EWR1 as a SCOOP peptide activates MIK2-dependent immunity in Arabidopsis

ABSTRACT Phytocytokines are plant peptide signals perceived by plasma membrane-localized receptors in regulating plant immunity. It was recently reported that the phytocytokine SERINE-RICH ENDOGENOUS PEPTIDE12 (SCOOP12) is recognized by the receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) and activates plant immune responses and resistance to pathogens in Arabidopsis. Here, we show that Arabidopsis ENHANCER OF VASCULAR WILT RESISTANCE 1 (EWR1) and four EWR1 close propeptide homologs encode functional SCOOP peptides, which are able to activate immune responses via MIK2 and BRASSINOSTEROID INSENSITVE 1 (BRI1)-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE 4 (SERK4).


Introduction
The perception of immunogenic patterns by pattern recognition receptors (PRRs) initiates pattern-triggered immunity (PTI), which plays a critical role in plant resistance to potential pathogens. Plant plasma membrane-resident PRRs activate plant immunity by recognizing microbe-associated molecular patterns (MAMPs), damage-associated molecular patterns (DAMPs), and phytocytokines (Gust et al. 2017;Hou et al. 2019;Zhou and Zhang 2020;Tanaka and Heil 2021;Hou et al. 2021a).
SCOOPs are a family of Brassicaceae-specific peptides, which are derived from the C-terminus of precursors of secreted peptide precursors, PROSCOOPs (Gully et al. 2019). At least 23 SCOOPs (SCOOP1-23) have been identified in Arabidopsis (Hou et al. 2021b). Most SCOOP peptides have been confirmed to trigger hallmark PTI responses or/and resistance to pathogens (Gully et al. 2019;Rhodes et al. 2021;Hou et al. 2021b). SCOOPs are recognized by the LRR-RLK MALE DISCOVERER 1-INTER-ACTING RECEPTOR-LIKE KINASE 2 (MIK2), induce the dimerization between MIK2 and BAK1/SERK4, and activate immune responses, such as MAPK activation, ROS production, and cytosolic Ca 2+ concentration increase (Rhodes et al. 2021;Hou et al. 2021b). Disruption of SCOOP-MIK2 signaling in Arabidopsis leads to a significant increase in susceptibility to the vascular wilt fungus Fusarium oxysporum (Van der Does et al. 2017;Coleman et al. 2021;Hou et al. 2021b).
In this study, we identified another five SCOOP peptides (SCOOP24-28), all which are active for activating plant immune responses in Arabidopsis but not in tobacco and tomato. The SCOOP activation of plant immune responses and/or resistance to F. oxysporum is disrupted by the lossof-function mutation of MIK2. Tobacco leaves expressing MIK2 obtain an ability to activate ROS production upon SCOOP treatments.

Peptide synthesis
Peptides were synthesized at Scilight-Peptide (Beijing, China). The sequences of synthesized peptides were listed in Table 1.

Root growth assay
Cold stratified seeds were surface-sterilized with 70% (v/v) ethanol for 5 min and were sown on 1/2MS plates with or without 1 μM peptides. Ten-day-old seedlings grown on plates vertically in a growth chamber were photographed, and the root length of seedlings was measured using Image J (http://rsb.info.nih.gov/ij/).

ROS assay
ROS burst was determined by a luminol-based assay. Leaf discs from four-week-old Arabidopsis or N. benthamiana plants or cotyledon fragments from one-week-old tomato seedlings were incubated in 200 μL ddH 2 O overnight in a 96-well plate. Then, ddH 2 O was replaced by 200 µL of reaction solution containing 50 µM of luminol, and 10 µg/mL of horseradish peroxidase (Sigma-Aldrich) supplemented with or without 100 nM peptide. Luminescence was measured immediately after adding the solution with a luminometer (Navigator, Promega) with a 30-second interval for 30 min.
The total values of ROS production were indicated as means of the relative light units (RLU).

Measurement of cytosolic Ca 2+ concentration
One-week-old seedlings expressing p35S::Aequorin grown vertically on ½MS plates were incubated in a 96-well plate containing 200 μL solution with 1 mM KCl and 1 mM CaCl 2 . Aequorin was reconstituted by treating the seedlings with coelenterazine-h (Promega, Beijing, China) in the dark overnight at a final concentration of 10 µM. Luminescence was measured with a luminometer (Navigator, Promega) with a 30-second interval for 20 min. The values for cytosolic Ca 2+ concentrations were indicated as means of RLU.

