Family-wide evaluation of RALF peptides in Arabidopsis ......2020/06/26 · (Gonneau et al., 65 2018). CrRLK1Ls, such as FER, ANX1/ANX2 and BUPS1/2, have been shown to work 66 together

1

Title: 1

Family-wide evaluation of RALF peptides in Arabidopsis thaliana 2

One sentence summary: 3

The majority of A. thaliana RALF peptides inhibit growth in a FERONIA-dependent manner 4

Authors: 5

Alicia Abarca1, Christina M. Franck1 and Cyril Zipfel1,2 6

Affiliations: 7

1Institute of Plant and Microbial Biology, Zurich-Basel Plant Science Center, University of 8 Zurich, 8008 Zurich, Switzerland. 9

2The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, NR4 7UH, 10 Norwich, UK. 11

*Corresponding author. Email: [email protected] 12

C.Z. conceived the original screening and research plan; A.A. and C.M.F. performed the 13 experiments; A.A. designed the experiments and analysed the data; A.A. wrote the first draft 14 of manuscript; C.Z. supervised and completed the writing. C.Z. agrees to serve as the author 15 responsible for contact and ensures communication. 16

17

Abstract 18

Plant peptide hormones are important players controlling various aspects of plants’ lives. 19 RAPID ALKALINIZATION FACTOR (RALF) peptides have recently emerged as important 20 players in multiple physiological processes. Numerous studies on RALF peptides focused on 21 broad phylogenetic analysis including multiple species. Thus, progress has been made in 22 understanding the evolutionary processes that shaped this family. Nevertheless, to date, 23 there is no comprehensive, family-wide functional study on RALF peptides. Here, we 24 analysed the phylogeny and function of the proposed multigenic RALF peptide family in the 25 model plant Arabidopsis thaliana, ecotype Col-0. Our phylogenetic analysis reveals that two 26 of the previously proposed RALF peptides are not genuine RALF peptides, which leads us to 27 propose a new consensus AtRALF peptide family annotation. Moreover, we show that the 28 majority of AtRALF peptides are able to induce seedling or root growth inhibition in A. 29 thaliana seedlings when applied exogenously as synthetic peptides. Additionally, we show 30 that most of these responses are dependent on the Catharanthus roseus RLK1-LIKE 31 receptor kinase FERONIA, suggesting a pivotal role in the perception of multiple RALF 32 peptides. 33

34

Introduction 35

Cell-to-cell communication is crucial for plants, as sessile organisms constantly exposed to 36 an ever-changing environment. Under this scenario, plant peptide hormones are key to 37 rapidly initiate, coordinate and integrate responses thanks to their large diversity in structure, 38 function and expression patterns (Matsubayashi, 2014; Olsson et al., 2019). 39

RAPID ALKALINIZATION FACTOR (RALF) peptides belong to a family of cysteine-rich plant 40 peptide hormones that are involved in multiple physiological and developmental processes, 41 ranging from pollen tube growth to modulation of immune responses (Murphy & De Smet, 42

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2014; Blackburn et al., 2020). They were discovered in a peptide hormone screen due to 43 their ability to cause medium alkalinisation of tobacco cell cultures (Pearce et al., 2001a). 44 Later, RALF peptides, with several conserved motifs, were found to be ubiquitous in 45 terrestrial plants highlighting their functional importance (Cao & Shi, 2012; Murphy & De 46 Smet, 2014; Campbell & Turner, 2017). 47

In Arabidopsis thaliana, more than 30 RALF peptides have been predicted. In the ecotype 48 Col-0, various studies have reported between 34 and 39 members, depending on the study 49 and criteria considered (Olsen et al., 2002; Cao & Shi, 2012; Haruta et al., 2014; Morato do 50 Canto et al., 2014; Sharma et al., 2016; Stegmann et al., 2017; Campbell & Turner, 2017). 51 This discrepancy calls for a careful examination of the RALF family annotation. 52

The majority of plant peptide hormones are perceived by plasma-membrane localized 53 leucine-rich repeat receptor kinases (LRR-RKs) or receptor proteins (LRR-RPs), which unlike 54 RKs lack an intracellular domain (Hohmann et al., 2017; Olsson et al., 2019). In contrast, 55 RALF peptides have recently been shown to be ligands of protein complexes involving 56 Catharanthus roseus RLK1-LIKE (CrRLK1L) receptor kinases, characterized by malectin-like 57 domains in their extracellular domain (Franck et al., 2018). For example, RALF1, RALF22 58 and RALF23 bind to the CrRLK1L FERONIA (FER) to regulate root growth, abiotic, and biotic 59 stress responses, respectively (Haruta et al., 2014; Stegmann et al., 2017; Zhao et al., 60 2018). RALF4 and RALF19 were shown to be ligands for the CrRLK1Ls ANXUR1 (ANX1), 61 ANX2 and BUDDHA’S PAPER SEAL (BUPS) 1 and BUPS2 in the context of pollen tube 62 growth and integrity (Ge et al., 2017). RALF34 was recently shown to bind the CrRLK1L 63 THESEUS1 (THE1) to regulate growth upon cellulose biosynthesis inhibition (Gonneau et al., 64 2018). CrRLK1Ls, such as FER, ANX1/ANX2 and BUPS1/2, have been shown to work 65 together with the glycosylphosphatidylinositol-anchored protein (GPI-AP) LORELEI or related 66 (LRE)-like-GPI-anchored proteins (LLGs) to perceive RALF peptides ( Xiao et al., 2019; Ge 67 et al., 2019; Feng et al., 2019). Notably, RALF peptides can also bind LEUCINE-RICH 68 REPEAT EXTENSINS (LRX) proteins (Mecchia et al., 2017; Zhao et al., 2018; Moussu et al., 69 2020). The biochemical relationship between RALF perception by CrRLK1L/LLG complexes 70 and LRXs is still unclear. 71

