Abstract
Pseudomonas syringae pv. syringae UMAF0158 (Pss0158) is a plant pathogen responsible for bacterial apical necrosis (BAN) disease in mango trees. This pathogenic bacterium exhibits resistance against various environmental stresses. The entry into the host plant occurs through microinjuries, favoured by lower temperatures and increased rainfalls. The disease symptoms are characterized by lesions on leaves and buds, which can eventually expand to the flower panicles. Understanding the infection mechanisms employed by Pss0158 is crucial for developing efficient strategies to control and prevent BAN. Previous work has identified a novel group of signaling molecules harbouring a diazeniumdiolate group. Leudiazen, a representative of this class identified in Pss0158, regulates the production of the major virulence factor mangotoxin. This PhD thesis aims to expand the knowledge on diazeniumdiolate signaling molecules in bacteria.
In this thesis, I mapped the stimulon controlled by leudiazen, using a transcriptomic approach. This analysis confirmed that leudiazen regulates the mangotoxin cluster (mbo). Additionally, genes related to metal homeostasis and amino acid transport were identified. I also investigated external stimuli which appear to influence leudiazen production. The results of this analysis suggest that plant-related compounds can induce the promoter of the leudiazen biosynthetic cluster.
Another line of research investigated upstream regulatory elements controlling leudiazen and mangotoxin production. This led to the identification of the transcriptional regulator GntR and the Gac two-component system. RNA-sequencing (RNA-seq) analyses showed that inactivation of either of the regulators affects global mRNA expression patterns. In fact, besides their involvement in leudiazen and mangotoxin regulation, the transcriptome profiles indicated their role in controlling diverse traits. Moreover, the promoter activity of the different players involved in this network, namely GntR, GacS, leudiazen and mangotoxin was examined by β-galactosidase assays. Furthermore, leudiazen was tested for its ability to modulate gntR and gacS gene expression. Finally, a transposon mutant library was constructed with the intent to identify putative leudiazen receptors and regulators.
The final chapter of this thesis presents an optimized protocol for the isolation and stabilisation of mangotoxin as a first step to determine its structure, which could provide a solid basis for the development of a novel plant protection strategy.
This work provides evidence that bacterial signaling networks can be modulated by plant-related compounds and unravelled a sophisticated regulatory network controlling mangotoxin production.
Paganini, Leonardo