The use of transcriptome and functional analysis to unravel the role played by PhoP and SlyA in the biology of Pectobacterium brasiliense1692

Abstract

Several members of the soft rot Pectobacteriaceae family such as Pectobacterium brasiliense 1692 (Pb1692) attack a wide range of crops worldwide. P. brasiliense is a major cause of black leg and soft rot disease in several important plants including beetroot, sugarbeet, cabbage, pepper, cucumber, tomato and potatoes. Currently, no chemical control exists to fight the spread of this disease only methods based on avoiding the pathogen. Despite all the research on Pectobacterium species, little is known on how P. brasiliense is able to colonise and cause disease in susceptible hosts. Therefore, as part of advancing knowledge on this organism, the objective of this study was to determine the role of two transcriptional regulators SlyA and PhoP in the biology of Pb1692. In order to better understand the role of the PhoP regulon during the infection process, transcriptional profiles of the wild type Pb1692 strain with its isogenic phoP deletion mutant were analysed using RNA-sequencing and phenotypic assays. Analysis of the transcriptome data demonstrates that the PhoP regulon is substantially broader than previously suspected affecting the differential expression of more than 400 genes. Additionally, the transcriptome data revealed the repression of T6SS and carbapenem genes, two of the most important traits involved in antibacterial competition, indicating that PhoP negatively regulates T6SS and carbapenem in planta. Apart from a role in antibacterial competition, PhoP was shown to play a role in the production of plant cell wall degrading enzymes (PCWDEs), with the repression and activation of some important PCWDEs, PELI/B/Z (pectate lyases) and PEH (polygalacturonase). The broad PhoP regulon included other transcriptional regulators such as EXPR (quorum sensing regulator). In addition to RNA-seq, the study functionally characterized phoP mutants based on virulence in susceptible potato tubers and their ability to outcompete Dickeya dadanti and attenuated motility. Furthermore, by implementing a time-course RT-qPCR analysis, the study revealed the importance of phoP gene in the survival of Pb1692 under acidic conditions encountered in the apoplast. The findings of the RT-qPCR revealed that the relative expression of phoP gene was low in the wild type when conditions are still acidic and gradually increased in expression over time when the environment supposedly becomes alkaline. The data demonstrates that PhoP functions as a global regulator in the survival and adaptation of Pb1692 in planta. Bioinformatics analysis was used to understand the evolutionary history and distribution of SlyA in different Enterobacteriaceae. In this thesis, multiple sequence alignments of SlyA revealed a 90-100% sequence similarity between the different Enterobacteriaceae, indicating that SlyA is conserved among closely related species. Furthermore, the transcriptional profiles of the wild type Pb1692 strain with its isogenic slyA mutant were analysed using RNA-sequencing and phenotypic assays. Findings from this study revealed a number of genes encoding important virulence factors such as, PCWDEs, biofilm formation as well as other important physiological processes, such as oxidative stress response, assimilation of carbohydrates, iron uptake are all SlyA regulated. The transcriptome data identified genes that are either downregulated or upregulated by slyA in planta, suggesting that SlyA activated and/or repressed a number of essential genes to adapt and proliferate in a stressful environment. Thus, highlighting that SlyA plays a key role in the adaptive responses of Pb1692. This study demonstrated that a functional SlyA is essential for full pathogenicity of Pb1692 in planta. Overall, the thesis sheds insights into the regulation of virulence by PhoP and SlyA transcriptional regulators in Pb1692 in planta.