There are 17 CrRLK1Ls, 11 LRXs and 4 LRE/LLGs in A. thaliana (Li et al., 2015; Franck et 72 al., 2018; Herger et al., 2020). As such, the diversity of potential assembly modules of the 73 RALF-perception/signalling axis could explain the functional plasticity of this family of 74 peptides. For instance, different RALF peptides might be secreted upon diverse stimuli in 75 different tissues and this, in turn, would trigger the formation of receptor complexes with a 76 combination of the above-mentioned assembly modules. There are numerous studies 77 analysing individual aspects of this complex signalling network (Blackburn et al., 2020). 78 However, the functional role of the majority of RALF peptides and their bioactivity is largely 79 unknown. In this study, we performed a family-wide analysis of AtRALF peptides using 80 seedling and root growth inhibition as read-outs. In addition to defining the core A. thaliana 81 Col-0 RALF family, we show that FER is required for full responsiveness to multiple RALFs, 82 suggesting a pivotal role of this receptor in potentially multiple RALF sensory complexes. 83

84

Results 85

Re-annotation of the Arabidopsis thaliana Col-0 RALF family 86

The AtRALF peptide family consists of more than 30 members, ranging from 34 to 39, 87 depending on the studies considered (Blackburn et al., 2020). In order to define the core 88 AtRALF family in the Col-0 ecotype, we compared the AtRALF peptide annotation used in 89 different publications and those available in the TAIR10 and UniProt databases (Table I). 90



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Notably, we found some inconsistency both in the number and identity proteins that comprise 91 the family. 92

Despite having low level of amino acid sequence similarity, RALF peptides, have diverse 93 conserved motifs, which have been shown to be important for diverse functions (Pearce et 94 al., 2001a; Matos et al., 2008; Srivastava et al., 2009; Pearce et al., 2010; Stegmann et al., 95 2017). For example, the dibasic site RR in the canonical AtS1P recognition site which is 96 important for processing of the peptide precursor (Fig 1A, Matos et al., 2008; Srivastava et 97 al., 2009). The N-terminal YI/LSY motif of the mature peptide which was shown to be 98 important for binding to the receptor and alkalinisation activity (residues 112 to 115, Fig 1A, 99 Pearce et al., 2010; Xiao et al., 2019). Additionally, there are other parts of the sequences 100 that show high conservation, such as the C-terminal RCRR(S) motif or the four di-sulfide 101 bond-forming cysteine residues that are important for the bioactivity of the peptide (residues 102 164 to 167, 130, 143, 159 and 165, respectively, Fig 1A, Pearce et al., 2001b; Haruta et al., 103 2008). Using these criteria, we re-analysed the published RALF family members and 104 propose the following updates to the annotation. 105

RALF17 is described as AT2G32890 in the aforementioned databases, while it corresponds 106 to AT2G32885 in some publications (Cao and Shi, 2012; Morato do Canto et al., 2014; 107 Stegmann et al., 2017). Protein sequence analysis however revealed that AT2G32890 lacks 108 the YI/LSY motif including the conserved internal I/L residues (Fig 1A). AT2G32890 109 additionally lacks the conserved RCRR motif (Fig1A), and notably also misses the four 110 cysteine residues that are positionally conserved across the RALF family (Fig 1A). 111 Furthermore, the phylogenetic tree inferred from the alignment of RALF aminoacid 112 sequences places AT2G32890 as an outgroup (Fig 1B). Altogether, we conclude that 113 AT2G32890 has been mistakenly annotated as a RALF peptide. 114

When searching for AT2G32885 (proposed in some publications as RALF17) in TAIR10 and 115 UniProt, we observed that this protein is annotated as RALF36. However, in other 116 publications RALF36 corresponds to AT2G32785 (Morato do Canto et al., 2014; Stegmann 117 et al., 2017). If AT2G32890 is not a RALF peptide as argued above, based on our phylogeny, 118 we propose that AT2G32885 actually corresponds to RALF17. This is in agreement with 119 previous studies (Morato do Canto et al., 2014; Stegmann et al., 2017). In contrast, we 120 propose to assign AT2G32785 to RALF36 (Table I). It has part of the YI/LSY motif and the 121 first cysteine bridge conserved, although it lacks the C-terminal RCRR motif (Fig 1A). 122 Phylogenetic analysis indicates that it is a distant RALF clustering with RALF37 (Fig 1B). 123 Furthermore, based on the information inferred from the alignment and the phylogenetic tree, 124 it is difficult to conclude definitely whether RALF36 (AT2G32785) and RALF37 (AT2G32788) 125 are genuine RALF peptides. As they present some of the important conserved residues, we 126 nevertheless included them in our list of core RALF family members (Table I, Fig S1). 127

RALF35 has two gene identifiers associated with it: AT4G14020 and AT1G60913 (Haruta et 128 al., 2014; Morato do Canto et al., 2014; Stegmann et al., 2017). Both have the highly 129 conserved I/L amino acid within the YI/LSY motif as well as the two flanking tyrosine residues 130 (Fig 1A). However, only AT1G60913 contains the four cysteine residues in the conserved 131 positions; while AT4G14020 has only one cysteine at a conserved position. Moreover, 132 AT4G14020 has part of the conserved RCRR motif, while AT1G60913 has not. Additionally, 133 AT4G14020 lacks a predicted signal peptide and is thus likely non-secreted; it also does not 134 cluster together with any other RALF in phylogenetic analysis (Fig 1B). Together, this 135 indicates that AT4G14020 is not a bona-fide RALF peptide (Table I). 136

