Light induces Phytochrome B SUMOylation to recruit the immune regulator NPR1 in nuclear condensates to control immunity in plants

35 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint Light, systemic acquired resistance, phytochrome, salicylic acid, plants, bacterial pathogens, chromatin, 36 nuclear condensates 37 38 39

40 The sessile nature of plants requires continuous monitoring and response to external 41 cues to optimize growth, development, and defence. Light is the paramount external 42 cue for plants, determining whether energy is devoted to growth and development, or 43 diverted to immunity which also represents a point of vulnerability for pathogens to 44 exploit. Photoreceptors form the core of light perception and there is emerging genetic 45 evidence that they play key roles in regulating plant immunity 1,2. Phytochrome B (PhyB) 46 perceives red light and is a necessary regulator of plant growth and development 3. A 47 striking observation made by several groups is the positive impact of PhyB mediated 48 molecular signalling on immunity against multiple pests and pathogens 4-9. 49 While defence at infected tissue is critical for limiting pathogen growth, plants also 50 possess the ability to prime and amplify immune responses at distal sites. This global 51 response is termed systemic acquired resistance (SAR) 10,11. SAR is critical for 52 preventing the spread of pathogens and protecting against subsequent infections 11. 53 The phytohormone salicylic acid (SA) activates NPR1 (Nonexpressor of Pathogenesis-54 Related 1 (PR1) genes), a master transcriptional regulator that underpins a cascade of 55 defence pathways. Disrupting NPR1 function prevents the activation of SA-mediated 56 defence pathways, renders the plant susceptible to pathogen attack and unable to 57 mount SAR 12,13. It has been reported that the activation of plant defence follows a 58 light/dark pattern of regulation where induction of SAR is highest when host plants are 59 inoculated at day time than at night 14. However, the molecular mechanism linking light 60 perception by photoreceptors to activation of systemic acquired resistance remains 61 elusive. 62 63

AND DISCUSSION 64 SUMOylation of PhyB is critical for SAR against bacterial pathogens 65 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint This study aimed to uncover the molecular mechanism that allows light perception by 66 photoreceptors to mediate immunity in plants. In previous work, we demonstrated that 67 light-activated P FR form of PhyB is specifically modified by the Small Ubiquitin-like 68 Modifier 1 (SUMO1) to underpin a key regulatory step in photomorphogenesis 15. 69 Furthermore, Arabidopsis PhyB mutant phyB-9 plants are also susceptible to pest and 70 pathogens including the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 71 (Pst) 1. To ascertain whether SUMO1 modification affected PhyB’s role in mediating 72 immunity we generated phyB-9 plants complemented with own promoter driven wildtype 73 PhyB (WT) and non-SUMOylated PhyB K996R fused to the Yellow Fluorescent Protein 74 (YFP). Arabidopsis Col-0 plants were susceptible to virulent Pst and our infection 75 assays indicated no significant difference in susceptibility in leaves expressing non-76 SUMOylatable PhyB K996R when compared to wildtype Col-0 plants or plants 77 complemented with WT PhyB (Fig. S1A). However, effector triggered immunity (ETI) 78 against avirulent Pst ( Pst (avrB)) was highly compromised in non-SUMOylatable 79 PhyBK996R lines. Plants expressing PhyBK996R showed more than fifteen times the levels 80 of avirulent bacterial growth in leaf tissues when compared to wildtype PhyB 81 complemented lines (Fig. S1B). Control plants inoculated in the dark were susceptible 82 to Pst (avrB) across the genotypes (Fig. S1C). Our data demonstrates that 83 SUMOylation of PhyB is critical for effector triggered immunity in plants. 84 A hallmark of ETI is the activation of SAR in uninfected adjacent (from now called 85 systemic) tissue, hence we wanted to ascertain whether PhyB K996R lines were also 86 compromised for immunity in systemic uninfected tissues. To trigger SAR in systemic 87 leaf tissue transgenic plants were infiltrated with the avirulent strain of Pst (Pst (avrB)) 88 after three days post infection (dpi) the systemic uninfected leaves were infected with 89 virulent Pst. Three days post-secondary infection the virulent Pst bacterial population 90 was measured along with defence responses such as callose deposition 16. To 91 ascertain that SAR is induced in systemic tissue, we compared defence against virulent 92 bacteria ( Pst) in systemic leaf tissue of plants originally infected with either avirulent 93 bacteria Pst avrB (to induce SAR) or virulent Pst (no induction of SAR). Indeed, 94 PhyBK996R plant lines show severely compromised defence against virulent Pst in 95 systemic leaf tissue of plants which were previously infected with avirulent Pst (avrB) 96 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint indicating that SAR was compromised in PhyB K996R lines (Fig. S2A and B). This 97 intriguing discovery led us to postulate that SUMOylation of PhyB could be a major 98 regulator of SAR. Light is known to regulate immunity where induction of SAR is higher 99 when host plants are inoculated at day-time 14. Once activated, SAR in leaf tissues is 100 maintained in the dark through a mechanism that is currently not well understood 17. 101 However, light is a prerequisite for PhyB SUMOylation and dark conditions suppress 102 PhyB SUMOylation 15. This observation led us to test whether phyB SUMOylation is 103 required for plants to maintain SAR in the dark 104 We found that systemic immune response to secondary virulent Pst infection in the light 105 or dark was compromised in plants expressing non-SUMOylated PhyBK996R compared to 106 controls. phyB-9 and non-SUMO mutant (PhyB K996R) plants are far more susceptible 107 regardless whether in the light or dark compared to WT controls. Remarkably the levels 108 of susceptibility in non-SUMOylated PhyB K996R plants was comparable to phyB-9 109 mutants where there is no PhyB protein 18, while as expected the wildtype (WT) PhyB 110 lines developed considerable resistance against secondary virulent Pst infection in light 111 and dark conditions in systemic tissues indicating efficient activation of SAR (Fig. 1A 112 and B). The activation of a robust defence response in PhyB WT plants was further 113 supported by higher callose deposition in infected tissue but this response was greatly 114 reduced in PhyBK996R plants (Fig. 1C and D). Our data demonstrates that the regulation 115 of PhyB mediated immunity including SAR activation in the light and maintenance in the 116 dark is dependent on SUMO modification of the photoreceptor. 117 SA production upon pathogen infection is dependent on PhyB SUMOylation 118 SA is the predominant phytohormone that accumulates and orchestrates the elaboration 119 of the defence response in plants against biotrophic pathogens. SA along with its 120 derivative, pipecolic acid (PA) act as systemic signals that accumulate in SAR activated 121 systemic tissues following local infection with avirulent pathogens to establish SAR 19,20. 122 Our phytohormone quantification analysis indicated that SA accumulation upon avirulent 123 pathogen infection in SAR tissues was significantly suppressed in the non 124 SUMOylatable PhyB K996R lines in both light and dark conditions (Fig. 1E). Whilst, as 125 expected SA levels increased significantly upon SAR activation in wild type PhyB 126 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint complemented lines, especially in dark (Fig. 1E). It is well established that the genes 127 encoding rate-limiting biosynthetic enzymes of SA ( ICS1) and PA (ALD1 ) are 128 upregulated during SAR. We further demonstrated that in PhyB K996R lines this 129 upregulation was abolished in indicating that SUMOylation of PhyB facilitates SA 130 accumulation by the upregulating the expression of genes encoding the rate-limiting 131 biosynthetic enzymes of SA (ICS1) and PA (ALD1) in SAR tissues in the light and dark. 132 (Fig. 1F). Our analysis reveals a novel link between SUMO modification of the PhyB 133 photoreceptor and regulation of SA production to underpin immunity. Although, it has 134 been known that SA levels spike, as a pre-emptive defence measure in SAR tissues at 135 night 17,21, the regulation of this process by PhyB photoreceptor has not been revealed 136 till now. 137 A common feature of PhyB is the ability to undergo nuclear body formation from 138 diffused to dense condensates in plant nuclei to fulfil their molecular role in the 139 biological processes it govern 22. We wanted to ascertain if SUMOylation facilitates the 140 formation of nuclear bodies that contain PhyB. First, we examined whether PhyB forms 141 nuclear condensates in immune primed systemic leaf tissues and whether SUMOylation 142 affects this process. We infected one half of 4-week-old plant leaf either expressing 143 PhyB-YFP or non-SUMOylatable PhyBK996R – YFP with Pst (avrB) and 5 hpi (hours post 144 inoculation) observed the occurrence of PhyB nuclear bodies in the light and dark 145 conditons. Indeed, we observed that wildtype PhyB complemented lines formed larger 146 nuclear bodies in the systemic tissues when compared to uninfected samples and the 147 non-SUMOylatable PhyBK996R (Fig. 1G). Non-SUMO PhyB K996R nuclear bodies were at 148 least 30% smaller compared to those of wildtype PhyB (Fig. 1H). This is most evident in 149 SAR tissue in dark conditions but not in non-SAR control tissues. Our data reveals that 150 SAR promotes nuclear body formation of photoreceptor PhyB in SAR tissues with 151 SUMOylation being a key facilitator of this process. 