Transcriptome analysis reveals role for WRKY70 in earlyN-hydroxy-pipecolic acid signaling

(250 words) 24 N-hydroxy-pipecolic acid (NHP) is a mobile metabolite essential for inducing and 25 amplifying systemic acquired resistance (SAR) following pathogen attack. Early phases 26 of NHP signaling leading to immunity have remained elusive. Here we report the early 27 transcriptional changes mediated by NHP and the role salicylic acid (SA) plays during this 28 response. We show that distinct waves of expression within minutes to hours of NHP 29 treatment include increased expression of WRKY transcription factors as the primary 30 transcriptional response, followed by the induction of WRKY-regulated defense genes as 31 the secondary response. The majority of genes induced by NHP within minutes were SA-32 dependent, whereas those induced within hours were SA -independent. These data 33 suggest that NHP induces the primary transcriptional response in a low SA environment 34 and new SA biosynthesis is dispensable for induction of the secondary transcriptional 35 response. We demonstrate that WRKY70 is required for the induced expression of a set 36 of genes defining some of the secondary transcriptional response, SAR protection, and 37 NHP-dependent enhancement of ROS production in response to flagellin treatment. 38 Taken together, our study highlights the key genes and pathways defining early NHP 39 responses and a role for WRKY70 in the regulation of NHP-dependent transcription. 40 41 42 43 44 45 46 47 48 49 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 3

50 Systemic acquired resistance (SAR) is a whole -plant defense response triggered by an 51 initial pathogen attack. During the establishment of SAR, mobile signals are sent from 52 infected tissue to uninfected tissue, initiating the priming of immune responses and 53 enhancing immunity against secondary pathogen infections (Fu and Dong, 2013; Tian 54 and Zhang, 2019; Kachroo and Kachroo, 2020). To achieve a primed state, plants 55 execute substantial transcriptional reprogramming of several hundred to a few thousand 56 genes, resulting in the increased accumulation of defense hormone salicylic acid (SA), 57 sensing and signaling components for pathogen detection, and production of 58 antimicrobial compounds (Jaskiewicz et al., 2011; Conrath et al., 2015; Bernsdorff et al., 59 2016). In addition, several small molecules produced by plants during infection help to 60 orchestrate this defense priming, including SA and the bioactive metabolite N-hydroxy-61 pipecolic acid (NHP) (Chen et al., 2018; Hartmann et al., 2018; Klessig et al., 2018). 62 63 NHP is a mobile metabolite that can move from one leaf to another to mediate long -64 distance cellular communication (Chen et al., 2018; Mohnike et al., 2021; Yildiz et al., 65 2021). In Arabidopsis thaliana (Arabidopsis), the enzyme FLAVIN -DEPENDENT 66 MONOOXYGENASE 1 (FMO1) catalyzes the N-hydroxylation of lysine-derived pipecolic 67 acid (Pip) in response to pathogen infection to produce NHP (Chen et al., 2018; 68 Hartmann et al., 2018). Notably, fmo1 null mutants are impaired in local disease 69 resistance during pathogen infection and fail to establish SAR (Chen et al., 2018; 70 Hartmann et al., 2018). Treatment of fmo1 mutant plants with exogenous NHP is 71 sufficient to induce systemic resistance against bacterial and oomycete pathogens (Chen 72 et al., 2018; Hartmann et al., 2018). Furthermore, NHP treatment of a single leaf is 73 sufficient to initiate and amplify defense signaling throughout the plant, resulting in the 74 transcriptional activation of a large subset of SAR -associated genes within 24 h of 75 treatment (Yildiz et al., 2021; Yildiz et al., 2023), including genes encoding the SA 76 biosynthetic enzyme ISOCHORISMATE SYNTHASE 1/SA -INDUCTION DEFICIENT 2 77 (ICS1/SID2), the Pip biosynthetic enzymes AGD2 -LIKE DEFENSE RESPONSE 78 PROTEIN 1 (ALD1) and SAR -DEFICIENT 4 (SARD4), as well as its own biosynthetic 79 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 4 enzyme FMO1 (Chen et al., 2018). Collectively, these findings highlight the importance of 80 NHP in initiating immune signaling to establish local and systemic plant defenses. 81 82 Induction of ICS1 transcription by NHP signaling results in the accumulation of SA in both 83 local and systemic tissues (Chen et al., 2018; Hartmann et al., 2018). Elevation of SA in 84 turn amplifies SAR-associated transcriptional changes (Bernsdorff et al., 2016). Signaling 85 downstream of SA accumulation is largely driven by the action of the SA receptor and 86 transcriptional coactivator NONEXPRESSER OF PR GENES 1 (NPR1), which interacts 87 with a number of TGA transcription factors (TFs) from the basic leucine zipper (bZIP) 88 family to alter gene expression (Zhang et al., 1999; Wang et al., 2006; Wu et al., 2012). 89 Members of the biotic and abiotic stress-response WRKY TF family are among the known 90 targets of the NPR1/TGA interaction (Thibaud‑Nissen et al., 2006; Wang et al., 2006). 91 WRKY TFs have been identified as key regulators of NPR1 -dependent and NPR1 -92 independent SAR responses. Additionally, some WRKY members play important roles in 93 feedback regulation of transcription (Pandey and Somssich, 2009; Fu and Dong, 2013). 94 For example, WRKY54 and WRKY70 modulate SA biosynthesis through repression of 95 ICS1 expression (Wang et al., 2006). 96 97 The interplay between SA and NHP appears to be interdependent and synergistic with 98 both metabolites being able to upregulate one another's biosynthetic pathways (Chen et 99 al., 2018; Hartmann et al., 2018; Sun et al., 2020). Notably, elevated endogenous levels 100 of Pip, the precursor of NHP, primes the expression of both NHP and SA biosynthetic 101 genes under low concentrations of SA by stabilizing NPR1 levels (Kim et al., 2020). This 102 points to an intriguing dynamic relationship between SA and NHP over the course of SAR 103 activation. Evidence suggests NHP accumulates to detectable levels in systemic tissues 104 roughly 24 h before SA accumulation is detected (Hartmann and Zeier, 2019). In such a 105 case, NHP would need to drive systemic signaling for a substantial period of time before 106 SA could further amplify SAR responses. It is currently unclear how the levels of SA in 107 leaves impact early NHP signaling. Furthermore, little is known about the very early phase 108 of NHP signaling in the tissues where it is first produced or in the distal tissues once NHP 109 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 5 arrives there. Key players of NHP signaling and the progression of NHP -dependent 110 transcriptional reprogramming require further investigation. 111 112 Presently, the majority of genome-wide transcriptomic studies of SAR have been carried 113 out on the timescale of 1 -2 days post induction; this includes pharmacological studies 114 using Pip and NHP treatment (Bernsdorff et al., 2016; Hartmann et al., 2018; Yildiz et al., 115 2021; Yildiz et al., 2023). These data have revealed that NHP ultimately activates SAR in 116 untreated tissue, but do not shed light on early signaling events that lead to the observed 117 SAR phenotype. The immediate consequences of NHP accumulation on gene expression 118 have not been described. Studying very early transcriptional changes, within minutes to 119 hours of elicitor treatment, has been an effective strategy elucidating key components of 120 signaling pathways for SA, jasmonic acid (JA), and brassinosteroid (Friedrichsen et al., 121 2002; Chini et al., 2007; Ding et al., 2018). Thus, in an effort to better understand NHP's 122 primary action in signal transduction, we determined the genome -wide transcriptional 123 responses in Arabidopsis from 15 minutes up to 6 hours after treatment with exogenous 124 NHP. 125 126 Here we report the early NHP transcriptional profile and the role SA plays during this 127 response. We present evidence indicating that increased expression of WRKY TFs define 128 the primary transcriptional responses mediated by NHP within minutes (i.e. 15 to 30 129 minutes) post -treatment, and WRKY TFs act as executors of the secondary 130 transcriptional responses within hours (i.e. 3 to 6 hours). The majority of genes induced 131 by NHP within minutes were SA -dependent, whereas those induced within hours were 132 SA-independent. These data suggest that NHP induces the primary transcriptional 133 response in a low SA environment and new SA biosynthesis is dispensable for induction 134 of the secondary transcriptional response. We also show that WRKY70 is required for 135 NHP induced transcription of a set of genes defining secondary transcriptional changes 136 as well as NHP enhancement of reactive oxygen species (ROS) and SAR. Taken 137 together, our study highlights the key genes and pathways defining early NHP responses 138 and a role for WRKY70 in the regulation of NHP-dependent transcription. 139 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 6 140

