNA find its DNA target? Why are some targets of RNA silencing sources of secondary siRNAs whereas others are simply degraded?

To address these questions we are using genetic and biochemical approaches in two model systems – Arabidopsis and Chlamydomonas (Molnar et al., 2007). The approach is the same in both systems – a transgene is designed to report on a certain aspect of silencing for example in a particular cell type, in response to a stimulus, in a defined subcellular compartment or with characteristics of a particular type of silencing target. The transgene is incorporated into the genome of the model organism and we screen for mutants with either reduced or enhanced silencing of the reporter. Mutants are characterized and the mutant genes identified. This approach has been highly successful in the past (Baurle et al., 2007; Dalmay et al., 2000; Dalmay et al., 2001; Hernandez-Pinzon et al., 2007; Herr et al., 2005; Herr et al., 2006; Smith et al., 2007) and in future we will develop novel mutant screens to dissect specific aspects of RNA silencing.

Having used this genetic approach to identify a protein involved in the silencing mechanism we then focus on the protein. We purify the protein encoded at the mutated locus – either in its mutant or wild type form – and we assay for biochemical activity or use mass spectrometry to identify interacting proteins. Our papers on a RNA processing cofactors (Herr et al., 2006) and Argonaute (Baumberger and Baulcombe, 2005) illustrate the types of approach that can be taken.

The outcome of this project will be knowledge about RNA silencing mechanisms that may be relevant to non plant systems. This information will help unravel the biological roles of RNA silencing. It can also be exploited in the development of RNA silencing technologies in biotechnology and functional genomics. The project will provide training in genetics, molecular biology and aspects of protein and nucleic acid biochemistry. There will also be the opportunity to participate in the development of novel approaches for mapping of mutations based on the use of high throughput DNA sequencing technologies.

References and recent relevant papers from the lab (key papers marked *)

  1. *Baulcombe, D. (2004) RNA silencing in plants. Nature, 431, 356-363.
  2. Baumberger, N. and Baulcombe, D.C. (2005) Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits micro RNAs and short interfering RNAs. Proceedings Of The National Academy Of Sciences Of The United States Of America, 102, 11928-11933.
  3. Baurle, I., Smith, L.M.A., Baulcombe, D. and Dean, C. (2007) Widespread role for the flowering time regulators FCA and FPA in siRNA-directed chromatin silencing. Science (in press)
  4. Dalmay, T., Hamilton, A.J., Rudd, S., Angell, S. and Baulcombe, D.C. (2000) An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell, 101, 543-553.
  5. Dalmay, T.D., Horsefield, R., Braunstein, T.H. and Baulcombe, D.C. (2001) SDE3 encodes an RNA helicase required for post-transcriptional gene silencing in Arabidopsis. EMBO Journal, 20, 2069-2078.
  6. Hernandez-Pinzon, I., Yelina, N.E., Schwach, F., Studholme, D.J., Baulcombe, D. and Dalmay, T. (2007) SDE5, the putative homologue of a human mRNA export factor, is required for transgene silencing and accumulation of trans-acting endogenous siRNA. The Plant Journal, 50, 140-148.
  7. *Herr, A.J., Jensen, M.B., Dalmay, T. and Baulcombe, D. (2005) RNA polymerase IV directs silencing of endogenous DNA. Science, 308, 118-120.
  8. Herr, A.J., Molnar, A., Jones, A. and Baulcombe, D.C. (2006) Defective RNA processing enhances RNA silencing and accelerates flowering in Arabidopsis. Proceedings Of The National Academy Of Sciences Of The United States Of America, 103, 14994-15001.
  9. *Molnar, A., Schwach, F., Studholme, D.J., Thuenemann, E. and Baulcombe, D. (2007) miRNAs control gene expression in single cell alga Chlamydomonas reinhardtii. Nature, 447, 1126-1129.
  10. Smith, L.M., Pontes, O., Searle, I., Yelina, N.E., Yousafzai, F.K., Herr, A.J., Pikaard, C. and Baulcombe, D. (2007) A novel SNF2 protein associated with nuclear RNA silencing and spread of a silencing signal between cells in Arabidopsis. Plant Cell, 19, doi: 10.1105/tpc.1107.051540

See Research or email: David.Baulcombe@plantsci.cam.ac.uk

 

Project Title: RNA silencing, natural variation and hybrid incompatability in plants.

Supervisor: Professor D.Baulcombe

Project outline:

There are tens or even hundreds of thousands of endogenous siRNAs (Nobuta et al., 2007; Zhang et al., 2007) in plants. Many of these endogenous siRNAs are derived from transposons and repeated sequence elements that are polymorphic – their sequence and presence is variable between genotypes of the same species or between closely related species. In hybrids between distantly related genotypes or species these transposon or repeat specific siRNAs could target essential genes of the unrelated parent in the cross and limit the viability or vigour of the hybrid genome. In this way it is likely that RNA silencing would influence natural phenotypic variation and the outcome of wide crosses in crop breeding programmes. These RNA silencing processes would also influence evolution in nature through their effects on vigour of hybrids between genotypes in different populations or between species.