Fusarium oxysporum disease assay
Fusarium oxysporum disease assay was followed as described previously with modifications (Wang et al. 2020). F. oxysporum strain Fo5176 was cultivated in liquid potato dextrose medium for 3 days at 28°C on a rotary shaker at 120 rpm. The cultivated fungi were filtered with eight layers of sterilized gauze and spores were collected by centrifugation at 1000 g. Spores were washed twice and resuspended with sterile water to adjust to a final concentration of 10 6 spores/mL. Ten-day-old Arabidopsis seedlings grown on 1/2MS plate were planted into a sterilized gas-permeable vessel chamber containing 160 g clay, 120 mL 1/2MS liquid medium, and 0.3 g sucrose (pH 5.7). After ten-day growth, each seedling was inoculated with 1 mL of spore suspension with or without 1 μM peptides by dripping around the root. The seedlings were imaged and the seedling survival rates were counted 10 days after inoculation.

Identification of new SCOOPs
In previous studies, 23 SCOOP family members have been identified in the Arabidopsis genome (Gully et al. 2019;Hou et al. 2021b). However, some SCOOP homologs may not have been identified through a comprehensive homology search using BLAST, due to the low identity of the amino acid sequence between SCOOPs. As reported previously, we treated ten-day-old Arabidopsis seedlings with 1 μM SCOOP12 for one and six hours and determined the gene expression change compared to non-treated seedlings through RNA sequencing analysis (Hou et al. 2021b). We identified 63 small genes encoding secreted peptide precursors which are upregulated by SCOOP12 peptide in Arabidopsis seedlings (Figure 1(a)). Eight of these genes encode PROSCOOP12 and PROSCOOP12 homologous proteins. Among the PROSCOOPs upregulated by SCOOP12, PROS-COOP12 is not most induced by SCOOP12, although PROSCOOP12 was reported to be highly induced by aggression by different pathogens (Gully et al. 2019). It suggests that SCOOP12 might regulate plant resistance to pathogens by initiating multiple SCOOP-mediated immune signaling.
In addition, we found four genes whose locus is closely linked (AT3G13432, AT3G13433, AT3G13435, and AT3g13437) in the list of the SCOOP12-upregulated genes. AT3g13437 was named EWR1 and has been reported to be involved in plant resistance to vascular wilt pathogens (Yadeta et al. 2014). Like SCOOPs, EWR1 is a Brassicaceaespecific gene. It promotes us to see if EWR1, as well as the three EWR1-linked proteins, belong to the SCOOP family. Sequence alignment does not show significant sequence similarity between these peptide precursors and PROS-COOPs. However, the four small proteins, like PROS-COOPs, harbor a typical SxS motif (where S is serine and x is any amino acid) in their C-termini (Figure 1(b)). These SxS motifs share high sequence similarity with SCOOPs ( Figure 1(c)), implying that they might belong to SCOOP family. Through blast searching using amino acid sequences of EWR1 and EWR1-linked genes in the TAIR database (https://www.arabidopsis.org/), we identified a homolog of AT3G13433 encoded by AT2g25297.2 transcripts. AT2g25297.2 also contains a SCOOP peptide (Figure 1(c)). Together, we identified another five SCOOP peptide candidates in Arabidopsis, and named them as SCOOP24 (AT3g13432), SCOOP25 (AT3g13433), SCOOP26 (AT3g13435), SCOOP27/EWR1 (AT3g13437), and SCOOP28 (AT2g25297) (Figure 1(c)).

SCOOP24-28 activate plant immune responses
To test if these peptides display activities similar to SCOOP12, we synthesized peptides corresponding to SCOOP24, SCOOP25, SCOOP26, SCOOP27, and SCOOP28, and analyzed their activities for immune activation. We found that all these peptides are able to differentially induce hallmarks of PTI responses, including ROS production (Figure 2(a,b)) and cytosolic calcium increase (Figure 2(c,d)), and root growth inhibition, at a concentration of 100 or 1000 nM (Figure 2(e,f)). In contrast to SCOOP12, the activities for ROS production and cytosolic calcium increase of all the peptides, especially SCOOP26, are lower (Figure 2(b,d)). The root growth inhibition by SCOOP27 is dose-dependent (Figure 2(g,h)). SCOOP27 activities for root inhibition are also weaker than Pep1 and SCOOP12 even at a higher concentration (Figure 2(i,j)). The SCOOP27 peptide is well dissolved. The relatively weak activity of the peptide may be determined by its sequence characteristics and stability. However, SCOOP25 and SCOOP26 show comparable activities for root growth inhibition to SCOOP12 and more potent activities than other peptides (Figure 2(e,f)). It is consistent with our previous report that the activities for immune activation by SCOOPs do not always match that of root growth inhibition (Hou et al. 2021b).