152 Previously we demonstrated that SUMOylation of PhyB is triggered by red light 15. 153 Intriguingly PhyB SUMOylation is almost undetectable in the dark yet SUMOylation of 154 PhyB is a prerequisite for SA accumulation in the dark in systemic tissues during SAR 155 (Fig. 1E). It has been reported that light pre-treatment induces SA accumulation in SAR 156 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint tissues 17,24. Therefore, we hypothesised that higher levels of SA that accumulates 157 during SAR in the light might be associated with promoting SUMOylation of PhyB in the 158 dark in effect, replacing light as a stimulus for promoting PhyB SUMOylation. Indeed, 159 through immunoblot analysis we observed that treatment of leaf tissue with exogenous 160 SA was sufficient to promote SUMOylation of PhyB in light (Fig. S3A) and further 161 enhanced SUMOylation of PhyB in the absence of light conditions (Fig. S3B). As 162 expected, the effect of SA promoting PhyB SUMOylation was also observed in SAR 163 activated leaf tissue in light and dark conditions (Fig. 2A and 2B). This observation was 164 more striking in SAR-activated systemic leaf tissues in the dark. To provide further 165 support that the observed phenotypes is through the posttranslational effect of PhyB, 166 we expressed PhyB/PhyB K996R under a constitutive promoter, Lip1 ( 17). As expected, 167 we observed increased resistance to virulent Pst infection in SAR tissues along with 168 accumulation of callose only in plants expressing WT PhyB and not the non-169 SUMOylatable PhyB K996R lines (Fig. S4). Alpha fold-based modelling showed that 170 SUMO1 binds to the C terminal of the HKRD (Histidine Kinase Related Domain) domain 171 of PhyB dimer (Fig. 2C). 172 It is well established that SUMO E2 alone is able to conjugate SUMO to target 173 substrates 25. To ascertain how PhyB is SUMOylated in the light we demonstrate using 174 coimmunoprecipitation assays that light promotes the interaction of the SUMO 175 conjugating enzyme SCE1 with PhyB thereby providing a mechanism for PhyB 176 SUMOylation in the light (Fig. 2D). However the mechanism for maintaining PhyB 177 SUMOylation in the dark is unclear. We previously demonstrated that PhyB is 178 deSUMOylated by the SUMO protease OTS1 15. OTS1 protein is degraded by SA 26. 179 We wanted to ascertain whether the stability of OTS1 SUMO Protease was affected in 180 SA enriched SAR activated leaf tissue in dark conditions and this allows for the 181 accumulation of SUMOylated PhyB in the absence of light. We generated our own 182 promoter OTS1-VENUS tagged transgenics and subjected them to avirulent Pst (avrB) 183 infection to trigger SAR. Immunoblot analysis with anti-VENUS antibody indicated that 184 in dark conditions OTS1-VENUS protein levels were undetectable when compared to 185 tissues that are not activated for SAR (Fig. 1E) and this degradation was proteasome 186 dependant. The levels of OTS1-VENUS protein levels in the light and dark were lower in 187 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint SAR tissue in comparison to non-infected tissues (Fig. S5). Further we determined that 188 OTS1 interacts with PhyB and this interaction is reduced in the presence of SA (Fig. 1F) 189 whilst there is enhanced SUMOylation of PhyB expressed in the ots1ots2 double mutant 190

which reinforces the importance of OTS SUMO proteases in regulating the 191 SUMOylation status of PhyB (Fig. 1G). ots1ots2 double mutants also show constitutive 192 SAR under both light and dark conditions which is genetically dependant on PhyB 193 further substantiating the role of OTS SUMO Proteases in regulating PhyB SUMO 194 mediated effects on immunity (Fig. 1 A and B). We demonstrate that PhyB SUMOylation 195 triggered by light ( via enhanced interaction with SCE1) mediates ETI triggered 196 biosynthetic accumulation of SA which in turn stimulates the degradation of OTS1 197 SUMO protease to increase the pool of SUMOylated PhyB in the dark in order to 198 maintain high SA levels and promote SAR in systemic tissues. We unravel a link 199 between light-SA hormone feedback loop mechanism through PhyB SUMOylation that 200 establishes and maintains SAR in systemic leaf tissues in the dark. 201 SUMO1-modified PhyB interacts with NPR1 to activate immunity 202 The primary target of SA is NPR1 (Nonexpressor of Pathogenesis-Related (PR) gene1), 203 a master transcriptional regulator of plant immunity 27. NPR1 plays an essential role in 204 activating SAR, which imparts a broad-spectrum immune response activated throughout 205 the plant upon pathogen attack. 206 SUMO modification of target proteins can confer interaction with partner proteins that 207 contain a SUMO interacting motif (SIM) 28. Based upon bioinformatic analysis, we 208 identified a potential SIM site specific for SUMO1 in NPR1 between amino acids 610-209 616 (Fig. S6). This SIM site was different to the SUMO3 interacting site previously 210 identified in NPR1 29. We hypothesised that this SIM site (from now on referred to as 211 SIM1) might confer interaction with SUMO1 modified PhyB. We mutated the Tyrosine 212 (Y611) and Methionine (M612) amino acid residues (positions 611 and 612 213 respectively) to Alanines to ascertain its function and renamed the mutant version 214 NPR1SIM1. In order to verify the interaction between SUMO1 and the new SIM1 site of 215 NPR1, we transiently co-expressed, HA epitope-tagged SUMO1 and C terminal- GFP 216 epitope-tagged NPR1 in Nicotiana benthamiana . Pull-down assays with NPR1-GFP 217 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint revealed a strong interaction between NPR1-GFP and HA-SUMO1 that is enhanced by 218 SA treatment but not with the SIM mutant NPR1 SIM1 (Fig. S7). Similarly in stable 219 transgenic lines NPR1 was found to bind to SUMO1 rather NPR1 SIM1 lines (Fig. 3A). At 220 elevated SA levels, NPR1 translocates into the nucleus where it undergoes nuclear 221 body formation to convert from a dilute to a condensed form 30. We found that m-Scarlet 222 labelled NPR1 is indeed localized in the nucleus in the SA treated tissue (Fig. 3B and 223 C). However, the nuclear body formation was strongly impaired in NPR1 SIM1 expressing 224 plants (Fig. 3B and C). Next, we wanted to ascertain whether SUMO1 affected the rate 225 of condensate formation for NPR1. Fluorescence Recovery After Photobleaching 226 (FRAP) analysis is commonly used to investigate interactions in binding complexes as 227 well as study the dynamics of phase separation in living tissues 31,32. To study the 228 consistency of the formation and dispersion of the NPR1 nuclear bodies, FRAP analysis 229 was performed after photobleaching a 1mm 2 region of nuclear body in SAR-activated 230 tissue of wildtype mScarlet-NPR1 as well as the NPR1 SIM1 mutant, the rate of recovery 231 of the fluorescence was studied over 30 seconds. The wildtype mScarlet-NPR1 nuclear 232 bodies demonstrated rapid reassociation post-photobleaching of the nuclear 233 condensates. However, the NPR1 SIM1 condensates showed a radical decrease in the 234 level of fluorescence, suggesting weak impaired nuclear body formation (Fig. 3D). Our 235 data reveals that the SIM1 motif is critical for NPR1 to assemble into nuclear 236 condensates. 237 We hypothesized that SUMO1-modified PhyB might interact with NPR1 through the 238 newly identified SIM1 motif in NPR1. Using immunoprecipitation assays in N. 239 benthamiana transient assays and in transgenics we demonstrated that SUMO1-240 modified PhyB interacted with NPR1 but not with NPR1 SIM1. Intriguingly this interaction 241 was more robust in the presence of SA (Fig. 3E and S8). As expected, the non-242 SUMOylatable PhyBK996R showed significantly reduced or weak interaction with NPR1. 