141 NHP induces expression of defense-associated genes within minutes 142 Previous studies in Arabidopsis have demonstrated that exogenous application of NHP 143 to a leaf (defined as “local tissue”) is sufficient to initiate and amplify defense signaling in 144 untreated leaves (defined as “distal tissue”), including the transcriptional activation of SAR 145 marker genes (Chen et al., 2018; Hartmann et al., 2018; Nair et al., 2021; Yildiz et al., 146 2021). Notably, these studies focused on gene expression changes in distal tissues at 147 late time points (24 to 48 h) following NHP elicitation. However, it was unknown which 148 gene expression changes occur immediately in response to NHP treatment. We 149 hypothesized that once present in the tissue, either via endogenous synthesis or 150 exogenous application, NHP initiates signaling that leads to transcriptional changes on a 151 timescale of minutes to several hours, which are essential for execution of NHP -152 dependent immune responses. 153 154 In order to test this hypothesis, we set out to identify NHP responsive transcriptional 155 markers and determine the earliest time frame when NHP could induce these genes. We 156 first questioned if NHP could induce the expression of classical SAR -associated marker 157 genes (i.e., FMO1, ICS1, and PATHOGENESIS-RELATED PROTEIN 1 (PR1)) within 158 several hours of treatment. Studies have shown FMO1, ICS1, and PR1 are induced 159 between 8 to 48 h following NHP treatment (Chen et al., 2018; Nair et al., 2021). 160 Therefore, we began by testing the response to NHP within 24 h of treatment. The leaves 161 of 4.5-week-old Arabidopsis Col-0 (wild type) plants were infiltrated with 1 mM NHP and 162 then treated leaves were collected for mRNA isolation at 2, 6 and 24 h. Quantitative real-163 time PCR (qRT-PCR) analysis showed significantly increased transcript abundance of all 164 three genes in NHP treated leaves at 24 h but not at 2 and 6 h compared to water (mock) 165 treated leaves (Fig. 1A). These data indicate that FMO1, ICS1, and PR1 are responsive 166 to NHP treatment but are likely not primary targets of NHP transcriptional activation. 167 168 Next, we tested the ability of NHP to induce expression of WRKY38 and UGT76B1, two 169 genes known to respond to SA within 2 h of treatment (Blanco et al., 2009). WRKY38 170 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 7 encodes a defense -associated TF and UGT76B1 encodes a UDP -dependent 171 glycosyltransferase shown to glycosylate SA and NHP (Eulgem et al., 2000; Bauer et al., 172 2021; Cai et al., 2021; Holmes et al., 2021; Mohnike et al., 2021). We found a significant 173 increase of mRNA abundance for both WRKY38 and UGT76B1 in NHP treated leaves at 174 2 h compared to mock treated leaves (Fig. 1A). WRKY38 transcript levels were also 175 elevated at 6 and 24 h after treatment with NHP (Fig. 1A). These data indicate that both 176 WRKY38 and UGT76B1 are early NHP responsive transcripts. 177 178 We used the WRKY38 gene as a marker to define the earliest time when NHP -induced 179 transcriptional changes could be measured. To this end, we implemented a 180 hydroponically grown seedling assay to synchronize chemical treatment and increase the 181 number of plants sampled, while minimizing mechanical stress. Wild type seedlings were 182 grown in liquid medium in six -well plates with 15-18 seeds per well. The seedlings were 183 treated with mock, 1 mM Pip, or 1 mM NHP at 10 -days-post germination and then 184 collected at 15 min, 30 min, 3 h, and 6 h post treatment to isolate mRNA (Fig. 1B). 185 WRKY38 transcript abundance was significantly elevated in Arabidopsis seedlings 186 treated with NHP as early as 15 min, peaking in abundance at 30 min, when compared 187 to those treated with mock or Pip (Fig. 1C). This trend continued through 3 h following 188 NHP treatment. The NHP precursor Pip did not elicit an increase in WRKY38 mRNA 189 within 3 h but showed a slight trend of increased abundance at the 6 h time point (Fig. 190 1C). These results indicate that exogenous NHP, but not Pip, is sufficient to elevate 191 WRKY38 transcript abundance within 15 minutes of application. 192 193 Since WRKY38 expression is known to be induced by SA treatment (Blanco et al., 2009), 194 we questioned if NHP-induced expression of WRKY38 is dependent on SA derived from 195 the ICS1/SID2 biosynthetic pathway by utilizing the well characterized sid2-2 mutant that 196 does not accumulate SA upon pathogen infection (Wildermuth et al., 2001). Wild type and 197 sid2-2 seedlings were grown hydroponically, treated with mock, 1 mM Pip, or 1 mM NHP, 198 and then collected 3 h after treatment. NHP treatment increased WRKY38 mRNA 199 abundance in the sid2-2 mutant relative to mock treatment, similar to wild type seedlings 200 (Supplemental Fig. S1). These findings indicate that NHP is sufficient to alter WRKY38 201 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 8 transcript abundance in the absence of SA accumulation, defining WRKY38 as an early 202 NHP induced, SA-independent transcript. 203 204 The early NHP-responsive transcriptome reveals distinct waves of expression 205 We next questioned how the presence of NHP impacts whole -genome transcriptional 206 changes within minutes to hours of treatment in a SA -dependent and -independent 207 manner. To address this, Arabidopsis wild type and sid2-2 mutant seedlings were grown 208 in the same hydroponic design as previously described for qRT -PCR experiments (Fig. 209 1B). The sid2-2 mutant was analyzed in order to identify transcriptional changes 210 dependent on NHP-induced SA biosynthesis (Chen et al., 2018). Each pool of seedlings 211 was treated with mock or 0.5 mM NHP for 15 min, 30 min, 3 h and 6 h. A reduced 212 concentration of NHP was used as we found that 0.5 mM NHP induced similar WRKY38 213 mRNA accumulation as 1 mM NHP (Supplemental Fig. S2). The abundance of mRNA in 214 the treated seedlings was assessed for each condition via RNA -sequencing (RNA-seq). 215 After general quality control and mapping to the Arabidopsis TAIR10 genome, genes 216 differentially expressed by NHP were determined relative to mock treatment for each time 217 point and genotype using DESeq2 (Love et al., 2014). Genes were considered 218 differentially expressed if they returned an adjusted P-value below 0.05 (Padj < 0.05). 219 220 In wild type seedlings, a total of 2,079 genes were differentially expressed in response to 221 NHP for at least one of the measured time points. To explore the set of early NHP -222 responsive genes, we first selected for genes showing a robust fold change over mock 223 by applying the cutoff of log2(fold change, FC) > 1 for upregulated genes and log 2(FC) < 224 -1 for downregulated genes. This identified 352 genes that were differentially expressed 225 in response to NHP for at least one time point, with 163 genes up and 189 genes down 226 (Supplemental Table S3). Next, the upregulated genes were hierarchically clustered 227 based on their log2(FC) values over the indicated time points and four well-defined gene 228 clusters were identified (Fig. 2A). For each group of genes in the defined clusters, the 229 log2(FC) values were then averaged by time point, revealing dynamic patterns of 230 expression. Two of the clusters were upregulated within minutes (designated as ‘early 231 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 9 transient’ and ‘early strong’) and two were upregulated by several hours (designated as 232 ‘late weak’ and ‘late strong’) (Fig. 2B, Table 1, Table 2, Supplemental Table S4). 233 234 The ‘early transient’ cluster includes 42 genes and is defined by elevated transcript 235 abundance 15-30 min after NHP treatment that did not persist by 3 h. Gene ontology (GO) 236 analysis for this cluster showed strong enrichment for biological processes associated 237 with responses to jasmonic acid (JA), wounding, and oxidative stress. Some of these 238 genes are associated with the regulation of defense as well as defense responses to fungi 239 and bacteria (Fig. 2C). Among the most highly expressed genes in this cluster are JA -240 responsive genes, which play key roles in the regulation of wounding responses, including 241 CML39 which encodes a calmodulin-like protein, JAZ10 which encodes a jasmonate-zim-242 domain protein, and MYC2 which encodes a jasmonate -inducible transcription factor 243 (Table 1, Table 2). Taken together, the early yet unsustained induction of these genes 244 within 30 min suggests a burst of JA - and wound-responsive gene expression that acts 245 as a primary wave of NHP signaling. 246 247 The ‘early strong’ cluster includes 10 genes and is defined by a high average log2(FC) 15 248 min after NHP treatment that was maintained for all subsequent time points. Of the 10 249 genes, two (AT1G06475 and AT5G40980) were strongly upregulated by NHP at 30 min 250 and no other time points. These two genes were removed from the average log 2(FC) 251 analysis and subsequent analyses because they did not behave like the other genes in 252 this cluster (Fig. 2B). The early strong cluster is notably enriched in biological processes 253 involved in the regulation of transcription, with over half the genes belonging to the WRKY 254 TF family, including four group III WRKY genes ( WRKY38, WRKY54, WRKY62 and 255 WRKY70) and two group II-c genes (WRKY51 and WRKY59) (Eulgem et al., 2000; Kalde 256 et al., 2003); (Table 1, Table2, Supplemental Table S4). Additional GO terms enriched in 257 the early strong cluster includes regulation of defense and regulation of hormonal 258 signaling pathways such as SA, JA, brassinosteroid, and ethylene (Fig. 2C). The early 259 and sustained induction of these genes, particularly the WRKYs, suggests they are 260 primary transcriptional target genes of NHP signaling. 261 262 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 10 The ‘late weak’ cluster includes the largest number of genes (96) that averaged a log2(FC) 263 around 1 (Fig. 2B). GO term analysis of the cluster revealed significant enrichment of 264 biological processes involved in response to bacteria and oomycetes, as well as SA -265 mediated signaling and regulation of SAR (Fig. 2C). This indicates NHP activation of SAR 266 and pathogen defense genes can be detected as early as 3 -6 h in treated tissues. 267 Notably, genes involved in the regulation and biosynthesis of NHP were observed in this 268 wave of transcription, including SARD1 (SAR DEFICIENT 1 ), NIMIN-1 (NIM-269 INTERACTING 1 ), ALD1, and UGT76B1 (Table 1, Table 2, Supplemental Table S4) 270 (Song et al., 2004; Zhang et al., 2010a; Holmes et al., 2021). 271 272 The ‘late strong’ cluster consists of 15 genes with log2(FC) values averaging between 3.5 273 - 4 (Fig. 2B). Response to SA was the only significantly enriched GO term for this cluster 274 (Fig. 2C). Despite having a role in the response to SA, the majority of genes classified as 275 late strong were upregulated by NHP in both wild type and sid2-2 (Fig. 3A, Table 1), 276 indicating that accumulation of these transcripts was at least partially SA-independent. Of 277 the 14 SA-independent genes identified in this cluster, only five have been found to be 278 upregulated 24 h after NHP treatment in an SA-independent manner (Yildiz et al., 2021). 279 Thus, our analysis has uncovered additional NHP -responsive genes that operate 280 independently of SA as early as 6 h post NHP signaling. 