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. 2026 Mar 22;28(5):1686–1697. doi: 10.1111/plb.70211

Chemical treatment rescues reduced growth of the autoimmune mutant chs3‐2D without compromising its immune responses

M Keijzer 1, M Liebers 1,4, J Seufer 2, L Voll 2, C Meier 3, S Hoth 1,✉
PMCID: PMC13358709  PMID: 41865387

Abstract

  • Plant development in many species including Arabidopsis relies on the accurate balance between growth and defence. A high degree of plant immunity is often accompanied by reduced growth. On the one hand, this growth–defence trade‐off depends on the redistribution of metabolic power to either growth or defence. On the other hand, its regulation requires distinct molecular signalling pathways. Reduced growth of the Arabidopsis autoimmune mutant chs3‐2D, which shows strong resistance to pathogens, has been rescued by the addition of the low molecular weight compound Ro 8‐4304. The application of Ro 8‐4304, however, also resulted in a decrease of immune responses to wild‐type levels.

  • We applied chemical modification of Ro 8‐4304 to identify a derivative that rescues growth of the autoimmune mutant chs3‐2D without compromising it immune capacity.

  • In a screening of Ro 8‐4304 derivatives, removal of a fluorine atom resulted in the derivative Ro‐A03 that rescued growth, but did not affect the increased immune gene expression in chs3‐2D. In the presence of Ro‐A03 chs3‐2D seedlings showed wild‐type‐like growth, but were more resistant to bacterial pathogens than wild‐type seedlings, in line with salicylic acid levels that were higher than in the absence of Ro‐A03.

  • We generated a compound by chemical modification that can efficiently rescue growth without decreasing the immune response of the chs3‐2D autoimmune mutant.

Keywords: Autoimmunity, chemical modification, chs3‐2D, growth–defence trade‐off, Ro 8‐4304, salicylic acid

We generated a compound by chemical modification that can efficiently rescue growth without decreasing the immune response of the chs3‐2D autoimmune mutants, thus chemically uncoupling its growth–defence trade‐off.

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INTRODUCTION

A central question during the life cycle of many plants is whether to grow or to defend against pathogens. Effective defence against pathogen infection is often associated with reduced growth in plants (Heil & Baldwin 2002; Tian et al. 2003; Kempel et al. 2011; Lozano‐Duran & Zipfel 2015). This apparent disadvantage of defence may be a reason for the high variability of immunity in plants (Todesco et al. 2010). However, plants are able to regulate the trade‐off between both growth and defence to define the optimal balance for their fitness and reproduction. This balance is also an important topic in plant breeding and thus agriculture, because breeding for maximal growth may compromise defence, whereas breeding for optimal defence may limit growth. As in both cases, yield would be decreased, it is important to find and understand mechanisms for the regulation of growth–defence trade‐offs in plants.

Plant defence relies on the recognition of pathogen infection on two different levels. At the plasma membrane, pattern recognition receptors (PRRs) recognize microbe‐associated molecular patterns (MAMPs) such as flagellin or chitin to initiate pattern‐triggered immunity (PTI). Signalling cascades are activated in PTI and induce various immune responses including an oxidative burst, changes in defence gene expression, production of antimicrobial phytoalexins, and callose appositions on the cell wall (Jones & Dangl 2006; Boller & Felix 2009; Monaghan & Zipfel 2012). Successful pathogens must suppress PTI and have evolved secretion systems to transfer effectors into the host cell to inhibit PTI (Galan et al. 2014; Macho & Zipfel 2015). The presence or the inhibitory function of effectors is recognized within the host cell by nucleotide‐binding leucine‐rich repeat receptors (NLRs). This recognition leads to the activation of effector‐triggered immunity (ETI), inducing the production of reactive oxygen species (ROS) and salicylic acid (SA) accumulation that eventually leads to a hypersensitve response (HR) and thus to cell death (Jones & Dangl 2006; Cui et al. 2015; Li et al. 2015; Sun et al. 2020). Activation of both PTI and ETI can thus reduce growth and eventually result in yield losses.

Considering a high demand of immune responses for energy, the growth–defence trade‐off may arise from the competition for resources, with reallocation from growth to immunity upon pathogen infection being a possible mechanism (Coley et al. 1985; Simms & Triplett 1994; Purrington 2000; Brown 2002). Indeed, the availability of carbon has been related to the susceptibility of plants to pathogens (Biemelt & Sonnewald 2006; Engelsdorf et al. 2013). This relationship between susceptibility and resource availability is influenced by environmental dynamics, including the availability of light in shaded locations or temperature fluctuations (de Vries et al. 2017). Although the competition for resources is important for growth–defence trade‐off, it has become clear in recent years that the decision to grow or to defend requires the crosstalk between growth and immune signalling, and that this decision for prioritization likely precedes the reallocation of nutrients. General molecular mechanisms are involved in the regulation of crosstalk between growth and immunity, such as chloroplast function, alternative splicing of NLR genes, miRNA‐dependent regulation, and hormone function (Huot et al. 2014; Eichmann & Schäfer 2015; Wasternack 2017; Figueroa‐Macias et al. 2021; Qi et al. 2024; Shen et al. 2024; Sun et al. 2024). Brassinosteroids (BRs) are hormones that regulate plant growth and development, but also influence immune responses in PTI (Albrecht et al. 2012; Belkhadir et al. 2012). The reception of BRs by its receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1) and of MAMPs by PRRs at the plasma membrane requires similar signalling components, including the co‐receptor BRI‐Associated Kinase 1 (BAK1) (Li et al. 2002; Nam & Li 2002; Chinchilla et al. 2007; Heese et al. 2007). Genetic analysis of Arabidopsis mutants lacking the expression of five repressors of jasmonic acid (JA) signalling and the light signalling receptor phyB suggested that growth and defence can be uncoupled, initiated by transcriptional rather than metabolic reprogramming (Campos et al. 2016). A frameshift mutation in one of the repressors, JAZ10, was sufficient to possibly uncouple the growth–defence trade‐off (Li et al. 2024). Upon pathogen infection, plants increase their SA content and SA‐dependent gene expression, resulting in increased defence and reduced growth (Pluharova et al. 2019; Peng et al. 2021). SA signalling requires complexes between the lipase‐like proteins ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN‐DEFICIENT 4 (PAD4), and SENESCENCE‐ASSOCIATED GENE 101 (SAG101) to transduce the signal into the nucleus (Wagner et al. 2013; Gantner et al. 2019; Lapin et al. 2019; Sun et al. 2021). Recently, it has been suggested that the interaction between EDS1 and DELLA proteins, which are repressors of GA signalling (Harberd 2003), contributes to the regulation of growth–defence trade‐off via feedback regulation (Li et al. 2019).

