Author ORCID Identifier

https://orcid.org/0000-0002-8091-2426

Year of Publication

2022

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department/School/Program

Plant Pathology

First Advisor

Dr. Pradeep Kachroo

Abstract

Plants have evolved a sophisticated immune system to defend themselves against pathogens. This immune response can be triggered in response to pathogen-associated molecular patterns (PAMP) or specialized effectors that are recognized by the plant resistance (R) proteins. The latter, commonly referred to as effector-triggered immunity (ETI), is well known to induce broad-spectrum resistance throughout the plants. This phenomenon known as systemic acquired resistance (SAR) is regulated by several chemical signals including salicylic acid (SA), and free radical nitric oxide (NO) and reactive oxygen species (ROS). These signals operate in two parallel branches with NO/ROS functioning downstream of pipecolic acid (Pip) and upstream of azelaic acid (AzA) and glycerol-3-phosphate (G3P). Earlier work has shown that optimum levels of NO/ROS are required for the induction of SAR with high concentrations inhibiting SAR. To characterize the role of NO in SAR, I evaluated SAR associated signaling in an Arabidopsis thaliana GSNOR1 (S-NITROSOGLUTATHIONE REDUCTASE 1) mutant that accumulates elevated levels of NO and shows compromised SAR. The pathogen inoculated gsnor1 plants accumulated wild type (WT) like levels of AzA and G3P, suggesting that increased NO levels were not associated with hyperactivation of the NO-AzA-G3P branch of the SAR pathway. Consistent with these results, the gsnor1 plants showed WT like levels of lipid transfer proteins DIR1 (DEFECTIVE IN INDUCED RESISTANCE 1) and AZI1 (AZELAIC ACID INDUCED 1), which operate in a feedback loop with G3P. Interestingly, the gsnor1 plants accumulated reduced levels of Pip, which in turn was associated with reduced expression of the gene encoding SARD1 (SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1) transcription factor. Since SARD1 regulates both SA and Pip biosynthetic genes, the gsnor1 plants also accumulated reduced levels of SA in infected leaves. Mutations in CAMTA (CALMODULIN BINDING TRANSCRIPTION ACTIVATOR) transcription factors, that negatively regulate SARD1 expression were able to partially elevate SARD1 expression and SA and Pip levels in gsnor1 plants but were unable to normalize the SAR phenotype. Petiole exudate transport assays showed normal transport of G3P, but defective transport of SA, in pathogen inoculated gsnor1 plants. Likewise, the gsnor1 plants showed reduced transport of exogenously applied cold or 14C labeled SA. Consequently, localized application of exogenous SA was unable to confer SAR on gsnor1 plants. Notably, SA delivered via root drench was able to confer SAR on gsnor1 plants, suggesting that these plants were specifically affected in SA transport. Reduced transport of SA in the presence of NO donor suggested that increased levels of NO affected SAR by impairing transport of SA. These results further reinforce the importance of SA transport in SAR.

To characterize NO-mediated signaling in relation to chloroplast-nucleus retrograde signaling, I next characterized the ssi2 mutant that accumulates elevated levels of NO in the chloroplast and shows constitutive defense phenotypes. SSI2 (SUPPRESSOR OF SA INSENSISTIVE 2) encodes stearoyl-ACP (acyl-carrier-protein) desaturase that catalyzes the conversion of 18:0 to 18:1 fatty acid. The ssi2-mediated activation of defense is associated with 18:1 levels and normalization of 18:1 levels restores their NO levels and defense phenotypes to wild-type like levels. Interestingly, a mutation in NO OVERPRODUCER 1 (NOX1), which encodes a plastid membrane localized phosphoenolpyruvate (PEP) phosphate translocator, normalizes wild-type-like defense phenotypes in ssi2 even though these plants accumulate elevated levels of NO in plastids. NOX1 facilitates import of PEP into the stroma, which in turn serves as a precursor to aromatic amino acid biosynthesis. Indeed, treatment with phenylalanine, but not tyrosine or tryptophane, was able to restore defense phenotypes in ssi2 nox1 plants. Together, these results suggest NO-mediated retrograde defense signaling in ssi2 is mediated via phenylalanine.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.77

Funding Information

National Science Foundation (IOS#051909)

Kentucky Soybean Board (3084113467)

National Institute of Food and Agriculture (3200004209)

Available for download on Saturday, April 27, 2024

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