137

Domain organization of the reannotated AtPRORALF family 138



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Like other plant polypeptide hormones, PRORALF genes encode pre-pro-peptides of 139 approximately 60-140 amino acids, which are predicted to undergo proteolytic processing to 140 release bioactive RALF peptides (Fig 2; Olsson et al., 2019). PRORALFs have a N-terminal 141 signal peptide for entry in the secretory pathway and the mature active peptide is located at 142 the C-terminal part (Matos et al., 2008). Although 11 PRORALF proteins have a predicted 143 subtilase cleavage site (RRXL, residues 101 to 104, Fig 1), so far only PRORALF23 and 144 PRORALF22 have been experimentally shown to be cleaved by the subtilase SITE-1 145 PROTEASE (S1P) (Srivastava et al., 2009; Stegmann et al., 2017; Zhao et al., 2018). The 146 protein domain organization of the PRORALFs that do not have the cleavage site suggests 147 that they might not need to undergo subtilase-mediated proteolytic cleavage in order to 148 release bioactive RALF peptides. Considering our proposed re-annotation of the AtRALF 149 family (Table 1), we depict the protein domain organization of the corresponding PRORALF 150 proteins in Figure 2. 151

The majority of the AtRALF peptides have growth inhibitory properties 152

One of the described functions of RALF peptides is to inhibit cell expansion and growth 153 (Blackburn et al., 2020), but this is based on testing of only a few family members (Morato do 154 Canto et al., 2014). Here, we screened 32 AtRALF peptides for their bioactivity using 155 seedling and root growth inhibition as read-outs (Fig 3). RALF11 and RALF12 were not 156 synthesized because of their high sequence similarity with RALF13 (identical mature 157 peptide), while RALF35, RALF36 and RALF37 could never be successfully synthesized, 158 despite several attempts. 159

We treated 5-day-old seedlings with different synthetic RALF peptides (Table II) for 7 days 160 before measuring seedling fresh weight or root length. We used the EF-Tu-derived peptide 161 elf18 and the plant-derived peptide AtPep1 as positive controls for the seedling and root 162 growth inhibition assays, respectively (Zipfel et al., 2006; Krol et al., 2010). Eighteen out of 163 32 (56 %) tested RALF peptides showed a significant seedling growth inhibition in the three 164 independent biological experiments (Fig 3A). 165

AtRALF genes and proposed receptor modules have a diverse expression patterns (Cao and 166 Shi, 2012) and seedlings fresh weight is primarily determined by shoot biomass; it is 167 therefore possible that no inhibition in the overall weight of the seedling may be observed if 168 the receptor of a specific RALF peptide is only expressed in roots. For this reason, we also 169 measured the primary root length after treatment with the different peptides. Strikingly, 20 out 170 of 32 (63%) of the tested RALF peptides were also able to induce root growth inhibition (Fig 171 3B). The 18 RALF peptides that inhibited whole seedling growth were also able to inhibit root 172 growth, while RALF22 and RALF25 only inhibited root growth. These data show that the 173 majority of exogenously applied RALF peptides have the ability to inhibit growth under the 174 conditions tested. 175

176

The majority of growth-inhibitory AtRALF peptides are FER-dependent 177

There are 17 CrRLK1L members in A. thaliana playing multiple and diverse roles, including 178 cell growth, reproduction and responses to the environment (Franck et al., 2018; Blackburn 179 et al., 2020). FER, the best characterized member of the family, is expressed throughout the 180 plant, and has already been shown to mediate recognition of RALF1, RALF23 and RALF22 181 in diverse physiological contexts (Haruta et al., 2014; Stegmann et al., 2017; Zhao et al., 182 2018). As such, we performed seedling and root growth inhibition assays in the knock-out 183 mutant line fer-4 in comparison with Col-0 (Fig 4). Surprisingly, fer-4 mutant seedlings were 184 insensitive to 15 out of 18 (83 %) RALF peptides that inhibit seedling growth in Col-0 (Figs 185 3A and 4A). In the case of root growth inhibition, fer-4 mutant seedlings were insensitive to 186



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16 out of 20 (75 %) RALF peptides that inhibit root length in Col-0 (Figs 3B and 4B). The 187 FER-independent RALF peptides that coincide between both assays are RALF28 and 188 RALF34, which have the same effect in Col-0 and in the mutant line fer-4 (Fig 4). In 189 comparison, RALF20 is still able to mildly inhibit seedling growth in fer-4 background but its 190 root growth inhibitory effect depends on FER (Fig 4). Interestingly, RALF32 and RALF33 are 191 FER-dependent in the seedling growth inhibition assay, but are still able to inhibit root length 192 in the mutant line fer-4 (Fig 4). Altogether, our data indicate that FER is involved in the 193 perception and/or signalling pathway of the majority of the RALF peptides tested in the 194 context of seedling and root growth inhibition. 195