To assess the effects of endogenous siRNAs we shall characterize individual genetic loci in related genotypes of Arabidopsis and tomato that are the templates of siRNA production. We shall generate mutant genotypes of these plants and their relatives that are defective in siRNA production so that comparison of crosses between these mutants and the corresponding wild type crosses will be informative about the overall extent to which siRNAs affect natural variation and hybrid vigour. We shall also assess the influence of individual siRNA loci through the characterization of plant lines that are mutant at one or more of these of these siRNA loci.

The projects will involve the use of novel high throughput sequencing technologies to characterize the endogenous siRNA populations of mutant and wild type plants. Correspondingly there will be the need to develop new computational methods of processing the data from these technologies and for comparing large and complex dataset. To follow up the siRNA characterization we will use genetic and molecular approaches to generate plant lines down regulated for proteins or siRNA loci. These plants and hybrids between them will then be characterized by careful assessment of growth, development and responses to stress and disease stimuli. The findings from this project will be relevant to our understanding of natural variation in plants and the role of RNA silencing in evolution.

References and recent relevant papers from the lab (key papers marked *)

  1. *Baulcombe, D. (2004) RNA silencing in plants. Nature, 431, 356-363.
  2. Baulcombe, D. (2005) RNA silencing. Trends In Biochemical Sciences, 30, 290-293.
  3. *Nobuta, K., Venu, R.C., Lu, C., Belo, A., Vemaraju, K., Kulkarni, K., Wang, W., Pillay, M., Green, P.J., Wang, G.-L. and Meyers, B.C. (2007) An expression atlas of rice mRNAs and small RNAsNature Biotechnology, 25, 473.
  4. Zhang, X., Henderson, I.R., Lu, C., Green, P.J. and Jacobsen, S.E. (2007) Role of RNA polymerase IV in plant small RNA metabolism. PNAS, 104, 4536-4541.

See Research or email: David.Baulcombe@plantsci.cam.ac.uk

 

Project Title: RNA silencing and heritable epigenetic changes.

Supervisor: Professor D.Baulcombe

Project outline:

RNA silencing is a recently discovered genetic mechanism in many eukaryotes (Baulcombe, 2004; Baulcombe, 2005). It involves double stranded RNA that is processed into short 21-24nt RNA molecules. These short interfering (si) RNAs are then recruited as the specificity determinant of an Argonaute nuclease. Argonaute proteins target RNA molecules with sequence complementarity to the bound siRNA (Baumberger and Baulcombe, 2005). It is thought that the role of RNA silencing in a primitive eukaryote was as a defense against viruses and transposons that produced double stranded RNA. In modern organisms this defense role remains but the process is also used to regulate aspects of chromosome structure and genetic regulation during normal growth and development. Correspondingly, in plants, nematodes and other organisms there may be tens or hundreds of thousands of endogenous siRNAs (Nobuta et al., 2007; Zhang et al., 2007).

The siRNA targeting mechanism is best understood in systems in which RNA is the target and the silencing is posttranscriptional. However there are also systems in which the targeting mechanism targets DNA and the silencing mechanism operates at the transcriptional level (Jones et al., 2001). In these systems the transcriptional silencing is associated with methylation of the target DNA or modification of its associated histones or both. The transcriptional silencing effect in these systems may persist for longer than the initiator silencing RNA and, in extreme examples, for many generations (Jones et al., 2001). These are examples therefore in which RNA triggers a transgenerational effect. Because this is a heritable effect without changes to the sequence of DNA it is epigenetic rather than genetic.

In this project the aim is to investigate the extent of transgenerational effects of endogenous siRNAs. To what extent do these endogenous RNAs initiate transgenerational effects? Even if the frequency is low this mechanism could be a potent inducer of epigenetic mutations that would be subject to natural selection in the same way as genetic mutations. Such a mechanism could influence evolution in natural populations.

The project involves two stages. The first stage is to design reporter constructs of transcriptional gene silencing. These constructs will comprise a reporter gene coding sequence coupled to a constitutive promoter. Adjacent to the promoter sequence we will incorporate DNA elements corresponding to endogenous siRNAs induced in response to a stress treatment. The expectation is that the stress treatment will initiate transcriptional silencing of the reporter gene and in some instances the silencing effect will persist for several generations in the absence of the stress.

The results of this stage of the project will allow us to identify RNA silencing target sequences that are particularly sensitive to transgenerational effects. Having identified such sequences elements it will then be possible to design mutant screens to detect genes involved in these transgenerational effects. This mutant screen will be a major activity in the second part of the project.

