Introducing Pattern Recognition Receptors in Potato Confers Enhanced Resistance to <scp> <i>Ralstonia solanacearum</i> </scp> but Not a Vector Borne Pathogen

Potato (Solanum tuberosum) is a major food crop that is vulnerable to various bacterial pathogens. A critical soil-borne bacterial pathogen is Ralstonia solanacearum (Rso), causing bacterial wilt that threatens potato production worldwide (Vailleau and Genin 2023). Another important pathogen is Candidatus Liberibacter solanacearum (Lso), a vector-borne pathogen that is phloem-limited, causes Zebra Chip disease in potato and reduces tuber yield and quality (Wenninger and Rashed 2024). Plants defend against microbes using cell-surface pattern recognition receptors (PRRs) that detect conserved microbial-associated molecular patterns (MAMPs) including flagellin epitopes and hydroxylated fatty acids (Li, Moreno-Pérez, and Coaker 2024). PRR distribution and recognition spectra vary across plants (Li, Moreno-Pérez, and Coaker 2024). FLAGELLIN-SENSING 2 (FLS2) is conserved and detects a 22-amino acid epitope flg22 from bacterial flagellin (Li, Moreno-Pérez, and Coaker 2024). Other PRRs, such as LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE), that detect bacterial 3-hydroxylated fatty acids (3-OH-FAs), are restricted to Brassicaceae (Kutschera et al. 2019). Interspecies PRR transfer has potential for boosting or conferring novel immune perception to restrict pathogens. For example, the transfer of the PRR ELONGATION FACTOR Tu RECEPTOR (EFR) to tomato confers resistance against Rso (Li, Moreno-Pérez, and Coaker 2024; Lacombe et al. 2010). Tobacco expressing FLS2XL from wild grape enables recognition of polymorphic flagellin variants, resulting in enhanced resistance against Agrobacterium tumefaciens (Fürst et al. 2020). A recent preprint stacked two PRRs in potato recognising Pep-13 and nlp20, resulting in increased resistance against the oomycete pathogen Phytophthora infestans (Ascurra et al. 2023). We sought to transfer two PRRs into cultivated potato enabling recognition and control of bacterial pathogens with distinct infection lifestyles. Screening the chipping cultivar ‘Atlantic’ with seven MAMPs revealed perception of Pseudomonas flg22 and chitin, but not elf18, csp22, 3-OH-FAs, Rso flg22 or Lso flg22 in juvenile plants (Figure 1a). To generate novel immune perception, we selected Arabidopsis thaliana LORE for its broad recognition of bacterial 3-OH-FAs (Kutschera et al. 2019), and Vitis riparia (riverbank grape) FLS2XL for its ability to detect polymorphic flg22 variants with similarity to Lso flg22 (Figure 1b) (Li, Bolaños, et al. 2024). However, FLS2XL cannot detect Rso flg22 (Li, Bolaños, et al. 2024). A dual PRR stack (LORE/FLS2XL) driven by separate medium-strength promoters was assembled using GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) and introduced into ‘Atlantic’ (Figure 1c; Figure S1a) (Collier et al. 2018). Two independent LORE/FLS2XL transgenic lines perceived 3-hydroxydecanoic acid (3-OH-C10:0) and Lso flg22, respectively (Figure 1d). This indicates both PRRs function in potato. We then tested whether the LORE/FLS2XL stack enhances resistance to Rso (IBSBF1503 strain) by soil drench. Transgenic lines exhibited reduced disease index, higher survival probabilities and lower Rso titers in roots and stems compared to control plants (Figure 1e–g). While all roots had detectable levels of Rso, LORE/FLS2XL plants were more likely to have undetectable titers in stems (Figure 1g), suggesting restriction of bacterial movement from root to stem. However, once Rso breached this barrier, pathogen titers in stems of LORE/FLS2XL were similar to controls (Figure 1g). Cultivated potato and the FLS2XL receptor cannot perceive Ralstonia flg22 (Figure 1a), suggesting that LORE presence accounts for the resistance phenotype (Li, Bolaños, et al. 2024). Next, we evaluated whether LORE/FLS2XL could restrict Lso, which is transmitted by piercing-sucking psyllids (Bactericera cockerelli). During feeding, psyllids secrete watery saliva containing bacterial MAMPs. Although LORE/FLS2XL lines can detect Lso flg22, they did not differ from controls in symptom severity or Lso load after a no-choice feeding assay with Lso-carrying psyllids (Figure 1h,i). To address whether promoter strength limited FLS2XL efficacy for Lso, we re-designed the FLS2XL construct under the stronger potato ubiquitin (StUbi7) promoter (Figure S1b). As expected, the ‘Atlantic’ lines expressing StUbi7::FLS2XL perceived both Agrobacterium flg22 and Lso flg22 (Figure S2a). However, they did not display any reduction in disease severity or Lso abundance (Figure S2b,c). This indicates that while FLS2XL can detect the Lso flg22 peptide, it is insufficient to inhibit Lso. This may be due to psyllid salivary effectors suppressing defence, Lso proliferation in the phloem and the identity of the feature perceived. This underscores the importance of testing pathogen resistance under natural infection modes and suggests additional traits may be needed to combat vector-borne bacteria. In summary, we introduced LORE and FLS2XL into cultivated potato, enabling perception of both 3-OH-C10:0 and polymorphic Lso flg22. These transgenic lines showed enhanced resistance to Rso by limiting pathogen movement from roots to stems. However, the same approach did not impede Lso, indicating that vector-borne pathogens might require different strategies or receptors. Introducing LORE into tomato plants conferred heightened resistance to Pseudomonas and Alternaria, and soil drenching with 3-OH-FA inhibited P. infestans, highlighting the utility of this PRR (Eschrig et al. 2024). Future research should tailor PRR stacks to pathogen ecology and infection routes for more robust disease control. Multi-location field trials will be an essential next step to evaluate the agronomic performance of PRR stacks and confirm resistance phenotypes. G.C. and J.G.T. conceived the study. S.R. assisted in designing the LORE construct. T.L., F.S.M., E.J.B. and B.G. performed the experiments. C.S.P. and K.K.M. independently generated transgenic lines and performed Lso infection assays with similar results. T.L. and G.C. wrote the manuscript, and all authors were involved in editing. T.L., F.S.M., J.G.T. and G.C. were supported by a USDA-NIFA grant (2019-70016-29796). G.C. and T.L. were supported by the NIH (2R35GM136402). S.R. was supported by funds from the German Research Foundation (SFB924/TP-B10) and the Swiss National Science Foundation (310030_208139 to S.R.). C.S.P. and K.K.M. were partially supported by USDA NIFA (2019-70016-29796 and 2021-70029-36056) and Texas A&M AgriLife Research Insect-vectored Disease Seed Grants (114190-96210). We thank Shirin Siefbarghi and Tania Toruño for assistance with receptor cloning. The data that support the findings are available in Zenodo at https://zenodo.org/records/15328569. The construct is available on Addgene (239620). Figure S1. Tables S1-S3. Data S1. Metrial and Methods Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.