The use of suitable systems to unravel molecular mechanisms that regulate growth–defence trade‐offs, such as the activation of ETI, are very promising. The strong activation of ETI responses in the absence of pathogen infection is referred to as plant autoimmunity. The accumulation and/or activation of NLRs in several autoimmune mutants that have a compromised homeostasis of PTI regulators, often lacking their expression due to mutation, led to reduced growth and increased defence. These responses can be induced by shifting autoimmune mutants to lower temperatures that lead to the activation of NLR signalling (Jacob et al. 2013; Li et al. 2015). The combination of inducibility and easy detection of phenotypic changes renders plant autoimmunity a very good system to apply chemical genetic screening for compounds that are able to uncouple growth and defence. Generally, chemical screening has identified several compounds with bioactivity related to plant immunity (Schreiber et al. 2008; Noutoshi et al. 2012; Joglekar et al. 2018; Halder et al. 2019). Huang et al. (2016) used chilling sensitive 3, 2D (chs3‐2D) autoimmunity mutants in chemical genetic screenings. CHS3 encodes an atypical Toll/Interleukin 1 Receptor (TIR)‐type NLR protein that regulates autoimmunity via the SAG101 and EDS1 pathway (Yang et al. 2010; Bi et al. 2011). In chs3‐2D plants, a gain‐of‐function mutation leads to the activation of constitutive defence responses (Bi et al. 2011). The chemical genetic screening on chs3‐2D seedlings identified the compound Ro 8‐4304, which suppressed autoimmune phenotypes. In the presence of Ro 8‐4304, however, low temperature‐dependent growth arrest and immune marker gene induction were both inhibited. Thus, Ro 8‐4304 did not uncouple the growth–defence trade‐off in autoimmunity of chs3‐2D (Huang et al. 2016). Interestingly, the activity of Ro 8‐4304 was dependent on the methylosome, a large protein complex that possesses an arginine methyltransferase activity. Target proteins of the methylosome are directed to the small nuclear ribonucleoprotein (snRNP) complex, which is involved in mRNA splicing (Meister & Fischer 2002). Mutations of the methylosome genes ICln or SmDb3 in chs3‐2D icln‐2 and chs3‐2D smdb3 mutants, respectively, resulted in a decreased Ro 8‐4304 activity when compared with the Ro 8‐4304 activity in chs3‐2D mutants (Huang et al. 2016).

Here, we aimed to generate Ro 8‐4304 derivatives by chemical modification that inhibit growth arrest without affecting the strong activation of defence responses in the autoimmune mutant chs3‐2D. We found that the derivative Ro‐A03 rescued the reduced growth phenotype while preserving increased SA levels and the increased expression of immune genes in chs3‐2D mutants. In the presence of Ro‐A03, chs3‐2D plants showed wild‐type‐like growth but enhanced resistance to bacterial pathogens, suggesting that growth and defence responses were indeed uncoupled.

MATERIALS AND METHODS

Plant material and growth conditions

Seeds of Col‐0 and chs3‐2D were surface‐sterilized by using chlorine gas and sown in 96‐well plates on ½ MS‐medium containing 0.245% Murashige and Skoog (Duchefa, Haarlem, The Netherlands), 0.5% sucrose (Roth, Karlsruhe, Germany), and 0.8% phytoagar (Duchefa). Stock solutions were made by dissolving the compound in either DMSO (VWR International GmbH, Darmstadt, Germany), or water. The compounds dissolved in water were filter sterilized before use. Each well in the 96‐wells plate contained 3 μl from a stock solution and 147 μl ½ MS‐medium. Seeds were stratified at 4 °C for 48 h in the dark. Hereafter, they were grown under long‐day conditions (16 h of light, 8 h darkness) at the indicated temperatures, including 18 °C, 20 °C, 22 °C, and 24 °C. Plants were analysed after 3 weeks.

Synthesis of compounds

All commercially available chemicals and reagents were used without any purification. Solvents were distilled before use, in contrast to dry solvents bought from Acros Organics (Geel, Belgium). The synthesis of Ro 8‐4304 and its analogues is described in the Data S1.