196

Discussion and Conclusions 197

The biological function of RALF peptides is an area of growing interest. Several studies on 198 this family of peptides in different plant species have generated extensive knowledge about 199 their roles and functions (Blackburn et al., 2020). Yet, even in the model plant A. thaliana, 200 there is no agreement about the exact AtRALF family composition (Olsen et al., 2002; Cao & 201 Shi, 2012; Morato do Canto et al., 2014; Sharma et al., 2016; Campbell & Turner, 2017). 202 Here, we gathered information from previous publications and databases to perform 203 phylogenetic analyses of AtRALF isoforms, and propose a new consensus annotation (Table 204 I, Fig S2). By focusing on the model plant A. thaliana, we wanted to provide a more accurate 205 annotation of the RALF peptide family rather than trying to provide evolutionary information 206 on the family, as has been previously done (Cao & Shi, 2012; Sharma et al., 2016; Campbell 207 & Turner, 2017). Based on previously identified conserved motifs (Pearce et al., 2001; Olsen 208 et al., 2002; Matos et al., 2008; Pearce et al., 2010; Cao & Shi, 2012), we conclude that 209 AT2G32890 and AT4G14020 are not genuine RALF peptides. AT2G32890 was probably 210 annotated in the family as a mistake due to its close proximity on Chromosome 2 with the 211 genuine RALF17 (AT2G32885). In turn, AT4G14020 was probably annotated in the family as 212 a result of some degree of sequence similarity. It has been previously shown that tandem 213 duplications played a dominant role in the evolution of A. thaliana RALF peptides (Cao & Shi, 214 2012; Campbell & Turner, 2017). It is possible that some of the newly duplicated genes have 215 evolved under positive selection, causing changes in the protein sequence, which could 216 explain why some of the proposed RALFs lack some of the conserved motifs. Future 217 structural and physiological work, for example via protein chimeras, could investigate the 218 biological relevance of the individual protein motifs as previously done for the conserved 219 YI/LSY motif (Pearce et al., 2010). 220

Our results show that the majority of RALF peptides induce inhibitory effects on seedling 221 weight and primary root length when exogenously applied (Fig 3). This result is consistent 222 with a previous study that showed biological activity for nine recombinant RALF peptides 223 (Morato do Canto et al., 2014). Notably, despite being closely related and varying only in 224 seven amino acids, RALF19, but not RALF4, induced growth inhibition (Fig 3). RALF19 and 225 RALF4 were previously tested for alkalinisation activity, which RALF19 possesses but 226 RALF4 does not (Morato do Canto et al., 2014). This might indicate that the growth inhibition 227 activity is linked to the ability of these peptides to increase the pH of the extracellular space. 228 Additionally, treatment with RALF1 suppresses cell elongation of the primary root by 229 activating FER, which in turns causes the phosphorylation of the plasma membrane H+-230 ADENOSINE TRIPHOSPHATASE 2 (AHA2), which inhibits proton transport (Haruta et al., 231 2014). Whether this pathway is transferable to the rest of the family remains elusive. Future 232 work expanded to the whole family should determine this unclear link between pH and 233 growth. 234



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FER is the best studied member of the CrRLK1L family, and has been shown to be involved 235 in numerous physiological processes (Franck et al., 2018; Blackburn et al., 2020). FER was 236 recently shown to recognise diverse RALFs, such as RALF1, RALF22 and RALF23 (Haruta 237 et al., 2014; Stegmann et al., 2017; Zhao et al., 2018; Xiao et al., 2019). Here, we show that 238 FER is required for the inhibitory activity of the majority of RALF peptides in the context of 239 growth inhibition (Fig 4). FER is widely expressed throughout the plant (Lindner et al., 2012). 240 As our assays rely on the exogenous treatment with synthetic RALF peptides, our results do 241 not necessarily imply that FER is the receptor for the corresponding endogenous RALF 242 peptides. Nevertheless, it is interesting that not all growth-inhibitory RALF peptides are FER-243 dependent, which would argue for some level of specificity. Additionally, some RALF 244 peptides, such as RALF32 and RALF33, are FER-dependent when inhibiting seedling growth 245 but are still able to inhibit root growth in the fer-4 mutant line (Fig 4). This suggests that these 246 RALF peptides might be perceived by different receptor complexes in the root and in the 247 shoot. Also, it is notable that different CrRLK1Ls have recently been shown to work together 248 as part of hetero-multimeric protein complexes to mediate RALF perception or control most 249 likely RALF-regulated processes. For example, RALF4 and 19 are proposed to be perceived 250 by a complex involving ANX1/2 and BUPS1/2 controlling pollen tube growth and integrity (Ge 251 et al., 2017), while ANJEA (ANJ) and HERCULES RECEPTOR KINASE 1 (HERK1) regulate 252

pollen tube reception (Galindo‐Trigo et al., 2020). Also, while THE1 is the receptor for 253

RALF34, fer-4 is also affected in RALF34 responsiveness (Gonneau et al., 2018), suggesting 254 that THE1 and FER might form a heteromeric complex to control responsiveness to cellulose 255 biosynthesis inhibition. In our hands, however, RALF34-induced growth inhibition was similar 256 in Col-0 and fer-4 mutant background (Fig 4). This suggests that, while THE1 is the primary 257 RALF34 receptor, it might form distinct heteromeric complexes with different CrRLK1L-family 258 members depending on the context. 259

Our results provide the basis for the future identification of RALF-CrRLK1L ligand-receptor 260 pairs. It will however be essential to determine overlapping expression patterns of different 261 PRORALF and CrRLK1L genes across different organs, tissues and cell types, and during 262 different developmental stages using either transcriptional reporters (Gonneau et al., 2018) 263 or capitalizing on recent quantitative proteomics studies of the A. thaliana proteome (Zhang 264 et al., 2019; Mergner et al., 2020; Bassal et al., 2020). These approaches will guide 265 downstream biochemical/biophysical characterizations of ligand-receptor binding and 266 potential heteromeric CrRLK1L complexes, as well as the genetic characterization of 267 PRORALF and CrRLK1L genes, which otherwise can suffer from functional redundancy and 268 pleiotropic issues. For example, recently-developed approaches such as cell-specific 269 CRISPR/Cas9-mediated genome editing could be used to generate high-order receptor and 270 ligand mutants (Decaestecker et al., 2019; Wang et al., 2019). Together, these integrated 271 approaches will be needed to decipher the complex signalling network that RALF peptides 272 and their corresponding receptors weave in their native contexts. 273

