
==== Front
Mol Plant Pathol
Mol Plant Pathol
10.1111/(ISSN)1364-3703
MPP
Molecular Plant Pathology
1464-6722
1364-3703
John Wiley and Sons Inc. Hoboken

10.1111/mpp.70005
MPP70005
MPP-OA-24-135.R2
Original Article
Original Article
Cross‐family transfer of the Arabidopsis cell‐surface immune receptor LORE to tomato confers sensing of 3‐hydroxylated fatty acids and enhanced disease resistance
Eschrig et al.
Eschrig Sabine 1
Kahlon Parvinderdeep S. 1 3
Agius Carlos 1
Holzer Andrea 1
Hückelhoven Ralph https://orcid.org/0000-0001-5632-5451
1
Schwechheimer Claus 1
Ranf Stefanie https://orcid.org/0000-0003-2262-2938
1 2 stefanie.ranf@tum.de
ranf@wzw.tum.de

1 TUM School of Life Sciences Technical University of Munich Freising‐Weihenstephan Germany
2 Department of Biology University of Fribourg Fribourg Switzerland
3 Present address: Plant Sciences Group, Laboratory of Plant Physiology Wageningen University and Research Wageningen Netherlands
* Correspondence
Stefanie Ranf, TUM School of Life Sciences, Technical University of Munich, Freising‐Weihenstephan, Germany.
Email: stefanie.ranf@tum.de; ranf@wzw.tum.de

05 9 2024
9 2024
25 9 10.1111/mpp.v25.9 e7000514 8 2024
25 4 2024
18 8 2024
© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

Plant pathogens pose a high risk of yield losses and threaten food security. Technological and scientific advances have improved our understanding of the molecular processes underlying host–pathogen interactions, which paves the way for new strategies in crop disease management beyond the limits of conventional breeding. Cross‐family transfer of immune receptor genes is one such strategy that takes advantage of common plant immune signalling pathways to improve disease resistance in crops. Sensing of microbe‐ or host damage‐associated molecular patterns (MAMPs/DAMPs) by plasma membrane‐resident pattern recognition receptors (PRR) activates pattern‐triggered immunity (PTI) and restricts the spread of a broad spectrum of pathogens in the host plant. In the model plant Arabidopsis thaliana, the S‐domain receptor‐like kinase LIPOOLIGOSACCHARIDE‐SPECIFIC REDUCED ELICITATION (AtLORE, SD1‐29) functions as a PRR, which senses medium‐chain‐length 3‐hydroxylated fatty acids (mc‐3‐OH‐FAs), such as 3‐OH‐C10:0, and 3‐hydroxyalkanoates (HAAs) of microbial origin to activate PTI. In this study, we show that ectopic expression of the Brassicaceae‐specific PRR AtLORE in the solanaceous crop species Solanum lycopersicum leads to the gain of 3‐OH‐C10:0 immune sensing without altering plant development. AtLORE‐transgenic tomato shows enhanced resistance against Pseudomonas syringae pv. tomato DC3000 and Alternaria solani NL03003. Applying 3‐OH‐C10:0 to the soil before infection induces resistance against the oomycete pathogen Phytophthora infestans Pi100 and further enhances resistance to A. solani NL03003. Our study proposes a potential application of AtLORE‐transgenic crop plants and mc‐3‐OH‐FAs as resistance‐inducing biostimulants in disease management.

Cross‐family transfer of the pattern recognition receptor LORE from Arabidopsis increases tomato resistance to bacterial and filamentous pathogens upon sensing pathogen‐derived or artificially applied 3‐hydroxydecanoic acid.

crop resistance engineering
cross‐family PRR transfer
LORE
pattern recognition receptor
pattern‐triggered immunity
plant immunity
tomato
Deutsche Forschungsgemeinschaft 10.13039/501100001659 Emmy Noether program RA‐2541/1 SFB924/TP B10 source-schema-version-number2.0
cover-dateSeptember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:05.09.2024
Eschrig, S. , Kahlon, P.S. , Agius, C. , Holzer, A. , Hückelhoven, R. , Schwechheimer, C. et al. (2024) Cross‐family transfer of the Arabidopsis cell‐surface immune receptor LORE to tomato confers sensing of 3‐hydroxylated fatty acids and enhanced disease resistance. Molecular Plant Pathology, 25 , e70005. Available from: 10.1111/mpp.70005

Sabine Eschrig and Parvinderdeep S. Kahlon contributed equally to this work.
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pmc1 INTRODUCTION

Since the early days of agriculture, plant diseases have affected food production and security for humankind. Despite substantial advances in agricultural practices, global crop production still suffers significant economic losses due to plant diseases (Oerke, 2005; Savary et al., 2019). Historic disease outbreaks illustrate the extent of devastation plant pathogens can cause (van Esse et al., 2020). The Irish potato famine caused by the oomycete Phytophthora infestans (Yoshida et al., 2013) claimed millions of lives and led to mass emigrations. Fusarium wilt disease almost wiped out banana cultivation (Pegg et al., 2019), while the papaya industry in Hawaii was massively threatened by infection with papaya ringspot virus (Gonsalves, 1998).

Tomato is one of the most important and widely consumed vegetable crops worldwide and is susceptible to several plant pathogens (Anders et al., 2021; Bozbuga et al., 2022; Panno et al., 2021). Bacterial diseases affecting tomato cultivation include bacterial speck caused by Pseudomonas syringae pv. tomato (Pst), bacterial spot caused by Xanthomonas species or bacterial wilt caused by Ralstonia solanacearum (Panno et al., 2021; Wang et al., 2018). The oomycete Phytophthora infestans (late blight) and the fungus Alternaria solani (early blight) are widespread in tomato cultivation and contribute to significant yield losses if not controlled (Adhikari et al., 2017; Mazumdar et al., 2021).

