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.

Candidatus Liberibacter solanacearum ( C Lso) 是一种仅限于韧皮部的挑剔细菌，与马铃薯 ( Solanum tuberosum ) 斑马片病有关。它由马铃薯木虱（ Bactericera cockerelli Šulc.）传播，并在全球造成重大经济损失（Mora等人， 2021 ）。通过传统育种开发抗病性的成功有限（Mora等人， 2022 ），因此需要新的基因工程或基因组编辑方法。

在植物中，发病机制相关 (NPR) 蛋白的非表达蛋白充当防御激素水杨酸 (SA) 的受体。虽然 NPR1 激活拟南芥 ( Arabidopsis thaliana ) 中 SA 介导的防御，但其同源物 NPR3 却负向调节 SA 防御。在甜橙（ Citrus sinensis ）和其他作物中表达拟南芥NPR1可增强 SA 介导的对多种病原体的耐受性（Peng等， 2021 ）。相反，下调拟南芥 (Ding et al ., 2018 ) 和可可 ( Theobroma cacao ) (Fister et al ., 2018 ) 中的NPR3分别增强对细菌和真菌病原体的抵抗力。我们之前表明，短暂下调马铃薯毛状根中的StNPR3会降低C Lso 滴度（Irigoyen等人， 2020 ）。在这里，我们证明StNPR3的基因组编辑通过激活 SA 介导的防御和 JA 分解代谢赋予马铃薯对C Lso 的抗性。

为了探索StNPR3在马铃薯中的功能，我们鉴定了NPR3的马铃薯直系同源物（NCBI# XM_006366563.2，表 S1），并设计了针对StNPR3开放阅读框 (ORF) 第一个外显子的向导 RNA（图 1a、b）。使用根癌农杆菌介导的马铃薯（cv.Atlantic）转化来产生多个StNPR3编辑的品系。根据扩增子测序，选择两个独立的品系进行进一步分析。行号1 号系是单等位基因纯合子，所有四个等位基因中都有 8 bp 缺失，且系号 1 是单等位基因纯合子。 2 是双等位基因杂合子，四个等位基因中的两个有 6/7-bp 缺失（图 1c）。编辑后的​​StNPR3 ORF 预计会产生具有部分 BTB 结构域且缺乏锚蛋白重复序列​​和 SA 结合核心的截短 NPR3 蛋白（Ding等人， 2018 ；Wang等人， 2020b ）。与载体对照（VC，仅表达Cas9）植物相比， StNPR3编辑的品系没有表现出异常生长或发育。

为了评估抗病性，用C Lso ( C Lso+) 攻击植物。两个StNPR3编辑的品系都显示出疾病症状减轻，而 VC 则表现出明显的叶片失绿，并在感染后 21 天 (dpi) 枯萎（图 1d）。与 VC 相比，来自StNPR3编辑品系的块茎的新鲜切割和油炸片显示变色减少（图 1e-g）。定量 PCR 分析显示StNPR3编辑的细胞系中C Lso 滴度显着降低 (>90%, P = 0.001)（图 1h）。此外，在未感染和C Lso 感染条件下， StNPR3编辑株系中多种防御相关标记基因（例如NPR1 、 WRKY6 、 PR1和PR3 ）的表达较高（图 1i-l）。总之，这些结果表明StNPR3的编辑增强了马铃薯对C Lso 的抗性。

接下来，我们通过转录组学和代谢组学研究了StNPR3编辑的马铃薯耐受的潜在机制。在 7 dpi 和 14 dpi 对StNPR3编辑的细胞系进行 RNA 测序，分别发现约 392 个和约 427 个常见上调基因。相比之下，~410 个和~204 个基因通常分别在 7 dpi 和 14 dpi 时下调（图 S1）。基于基因本体论 (GO) 的 DEG 功能分析揭示了生物过程的显着富集，例如生物应激和防御反应（图 1m）。值得注意的是，编码乙烯反应因子的几个基因被下调，表明StNPR3编辑的品系中乙烯介导的反应受到损害（图 S2A）（Spoel等人， 2007 ）。在生物应激相关基因中，氧脂素生物合成和JA分解代谢酶，例如脂氧合酶（LOX2）和细胞色素P450，分别上调（图S2B；Zhang等人， 2023 ）。 DEG 的 MapMan 代谢图谱还显示了属于WRKY 、 MADS 、 AP2和bZIP家族的几种过氧化物酶、谷胱甘肽 S 转移酶和转录因子的激活（图 S3）。

进行靶向 LC-MS/MS 分析以确定StNPR3编辑品系中受影响的激素和代谢物的水平（图 1n）。在 7 和 14 dpi 阶段，与 VC 相比， StNPR3编辑品系中 SA 的累积水平显着更高（ P ≤ 0.05） （图 1o）。 JA-Ile（JA 的生物活性形式）在大多数组织中通常较低或检测不到（图 1p）。值得注意的是，JA-Ile 分解代谢物（12OH-JA-Ile 和 12COOH-JA-Ile）和几种在植物防御中具有推定作用的氧脂素（9-HOD、13-HOD、9-HOT、13-HOT、9-KOT 和 13） -KOT）（Wang等人， 2020a ）在StNPR3编辑的品系中显着较高（ P ≤ 0.05）（图 1q、r 和 S4）。

总之，我们提出了一个工作模型，在马铃薯中，敲低或完全去除 NPR3 可激活 SA 信号传导和对C Lso 的抵抗（图 1s）。 NPR3 的去除还会激活 JA-Ile 分解代谢和周转，以避免 JA 防御同时过度激活，从而导致细胞不受限制地死亡。我们的结果强调了马铃薯 NPR3 在调节 SA-JA 稳态中的关键作用，并提出了一种通过基因组编辑技术破坏其功能来获得抗病性的策略。