Dr Hilary Rogers - PhD
My research focuses on gene expression changes elicited by stress in plants and fungi. In particular I am interested in interactions between stress and senescence/ cell death. This work has focussed on three areas:
- Plant organ senescence and responses to stress. This includes work on petal senescence in ornamental species, and interactions between stress and senescence responses in the model plant Arabidopsis thaliana.
- Genes regulating the effects of stress on plant cell division. Of particular interest have been the effects DNA damage/ DNA replication checkpoint controlling entry of cells into mitosis.
- Stress induced during mycelial interactions between competing fungi. This has focussed on wood rotting fungi, following gene expression and enzyme production as competing mycelia interact.
I also collaborate on other projects to understand microbial/plant interactions relating to fungal and bacterial endophytes, and in the use of molecular markers to assist in fungal taxonomy and ecology.
I am part of a number of collaborative groups and further information on our projects may be available on the web-sites of my collaborators.
1) Plant Organ senescence
A) SAG21/AtLEA5 a gene at the interface between stress responses and senescence
SAG21 belongs to the late embryogenesis-associated (LEA) protein family. Although it has been implicated in growth and redox responses, its precise roles remain obscure. To address this problem, in collaboration with Prof Christine Foyer (Leeds) and Dr Frederica Theodoulou (Rothamsted) we characterised root and shoot development and response to biotic stress in SAG21 over-expressor (OEX) and antisense (AS) lines. AS lines exhibited earlier flowering and senescence and reduced shoot biomass (Fig 1) (Mohd Salleh et al, 2011).
Fig 1: A: Arabidopsis lines in which SAG21 is over-expressed (OEX) B: and down regulated by antisense (AS) showing effects on (A) growth and (B) senescence (Mohd Salleh et al., 2012)
Expression of SAG21 is induced by numerous abiotic stresses and in collaboration with Dr Luis Mur (Aberystwyth) we also investigated whether perturbation of SAG21 affected growth of pathogens. We found that growth of the fungal nectroph, Botrytis cinerea and of a virulent bacterial pathogen (Pseudomonas syringae pv. tomato) was affected by SAG21 expression, however growth of an avirulent P.syringae strain was unaffected (Fig. 2). In collaboration with Dr John Runions (Oxford Brookes) we showed that a SAG21 -YFP fusion was localised to mitochondria, raising the intriguing possibility that SAG21 interacts with proteins involved in mitochondrial ROS signalling which in turn, impacts on root development and pathogen responses.
B) Floral Senescence
Fig. 1 Stages of Alstroemeria flower development and senescence and microarray analysis showing significant changes in gene expression (Breeze et al. 2004)
Floral senescence in many species is largely controlled by the plant growth regulator ethylene. However, the senescence of many economically important species is not ethylene sensitive and therefore the techniques presently available are ineffectual at prolonging their storage or vase life. How floral senescence in these species is regulated remains an interesting biological question. In collaboration with Dr A Stead (Royal Holloway), Dr B Thomas, and Dr V Buchanan-Wollaston (University of Warwick HRI), we investigated the biochemical and molecular events occurring during floral senescence in an important UK cut flower crop Alstroemeria (Wagstaff et al. 2002a,b; 2003; Leverentz et al., 2003). This species shows ethylene independent floral senescence (Wagstaff et al, 2005) with flowers lasting up to 15 days under optimal conditions. Using microarrays we have shown that large numbers of transcripts are up-and down-regulated during senescence (Breeze et al. 2004) (Fig. 3). We are currently studying the expression of our EST collection in shorter lived varieties of Alstroemeria and closely related species.
Fig 4 - Microarray analysis of changes in gene expression in Alstroemeria petals during senescence and following stress treatments at bud stage (S2) Wagstaff et al. (2010)
We were also interested in discovering what the overlap in gene expression is between developmental senescence, and premature senescence induced by environmental stress such as ambient dehydration and cold storage. This has implications both for understanding the regulation of senescence regulatory networks and has practical implications in the cut flower industry where flowers are often stored in suboptimal conditions during the transport chain. Microarray analysis revealed that there was significant sharing of gene expression between developmental senescence and an ambient dry stress treatment, whereas cold induced a distinct profile of transcripts (Wagstaff et al., 2010) (Fig. 4).
One of the transcripts whose expression changed during cold treatments is a terpene synthase-like gene and we are currently studying its expression during development in scented and non-scented varieties of Alstroemeria.
Petals and leaves are considered to be of common evolutionary origins, and both senesce as their final stage in development. We therefore asked to what extent gene expression changes during senescence were shared between these two organs. We chose to use wallflowers (Erysimum linifolium) as our model, a member of the Brassicaceae in which flowers follow a clear developmental pattern lasting 8 days from bud to petal abscission (Fig. 5). Using microarrays we have shown that over half of the transcripts in an EST collection changed in expression in the same way in the two organs whereas expression of a class of chitinase related genes and GSTs was specific to petal senescence (Price et al., 2008) (Fig. 5). The role of reactive oxygen species (ROS) in petal senescence is far from clear (Rogers, 2011) and we are currently studying genes related to ROS and stress during petal senescence in both wallflowers and Arabidopsis.