243 Conversely, Co-IP assays showed a lack of interaction between wildtype PhyB with 244 NPR1SIM1 lines even in the presence of SA (Fig. 3E and Fig. S8). Next, we ascertained 245 that this SUMO1 dependant interaction between PhyB and NPR1 occurs in SAR 246 activated systemic leaf tissue of transgenic lines expressing PhyB-YFP and NPR1-247 mScarlet in light and dark conditions (Fig. 3F, G and Fig. S9). These observations 248 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint indicate that the SUMO1 modification of PhyB is critical for the photoreceptor to interact 249 with the SIM1 motif in NPR1 in the presence of SA and this underpins a molecular 250 conduit for light to activate immunity in plants. 251 We wanted to ascertain the impact of the SIM1 motif in NPR1 on its function during 252 immune activation. Arabidopsis npr1-1 mutants that are susceptible to pathogens were 253 complemented with NPR1 (WT) and NPR1 SIM1 to study the systemic immune response 254 to virulent Pst infection under light and dark regimes after activation of SAR with the 255 avirulent strain of Pst (avrB). We found that npr1-1 mutants complemented with wildtype 256 NPR1 developed robust resistance in systemic leaves against infection with virulent Pst 257 similar to wildtype plants both in the light and dark (Fig. 3H). This activation of robust 258 defence is supported by higher callose deposition in the infected tissue (Fig. 3I and 3J). 259 However, the NPR1SIM1 lines show enhanced disease susceptibility to Pst infection akin 260 to npr1-1 mutants in light and dark conditions. Our data indicates that the SIM1 motif in 261 NPR1 that allows interaction with SUMO1-modified PhyB is a key determinant 262 mediating an effective immune response in Arabidopsis. 263 PhyB mediates NPR1 nuclear bodies formation 264 A major activity of PhyB and NPR1 is the ability to undergo nuclear body formation from 265 diffused to dense condensates in plant nuclei to fulfil their molecular role in the 266 respective biological processes they govern 30. So far, our data indicated that 267 SUMOylation is a key process for light to regulate immunity through promoting PhyB-268 NPR1 interaction. We therefore hypothesised that SUMOylation may facilitate the 269 formation of nuclear bodies that contain PhyB and NPR1 to integrate environmental 270 signals such as light for effective immune responses. 271 We found that m-Scarlet labelled NPR1 is indeed localized in the nucleus in the SAR-272 activated systemic tissues not only in the light but also dark conditions but also forms 273 nuclear bodies (Fig. 4A and C). However, the nuclear body formation was strongly 274 impaired in the systemic tissues of NPR1SIM1 expressing plants (Fig. 4C). The size of the 275 nuclear bodies was estimated to be significantly higher in wildtype NPR1 tissue than 276 NPR1SIM1 tissue upon the activation of SAR (Fig. 4B and D). Strikingly the mScarlet-277 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint NPR1 nuclear condensates only overlapped with those of PhyB-YFP in systemic tissue 278 primed for SAR (Fig. 4A and C). This co-localisation was not seen when either the 279 SUMO or the SIM sites were disrupted in either PhyB or NPR1 respectively (Fig. 4A and 280 C). This was further validated by BiFC (Bi-molecular Flourescence Complementation) 281 assay which revealed the interaction between wild type PhyB and NPR1 resulted in 282 reconstitution and stable expression of YFP in the nucleus (Fig. S11). Active PhyB (P FR 283 form) can be rapidly converted to its inactive (P R form) state under low red/far red 284 (R:FR) ratio 18. We previously showed that far-red suppressed PhyB SUMOylation ( 13). 285 We demonstrate that WT PhyB complemented lines not only fail to show SAR response 286 against Pst infection under FR but also PhyB shows reduced interaction with NPR1 287 (Fig. S12A and B). Upon far red treatment the nuclear localization of NPR1 is lost 288 showing the importance of PhyB in stabilizing the condensate formation of NPR1 (Fig. 289 4E and F). This evidence reinforces the importance FR light in regulating SAR through 290 PhyB SUMOylation. These findings indicate that nuclear condensate formation of PhyB 291 and NPR1 relies on SUMO modification thereby promoting efficient defence responses 292 in systemic tissues. The data also indicates that the photobody formation of PhyB has 293 implications for the activation of immune signaling during SAR in effect generating a 294 new kind of immune-related photobodies. Our bioimaging and biochemical analysis 295 unravel a new dimension of how SUMOylation of PhyB photoreceptor influences NPR1 296 condensates to activate defence signalling through immune photobodies. 297 SUMOylated PhyB enables NPR1 interaction with TGA to activate defence 298 A key mechanism by which NPR1 mediates immune genes expression is through its 299 interaction with TGA transcription factors which also form part of NPR1 nuclear 300 condensates. To ascertain whether PhyB-SUMO interacting SIM1 on NPR1 regulated 301 its interaction with-TGAs, we co-expressed PhyB or PhyB K996R fused with YFP, TGA3 302 fused with myc, NPR1 or NPR1 SIM1 fused with HA in N. benthamiana . 303 Immunoprecipitation with GFP MicroBeads followed by immunoblot analysis revealed a 304 strong interaction of PhyB with TGA3 and NPR1 in the presence of SA but not with the 305 NPR1SIM1 (Fig. 4G). The data indicates that SUMOylated PhyB interaction with the SIM1 306 motif in NPR1 facilitates NPR1-TGA transcription factor association to regulate immune 307 gene expression. 308 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint So far our data indicates that SUMOylation of PhyB allows light to regulate immune 309 gene expression through physical interaction with NPR1 and TGA in immune 310 photobodies. To ascertain whether PhyB immune photobodies is associated with 311 defence gene chromatin we performed Chromatin immunoprecipitation (ChIP) assays 312 with PhyB-YFP expressing systemic leaf tissues activated for SAR. The PhyB 313 interacting DNA fragments were pulled with GFP MicroBeads and enriched promoter 314 elements were quantified using qPCR analysis. Our immunoprecipitation analysis 315 revealed that PhyB was associated with the defence gene, PR1 (archetypical defense 316 gene) promoter elements and this was diminished in the lines expressing the non-317 SUMOylatable PhyBK996R, especially in the dark (Fig. 5A). This was further correlated 318 with higher levels of PR1 gene expression in SAR activated systemic tissues of wildtype 319 PhyB-YFP complemented phyB-9 mutants when compared to non-SUMOylatable 320 PhyBK996R levels (Fig. 5B). These findings indicates that PhyB associated immune 321 photobodies play a crucial role in regulating expression of defense-related genes, such 322 as PR1, during SAR in systemic tissues and this effect was enhanced in the dark (Fig. 323 5A). We also performed ChIP assays in SAR tissues of plants expressing NPR1-HA 324 and NPR1SIM1 -HA in npr1-1 mutant plants. ChIP assays indicated higher occupancy of 325 wildtype NPR1 in the promoters of PR1 in the light and dark than NPR1 SIM1 leaf tissue 326 (Fig. 5C) along with higher expression of PR1 mRNA in NPR1 overexpression lines 327 (Fig. 5D). Furthermore ChIP-qPCR assay of NPR1 overexpressed in phyB-9 328

shows lack of binding to the PR1 promoter even in the presence of SA (Fig. 329 S12). These results conclusively indicate that PhyB interaction with NPR1 mediated by 330 SUMO1 is directly involved in the regulation of defense-related gene expression at the 331 chromatin level in these immune photobodies. Our data reveal a molecular mechanism 332 directly connecting light perception to immune gene expression through SUMOylation of 333 the photoreceptor PhyB. Coimmunoprecipitation assays of NPR1 and NPR1 SIM1 along 334 with CUL3 revealed similar binding affinity suggesting that NPR1 E3 ligase activity is not 335 affected by the SIM1 mutation. (Fig. S13). 336 SUMOylated PhyB and NPR1 share common a defence transcriptome 337 We have demonstrated that SUMOylated PhyB binds to NPR1 to activate expression of 338 the archetypical defence gene PR1. Given that PhyB SUMOylation is also a critical 339 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint regulator of photomorphogenesis 15 we sought to identify at the whole transcriptome 340 level differentially expressed immune-related genes that rely on SUMO dependant 341 interaction between PhyB and NPR1. Therefore, we performed RNA-Seq analysis of 342 SAR activated distal tissue using avirulent Pst (avrB) infection at 3 days post infection 343 when immune gene expression is relatively high 33. 