Early N-hydroxy-pipecolic acid transcriptional profile

N-hydroxy-pipecolic acid (NHP) is a mobile metabolite essential for inducing and amplifying systemic acquired resistance (SAR) following pathogen attack. In Arabidopsis thaliana (Arabidopsis), exogenous NHP is sufficient to activate SAR-related immune responses, including salicylic acid (SA) accumulation and changes in global transcriptional profile. Previous studies have tracked these changes 1-2 days after an initial NHP treatment, resulting in little knowledge of the very early phases of NHP signaling and transcriptional responses leading to immunity. Here we show NHP elicits transcriptional changes within minutes of treatment. We report distinct waves of expression over the course of minutes to hours defined by transient induction of jasmonic acid/wound-related responses, a primary induction of WRKY transcription factor expression, and subsequent induction of WRKY-regulated defense genes. The upregulation of WRKYs and the majority of defense-related genes occurred in the sid2-2 mutant, which is unable to accumulate SA upon NHP treatment, suggesting NHP is sufficient to drive the early phase of transcriptional changes in a low SA environment. We also show that WRKY70 is required for the expression of a set of genes defining the secondary transcriptional changes, as well as NHP enhancement of ROS production and SAR. To investigate how the presence of NHP affects transcriptional changes within minutes to hours of treatment, six-well plates containing 10-day-old hydroponically grown Arabidopsis wild type, sid2-2, and fmo1-1 seedlings were treated with mock or NHP. The sid2-2 and fmo1-1 mutants were analyzed to identify transcriptional changes dependent on NHP-induced salicylic acid biosynthesis and endogenous NHP production, respectively. All seedlings from a single well were pooled and collected 15 min, 30 min, 3 h, and 6 h following treatment for a single sample, this was repeated for a total of three samples per condition, n = 3.