Recording of spectra

The 1H‐, 13C‐, 19F‐, and 2D‐NMR spectra were recorded on the following Bruker (Billerica, Massachusetts, United States of America) instruments: Fourier‐300 (300 MHz/75 MHz), AV‐400 (400 MHz/100 MHz), DRX‐500 (500 MHz/125 MHz), and AVIII6000 (600 MHz/150 MHz). Chemical shifts were referenced to the solvent: Chloroform‐d (δ = 7.26 ppm in 1H‐NMR, δ = 77.16 ppm in 13C‐NMR), Methanol‐d4 (δ = 3.31 ppm and 4.87 ppm in 1H‐NMR, δ = 79.00 ppm in 13C‐NMR), DMSO‐d6 (δ = 2.50 ppm and 3.33 ppm in 1H‐NMR, δ = 39.52 ppm in 13C‐NMR), D2O (δ = 4.79 ppm in 1H‐NMR). Data were analysed with the MestReNova software (Mestrelab Research, Santiago de Compostela, Spain), and multiplicities are abbreviated as: s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublet of doublets, td = triplet of doublets, dq = doublet of quartets, t = triplet, tt = triplet of triplets, quint = quintet and m = multiplet.

ESI‐MS spectra were recorded on an Agilent 6224 ESI‐TOF mass spectrometer attached to an Agilent HPLC 1200 Series (Agilent Technologies, Santa Clara, California, USA). The EI‐MS spectra were recorded on a Thermo ISQ LT EI attached to a Thermo Trace 1300 (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The data were processed using MestReNova (Mestrelab Research).

Gene expression analysis

Plant material for quantitative PCR (qPCR) was collected after 21 days. The samples were immediately frozen in liquid nitrogen and stored at −80 °C until use. RNA was isolated using the innuPREP Plant RNA kit (Analytik Jena AG, Jena, Germany). The RNA was transformed to cDNA using the Qiagen QuantiTect® reverse transcription kit (Qiagen, Hilden, Germany). For analysis of immune gene expression, qPCR was performed on RT2 SYBR® Green qPCR master mix (Qiagen) treated samples using a Rotor‐Gene® Q (Qiagen). Gene specific primers (Table S1) were synthesized by Eurofins (Hamburg, Germany). The qPCR data were analysed using qbase+ software, version 3.0 (Biogazelle, Zwijnaarde, Belgium) (www.qbaseplus.com). All samples were standardized to transcription levels of the reference genes GAPDH (AT1G13440) and EIFα (AT5G60390). Student's t‐test analysis was used to determine the significance of different values.

Determination of salicylic acid (SA) levels

For the determination of SA levels, plants were grown for 3 days or 3 weeks on ½ MS‐medium in long‐day conditions at 18 °C in the presence of DMSO, Ro 8‐4304, and Ro‐A03, respectively. At the respective days of harvesting, 12 seedlings were pooled for each sample and stored at −80 °C after shock‐freezing in liquid nitrogen. Free SA and SAG were extracted as described previously (Sangster et al. 2007). HPLC separation of SA and the internal standard o‐anisic acid was performed with a binary gradient on an Agilent 1260 Infinity II system (Agilent Technologies, Inc.) equipped with a SecurityGuard Ultra guard Column (3 mm; Phenomenex) followed by an Agilent Poroshell 120 EC‐C18, 150 × 3 mm, 2.7 μm (Agilent Technologies, Inc.) at 40 °C. SAG samples were diluted 1:10 in the starting mobile phase prior to analysis. Elution began with 10% eluent A (25 mM KH2PO4, pH 2.6) and 90% eluent B (99:1 Acetonitrile:H2O) at a flow rate of 1 mL min−1, and the following gradient was applied: 0–1 min – 10% eluent B; 1–6 min – 10%–25% B; 6–9 min – 25%–50% B; 9–9.5 min – 50%–80% B; 9.5–11.5 min – 80% B; 11.5–12 min – 80%–10% B; 12–15 min – 10% B. The fluorescence detector was programmed to measure o‐anisic acid (excitation 305 nm, emission 365 nm, retention time: 5.3 min) and SA (excitation 305 nm, emission 407 nm, retention time 6.4 min). Peak identification and quantification were performed by comparison to authentic standards in the range of 1.25 to 125 ng.

Bacterial infection assays

Arabidopsis thaliana Col‐0 and chs3‐2D plants were grown for 14 days on ½ MS‐medium at 20 °C and long‐day conditions. The growth medium contained 50 μM DMSO, Ro 8‐4304, or Ro‐A03, respectively. Plants were vacuum infiltrated with the fluorescent strain PstDC3000‐GFP of Pseudomonas syringae pv tomato at OD600 = 0.002 (Oh et al. 2010), and leaf discs were collected 0 and 3 days after infection. These discs were ground in a 1.5 ml tube containing 10 mM MgCl2 (Roth). The bacterial solutions were serial diluted and plated on NYG‐agar plates containing tetracycline (Molekula GmbH, München, Germany). They were incubated at 28 °C for 48 h. Hereafter, colony‐forming units were counted and titres were calculated.

RESULTS

Suppression of autoimmunity by Ro 8‐4304 is temperature‐independent

The small molecule Ro 8‐4304 was able to suppress autoimmune responses, namely reduced growth and induced immune gene expression of chs3‐2D mutants grown at low temperature (Huang et al. 2016). We examined this effect for different temperatures and showed that this suppression, which can rescue growth of chs3‐2D to wild‐type levels, was independent of the applied temperature (Fig. 1). Ro 8‐4304 rescued the reduced growth of chs3‐2D at 18 °C, 20 °C, and 22 °C, with plants generally growing larger at higher temperatures. The observed morphology and the determined relative fresh weight indicated that Ro 8‐4304 was active in this temperature range (Fig. 1A,B). Note that chs3‐2D did not show growth arrest at 24 °C. Additionally, we tested whether the effect of Ro 8‐4304 was stable across the development of chs3‐2D plants. While growth of chs3‐2D mutants was greatly reduced in the absence of Ro 8‐4304, plant development was fully recovered in the presence of Ro 8‐4304 when grown at 22 °C for 3 weeks (Fig. 1C). Growth and development of wild‐type Col‐0 plants were not affected by Ro 8‐4304 treatment over the long term, indicating that Ro 8‐4304 is not a general growth promoting molecule (Fig. 1C and Huang et al. 2016).