The basis for the effective and sustainable control of plant diseases is a detailed molecular understanding of the interactions between plants and pathogens. The last 30 years have seen immense advances in research investigating the plant immune system (Ngou et al., 2022). Plants evolved an effective, genetically determined immune system consisting of preformed barriers and induced defence mechanisms (Dangl & Jones, 2001; Jones & Dangl, 2006; Ngou et al., 2022). Pattern‐triggered immunity (PTI) is based on the recognition of conserved microbe‐ or damage‐associated epitopes and phytocytokines by pattern recognition receptors (PRRs) (Ngou et al., 2022). PRR activation induces a broad‐spectrum defence response via transcriptional, metabolic and hormonal reprogramming at the site of infection and systemically in distal tissues (Ngou et al., 2022; Tsuda & Somssich, 2015; Vlot et al., 2021). Adapted pathogens secrete effector molecules into their hosts to manipulate PTI and host physiology to their advantage. Detection of these microbial manipulations by plant resistance (R) proteins activates effector‐triggered immunity (ETI) (Ngou et al., 2022). PTI and ETI are strongly intertwined and cooperatively contribute to disease resistance (Ngou et al., 2021; Yuan, Ngou, et al., 2021). Basic molecular research in the field of plant immunity has improved our understanding of the complex interaction between plants and pathogens and paved the way for new strategies to combat crop diseases.

Effective crop protection combines cultural, chemical, biological and genetic control methods (Sharma et al., 2022). While chemical control has become more challenging due to increasing pesticide resistance of the pathogens and rising concerns regarding their potential toxicity and environmental impact, genetic crop protection offers a more environmentally friendly and less labour‐ and cost‐intensive strategy (Dangl et al., 2013; Isman, 2019; van Esse et al., 2020). Historically, genetic host resistance has been achieved by identification of new quantitative trait loci (QTLs) for basal resistance or R genes in the gene pools of wild relatives and their introgression into elite crop cultivars. However, lack of sexual compatibility, long generation times and difficult introgression into polyploid species limit the breeding processes (Dangl et al., 2013). Moreover, because race‐specific R genes drive the adaptation of pathogen populations to overcome resistance, R gene‐based breeding strategies often lead to short‐lived resistance in the field (Dangl et al., 2013; Huang & Zimmerli, 2014). Besides advanced marker‐assisted resistance breeding, genome editing and transgenic approaches became a newly emerging field in genetic crop protection (Pickett, 2016; Wang et al., 2019). They can effectively enhance disease resistance by the addition or modulation of defence components and have shorter development times compared to conventional breeding techniques (Gurr & Rushton, 2005; Sharma et al., 2022; van Esse et al., 2020). Although the application of genetically modified organisms is subject to strict legal regulations and public acceptance across the globe varies and can be locally low, proof‐of‐concept studies demonstrate the efficacy of such genetic approaches and their relevance for future crop protection measures (Anders et al., 2021; Bubolz et al., 2022; Pickett, 2016; Wang et al., 2019). For instance, genome editing of the resistance gene mlo in tomatoes provided resistance to powdery mildew (Nekrasov et al., 2017). Transgenic Rainbow papaya is a commercially available ringspot virus‐resistant variety overexpressing a viral coat protein (Ferreira et al., 2002; Fitch et al., 1992). Interspecies transfer of the resistance gene Bs2 from pepper to tomato improved resistance against bacterial spot caused by Xanthomonas spp. (Horvath et al., 2012).

As PTI is critical for plant health (Yuan, Ngou, et al., 2021) and recognized elicitors are considered less likely to adopt mutations to evade PRR recognition (Huang & Zimmerli, 2014), PTI‐based crop engineering displays a great alternative to R gene transfer and may lead to broad‐spectrum, and hence more durable, resistance. PRRs are attractive targets for genetic resistance engineering as they can be easily transferred into host plants to expand their receptor repertoire (Huang & Zimmerli, 2014; Ranf, 2018). The great potential of cross‐family PRR transfer was firstly demonstrated by the integration of the Brassicaceae‐specific PRR EF‐Tu Receptor (EFR) from Arabidopsis thaliana into Nicotiana benthamiana and tomato, conferring increased disease resistance against various bacterial pathogens (Lacombe et al., 2010). AtEFR was later integrated into rice, wheat, potato, orange and apple (Boschi et al., 2017; Lacombe et al., 2010; Lu et al., 2015; Mitre et al., 2021; Piazza et al., 2021; Schoonbeek et al., 2015; Schwessinger et al., 2015). The effectiveness of AtEFR‐transgenic tomato against bacterial wilt disease caused by Ralstonia solanacearum and bacterial spot caused by Xanthomonas perforans was confirmed in field trials in the United States (Kunwar et al., 2018). Another example is the transfer of the rice immune receptor kinase Xa21 to oranges, tomatoes and bananas to enhance resistance against wilt diseases (Afroz et al., 2011; Mendes et al., 2010; Tripathi et al., 2014). Expression of the extracellular ATP (eATP) receptor DOES NOT RESPOND TO NUCLEOTIDES 1 (DORN1 or LecRK‐1.9) of A. thaliana in Solanum tuberosum and N. benthamiana increased resistance to P. infestans (Bouwmeester et al., 2014; Wang et al., 2016). Finally, citrus species expressing the FLS2 receptor from N. benthamiana are more resistant to citrus canker caused by Xanthomonas citri (Hao et al., 2016).