281 282 We also performed a similar clustering analysis on the 189 NHP -downregulated 283 transcripts and identified three gene clusters (Supplemental Fig. S3A, B). In contrast to 284 the upregulated gene clusters, the downregulated gene clusters had less distinct patterns 285 of expression and averaged log 2(FC) values around -1 to -1.5. Two clusters were 286 downregulated within 15-30 min and persisted 3 -6 h hours after NHP treatment. One of 287 these showed weaker downregulation and was classified as an ‘early weak’ cluster. The 288 other showed stronger downregulation and was classified as an ‘early strong’ cluster. The 289 third did not show decreased transcript abundance until 3 -6 h after treatment and thus 290 was labeled as a ‘late’ cluster (Supplemental Fig. S3B). All three clusters were enriched 291 for biological processes involving growth and development such as root morphogenesis, 292 plant epidermis development, plant -type cell wall organization, and root hair cell 293 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 11 differentiation (Supplemental Fig. S3C). Given the NHP precursor Pip is known to inhibit 294 root growth of seedlings (Wang et al., 2018), it is likely that NHP also inhibits root growth. 295 These clusters shed new light on the genes that may be associated with altered plant 296 growth and development in response to elevated levels of NHP. 297 298 SA biosynthesis is dispensable for the early induction of a majority of NHP 299 upregulated genes 300 Interrogation into the late strong cluster suggested NHP may be sufficient to activate a 301 subset of SA-responsive genes in the absence of NHP-induced SA biosynthesis (Fig. 2C, 302 Fig. 3A, 'late strong'). This was an intriguing possibility given NHP likely accumulates in 303 distal tissues prior to SA accumulation following a local tissue infection (Bernsdorff et al., 304 2016; Hartmann et al., 2018; Hartmann and Zeier, 2019). We therefore hypothesized that 305 NHP would need to drive transcriptional reprogramming as SA levels rise from low to 306 high. To investigate this, we assessed the impact of SA biosynthesis on NHP -induced 307 gene expression from 15 min to 6 h. For each time point of the wild type gene clusters 308 described in Fig. 2, the percentage of genes upregulated in both wild type and the sid2-2 309 mutant was determined. This group of genes was defined as ‘SA -independent’. Genes 310 upregulated only in wild type and not in sid2-2 at the defined time point were conversely 311 defined as ‘SA-dependent’. 312 313 Notably, the majority of genes upregulated by NHP at 15 min in the early transient cluster 314 were SA-dependent. For example, 87% of the genes (27 out of 31) were upregulated at 315 15 min in wild type but not in sid2-2 seedlings (Fig. 3A) and similar trends were seen at 316 30 min. By contrast, 100% of the genes (8 out of 8) in the early strong cluster upregulated 317 3-6 h after NHP treatment were SA -independent (Fig. 3A). 69% of the genes (36 out of 318 52) in the late weak cluster were SA-independent at 3h, which dropped to 53% (37 out of 319 70 genes) at 6 h (Fig. 3A). 320 321 A similar pattern of gene expression was observed when we analyzed the total number 322 of NHP upregulated genes reaching a log2(FC) > 1 without gene clustering (Fig. 3B). The 323 majority of genes induced by NHP between 15-30 min were SA-dependent (i.e., 36 genes 324 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 12 up in wild type and 9 up in both wild type and sid2-2 at 15 min) and the majority of genes 325 induced by NHP between 3-6 h were SA-independent (i.e., 20 genes up in wild type and 326 55 up in both wild type and sid2-2 at 3 h) (Fig. 3B). Taken together, this analysis highlights 327 an important temporal role of SA on NHP induced transcription. Our data suggest that 328 new SA biosynthesis is required for the induction of NHP -responsive genes within 329 minutes of exposure to NHP, whereas SA biosynthesis is dispensable for NHP -induced 330 expression several hours later. 331 332 We also analyzed the downregulated genes using the same approach and found a slight 333 trend of SA-dependence for these genes following NHP treatment. For example, the early 334 weak and late clusters defined in Supplemental Fig. S3 showed that a majority of genes 335 are downregulated in wild type but not the sid2-2 mutant at each time point (Supplemental 336 Fig. S4A). By contrast, the genes of the early strong cluster were less dependent on SA 337 biosynthesis, such that by the 6 h time point only 15% of genes (8 out of 44) were 338 downregulated in wild type and not in sid2-2 seedlings (Supplemental Fig. S4A). 339 Independent of clustering, greater than 60% of the downregulated genes were found to 340 be SA-dependent at 15 min and 3 h and approximately 40% were SA -dependent at 30 341 min and 6 h (Supplemental Fig. S4B). Taken together, these findings suggest a stronger 342 dependence on SA biosynthesis for NHP-dependent downregulation of gene expression 343 over the time courses studied. 344 345 SA biosynthesis antagonizes NHP-elicited gene expression at 3 to 6 h 346 Looking at the genes upregulated only in the sid2-2 mutant and not wild type, we 347 discovered an unexpectedly large number of transcripts that increased in abundance 348 upon NHP treatment at 3 and 6 h post treatment (Fig. 3B). This includes 151 genes at 3 349 h and 98 genes at 6 h that were significantly upregulated by NHP in the sid2-2 mutant but 350 not in wild type (Fig. 3B). GO-term enrichment analysis of these genes showed significant 351 enrichment for a number of biotic and abiotic stress responses, such as defense response 352 to bacteria and fungi, SAR, response to abscisic acid, and response to hypoxia (Fig. 3C). 353 These data suggest that SA may play an important role in modulating the transcription of 354 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 13 a suite of genes, through an unknown mechanism, once NHP -mediated transcriptional 355 reprogramming has been initiated. 356 357 TGA and WRKY transcription factor cis-regulatory elements are enriched in the 358 promoters of early NHP upregulated genes 359 To identify TFs that might directly control the expression of early NHP-responsive genes, 360 we analyzed the promoter regions of all genes upregulated after NHP treatment in both 361 wild type and sid2-2 mutant seedlings to identify putative TF bindings sites (i.e., cis-362 regulatory elements, CREs). A less stringent cutoff of log 2(FC) > 0 and Padj < 0.05 was 363 used for the upregulated genes to capture putative transcriptional regulators of all genes 364 significantly elevated in transcript abundance, regardless of the magnitude of expression 365 change. Following a modified workflow detailed by Bjornson et al., 2021, the NHP 366 upregulated genes were then separated by the time points in which they were 367 differentially expressed. Enrichment of transcription factor CREs was then determined for 368 each time point using the DAPseq database (O’Malley et al., 2016) (Fig. 4A). 369 Supplemental Fig. 5 shows all the CREs found to be significantly enriched in the 370 promoters of NHP-upregulated genes in both wild type and sid2-2 seedings between 15 371 min and 6 h following NHP treatment. 372 373 We found that CREs for bZIP family members, specifically TGA TFs, were enriched in the 374 promoters of genes upregulated by NHP as early as 15 min. For some TGAs (e.g., TGA2, 375 TGA5, and TGA7), the enrichment of their TF binding sites was observed for all time 376 points (Fig. 4B). Enrichment of TGA binding sites in the promoters of early NHP 377 upregulated genes is consistent with previous work demonstrating TGA2/5/6 and TGA1/4 378 are required for the induction of NHP responsive genes at 24 -48 h post NHP treatment 379 (Nair et al., 2021; Yildiz et al., 2023). TGA TFs are known to interact with the 380 transcriptional coactivator NPR1 to drive SAR - and SA-induced transcriptional changes 381 (Zhang et al., 1999). The enrichment of TGA binding sites in the promoters of NHP 382 upregulated genes as early as 15 min implicates TGAs and NPR1 among the primary 383 transcriptional regulators of NHP signaling. Furthermore, enrichment of TGA CREs was 384 not observed in sid2-2 seedlings until 30 min and was also not significantly enriched at 3 385 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 14 h (Fig. 4B). These findings suggest that SA biosynthesis and likely elevated SA levels are 386 required for rapid and stable induction of some TGA -regulated genes induced by NHP 387 signaling. 388 389 Moreover, we discovered that a large number of WRKY CREs were highly enriched in the 390 promoter regions of NHP upregulated genes at 3 -6 h in both wild type and sid2-2 391 seedlings (Fig. 4C). This correlates with the majority of genes identified in the NHP 392 upregulated early strong cluster belonging to the WRKY TF family (Fig. 2, Table 1). This 393 finding suggests a model where NHP induces the expression of WRKY genes as a 394 primary transcriptional response, followed by WRKY-regulated gene expression changes 395 that act as a secondary wave of transcriptional control. 396 397 It is striking that the confidence in WRKY CRE enrichment was greater (i.e., lower P value) 398 at 3 and 6 h in the promoters of genes upregulated in the sid2-2 mutant compared to 399 those of wild type (Fig. 4C). These data suggest that a substantial number of NHP 400 upregulated genes are directly regulated by WRKY TFs, and such regulation is influenced 401 by SA levels. A similar pattern emerged for members of the NAC TF family, where NAC 402 CREs were enriched in the promoters of NHP upregulated genes at 3 h in sid2-2 but not 403 in the promoters of genes upregulated in wild type (Supplemental Fig. S5). Both WRKY 404 and NAC TFs are involved in the regulation of a number of biotic and abiotic stress 405 responses (Eulgem et al., 2000; Mohanta et al., 2020). Notably, the set of genes 406 upregulated by NHP at 3-6 h only in the sid2-2 mutant but not in wild type were enriched 407 in biological processes associated with biotic and abiotic stress responses (Fig. 3B, C). 408 Taken together, these data suggest SA may antagonize the transcription of a subset of 409 NHP responsive genes by regulating the activities of WRKY and NAC TFs. 410 411 The same CRE enrichment analysis was also carried out on all NHP downregulated 412 genes, regardless of the magnitude of fold change (log 2(FC) < 0 and Padj < 0.05). We 413 found multiple WRKY CREs enriched in the promoter regions of NHP downregulated 414 genes at 15 min, 3 h, and 6 h, in wild type seedlings (Supplemental Fig. S6), further 415 supporting a role for WRKY TFs in relaying NHP-induced transcriptional changes. 