Fig. 1.

Fig. 1

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Temperature dependence of the Ro 8‐4304 effect on chs3‐2D growth. (A) Morphology of chs3‐2D plants grown in in vitro cultures for 21 days on solid MS media containing 15 μM Ro 8‐4304 or DMSO for control, at the indicated temperatures. The scale bars represent 0.5 cm. (B) The measured fresh weight of chs3‐2D plotted against the mean value of chs3‐2D grown on DMSO at 22 °C. The data shows the means of three replicates, and error bars represent mean ± SD (n = 3, with 10 ± 2 plants each). An asterisk indicates a significant difference to the respective DMSO control (Student's t‐test, P < 0.05). (C) Morphology of wild‐type Col‐0 and chs3‐2D mutant plants grown for 3 weeks on solid medium in a sterile preserving jar at 22 °C in the absence and presence of Ro 8‐4304. The scale bars represent 2 cm.

Synthesis of Ro 8‐4304 derivatives by chemical modification

To identify derivatives of the small molecule Ro 8‐4304 that restore the reduced growth of chs3‐2D but do not suppress the expression of immune genes, we performed chemical modification of Ro 8‐4304 to alter different parts of the molecule. As a first step, we established the synthesis of Ro 8‐4304 following the protocol reported in the United States Patent no. 3674799A (Edenhofer & Spiegelberg, 1972). 4‐Hydroxybenzamide and epichlorohydrine were reacted overnight at room temperature (RT) in a NaOH solution to prepare 4‐oxiranylmethoxybenzamide. The product was reacted with 4‐(4‐fluorophenyl)‐1,2,3,6‐ tetrahydropyridine·HCl (PTP‐F) in EtOH, pre‐treated with triethylamine. This mixture was heated to reflux for 4 h, and Ro 8‐4304 precipitated upon cooling (Fig. 2A and Fig. S1). The Edenhofer reaction was used to synthesize various analogues by exchanging the starting materials. For example, replacement of PTP‐F by 4‐(4‐bromophenyl)‐1,2,3,6‐tetrahydropyridine·HCl gave the derivative Ro‐A01. By various changes, we were able to synthesize 14 different Ro 8‐4304 derivatives (Table S2 and Fig. 2B). A different synthetic route was used to prepare analogues Ro‐A13 to Ro‐A16 (Fig. S1). This route was adapted from Kubota et al. (2003) and the patent WO 2008096093A1 of (Ray et al. 2008). In this synthetic route, 4‐hydroxybenzamide, and for example, 1,3‐dibromopropane, were combined in ACN and K2CO3 was added. This was refluxed overnight to synthesize 4‐(4‐bromopropoxy)benzamide. After purification, this product was reacted with PTP‐F, similar to the reaction used to synthesize Ro 8‐4034. By changing the length of the alkane chain, for example, in 1,3‐dibromopropane, we were able to synthesize 4 derivatives with different chain lengths (Table S2 and Fig. 2B). The differences in structural elements or functional groups of Ro 8‐4304 in the 18 derivatives Ro‐A01 to Ro‐A18 could be used to identify parts that might be important for the activity of Ro 8‐4304.

Fig. 2.

Fig. 2

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Chemical structure of Ro 8‐4304 (A) and the synthesized Ro 8‐4304 derivatives (B). The name for each derivative is given below the respective structure. The chemical change in comparison to Ro 8‐4304 is indicated by the coloured parts.

Compound Ro‐A03 rescues growth while immune gene induction and salicylic acid levels remain high

The 18 newly synthesized Ro 8‐4304 derivatives were used in concentrations of 5 μM, 15 μM, 30 μM, 50 μM, 100 μM, and 200 μM to treat chs3‐2D mutant seedlings grown at 18 °C. While in the absence of Ro 8‐4304 in the DMSO control chs3‐2D showed the expected reduction of growth, Ro 8‐4304 restored growth at all concentrations (Fig. 3A). Growth in the presence of the two building blocks of Ro 8‐4304, namely, 4‐oxiranylmethoxy benzamide and PTP‐F (c.f. Fig. S1), as well as in the presence of the derivatives Ro‐A05, Ro‐A06, Ro‐A08, and Ro‐A13 to Ro‐A17 did not result in morphological changes when compared with growth in the DMSO control. Slight changes in morphology were observed in the presence of Ro‐A01, Ro‐A02, Ro‐A07, Ro‐A09 to Ro‐A12. The appearance of chs3‐2D plants in the presence of Ro‐A03, Ro‐A04, and Ro‐A18 suggested that the development was restored to a similar degree as for Ro 8‐4304 (Fig. 3A). To quantify these morphological changes, we determined the fresh weight of chs3‐2D seedlings grown for 3 weeks in the presence of the respective small molecules and concentrations (Fig. 3B). While in DMSO chs3‐2D seedlings were strongly reduced in growth, the presence of Ro 8‐4304 was sufficient to restore growth, indicated by a significant increase in the fresh weight (Fig. 3B). A similar increase in fresh weight was also determined for chs3‐2D seedlings grown in the presence of Ro‐A03, Ro‐A04, Ro‐A11, and Ro‐A18 (Fig. 3B). Except for the two starting compounds for the synthesis of Ro 8‐4304, which could not change growth of chs3‐2D, all other Ro 8‐4304 derivatives still restored growth but less effective than Ro 8‐4304. For better resolution, the significances were given in separate graphs (Fig. S2).

Fig. 3.

