A novel plant pathogenesis regulatory system in Erwinia: functional analysis of a new post-transcriptional input to bacterial quorum sensing control.

This proposed research involves the study of a new gene in a bacterial pathogen of plants. The bacterial pathogen (Erwinia) causes rotting diseases of potato and other plants and is important in potato crop production. The bacteria attack the plant by producing a spectrum of enzymes that can degrade the cell walls of the plant leading to the commercially-significant rotting disease symptoms. The ability of the bacteria to make these plant cell wall degrading enzymes is affected by the cell population density of the bacteria; the enzymes are only made in abundance when the bacterial density if high. The bacteria make a small diffusible chemical signal called OHHL and the concentration of this molecule is a direct reflection of bacterial density. In this process (quorum sensing) the bacteria use the chemical signal to communicate with each other and thereby link cell density to the ability to aggressively rot the plant. If the ability to enact the process of quorum sensing is blocked then the bacteria are no longer capable of causing disease. Consequently, a deeper understanding of the mode of action of quorum sensing in this potato pathogen is important for our fundamental appreciation of how bacterial pathogens communicate with each other and, if we can understand the process, how we might intervene in the disease. There are no chemical control systems available for potato diseases caused by Erwinia and so fundamental understanding of how it causes infection is the only route to an eventual rational therapy. In this proposal we will investigate the role of a new gene (ECA2020) that we have shown recently to be involved in the process of quorum sensing. The product of this gene affects the expression/functionality of one of the key proteins (VirR) involved in quorum sensing. We will study the way ECA2020 operates by investigating which other proteins it interacts with and we will test the hypothesis that it may have some functions similar to proteins from higher organisms that are involved in messenger RNA processing. We will look for bacterial mutants that can bypass their dependence on the ECA2020 system and we will study the precise impact on production of the fully active VirR protein. We will also test the possibility that the contiguous gene (ECA2019) might be involved in a related process and we will investigate how the ECA2020 gene is itself regulated. The overall aim is to try to work out how this new gene modulates the function of the quorum sensing system during plant infection and disease initiation because this might be a target in the longer term for control of potato rotting diseases caused by Erwinia.

Erwinia carotovora subspecies atroseptica (Eca) causes potato blackleg disease. Eca produces >20 plant cell wall degrading enzymes and multiple additional determinants that affect virulence, including secondary metabolite toxins, Type VI targeting systems and proteins of unknown function. All known virulence factors are regulated by quorum sensing (QS) during plant infection. In QS the expI/carI gene encodes a LuxI-type enzyme that makes OHHL (the N-acyl homoserine lactone intercellular chemical communication signal for Eca). At high cell density the concentration of OHHL reaches the threshold level that switches on virulence factor elaboration. The main regulator of virulence in Eca is VirR. VirR is a LuxR-type regulator and the central repressor of virulence gene expression. The current model is that OHHL binds to the VirR repressor leading to de-repression of virulence genes, although probably indirectly rather than by direct action on virulence genes. We wanted to identify new inputs to virR regulation. Using a virR::lacZ fusion screen we identified a novel regulatory input to the QS process in Eca. Mutation of the ECA2020 gene led to down-regulation of virR expression. The ECA2020 protein shows some sequence relatedness with eukaryotic adenosine deaminases (ADARs) that control gene expression via edited base substitutions in target mRNA, affecting function. Preliminary evidence suggests that virR mRNA may be processed in the ECA2020 mutant leading to truncated VirR production and loss of functionality. We will test the hypothesis directly that ECA2020 acts in this post-transcriptional way; a mechanism that would be unique in a eubacterium. We will assess the impact of mutation of the ECA2020 residues conserved in ADARs, search for ECA2020-interacting proteins that may be partners in the regulation, and determine if ECA2019 has any related role in virR expression. We will study regulation of ECA2020 by bypass mutagenesis to define hierarchical controls.

Who will benefit from this research? Potential beneficiaries include potato growers and distributers and, ultimately, the consumer. Furthermore, the basic principles of QS control of pathogenesis radiates across plant, animal and human pathogens. Knowledge gleaned from our studies might have relevance and wider applications in plant pathology, and in control/therapy of some animal/human diseases due to Gram-negative bacteria that use a QS mode of virulence regulation. Thus the study has longer term implications for agricultural productivity and, potentially, for medicine. QS systems could also have potential applications in industrial fermentations as easily manipulated gene induction and repression control systems in the manufacture of biotechnologically useful products. Such QS systems are also potentially useful in chemical biology and synthetic biology arenas where simple, modular gene regulation cassettes might find applications where there is a need to engineer metabolic process controls or report on physiological events in bacteria. Thus, our studies in the longer term could have applicability in far wider areas than potato crop disease. In effect a wide spectrum of possibilities might emerge from a basic understanding of how Erwinia uses intercellular chemical communication to regulate disease of a crop plant. How will they benefit from this research? Crop losses due to plant disease and biodeterioration are significant, on a global scale. Indeed, it is estimated that the latter crop losses may account for 10-30% of bulk food production, globally; this is obviously a major issue. If our studies lead eventually to new potato disease control procedures (for blackleg in the field and soft rot in storage) this could diminish crop losses and enhance profitability in agriculture and aspects of the food production and distribution chain in the UK and globally. Decreasing potato crop losses could also enhance our domestic (UK) food security and so, even marginal impacts on our ability to control potato crop pathogenesis has value. On a global scale, improvement in potato crop productivity through decreased deterioration could have impacts on food availability and health in poorer countries through knowledge transfer. The potential biotechnological applications of QS research would require agricultural and industrial sector participation and exploitation with investment over a longer time scale than envisaged in this current proposal. The PDRA employed on this project will acquire diverse skills in molecular microbiology, cutting edge 'omics technologies, bioinformatics, and experience in teaching and science communication. What will be done to ensure that they have the opportunity to benefit from this research? The research outcomes will be disseminated to scientists (from universities, institutes and commercial organisations) via peer reviewed international publications and lectures and posters presented at domestic and international symposia. When publishing in open access journals, there will be a global accessibility of the knowledge generated from this work. We have a track record over the past 20 years of BBSRC-CASE studentships (two currently running with UK companies in other projects) and collaboration with UK research institutes. We will try to further extend such associations as a way of leveraging additional funding for our research programmes and to generate added value through synergy of research interests. Cambridge university takes a very pragmatic and enlightened industry-friendly stance that encourages commercial collaborations and the generation of spin-out companies, after the establishment of solid IP positions. We have a track record of lodging patent applications on carbapenem antibiotics and on cryptic gene regulation e.g. Salmond et al (US Patent 5821077 - issued 1998); Salmond et al (WO/1995/032294) and have recently filed a patent application on phage abortive infection in late 2008.