416 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 15 417 Finally, we found the CREs of Homeobox gene family members to be abundant in the 418 promoters of NHP downregulated genes spanning from 15 min through 6 h, with the 419 greatest confidence of enrichment found in the promoters of genes downregulated by 420 NHP at 3 -6 h in an SA -dependent manner (Supplemental Fig. S6). Among these was 421 ATHB5, a homeodomain leucine zipper (HDZip) protein linked to the abscisic acid (ABA)-422 driven repression of germination and root growth in seedlings (Johannesson et al., 2003). 423 Given that NHP precursor Pip inhibits seedling root growth, we now hypothesize that NHP 424 may drive the repression of root growth and development genes (Supplemental Fig. 3C) 425 through the action of HDZip Homeobox proteins like ATHB5. 426 427 Early NHP-responsive gene WRKY70 is required for NHP-elicited SAR 428 To further dissect the NHP response pathway, we carried out a reverse genetic screen to 429 identify Arabidopsis mutants with compromised NHP -elicited SAR. Given WRKY CREs 430 were the predominant regulatory elements identified in genes responsive to NHP 3 -6 h 431 following treatment (Fig. 4C, Supplemental Fig. S6), we selected the six WRKYs 432 (WRKY38, WRKY51, WRKY54, WRKY59, WRKY62, WRKY70) from the early strong 433 NHP upregulated cluster (Figure 2B, Table 1, Table 2) for mutant analysis. 434 435 To measure NHP -induced SAR, we obtained homozygous Arabidopsis mutants and 436 quantified bacterial growth in their leaves after treatment with exogenous NHP (Chen et 437 al., 2018). Specifically, three lower leaves of 4.5 -week-old wild type and mutant plants 438 were infiltrated with water (mock) or 0.5 mM NHP. One day later, one distal upper leaf 439 was inoculated with a 1 x 105 CFU/mL suspension of the virulent bacterium Pseudomonas 440 syringae pathovar maculicola strain ES4326 ( Psm). Bacterial growth of Psm was 441 quantified three days post infection (dpi) to determine if the mutants were altered in their 442 resistance to pathogen infection. Mutants were considered to exhibit NHP-elicited SAR if 443 bacterial titer was significantly lower in NHP treated plants compared to mock treated 444 plants. By contrast, mutants were considered insensitive or partially insensitive to NHP if 445 the bacterial titer of NHP treated mutants was higher than NHP treated wild type plants. 446 447 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 16 Of the six wrky mutants screened, five ( wrky38, wrky62, wrky54, wrky51, and wrky59) 448 exhibited normal NHP -elicited SAR (Fig. 5B -E). Only one, wrky70-1 (T-DNA insertional 449 line SALK_025198) (Li et al., 2006), displayed compromised NHP-elicited SAR (Fig. 5A). 450 That is, NHP treatment did not decrease bacterial growth in distal leaves of wrky70-1 451 plants compared to mock treatment. We also observed that wrky70-1 mutants treated 452 with mock contained less bacteria than wild type plants treated with mock, demonstrating 453 that basal resistance is enhanced in the wrk70-1 mutant (Fig. 5A). Notably, distal leaves 454 of wrky70-1 plants treated with NHP contained higher levels of bacteria than distal leaves 455 of wild type plants treated with NHP (Fig. 5A). The inability of wrky70-1 plants to further 456 restrict bacterial growth following NHP treatment suggests that the mutant may be 457 insensitive to NHP or defective in NHP signaling. Taken together, these data indicate that 458 WRKY70 is required for full NHP-elicited systemic resistance. 459 460 We next questioned if systemic resistance to Psm was similarly compromised in wrky70-461 1 plants primed with the avirulent pathogen P. syringae pathovar tomato (Pst) strain 462 DC3000 carrying avrRpt2 (Pst avrRpt2). Localized infection with Pst avrRpt2 is known to 463 elicit a strong immune response that leads to SAR and protection against Psm in distal 464 leaves (Kohler et al., 2002). We found that the Psm titer in distal leaves reached similar 465 levels for wrk70-1 plants treated with mock or Pst avrRpt2 and this level was comparable 466 to Psm growth in wild type leaves of plants treated with Pst avrRpt2 (Supplemental Fig. 467 S7A). These data indicate that wrky70-1 mutants exhibit a basal-level of defense priming 468 and Pst avrRpt2 primary infection does not further enhance this resistance. 469 470 We also examined pathogen growth in local wrky70-1 leaves. We found that the titer of 471 Psm in infected wrky70-1 leaves was lower than in Psm infected wild type leaves 472 (Supplemental Fig. S7B). This phenotype corroborates previous work demonstrating loss 473 of WRKY70 expression enhances resistance to Psm infection (Zhou et al., 2018). 474 475 Expression of SARD1 and PR genes is elevated in wrky70-1 independent of NHP 476 Previous studies in Arabidopsis have demonstrated a role for WRKY70 in the 477 transcriptional regulation of several genes required for SAR signaling and disease 478 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 17 resistance, including SARD1 and the PATHOGENESIS-RELATED genes PR1, PR2, and 479 PR5 (Li et al., 2004; Zhou et al., 2018; Liu et al., 2021). These genes were also shown to 480 be upregulated in wild type Arabidopsis plants 24 h after treatment with NHP (Yildiz et al., 481 2021). To determine if WRKY70 is required for NHP induced expression of SARD1, PR1, 482 PR2, and PR5 at 24 h, we quantified transcript abundance in wild type and wrky70-1 483 plants treated with water or 1 mM NHP. As expected, qRT -PCR showed increased 484 abundance of SARD1, PR1, PR2, and PR5 mRNA in wild type plants treated with NHP 485 compared to water (Supplemental Fig. S7C). However, NHP treatment did not further 486 increase transcript levels in wrky70-1 plants (Supplemental Fig. S7C). Notably, all four 487 genes showed elevated transcript abundance in water treated wrky70-1 mutants 488 compared to water treated wild type. The elevated expression of SARD1 and PR1 in 489 water treated wrky70-1 plants is consistent with previous studies demonstrating WRKY70 490 negatively regulates these two genes in the absence of a pathogen (Zhou et al., 2018; Liu 491 et al., 2021). 492 493 WRKY70 is required for NHP induction of genes in the late weak and late strong 494 clusters 495 Given wrky70-1 mutants exhibited enhanced basal resistance but were still unable to 496 achieve the same level of resistance as NHP treated wild type plants (Figure 5A), we 497 hypothesized that a subset of early NHP-responsive genes containing putative WRKY70 498 CREs in their promoters may be dependent on WRKY70 function for full, wild type gene 499 expression levels following NHP treatment. To investigate this, we selected three genes 500 from the late weak and late strong clusters (Fig. 2, Table 1), specifically BDA1, 501 PROSCOOP4, and CML10, and examined their transcript accumulation in response to 1 502 mM NHP over a time course of 0 to 12 h. The promoters of these genes contain one or 503 more WRKY70 CREs (i.e., W -box (TTGACY), WT -box (YGACTTTT), and WRKY70 504 DAPseq motif; (Rushton et al., 2010; Machens et al., 2014; O’Malley et al., 2016)) within 505 2000 base pairs upstream of their respective putative transcriptional start site (Fig. 6A). 506 507 We found that BDA1 (bian da; “becoming big” in Chinese), a gene encoding an ankyrin -508 repeat transmembrane protein (Yang et al., 2012), exhibited the strongest dependence 509 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 18 on WRKY70 for NHP-induced expression. BDA1 transcripts were significantly increased 510 at 6, 9, and 12 h post NHP treatment in wild type leaves but not wrk70-1 leaves (Fig. 6B). 511 Transcript levels for PROSCOOP4, a gene encoding the precursor of secreted peptide 512 SCOOP4/STMP10 (Gully et al., 2019; Hou et al., 2021), were also significantly higher at 513 6 and 9 h post NHP treatment in wild type leaves compared to that observed for wrk70-1 514 plants (Fig. 6C). Unlike BDA1, we found that PROSCOOP4 transcripts were higher in 515 NHP-treated wrk70-1 leaves at 6 and 9h compared to the 0h timepoint (Fig. 6C). These 516 data show that wrk70-1 plants responded to NHP treatment, suggesting that they can 517 sense NHP but are impaired in NHP signaling. Similar trends were observed for CML10 518 (CALMODULIN-LIKE 10/CaBP22; (Luan et al., 2002)) at the 9 h timepoint (Fig. 6D). We 519 confirmed these results by analyzing a second wrky70 allele, wrky70-2 (Supplementary 520 Fig. S8). 521 522 Taken together, our findings reveal that WRKY70 is required for the proper expression of 523 NHP responsive genes from the late weak and late strong clusters, highlighting a role for 524 WRKY70 in the transcriptional response to NHP several hours following treatment. 525 526 NHP pretreatment enhances flg22 -elicited ROS production and WRKY70 527 contributes to the full response 528 We next questioned if WRKY70 regulates a specific branch of NHP defense signaling. 529 One such branch involves the response to common molecular features of microbes (e.g., 530 bacterial flagellin or fungal chitin) known as microbe -associated molecular patterns 531 (MAMPs) and the response to plant -derived damage -associated molecular patterns 532 (DAMPs), a process collectively referred to as pattern -triggered immunity (PTI). The 533 WRKY70-dependent gene BDA1 is known to mediate signaling in response to MAMP 534 detection and interacts with receptor -like protein SNC2 (Suppressor of NPR1, 535 Constitutive2) to relay MAMP -triggered defense responses (Yang et al., 2012). 536 Production of reactive oxygen species (ROS) is a MAMP -triggered defense response 537 which can act directly or indirectly as an antimicrobial agent and serves as a secondary 538 signal to activate further defense responses (Boller and Felix, 2009; Couto and Zipfel, 539 2016). Similarly, DAMPs, such as secreted peptides from the PROSCOOP family, are 540 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 19 known to elicit an increase in ROS production (Gully et al., 2019; Hou et al., 2021; 541 Rhodes et al., 2021). Incidentally, CML10 encodes a calmodulin -like protein known to 542 interact with phosphomannomutase (PMM) in order to modulate ascorbic acid synthesis 543 and cellular homeostasis of ROS (Cho et al., 2016). 544 545 We thus investigated how NHP alters MAMP -triggered ROS production using bacterial 546 flagellin as the elicitor in the presence and absence of NHP pretreatment and asked if 547 WRKY70 is required for the response. Leaf discs of wild type and wrky70-1 plants were 548 floated on solutions of water, NHP, and SA. SA was included as a positive control, as it 549 is known to enhance the MAMP-triggered ROS burst (Yi et al., 2014). After 24 h, leaf discs 550 were treated with a 100 nM solution of flagellin peptide (flg22) and then ROS was 551 measured using a luminol-based assay. We found ROS levels from mock treated wrky70-552 1 plants trended lower than water treated wild type, suggesting WRKY70 is required for 553 maximal ROS accumulation (Fig. 7, upper panel). NHP treatment increased ROS 554 generation in both wild type and wrky70-1 tissue exposed to flg22 when compared to 555 water treated tissues. The average ROS production in NHP treated wrky70-1 leaf discs 556 was lower than NHP treated wild type leaf discs (Fig. 7, lower panel). Similar results were 557 found when analyzing wrky70-2 plants (Supplementary Fig. S9). Collectively, these data 558 indicate that NHP is able to enhance the flg22-elicited ROS burst, and this enhancement 559 requires WRKY70 for full ROS generation. 560 561