Material and Methods 274

Plant growth and conditions 275

Arabidopsis thaliana seeds were surface-sterilized using 70 % and 100 % ethanol for 20 276 minutes and grown on ½ Murashige and Skoog (MS) media with 1% sucrose, adjusted to pH 277 5.8 using KOH, with or without 0.9 % agar at 20 °C and a 16h-photoperiod. The fer-4 seeds 278 were kindly provided by Alice Cheung (University of Massachusetts). 279

Peptides 280



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RALF peptides were synthesized (Table II) by SciLight Biotechnology LLC (www.scilight-281 peptide.com) with a purity of >85 %. All peptides were dissolved in sterile pure water for 282 usage and stored at -20 °C at a concentration of 1 mM. 283

Phylogenetic analysis 284

Multiple sequence alignments of the full-length or mature peptide sequences were created 285 using the MUSCLE algorithm with the MEGA X software (Kumar et al., 2018). Sequence 286 alignment was coloured according to sequence conservation and amino acid type using the 287 software Jalview. Phylogenetic rooted trees were constructed with the MEGA X software by 288 using the UPGMA algorithm with the default parameters. Bootstrapping was performed 1000 289 times. The inferred trees were visualized using iTOL (https://itol.embl.de/) (Letunic and Bork, 290 2019). 291

Seedling growth inhibition assay 292

Seeds were surface-sterilized and grown on MS agar plates for 5 days before transferring 293

individual seedlings in each well of a 48-well plate containing 500 L per well of MS medium 294

containing 1 M RALF, or 5 nM elf18 as control. Seedlings weight was measured 7 days 295

later. Control seedlings were grown under identical conditions in a peptide-free medium. The 296 experiments were repeated 3 times using independent biological replicates. Twelve 297 seedlings for each treatment were measured. 298

Root growth inhibition assay 299

Seeds were surface-sterilized and vertically grown on MS agar plates for 5 days before 300 transferring 6 seedlings to each well of a 12-well plate containing 4 mL per well of MS 301

medium containing 2 M RALF using 10 nM AtPep1 as control. Seedlings were transferred 3 302

days later to solid MS plates. Control seedlings were grown under identical conditions in a 303 peptide-free medium. Primary root length was measured by scanning the plates and 304 quantified using the software Fiji (https://imagej.net/Fiji) (Schindelin et al., 2012). 305 Experiments were repeated 3 times using independent biological replicates. Roots from 306 approximately 6 seedlings for each treatment and genotype were measured. 307

Statistical analysis 308

Statistical analysis was performed applying non-parametric Kruskal-Wallis multiple 309 comparison test, comparing every treatment to its respective mock control using Prism 8.0 310 (GraphPad Software). Similar significance levels were obtained when transforming the data 311 to normal distribution and performing a Two-way ANOVA test followed by Dunnett’s post-hoc 312 test using the software R. Two-tailed t-tests were performed to assess the significant 313 differences between Col-0 and fer-4 in growth inhibition experiments, using Prism 8.0 314 (GraphPad Software). 315

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Zipfel C, Kunze G, Chinchilla D, Caniard A, Jones JDG, Boller T, Felix G (2006) Perception 427 of the Bacterial PAMP EF-Tu by the Receptor EFR Restricts Agrobacterium-Mediated 428 Transformation. Cell 125: 749–760 429

430

Acknowledgments: 431

We thank all the members of the Zipfel group, and most particularly Marta Bjornson and 432 Isabel Monte, for fruitful discussions and guidance. We thank Joop Vermeer and Vinay 433 Shekhar for providing access to the plate scanner. This work was supported by the European 434 Research Council under the Grant Agreement no. 773153 (grant ‘IMMUNO-PEPTALK’), the 435 University of Zürich, and the Swiss National Science Foundation grant no. 31003A_182625. 436 C.M.F. is supported by a post-doctoral fellowship (EMBO ALTF 512-2019) from the 437 European Molecular Biology Organization. Authors have no competing interests. 438

All data is available in the manuscript or the supplementary materials 439



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predicted S1P cleavage site YI/LSY motif Conserved Cysteines(RRXL)

A

B

Fig. 1. Re-evaluation of the A. thaliana RALF family. (A) Alignment of AtPRORALFs, AT2G32890 andAT4G14020. Colour-code based on sequence conservation. The darker the colour, the more conserved the residue.Pink boxes indicate conserved motifs (B) Phylogenetic rooted tree of the AtRALF peptides, AT2G32890 andAT4G14020. UPGMA tree inferred from the MUSCLE alignment displayed in Fig 1A. SCR/SP11 sequence was usedto root the tree. RALFs highlighted in pink indicated those predicted to be cleaved by the protease S1P. Bootstrapvalues (1000 repetitions) above 50 % are represented by purple circles in the corresponding branches.

