Plant growth is driven by photosynthesis. Yet how plants produce their photosynthetic proteins is not well understood. The chloroplast contains a genome that encodes key photosynthetic proteins and a unique molecular machinery that expresses them. Despite their importance, how the chloroplast gene expression machinery functions has not been characterised in detail. This project focuses on the first stage of gene expression in the chloroplast, in which genes are transcribed to produce messenger RNAs (mRNAs) that encode photosynthetic proteins. Regulation of chloroplast transcription underpins a key stage of plant development: the maturation of chloroplasts in response to light. This process is observable in the plant turning green and allows photosynthetic proteins to be selectively produced when solar energy is available for them to collect. Yet how chloroplast transcription is activated is not well understood. To advance our understanding of chloroplast transcription and the mechanism of its activation, we will characterise the enzyme that performs this process: the plastid-encoded polymerase (PEP). PEP is a large molecular assembly with at least 16 protein subunits. PEP is remarkable amongst transcription enzymes in that it contains subunits of two evolutionary origins. The core resembles bacterial enzymes and was inherited with the chloroplast genome from a cyanobacterial ancestor. By contrast, the twelve or more proteins that stably bind to the core are encoded in the nuclear genome and imported to the chloroplast. We therefore expect that these proteins, known as PAPs (PEP-associated proteins), orchestrate key regulatory processes unique to the chloroplast. In this project we will visualise the molecular architecture of the chloroplast transcription machinery using cryogenic electron microscopy (cryo-EM). Atomic models of PEP will shed light on how each subunit regulates transcription in response to the needs of the chloroplast. The level of detail provided by modern cryo-EM is immensely valuable to developing new hypotheses, as precise modifications can be designed with predictable changes in activity. We will examine the consequences of making specific changes, using transcription reactions reconstituted with purified components and plant genetic complementation experiments. The outcome will be a better understanding of what role each component of PEP has, how it performs it, and why these processes are essential to chloroplast development and photosynthesis. This project is expected to deepen our fundamental understanding of the biochemical basis of transcription. Decades of detailed study have been performed on the proteins that perform transcription in the eukaryotic nucleus and bacteria. This has shown that collating information about diverse proteins is essential to inferring general principles of how gene expression is regulated. Understanding the unique set of proteins that act on chloroplast genes therefore represents an exciting opportunity to advance this. Transcription regulation is a key component to human health and disease, and this research consequently has diverse potential uses. Photosynthesis has a central role in producing the oxygen and energy that sustains much of life on earth. Detailed structural and biochemical studies on the photosynthetic proteins have revealed in detail how they harness solar energy, and this has provided a valuable foundation for crop improvement and development of diverse biotechnologies. By contrast, equivalent mechanistic studies of the gene expression processes that underpin production of the photosynthetic proteins are largely lacking. This project will answer a complementary set of questions: what determines the timing and level of photosynthetic protein production, and how could we modify this to develop more robust crops and new biotechnological applications?

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Microbial pathogens invade their hosts via a range of infection strategies that allow the pathogen to grow and reproduce. Infection can include physical processes that transform host cells and tissues to accommodate the invader, and molecular warfare in which proteins and small molecules are exchanged to impede and manipulate the other organism. At the molecular level, pathogens are armed with a repertoire of proteins and small molecules that can be delivered into host cells, targeting specific physiological processes to control cellular function. Microbial proteins that are delivered into host cells are referred to as effectors and while there are common themes amongst their function in targeting immune suppression and resource distribution, they have a wide variety of molecular targets, specific to a given microbe. Pathogen effectors from different kingdoms target host plasmodesmata, the cytoplasmic connections between cells. Plasmodesmata offer a pathway for some pathogens to pass between cells and spread through host tissues, as well as acting as conduits by which molecules can pass to sites where they are deployed in infection; effectors can pass from infected cells into uninfected cells and nutrients can pass freely from host sources to the site of infection. As might be expected, host cells usually try to close their plasmodesmata as a defence mechanism. However, several effectors that target plasmodesmata can prevent this response and maintain connectivity between host cells. Thus, plasmodesmata have emerged as a critical battleground between host and pathogen. There have been several observations of effectors from viral and fungal pathogens that target heavy metal associated (HMA) domain proteins located at plasmodesmata. That such diverse pathogens target the same class of proteins located at intercellular bridges suggests that HMA domain proteins offer significant gains during infection. Further, in many plant species HMA domains are integrated into immune receptor sequences where they act as decoys to bind the relevant effector and activate the immune receptor, triggering cell death and consequent resistance. Unfortunately, while immune receptor hijacking of effector-HMA domain interactions points to the significance of the association, it also impedes research into the role of the effector and the HMA target as it becomes masked by immune receptor activation. We recently showed that the Arabidopsis fungal pathogen Colletotrichum higginsianum produces an effector that targets a plasmodesmata-located HMA domain protein in the host. Arabidopsis does not produce immune receptors with integrated HMA domains, allowing us to investigate the role and mechanism of this interaction in infection. This will also allow us to ask how and why these effectors target plasmodesmata. As the C. higginsianum effector not only targets plasmodesmata but moves cell to cell and modifies plasmodesmata to allow large proteins to move between cells more frequently, it suggests that one effector function is to increase the capacity for molecular exchange between host cells. This proposal will use the Arabidopsis-Colletotrichum interaction to determine what function the effector and host target each play in infection. We will use structural biology to compare the interactions between the effector and target HMAs from diverse species and identify any conservation between the mechanisms by which this occurs. We will also exploit any conservation to determine if we can exchange the HMA domain in immune receptors from rice with the HMA domain from Arabidopsis targeted by Colletotrichum, and thus engineer an immune receptor that recognises the Colletotrichum effector and confers novel resistance.