562 NHP has emerged as an essential signaling molecule required for the establishment and 563 amplification of SAR in Arabidopsis and crop plants (Chen et al., 2018; Holmes et al., 564 2019), though the early signaling events mediated by NHP had yet to be described. Our 565 study provides the first insights into the primary transcriptional responses to NHP, as well 566 as the role that NHP -induced SA biosynthesis plays during this response. We also 567 discovered that TGA and WRKY CREs are enriched in the promoters of early NHP 568 induced genes, providing a framework for dissecting primary and secondary 569 transcriptional responses. Further, we show that early transcriptional expression of 570 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 20 WRKY70, within minutes of NHP treatment, mediates secondary transcriptional 571 reprogramming that is required for NHP signaling, ROS production, and SAR. 572 573 Early short-lived NHP transcriptional responses associate with general stress 574 In the early response to NHP, we identified a primary wave of NHP signaling that was 575 short lived and activated transcriptional responses mirroring wounding/DAMP and JA 576 signaling in a partially SA -dependent manner. Of the pathways activated by NHP, we 577 found a considerable enrichment of known JA -response genes, including MYC2 and 578 JAZ10 (Fig. 2C, Table 1, Table 2). Given the established antagonism between JA and SA 579 signaling pathways (Pieterse et al., 2012), it was surprising to find the majority of genes 580 in the early transient cluster were SA -dependent (Fig. 3A). These findings suggest that 581 part of the early NHP transcriptional response depends on synergism between JA 582 signaling components and pre -infection levels of SA. Our data support a model where 583 general stress response signatures are activated by NHP treatment in an early pulse of 584 gene expression that acts synergistically with SA to modulate activation of defense 585 pathways. 586 587 Previous work has described a strong, systemic JA response in Arabidopsis leaves 588 infected with an avirulent Pst strain after 4 h, suggesting JA signaling pathways are 589 involved in SAR development (Truman et al., 2007). The JA response detected by 590 Truman et al. in the systemic leaves of avirulent Pst-treated plants after 4 h may be 591 reflective of the early and transient response to NHP observed in our transcriptional 592 analysis. These JA - and early NHP -responsive marker genes may prove useful for 593 determining optimal time points in testing the systemic translocation of NHP. Furthermore, 594 although genetic studies contest the requirement for JA biosynthesis and signaling in SAR 595 (Attaran et al., 2009), it remains a possibility that differences in primary pathogen 596 treatment or secondary pathogen infection may shift the requirement for JA signaling in 597 SAR establishment and warrant further study. 598 599 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 21 Early strong NHP responses activate a primary wave of WRKY transcription 600 We discovered another primary wave of early NHP signaling that was partially SA -601 independent and exhibited strong induction of WRKY gene transcription within minutes 602 of NHP treatment, persisting several hours after treatment. All six WRKY TF genes in the 603 early stable upregulated gene cluster were activated by NHP treatment in the sid2-2 604 mutant, and although some showed delays in the timing of induction in sid2-2 seedlings, 605 all six were strongly upregulated by 3 h (Table 1, Table 2). These findings are intriguing 606 given that the expression of these WRKY TFs is known to be induced by SA and SA -607 analogs in an NPR1 -dependent manner (Kalde et al., 2003; Wang et al., 2006). The 608 ability of NHP to induce an early wave of WRKY expression, independent of elevated SA, 609 suggests NHP and not SA is the primary signal driving this early response. 610 611 The early and strong induction of WRKY transcription, including two sets of homologs 612 belonging to WRKY group III (WRKY38 and WRKY62 as well as WRKY54 and WRKY70), 613 suggested WRKY TFs may play a role in driving the secondary transcriptional response 614 to NHP. Members of WRKY group III are known to play both positive and negative 615 regulatory roles in defense responses (Kalde et al., 2003; Pandey and Somssich, 2009). 616 Consistent with this, we found CREs of WRKY TFs to be abundant in the promoters of 617 genes, both up and downregulated, several hours after NHP treatment (Fig. 4C, 618 Supplemental Fig. 5). For example, the CRE bound by WRKY70, as defined by the 619 DAPseq Plant Cistrome Database (O’Malley et al., 2016) , was enriched in the promoters 620 of genes upregulated by NHP at 3 and 6 h (Fig. 4C). Other canonical motifs bound by 621 WRKY70, such as the W -box and WT -box (Rushton et al., 2010; Machens et al., 2014), 622 were also found in the promoters of genes upregulated several hours after NHP treatment 623 (Fig. 6A). Taken together, these findings link the early NHP -driven induction of WRKY 624 expression to the subsequent up and downregulation of WRKY-targeted genes 3-6 h after 625 initial NHP treatment. Based on these findings, we hypothesize a model of early NHP 626 signaling wherein activation of WRKY transcription is a primary response to NHP 627 signaling that is followed by a secondary wave of expression driven through WRKY 628 regulation. 629 630 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 22 While not the focus of this study, it is noteworthy to mention that TGA TFs are also 631 associated with the primary wave of NHP mediated transcription. CREs of TGAs were 632 abundant in the promoters of early NHP upregulated genes across all time points 633 sampled, particularly those in the early strong cluster (Fig. 4B). These findings are in 634 agreement with recent studies demonstrating TGAs are redundantly required for NHP -635 induction of SAR and SAR marker genes (Nair et al., 2021; Yildiz et al., 2023). Our data 636 provide further evidence highlighting the importance of TGAs in NHP-driven signaling and 637 suggest that a NPR1/TGA complex, or a key interactor of this complex, directly relays the 638 NHP signal. 639 640 Late weak and late strong genes define the secondary wave of transcription 641 Finally, our RNA-seq analysis also revealed a secondary wave of NHP signaling defined 642 by late weak and late strong upregulated genes. These genes are associated with 643 pathogen defense, SAR, and SA responses, and showed features of SA-dependent and 644 SA-independent transcriptional induction (Fig. 2C, Fig. 3A). Upregulation of SAR -645 associated genes by NHP is consistent with previous studies (Bernsdorff et al., 2016; 646 Yildiz et al., 2021; Yildiz et al., 2023). Our study reveals that transcriptional activation of 647 these SAR-associated genes occurs as early as 3 -6 h following detection of the NHP 648 signal. Interestingly, 24 genes from the late weak cluster and 4 genes from the late strong 649 cluster are SA-independent within 6 h of NHP treatment but SA-dependent 24 h post NHP 650 (Yildiz et al., 2021). Among this group are key SA and NHP biosynthetic and regulatory 651 genes including ALD1, UGT76B1, SARD1 and NPR3. Together, these data suggest NHP 652 is sufficient to drive gene expression over a short time frame but requires SA 653 accumulation for sustained expression, thus highlighting the importance of interrogation 654 into early NHP transcriptional changes. Further elucidation of a timeframe for early NHP 655 responses should prove useful in studying other aspects of NHP biology, such as the 656 biosynthesis and translocation of NHP and the identification of NHP sensors and their 657 respective downstream signaling pathways. 658 659 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 23 Primary NHP -responsive gene WRKY70 mediates secondary transcriptional 660 changes required for NHP-elicited SAR, signaling, and ROS production 661 We found WRKY70 expression is induced within 15 min of NHP treatment and is required 662 for full NHP -elicited SAR, as NHP did not enhance resistance in distal leaves of the 663 wrky70-1 mutant and could not achieve the same level of resistance observed in NHP -664 treated wild type plants (Fig. 5A). WRKY70 is known to repress many pathogen-inducible 665 genes in the absence of infection, but is also required for full activation of pathogen -666 inducible genes during infection (Zhou et al., 2018). We observed enhanced resistance 667 to Psm infection and enhanced transcript abundance of defense genes SARD1, PR1, 668 PR2, and PR5 after mock treatment (Supplement Fig. S7), consistent with a repressive 669 role for WRKY70. However, NHP treatment did not enhance resistance nor increase 670 transcript abundance of these genes in wrky70-1 (Fig. 5A, Supplement Fig. S7). 671 Furthermore, expression of the NHP-upregulated early strong gene BDA1 was completely 672 abolished in wrky70 mutant lines (Fig. 6B, Supplemental Fig. S8), demonstrating early 673 expression of WRKY70 is required to drive and modulate a subset of the secondary 674 transcriptional responses of NHP signaling. 675 676 Evidence supports a SA - and NPR1-independent defense pathway driven by WRKY70 677 (Shah et al., 2001; Zhang et al., 2010b). We found that NHP was sufficient to induce 678 expression of WRKY70 in the sid2-2 mutant within 30 min of treatment (Table 1), 679 suggesting SA biosynthesis is dispensable for the early transcriptional activation of 680 WRKY70. Likewise, WRKY70-regulated genes BDA1 and CML10 are induced within 3-6 681 h of NHP signaling partially independent of SA (Table1). Notably, 24 h after NHP 682 treatment, BDA1 and CML10 are also upregulated in both sid2-1 and npr1-3 mutants 683 (Yildiz et al., 2021). It is therefore possible that NHP signaling activates a SA- and NPR1-684 independent defense pathway through the action of WRKY70. 685 686 Studies into the MAMP -signaling pathway functioning through the receptor -like protein 687 SNC2 suggest a role for WRKY70 and BDA1 in PTI responses such as ROS production 688 (Yang et al., 2012). While flg22 -elicited ROS production was not significantly different 689 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 24 between mock treated wild type and wrky70 mutants, the enhanced production of flg22 -690 elicited ROS following NHP and SA pretreatment was compromised in wrky70 mutants 691 (Fig. 7, Supplemental Fig. S9). Our findings indicate WRKY70 positively contributes to 692 the NHP and SA signaling pathways that prime PTI. Further, our discovery that NHP 693 primes plants for enhanced production of flg22-elicited ROS is consistent with reports of 694 NHP priming Arabidopsis for enhanced metabolic and transcriptional responses following 695 flg22 treatment (Löwe et al., 2023). Our work links WRKY70 -dependent signaling 696 pathways to this NHP-primed PTI response. 697 698