344 345 We found more than 50% of differentially expressed genes (DEGs) in PhyB vs 346 PhyBK996R lines to be common to those DEGs in NPR1 vs NPR1SIM1 with Padj<0.05 (Fig. 347 5E and F). The GO analysis of biological processes and molecular function associated 348 with these DEGs showed enrichment of terms related to external biotic stimulus and 349 endogenous phytohormone signaling including SA, JA and Ethylene (Fig. S14A and 350 B). Further, we observed that biosynthesis of secondary metabolites, plant-pathogen 351 interaction, circadian rhythm and plant hormone signal transduction were the 352 predominant KEGG pathways that were enriched commonly between PhyB vs 353 PhyBK996R and NPR1 vs NPR1 SIM1 (Fig. S14C). Our global RNA-Seq analysis 354 demonstrates that SUMOylated PhyB facilitates the recruitment and activation of 355 defence gene expression during SAR by interacting with the SIM1 domain of NPR1 356 revealing a key role for SUMOylated PhyB in mediating immune responses at the whole 357 transcriptome level. 358 359 We unravel a mechanism where light establishes and maintains systemic immunity in a 360 light/dark cycle in plants through SUMOylation of PhyB photoreceptors (Fig. 5G). PhyB 361 SUMOylation in the light leads to the upturn of SA production which in turn suppresses 362 PhyB deSUMOylation in the night by inducing the turnover of OTS SUMO Proteases. 363 The maintenance of PhyB SUMOylation in distinct light and dark regimes enables the 364 recruitment of NPR1 to TGA transcription factors to immune related nuclear 365 condensates to activate immune gene expression. 366 367 RESOURCE AVAILABILITY 368 Lead contact 369 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint Further information and requests for resources and reagents should be directed to and will be fulfilled by 370 the lead contact, Ari Sadanandom ([email protected]). 371 Data and code availability 372 All data are available in the main text or the supplementary materials. The transcriptome data have been 373 submitted in NCBI under the Bioproject ID PRJNA1062808. 374 ACKNOWLEDGMENTS 375 The authors acknowledge Tim Hawkins and Joanne Robson from Bioimaging Facility, Department of 376 Biosciences, Durham University for assisting in setting up protocols for confocal microscopy. The authors 377 acknowledge Rachael Dack and Bethany Lowes for assisting in LC-MS/MS facility of the Department of 378 Biosciences, Durham University. The assistance from the Genomics facility Department of Biosciences, 379 Durham University for providing sequencing results through Sanger sequencing. The authors thank 380 Novogene for providing RNAseq sequencing data. 381 AUTHOR CONTRIBUTIONS 382 Conceptualization: SG, AS; Methodology: SG, AS; Investigation: SG, SG, XL, AS, SK, LC, MM, BO, MB, 383 PK, CG, CZ; Visualization: SG, AS; Funding acquisition: AS; Project administration: AS; Supervision: AS; 384 Writing – original draft: SG, XL, AS; Writing – review & editing: SG, AS 385 DECLARATION OF INTERESTS 386 Authors declare that they have no competing interests. 387 DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES 388 The authors did not use any Generative AI or AI-Assisted technologies during the preparation of this 389 work. 390 SUPPLEMENTAL INFORMATION 391 Document S1. Figures S1–S16 and Table S1-S3 392 393 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint FIGURE TITLES AND LEGENDS 394 Figure 1. SUMOylation of PhyB is required for light mediated stimulation of salicylic acid 395 (SA) production and nuclear condensate formation to underpin systemic acquired 396 resistance (SAR) in Arabidopsis. 397 PhyBK996R plant lines are compromised for SAR against Pseudomonas syringae pv. tomato 398 DC3000 during infection at (A) light and (B) dark. Callose deposition assay calculated as 399 deposit count per field area (FA) showing activation of defense responses in PhyB (WT) in 400 comparison to PhyB K996R in (C) light and (D) dark. (E) Salicylic acid levels in SAR activated 401 systemic leaves of plants infected with Pst (avrB). (F) qRT-PCR analysis showing levels of SA 402 biosynthetic genes (ICS1 and ALD1) during SAR. Bars represent mean log fold change when 403 compared to PhyB K996R samples. Each experiment was done at least three times with the 404 representative data shown. Bars represent mean log fold change when compared to untreated 405 samples. (G) YFP fused PhyB complemented phyB-9 plants were treated with Pst (avrB) under 406 light and dark and images were taken from SAR leaves under YFP channel of confocal 407 microscope. Untreated plants were used as control. At 3dpi we observe PhyB forms large 408 photobodies. However, in PhyBK996R the formation of photobodies were impaired in SAR tissues. 409 (H) Graph shows average mean size of photobodies under light and dark in the SAR tissues. 410 The size of the photobodies were relatively high especially under dark. Error bars show the 411 standard error of three biological replicates. Different alphabets and * indicates significant 412 difference at p-value ≤ 0.05. 413 414 Figure 2. Systemic acquired resistance and Salicylic acid regulates PhyB SUMOylation in 415 the dark through SUMO E2 (SCE1) and the SUMO Protease OTS1. Total protein was 416 extracted from infiltrated leaves post 3hrs and immunoprecipitated (IP: α GFP). ( A and B ) 417 Immunoblots analysis of PhyB-GFP SUMOylation. The blots were probed with α GFP and 418 α SUMO1. PhyB and PhyB K996R transgenic lines were infiltrated with Pst DC3000 avrB. Total 419 protein was extracted from SAR leaves at 3dpi and immunoprecipitated (IP: α GFP). The blots 420 were probed with α GFP and α SUMO1. The SUMOylation levels of PhyB were checked under 421 (A) light and (B) dark. No SUMOylation was detected in PhyB K996R -GFP lines. The presence of 422 an immunoprecipitated band for PhyB and a band for SUMO1 on the blot demonstrated this. In 423 contrast, the PhyB K996R transgenic line did not show any SUMOylation of PhyB, indicating that 424 the SUMOylation is specific to the wild-type PhyB protein. In response to infection with avrB, 425 these results show that increased SA levels upon SAR activation causes PhyB to become 426 SUMOylated in light and dark as a part of the plant's defence response. Under non-SAR control 427 conditions SUMOylation was only detectable in light plants albeit in reduced levels compared to 428 SAR activated light tissue. (C) The PhyB protein (depicted in red) forms a dimer and conjugates 429 with SUMO1 (illustrated in green). This interaction occurs at the lysine residue at position 996 of 430 PhyB, which binds to the glycine motif located at position 93 of SUMO1. The interaction sites 431 have been shown as spheres. (D) Increased PhyB interaction with SUMO conjugating enzyme, 432 SCE1 facilitates its SUMOylation in light conditions. There is redu ced interaction in dark. PhyB-433 YFP was co-infiltrated with SCE1-HA and transiently expressed in N. benthamiana and samples 434 collected at time of day as indicated. The total protein was extracted at 3dpi and 435 immunoprecipitated with α GFP. The blots were probed with α GFP and α HA. Each blot was 436 repeated atleast three times, and the best representative image is shown. (E) OTS1-Venus 437 fused expression lines were infiltrated SA and OTS1 protein levels were analyzed in infiltrated 438 tissues via immunoblotting with GFP antibody. We observed a reduction of OTS1 under light but 439 increased upon proteasomal inhibitor, Bortezomib (Bz) treatment comparatively less protein is 440 detected. (F) PhyB and OTS1 interact in planta. PhyB-YFP was coinfiltrated with OTS1-HA and 441 transiently expressed in N. benthamiana. The total protein was extracted at 3dpi and 442 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint immunoprecipitated with α GFP. The blots were probed with α GFP and α HA. The results 443 showed that PhyB interaction with OTS1 is reduced in the presence of SA. The OTS1-HA levels 444 were normalized in the input lanes (G) PhyB SUMOylation is enhanced in the ots1ots2 double 445 mutant genetic background. PhyB- YFP and PhyB K996R-YFP transgenic lines in phyB-9 was 446 crossed with ots1ots2 double mutants and treated with SA and samples were harvested 3 hrs 447 post treatment. Total protein was extracted from the treated leaves and immunoprecipitated (IP: 448 α GFP). The blots were probed with α GFP and α SUMO1. GFP only control was used for the 449 pulldowns. Each experiment was done at least three times with the representative data shown. 450 451 Fig. 3. SUMO1 conjugated PhyB mediates light regulated immunity through interaction 452 with the SUMO interacting motif in NPR1. 