CRISPR/Cas9‐guided editing of a novel susceptibility gene in potato improves Phytophthora resistance without growth penalty

Potato (Solanum tuberosum L.), one of the most important food crops in the world, is affected by many pathogens, among which the oomycete Phytophthora infestans cause late blight and thereby lead to the most striking yield losses in potato production. Currently, the prevention and control of potato late blight mainly relies on the use of chemical pesticides, which cause environmental pollution problems and seriously threaten food security. Therefore, breeding P. infestans-resistant potato varieties is the most cost-effective and effective way to control late blight disease. To achieve this goal, it is prerequisite to dissect the molecular mechanism underlying the resistance and susceptibility of potato to P. infestans. Plants have evolved a two-tiered innate immune system comprising pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Pattern-triggered immunity is initiated by pattern recognition receptors (PRRs) upon recognition of microbe/herbivore-associated molecular patterns (MAMPs/HAMPs) or plant-derived endogenous damage-associated molecular patterns (DAMPs). To mount a successful infection, pathogens secrete effector proteins into the apoplast or host cells to interfere with plant immune system. As a countermeasure, plants have evolved ETI mediated by nucleotide-binding domain leucine-rich repeat (NLRs) proteins that recognize pathogen effectors directly or indirectly. Recent studies have shown that the boundaries between PTI and ETI become increasingly blurred. Some core PTI components are necessary for full exploitation of ETI, and vice versa. Pattern-triggered immunity and effector-triggered immunity also mutually potentiate each other (Ngou et al., 2021; Pruitt et al., 2021; Tian et al., 2021; Yuan et al., 2021). The bottleneck in engineering plant disease resistance is to obtain key useful genes. Nucleotide-binding domain leucine-rich repeat genes are frequently adopted for breeding resistance against a specific pathogen. However, NLRs-mediated plant disease resistance is usually race-specific and often overcome by pathogens through mutation or deletion of their effectors recognized by plant NLRs. Therefore, utilizing the susceptibility (S) genes that negatively regulate plant immunity has become an attractive breeding strategy for engineering plant disease resistance. Disease resistance conferred by S gene mutations is often nonrace-specific, durable and broad-spectrum. For instance, knockout of the S gene MLO in wheat via precise genome editing confers robust powdery mildew resistance (Li et al., 2022). Additionally, mutation of the kinase gene TaPsIPK1 (another S gene) in wheat via CRISPR/Cas9-mediated gene editing confers robust rust resistance without growth and yield penalty (Wang et al., 2022). To date, several S genes have been characterized in the interaction between P. infestans and potato (He et al., 2020). For example, BRI1-SUPPRESSOR1-like (BSL) family members act as susceptibility factors to promote P. infestans virulence (Turnbull et al., 2019). However, the utilization of S genes in potato to engineer disease resistance is much less exploited. In this study, we identified a novel potato susceptibility factor, Solanum tuberosum PLASMA MEMBRANE PROTEIN 1 (StPM1), encoded by a gene of ABA-induced Wheat Plasma Membrane Polypeptide-19 (AWPM-19)-like family and involved in the defence response to P. infestans. ABA-induced Wheat Plasma Membrane Polypeptide-19 protein was first isolated from plasma membranes of ABA-treated wheat suspension cells and AWPM-19 homologues play essential roles in developmental processes and abiotic stresses (Yao et al., 2018). However, the biological roles and the underlying molecular functions of AWPM-19 in plant–pathogen interactions remain unclear. The expression of StPM1 was significantly down-regulated in S. tuberosum upon infection by P. infestans (Figure 1a). In order to determine the role of StPM1 during potato response to P. infestans, we generated a CRISPR/Cas9 construct with two single guide RNA (sgRNA) containing a 20-bp target sequence and transformed this construct into the diploid self-incompatible S. tuberosum group Phureja S15-65 clone (Figure 1b). Two homozygous StPM1gene-edited lines containing 1 bp or 3 bp deletions and the consequent frameshift mutations in StPM1 were identified for subsequent analysis (Figure 1b). These two lines were inoculated with P. infestans, and the stpm1 mutants exhibited less pronounced disease symptom and reduced lesion area compared with the wild-type plants (Figure 1c,d), indicating that StPM1 acts as a negative regulator in potato resistance to P. infestans. Moreover, stpm1 mutants displayed the elevated expression of several defence-related genes, including StPR1, StPR5, StWRKY7 and StWRKY8 after P. infestans infection, compared with wild-type plants (Figure 1e). Importantly, the StPM1 gene-edited