SCOOP27 trigger plant immunity through MIK2 and BAK1/SERK4
To determine if the SCOOP-triggered immune activation relies on the SCOOP receptor MIK2 and coreceptor BAK1/ SERK4, we tested SCOOP27-induced ROS production in mik2 and bak1/serk4 mutants. The results indicated that the SCOOP27-induced ROS production is fully abolished in mik2-1, mik2-2, and bak1-5/serk4 mutants (Figure 3(a)). SCOOP27 as well as SCOOP12 cannot induce ROS production in tobacco and tomato (Figure 3(b)). In contrast, flg22 strongly induced ROS in the Solanaceous plants as reported previously (Figure 3(b)) (Robatzek et al. 2007). Then, we constructed a binary vector harboring carboxylterminally FLAG-tagged MIK2 under CaMV 35S promote and transiently expressed MIK2 in tobacco leaves using Agrobacterium-mediated transient transformation. We found that SCOOP12 and SCOOP27 are able to dramatically induce ROS production in tobacco leaves expressing MIK2 but not in control plants (Figure 3(c)). Moreover, we found that the SCOOP27 suppression on root growth inhibition is also abolished in mik2 and bak1-5/serk4 mutants (Figure 3(d,e)). Together, our results suggest that SCOOP27 functions in a MIK2-and BAK1/SERK4-dependent manner.
A previous report showed that SCOOP27/EWR1 overexpression confers Arabidopsis resistance to vascular wilt pathogens, including Verticillium dahlia and F. oxysporum (Yadeta et al. 2014). The mik2 mutants are also markedly Figure 2. SCOOP24-28 activate hallmark PTI responses in Arabidopsis. (a) SCOOP27/EWR1 induces ROS production. Leaf discs of four-week-old Arabidopsis plant were treated with or without 100 nM indicated peptides, and ROS production was measured by a luminometer over 60 min. A real-time ROS production is presented as relative light units (RLU). Data are shown as mean ± s.e.m. (n = 8). (b) SCOOP24-28 induce ROS production. The experiment was performed as in A, and total ROS productions are presented as RLU. Data are shown as mean ± s.e.m. (n = 8). Different letters indicate a significant difference from others (P < 0.01, Oneway ANOVA followed by Tukey's test). (c) SCOOP27/EWR1 triggers cytosolic Ca 2+ increases. One-week-old transgenic Arabidopsis seedlings carrying p35S::Aequorin grown on ½MS plates were pretreated with coelenterazine in the dark overnight, followed by treatment with or without 100 nM SCOOP peptides. The luminescence units were detected by a luminometer over 15 min. The value for real-time cytosolic Ca 2+ concentration is indicated as means of RLU (n = 5). (d) SCOOP24-28 trigger cytosolic Ca 2+ increases. The experiment was performed as in C and maximum cytosolic Ca 2+ concentrations are indicated as means of RLU (n = 5). Different letters indicate a significant difference with others (P < 0.01, One-way ANOVA followed by Tukey's test). (e) Arabidopsis representative seedlings upon treatment with H 2 O or SCOOP27 peptides. WT seedlings were grown on ½MS plates with or without 1 µM peptides for 10 days. Scale bar, 1 cm. (f) SCOOP24-28 inhibit Arabidopsis root growth. WT seedlings were grown on ½MS plates with or without 1 µM of different peptides for 10 days. Quantification data of seedling root length are shown as mean ± s.e.m. (n ≥ 10). Different letters indicate a significant difference with others (P < 0.01, One-way ANOVA followed by Tukey's test). (g,h) SCOOP27 inhibits Arabidopsis seedling root growth in a dose-dependent manner. WT seedlings were grown on ½MS plates with SCOOP27 of indicated concentrations for 10 days. Seedling were imaged (G) and seedling root lengths (H) are measured after ten-day growth. Quantification data of seedling root length are shown as mean ± s.e.m. (n ≥ 10). Different letters indicate a significant difference with others (P < 0.01, One-way ANOVA followed by Tukey's test). Scale bar, 1 cm. (i,j) SCOOP27 activities for Arabidopsis root growth inhibition are weaker than that of Pep1 and SCOOP12. WT seedlings were grown on ½MS plates with 100 nM Pep1, 100 nM SCOOP12, or 1 µM SCOOP27 for 10 days. Seedling imaging and quantification data of seedling root length are shown as in G and H. Scale bar, 1 cm. more susceptible to F. oxysporum infections compared to WT plants ( Van der Does et al. 2017;Coleman et al. 2021;Hou et al. 2021b). To determine if SCOOP27/EWR1 triggers a MIK2-dependent pathogen resistance, we pretreated roots of Arabidopsis WT and mik2 seedlings with or without SCOOP27 peptides and tested the seedling resistance to F. oxysporum f. sp. conglutinans strain Fo5176. mik2 seedlings show no different growth phenotype and survival rate compared to WT plants in a sterile growth state (Figure 3  (f,g)). As reported previously, mik2 mutants are more susceptible to Fo5176 than WT plants (Figure 3(f,g)). SCOOP27 co-inoculation significantly enhanced the plant resistance to Fo5176 in WT plants but not mik2 mutants (Figure 3(f,g)). Therefore, SCOOP27-triggered a MIK2-depdendent resistance to Fo5176 in Arabidopsis.