PRORALF21

PRORALF29

PRORALF15

PRORALF9

PRORALF13

PRORALF28

PRORALF2

PRORALF30

PRORALF25

PRORALF23

PRORALF31

PRORALF8

PRORALF33

SCR/SP11

PRORAL

F17

PRORALF11

PRORALF6

PRORALF12

AT4G14020

PRORALF14PRORALF24

PRORALF18

PRORALF32

PRORALF5

PRORALF

20

AT2G32890

PRORALF27

PRORALF34

PRORALF3

PRORALF26

PRORALF4

PRORALF10

PRORALF36

PRORAL

F19

PRORALF37

PRORALF35

PRORALF1

PRORALF22

PRORALF7

PRORALF16

RCRR motif



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1817 73117 18 721

21 22 1015756128 29 791

1817 7211817 721

29 30 8112928 821

28 29 751

2625 8913029 811

29 30 901

29 95301

Signal peptidePro-domainMature peptide

23 24 56 57 1101

27 28 72130 31 1051

23 24 1197170128 29 88 89 1381

22 23 6362 118122 23 74122 23 76127 28 117131 32 851

25 26 90122 23 76121 1135722 581

28 29 65 11764127 28 68 11667125 26 1297473129 30 791

22 23 71131 32 641

amino acid (aa) length

25 26 97126 27 71 72 1201

27 2832 33

23 24 57 58 1101036059

72111

PRORALF1PRORALF2PRORALF3PRORALF4PRORALF5PRORALF6PRORALF7PRORALF8PRORALF9PRORALF10PRORALF11PRORALF12PRORALF13PRORALF14PRORALF15PRORALF16PRORALF17PRORALF18PRORALF19PRORALF20PRORALF21PRORALF22PRORALF23PRORALF24PRORALF25PRORALF26PRORALF27PRORALF28PRORALF29PRORALF30PRORALF31PRORALF32PRORALF33PRORALF34PRORALF35PRORALF36PRORALF37

120 140100806040200

Figure 2. Schematic representation of A. thaliana PRORALFs domains.RALF peptides are expressed as protein precursors that require further processingsteps.They contain an N-terminal SP (in green), variable pro domain for thosewhich are predicted to be cleaved (pink) and a mature C-terminal part (blue).



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A

B

Fig. 3. Effect of AtRALF peptides on seedling and root growth inhibition. (A) Fresh weight of 12-day-oldseedlings grown in the absence (mock) or presence of 1 �M RALF peptides (n=12) using elf18 (100 nM) ascontrol. (B) Primary root length of 8-day-old seedlings grown in the absence (mock) or presence of 2 �MRALF peptides (n=6) using pep1 (10 nM) as control. Data from three independent experiments are shown(colours indicate different replicates). Upper and lower whiskers represent 1.5 times and −1.5 timesinterquartile range; upper and lower hinges represent 25% and 75% quartiles; middle represents median or50% quartile. Asterisks indicate significance levels of a Kruskall-Wallis’s multiple comparison test, eachtreatment was compared to their corresponding mock: ns (p-value > 0.05), * (p-value ≤ 0.05), ** (p-value ≤0.01), *** (p-value ≤ 0.001) and **** (p-value ≤ 0.0001).

0

5

10

15

mockelf18

RALF1

RALF2

RALF3

RALF4

RALF5

RALF6

RALF7

RALF8

RALF9

RALF10

RALF13

RALF14

RALF15

RALF16

RALF17

RALF18

RALF19

RALF20

RALF21

RALF22

RALF23

RALF24

RALF25

RALF26

RALF27

RALF28

RALF29

RALF30

RALF31

RALF32

RALF33

RALF34

weight(mg)

rep1

2

3****

****

******

******

***

***

****

****

**

**

**

**** **nsns

ns

ns

ns

nsns

ns

ns

ns

ns

nsns

****

** *

ns

0

1

2

3

4

5

mockpep1

RALF1

RALF2

RALF3

RALF4

RALF5

RALF6

RALF7

RALF8

RALF9

RALF10

RALF13

RALF14

RALF15

RALF16

RALF17

RALF18

RALF19

RALF20

RALF21

RALF22

RALF23

RALF24

RALF25

RALF26

RALF27

RALF28

RALF29

RALF30

RALF31

RALF32

RALF33

RALF34

length(cm)

rep1

2

3

**** ****

****

****

****

****

****

****

**** ****

********

****

****

**** ****

*** **

***

*

ns

nsns

ns ns ns nsns ns

ns

ns

ns

*



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A

B

Fig. 4 FER-dependency of AtRALF peptides inducing growth inhibition. (A) Fresh weight of 12-day-oldseedlings grown in the presence of 1 �M RALF peptides relative to mock (B) Primary root length of 8-day-oldseedlings grown in the presence of 2 �M RALF peptides relative to mock. Mock primary root length of Col-0(blue) and fer-4 (pink) correspond to 100 % in the y axis. Data from three independent repetitions are shown(colours indicate different replicates). Upper and lower whiskers represent 1.5 times and −1.5 timesinterquartile range; upper and lower hinges represent 25% and 75% quartiles; middle represents median or50% quartile. Asterisks indicate significance levels of a two-tailed T-test comparing each treatment in fer-4 tothe corresponding in Col-0: ns (p-value >0.05), * (p-value ≤ 0.05), ** (p-value ≤ 0.01), *** (p-value ≤ 0.001)and **** (p-value ≤ 0.0001).

