Genome editing of NPR3 confers potato resistance to Candidatus Liberibacter spp.

Candidatus Liberibacter solanacearum (CLso) is a phloem-limited, fastidious bacterium associated with the potato (Solanum tuberosum) zebra chip disease. It is transmitted by the potato psyllid (Bactericera cockerelli Šulc.) and causes significant economic losses globally (Mora et al., 2021). Developing disease resistance by conventional breeding has shown limited success (Mora et al., 2022), thus necessitating new genetic engineering or genome editing approaches. In plants, non-expressor of pathogenesis-related (NPR) proteins act as receptors of the defence hormone, salicylic acid (SA). While NPR1 activates SA-mediated defences in Arabidopsis (Arabidopsis thaliana), its homologue, NPR3, negatively regulates SA defences. Expressing Arabidopsis NPR1 in sweet oranges (Citrus sinensis) and other crops enhances SA-mediated tolerance to multiple pathogens (Peng et al., 2021). Conversely, down-regulating NPR3 in Arabidopsis (Ding et al., 2018) and cacao (Theobroma cacao) (Fister et al., 2018) enhances resistance to bacterial and fungal pathogens, respectively. We previously showed that transiently down-regulating StNPR3 in potato hairy roots reduces CLso titer (Irigoyen et al., 2020). Here, we show that genome editing of StNPR3 confers potato resistance to CLso by activating SA-mediated defences and JA catabolism. To explore the StNPR3 function in potatoes, we identified a potato orthologue of NPR3 (NCBI# XM_006366563.2, Table S1) and designed a guide RNA targeting the first exon of the StNPR3 open reading frame (ORF) (Figure 1a,b). Agrobacterium tumefaciens-mediated transformation of potato (cv. Atlantic) was used to generate multiple StNPR3-edited lines. Based on amplicon sequencing, two independent lines were selected for further analyses. Line no. 1 is mono-allelic homozygous with an 8-bp deletion in all four alleles, and line no. 2 is bi-allelic heterozygous with a 6/7-bp deletion in two of the four alleles (Figure 1c). The edited StNPR3 ORFs are predicted to produce truncated NPR3 protein with partial BTB domain and lacking the Ankyrin-repeat and SA-binding core (Ding et al., 2018; Wang et al., 2020b). The StNPR3-edited lines exhibited no abnormal growth or development compared with vector control (VC, expressing Cas9 alone) plants. To evaluate disease resistance, plants were challenged with CLso (CLso+). Both StNPR3-edited lines showed reduced disease symptoms, while the VC exhibited prominent leaf chlorosis and wilted by 21 days post-infection (dpi) (Figure 1d). Freshly cut and fried chips from tubers from StNPR3-edited lines showed reduced discoloration compared with VC (Figure 1e–g). Quantitative PCR analysis revealed a significant reduction in CLso titer (>90%, P = 0.001) in StNPR3-edited lines (Figure 1h). Furthermore, expression of multiple defence-related marker genes (e.g., NPR1, WRKY6, PR1 and PR3) was higher in StNPR3-edited lines in uninfected and CLso-infected conditions (Figure 1i–l). Together, these results demonstrate that editing of StNPR3 enhanced potato resistance to CLso. We next examined the underlying mechanisms of tolerance of StNPR3 edited potato via transcriptomics and metabolomics. RNA sequencing of the StNPR3 edited lines at 7 and 14 dpi uncovered ~392 and ~427 commonly up-regulated genes, respectively. In comparison, ~410 and ~204 genes were commonly down-regulated at 7 and 14 dpi, respectively (Figure S1). Gene Ontology (GO)-based functional analysis of the DEGs revealed significant enrichment in biological processes such as biotic stress and defence responses (Figure 1m). Notably, several genes encoding ethylene response factors were down-regulated, suggesting a compromise of ethylene-mediated responses in the StNPR3-edited lines (Figure S2A) (Spoel et al., 2007). Among the biotic stress-related genes, oxylipin biosynthesis and JA catabolism enzymes, such as lipoxygenases (LOX2) and cytochrome P450s, respectively (Figure S2B; Zhang et al., 2023), were up-regulated. MapMan metabolite mapping of the DEGs also showed activation of several peroxidases, glutathione S-transferases and transcription factors belonging to WRKY, MADS, AP2 and bZIP families (Figure S3). Targeted LC–MS/MS analysis was performed to determine the levels of hormones and metabolites affected in the StNPR3-edited lines (Figure 1n). Levels of SA accumulated significantly higher (P ≤ 0.05) in the StNPR3-edited lines compared with VC at the 7 and 14 dpi stages (Figure 1o). JA-Ile (the biologically active form of JA) was generally low or undetectable in most tissues (Figure 1p). Remarkably, JA-Ile catabolites (12OH-JA-Ile and 12COOH-JA-Ile) and several oxylipins with putative roles in plant defences (9-HOD, 13-HOD, 9-HOT, 13-HOT, 9-KOT and 13-KOT) (Wang et al., 2020a) were significantly (P ≤ 0.05) higher in the StNPR3-edited lines (Figures 1q,r and S4). In summary, we propose a working model that, in potatoes, knockdown or complete NPR3 removal activates SA signalling and resistance to CLso (Figure 1s). NPR3 removal also activates JA-Ile catabolism and turnover to avoid hyperactivation of JA defences concomitantly that could lead to unrestricted cell death. Our results underscore the critical role of potato NPR3 in regulating SA-JA homeostasis and present a strategy to attain disease resistance by disrupting its function with genome editing technology. This study was partially supported by funds from USDA-NIFA (2021-70029-36 056; HATCH TEX0-9621, TEX0-7790), Texas A&M AgriLife Research Insect-vectored Disease Seed Grants (124190-96210), and the Texas A&M AgriLife IAHA to KM. Metabolite analyses were partially supported by a USDA-NIFA grant (2021-67013-33568) to MVK. We thank D. Rossi, V. Mora, V. Garza and R. Mireles (Texas A&M AgriLife Research) for various technical assistance. All authors declare no competing interests. K.M. and M.V.K. designed and supervised the experiments. M.R., M.S.R., S.I., R.B. and K.B.-F. performed the experiments and analysed the data. All authors contributed to the preparation and review of the manuscript. The data that supports the findings of this study are available in the supplementary material of this article. Table S1 The StNPR3 genomic, coding, and protein sequences are based on the Phytozome, S. tuberosum (v4.03) genome. Table S2 List of primers used in this study. Figure S1 Co-differentially expressed genes among the two StNPR3-edited lines. Figure S2 Heat maps of up- and down-regulated genes in the StNPR3-edited lines. Figure S3 Pathway analysis of the differentially expressed genes in the StNPR3-edited lines. Figure S4 LC-MS/MS quantification of hormones and defense-related metabolites in StNPR3-edited lines during CLso infection. Method S1 Design of CRISPR-CAS9 gene constructs. 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.