Discussion
Twenty-three SCOOP peptides have previously been identified as phytocytokines, which activate MIK2-mediated immunity in Arabidopsis. In this study, we identified another five SCOOP peptides through analysis of the SCOOP12-upregulated genes and homologous blast using new identified SCOOPs. All these newly identified SCOOPs are active in the activation of plant immune responses. Therefore, a total of 28 SCOOPs have been identified in Arabidopsis so far. All these SCOOP peptides are about 15-20 amino acids in length with a SxS motif and are derived from the carboxyl ends of corresponding secreted peptide precursors. However, these SCOOP precursor genes seem to belong to different gene families although they all share a relatively conserved SxS motif. Even for the SxS motifs, sequence similarity among different SCOOP members is also low except for two completely conserved serines. That is why the five SCOOP members have not been identified previously. Likewise, it is possible that other unidentified SCOOPs may exist in other peptide precursors or other protein families in Arabidopsis.
Peptide signals are usually perceived by LRR-RLKs or LRRreceptor-like proteins (LRR-RLPs) (Olsson et al. 2019). In Arabidopsis, more than 10 peptide-receptor pairs have been identified to be involved in the regulation of various plant developmental processes and plant stress responses. Most of these peptide families have fewer than 10 members, and different peptide members in the same family are often recognized by more than one receptor. For example, eight Pep1 homologous peptides are perceived by two receptors, PEPR1 and PEPR2, and nine ROOT MERISTEM GROWTH FACTORs (RGFs) are perceived by five receptors, RGI1-RGI5 (Huffaker et al. 2006;Yamaguchi et al. 2006;Matsuzaki et al. 2010;Yamaguchi et al. 2010;Ou et al. 2016). However, Arabidopsis encodes a big Figure 3. SCOOP27/EWR1 triggers plant immunity and resistance to Fusarium oxysporum through MIK2. (a) SCOOP27/EWR1 induction of ROS production is compromised in mik2 and bak1/serk4 mutants. Leaf discs of four-week-old WT, mik2, and bak1/serk4 plants were treated with 100 nM SCOOP27 peptides, and ROS production was measured with a luminometer over 50 min. A real-time ROS production is presented as RLU. Data are shown as mean ± s.e.m. (n = 8). (b) SCOOP peptides are unable to induce ROS production in tobacco and tomato. Leaf discs of three-week-old tobacco discs or cotyledon pieces of one-week old tomato seedlings were treated with 100 nM flg22, SCOOP12, and SCOOP27 peptides, and ROS production was detected over 30 min. Maximum ROS productions as RLU are shown as mean ± s.e.m. (n = 8). (*P < 0.01, One-way ANOVA followed by Tukey's test). (c) SCOOP peptides induce ROS production in MIK2 expressed tobacco. Leaf discs of three-week-old tobaccos injected with A. tumefaciens GV3101 carrying pCAMBIA1300-Ubi::MIK2-FLAG (MIK2) or a blank vector (Vector) were treated with 100 nM SCOOP12 or SCOOP27 peptides, and ROS production was detected over 30 min. Maximum ROS productions as RLU are shown as mean ± s.e.m. (n = 8). (*P < 0.01, One-way ANOVA followed by Tukey's test). (d,e) SCOOP27/EWR1 inhibition of root growth is compromised in mik2 and bak1/serk4 mutants. WT, mik2, and bak1/serk4 mutant seedlings were grown on ½MS plates with or without 1 µM SCOOP peptides for 10 days (Scale bar, 1 cm) (D). Quantification data of seedling root length are shown in bar charts (E). Different letters indicate a significant difference from others (P < 0.01, One-way ANOVA followed by Tukey's test). Scale bar, 1 cm. (f,g) SCOOP27/EWR1 induces Arabidopsis resistance to Fo5176 through MIK2. The symptom of WT and mik2 seedlings with indicated treatments (F). Survival rates of seedlings are shown as mean ± s.e.m. (n = 12) (G). Each seedling of WT and mik2 was inoculated with 1 mL Fo5176 spore suspension (10 6 /mL) together with H 2 O or 1 μM SCOOP27 peptides. Different letters indicate a significant difference from others (P < 0.01, One-way ANOVA followed by Tukey's test). The experiment was repeated twice with similar results. SCOOP family which contains 28 members, and all SCOOP peptides tested previously and in this study trigger responses through one LRR-RLK, MIK2, though Arabidopsis also encodes a close MIK2 homolog MIK2L (Hou et al. 2021b). In addition, SCOOPs are orphan genes and evolved through rapid duplication (Arendsee et al. 2014;Gully et al. 2019). These clues indicate that SCOOPs play very important roles in plants and thus are under strong selection pressures. However, the SCOOP-MIK2 function in the regulation of plant immunity and other physiological processes remains largely unknown. Therefore, much effort is needed to invest toward understanding the SCOOP-MIK2 function in further studies.
SCOOP27 is also known as EWR1, which was identified as an enhancer of plant resistance to vascular wilt pathogens. In line with this, our study indicated SCOOP27 triggers Arabidopsis resistance to the vascular wilt pathogen F. oxysporum through MIK2 (Figure 3(f)). It was reported that SCOOP27/EWR1 overexpression enhances tobacco resistance to vascular wilt pathogens (Yadeta et al. 2014). However, no ROS production is induced by the SCOOP27 peptide in tobacco and tomato (Figure 3(b)), suggesting that SCOOP27 may not trigger pathogen resistance in Solanaceae plants. It also suggests that SCOOP27 might not be equivalent to the full length of EWR1 in plant resistance. It is possible that another peptide motif in EWR1 is perceived by tobacco to activate immunity or EWR1 owns antimicrobe activities as suggested previously (Yadeta et al. 2014).