In the model plant A. thaliana, we previously identified the S‐domain receptor‐like kinase (SD‐RLK) LIPOOLIGOSACCHARIDE‐SPECIFIC REDUCED ELICITATION (AtLORE, SD1‐29, AT1G61380) as a PRR (Ranf et al., 2015) that senses bacterial 3‐hydroxylated fatty acids (mc‐3‐OH‐FAs) and 3‐hydroxyalkanoates (HAAs) of medium‐chain‐length ranging from C8 to C12 (Kutschera et al., 2019; Schellenberger et al., 2021). Free 3‐hydroxydecanoic acid (3‐OH‐C10:0) is the strongest elicitor of AtLORE‐dependent immunity (Kutschera et al., 2019). AtLORE promotes resistance against Pst DC3000 and R. solanacearum in A. thaliana (Kutschera et al., 2019; Wang et al., 2023). Pst DC3000 resistance can be further enhanced by pretreatment with 3‐OH‐C10:0 or HAAs before infection (Kutschera et al., 2019; Schellenberger et al., 2021).

LORE is phylogenetically restricted to Brassicaceae (Eschrig et al., 2024; Ranf et al., 2015) and thus an excellent model for application in genetic crop disease management via cross‐family gene transfer. We have previously shown that transient expression of AtLORE in solanaceous N. benthamiana confers sensitivity to mc‐3‐OH‐FAs (Eschrig et al., 2024; Kutschera et al., 2019). In this study, we generated transgenic AtLORE‐overexpressing S. lycopersicum ‘M82’ to investigate the feasibility of functionally transferring an SD‐type PRR across plant families into an agriculturally relevant crop. We tested whether AtLORE‐transgenic tomato is more resistant to the adapted tomato pathogen Pst, a prominent producer of 3‐OH‐C10:0. Furthermore, we investigated whether soil application of 3‐OH‐C10:0 induces systemic resistance of AtLORE‐transgenic tomato to other tomato pathogens, which might not directly activate LORE‐dependent immunity, such as the fungus A. solani or the oomycete P. infestans.

2 RESULTS

2.1 Generation of stable transgenic S. lycopersicum‐overexpressing AtLORE

The finding that LORE confers resistance to P. syringae and R. solanacearum in A. thaliana (Kutschera et al., 2019; Wang et al., 2023) and that AtLORE is functional in N. benthamiana upon transient expression (Eschrig et al., 2024; Ranf et al., 2015) raised the question of whether stable expression of AtLORE in S. lycopersicum also confers gain of mc‐3‐OH‐FA immune sensing and increased resistance to tomato pathogens. Therefore, we generated transgenic S. lycopersicum ‘M82’ via agrobacteria‐mediated transformation, callus culture and regeneration of transgenic plants. We ectopically overexpressed AtLORE under control of the strong cauliflower mosaic virus 35S (CaMV 35S) promoter (AtLORE‐OE lines) or integrated the T‐DNA from an empty vector (EV lines) as negative control (Figure 1a). Four regenerated AtLORE‐OE plants from different calli and one EV plant were grown until fruiting in the greenhouse and seeds were harvested from ripe tomato fruits (Figure S1). No obvious visible differences in growth or fruiting behaviour were observed for any of the lines, apart from line AtLORE‐OE7‐1 (Figure S1). AtLORE‐OE7‐1 was excluded from further analysis because it exhibited a dwarf, sterile phenotype directly after regeneration from the callus and remained developmentally impaired (Figures S2 and S3). Although leaf shapes were comparable between all lines, AtLORE‐OE3‐4 displayed a slightly sharper serration than the wild type and other AtLORE‐OE or EV lines (Figure S3). For further analysis, the three independent AtLORE‐OE lines and one EV line were grown from harvested seeds. To select transgenic progeny in the segregating T1 generation, we genotyped up to eight seedlings per line by PCR on extracted genomic DNA (gDNA) for the presence of AtLORE or EV T‐DNA (Figure 1b). The housekeeping gene ELONGATION FACTOR 1α from S. lycopersicum (SlEF1‐α) served as control. For these lines, we evaluated overall macroscopic growth phenotypes (Figure 2a–c) and germination rates (Figure 2d) to determine whether the transformation process or AtLORE overexpression negatively affected their development. All transgenic OE plants grew similarly to M82 wild‐type and EV control lines.

FIGURE 1 Expression cassettes and genotyping results of transgenic AtLORE‐OE and empty vector (EV) Solanum lycopersicum lines. (a) Schematic illustration shows the expression cassettes of the empty vector (EV, pART27) and for AtLORE overexpression (OE), including primers (red) used for genotyping via PCR. AtLORE coding sequence (CDS, lilac), cauliflower mosaic virus 35S promotor sequence (CaMV 35S, grey) and an octopine synthase terminator sequence (TERM, grey) were integrated into pART27 via NotI and disrupted the LacZ selection marker cassette (blue). The T‐DNA furthermore contains a kanamycin resistance cassette (KanR, green), and left (LB) and right borders (RB, orange). Scheme was created with BioRender.com. (b) Image shows agarose gel analysis of genotyping PCR from segregating progeny of one EV and three independent AtLORE‐OE lines; untransformed wild‐type M82 served as control. DNA fragments specific to the T‐DNA insertions (EV or AtLORE) and PCR amplified from extracted gDNA are shown. The wild‐type housekeeping gene SlEF1‐α was amplified as control.