plants exhibited normal growth and development, compared with wild-type plants (Figure 1f). To further confirm the role of StPM1 in plant immunity, we also generated transgenic plants overexpression a HA-tagged StPM1 under the control of the 35S promoter in potato and Nicotiana benthamiana, respectively (Figure S1). The pathogen inoculation assays showed that overexpression of StPM1 led to increased susceptibility to Phytophthora infestans and Phytophthora capsici in transgenic potato and N. benthamiana plants, respectively (Figures S2a,b and S3a,b), which was accompanied by reduced induction of defence-related genes in these transgenic lines compared with that in wild-type plants (Figure S2c), further indicating that StPM1 negatively regulate potato immunity and resistance to Phytophthora. Transient expression of the StPM1-green fluorescent protein (GFP) fusion protein in N. benthamiana leaves revealed that StPM1 is localized to the plasma membrane (Figure 1g). In order to further explore the potential mechanism underlying the negative regulation of potato immunity by StPM1, we performed immunoprecipitation in combination with mass spectrometry (IP-MS) analyses in N. benthamiana and found that StPM1 associates with the NADPH oxidase NbRbohC, a homologue of StRbohC in S. tuberosum (Figure S4). We further showed the association between StPM1 and StRbohC by co-immunoprecipitation and split-luciferase assays (Figures 1h,i and S5). Interestingly, we observed that, when transiently expressed in N. benthamiana, the StRbohC protein accumulation levels detected by immunoblotting or luciferase imaging were significantly reduced upon coexpression of StPM1 (Figure 1j,k). It has been reported that AtRbohD, the homologue of StRbohC, is regulated by vacuolar-mediated protein degradation in Arabidopsis (Lee et al., 2020). To test whether StRbohC is also regulated by vacuolar degradation, Concanamycin A (ConA) was used to inhibit vacuolar degradation. As shown in Figure 1l, ConA treatment of N. benthamiana leaves expressing FLAG-StRbohC increased the accumulation of StRbohC, and blocked the StPM1-induced degradation of StRbohC (Figure 1l). Together, these results suggest that StPM1 interacts with StRbohC to promote its degradation, probably through the vacuolar degradation pathway. Consistent with the negative regulation of StRbohC protein stability by StPM1, overexpression of StPM1 inhibited fungal PAMP chitin-induced ROS production and the P. infestans effector Avr3a and its cognate NLR protein R3a-induced cell death in N. benthamiana (Figure S6). Additionally, silencing NbRbohC, the homologue of StRbohC in N. benthamiana diminished chitin-induced ROS production and reduced resistance to P. capsici and P. infestans colonization (Figure S7). Collectively, we identified a novel susceptibility factor StPM1 in Solanum tuberosum and showed that CRISPR/Cas9-mediated knockout of StPM1 gene in potato improved its resistance to Phytophthora without affecting potato growth and development. This further substantiate that modification of S genes in crops would offer a new avenue to improve plant disease resistance. Our further studies suggest that StPM1 negatively regulates plant immunity by promoting vacuolar-mediated degradation of StRbohC. StPM1 is a basic protein and abundant in hydrophobic amino acids, which are predicted to form membrane-spanning α-helices. However, the precise mechanism of how StPM1 acts on and modulates StRbohC stability remains future investigation. Importantly, the phylogenetic analysis showed that StPM1 is conserved across many species, including monocot and dicot (Figure 1m), indicating that StPM1 may be functionally conserved and good candidate to be used in other crop plants for improving the disease resistance without growth penalty. We thank Drs. Sanwen Huang from Chinese Academy of Agricultural Sciences and Jianjian Qi from Inner Mongolia University for sharing the diploid potato cultivar S. tuberosum group Phureja S15-65 clone and transformation protocol for potato, respectively. This work is supported by the National Natural Science Foundation of China (32000200) and the 2115 Talent Development Program of China Agricultural University to G.X. The authors declare no conflict of interest. G.X. and D.D. coordinated the research and wrote the manuscript. W.B. performed the majority of the experiments and draft the manuscript. J.L. prepared StPM1gene-edited potato lines. Y.L. prepared StPM1 overexpression lines in N.benthamiana. Z.H. and Y.C. performed subcellular localization for StPM1. T.Z. performed cell death and ion leakage assays. W.B., X.L., X.W., X.M., D.D. and G.X. discussed and interpreted the data. Figure S1-S7. Supplementary Figures transgenic lines of StPM1 used for phenotypic assay were analyzed by immunoblotting for protein expression. Top panel: the protein expression in S. tuberosum. Bottom panel: the protein expression in N.benthamiana. 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.