699 Our work describes the early transcriptional response to NHP and highlights key genes 700 and signaling pathways that may contribute to the physiological processes regulated by 701 this important bioactive molecule. It also provides a foundation for better understanding 702 the transcriptional regulators, particularly the role of WRKY70, and gene networks that 703 define early NHP signal transduction. 704 705

706 Plant materials and growth conditions 707 Arabidopsis thaliana (Arabidopsis) lines used in this study were in the Col-0 ecotype (wild 708 type) with mutant lines as follows: sid2-2 (Wildermuth et al., 2001) and T-DNA insertion 709 lines wrky70-1 (SALK_025198) and wrky70-2 (GABI_324D11) (Li et al., 2006), wrky38 710 (WiscDsLox489-492C21), wrky54 (SALK_017254), and wrky59 (SALK_039436) (Wang 711 et al., 2006), wrky51 (SALK_022198) (Yan et al., 2018), and wrky62 (SM_3_38820) (Kim 712 et al., 2008). All mutant lines were obtained from the Arabidopsis Biological Resource 713 Center (ABRC). The presence of T -DNA insertions were confirmed with the genotyping 714 primers listed in Supplemental Table S1. Plants were grown on soil in a controlled growth 715 chamber at 80% humidity and 22°C on a 10 h light/14 h dark cycle for gene expression 716 and bacterial growth assays. For seedlings grown hydroponically, seeds were surface 717 sterilized and stratified at 4°C in darkness for 3 days. Seeds were placed into sterile six -718 well plates (15-18 seeds per well) with half-strength Murashige and Skoog (MS) medium, 719 0.5% (w/v) sucrose, 0.05% (w/v) MES at pH 5.7. Plates were covered with a lid, sealed 720 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 25 with micropore tape (3M), and placed in a chamber on a 10 h light/14 h dark cycle. Liquid 721 medium was agitated twice daily to facilitate gas exchange and after five days of growth, 722 spent medium was replaced with fresh MS. 723 724 Bacterial strains and growth conditions 725 P. syringae pv. maculicola strain ES4326 (Psm) and P. syringae pv. tomato DC3000 (Pst 726 DC3000) carrying pVSP61 + avrRpt2 (Pst avrRpt2) were used in this study. Growth 727 conditions for all bacterial strains are as previously described (Holmes et al., 2021). 728 729 Chemical stocks 730 Two sources of NHP were used in this study, one synthesized by Elizabeth Sattely’s lab 731 as previously described (Chen et al., 2018) and the second obtained from Med Chem 732 Express. Gene expression and NHP-elicited SAR assays showed similar results for both 733 sources of NHP. Pipecolic acid stocks were made from L -pipecolic acid (Oakwood 734 Chemical). Salicylic acid stocks were made from sodium salicylate (Mallinckrodt). 735 736 Chemical treatment of plants for qRT-PCR assays 737 For gene expression by qRT-PCR of 4.5-week-old, soil-grown Arabidopsis, three leaves 738 were infiltrated with sterile water or NHP dissolved in water at the indicated 739 concentrations. The three treated leaves were collected and flash frozen at the indicated 740 time points. For the time course of gene expression in wrky70-1 and wrky70-2 mutants, 741 three leaves were treated with sterile water or 1 mM NHP. One leaf from the same plant 742 was sampled at 6, 9, and 12 h and pooled with a second plant of the same condition for 743 one replicate. Samples collected at 0 h were untreated before collecting. 744 745 For gene expression in Arabidopsis seedling -based assays, plants were grown as 746 described for seedling hydroponics and treated at 10 -days-post-germination. Before 747 addition of treatment, all liquid medium was removed from plate wells and replaced with 748 fresh MS medium, seedlings were allowed to recover under lights for at least 1 h, followed 749 by application of 2x solutions of Pip, NHP, or mock in MS medium. The 15 -18 seedlings 750 within a single well were pooled and treated as a single replicate, with three replicates 751 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 26 sampled per condition. Following treatment, seedlings were blotted dry, and flash frozen 752 at the indicated time points. 753 754 RNA isolation and qRT-PCR 755 Total RNA was isolated from leaves and seedlings using TRIsure ™ reagent (Meridian 756 Bioscience) according to the manufacturer’s instructions. 5 µg of RNA was used to 757 synthesize cDNA by oligo dT (New England Biolabs) and reverse transcriptase (Thermo 758 Fisher), followed by dilution of cDNA. The qRT -PCR reactions were performed on a MJ 759 Opticon 2 (Bio-Rad) using Green Taq DNA polymerase (GenScript) with EvaGreen Dye 760 (Biotium) for amplification and detection. Three technical replicates were performed per 761 sample and three to four biological replicates were included for each condition. 762 Expression values were normalized to the reference gene UBC21 (AT5G25760) for 763 relative expression determined using 2 -ΔCt and normalized to both UBC21 and mock 764 treated samples for fold change using 2 -ΔΔCt. Genes assayed include FMO1 765 (AT1G19250), ICS1/SID2 (AT1G74710), UGT76B1 (AT3G11340), WRKY38 766 (AT5G22570), SARD1 (AT1G73805), PR1 (AT2G14610), PR2 (AT3G57260), PR5 767 (AT1G75040), BDA1 (AT5G54610), PROSCOOP4 (AT5G44568), and CML10 768 (AT2G41090). Primers used in these experiments can be found in Supplemental Table 769 S1. 770 771 RNA-seq treatment and library preparation 772 Seedlings used for mRNA isolation were grown hydroponically as described above. Six -773 well plates contained equal numbers of Col -0 and sid2-2 seedlings, with one genotype 774 per plate and 15 seedlings per well. At 10 -days-post-germination, all liquid medium was 775 removed from each well and fresh MS medium was added to the seedlings. Seedlings 776 were placed back into the growth chamber for an hour to acclimate, followed by treatment 777 with 2x NHP (final concentration 0.5 mM) or mock (MS medium). Three wells of pooled 778 seedlings were collected for each genotype, treatment, and time (15 min, 30 min, 3 h, 779 and 6 h) combinations, blotted dry, and flash frozen for mRNA isolation. 780 781 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 27 Total RNA was extracted from pooled seedlings using TRIsure ™ reagent (Meridian 782 Bioscience) according to the manufacturer’s instructions. Total RNA was then divided 783 with 5 µg used for cDNA synthesis and marker gene expression to assess quality of 784 treatment, as described for qRT -PCR experiments, and 5 µg was used for cDNA library 785 preparation. The cDNA library was prepared using NEBNext® Ultra™ II RNA Library Prep 786 Kit for Illumina® with NEBNext® Poly(A) mRNA Magnetic Isolation Module and then 787 multiplexed via PCR amplification using NEBNext® Multiplex Oligos for Illumina® 788 according to the manufacturer’s instructions (New England Biolabs). Library 789 concentration was quantified with Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific) 790 followed by measurement of quality and average cDNA length using the DNA 1000 Series 791 II chip on a 2100 Bioanalyzer (Agilent Technologies). The multiplexed cDNA libraries 792 were then pooled (two sets of 36 libraries) and sequenced using both lanes of the 793 NovaSeq 6000 (Illumina) S1 flow cell with run type 100 bp paired -end reads at the 794 Genome Sequencing Service Center by the Stanford Center for Genomics and 795 Personalized Medicine, supported by the grant award NIH S10OD025212. 796 797 RNA-seq data analysis 798 Read data was initially assessed using FastQC. CLC Genomics Workbench version 799 11.0.2 (QIAGEN) was used for trimming, discarding reads trimmed below 20 bp, and then 800 mapped to the Arabidopsis TAIR10 genome with the auto-detect paired distances option 801 deselected. Differential expression analysis was performed using DESeq2 (Love et al., 802 2014) with mock treated samples compared to NHP treated samples for each respective 803 time point and genotype. Genes were considered differentially expressed if they returned 804 an adjusted P-value below 0.05 (Padj < 0.05). The complete list of early NHP-responsive 805 transcripts is provided in Supplemental Table S3 and the raw sequencing data deposited 806 in the Gene Expression Omnibus (GEO) under accession code GSE263276. 807 808 Exploratory analysis of differentially expressed genes 809 To define patterns of early expression in response to NHP treatment, differentially 810 expressed genes were considered upregulated in wild type if they had a log 2(FC) > 1 at 811 any time point and downregulated with a log 2(FC) < -1 at any time point. The sets of up 812 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 28 and downregulated genes were then hierarchically clustered by log 2(FC) over the four 813 timepoints using the hclust function and dendextend packages in R, with Euclidean 814 distances and Ward.D2 linkage (Galili, 2015). For each defined cluster in wild type, the 815 log2(FC) values were averaged at each time point to visualize broad trends in expression. 