453 (A) NPR1 interacts with SUMO1. NPR1-HA expressing transgenic lines were used to extract 454 total protein after SA treatment and immunoprecipitated with α HA. The blots were probed with 455 α HA and α SUMO1. The results showed that NPR1 interaction with SUMO1 requires its SIM 456 motif. (B) NPR1-mScarlet and NPR1 SIM1 mScarlet were agroinfiltrated in N. benthamiana with 457 and without SA treatment and observed under RFP channel of confocal microscope. In the 458 presence of SA, NPR1 translocates into the nucleus and undergoes nuclear condensates 459 formation. However, the condensate formation was impaired in NPR1 SIM1 plants. (C) The mean 460 size of SA regulated nuclear condensates of NPR1 are presented as a bar graph. (D) The 461 nuclear condensates of NPR1 were subjected to photobleaching (10 pulses) and the recovery of 462 the bleached region was studied over a 120 seconds time period (measured in log2 463 milliseconds). The fluorescence of NPR1 bleached region recovered rapidly in contrast to 464 NPR1SIM1. Data points represent mean fluorescence recorded with standard error bars. (E) 465 Stable transgenic Arabidopsis plants expressing PhyB/ PhyB K996R -YFP along with NPR1/ 466 NPR1SIM1 -mScarlet treated with SA was used for interaction studies. The total protein was 467 extracted at 3dpi and immunoprecipitated with α GFP. The blots were probed with α GFP and 468 α RFP (for detecting mScarlet). The results showed that NPR1 interacts with PhyB via its SIM 469 motif in NPR1 in the presence of SA. However, the interaction was weak in case of PhyB-470 NPR1SIM1 and PhyB K996R-NPR1. The interaction was totally abolished in PhyB K996R-NPR1SIM1 471 interaction. PhyB interacts with NPR1 in SAR activated systemic leaves of plants pre-infected 472 with avrB in (F) light, (G) dark. Controls blots from non-SAR activated tissues are indicated in 473 Fig. S11. (H) NPR1 SIM1 complemented lines are compromised in SAR response against Pst 474 DC3000 during infection at day (light) and night (dark). Callose deposition assay calculated as 475 deposit count per field area (FA) showing activation of defence response in NPR1 (WT) in 476 comparison to NPR1 SIM1 at (I) light and (J) dark. Bars represent mean log fold change when 477 compared to untreated samples. Error bars show standard error of three biological replicates. 478 Different alphabets indicate significant difference at p-value ≤ 0.05. The western blot of each 479 experiment was done more than three times with the best representative image been shown. 480 Fig. 4. Light induced SUMOylation of PhyB promotes nuclear body formation to promote 481 interaction of NPR1 with TGAs to activate immunity. 482 (A) Plants overexpressing mScarlet fused to NPR1 were infiltrated with avrB and localization of 483 mScarlet was observed in SAR tissues under RFP channel. We observed NPR1 localizes in the 484 nucleus as it overlaps with Hoechst 33342 that preferentially stains nucleic acid. Upon activation 485 of SAR, NPR1 translocates into the nucleus and form nuclear bodies. However, this was 486 impaired in NPR1SIM1 plants under light and dark (C). The mean size of nuclear condensates of 487 NPR1 under (B) light and (D) dark are presented as a bar graph. (E) Confocal imaging showing 488 lack of PhyB and NPR1 interaction in low red:far red ratio light. (F) The total protein from SAR 489 activated tissue was extracted at 3dpi and immunoprecipitated with α GFP. The blots were 490 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint probed with α GFP and α RFP. The results showed that NPR1 interact with PhyB via its SIM 491 motif under light but not under Far red conditions (low red: far red ratio). (G) PhyB-YFP was co-492 infiltrated with TGA3-myc and NPR1-HA and transiently expressed in N. benthamiana. The total 493 protein was extracted at 3dpi and immunoprecipitated with α GFP. The blots were probed with 494 α GFP, α myc and α HA. The results showed that PhyB interacts with NPR1 and TGA3 in the 495 presence of SA. The interaction is abolished when PhyB K996R is expressed with TGA3 and 496 NPR1. The immunoblot analysis was performed at least 3 times, and the best representative 497 image is shown. Error bars show standard error of three biological replicates. Different 498 alphabets indicate significant difference at p-value ≤ 0.05. 499 500 Figure 5. SUMO conjugated PhyB is critical for NPR1 to associate with defence gene 501 chromatin to regulate immune gene expression. 502 (A) ChIP assay was performed in SAR tissues of PhyB and PhyB K996R plants at light and dark 503 using anti-GFP microbeads. ChIP-qPCR revealed higher fold change in PhyB at dark than 504 PhyBK996R which suggests that SUMOylated PhyB binds to PR1 promoter in SAR tissues. (B) 505 qPCR-based expression profile shows upregulation of PR-1 levels in SAR tissue of PhyB plants 506 at dark. ChIP assay was performed in SAR tissues of NPR1 and NPR1 SIM1 plants at light and 507 dark using anti-HA microbeads. (C) ChIP-qPCR revealed higher fold change in NPR1 at dark 508 than NPR1 SIM1 which suggests that SUMO1-NPR1 interaction at the SIM site promotes PR1 509 promoter binding activity in SAR tissues. Bars indicate mean percentage input of ChIP samples 510 with error bars calculated across three replicates. (D) qPCR-based expression analysis shows 511 increased accumulation of PR1 transcripts in NPR1 in light and dark. (E) Venn Diagram 512 showing common upregulated differentially expressed genes (DEGs) in SAR tissues of PhyB vs 513 PhyBK996R and NPR1 vs NPR1 SIM1 at light and dark. (F) Heat map showing upregulation of 514 common defense-related DEGs in PhyB vs PhyBK996R and NPR1 vs NPR1SIM1 at light. Error bars 515 show standard error of three biological replicates. 516 (G) Model showing PhyB mediated fine-tuning of defence responses via NPR1 in light and dark. 517 In the light in SAR tissues PhyB is SUMOylated by enhanced interaction with SCE1- SUMO 518 conjugating enzyme and reduced interaction with OTS1 deSUMOylase as SA levels go up. 519 This facilitates SUMOylated PhyB interaction with NPR1 via its SIM motif which together forms 520 a complex with TGA transcription factors in nuclear condensates and drives the expression of 521 defence genes. Once SAR is activated, in the dark high salicylic acid levels promote OTS1 522 degradation to prevent deSUMOylation of PhyB. Hence PhyB SUMOylation and its interaction 523 with NPR1-TGA complex in the nuclear condensates is maintained to drive immune responses. 524 525 Supplementary Figure Legend 526 Fig. S1. Disease susceptibility of PhyB and its mutant variants upon Pseudomonas 527 syringae pv. tomato DC3000 ( Pst) inoculation. (A) Bacterial count from 4-week-old 528 Arabidopsis plants infected with virulent Pst DC3000 at 3dpi (B) Bacterial count from 4-week-old 529 Arabidopsis plants infected with avirulent Pst (avrB) at 3dpi under light. PhyBK996R lines are more 530 susceptible when infected with avirulent Pst indicating that they are impaired in effector 531 triggered immunity. (C) Bacterial count from 4-week-old Arabidopsis plants infected with 532 avirulent Pst (avrB) at 3dpi under dark. Bar graph shows mean value with error bars 533 representing standard error. Different alphabets indicate significant difference at p-value ≤ 0.05. 534 Each experiment was done at least three times with the representative data shown. 535 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint Fig. S2. Disease susceptibility in systemic tissues of PhyB, NPR1 and OTS SUMO 536 Proteases and their mutant variants to virulent Pst infection upon SAR activation. (A) 537 SAR activated (by preinoculation with avirulent Pst (avrB)) 4-week-old Arabidopsis plants 538 followed by Pst DC3000 infection at 3dpi and bacterial count was done at 3dpi. These plants 539 were SAR induced. (B) Control inactivated 4-week-old Arabidopsis plants infected with Pst 540 DC3000 infection followed by infection with virulent Pst DC3000 and bacterial count was done 541 at 3dpi. These plants were SAR uninduced. Bar graph shows mean value with error bars 542 representing standard error. Different alphabets indicate significant difference at p-value ≤ 0.05. 543 Each experiment was done at least three times with the representative data shown. 544 Fig. S3. PhyB is SUMOylated in the presence of SA . Total protein was extracted from SA 545 infiltrated leaves post 3hrs and immunoprecipitated (IP: α GFP). The blots were probed with 546 α GFP and α SUMO1. PhyB and PhyB K996R transgenic leaves were infiltrated with SA light and 547 dark. Total protein was extracted from leaves at 1, 2 and 3 hrs post treatment and 548 immunoprecipitated (IP: α GFP). The blots were probed with α GFP and α SUMO1. PhyB gets 549 SUMOylated in light when treated with SA (1mM) for a period of 1 to 3 hrs at (A) light and (B) 550 dark in PhyB and PhyB K996R transgenic lines. The results showed that treating with SA caused 551 PhyB to be SUMOylated at light and dark. The presence of an immunoprecipitated band for 552 PhyB and a band for SUMO1 is indicated by arrows. In contrast, the PhyB K996R transgenic line 553 did not show any SUMOylation of PhyB, indicating that the SUMOylation is specific to lysine at 554 position 996. 