FIGURE 2 Wild‐type and AtLORE‐transgenic Solanum lycopersicum are phenotypically indistinguishable. (ac) Photographs of 8‐week‐old plants of the specified genotypes. Scale bar represents 10 cm. (d) Graph displaying the germination rates (number of germinated seeds/number of seeds sown) of AtLORE‐transgenic overexpression (OE) and empty vector (EV) lines compared to the M82 wild‐type control. Bar graphs show mean with SD of pooled data from at least three biological replicates; each data point corresponds to an individual set of 10 seeds of the indicated line. Germination rates of seeds were assessed 1 week after sowing. Data do not show significant differences (one‐way analysis of variance with Tukey's multiple‐comparisons test, α = 0.05).

2.2 Overexpression of AtLORE in S. lycopersicum confers 3‐OH‐C10:0 sensing

To test whether AtLORE overexpression renders tomato sensitive to 3‐OH‐C10:0, we assessed the production of the phytohormone ethylene as a typical PTI output (Anver & Tsuda, 2015) upon elicitation with 3‐OH‐C10:0. We analysed three individual genotyping positive plants of different AtLORE‐OE lines, EV control or M82 wild type (Figure 3). Leaf discs from AtLORE‐OE3‐4 and AtLORE‐OE5‐1 plant lines produced high amounts of ethylene when treated with 3‐OH‐C10:0 compared to the control treatment, while the EV control line showed no detectable ethylene production. For AtLORE‐OE2‐1, only two of three plants responded with rather weak ethylene production compared to AtLORE‐OE3‐4 and AtLORE‐OE5‐1 (Figure 3A). To evaluate the specificity of AtLORE sensing in S. lycopersicum, we tested the ethylene response of the highest responding lines AtLORE‐OE3‐4#1 and AtLORE‐OE5‐1#7 upon administration of different concentrations of 3‐OH‐FAs of different chain lengths. We treated plants with 1, 5 or 10 μM 3‐OH‐C10:0, 3‐OH‐C14:0 or the same volume of ethanol as control. For both lines, we observed a strong concentration‐dependent and chain length‐specific response (Figure 3B), resembling the response characteristics described for AtLORE in A. thaliana (Kutschera et al., 2019). This confirms that 3‐OH‐C10:0‐sensing specificity by AtLORE is transferable from A. thaliana to S. lycopersicum. Plants OE2‐1#17, OE3‐4#7 and OE5‐1#1, which reacted with high ethylene production, as well as the control line EV2‐2#2 were maintained and new stem cuttings were taken from them for further experiments as required.

FIGURE 3 AtLORE overexpression in Solanum lycopersicum confers chain length‐specific and concentration‐dependent 3‐OH‐FA sensing. (A) Graph displays ethylene production (pmol/mL air) of leaf discs from three independent genotyping‐positive plants of each AtLORE‐transgenic tomato line 3 h after elicitation with 5 μM 3‐OH‐C10:0 or the same volume of ethanol as control. (B) Graph displays ethylene production of the most strongly responding lines from (A), OE3‐4 #1 and OE5‐1 #7, and an empty vector (EV) control to different concentrations of 3‐OH‐FAs of different chain lengths. Leaf discs were treated with 1, 5 or 10 μM of 3‐OH‐C10:0, 3‐OH‐C14:0 or ethanol as a control for 3 h. (A and B) Bar graphs show mean with SD of pooled data from two biological replicates. Individual ethylene measurements per line are represented by black dots (n ≥ 6). Statistical differences between treatments among individuals of a line (A) or within lines (B) were analysed by two‐way analysis of variance with multiple‐comparisons test corrected for false discovery rate via two‐stage linear step‐up procedure of Benjamini, Krieger and Yekutieli, q (desired false discovery rate) = 0.01. Data not sharing the same letter are significantly different.

2.3 Overexpression of AtLORE in S. lycopersicum increases resistance towards pathogenic Pst DC3000

To test whether overexpression of AtLORE leads to increased disease resistance in tomatoes, we performed pathogen infection assays by spray inoculation of AtLORE‐OE and EV plants with Pst DC3000. In line with the ethylene accumulation data (Figure 3), bacterial titres were significantly lower in the strongly responding lines OE3‐4#1 and OE5‐1#7 compared to the EV control line 3 days after inoculation (Figure 4A). These strongly responding lines also showed fewer disease symptoms on leaves (Figure 4B) compared to the EV control. No statistically significant reduction in bacterial titres was measurable in the low ethylene‐producing line OE2‐1#17 3 days after infection. However, slightly reduced macroscopic disease symptoms were repeatedly observed in this line compared to the EV control (Figure 4). Our results demonstrate that overexpression of AtLORE in tomatoes might be an effective disease management strategy to increase resistance to bacterial pathogens without compromising plant growth and development.

FIGURE 4 At LORE overexpression enhances resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. Pst DC3000 infection of AtLORE‐transgenic Solanum lycopersicum lines was assessed 3 days after spraying leaves of the indicated lines with a bacterial suspension of an optical density OD600 of 0.002. Experiments were performed on cuttings 4–6 weeks after their propagation. (A) Graph displays the bacterial titres in leaflets from three cuttings per transgenic line. Bar graphs show mean with SD of pooled data from two independent biological replicates with black dots representing individual data points (n = 12 samples from four leaves per cutting and replicate). Statistical differences were analysed by one‐way analysis of variance and Tukey's multiple‐comparisons test, α =0.01. Data not sharing the same letter are significantly different. (B) Photographs show leaflets of the indicated transgenic tomato lines 3 days after Pst DC3000 infection and illustrate the macroscopic disease symptoms.

