Analysis of the regulation of ICS1-independent SAR gene expression by N-hydroxy-pipecolic acid
by Isha Goyal
Date of Examination:2023-10-11
Date of issue:2024-10-08
Advisor:Prof. Dr. Christiane Gatz
Referee:Prof. Dr. Christiane Gatz
Referee:Prof. Dr. Marcel Wiermer
Persistent Address:
http://dx.doi.org/10.53846/goediss-10781
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Abstract
English
Systemic acquired resistance (SAR) is a plant immune response that ripostes a local pathogen attack. A mobile signal, induced in infected plant cells, travels from these infected tissues to systemic leaves. This prepares the uninfected leaves to mount a much more effective response to a future pathogen encounter compared to naïve plant parts. Recent work has presented the amino acid-derived metabolite N-hydroxypipecolic acid (NHP) as the potential mobile signal which establishes SAR. Once perceived by the uninduced systemic leaves, NHP activates salicylic acid (SA) biosynthesis through the isochorismate pathway, which in turn activates the expression of NHP biosynthesis genes further increasing NHP levels. NHP and SA work synergistically to establish this primed state in the systemic leaves. Increased SA levels are essential for induction of a large set of SAR marker genes and full SAR establishment. The SA induction-deficient 2 (sid2/isochorismate synthase1 (ics1)) mutant, which is devoid of ICS1-derived SA, can establish an SAR response, though much weaker compared to wild-type plants. In this study, we wanted to decipher the molecular components involved in the establishment of SAR in pathogen-induced SA devoid sid2 plants. A secondary pathogen infection in Psm-pretreated sid2 plants, resulted in the induction of SAR marker gene FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) which cannot be induced in SA and NHP biosynthesis mutant sid2 ald1. This presented NHP as the candidate molecule required for establishing the weak SAR response in sid2 plants. We showed that pharmacological treatment of Arabidopsis thaliana with NHP can induce SAR-related genes even in the sid2 mutant in the absence of extravagant effects of NHP-induced SA. This requires the SA receptor NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and its interacting TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors. However, this is not mediated by direct binding of NHP to NPR1 protein. Also, the SA-binding motif R432 is required for the activation of NHP-mediated processes. This suggests the requirement of direct binding of basal SA levels to NPR1 in sid2 plants. Absence of avrPPHB SUSCEPTIBLE3 (PBS3), which converts ICS-derived isochorismate to SA, in addition to sid2, severely affected NHP-responsive gene expression. NHP-mediated gene induction was abolished in sid2-1 NahG plants, in which basal SA levels are degraded by constitutively accumulating SA hydroxylase, NahG. The induction of both SA and NHP biosynthesis genes takes 48 h to reach full expression potential, however, the SAR master regulator, SAR DEFICIENT 1 (SARD1), was expressed as early as 8 h after NHP treatment. This is accompanied by the induction of other early regulatory genes which are potentially required for the establishment of a primed SAR state. These early NHP-induced genes, in contrast to the hormone biosynthesis genes, do not require SARD1 for their expression. The nucleocytoplasmic protein PHYTOALEXIN DEFICIENT4 is required for NHP-induced expression of a subset of these genes. NHP resulted in a slight increase in NPR1 protein levels at 8 h, which is only detected in extracts enriched in nuclear proteins. This increase in NPR1 protein amount was more clearly observed 48 h after NHP treatment in total cell extracts. NHP-mediated NPR1 protein accumulation correlates with slightly increased NPR1 transcript levels, which were dependent on PAD4. While exogenously applied SA, as well as its synthetic analogues, INA and BTH, led to a clear stabilization of NPR1 protein, this was not as pronounced in response to NHP treatment. Our work shows the requirement of basal SA in aiding NPR1 to promote NHP-induced gene expression. Regulatory components of the SA signalling pathway are required to relay this signal initiated by NHP via basal SA-bound NPR1 which results in the establishment of SAR. Still, the mechanism of NHP perception and NHP-mediated NPR1 activation remains enigmatic.
Keywords: Systemic Acquired Resistance; N-hydroxy-pipecolic acid; NPR1; TGA transcription factors; Salicylic acid; Plant Immunity