555 Fig. S4. Constitutive expression of PhyB (WT) under Lip1 promoter complements phyB-9 556 mutants. (A) Bacterial count from 4-week-old PhyB under Lip1 promoter shows SAR immunity 557 against Pst similar to that observed in PhyB under native promoter. (B) Increased callose 558 deposit count per field area (FA) observed in PhyB complemented lines under Lip1 and native 559 promoter compared to PhyB K996R complemented and phyb9 mutant. (C) Lip1:PhyB and 560 Lip1:PhyBK996R transgenic lines were treated with SA and sample was harvested 3 hrs post 561 treatment. Total protein was extracted from the treated leaves and immunoprecipitated (IP: 562 α GFP). The blots were probed with α GFP and α SUMO1. PhyB is SUMOylated just as observed 563 in the lines expressing PhyB under native promoter. Different alphabets indicate significant 564 difference at p-value ≤ 0.05. Each experiment was done at least three times with the 565 representative data shown. 566 Fig. S5. Confocal image analysis of OTS1-mVenus tagged lines showing mVenus fused 567 SUMO protease OTS1 protein levels. Upon SA treatment and in SAR activated leave tissue 568 OTS1 levels is drastically reduced. Each experiment was done at least three times with the 569 representative image shown. 570 Fig. S6. Schematic of the domain structure of NPR1 showing SUMO Interacting Motif (SIM 571 motif). NPR1 exists as a homodimer consisted of 4 domains namely, Bric-à-brac (BTB) domain, 572 a BTB and carboxyterminal Kelch helix bundle (BHB), four ankyrin repeats (ANKs) and a 573 disordered salicylic-acid-binding domain at the C terminal. A T-coffee based multiple sequence 574 alignment of NPR1 protein across multiple plant species was performed. The putative SUMO 575 interacting motif was found at the position RYMEIQE. In the mutated NPR1 SIM1 the site was 576 mutated to RAAEIQE, wherein the hydrophobic re sidues tyrosine and methionine residues were 577 changed to alanine. 578 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint Fig. S7. NPR1 interacts with SUMO1 through its SIM motif. NPR1-GFP and SUMO1-HA was 579 coinfiltrated in N. benthamiana and post 3 days the leaves were infiltrated with SA. The total 580 protein was extracted from the infiltrated leaves and immunoprecipitated with anti-GFP 581 microbeads. The blots were probed with α GFP and α HA antibody. The result shows NPR1 582 interaction with SUMO1 is enhanced significantly without SA treatment and requires the SIM for 583 this interaction. Each experiment was performed at least three times, and the best 584 representative image is shown. 585 Fig. S8. SUMO-SIM dependant interaction of PhyB-YFP with NPR1-HA in transient assays 586 in N. benthamiana. The plants were treated with SA at 3dpi. The total protein was extracted at 587 3dpi and immunoprecipitated with anti-GFP microbeads. The blots were probed with α GFP and 588 α HA antibody. The result showed that NPR1 interacts via its SIM motif with PhyB in the 589 presence of SA. The interaction is abolished in PhyB K996R and NPR1 SIM1mutant proteins. Each 590 experiment was performed at least three times, and the best representative image is shown. 591 Fig. S9. SAR is required for PhyB interaction with NPR1 in stable transgenic Arabidopsis 592 plants. The total protein was extracted from leaves of non-SAR activated plants at 3dpi and 593 immunoprecipitated with α GFP. The blots were probed with α GFP and α RFP. The results 594 showed that under control conditions NPR1 interact with PhyB via its SIM motif at (A) light but 595 not in (B) dark. However, the interaction was weak in case of PhyB-NPR1 SIM1 and PhyB K996R-596 NPR1. The interaction was totally abolished in PhyBK996R-NPR1SIM1 interaction. Each experiment 597 was performed at least three times, and the best representative image is shown. 598 Fig. S10. Bi-molecular Fluorescence Complementation (BiFC) showing interaction 599 between PhyB and NPR1 in the nucleus. Split YFP fused to PhyB and NPR1 is brought 600 together to form stable YFP protein through PhyB-NPR1 SUMO mediated interaction. Mutating 601 the SUMO site (PhyB K996R) or SIM (NPR1 SIM1). Inset shows zoomed image of nuclear 602 localization of PhyB-NPR1. Each experiment was performed at least three times, and the best 603 representative image is shown. 604 Fig. S11. phyB-9 complemented lines shows higher susceptibility to Pst DC3000 in SAR 605 tissue when treated under low red:far red ratio light. (A) Bacterial count of PhyB 606 complemented lines in SAR tissues under low red:far red ratio light. (B) Callose deposit count 607 per field area (FA) in SAR tissues. Error bars show the standard error of three biologicl 608 replicates. Different alphabets indicate significant difference at p-value ≤ 0.05. Each experiment 609 was performed at least three times. 610 Fig. S12. NPR1 does not bind to PR1 promoter in absence of PhyB. ChIP assay was 611 performed in SA treated and control tissues of NPR1 and NPR1 SIM1 phyB-9 plants in light using 612 anti-HA microbeads. ChIP-qPCR revealed low fold change in NPR1 which suggests that in the 613 absence of PhyB, NPR1 does not binds to PR1 promoter even in the presence of SA. Each 614 experiment was done at least three times with the representative data shown. 615 Fig. S13. SIM1 mutation in NPR1 does not alter its E3 Ligase complex formation. Both 616 NPR1 and NPR1 SIM1 interacts with CUL3 with equal affinity. NPR1-GFP or NPR1 SIM1-GFP was 617 coinfiltrated with CUL3-HA and transiently expressed in N. benthamiana. The total protein was 618 extracted at 3dpi and immunoprecipitated with α GFP. The blots were probed with α GFP and 619 α HA. Each experiment was performed at least three times, and the best representative image is 620 shown. 621 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint Fig. S14. GO Enrichment of common DEGs in PhyB vs PhyB K996R and NPR1 vs NPR1 SIM1 622 in SAR tissues under light. The top 20 GO terms have been represented as dotplot matrix 623 with size of dots representing number of genes and X axis indicating Fold Enrichment. The 624 color coding of the dots represents log of FDR (False Discovery Rate) with FDR<0.05 taken as 625 the cutoff. (A) Enriched GO terms under Biological Process along with (B) Molecular Function 626 and (C) Common enriched KEGG pathways. 627 Fig. S15. Expression profile of transgenes overexpressed in the respective transgenic 628 lines under control conditions. Bars indicate average relative expression calculated with 629 respective to endogenous control Actin. Error bars show standard error of three biological 630 replicates. (A) Relative expression of PhyB transgenic lines. (B) Relative expression of NPR1 631 transgenic lines. (C) Relative expression of NPR1 transgenic lines in PhyB background. (D) 632 Relative expression of OTS1 transgenic lines. 633 Fig. S16. phyB-9 linked mutation ven4 shows no altered SAR response. (A) Bacterial count 634 comparing SAR response in ven4, phyB-9OG (original mutant with ven4 linked mutation) and 635 phyB-9BC (new mutant without ven4 mutation). (B) Callose deposits per field area (FA) in SAR 636 tissues. phyB-9OG (original mutant) shows similar phenotype phyB-9BC (new mutant) during 637 SAR response in light and dark. ven4 shows defence responses similar to Col-0. Different 638 alphabets indicate significant difference at p-value ≤ 0.05. 639 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint

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Plant Cell 34, 2892-2906. 748 10.1093/plcell/koac142. 749 750 STAR/i1METHODS 751 KEY RESOURCES TABLE 752 753 REAGENT OR RESOURCE SOURCE IDENTIFIER Antibodies Anti-GFP Antibody Abcam AB_305564 Anti-HA-Biotin Antibody Roche AB_390915 Rabbit Anti-RFP Polyclonal Antibody Abcam AB_945213 Anti-SUMO1Antibody Deposited data RNAseq data NCBI PRJNA1062808 754