2.4 Pretreatment with 3‐OH‐C10:0 activates systemic and broad‐spectrum disease resistance

To investigate whether pretreatment of AtLORE‐transgenic tomato with 3‐OH‐C10:0 before infection induces systemic resistance against filamentous pathogens, we treated cuttings of EV2‐2#2 and OE3‐4#1 with 10 μM 3‐OH‐C10:0 dissolved in water or a water control via soil irrigation. After 48 h, we spray‐inoculated leaves with sporangia solutions of P. infestans Pi100 or spore solutions of A. solani NL03003 and quantified the infection frequency 14 days post‐inoculation (Figure 5). Compared to control‐treated plants or the EV control (EV2‐2#2), 3‐OH‐C10:0‐treated AtLORE‐OE3‐4 plants were significantly more resistant to P. infestans. Interestingly, for A. solani, we observed reduced infection of AtLORE‐OE3‐4 independently of the pretreatment with 3‐OH‐C10:0. The infection score of the water‐treated AtLORE‐OE3‐4 plants was significantly lower than that of the EV control. Pretreatment of plants with 3‐OH‐C10:0 further significantly enhanced resistance to A. solani compared to the water‐treated plants. These data indicate that overexpression of AtLORE enables tomatoes to activate systemic resistance by sensing 3‐OH‐C10:0, thereby enhancing resistance against filamentous pathogens.

FIGURE 5 Pretreatment with 3‐OH‐C10:0 induces resistance against Phytophthora infestans and Alternaria solani. Graph displays the infection frequency (number of symptomatic leaves/infected leaves) 14 days after spray inoculation with P. infestans Pi100 or A. solani NL03003 of two to three cuttings from empty vector (EV) or AtLORE‐OE3‐4 that were pretreated for 48 h via soil irrigation with 10 μM 3‐OH‐C10:0 dissolved in water or a water‐only control. Experiments were performed 6–7 weeks after propagation by cutting. Pooled data from two biological replicates are shown, which were obtained from five or six stem cuttings each (n = number of assessed leaves, n ≥ 34 for A. solani, n ≥ 50 for P. infestans). Statistical differences were analysed by one‐way analysis of variance and Tukey's multiple‐comparisons test, α = 0.05, for A. solani and P. infestans respectively. Data not sharing the same letter are significantly different.

3 DISCUSSION

Historically, resistance breeding has relied primarily on the introduction of major R genes from cultivars or landraces of the same species into a variety lacking the desired trait. In recent years, however, several studies have highlighted the potential of cross‐family PRR transfer as a promising alternative for engineering disease resistance in crops (Afroz et al., 2011; Boschi et al., 2017; Bouwmeester et al., 2014; Hao et al., 2016; Kunwar et al., 2018; Lacombe et al., 2010; Lu et al., 2015; Mendes et al., 2010; Mitre et al., 2021; Piazza et al., 2021; Schoonbeek et al., 2015; Schwessinger et al., 2015; Tripathi et al., 2014, 2017; Wang et al., 2016). These studies have demonstrated that signalling networks downstream of PTI are often sufficiently conserved, even between monocot and dicot plant families, to allow foreign PRRs to fit seamlessly into endogenous signalling pathways of a given species (Afroz et al., 2011; Holton et al., 2015; Schoonbeek et al., 2015; Schwessinger et al., 2015; van Esse et al., 2020).

Our study shows that the cross‐family PRR transfer of the Arabidopsis PRR AtLORE to tomato enhances resistance against three major tomato diseases, bacterial speck, early blight and late blight, either directly or upon application of synthetic 3‐OH‐FAs. This suggests that all essential signalling components required for LORE‐mediated immunity are present in tomatoes, although LORE is a Brassicaceae‐specific PRR (Ranf et al., 2015). Indeed, the signalling components downstream of LORE known to date, PBS1‐like (PBL) receptor‐like cytoplasmic kinases AtPBL34/35/36, RPM1‐INDUCED PROTEIN KINASE (AtRIPK) and LORE‐ASSOCIATED PROTEIN PHOSPHATASE (AtLOPP) all have putative tomato orthologues (Li et al., 2021; Luo et al., 2020; Wang et al., 2023). Most of the known PRRs require universal co‐receptors for ligand binding and signalling, such as members of the SERK (somatic embryogenesis receptor‐like kinase) family, which are usually sufficiently conserved between species (Chen et al., 2014; Holton et al., 2015). In contrast, no co‐receptors have been described for AtLORE signalling. Instead, we previously showed that receptor homomerization is essential for 3‐OH‐C10:0‐induced immunity in A. thaliana (Eschrig et al., 2024). The concept of receptor homomerization could be advantageous to ensure its functionality when transferred into taxonomically different plant families, as co‐receptors of phylogenetically restricted receptors may have co‐evolved and be absent in other species.