During reproduction evolutionary theory has shown that parents can have conflicting optimal strategies for maximising fitness of their offspring. One example where this situation arises is when males give rise to a fraction of the female brood: in this case the optimal maternal strategy is to distribute resources among progeny and minimise competition between kin, while fathers gain an advantage by allowing progeny to sequester resources and outcompete kin. In plants, even in predominantly selfing species, there are clear examples of parental conflict such as in the regulation of seed size. Increasing the maternal genome dose in Arabidopsis seeds leads to smaller seeds, whereas increasing the paternal genome dose leads to larger seeds. These conflicts do not play out in the embryo but in a second seed tissue called the endosperm. Flowering plants are distinguished from other plant lineages by the process of double fertilisation: two sperm cells in each pollen grain fertilise the egg cell to create the embryo, but also the central cell of the ovule to create the endosperm. The endosperm plays a key role in the control of seed size, and the conflicting influences of the mother and father on seed size are played out through distinctive epigenomes which are established in the gametes and inherited into the endosperm. These epigenomes lead to so-called 'imprinted' gene expression where the allele inherited from only one parent is expressed during endosperm development. For instance paternally expressed imprinted genes arise if the paternal allele is inherited in an epigenetic 'on' state, whereas the maternal alleles are inherited in an epigenetic 'off' state. The endosperm is retained in mature seeds of most species and plays a key role in seed dormancy and germination, after which it undergoes programmed cell death. Germination terminates seed dispersal and so plays a role in distributing progeny in space and time. One further prediction of mathematical theories of parental conflict during plant reproduction is that mothers should favour increased dormancy to disperse progeny and reduce competition, whereas fathers should favour fast germination to outcompete progeny and secure favourable niches closer to the mother plant. It remains an open question whether parental conflicts really occur in the control of seed dormancy but recently it has been shown that imprinted genes can play a role in seed dormancy control in the endosperm. Recently we have shown that the maternal epigenome is retained stably in the endosperm even after seed dispersal and is essential for the development of seed dormancy and the control of embryo behaviour, and identified specific chromatin remodelling complexes that impose seed dormancy from the maternal genome. Here we also show that increasing the paternal genome dose in the endosperm leads to an identical low dormancy phenotype. This raises the novel hypothesis that parental epigenomes compete in the endosperm to control progeny seed dormancy and germination behaviour. This proposal describes a detailed characterisation of the roles of parental epigenomes in the endosperm that regulate seed dormancy and aims to identify the target genes and processes by which the father's epigenetic state promotes low dormancy, via the study of imprinted genes at the critical developmental stages. Because seed dormancy is promoted by changes in environmental temperature during seed set, this requires understanding how temperature and whether temperature acts through the same chromatin remodelling processes to affect seed dormancy and germination.

Viruses constantly threaten humans, livestock, and crops. Many viruses have RNA genomes. In the most extreme case, noncoding RNAs alone (i.e., viroids) can infect crops leading to production losses. Taking oil palm and coconut palm as an example, ~40 million palms were killed by viroids between 1980 to 2007 due to the lack of effective treatments. Additionally, varying temperatures can affect viroid titre and symptom severity. Warm temperatures lead to more severe foliage symptoms and significant potato yield loss by viroid infection. It has been projected that climate change increases cross-species viral transmission risks and poses risks of new viroid and virus outbreaks. Therefore, it is timely to explore potential solutions for informing sustainable agriculture in the future. This proposal builds on recent breakthroughs in both UK and US groups and aims to transform the current knowledge on viroid RNA structure architecture. This proposal will develop advanced methods to decipher both spatial and temporal RNA structure landscapes of viroids in living plant cells. Then we will assess the functional importance of these RNA structure features in viroid infection. We will deliver novel RNA structure-mediated regulation of viroid infection, offering new knowledge for combating viroid disease to ultimately lay a solid foundation for future screening of anti-viroid drugs. The fundamental knowledge of structure-mediated intracellular and intercellular viroid transmission will provide a general understanding of RNA movement and host-virus interactions. This proposed research will establish a series of advanced novel methods where these methods could be applied to other organisms. The novel framework generated from this proposed research will be readily applicable in studying other plant or animal viral RNAs.