DETAILS 755 Plant materials and growth conditions 756 Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wildtype control plants in all 757 experiments. Arabidopsis plants in soil (Levington seed & modular F2S (with sand)) were grown 758 in environmentally controlled chambers (SANYO, Panasonic) in 60% relative humidity, following 759 the temperature and photoperiod of 22 °C for 16 h (dark) and 20 °C for 8 h (light). For light 760 treatment, samples were infected and harvested at 11 am, i.e. middle of day. Another batch of 761 Arabidopsis plants was grown under the same conditions as the middle of night treatment. For 762 dark experiments samples were infected and harvested at 9pm i.e. middle of night. The mutant 763 plants were obtained from the Nottingham Arabidopsis Stock Centre (NASC; 764 https://arabidopsis.info/) and the homozygous plants were selected by genotyping. 765 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint Nicotiana benthamiana plants were used as models for transient protein expression and were 766 grown at 28 °C with a fixing light-dark period (14 h light and 10 h dark). 4-week-old plants were 767 used for agroinfiltration. 768 Generation of plasmids and plant transformation 769 The constructs were generated using different cloning strategies. NPR1 was cloned in entry 770 vector dTOPO and different destination vectors using Gateway cloning technology under 35S 771 promoter. NPR1 was cloned into pEG103 and pEG201 destination binary vectors for C terminal 772 GFP and HA tag respectively. NPR1 was fused with mScarlet using Gibson Cloning Assembly 773 and cloned into pEG100 binary vector. To generate the PhyB (PhyB genomic DNA along with 774 native promoter) constructs 2 kb PhyB promoter was amplified along with PhyB and cloned in 775 pPCV vector using restriction digestion based cloning strategy. The OTS1 native promoter 776 (1000kb) cloned along with its genomic DNA in pMDS vector. OTS1 gene was synthesised and 777 cloned by Genscript company. The cds of TGA3, CUL3 and SCE1 were cloned from cDNA of 778 SAR leaves of Col-0 (3dpi) and were cloned in entry vector pENTR4 followed by destination 779 vector pEG203 and pEG 201 respectively. To generate the Bimolecular Flourescence 780 Complementation (BiFC) constructs PhyB and NPR1 were cloned into destination vector 781 pYFC43, containing C terminal end of YFP and YFN43, containing N terminal end of YFP using 782 LR clonase. The verification of the final constructs was achieved by Sanger Sequencing. The 783 primers used have been enlisted in Supplementary Table S1. 784 To generate PhyB/PhyB K996R transgenic plants phyB-9 background, the constructs were 785 introduced into the Agrobacterium tumefaciens strain GV3101 via the floral dip method that was 786 previously described 34. The positive transformant plants were selected using Hygromycin. To 787 generate NPR1/NPR1SIM transgenic plants npr1-1 background, the constructs were introduced 788 into the Agrobacterium tumefaciens strain GV3101 via the floral dip method that was previously 789 described. The positive transformant plants were selected using BASTA. The construct name, 790 plasmid generated along with plant genotype has been tabulated in Supplementary Table S2 791 and Table S3. The expression profile of the respective transgenic lines has been shown in Fig. 792 S15. 793 Bacterial Growth Assays and Systemic Immune Responses 794 The Pseudomonas syringae pv. tomato DC3000 ( Pst; virulent strain) and Pst avrB (avrB; 795 avirulent strain) were used in this study. Each strain was streaked out on Kings medium B agar 796 plate and cultured at 28 /i1 °C for two days. The inoculum was made by 28 /i1 °C overnight cultured 797 bacterial strains in Kings B Broth (20 g/L Proteose Peptone, 1.5 g/L Magnesium Sulphate 7 798 H2O, 10% Glycerol and 1.5 g/L Dipotassium Hydrogen Phosphate). For each primary infection 799 assay, the bacterial suspension was standardized to a concentration of 0.002 OD in a solution 800 comprising 10 mM MgCl 2. Four-week-old Arabidopsis plants were syringe infiltrated with the 801 bacterial suspension (Pst) for inoculation. The infection rate was scored at 3 days post infection 802 (dpi) based on the CFU and callose spot counts on the leave surface. Three samples from ten 803 independent plants were used as one replicate for spot count and CFU count respectively. The 804 whole experiment was repeated more than three times. 805 For systemic infection, four oppositely positioned leaves were syringe infiltrated with avrB in a 806 suspension standardized to a concentration of 0.01 OD in a solution comprising 10 mM MgCl 2. 807 At 3 dpi the adjacent leaves of either side of the avrB infiltrated leaves were injected with Pst at 808 a concentration of 0.002 OD. The infection rate in the systemic tissues were scored at 3 dpi 809 based on the CFU and callose spots on the leaf surface. Three samples from ten independent 810 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint plants were used as one replicate for spot count and CFU count respectively. The whole 811 experiment was repeated at least three times. It is important to note that the phyB-9 mutant 812 (phyB-9OG) contains a venosa mutation (ven4) associated with it 35. As the transgenics were 813 done in the phyB-9OG background we compared it with phyB-9BC (phyB-9 without ven4 mutation) 814 and found similar level of susceptibility in both the mutants negating the role of ven4 in phyB-815 9OG (Fig. S16). 816 Site-directed mutagenesis for generating mutants 817 NPR1 was cloned in entry vector pENTR-dTOPO. The entry clone was (mentioned previously) 818 used as template for generating the mutated version of NPR1 gene. Oligonucleotide primers 819 utilized for the introduction of mutations have been listed in Supplementary table S1. The PCR 820 product was treated with DpnI digestion overnight to get rid of the template DNA before 821 transforming it into DH5a. The positive transformants were selected on selection plates. All the 822 mutations were confirmed by Sanger sequencing, which was performed before and subsequent 823 to the introduction of the mutated NPR1 gene into the pEG103 destination vector via the 824 Gateway LR cloning method. 825 Salicylic acid and Far-red light treatment 826 Salicylic acid (SA) treatment was given to the plants by spraying with 1mM SA and samples 827 were harvested 3hrs post treatment. For different timepoints of SA treated samples were 828 harvested 1, 2 and 3 hours post treatment at day or night depending on the experimental 829 conditions. For far-red light treatment, plants were treated with low red/far red light (Ratio of 830 Red-2.3 μ mol/m2/s to Far red-11.3 μ mol/m2/s) provided using a Heliospectra growth light 831 (Heliospectra, Sweden) during SAR response. 832 Salicylic acid extraction and estimation 833 For evaluating the salicylic acid levels of the leaves, a total of 150 mg of plant leaf tissue was 834 crushed into fine powder in liquid nitrogen and was transferred into a screwcap tube. The 835 samples were then homogenized within 1.5 ml extraction solution (20 ml isopropanol, 10 ml 836 water and 20 ul HCl) and the internal standard salicylic acid-D4 (SUPELCO) by vortexing. After 837 this initial phase, 2 ml of Dichloromethane (DCM) was added followed by another vigorous 838 vortexing. The centrifugation of the samples was performed at 1000 g for 15 min at 4 °C. The 839 lower phase was transferred to a clean glass tube and followed by a second extraction with 1 ml 840 of DCM. Subsequently, the fractions obtained from initial and secondary extraction were 841 combined and were subjected to a drying process by placing them under liquid nitrogen. 842 LC-MS/MS analysis of phytohormones 843 Samples were dried under a stream on N 2 and reconstituted in 300µL LC-MS grade MeOH 844 (Radnor, USA). Subsequently, these samples were centrifuged at 9500 rcf for 2 minutes and 845 placed in 200µL glass inserts. Phytohormone measurements were performed on a Shimadzu 846 Nexera X2 Ultra-Fast Liquid Chromatography system consisting of binary pump, an on-line 847 degassing unit, autosampler, and a column oven (Shimadzu Corporation, Kyoto, Japan), 848 coupled with an AB Sciex 6500 QTRAP mass spectrometer consisting of an electrospray 849 ionization (ESI) source (AB SCIEX, Framingham, MA, USA). Samples were held at 4°C in the 850 autosampler and 5 µl of sample was injected on to an Atlantis premier AX C18 column (2.1 x 851 100mm, Waters, Milford, MA, USA), maintained at 40°C, with a flow rate of 0.2 mL/min. The 852 mobile phases consisted of Solution A (10nM ammonium bicarbonate pH6.7) and Solution B 853 (90:10 MeOH: 100nM ammonium bicarbonate pH6.7). Honeywell ammonium bicarbonate was 854 from Fisher Scientific (Waltham, USA). Solvents were prepared fresh on the day of analysis and 855 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint filtered through a 0.2µm nylon filter (Phenomenex). A gradient elution was used as follows: 0.0-856 2.0 min, 5% B, 2-7.0 mins, 95% B, 7.0-9.9 mins, 95% B, 9.9-10.15 mins, 5% B, and held for 3.1 857 mins. The ion source was operated in negative ionization mode with the following conditions: 858 curtain gas, 40 psi; nebulizer gas , 50 psi; auxiliary gas, 60 psi; ion spray voltage, -4500 V; and 859 temperature, 550°C. Quantification was performed by external calibration curve with standards 860 prepared in LCMS grade methanol. 861 Callose deposition Assay 862 The infected Arabidopsis leaves were collected and subjected to overnight clearing and fixing in 863 a solution, consisting of 95% ethanol and a lactophenol solution in a 1:2 ratio. The lactophenol 864 solution was composed of phenol, 100% glycerol, lactic acid, and water in a 1:1:1:1 ratio. 