Overexpression or constitutive activation of immunity components is often associated with fitness costs, as wild‐type plants need to fine‐tune the division of their limited resources between immunity and growth (Karasov et al., 2017; Wang et al., 2021; Zhang et al., 2023). Such observations were, for example, made for overexpression of the immune regulator NONEXPRESSOR OF PR1 (NPR1) in rice, which not only increases disease resistance but also causes growth retardation and spontaneous cell death (Chern et al., 2005). Cross‐family transfer of PRRs seems to be largely tolerated by recipient plants (Huang & Zimmerli, 2014; Ranf, 2018; van Esse et al., 2020). However, for DORN1‐transgenic potatoes, impaired plant and tuber development were reported (Bouwmeester et al., 2014). Overexpression of AtLORE in tomatoes does not alter growth, development or reproduction, indicating that LORE expression and signalling are sufficiently controlled in this heterologous system and do not cause autoimmunity or cell death. Interestingly, we previously reported that strong transient overexpression of AtLORE in agroinfiltrated N. benthamiana leaves leads to receptor auto‐activation and induces cell death (Eschrig et al., 2024). Thus, although N. benthamiana and S. lycopersicum both belong to the Solanaceae family, our data show that tomato tolerates AtLORE overexpression without apparent adverse side effects. This may be due to more moderate expression levels from stably integrated transgenes, or due to the effects of negative regulators of unknown identity that are present in tomato, but absent, diversified or expressed at lower levels in N. benthamiana.

We found that 3‐OH‐C10:0 pretreatment of roots via soil drainage induces resistance against the major tomato pathogens P. infestans and A. solani in AtLORE‐transgenic tomato (Figure 5). Fighting plant diseases through defence priming strategies is a widely discussed disease management approach and offers a great addition to genetic crop protection (Abbasi et al., 2021; Alexandersson et al., 2016; Conrath et al., 2015; Sandroni et al., 2020). Primary infection, application of beneficial microbes and treatment with natural or synthetic chemicals activate systemic resistance and switch the plant to a primed state, leading to a more rapid and enhanced resistance activation against subsequent secondary infections (Abbasi et al., 2021; Conrath et al., 2015; Vlot et al., 2021). Similar to 3‐OH‐C10:0 shown here, other fatty acids, such as eicosapolyenoic fatty acids, have been described as systemic resistance inducers. These fatty acids are released during oomycete infection (or are produced by brown seaweed Ascophyllum nosodum) and induce resistance against Phytophthora capsici in tomatoes and peppers (Dye & Bostock, 2021; Lewis et al., 2023). Furthermore, soil application of hexanoic acid induces resistance against Botrytis cinerea and P. syringae in A. thaliana and S. lycopersicum (Kravchuk et al., 2011; Scalschi et al., 2013; Vicedo et al., 2009). However, for most stimulants, the exact resistance mechanisms are not understood, which is why their efficacy in different plant species can hardly be predicted. In contrast, the elicitor, receptor and parts of the immune signalling pathways are characterized for LORE, which could make mc‐3‐OH‐FAs superior to other known resistance inducers. Transgenic expression of AtLORE and mc‐3‐OH‐FA soil application could be combined in several important crop species. Yet, potential effects of mc‐3‐OH‐FAs on plant growth, yield, consumers and the environment require further evaluation.

Mc‐3‐OH‐FAs, as sensed by LORE, are widespread in nature and can be naturally found in soils, humans, animals, insects, plants, microorganisms and dairy foodstuffs (Jenske & Vetter, 2009; Jones & Bennett, 2011; Keinänen et al., 2003; Kodai et al., 2011; Mikkelsen et al., 2022; Nagahashi et al., 2010; Nagahashi & Douds, 2011; Schildknecht & Koob, 1971; Sjögren et al., 2003; Suzuki et al., 2013). Therefore, adverse effects on the environment by 3‐OH‐FA application might be low. With few exceptions, gram‐negative bacteria contain large amounts of 3‐OH‐FAs which are used as building blocks in various compounds, such as lipopolysaccharide (LPS) (Alexander & Rietschel, 2001), lipopeptides (Raaijmakers et al., 2006; Souza et al., 2003), HAAs and rhamnolipids (Abdel‐Mawgoud et al., 2010), N‐acyl‐homoserine lactone‐type quorum sensing molecules (Thiel et al., 2009; Williams, 2007) or polyhydroxyalkanoates (Paduvari et al., 2024; Raza et al., 2018). However, most of these compounds do not activate LORE‐mediated immunity directly (Kutschera et al., 2019) but may release mc‐3‐OH‐FAs sensed by LORE (Ernst et al., 2006; Gerster et al., 2022; Geurtsen et al., 2005; Kutschera et al., 2019; Zheng, Gong, et al., 2004; Zheng, Zhang, et al., 2004). Free 3‐OH‐C10:0 FAs are, for example, prevalent in the secretome of Pseudomonas spp. (Schellenberger et al., 2021) and the culture medium of Escherichia coli (Zheng, Gong, et al., 2004; Zheng, Zhang, et al., 2004). Thus, we assume that AtLORE confers resistance against P. syringae upon release of free 3‐OH‐C10:0 from the pathogen. So far, the release of free mc‐3‐OH‐FAs has not been reported for fungi or oomycetes, which are rather known to produce complex, long‐chain hydroxy fatty acids (Ivanova et al., 2010; Neri et al., 2023). However, we found that A. solani infection was decreased in AtLORE‐transgenic tomato independent of the elicitor pretreatment. Thus, A. solani potentially directly activates LORE‐dependent immunity, possibly by releasing mc‐3‐OH‐FAs. Interestingly, it has been shown that 3‐OH‐C10:0 itself has antifungal properties against yeasts and moulds (Sjögren et al., 2003). In our study, however, synthetic 3‐OH‐C10:0 was applied to the soil 2 days before the leaves were infected with A. solani and P. infestans. This spatial and temporal separation makes it unlikely that direct contact of 3‐OH‐C10:0 with the pathogens led to the observed resistance phenotypes. Instead, we assume that the soil application of 3‐OH‐C10:0 triggers systemic immunity in AtLORE‐transgenic tomatoes and subsequently enhances pathogen resistance. Additionally, independent of treatment with synthetic 3‐OH‐C10:0, the release of mc‐3‐OH‐FAs from soil/plant microbiota or root exudates may lead to low constitutive immune activation in AtLORE‐transgenic tomatoes. This may contribute to the observed ligand‐independent resistance to A. solani and P. syringae but may be insufficient for the more aggressive P. infestans.