Fig. 3

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Activity of the Ro 8‐4304 derivatives on growth and immune response in chs3‐2D seedlings. Plants were grown for 21 ± 1 days at 18 °C and at concentrations of 5 μM (dark blue bars in (B)), 15 μM (red), 30 μM (grey), 50 μM (yellow), 100 μM (light blue), and 200 μM (green) of the respective derivative. (A) Morphology of chs3‐2D seedlings after treatment with different concentrations of the Ro 8‐4304 derivatives. (B) Fresh weight of chs3‐2D relative to the mean fresh weight of chs3‐2D grown in the presence of 0.01% DMSO. The data show the mean values of three replicates, and error bars represent mean ± SD (n = 3, with 8–12 plants each). Statistical significances of the differences are given in Fig. S2. (C) Relative expression of the PR1 gene in chs3‐2D seedlings grown at 18 °C for 21 days and treated with the respective derivative at a concentration of 15 μM. Mean values of three replicates each are given, and error bars represent the standard deviation. The carets indicate a significant difference of P < 0.05 in comparison to the values of chs3‐2D treated with 0.01% DMSO. The asterisks indicate a significant difference of P < 0.05 in comparison to the values of chs3‐2D treated with 15 μM Ro 8‐4304. The hashtags indicate a significant difference of P < 0.05 in comparison to both, to the values of chs3‐2D treated with 0.01% DMSO and to the values of chs3‐2D treated with 15 μM Ro 8‐4304. The significances were calculated using the Student's t‐test.

In addition to the described morphological changes, we analysed the expression of the immune genes PR1 and NIMIN1 in chs3‐2D seedlings grown in the presence of the respective synthesized compounds to investigate immune responses on the molecular level (Fig. 3C and Fig. S3). No significant expression of both genes was observed in wild‐type plants. As expected, the expression of both PR1 and NIMIN1 was on a high level in chs3‐2D seedlings grown in the DMSO control, because autoimmunity was induced at 18 °C. The presence of Ro 8‐4304 in the growth medium reduced the expression of PR1 to almost wild‐type levels and significantly diminished the expression of NIMIN1. While most Ro 8‐4304 derivatives that restored growth of chs3‐2D only partially also mediated reduced expression of immune genes, the derivative Ro‐A03 was an exception. Ro‐A03 restored growth of chs3‐2D to comparable levels as Ro 8‐4304 did (Fig. 3B). However, in the presence of Ro‐A03 the expression of PR1 and NIMIN1 was similar to the expression in the DMSO control (Fig. 3C and Fig. S3). In a next step, we tested whether a higher concentration of Ro‐A03 of 50 μM, which restored growth more efficiently than a concentration of 15 μM (Fig. 3B), resulted in identical expression of immune genes. Indeed, the expression of all tested genes PR1, NIMIN1, SID2, EDS1, and PAD4 was identical in chs3‐2D seedlings grown in the presence of DMSO or Ro‐A03, respectively (Fig. 4A). Only the expression of PR2 was slightly lower in the presence of Ro‐A03. As the induction of the studied immune genes is triggered by SA signalling, the SA pool was examined in wild‐type and chs3‐2D plants grown in the absence and in the presence of Ro 8‐4304 or Ro‐A03, respectively. Whereas in all conditions SA levels stayed low in Col‐0 wild‐type plants, free and conjugated SA increased in chs3‐2D seedlings (Fig. 4B). Incubation for 3 days on Ro 8‐4304 or Ro‐A03 resulted in a slight decrease of SA when compared with the DMSO control. Whereas the SA content was further increased in chs3‐2D plants grown for 21 days on DMSO, the SA content in chs3‐2D plants was fully reduced back to wild‐type levels after 21 days of growth in the presence of Ro 8‐4304. In chs3‐2D plants grown for 21 days in presence of Ro‐A03, the SA content was also reduced with respect to the DMSO controls. However, the SA content stayed on a level that was about 45‐fold and 10‐fold higher when compared with the levels in wild‐type plants or in chs3‐2D plants grown for 21 days in the presence of Ro 8‐4304, respectively (Fig. 4B).

Fig. 4.

Fig. 4

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Immune responses in chs3‐2D seedlings in the absence or presence of 50 μM Ro‐A03. (A) Defence gene expression in chs3‐2D seedlings in the absence or presence of 50 μM Ro‐A03. Plants were grown at 18 °C for 21 days. The relative expression is shown for the indicated immune genes PR1, NIMIN1, PR2, SID2, EDS1, and PAD4. The data represent mean ± SD (n = 3). Different letters indicate significant differences between groups, whereas identical letters denote no significant differences (Student's t‐test, P < 0.05). (B) SA content in 3‐week‐old chs3‐2D and Col‐0 wild‐type seedlings grown at 18 °C in the absence or presence of 50 μM Ro‐A03 or Ro 8‐4304. Levels of free SA (white) and conjugated SA (SAG) (grey) are shown for Col‐0 and chs3‐2D plants that were incubated for 3 and 21 days, respectively. The data represent the mean ± SE of five biological replicates with 12 plants each. Different letters on top of the bars indicate significant differences, whereas identical letters denote no significant differences (two‐way Anova, LSD test, P < 0.05).

Apparently, the restoration of growth by Ro‐A03 was uncoupled from immune gene induction and SA content in chs3‐2D seedlings, thus suggesting that chs3‐2D plants with restored growth may be more resistant to pathogens.

