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. 2026 Feb 13;15(4):601. doi: 10.3390/plants15040601

Multiplex Gene Editing Creates Triple-Resistant Rice Against Both Insect Herbivores and Pathogens

Guannan Qin 1,2,†, Quanlei Shentu 1,2,†, Jingling Pan 1,2,†, Lizhou Lin 1,2, Caili Xie 1,2, Jiarou Ji 1,2, Huaying Du 1,2, Tingyi Chen 1,2, Chunmei Liu 3, Rensen Zeng 1,2,*, Yuanyuan Song 1,2,*
Editors: Xinwu Pei, Lu Gan
PMCID: PMC12944508  PMID: 41754307

Abstract

Rice (Oryza sativa) production faces serious threats from multiple biotic stresses, particularly the brown planthopper, rice blast, and bacterial blight. Developing resistant cultivars is the most sustainable control strategy. Compared to race-specific resistance genes, disrupting susceptibility genes often confers broader and potentially more durable resistance. However, engineering broad-spectrum resistance against both insect pests and pathogens by editing susceptibility genes remains challenging. In this study, we employed multiplex CRISPR/Cas9 editing to simultaneously disrupt key susceptibility genes involved in distinct defense pathways: ACS2 (for brown planthopper), Bsr-D1, ERF922 or Pi21 (for fungal blast), and Xa5 (for bacterial blight). Three triple-mutant lines (abx, aex, and apx) were successfully generated, and all exhibited significantly enhanced resistance to brown planthopper, blast, and bacterial blight without compromising major agronomic traits compared to the wild type. Our work demonstrates the feasibility of multiplex susceptibility gene editing as a precise and efficient strategy for breeding rice varieties with synchronized, broad-spectrum resistance to both insect pests and pathogenic diseases.

Keywords: rice, gene editing, brown planthopper, rice blast, bacterial blight

1. Introduction

Rice production is threatened by multiple biotic stresses, among which the brown planthopper (Nilaparvata lugens), bacterial blight (caused by Xanthomonas oryzae pv. Oryzae, Xoo), and blast fungus (Magnaporthe oryzae) are the most devastating. Development of resistant cultivars is considered the most economical and effective control strategy. Plant immunity against pathogens and insect pests is primarily governed by two classes of genes: resistance (R) genes and susceptibility (S) genes.

R genes activate downstream defense responses by recognizing specific effectors from pathogens or pests, and their resistant alleles are typically dominant. However, R gene-mediated resistance is often race-specific, dependent on the host plant’s intraspecific genetic variation, and can be rapidly overcome by pathogen evolution. Traditional resistance breeding, which mainly relies on crossing to pyramid natural R genes, is time-consuming and often hampered by linkage drag [1]. Advances in genetic engineering have opened new avenues for R gene application, including transgenic stacking [2], precise editing to activate silent R genes [3,4], and structural modification to broaden the resistance spectrum [5].

S genes are essential host factors required for successful pathogen infection; loss of their function often confers broad-spectrum and durable resistance [6]. Knocking out S genes via editing is technically straightforward and can avoid regulatory hurdles associated with transgenic approaches [7,8]. Editing individual S genes that affect multiple diseases [9,10], or simultaneously targeting multiple S genes, can enhance resistance against a wide range of pathogens. Nevertheless, compared to the extensive research on disease-related S genes [11] and insect-related R genes [12,13], the identification and application of insect-related S genes lag significantly, with only a few functionally characterized genes reported to date [14,15,16]. Consequently, although multiple S gene editing has been successfully demonstrated for multi-disease resistance [17,18], molecular breeding for broad-spectrum resistance against both insects and diseases remains rare and challenging.

Plant defense responses rely on complex signaling networks, in which salicylic acid (SA), jasmonic acid (JA), and ethylene are three key signaling pathways of phytohormones. In most instances, the SA pathway primarily regulates plant resistance against biotrophic pathogens, whereas JA and ethylene signaling pathways play central roles in plant defense against insect herbivores and necrotrophic pathogens [19,20,21]. While SA and JA generally positively regulate resistance, ethylene exhibits a “dual role”: it positively regulates resistance to blast and the striped stem borer but negatively regulates resistance to brown planthopper [22,23,24].