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The search was supported by the Youth Innovation Technology Project of Higher School in Shandong Province [2020KJF013], and the Natural Science Foundation of Shandong Province [ZR2020MC022] to S.H.

Notes on contributors
Jie Zhang is a senior technician in biology. She is good at plant tissue culture and plant transformation. She has developed a variety of transgenic plants, including Arabidopsis, potatoes, and tomatoes.
Jinxiu Zhao is an undergraduate student at Shandong Jianzhu University in China. She has completed biochemistry and plant physiology during her college studies in the past two years. She likes to explore the mysteries of biology and hopes to be a biologist in the future.
Yifei Yang has isolated and identified lots of microorganism species from soil and wastewater in the past several years. His research interests are in the role of microorganisms in soil improvement and wastewater treatment.
Qixin Bao is a Masters's student at Shandong Jianzhu University in China. She is conducting his master's research on the role of microbial-plant symbiosis in soil remediation.
Yuxi Li is a technician in biology. He is good at culture and isolation. He has isolated a large number of plant pathogens, such as Fusarium oxysporum and Sclerotinia sclerotiorum.
Hongbo Wang has been working in the field of bioinformatics analysis. He is focusing on the migration of microorganisms in water and on land.
Shuguo Hou has broad expertise in plant molecular biology and biochemistry. He is particularly interested in the molecular mechanism of endogenous peptide signals involved in plant immune regulation. Some recent work in his group concerning the SCOOP and SCREW peptide functions in plant immune modulations has been published in Nature and Nature Communications.