0

100

200

300

RALF1

RALF2

RALF3

RALF4

RALF5

RALF6

RALF7

RALF8

RALF9

RALF10

RALF13

RALF14

RALF15

RALF16

RALF17

RALF18

RALF19

RALF20

RALF21

RALF22

RALF23

RALF24

RALF25

RALF26

RALF27

RALF28

RALF29

RALF30

RALF31

RALF32

RALF33

RALF34

weight

(normalize

dto

mock)

genotype Col−0 fer−4

rep 1 2 3****

**

****

****

****

****** ***

*

*

ns

ns

****

****

****

****

**

**

***

*

nsns

****

**** ****

** *

*

ns

ns

ns

ns

0

50

100

150

RALF1

RALF2

RALF3

RALF4

RALF5

RALF6

RALF7

RALF8

RALF9

RALF10

RALF13

RALF14

RALF15

RALF16

RALF17

RALF18

RALF19

RALF20

RALF21

RALF22

RALF23

RALF24

RALF25

RALF26

RALF27

RALF28

RALF29

RALF30

RALF31

RALF32

RALF33

RALF34

leng

th(normalize

dto

mock)

genotype Col−0 fer−4

rep 1 2 3

ns

ns

ns

ns ns

ns

nsns

ns

ns

ns

****

****

****

**** ****

****

***

***

***

*

**

ns

ns

**** **** ****

****

****

**

ns

ns



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Table I. Re-annotation of AtRALFs. Literature comparison of the AtRALF peptide annotation used indifferent publications (listed in chronological order) and databases. Pink rows indicate inconsistency inthe annotation and the final column coloured un green shows our proposed consensus AtRALFannotation.

