Dissertation/Thesis Abstract

Characterization of components involved in tomato innate immunity against bacterial speck disease
by Schwizer, Simon, Ph.D., Cornell University, 2017, 168; 10168958
Abstract (Summary)

The Pti1 kinase was identified from a reverse genetic screen as contributing to pattern-triggered immunity (PTI) against the bacterial speck pathogen Pseudomonas syringae pv. tomato (Pst). This was unexpected because Pti1 was originally identified as an interactor of the Pto kinase that confers effector-triggered immunity to Pst strains secreting either of the type III effectors AvrPto or AvrPtoB. A hairpin-Pti1 (hpPti1) construct was developed and used to generate stable transgenic tomato lines with reduced expression of Pti1. These hpPti1 plants were more susceptible to infection with Pst strains lacking both AvrPto and AvrPtoB and had reduced transcript accumulation of PTI-associated genes compared to wild-type plants. The hpPti1 plants produced less reactive oxygen species (ROS), but showed no difference in mitogen-activated protein kinase (MAPK) activation in response to two flagellin-derived peptides. Synthetic Pti1 genes designed to avoid silencing were transiently expressed in hpPti1 plants and restored the ability of the plants to produce wild-type levels of ROS. This work identifies a new component of PTI in tomato which, because it affects ROS production but not MAPK signaling, appears to act early in the immune response. We speculate that Pti1 was identified originally as a Pto interactor because it may interact with a Pto-like kinase that plays a role in PTI.

We identified several putative receptors in tomato that consist of extracellular malectin-like domains and intracellular kinase domains that show high similarity to Pto. Interestingly, the starting sequence of Pto and some of its family members is conserved in two of the identified malectin-like proteins, Mal1 and Mal2, at approximately the beginning of their kinase domains and is not found in any other predicted tomato protein. Silencing the orthologs of Mal1 and Mal2 in Nicotiana benthamiana resulted in compromised induction of immune responses and thus increased susceptibility to Pst infection. We found that AvrPtoB interacts with the kinase domains of both Mal1 and Mal2 and we speculate that these putative receptors are targeted by AvrPtoB to suppress plant immunity. Furthermore, we propose that Pto evolved from the kinase domain of either Mal1 or Mal2 (or a progenitor protein) to sense the presence of AvrPtoB inside the plant cell and induce robust defense responses.

We describe a rapid method to detect two major forms of fatty acylation, N-myristoylation and S-acylation, of candidate proteins using alkyne fatty acid analogs coupled with click chemistry. We applied our approach to confirm and decisively demonstrate that AvrPto, Pto, and the FLS2 receptor all undergo plant-mediated fatty acylation. In addition to providing a means to readily determine fatty acylation, particularly myristoylation, of candidate proteins, this method is amenable to a variety of expression systems. We demonstrate this using both Arabidopsis protoplasts and stable transgenic Arabidopsis plants and we leverage Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves as a means for high-throughput evaluation of candidate proteins. The metabolic labeling approach leveraging alkyne fatty acid analogs and click chemistry described here has the potential to provide mechanistic details of the molecular tactics used at the host plasma membrane in the battle between plants and pathogens.

Indexing (document details)
Advisor: Martin, Gregory
Commitee: Collmer, Alan, Mazourek, Michael
School: Cornell University
Department: Plant Path and Plant-Microbe Bio
School Location: United States -- New York
Source: DAI-B 78/03(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Genetics, Plant sciences, Plant Pathology, Biochemistry
Keywords: Bacterial speck disease, Components, Innate immunity, Tomato
Publication Number: 10168958
ISBN: 9781369231816
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