Our study suggests that mc‐3‐OH‐FAs seem to be potent resistance‐inducing biostimulants on AtLORE‐expressing plants. In combination, this might form an effective, transferable resistance module and add to the list of successful examples of resistance engineering in crops. LORE might thereby be especially applicable for PRR transfer, as the receptor is not widespread in the plant kingdom compared to others, such as FLS2 (Yue et al., 2012), and pre‐existing natural adaptation of pathogens may be low. This may delay the potential selection of pathogens with altered mc‐3‐OH‐FA profiles that would evade LORE‐dependent immunity, potentially leading to more durable resistance of LORE‐transgenic crops in the field. In the future, infection assays with Clavibacter michiganensis subsp. michiganensis and R. solanacearum on AtLORE‐transgenic tomato could confirm its effectiveness against other devastating bacterial tomato pathogens. It would be interesting to transfer LORE into other crops often threatened by bacterial pathogens, such as potato or rice, which could profit from direct activation of LORE‐dependent immunity or the combination with mc‐3‐OH‐FAs application. On the pathogen side, analysis of mc‐3‐OH‐FA contents in fungi and oomycetes would give new insights into whether LORE‐dependent immunity is more widely triggered by pathogens from different kingdoms. As advances in plant immunity research have shown that ETI and PTI mutually enhance plant immunity, another benefit of cross‐family PRR transfer could be the potentiation of ETI (Ngou et al., 2021; Tian et al., 2021; Yuan, Jiang, et al., 2021; Yuan, Ngou, et al., 2021). This suggests that combining AtLORE expression with QTLs or R genes may promote even more profound and durable resistance. In potatoes, an introgressed QTL from wild potato combined with an AtEFR transgene demonstrated an additive effect of quantitative resistance and heterologous PRR expression over QTLs or EFR alone (Boschi et al., 2017). In this respect, transgenic approaches and gene‐editing tools hold great potential to further engineer durable resistance or circumvent growth–immunity trade‐offs (Cadiou et al., 2023; Luo et al., 2021; van Esse et al., 2020). In conclusion, our study shows that the Brassicaceae‐specific PRR AtLORE is a great model for investigating genetic resistance engineering via cross‐family gene transfer. It further emphasizes the advances and importance of basic molecular research in the field of plant immunity and underlines its great potential for application in modern and sustainable agriculture in the future.

4 EXPERIMENTAL PROCEDURES

4.1 Molecular cloning

Coding sequence (CDS) of AtLORE (AT1G61380) was amplified from cDNA before (Ranf et al., 2015) and cloned via a binary vector system including pART7 and pART27 (Gleave, 1992) for Agrobacterium‐mediated transformation. AtLORE CDS was amplified via PCR with adapters containing enzymatic restriction sites for integration into the multiple cloning site of the primary cloning vector pART7 via XhoI and EcoRI (Thermo Fischer Scientific). The CaMV 35S expression cassette of pART7 including the desired CDS was transferred into the binary vector pART27 via NotI (Thermo Fischer Scientific). The empty vector (EV) pART27 or pART27‐AtLORE was transferred into Agrobacterium tumefaciens GV3101 (pMP90) for Agrobacterium‐mediated transformation of tomato.

4.2 Generation of stable transgenic S. lycopersicum

Stable transgenic S. lycopersicum ‘M82’ containing the empty vector pART27 or pART27‐AtLORE were generated under sterile conditions by agrobacteria‐mediated transformation of cotyledons, callus formation and regeneration of transgenic plants as described (Wittmann et al., 2016). Fully regenerated sterile transgenic T0 plants were transferred to soil, genotyped via PCR and grown in the greenhouse until seed set.

4.3 Genotyping by PCR

Presence of T‐DNA in transgenic plants was confirmed via extraction of genomic DNA and genotyping by PCR with the REDExtract‐N‐Amp Plant PCR‐Kit (Merck). Primer sequences are listed in Table S1.

4.4 Growth conditions

After transformation and in vitro callus regeneration, transgenic tomatoes were transferred to potting soil (Einheitserde CL ED73) mixed with vermiculite (1:8). Plants were grown in climate chambers (Fitotron SGC120‐H, Weiss Technik) under long‐day conditions (16 h light/8 h dark) at 23°C and 60% relative humidity and transferred to the greenhouse for seed set. Plants grown from seeds were sterilized, germinated on Jiffy pellets (Jiffy‐7, 44 mm; Jiffy Products International AS) and transferred to potting soil/vermiculite (1:8). Plantlets were grown in climate chambers as described above and propagated for experiments using stem cuttings.

4.5 Seed extraction and sterilization

Fruits of T0 tomato were cut open, and fruit flesh was removed, mixed 1:1 with 3 M HCl and incubated with stirring for 20–30 min to remove seed coats. Seeds were washed with water, neutralized in 10% (wt/vol) Na3PO4 for 30 min, washed again with water and dried on filter paper overnight. Before planting, all seeds were surface sterilized with 3% sodium hypochlorite for 10 min and washed three times with water.