Compound Ro‐A03 conferred higher pathogen resistance to chs3‐2D plants with restored growth

The maintained induction of immune gene expression in chs3‐2D, in which growth was restored by Ro‐A03, prompted us to test for pathogen resistance of chs3‐2D in the presence of Ro‐A03. For that purpose, we grew chs3‐2D seedlings for 14 days at 20 °C in the presence of 50 μM Ro‐A03, of Ro 8‐4304 as positive control, and of DMSO as negative control. At day 14, seedlings were infected with the Pseudomonas syringae pv tomato strain P.s.t. DC3000‐GFP (Oh et al. 2010). In the presence of Ro 8‐4304 plant growth was restored in chs3‐2D (Fig. 1). However, the reduction of immune gene expression to wild‐type levels by Ro 8‐4304 that was previously published and confirmed for additional immune genes (c.f. Figs 3 and 4; Fig. S3; Huang et al. 2016) resulted in enhanced growth of P.s.t. DC3000‐GFP and thus in decreased resistance in chs3‐2D seedlings (Fig. 5). In contrast to Ro 8‐4304, its derivative Ro‐A03 restored growth but allowed for higher expression of immune genes (Figs 3 and 4; Fig. S3). In line with that bacterial growth in chs3‐2D seedlings grown in the presence of Ro‐A03 was reduced, indicating resistance to P.s.t. DC3000‐GFP (Fig. 5). While bacterial growth in chs3‐2D in the presence of Ro 8‐4304 was as high as in wild‐type plants, the addition of Ro‐A03 significantly reduced bacterial growth (Fig. 5), indicating that immune responses stayed high although growth was restored to wild‐type‐like growth.

Fig. 5.

Fig. 5

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The effect of Ro‐A03 on resistance of chs3‐2D to the bacterial pathogen P.s.t. DC3000. Col‐0 and chs3‐2D plants were infected with the Pseudomonas syringae pv tomato strain P.s.t. DC3000‐GFP in the presence of DMSO, 50 μM Ro 8‐4304, or 50 μM Ro‐A03. Plants were grown for 2 weeks at 20 °C prior to vacuum infiltration with P.s.t. DC3000‐GFP. Quantification of colony‐forming units (CFU) at 0 and 3 days post‐inoculation. Data represent means of three replicates with four plant discs per replicate. Error bars are ± SD. Different letters indicate significant differences between groups, whereas identical letters denote no significant differences (Student's t‐test, P < 0.05).

The dependence of Ro 8‐4304 activity on the methylosome was marginally changed by the chemical modification in Ro‐A03

The rescue of growth in chs3‐2D mutants in the presence of Ro 8‐4304 required an intact methylosome. The effect was lost in chs3‐2D smd3b and chs3‐2D icln‐1 double mutants that lack expression of the methylosome components SmD3b and AtICln, respectively (Huang et al. 2016). To investigate whether this dependence was maintained for the effect of Ro‐A03, we investigated the growth of icln‐2 single and chs3‐2D icln‐2 double mutants in the presence of Ro‐A03 in comparison to chs3‐2D. In line with previous data, growth of icln‐2 mutants was not affected by Ro 8‐4304 (Huang et al. 2016), and was also not changed upon treatment with Ro‐A03 (Fig. 6A,B). The growth rescue in chs3‐2D icln‐2 double mutants in the presence of Ro 8‐4304 and Ro‐A03 was reduced for both treatments to almost the same level (Fig. 6A,B), indicating that the dependence on the methylosome did not generally change. Due to the small but significant difference between both treatments we can, however, not exclude that the chemical modification in Ro‐A03 may slightly affect methylosome function.

Fig. 6.

Fig. 6

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The impact of Ro‐A03 on the methylosome mutant icln‐2. (A) Morphology of Col‐0, chs3‐2D, icln‐2, and chs3‐2D icln‐2 seedlings grown for 21 days at 18 °C on DMSO control or in the presence of Ro 8‐4304 and Ro‐A03, respectively, in a concentration of 50 μM. (B) The measured fresh weight of the mutants plotted relative to the mean value of Col‐0 grown on 0.02% DMSO. The data show the means of three replicates, and error bars represent ± SD (n = 3, with 8–12 plants each). Different letters indicate significant differences between groups, whereas identical letters denote no significant differences (Student's t‐test, P < 0.05).

DISCUSSION

Plant growth and immunity are regulated by small organic molecules, including plant hormones. Chemical genetic screening has proven to be a powerful tool for identifying chemical compounds that regulate growth and immunity (Lepri et al. 2023). The inhibitor of cell expansion hypostatin, compounds for priming immune responses or altering growth, regulators of hypersensitive cell death, PTI regulators, effectors of hormone signalling, small molecules affecting strigolactone accumulation, and the seed germination inhibitor pyrabactin, which was used to identify the abscisic acid (ABA) receptor PYRABACTIN RESISTANCE 1, were identified in such screens (Serrano et al. 2007; Zhao et al. 2007; Park et al. 2009; Tsuchiya et al. 2010; Noutoshi et al. 2012; Noutoshi & Shirasu 2018; Chini et al. 2021; Emmenecker et al. 2025). High‐throughput screening also led to the identification of Ro 8‐4304 that has been shown to rescue reduced growth of the autoimmune mutant chs3‐2D to wild‐type growth and development (Huang et al. 2016; Fig. 1). Here, we aimed to change and eventually improve the properties of Ro 8‐4304 and synthesized 18 derivatives to study their properties. The synthesis and targeted modification of growth‐ and immunity‐affecting compounds is a promising approach in fundamental research and in agriculture (Zhou & Wang 2018). Chemical modification generated various halogenated derivatives of SA by the addition of chloro and fluoro atoms, respectively, that induced higher resistance to pathogens than SA (Conrath et al. 1995; Silverman et al. 2005). The SA derivative benzoylsalicylic acid (BzSA) was modified by chemical synthesis to produce different compounds that were slightly more active than BzSA (Kamatham et al. 2017). Activity of several synthesized derivatives of the defence‐regulating plant hormone jasmonic acid (JA) or of the JA mimic coronatine was also proven (Krumm et al. 1995; Pluskota et al. 2007; Monte et al. 2014). Synthetic JA agonists were recently shown to promote small changes in defence without reducing plant yield in rice, and may open new avenues for disease control (Xiao et al. 2025).