865 Following the cleaning step, the leaves underwent rinsing with a rinsing solution (3:1 95% 866 Ethanol: Glacial acetic acid). The staining step was achieved by immersing the leaves in a 867 staining solution containing 0.01% aniline blue in 0.15 M phosphate buffer adjusted to a pH of 868 9.5. The stained leaves were subsequently observed using a fluorescence microscope (Zeiss 869 Apotome). 870 Total RNA extraction and quantitative RT-PCR 871 The total RNA extraction was conducted from 100 mg leaf tissue of four-week-old plants using 872 the RNA isolation kit (Spectrum™ Plant Total RNA Kit, Merck) following the manufacturer’s 873 protocol. The quantity and purity of the total RNA were measured with a NanoDrop 874 Spectrophotometer (NanoDrop One, Thermo Fisher). The High-Capacity cDNA synthesis kit 875 (ABI) was used to generate the cDNA following the manufacturer’s protocol, using 2 µg of total 876 RNA. 877 The relative abundance of the mRNA was quantified via quantitative real-time PCR (qCFX 878 Connect, Biorad) in a total reaction of 10 µl, using Brilliant III Ultra-Fast SYBR qPCR master mix 879 (Agilent). Actin7 (gene code: At5g09810) was used as the reference gene for normalization. 880 Primers used in the RT-qPCR are documented in supplementary table S1. 881 Chromatin Immunoprecipitation and qPCR 882 For ChIP assay the nuclei were isolated followed by extraction of bound chromatin. The nucleus 883 was isolated from fixed plant samples using Nuclei extraction kit (CelLytic PN 884 isolation/extraction kit, Merck) using manufacturers’ protocol. The chromatin was isolated using 885 the method as previously described in Srivastava et al 2021 36. The qPCR was performed with 886 was quantified via quantitative real-time PCR (qCFX Connect, Biorad) in a total reaction of 10 887 µl, using Brilliant III Ultra-Fast SYBR qPCR master mix (Agilent). Actin7 (gene code: At5g09810) 888 was used as the reference gene for normalization. Primers used in the RT-qPCR are 889 documented in supplementary table S1. The log fold change was calculated as percentage 890 input for each sample normalized with input samples. 891 Immunoprecipitation and Coimmunoprecipitation Assays 892 1.5 g of Arabidopsis leaf tissue was collected and grounded with 1.5 ml of protein extraction 893 buffer containing 1 tablet (per 10 ml buffer) of protease inhibitor cocktail (Roche), 0.1% SDS, 894 0.5% sodium deoxycholate, 100 mM Tris-HCl (pH 8.0), 1% glycerol, 20 mM N-ethylmaleimide 895 (NEM) and 50 mM sodium metabisulfite. Samples were incubated in protein extraction buffer for 896 15 mins and centrifuged twice at 14,000 rpm for 10 mins each to remove the cellular debris. 897 Total protein was subsequently incubated with anti-GFP microBeads (Miltenyi) at 4 °C for 30 898 mins. The beads were passed through Miltenyi columns and washed for three times with 200 µl 899 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint of extraction buffer. After three washes, the immuno-complex was eluted by 95 µl of 95 °C 900 preheated 1 x lamellae buffer and analyzed on 10 % SDS-PAGE using immunoblotting method. 901 Anti-GFP (1:5000; Antirabbit, Merck), anti-HA (1:2500; Anti-Rat, Merck) and anti-Rabbit 902 (company, dilution factor) were used as primary and secondary antibodies respectively. 100 ul 903 of input fractions were loaded as loading control. 904 N. benthamiana plants were infiltrated with salicylic acid (1mM), 10 mM MgCl 2 180 min prior to 905 sample collection. The total protein for co-IP was extracted using an extraction buffer consisting 906 of 1mM DTT, 1 mM EDTA, 50 mM Tris (pH 8) and 0.5% Trition X-100. Total protein samples 907 were incubated in protein extraction buffer for 15 mins and centrifuged twice at 14,000 rpm for 908 10 mins each to remove the cellular debris. Total protein was subsequently incubated with anti-909 GFP microBeads (Miltenyi) at 4 °C for 30 mins. The beads were passed through Miltenyi µ 910 columns and washed for thrice times with 200 µl of extraction buffer. After three washes, the 911 immuno-complex was eluted by 95 µl of 95 °C preheated 1 x lamellae buffer and analyzed on 912 10 % SDS-PAGE using immunoblotting method. Anti-GFP (1:5000; Anti-Rabbit, Merck), anti-HA 913 (1:2500; Anti-Rat, Merck) and anti-Rabbit (company, dilution factor) were used as primary and 914 secondary antibodies respectively. 915 Protein extraction and western blot 916 1g of Arabidopsis leaves were grounded in liquid nitrogen and 1 ml protein extraction buffer (4% 917 SDS, 50 mM Tris-HCl (pH 8.5), 2% β -mercaptoethanol, 10 mM EDTA and 1 tablet protease 918 inhibitor). The mixture was centrifuged at 14,000 rpm for 15 mins and the following procedures. 919 Total protein was diluted with 4 x laemmli dye and boiled at 98 °C for 10 mins and loaded on 8% 920 polyacrylamide gels. Later, a polyvinylidene difluoride (PVDF) membrane was used for 921 transferring the separated protein from the gels to the membrane. The membrane was then 922 blocked with 5% skimmed milk (brand) for 1 h at room temperature (RT) and proceeded with 923 primary antibody incubation for 2 hrs at RT. Washes after each incubation were 10 min for three 924 times. Secondary antibody coupled with HRP was used for incubation at RT for 1 h followed by 925 the wash cycles mentioned above. The ECL solution 1 and 2 (Biorad) was mixed in an equal 926 volume and incubated with the membrane in a light-proof cassette. The blots were then 927 developed with X-ray using a film developer instrument Xograph Compact 4x Automated 928 Processor (Xograph Imaging Systems) in a dark room. 929 Confocal microscopy imaging 930 Four-week-old N. benthamiana plants were infiltrated with Agrobacterium tumefaciens strain 931 GV3101 harboring expression constructs suspended in infiltrating buffer containing 10 mM 932 MgCl2 and 150 ug/ml acetosyringone at an OD600 of 0.4 for transient assays. For stable 933 expression analysis samples were harvested from transgenic Arabidopsis plants. At 3dpi, 3mm 934 diameter leaf disks were extracted using a cork borer and were subsequently stained with 935 Hoechst 33342 dye for 10 minutes to stain the nucleus. Imaging was conducted using confocal 936 microscopy (Zeiss LSM 800) under 20X magnification. YFP signal was detected using 514nm 937 excitation and 520nm-590nm emission channel. For detection of mScarlet the excitation was 938 adjusted to 561nm and 590-650nm emission spectra. For detection Hoechst 33342 nuclear 939 stain, the excitation was adjusted to 405nm and 410-510nm emission. All the images were 940 taken in Airyscan scanning mode for super-resolution. For BiFC analysis, different (PhyB-NPR1) 941 construct combinations in A. tumefaciens were infiltrated in N. benthamiana plants and the 942 plants were images at 3dpi. Imaging was done using confocal microscopy (Zeiss LSM 800) 943 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint under 20X magnification. YFP signal was detected using 514nm excitation and 520nm-590nm 944 emission channel. 945 Transcriptome sequencing and differential expression analysis 946 RNAs were isolated from 150 mg of systemic leaves post 3dpi from Pst avrB infiltrated plants 947 using RNeasy Plant Mini Kit (Qiagen). On-column DNase digestion was done to get rid of 948 contaminating DNA. Transcriptome sequencing was performed using Paired-end (PE) 949 2/i1 ×/i1 150/i1 bp library on Illumina Sequencing PE150. The quality check of the RNA samples 950 was run in a bioanalyzer. The mRNA was enriched in the samples using Poly A enrichment kit. 951 Purification of messenger RNA (mRNA) was achieved by using poly-T oligo-attached magnetic 952 beads to isolate it from total RNA. Following fragmentation, the initial strand of cDNA was 953 created using random hexamer primers, which was then followed by the synthesis of the second 954 strand of DNA. Following the end repair, purification, A-tailing, adapter ligation, size selection, 955 and amplification, the library was complete. Following library preparation, the reads were 956 processed using Trimmomatic to remove pair-end adapter sequences. Using StringTie the 957 reads were aligned to the Arabidopsis genome (TAIR Version 10) alignment for all samples. The 958 read count for transcript was calculated using RNASTAR software. The log fold change for the 959 samples were calculated using DESEQ2 and padj <0.05 were removed from the analysis. Each 960 sample was sequenced in three biological replicates and the DEG (differentially expressed 961 genes) reveal the mean log fold change. The GO and KEGG enrichment of the DEGs was 962 performed using ShinyGO tool. The heat map was generated using Morpheus software. 963 .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which 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 preprintthis version posted January 22, 2025. ; https://doi.org/10.1101/2025.01.19.633791doi: bioRxiv preprint