4.6 Assessment of tomato seed germination rates

Surface‐sterilized tomato seeds were germinated under sterile conditions on water‐soaked filter paper in Petri dishes and incubated in a long‐day climate chamber (16 h light/8 h dark). The germination rates (number of germinated seeds/number of sown seeds) were assessed on the basis of radical and hypocotyl emergence 1 week after sowing.

4.7 Ethylene measurement

A leaf disc‐based method was performed to evaluate ethylene accumulation in the different plant lines as previously described (Kahlon et al., 2023). Leaf discs were sampled with a 4‐mm‐diameter biopsy puncher and incubated overnight at room temperature while floating on water to allow wounding reactions to decline. Three leaf discs were transferred to 5 mL glass vials containing 300 μL water. Elicitors (3‐hydroxydecanoic acid, Manchester Organics; 3‐hydroxytetradecanoic acid, Cayman Chemical; both dissolved in ethanol) or the corresponding volume of ethanol as control was added to the vials, which were immediately sealed with septa (Carl Roth GmbH). Upon 3 h of incubation with constant shaking (50 rpm, Polymax 2040; Heidolph Instruments GmbH & Co. KG), 1 mL of air was sampled with a 1 mL syringe and injected into a gas chromatograph (Varian 3300; Waters) equipped with an AlO3 column (length 1 m). The detector was set to a temperature of 225°C and the column and injector to 80°C. The gases H2, N2 and O2 at 0.5 MPa each were used to separate ethylene from the sample. The amount of ethylene was calculated based on the standard calculation developed by Von Kruedener et al. (1995) using the area under the curve (AUC).

4.8 Infection assays with Pst DC3000

Pst DC3000 was grown on King's B (KB) agar with 50 μg/mL rifampicin at 28°C for 2 days. Bacteria were scratched from plates and diluted in 10 mM MgCl2 and 0.04% Silwet L77 (Kurt Obermeier GmbH & Co. KG) to an OD600 of 0.002. Spray infection assays were performed on 4‐ to 6‐week‐old stem cuttings of AtLORE‐OE and EV lines. Four cuttings per line were prepared, from which three were sprayed with Pst DC3000 and one with mock (10 mM MgCl2, 0.04% Silwet L‐77). Plants were covered with bags for 2 days to maintain high humidity. Three days post‐infection, disease symptoms were assessed by photography and bacterial colony counting. For each of the three infected cuttings, four leaves were sampled by punching one leaf disc (diameter 4 mm) from the same position of three random leaflets, leading to 12 technical replicates. After adding 100 μL MgCl2 (10 mM) and two glass beads per sample, all samples were ground for 2 min at 25 Hz (TissueLyser II; Qiagen). A dilution series was performed on this mixture with MgCl2 (10 mM) to a dilution of up to 10−8. Ten microlitres of each dilution was drop inoculated on KB agar plates (10 μg/mL rifampicin) and incubated at 28°C for about 36 h. Colony‐forming units were counted, normalized to the leaf area and log10 transformed.

4.9 Induced resistance infection assay with A. solani and P. infestans

To induce resistance, 6‐ to 7‐week‐old stem cuttings of EV and AtLORE‐OE lines were irrigated with 10 μM 3‐OH‐C10:0 dissolved in water (Manchester Organics) or a water control for 48 h prior to infection. The whole leaf area was spray infected with spore solutions of P. infestans isolate Pi100 (3000 sporangia/mL) as described (Kahlon et al., 2021) or A. solani NL03003 (5000 spores/mL). Leaf infection frequencies (symptomatic leaflets/inoculated leaflets) were assessed 14 days after infection as described (Kahlon et al., 2021).

Supporting information

Figure S1. Propagation of AtLORE‐transgenic Solanum lycopersicum ‘M82’ in the greenhouse. Transgenic tomatoes overexpressing AtLORE or harbouring an empty vector control were regenerated from callus culture and were grown in the greenhouse (a). Most transgenic lines did not show obvious growth, fruiting or yield alterations (b, fruits of OE2‐1).

Figure S2. AtLORE‐transgenic tomato line OE7‐1 exhibits an impaired growth phenotype. Line OE7‐1 shows a dwarf, developmentally impaired phenotype (a and b). Flowers of OE7‐1 appeared to be sterile due to an exerted style which outgrows the stigma from the anthers and prevents self‐pollination (c and d).

Figure S3. Leaf shape phenotypes of AtLORE‐transgenic tomato lines (T0).

Table S1. Primer sequences for genotyping PCR.

ACKNOWLEDGEMENTS

We thank Bert Evenhuis for kindly providing the A. solani isolate NL03003 and Remco Stam for providing the P. infestans isolate Pi100. We thank Kai Steinmetz, Sabine Zuber and Bärbel Breulmann from the TUM Plant Technology Center for maintenance of tomatoes in the greenhouse. Research was supported by grants from the German Research Foundation to S.R. (SFB924/TP B10 and Emmy Noether programme RA‐2541/1). Technical University of Munich has filed a patent application to inventors S.R. and R.H. The authors state they have no competing interests or disclosures. Open Access funding enabled and organized by Projekt DEAL.

DATA AVAILABILITY STATEMENT

All data supporting the findings of this study are available within the article and its Supplementary Material. Raw data are available from the corresponding author on request.
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