ATG number TAIR Proteinaccession

Olsen etal.,2002

Cao and Shiet al., 2012

Haruta etal., 2014

Morato doCanto et al.,2014

Stegmannet al., 2017

Campbell &Turner etal.,2017

Consensus

AT1G02900 RALF1 Q9SRY3 RALF1 AT1G02900 RALF1 RALF1 RALF1 AT1G02900 RALF1

AT1G23145 RALF2 A8MQ92 RALF2 AT1G23145 RALF2 RALF2 RALF2 AT1G23145 RALF2

AT1G23147 RALF3 A7REE5 RALF3 AT1G23147 RALF3 RALF3 RALF3 AT1G23147 RALF3

AT1G28270 RALF4 Q6TF26 RALF4 AT1G28270 RALF4 RALF4 RALF4 AT1G28270 RALF4

AT1G35467 RALF5 A8MQI8 RALF5 AT1G35467 RALF5 RALF5 RALF5 AT1G35467 RALF5

AT1G60625 RALF6 A8MQM2 RALF6 AT1G60625 RALF6 RALF6 RALF6 AT1G60625 RALF6

AT1G60815 RALF7 A8MRD4 RALF7 AT1G60815 RALF7 RALF7 RALF7 AT1G60815 RALF7

AT1G61563 RALF8 Q1ECR9 RALF8 AT1G61563 RALF8 RALF8 RALF8 AT1G61563 RALF8

AT1G61566 RALF9 Q3ECL0 RALF9 AT1G61566 RALF9 RALF9 RALF9 AT1G61566 RALF9

AT2G19020 RALF10 O65919 RALF10 AT2G19020 RALF10 RALF10 RALF10 AT2G19020 RALF10


AT2G19040 RALF12 F4ISE1 RALF12 AT2G19040 RALF12 RALF12 RALF12 AT2G19040 RALF12

AT2G19045 RALF13 F4ISE2 RALF13 AT2G19045 RALF13 RALF13 RALF13 AT2G19045 RALF13

AT2G20660 RALF14 Q9SIU6 RALF14 AT2G20660 RALF14 RALF14 RALF14 AT2G20660 RALF14

AT2G22055 RALF15 A8MQM7 RALF15 AT2G22055 RALF15 RALF15 RALF15 AT2G22055 RALF15

AT2G32835 RALF16 A8MRM1 RALF16 AT2G32835 RALF16 RALF16 RALF16 AT2G32835 RALF16

AT2G32885 RALF36 A8MR00 - AT2G32885 - RALF17 RALF17 AT2G32885 RALF17

AT2G32890 RALF17 O48776 RALF17 - RALF17 - - AT2G32890 AT2G32890


AT2G33775 RALF19 Q6NME6 RALF19 AT2G33775 RALF19 RALF19 RALF19 AT2G33775 RALF19

AT2G34825 RALF20 A8MQL7 RALF20 AT2G34825 RALF20 RALF20 RALF20 AT2G34825 RALF20

AT3G04735 RALF21 A8MRF9 RALF21 AT3G04735 RALF21 RALF21 RALF21 AT3G04735 RALF21

AT3G05490 RALF22 Q9MA62 RALF22 AT3G05490 RALF22 RALF22 RALF22 AT3G05490 RALF22

AT3G16570 RALF23 Q9LUS7 RALF23 AT3G16570 RALF23 RALF23 RALF23 AT3G16570 RALF23

AT3G23805 RALF24 Q9LK37 RALF24 AT3G23805 RALF24 RALF24 RALF24 AT3G23805 RALF24

AT3G25165 RALF25 Q9LSG0 RALF25 AT3G25165 RALF25 RALF25 RALF25 AT3G25165 RALF25

AT3G25170 RALF26 Q0V822 RALF26 AT3G25170 RALF26 RALF26 RALF26 AT3G25170 RALF26

AT3G29780 RALF27 Q9LH43 RALF27 - RALF27 RALF27 RALF27 AT3G29780 RALF27

AT4G11510 RALF28 Q9LDU1 RALF28 AT4G11510 RALF28 RALF28 RALF28 AT4G11510 RALF28

AT4G11653 RALF29 A8MQP2 RALF29 AT4G11653 RALF29 RALF29 RALF29 AT4G11653 RALF29

AT4G13075 RALF30 A7REH2 RALF30 AT4G13075 RALF30 RALF30 RALF30 AT4G13075 RALF30

AT4G13950 RALF31 Q2HIM9 RALF31 AT4G13950 RALF31 RALF31 RALF31 AT3G16570 RALF31


AT4G15800 RALF33 Q8L9P8 RALF33 AT4G15800 RALF33 RALF33 RALF33 AT4G15800 RALF33

AT5G67070 RALF34 Q9FHA6 RALF34 AT5G67070 RALF34 RALF34 RALF34 AT5G67070 RALF34

AT1G60913 - A8MRK3 - - - RALF35 RALF35 AT1G60913 RALF35

AT4G14020 - O23263 - AT4G14020 RALF35 - - AT4G14020 AT4G14020

AT2G32785 - A8MR74 - - - RALF36 RALF36 - RALF36

AT2G32788 - A8MRN0 - - - RALF37 RALF37 - RALF37



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Pep�de Sequence of synthesized RALF pep�desRALF1 ATTKYISYQSLKRNSVPCSRRGASYYNCQNGAQANPYSRGCSKIARCRSRALF2 AQKVIGYPAIGRDGARGCSPKDPSCPQQPEKPYKRGCEKITRCERDRRKQAHLRNPRKVLDVVAVMAKAKQLYRALF3 GYPKQRFGEDRTNPYEEITPPLIGGCDPKNPQTCLPKQPANPYRRGCLKITRCQRDVRALF4 ARGRRYIGYDALKKNNVPCSRRGRSYYDCKKRRRNNPYRRGCSAITHCYRYARRALF5 AKRYIEYPPWQKHPCNPRFPTPDCYKRTPANPYRRGCTCISRCRRDCGGLSTWKKLLDTILKIPVRALF6 QTYINYNGMKGDIIPGCSSKNPKECVKIPAYSYNRGCEISTRCQRQQHSSSSRALF7 IKQINYKDLIKDTIPGCTSKNPKECVKVPANTYHRGCEISTRCHREQHSSSGRALF8 SVRYITYPAIDRGDHAVHCDKAHPNTCKKKQANPYRRGCGVLEGCHRETGPKPTRALF9 TRYITYPAIDRGDHAVHCDKAHPNTCKKKEANPYQRGCEKINRCRGGRALF10 VPVESRRKHLDYGVITKCAGPNPPPGCYPPGAQQKNPTPANEYRRGCSKITRCKRDRALF13 EPVESRNYIEYGAINKCAGPNPPPGCNPPGAEQKNPNPVNEYSRGCSKIHRCRRDRALF14 QASRYISYEALKKNLPDNRRGEPDQRDNPYRRSCDVHSHCYRFTNRALF15 TRYISYRGMNHGDHAIHCDKAHPNTCKKQVANPYRRGCGTIERCRRDTGRKRALF16 RTLGYGSIKGDRIPACGYKNPNSCVKQPVNHYHRGCEKITRCARDAARYTESFNVDDDESPIINLHRALF17 NSIGAPAMREDLPKGCAPGSSAGCKMQPANPYKPGCEASQRCRGGRALF18 AKRFIDYEALKKNLPAKPDGKPDKPDNKYRRGCSAATGCYRFTNRALF19 AARRSYISYGALRKNNVPCSRRGRSYYDCKKRKRANPYRRGCSVITHCYRQTSRALF20 AKTIGNPAMREDEPKGCPPGSPASCKMQPANPYKPGCEASQRCRGTRALF21 KVIGYPGLKPDLPCDHHRYPSACAPSEQPVNPYRRGCSKIHRCRRDSPPAPISRKMLIRGQLIYNNAYNAYIQYPRALF22 AQKKYISYGAMRRNSVPCSRRGASYYNCQRGAQANPYSRGCSTITRCRRRALF23 ATRRYISYGALRRNTIPCSRRGASYYNCRRGAQANPYSRGCSAITRCRRSRALF24 MMRKQYISYETLRRDMVPCQKPGASYYACRSGQANAYNRGCSVITRCARDTNDIKTRALF25 NDNKRKYLLLDPCLRPNAPPGCHRQPYKPRTPVNVYSRGCTTINRCRRVQNPRALF26 KYLNPGVLDRCRGPNPPAGCHPHNSHHKPRVPVHNYSRGCSRITRCRRDARALF27 SYKTLQKQPTCDGRIAGNCIGTVNPKGATCTYYQRCKRAARALF28 NEIGYPGMGRGDRQPGCDHGNCPPDQPANPYHRGCEKSKRCRGPDPPALPRKMIRALF29 AKRYIEYPIRLDLGKGCDPRFPTAACYKRTPANPYRRPCTTANRCRRSTSSTRVPSLKTFVEIPPMRALF30 AGGGKFLNPGVLDPCLRPNPPPECQAPGSAGKPRERVNEYKVGCSKLTRCDRVGRALF31 MAQKRYIGYETLRRDMVPCQKPGASYYDCRSGQANSYSRGCDTITRCARDTNDINTRALF32 QAHKLSYGALRRNQPACDGGKRGESYSTQCLPPPSNPYSRGCSKHYRCGRDSRALF33 ATTKYISYGALRRNTVPCSRRGASYYNCRRGAQANPYSRGCSAITRCRRRALF34 YWRRTKYYISYGALSANRVPCPPRSGRSYYTHNCFRARGPVHPYSRGCSSITRCRR

Table II. Sequences of RALF peptides synthesized. Aminoacidic sequence of the synthesizedRALF peptides.



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Acknowledgments:

We thank all the members of the Zipfel group, and most particularly Marta Bjornson and Isabel Monte, for fruitful discussions andguidance. We thank Joop Vermeer and Vinay Shekhar for providing access to the plate scanner. This work was supported by theEuropean Research Council under the Grant Agreement no. 773153 (grant 'IMMUNO-PEPTALK'), the University of Zürich, and theSwiss National Science Foundation grant no. 31003A_182625. C.M.F. is supported by a post-doctoral fellowship (EMBO ALTF 512-2019)from the European Molecular Biology Organization. Authors have no competing interests.


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