Genome-wide studies of virulence-associated mechanisms in Erwinia amylovora

Erwinia amylovora is the causal agent of fire blight, a devastating disease that threatens apple and pear production. The pathogen utilizes two virulence factors, the type III secretion system (T3SS) and the exopolysaccharide (EPS) amylovoran, to cause disease. Previous studies have reconstructed regulatory networks controlling the expression of genes related to T3SS and amylovoran production. As an example, HrpL, the master regulator of T3SS gene expression, is activated by a sigma factor RpoN, along with a modulation protein YhbH, a bacterial enhancer-binding protein (bEBP) HrpS, and an integration host factor (IHF). The RpoN-HrpL sigma factor cascade is then regulated by the bacterial alarmone (p)ppGpp. However, genome-wide studies to reveal the global effects of these regulatory systems remain elusive. In this dissertation, my main goal is to understand the gene regulatory network through transcriptomic, comparative genomic, and genetic studies to understand the coordination of virulence-associated mechanisms for better disease management. First, transcriptomic analyses uncovered the overall effect of (p)ppGpp-mediated stringent response on how E. amylovora mediate virulence mechanisms in the hrp-inducing medium (HMM), which mimics the stress environment during infection. Results revealed that about one third (n = 1314, 36.8%) of all genes in the E. amylovora genome were regulated by (p)ppGpp. Our results also indicate that (p)ppGpp served as a signal to activate virulence gene expression for colonization, and simultaneously to mediate the balance between virulence and survival by negatively regulating DNA replication, translation, cell division, as well as biosynthesis of nucleotides, amino acids, fatty acids, and lipids. Second, the regulon of RpoN in E. amylovora was determined by combining RNA-seq transcriptomic analysis with in silico binding site analysis. Results revealed that RpoN regulated about 7.5% genes (n = 262) in the E. amylovora genome. Genes associated with virulence, motility, nitrogen assimilation, the PspF system, stress response, and arginine biosynthesis are positively regulated by RpoN, whereas genes associated with biosynthesis of amino acids and sorbitol transport are negatively regulated by RpoN. In addition, in silico binding site analysis identified 46 potential target genes with putative RpoN binding site. Among them, 6, 3, and 3 genes also contain putative GlnG, PspF, and YfhA bEBP binding site, respectively. Overall, RpoN directly regulates genes associated with virulence, nitrogen assimilation, the PspF system, motility and the YfhA/YfhK two component regulatory system. Third, the role of the purA/purB and guaA/guaB genes, which are involved in the AMP/adenine and GMP/guanine biosynthesis, respectively, was characterized. Both AMP and GMP are precursors of (p)ppGpp. Our results showed that growth of the purA, purB, and guaAB mutants was much slower in minimal media, but their growth could be restored by addition of exogenous AMP/adenine and GMP/guanine, respectively. However, mixed results were obtained for their contribution to virulence on immature pear fruits. Overall, our results suggest that the de novo purine biosynthesis pathway might be required for proliferation, colonization, and infection of E. amylovora. Finally, comparative genomics studies uncovered large chromosome inversions (LCIs) in the Spiraeoideae-infecting (SI) strains/isolates of E. amylovora and PCR-based molecular markers were developed for detecting and identifying LCIs. Different LCI types were revealed and confirmed by PCR and whole-genome sequencing. In addition, the evolutionary history and physical/estimated replichore imbalance of LCIs were also examined. Among the 117 SI strains tested, the LCI types of Ea1189, CFBP1430, and Ea273 are the most dominant ones. Furthermore, the inter- and intra- variations in the internal transcribed spacer (ITS1 and ITS2) regions among rRNA Operons (rrns) were examined, and results indicated that LCIs tend to occur between rrns transcribed in opposite directions and with the same tRNA content (tRNA-Glu or tRNA-Ile/Ala) in ITS1. These findings may provide clues for the spread of fire blight disease and its potential evolutionary history based on prevalent LCI types in different geographical regions.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Crop Sciences

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Yang, Ho-Wen

Contributors dc:contributor

• Zhao, Youfu Frank
• Villamil, Maria Bonita
• Hind, Sarah Refi
• Mideros, Santiago

• Erwinia amylovora
• Virulence
• ppGpp
• RpoN
• Bacterial genome

Statement dc:rights

• Copyright 2022 Ho-Wen Yang

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/115732

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F115732