The projects will involve the use of novel high throughput sequencing technologies to characterize the endogenous siRNA populations in stressed plants. Correspondingly there will be the need to develop new computational methods of processing the data from these technologies and for comparing large and complex datasets. Genetic and molecular approaches will then be used to identify genes required for transgenerational gene silencing. The outcome will be understanding of mechanisms that may be fundamentally important in adaptive responses to stress and in evolution.

References and recent relevant papers from the lab (key papers marked *)

  1. *Baulcombe, D. (2004) RNA silencing in plants. Nature, 431, 356-363.
  2. Baulcombe, D. (2005) RNA silencing. Trends In Biochemical Sciences, 30, 290-293.
  3. *Herr, A.J., Jensen, M.B., Dalmay, T. and Baulcombe, D. (2005) RNA polymerase IV directs silencing of endogenous DNA. Science, 308, 118-120.
  4. *Jones, L., Ratcliff, F. and Baulcombe, D.C. (2001) RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for maintenance. Current Biology, 11, 747-757.
  5. Nobuta, K., Venu, R.C., Lu, C., Belo, A., Vemaraju, K., Kulkarni, K., Wang, W., Pillay, M., Green, P.J., Wang, G.-L. and Meyers, B.C. (2007) An expression atlas of rice mRNAs and small RNAs Nature Biotechnology, 25, 473.
  6. Smith, L.M., Pontes, O., Searle, I., Yelina, N.E., Yousafzai, F.K., Herr, A.J., Pikaard, C. and Baulcombe, D. (2007) A novel SNF2 protein associated with nuclear RNA silencing and spread of a silencing signal between cells in Arabidopsis. Plant Cell, 19, doi: 10.1105/tpc.1107.051540

See Research or email: David.Baulcombe@plantsci.cam.ac.uk

 

Project Title: Artificial evolution of disease resistance.

Supervisor: Professor D.Baulcombe

Project outline:

Plant disease resistance can be enhanced by random mutagenesis of a disease resistance gene. In this project the aim will be to further explore the potential for this artificial evolution of disease resistance in agriculture and as a means of exploring factors that may influence the evolutionary dynamics of host parasite interactions in wild populations.

In our previous work we showed that a protein in potato – Rx – confers resistance against certain strains of potato virus X: other strains of virus overcame this resistance (Bendahmane et al., 1999). However after random mutagenesis of Rx we could identify mutants of Rx that were able to confer resistance against the strains that overcame the wild type resistance (Farnham and Baulcombe, 2006). This enhanced resistance after artificial evolution may be useful in agriculture.

In the proposed project we will use the Rx system to further explore the evolutionary dynamics of disease resistance in both the host and the pathogen. To explore the host side we will target mutagenesis to different domains in the Rx protein to identify regions where mutation has the greatest enhancing effect. To explore the pathogen side of the interaction we will randomly mutagenize genes in the viral pathogen to find out whether resistance-breaking strains can evolve equally against the wild type or mutant versions of Rx.

To investigate practical potential of artificial evolution we will randomly mutagenise a nematode resistance gene (Gpa2) that confers resistance against specific strains of Globodera pallida (van der Voort et al., 1999). Gpa2 is similar to Rx so that many of the findings from our virus resistance work will be transferable between genes. The mutant versions of Gpa2 will be tested for effectiveness against different strains of G. pallida and the most effective mutants will be transformed into nematode susceptible cultivars of potato. This part of the project will involve collaboration with Dr Pete Urwin at the University of Leeds who is a nematode expert and it may be necessary to spend time in Leeds for parts of the project.

The project will involve use of techniques in plant molecular biology and virology. The student will become familiar with the literature on disease resistance in plants, host-parasite interactions and with aspects of protein structure and function. The project will also introduce issues related to the use of genetically modified plants in agriculture and the distinctions between transgenic plants carrying non plant genes and cisgenic plants carrying genetically modified genes transferred between closely related plant species.

References and recent relevant papers from the lab (key papers marked *)

  1. Bendahmane A, Kanyuka K, Baulcombe DC (1999) The Rx gene from potato controls separate virus resistance and cell death responses. Plant Cell 11: 781-791
  2. *Farnham G, Baulcombe D (2006) Artificial evolution extends the spectrum of viruses that are targeted by a disease-resistance gene from potato. Proc Natl Acad Sci USA 103: 18828-18833
  3. van der Voort JR, Kanyuka K, van der Vossen E, Bendahmane A, Klein-Lankhorst R, Stiekema W, Baulcombe DC, Bakker J (1999) Tight physical linkage of the nematode resistance gene Gpa2 and the virus resistance gene gene Rx on a single segment introgressed from the wild species Solanum tuberosum subsp. andigena CPC 1673 into cultivated potato. Mol PlMicrobe Int 12: 197-206

See Research or email: David.Baulcombe@plantsci.cam.ac.uk