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Genome editing of NPR3 confers potato resistance to Candidatus Liberibacter spp.
Monday, 2024/06/03 | 08:23:17

Manikandan RamasamyMohan Singh RajkumarRenesh BedreSonia IrigoyenKatherine Berg-FalloureMichael V. KolomietsKranthi K. Mandadi

Plant Biotechnology Journal; First published: 22 May 2024. https://doi.org/10.1111/pbi.14378

 

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. 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.

 

See https://onlinelibrary.wiley.com/doi/10.1111/pbi.14378

 

Fig.1: CRISPR-Cas9-mediated editing of NPR3 confers tolerance to potato zebra chip disease. (a) Schematic diagram of the potato NPR3 (XM_006366563.2) and position of single guide (sg) RNA. (b) Binary vector expressing sgNPR3 and Cas9. (c) Amplicon sequencing of StNPR3-edited lines. (d) Zebra chip disease symptoms in aboveground parts of StNPR3-edited and VC plants and corresponding whole tubers (e), freshly cut (f) and fried (g) chips. (h) Quantification of CLso titer in StNPR3-edited lines. Relative expression of SA-mediated defence marker genes NPR1-like (i), WRKY6-like (j), PR1-like (k) and PR3-like (l) in StNPR3-edited and VC plants. (m) Bubble plot of significantly enriched GO terms in the StNPR3-edited lines. Scales depict significance level (P-value) and number of genes for each enriched GO term. (n) Heatmap of metabolites modulated in StNPR3-edited lines, as determined by LC–MS/MS. Quantification of SA (o), JA-Ile (p), 12OH-JA-Ile (q) and 12COOH-JA-Ile (r) in StNPR3-edited lines compared with VC. (s) Proposed mechanism of CLso resistance in StNPR3-edited lines. Graphic created with Biorender.com. Error bars in h–l and o–r represent ± standard error of the mean (n = 3–5). *, **, Student's t-test P ≤ 0.05 and P ≤ 0.01, respectively.

 

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