816 817 The PANTHER classification system accessed via TAIR was used for gene ontology 818 enrichment analysis ( https://www.arabidopsis.org/tools/go_term_enrichment.jsp; (Mi et 819 al., 2021)). Version 17.0 of the PANTHER overrepresentation test was used (Fisher’s 820 exact test with FDR correction) and matched to the GO database 2021_03 release. 821 822 Promoter and cis-regulatory element enrichment analysis 823 Araport11 sequences 1000 bp upstream of the TSS of NHP -responsive genes were 824 downloaded from TAIR ( https://www.arabidopsis.org/tools/bulk/sequences/index.jsp) for 825 each time point and genotype grouping of the NHP upregulated and downregulated 826 genes. A less stringent cut -off of log 2(FC) > 0 (upregulated) or log 2(FC) < 0 827 (downregulated) with a Padj < 0.05 was used to select for NHP -responsive genes. Motif 828 identification and enrichment of cis-regulatory elements (CREs) was performed using 829 SEA, part of the MEME suite (Bailey et al., 2015; Bailey and Grant, 2021) and compared 830 to the published library of TF binding sites found within the DAPseq database (O’Malley 831 et al., 2016). Only CREs from the DAP-seq library were used in this study, omitting CREs 832 from the ampDAP-seq library (where DNA modifications have been removed). Following 833 the workflow detailed by Bjornson et al. (2021), relative enrichment was determined 834 against the same three sets of randomly sampled genes detected in this RNA -seq 835 experiment for all genotypes and time points tested. The identified motif was considered 836 enriched if it returned a significant P-value (< 0.05) relative to at least two of the three 837 randomly sampled gene sets. P-values for significantly enriched TF-binding motifs were 838 then averaged for visualization. 839 840 Measurement of bacterial growth in plants 841 Chemical treatment of leaves for NHP-elicited SAR assays were performed as described 842 in (Chen et al., 2018) with minor alterations. At 4.5-weeks-old, three lower leaves of wild 843 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 29 type and mutant Arabidopsis plants were infiltrated with sterile water, 0.5 mM NHP or 1 844 mM NHP. After 24 h, one untreated distal leaf of each plant was inoculated with a 1 x 105 845 CFU/mL suspension of Psm. The distal infected leaf was always separated by one leaf 846 from the original three treated (i.e., primary treatment in leaf no. 7 -9 then infected leaf is 847 no. 11). Inoculated plants were covered with a dome to increase humidity and 3 dpi the 848 titer of Psm in the distal leaves was quantified. Leaf discs were homogenized in 1 mL of 849 10 mM MgCl 2, plated in triplicate in a dilution series on nutrient yeast glycerol medium 850 supplemented with 1.5% wt/vol agar (NYGA) with rifampicin (100 μg/mL), incubated at 28 851 °C for 1.5 d, and bacterial colonies counted. For each individual experiment, 4 -12 plants 852 were tested per condition. 853 854 Pathogen priming of leaves for SAR assays were performed as previously described 855 (Chen et al., 2018). Three lower leaves of wild type and mutant Arabidopsis plants (4.5 -856 weeks-old) were infiltrated with 10 mM MgCl 2 or a 5 x 10 6 CFU/mL suspension of Pst 857 avrRpt2 in 10 mM MgCl2. After 48 h, one distal untreated leaf of each plant was inoculated 858 with a 1 x 10 5 CFU/mL suspension of Psm and plants were covered with a dome to 859 increase humidity. At 3 dpi, the titer of Psm in the upper leaves was quantified as 860 described above for NHP -elicited SAR. For each individual experiment, 8 plants were 861 tested per condition and experiments repeated twice with similar results. 862 863 For bacterial growth measurements in unprimed leaves of Arabidopsis wild type and 864 mutant plants, a suspension of 1 x 105 CFU/mL Psm was infiltrated into two young leaves 865 per plant. Plants were placed under a dome and 3 dpi the two infected leaves were pooled 866 and bacterial titer quantified for 12 plants per genotype. 867 868 Oxidative burst assay 869 Four leaf discs (4 mm diameter) from 5 -week-old Arabidopsis plants (n = 12) were 870 incubated on water, 500 µM NHP, or 100 µM SA in a 96-well plate (one leaf disc per well) 871 for 24 h. After pretreatment, ROS was measured by addition of flg22 (100 nM) in a solution 872 of 20 mg/mL horseradish peroxidase and 200 µM Luminol (Sigma) (Gómez‑Gómez et al., 873 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 30 1999), followed by immediate measurement of luminescence in a Synergy H1 Microplate 874 Reader (Biotek). Relative luminescence units (RLU) are reported. 875 876 Statistical analyses 877 Two-tailed Student’s t tests were performed to determine statistically significant 878 differences between the different conditions for measurements of bacterial growth using 879 the stat_compare_means function of the ggpubr package (Kassambara, 2023). P values 880 < 0.05 were considered statistically significant and all significance levels are indicated in 881 the figure legends (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Statistical 882 significance of qRT-PCR assays was determined using Mann-Whitney U test or one-way 883 ANOVA with post hoc Sidak test using the ggpubr and lsmeans R packages, respectively 884 (Lenth, 2016). 885 886

887 We thank the Mudgett laboratory for feedback on the research and manuscript. We also 888 thank Zhiyong Wang, Dominique Bergmann, Sharon Long, and Virginia Walbot for 889 instrument use and intellectual discussion. This work was supported by National Science 890 Foundation IOS -2026368 (to M.B.M. and E.S.S) and National Science Foundation 891 Graduate Research Fellowship DGE-1656518 (to J.F.). 892 893 Author contributions 894 J.F., J. -G.K., E.S.S. and M.B.M contributed to the study design; J.F. and J. -G.K. 895 performed research; J.F., J. -G.K., E.S.S. and M.B.M analyzed data; J.F., and M.B.M. 896 wrote the manuscript; J.F., J. -G.K., E.S.S. and M.B.M. reviewed and revised the 897 manuscript for accuracy and final approval. 898 899

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It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 37 Zhou M, Lu Y, Bethke G, Harrison BT, Hatsugai N, Katagiri F, Glazebrook J (2018) 1107 WRKY70 prevents axenic activation of plant immunity by direct repression of SARD1. 1108 New Phytol 217: 700–712 1109 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 38 Tables 1110 Table 1. SA-independent/partially independent NHP upregulated genes by cluster. Log2(FC) Col-0 sid2-2 AGI Name Annotation 15m 30m 3h 6h 15m 30m 3h 6h Early transient AT3G44870 FAMT-L FARNESOIC ACID METHYL TRANSFERASE-LIKE 4.3 4.4 - - 2.3 2.7 - - AT2G44840 ERF13 Ethylene-responsive transcription factor 3.3 2.1 - - 1.4 - - - AT1G76640 CML39 CALMODULIN LIKE 39 2.7 2.7 - - 1.7 - - - AT5G13220 JAZ10 JASMONATE-ZIM-DOMAIN PROTEIN 10 1.6 1.1 - 0.6 1.2 1.0 - - AT1G80840 WRKY40 WRKY Transcription Factor; Group II-a 1.8 1.1 - 0.5 - 1.4 1.4 0.7 AT3G17690 CNGC19 Cyclic nucleotide-gated ion channel 1.6 1.6 - - - 1.2 - - AT4G21840 MSRB8 Methionine sulfoxide reductase B8 3.1 - - 1.5 - 2.2 - - Early strong AT5G22570 WRKY38 WRKY Transcription Factor; Group III 5.8 5.2 4.7 3.6 3.9 6.2 5.8 2.9 AT5G01900 WRKY62 WRKY Transcription Factor; Group III 3.9 5.0 4.4 2.6 - 5.8 4.1 4.4 AT1G28480 GRXC9 GRX480/ROXY19, glutaredoxin family 3.2 2.2 2.7 1.8 3.2 3.1 2.3 2.1 AT2G40750 WRKY54 WRKY Transcription Factor; Group III 2.7 3.7 2.8 2.9 3.0 3.6 3.5 3.0 AT5G64810 WRKY51 WRKY Transcription Factor; Group II-c 2.6 3.4 3.3 3.0 1.7 3.2 3.2 3.5 AT3G56400 WRKY70 WRKY Transcription Factor; Group III 1.6 1.6 2.4 2.1 - 1.3 2.3 1.8 AT3G25882 NIMIN-2 NIM1-INTERACTING 2 1.4 1.5 1.8 1.6 1.2 1.1 1.0 1.6 Late weak AT3G60470 - Transmembrane protein, putative (DUF247) - - - 3.3 - - - 5.1 AT2G13810 ALD1 AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 - - - 2.3 - - - 6.6 AT1G01680 PUB54 Plant U-box type E3 ubiquitin ligase - - - 2.2 - - - 1.4 AT3G12220 SCPL16 Serine carboxypeptidase-like - - 1.1 2.0 - - - 2.0 AT1G78340 GSTU22 Glutathione S-transferase U22 - - 2.0 2.0 - - 1.6 1.7 AT5G03350 LLP Lectin-like protein - - 2.1 1.8 - - 2.1 1.9 AT5G24530 DMR6 DOWNY MILDEW RESISTANCE 6 0.7 1.2 1.6 1.7 0.5 1.6 2.0 1.7 AT2G41090 CML10 Calmodulin-like protein - - 1.1 1.7 - - 0.3 1.2 AT4G14400 ACD6 ACCELERATED CELL DEATH 6 - - 2.1 1.6 - - 1.4 1.4 AT2G15490 UGT73B4 UDP-DEPENDENT GLYCOSYLTRANSFERASE 73B4 - 0.7 1.2 1.5 - 0.9 2.7 1.5 AT4G08040 ACS11 1-aminocyclopropane-1-carboxylate synthase 11 - 1.0 1.5 1.5 - - 1.0 1.2 AT1G21250 WAK1 CELL WALL-ASSOCIATED KINASE 1 0.7 - 1.8 1.5 - - 1.7 2.0 AT1G17170 GSTU24 Glutathione S-transferase U24 - - - 1.4 - - 1.6 1.5 AT3G11340 UGT76B1 UDP-DEPENDENT GLYCOSYLTRANSFERASE 76B1 - 1.0 0.9 1.1 - 0.7 1.1 1.0 AT1G73805 SARD1 SAR DEFICIENT 1 1.0 0.7 1.5 1.0 - - 1.3 1.3 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 39 Late strong AT3G12230 SCPL14 Serine carboxypeptidase-like - - 5.9 6.9 - - 5.4 4.6 AT1G15610 - Transmembrane protein - - 5.5 5.9 - - - 5.1 AT3G28510 - AAA-type ATPase family protein - - 3.2 5.6 - - - 4.9 AT2G26400 ARD ACIREDUCTONE DIOXYGENASE 3 - 2.2 4.4 5.0 - - 5.1 5.8 AT4G19750 - Glycosyl hydrolase family with chitinase insertion domain - - 3.9 4.8 - - 6.7 5.3 AT1G19960 - Putative uncharacterized protein 1.2 1.6 4.0 4.0 - 2.9 5.5 4.4 AT4G10500 DLO1 DMR6-LIKE OXYGENASE 1 - 1.4 3.1 3.8 - - 3.4 4.2 AT5G41280 CRRSP57 Receptor-like protein kinase-related family protein - - 2.6 3.2 - - 3.2 2.2 AT2G14560 LURP1 late upregulated in response to Hyaloperonospora parasitica - - 3.0 3.1 - - - 4.5 AT1G02230 NAC004 NAC DOMAIN CONTAINING PROTEIN 4 - 2.8 3.8 3.0 - 2.5 3.5 4.3 AT5G54610 BDA1 Ankyrin repeat protein family - - 2.2 3.0 - - 3.0 6.4 Selection of genes upregulated by NHP in Col -0 and sid2-2 seedlings. Clusters defined for wild type expression are noted (i.e., early transient, early strong, late weak, late strong) . Indicated log2 fold change (FC) values determined relative to mock treatment for each genotype and time point. Genes are categorized as SA-independent/partially independent if significant upregulation was observed in sid2-2 within 15-30 min for the early clusters and within 3-6 h for the late clusters. Not all NHP upregulated genes are shown in this table, the complete list can be found in Supplemental Table S3. 