Fig 3 - Stages of wallflower petal and leaf senescence, and functional analysis of genes specifically up-regulated during petal senescence (Price et al. 2008)
2) Effects of stress on cell division
Fig 4 - Induction of Arath; WEE1 in yeast cells resulting in G2/M arrest and increased cell size
Plants are subject to numerous stresses including DNA damaging agents such as uv, soil pollutants and saline environments. Many of these agents cause an arrest in cell division until favourable conditions allow growth to resume. In collaboration with Dr Dennis Francis, and Dr RJ Herbert (University of Worcester) we have been studying genes that regulate these responses. DNA damage induces a cell cycle checkpoint arresting cells at G2/M One of the key regulators of this process in plants is WEE1 kinase which inactivates the the cyclin dependent kinase (CDK) by phosphorylation (Sorrell et al., 2002) (Fig 6.). We have recently shown that Arabidopsis WEE1 binds to 14-3-3 proteins (Lentz, 2009). In other eukaryotes this interaction is mediated by phosphorylation of WEE1 and we also showed that mutation of a predicted phosphorylation target in Arath;WEE1abolishes interaction to GF14w (Fig. 7).
Fig 5 - Interaction between Arath; WEE1 and GF14 using bimolecular fluorescence: YFP signal indicates interacting proteins. Mutagenesis of Arath;WEE1 at S485A abolishes 2-hybrid interaction between the two proteins as shown by the lack of LacZ reporter gene activity (blue)
We are currently studying the regulatory networks of interacting proteins that modulate WEE1 activity.
We are also interested in how cell division recovers from stress. In other eukaryotes CDC25 is a phosphatase that releases the block on cell division imposed by WEE1. A full length CDC25 is not present in higher plant genomes. However shorter gene lacking the regulatory domain can induce in a small cell size phenotype when expressed in fission yeast, suggesting a role in cell cycle regulation (Sorrell et al, 2004) (Fig. 8). We are further studying the role of Arath;CDC25 in the plant cell cycle (Spadafora et al, 2010).
Fig 6 - Expression of pREP::Arath;CDC25 in fission yeast results in small cell size
Other projects in the lab focus on the effect of other stresses on the cell cycle such as salinity and toxic metals e.g. cadmium.
3) Mycelial interactions between competing fungi and fungal ecology
Fig 7 - Microarray analysis of gene expression changes in Trametes versicolor mycelium close to the interaction zone with competitors: Stereum gausapatum (Sg) Bjerkandera adusta (Bk) and Hypholoma fasciculare (Hf)
In collaboration with Prof Lynne Boddy, we have been using molecular approaches including microarrays to study gene expression during inter-species fungal interactions. Fungal interactions can result in deadlock or overgrowth by one of the competitors. Our microarray analysis indicates that changes in gene expression are related to the outcome (Eyre et al 2010; Fig. 9). We have also focussed on ROS –related enzymes and shown that the activity of these enzymes is up-regulated close to the interaction zone between competing fungi in all interactions irrespective of outcome (Hiscox et al. 2010) (Fig. 10).
We are currently analysing expression of genes related to interactions through over-expression in T. versicolor.
Fig 8 - laccase, manganese peroxidase and other peroxidases are up-regulated at the interaction zone between Trametes versicolor (Tv) and Stereum gausapatum (Sg) (visualised by staining) as well as in interactions between Tv and Bjerkandera adusta (B) and Hypholoma fasciculare (Hf) measured by enzyme activity
Other collaborative projects
A) Fungal taxonomy
In collaboration with Prof Lynne Boddy and Dr Martyn Ainsworth (Kew Gardens) we have been using targeted PCR primers in support of conservation efforts of rare UK fungi. We were able to show that Hericium species that fruit rarely are present as endophytes in the sapwood of many tree species (Parfitt et al, 2007). We have also used ITS sequencing to help to clarify taxonomic relationships within Hydnellum and Phellodon, which are often difficult to distinguish based on morphology(Fig 11), revealing the presence of cryptic species. Current work is aimed at extending our taxonomic understanding of these species.
Fig 9 - Phellodon melaleucus, B: Phellodon confluens, showing very similar morphology
B) Bacterial endophytes
In collaboration with Dr Esh Mahenthiralingam and Dr Colin Berry we are exploring further the movement of bacterial endophytes within plants and its biotechnological applications.
Staff associated with research:
David Parfitt, Julie Hunt, Jennifer Hiscox (post-doctoral researchers on on fungal populations and ecology)
Cristian Vidal Quist (post-doctoral researcher on plant-bacterial interactions)
Natasha Spadafora (post-doctoral researcher on post-harvest biology)
Adel Elmaghrabi (current postgraduate on stress and cell division)
Faezah Mohd Salleh and Danilo Aros (recent postgraduates on floral development and senescence)
Golnaz Rafiei and Gemma Cook (recent postgraduates on stress and cell division)
Wafa Muftah (recent postgraduate on fungal populations and ecology)
Other international collaborations
Prof Nello Ceccarelli and Prof Piero Picciarelli (University of Pisa, Italy) on floral senescence
Dr Lien Gonzalez (University of Havan Cuba) on the cell cycle
Prof Diego Albani (University of Sassari, Sardinia, Italy) on the cell cycle
Prof M. Beatrice Bitonti (University of Calabria, Italy) on the cell cycle
Prof Antonio Ferrante (University of Milano, Italy) onstress responses
Dr Wan Liu (China) onstress responses