The results of chemical modification of Ro 8‐4304 shown in Figs 2 and 3; Figs S2 and S3 will be discussed in the following paragraph. Ro 8‐4304 consists of two major parts that contain ring structures and that are connected with each other via a linker. Both parts were important for the efficiency of Ro 8‐4304, because both building blocks 4‐oxiranylmethoxy benzamide and PTP‐F, which resemble the two major parts, failed to achieve rescue of chs3‐2D autoimmunity. In derivatives Ro‐A13 to Ro‐A16, we changed the length of the linker and deleted also the hydroxy group comprised in the linker. These changes resulted in the loss of activity with respect to the reduction in growth, and immune gene expression was also not significantly changed. Derivative Ro‐A17 has a slightly longer linker, but still comprised the hydroxy group. Treatment with Ro‐A17 resulted in an intermediate rescue of growth, and immune gene expression upon Ro‐A17 was comparable to Ro 8‐4304. Apparently, the hydroxy group in the linker region is important for Ro 8‐4304 activity. Introducing an additional methyl group next to the hydroxy group increased the capacity of Ro‐A18 to rescue growth and to decrease immune gene expression. The electron donating property or the steric effects of the methyl group thus did not generally change the efficiency of the hydroxy group Ro 8‐4304. Chemical modifications of the benzamide side of Ro 8‐4304 changed Ro 8‐4304 properties differently. Removing the oxygen from the benzamide (Ro‐A07) and exchanging the acid amide by fluorine (Ro‐A09) or an ethyl group (Ro‐A12), respectively, rescued growth of chs3‐2D, but less effective than Ro 8‐4304, although the expression of immune genes appeared slightly reduced compared with Ro 8‐4304. Changing the amine for a methyl group (Ro‐A11) led to almost the same degree of growth rescue. Taken together, chemical modifications of the benzamide side did not strongly affect the properties of Ro 8‐4304. Only when introducing a chemical group for the acid amide that was much larger (Ro‐A08), the reduced growth could not be rescued. Correspondingly, the expression of PR1 was still high upon Ro‐A08 treatment. Finally, we introduced changes to the fluorine atom in Ro 8‐4304. This electron withdrawing atom was changed for bromine (Ro‐A01), which has a weaker electron withdrawing effect, but a higher atomic radius. Nonetheless, this modification was not very effective. Changing the fluorine to an electron donating alcohol group (Ro‐A02) also resulted in properties comparable to Ro‐A01, indicating that the electronic properties were not important for Ro 8‐4304 activity. Introducing larger groups in Ro‐A05 and Ro‐A06 did also not strongly change the properties of Ro 8‐4304, except for a less pronounced growth rescue, suggesting that the increased size prevents these compounds from binding to a putative binding protein. Reduced growth, however, was rescued as efficiently as in Ro 8‐4304 when removing the fluorine (Ro‐A03) or even a larger part (Ro‐A04), showing that this side is less important for the impact of Ro 8‐4304 on growth. Interestingly, although still rescuing growth, Ro‐A03 did not reduce immune gene expression (Figs. 3 and 4; Fig. S2), indicating that growth and immunity were not coupled anymore. In line with that defence of chs3‐2D was as effective in the presence of Ro‐A03 as in the presence of DMSO only (Fig. 5). Taken together, chemical modification of Ro 8‐4304 generated changes in its properties, in particular in the derivative Ro‐A03 lacking the fluorine atom. A similar effect on activity was seen in SA signalling, where the addition of a fluorine atom to SA increased its potential for plant defence (Silverman et al. 2005).

The mechanism for recognition of Ro 8‐4304 is not known, as no binding protein was found so far. It has been previously shown that activity of Ro 8‐4304 on chs3‐2D depends on components of the methylosome, which in eukaryotic cells plays a role in the biogenesis of small nuclear ribonucleoproteins required for spliceosome function, and splicing has been suggested to regulate growth–defence trade‐offs (Meister & Fischer 2002; Huang et al. 2016; Sun et al. 2024). We determined that Ro 8‐4304 and Ro‐A03 likely require the same molecular mechanism for the rescue of growth in chs3‐2D plants (Fig. 6). To unravel the signalling pathway that is used by Ro 8‐4304 and Ro‐A03, it will be important to identify a binding protein. This may also help to investigate why Ro‐A03 is able to rescue growth of chs3‐2D without reducing the expression of immune genes and thus resistance. We could show that in the presence of Ro 8‐4304 and Ro‐A03, respectively, growth of chs3‐2D plants was rescued. The accumulation of SA to 574.2 μg/gFW−1, which was observed in chs3‐2D autoimmunity under control conditions, was less pronounced already after 3 days of growth in the presence of Ro 8‐4304 or Ro‐A03 and was compromised to a large extent after 21 days of growth in the presence of either of the two chemicals (Fig. 4). This indicated that treatment with Ro 8‐4304 or Ro‐A03 generally leads to suppression of SA accumulation in the autoimmune mutant chs3‐2D, regardless of whether treatment is short (3 days) or long (21 days) and apparently more pronounced over the course of treatment. Interestingly, longer treatment resulted in differences in the SA content between Ro 8‐4304‐ and Ro‐A03‐treated chs3‐2D plants. When grown in the presence of Ro‐A03 for 21 days, the SA content of chs3‐2D mutants was still significantly increased (63.7 μg/gFW−1) compared with the SA content in chs3‐2D seedlings grown for 21 days in the presence of Ro 8‐4304 (6.3 μg/gFW−1) or in wild‐type plants (1.4 μg/gFW−1) (Fig. 4B). Such a moderate but significant SA accumulation suggested that Ro‐A03‐treated chs3‐2D plants, in contrast to Ro 8‐4304‐treated chs3‐2D plants, may have higher resistance to pathogen infection, because it has been shown previously that only 10 μg/g−1 fresh weight (gFW) of total SA or 50 μg/gFW−1 of SAG were necessary for an effective immune response in Arabidopsis (Wildermuth et al. 2001; Yang et al. 2010). Based on the measurements taken after 3 and 21 days of treatment, we decided to test resistance of Ro‐A03‐treated plants after 14 days of growth and exposed the plants to bacterial pathogens for 3 days in accordance with standard infection protocols. We were indeed able to show that the chs3‐2D plants after longer treatment with Ro‐A03 for these 17 days were more resistant to a bacterial pathogen than chs3‐2D plants treated with Ro 8‐4304 (Fig. 5). However, the suggested correlation between the higher SA content with higher resistance in Ro‐A03‐ versus Ro 8‐4304‐treated chs3‐2D plants has still to be proven. The exact involvement of SA‐dependent processes, which may lead to the sustained immune gene expression and resistance of Ro‐A03‐treated compared with Ro 8‐4304‐treated chs3‐2D plants, needs to be investigated in future studies. This could also help to clarify how the effect at the molecular level differs between Ro 8‐4304 and its derivative Ro‐A03. Taken together, Ro‐A03 mediated uncoupling may be the starting point to unravel additional regulations of growth–defence trade‐offs.