1111 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 40 Table 2. SA-dependent NHP upregulated genes by cluster. Log2(FC) Col-0 sid2-2 AGI Name Annotation 15m 30m 3h 6h 15m 30m 3h 6h Early transient AT1G32970 SBT3.2 Subtilisin-like protease 2.8 3.2 - - - - - - AT1G21240 WAK3 Wall-associated receptor kinase 2.5 - - 1.4 - - - 2.0 AT5G44430 PDF1.2C PLANT DEFENSIN 1.2C 2.4 - - - - - - - AT2G14610 PR1 PATHOGENESIS-RELATED GENE 1 2.3 - - - - - - - AT4G34410 ERF109 Ethylene-responsive transcription factor 2.1 - - - - - - - AT2G32140 - Transmembrane receptor 2.1 - - - - - 2.0 - AT5G44420 PDF1.2A PLANT DEFENSIN 1.2 2.0 - - - - - - - AT3G14260 - LURP-one-like protein (DUF567) 1.8 - - - - - - - AT2G26020 PDF1.2B PLANT DEFENSIN 1.2B 1.6 - - - - - - - AT5G64905 PROPEP3 ELICITOR PEPTIDE 3 PRECURSOR 1.5 - - - - - - - AT1G32640 MYC2 JASMONATE INSENSITIVE 1 1.4 - - - - - - - AT2G18660 EGC2 PLANT NATRIURETIC PEPTIDE A 1.4 - - - - - - - Early strong AT2G21900 WRKY59 WRKY Transcription Factor; Group II-c 1.9 2.4 2.3 2.3 - - 2.2 2.7 Late weak AT2G28850 CYP710A3 Cytochrome P450 710A3 - - 2.4 2.1 - - - - AT1G15630 - Transmembrane protein - - 1.9 1.4 - - - - AT5G44568 PROSCOOP4 SERINE RICH ENDOGENOUS PEPTIDE - - 1.3 1.3 - - 0.7 1.0 AT1G18830 SEC31A Protein transport protein SEC31 homolog A - - - 2.8 - - - - AT2G45550 CYP76C4 Cytochrome P450 76C4 - - - 2.6 - - - - AT1G10155 ATPP2-A10 PHLOEM PROTEIN 2-A10 - - - 2.3 - - - - AT1G02450 NIMIN-1 NIM1-INTERACTING 1 - - - 2.3 - - - - AT3G45860 CRK4 Cysteine-rich receptor-like protein kinase - - - 2.1 - - - - AT5G51500 PME60 Pectin methylesterase inhibitor - - - 1.7 - - - - AT2G29350 SAG13 SENESCENCE-ASSOCIATED GENE 13 - - - 1.3 - - - - Late strong AT3G22231 PCC1 PATHOGEN AND CIRCADIAN CONTROLLED 1 - - 3.8 3.9 - - - - Selection of genes upregulated by NHP in Col-0 and sid2-2 seedlings. Clusters defined for wild type (Col- 0) expression are noted. Indicated log2 fold change (FC) values were determined relative to mock treatment for each genotype and time point. Genes are categorized as SA -dependent if significant upregulation was not observed in sid2-2 within 15-30 min for the early clusters and within 3 -6 h for the late clusters. Not all NHP upregulated genes are shown in this table, the complete list can be found in Supplemental Table S3. 1112 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 41 1113 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 42 Figure Legends 1114 1115 Figure 1 NHP induces WRKY38 expression at early time points. 1116 A, Transcript abundance of SAR marker genes FMO1, ICS1, and PR1 and early SA -1117 responsive marker genes WRKY38 and UGT76B1 in 4.5 -week-old wild type (Col -0) 1118 Arabidopsis plants. Three leaves were infiltrated with sterile water (mock) or 1 mM NHP 1119 and collected after 2, 6, and 24 h for mRNA isolation. Transcript abundance was 1120 measured via quantitative real -time PCR (qRT-PCR) and normalized relative to UBC21 1121 (Ubiquitin-Conjugating Enzyme 21; At5g25760). Fold change was determined relative to 1122 mock (2-∆∆Ct). Asterisks indicate a significant difference between mock and NHP per time 1123 point (n = 4; Mann-Whitney U test; *P < 0.05, ns = not significant). B, Design of seedling-1124 based assay. Arabidopsis seedlings (15-18 per well) were hydroponically grown, treated 1125 with MS medium (mock), 1 mM Pip, or 1 mM NHP in MS medium then collected at 15 1126 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 43 min, 30 min, 3 h, and 6 h for qRT-PCR analysis of WRKY38 transcript abundance in wild 1127 type. C, Expression of WRKY38 in seedlings from the experiment described in B. 1128 Transcript abundance was normalized relative to UBC21 and mock treated samples for 1129 each condition (2 -∆∆Ct). Asterisks indicate a significant difference between mock and Pip 1130 or NHP at each time point (n = 4-6; Mann-Whitney U test; **P < 0.01, ns = not significant). 1131 1132 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 44 1133 Figure 2 Profile of early NHP-upregulated genes in wild type seedlings. 1134 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 45 A, Heatmap of genes upregulated (log2(FC) > 1, Padj < 0.05) in response to NHP treatment 1135 in wild type seedlings. One biological replicate consists of 15 pooled seedlings per 1136 condition (n = 3). Euclidean distances determined from the log 2(FC) values were 1137 hierarchically clustered across the indicated time points (15 min, 30 min, 3 h and 6 h), 1138 resulting in four distinct clusters of gene expression. B, Average log 2(FC) of all genes 1139 within each cluster defined in (A) showing the expression trends across the four time 1140 points. Note, the two genes expressed only at 30 min, and no other time, were removed 1141 from the 'early strong' cluster average. Error bars represent the ± SEM. C, Biological 1142 processes of significantly enriched (FDR < 0.05) Gene Ontology (GO) terms for the genes 1143 within each cluster are shown. Bars represent the -log(FDR) of each significantly enriched 1144 GO term. Bars were omitted if the term was not significantly enriched for the defined 1145 cluster. The most significantly enriched GO term for each cluster is indicated in bold. 1146 1147 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 46 1148 Figure 3 Comparison of NHP upregulated genes in wild type and sid2-2 seedlings. 1149 A, Percent of NHP upregulated genes expressed only in wild type (Col-0, SA-dependent) 1150 and in both wild type and the sid2-2 mutant (SA -independent) for the wild type gene 1151 expression clusters defined in Figure 2. Total number of genes in each condition is 1152 indicated. B, Total number of NHP upregulated genes (log2(FC) > 1, Padj < 0.05) per time 1153 point unique to wild type (Col -0), shared between wild type and sid2-2, and unique to 1154 sid2-2. The number of upregulated genes in each grouping is indicated. C, Biological 1155 processes of significantly enriched (gray bar) or depleted (white bar) (FDR < 0.05) GO 1156 terms for the set of genes upregulated 3 and 6 h after NHP treatment that were 1157 upregulated only in the sid2-2 mutant and not wild type. Bars represent the negative 1158 log(FDR) of each significantly enriched GO term, bars were omitted if the term was not 1159 significantly enriched or depleted for the defined time point. 1160 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 47 1161 Figure 4 Presence of TGA/bZIP and WRKY TF cis-regulatory elements (CREs) in the 1162 promoters of NHP-upregulated genes in wild type (Col-0) and sid2-2 seedlings. 1163 A, Schematic of the TF binding motif (i.e., CRE) enrichment analysis performed. All genes 1164 with increased transcript abundance upon NHP treatment (log 2(FC) > 0 and Padj < 0.05) 1165 were grouped by time point for the indicated genotype, these groups are represented as 1166 “test promoter group” in the diagram. CREs of known Arabidopsis TFs from the DAPseq 1167 database (O’Malley et al., 2016) were identified in the promoters (1 kb upstream of the 1168 transcriptional start site) of each group of promoters. Enrichment of identified CREs was 1169 determined relative to CREs found in promoters pulled from a random sampling of genes 1170 detected in this RNA -seq experiment. Schematic design is modified from Mariani et al., 1171 2017. Enrichment analysis of TGA/bZIP (B) and WRKY (C) CREs. The name of the TF 1172 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 48 known to bind the enriched CRE is listed on the right and the TF family name is indicated 1173 above (i.e., bZIP or WRKY). The -log2(P value) of the enrichment analysis is indicated by 1174 the scale bar with black indicating no significant enrichment of the CRE and purple to 1175 yellow denoting an enriched CRE. 1176 1177 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 49 1178 Figure 5 WRKY70 is involved in NHP-elicited SAR 1179 Bacterial growth in the distal leaves of mock and NHP treated wild type (Col-0) and wrky 1180 mutant plants. WRKY genes for mutant analysis were selected from the early stable 1181 cluster defined in Figure 2 and include WRKY70 (A), WRKY38 and WRKY62 (B), 1182 WRKY54 (C), WRKY51 (D), and WRKY59 (E). Three lower (1°) leaves were infiltrated 1183 with water (mock), 0.5 mM NHP, or 1 mM NHP (only for wrky59) and 1 d later one upper, 1184 distal leaf was inoculated with a 1 x 10 5 CFU/mL suspension of Psm, followed by 1185 quantification of bacterial titer 3 dpi (n = 4-8). Asterisks indicate significant differences in 1186 bacterial titer (two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = 1187 not significant). 1188 1189 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 50 1190 Figure 6 Loss of WRKY70 function impacts upregulation of early NHP-responsive genes 1191 A, Diagram of putative WRKY70 CREs, including W -box, WT-box, and WRKY70 (W70) 1192 DAPseq motifs in the promoters (-2 Kb upstream of the transcriptional start site; TSS) of 1193 NHP-responsive genes BDA1, PROSCOOP4, and CML10. Note W70 DAPseq sites 1194 adjacent to a W - or WT-box overlap. Expression of BDA1 (B), PROSCOOP4 (C), and 1195 CML10 (D) in wrky70-1 mutant plants. Three leaves of 4.5-week-old wild type (Col-0) and 1196 wrky70-1 mutant plants were infiltrated with water or 1 mM NHP. Samples collected at 0 1197 h were untreated before collection. Transcript abundance was determined relative to 1198 UBC21 for each condition (2-∆Ct). Asterisks indicate a significant difference between wild 1199 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 51 type and wrky70-1 at each time point (two -tailed t-test; *P < 0.05, ** P < 0.01, ns = not 1200 significant). 1201 1202 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint 52 1203 Figure 7 NHP enhances ROS production and requires WRKY70 for full ROS levels. 1204 Flg22-elicited ROS quantification in wrky70-1 plants. Four leaf discs from 4.5 to 5-week-1205 old wild type and wrky70-1 plants were pretreated by floating leaf discs on water, 0.1 mM 1206 SA, or 0.5 mM NHP for 24 h before treating with 100 nM flg22 in horseradish peroxidase 1207 and Luminol. Top, traces of the average relative luminescence units (RLU) over the 1208 indicated run time. Each point represents the average of 6 plants (n = 24) from two 1209 experiments. Bottom, total quantification of RLUs from top plot. For each leaf disc, RLUs 1210 from each time point were summed by condition over the run time and averaged with 1211 error bars representing the standard error of the mean. Statistical analysis was performed 1212 using a one -way ANOVA and post hoc Sidak test, different letters indicated statistical 1213 differences between means with P < 0.05. 1214 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 28, 2024. ; https://doi.org/10.1101/2024.04.23.590810doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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