Generally, plant hormones are small organic molecules that mediate the regulation of growth and defence (Huot et al. 2014; Berens et al. 2017; Aerts et al. 2021). The performance of plants depends on the successful recognition of changes in the environment and the implementation into an integrated response to abiotic and biotic stimuli. This often involves the synthesis of hormones and changes in hormone signalling. As plant hormones not only regulate growth and development, but are also involved in the regulation of immune responses (Tsuda et al. 2009), hormone crosstalk may be central to the regulation of growth–defence trade‐off. This has been observed in several interactions between the classical defence‐related hormones JA or SA and hormones, which were described as growth‐regulating hormones, including ABA, GA, and ethylene (Pieterse et al. 2009; Shigenaga & Argueso 2016). For example, the MYC transcription factor MYC2 and the ERF transcription factor ORA59 are hubs in JA signalling. Both transcription factors represent important points of intersection with other hormone signalling pathways (Aerts et al. 2021), such as ABA signalling through the interaction of MYC2 with the ABA receptor PYL6 (Aleman et al. 2016). In Arabidopsis and rice the antagonism between GA and JA signalling has been shown to determine the trade‐off between growth and defence (Campos et al. 2016). Recently, it has been suggested that the antagonism between the kinase complexes SnRK1 and TOR may effectively regulate the balancing between growth and defence (Margalha et al. 2019). Generally, the regulation of growth–defence trade‐off may also involve changes in cell cycle control that is largely determined by hormone signalling networks (Eichmann & Schäfer 2015). It will be interesting to investigate the effect of Ro 8‐4304 and Ro‐A03 on the different molecular pathways that appear to decide about how to balance growth and defence for optimal plant performance and reproduction.

AUTHOR CONTRIBUTIONS

SH designed the study. MK, CM, LV, and SH designed the experiments. MK, ML, and JS performed the experiments and data analyses. MK and SH drafted the manuscript. All authors discussed and interpreted results, revised the manuscript, and approved it.

Supporting information

Table S1. Primers used for PCR experiments.

Table S2. Starting compounds for synthesis of Ro 8‐4304 derivatives.

Fig. S1. Synthesis of derivatives of Ro 8‐4304.

Fig. S2. The relative fresh weight of chs3‐2D treated with different concentrations of DMSO, Ro 8‐4304, 4‐oxiranyl methoxybenzamide, and PTP‐F, as well as the derivatives Ro‐A01 to Ro‐A18.

Fig. S3. Relative expression of the NIMIN1 gene in chs3‐2D seedlings grown at 18 °C for 21 days and treated with the respective compound at a concentration of 15 μM.

PLB-28-1686-s001.pdf (1.7MB, pdf)

ACKNOWLEDGEMENTS

We thank Xin Li (University of British Columbia, Canada) for providing chs3‐2D, icln‐2, and chs3‐2D icln2 seeds, Thomas Hackl und Young‐Joo Lee (Universität Hamburg, Germany) for help with recording of NMR spectra, and Maria Riedner (Universität Hamburg) for help with mass spectrometry. This work has been funded by a grant of the Bundesministerium für Bildung und Forschung to S.H. (031B0499). We acknowledge financial support from the Open Access Publication Fund of Universität Hamburg. Open Access funding enabled and organized by Projekt DEAL.

Editor: D. Geiger

DATA AVAILABILITY STATEMENT

All data supporting the findings of this study are available within the paper and within its Supporting Information – S1.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1. Primers used for PCR experiments.

Table S2. Starting compounds for synthesis of Ro 8‐4304 derivatives.

Fig. S1. Synthesis of derivatives of Ro 8‐4304.

Fig. S2. The relative fresh weight of chs3‐2D treated with different concentrations of DMSO, Ro 8‐4304, 4‐oxiranyl methoxybenzamide, and PTP‐F, as well as the derivatives Ro‐A01 to Ro‐A18.

Fig. S3. Relative expression of the NIMIN1 gene in chs3‐2D seedlings grown at 18 °C for 21 days and treated with the respective compound at a concentration of 15 μM.

PLB-28-1686-s001.pdf (1.7MB, pdf)

Data Availability Statement

All data supporting the findings of this study are available within the paper and within its Supporting Information – S1.

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