To develop novel rice varieties with synchronized, broad-spectrum, and durable resistance to brown planthopper, blast, and bacterial blight via S gene editing, the ethylene biosynthesis gene ACS2 was firstly targeted using CRISPR/Cas9 to confer brown planthopper resistance. Previous studies showed that RNAi-mediated suppression of OsACS enhanced brown planthopper resistance but compromised blast resistance [24]. To overcome this dilemmatic trade-off, Bsr-D1 [25], ERF922 [26] and Pi21 [27], whose loss-of-function alleles confer blast resistance [28], were then edited separately to improve blast resistance. Additionally, the Xa5 locus was edited [29] to enhance bacterial blight resistance. Ultimately, three triple-mutant lines, i.e., acs2/bsr-d1/xa5, acs2/erf922/xa5 and acs2/pi21/xa5 were successfully generated. All three lines showed significantly enhanced combined resistance to brown planthopper, blast, and bacterial blight without compromising yield. The novel triple-resistant cultivars developed in this study provide valuable genetic resources and a feasible technical strategy for composite resistance breeding in rice.

2. Methods and Materials

2.1. Plants, Bacteria, Insect and Plasmids

The japonica rice variety Nipponbare (Oryza sativa L. geng cv. Nipponbare) was used as the recipient for genetic transformation and as the susceptible control in resistance evaluations. The brown planthopper susceptible variety Taichung Native 1 (TN1) was used for brown planthopper rearing. Competent cells of Escherichia coli strain DH10B and Agrobacterium tumefaciens strain AGL1 were purchased from Biomed Co., Ltd. (Beijing, China). The brown planthopper population was initially collected from the field. The M. oryzae isolate Guy11 and the Xoo strain ZHE173 were preserved in our laboratory. The plasmids used for vector construction, namely pYLCRISPR/Cas9Pubi-H, pYLgRNA-POsU6a, pYLgRNA-POsU6b, and pYLgRNA-POsU6c, were kindly provided by Professor Yaoguang Liu of South China Agricultural University.

2.2. Vector Construction

The S genes selected for knockout included the brown planthopper-sensitive gene ACS2 (LOC_Os04g48850), the three blast-sensitive genes either Bsr-D1 (LOC_Os03g32230), ERF922 (LOC_Os01g54890), or Pi21 (LOC_Os04g32850), and the bacterial blight-sensitive gene Xa5 (LOC_Os05g01710). The structures of these five S genes, along with the locations and sequences of the designed CRISPR target sites, were presented in Figure 1.

Figure 1.

Figure 1

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Susceptibility (S) genes selected for editing in rice. Exons, untranslated regions, and introns are depicted by grey boxes, white boxes, and solid lines, respectively. The diagram is drawn to scale, with a scale bar provided above. Short green vertical lines within each gene mark the locations of the CRISPR target sites. The orientation of genes and target sites is indicated by arrows. In the displayed target sequences, the protospacer adjacent motif (PAM) is highlighted in red.

Binary vectors for gene editing were constructed as described by Ma [30]. Restriction enzyme BsaI and T4 DNA ligase were purchased from Biorun Co., Ltd. (Wuhan, China). Primers used are listed in Supplementary Table S1. The constructed binary vectors were introduced into the A. tumefaciens for plant transformation.

2.3. Agrobacterium-Mediated Rice Transformation

Dehusked Nipponbare seeds were surface-sterilized and cultured on MS medium (Coolaber Co., Ltd., Beijing, China; Cat. No. PM1015-30) containing 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for one month to induce callus formation. Induced calli were subcultured for 7 days. Healthy calli were selected and immersed in an A. tumefaciens suspension resuspended in AAM liquid medium (Coolaber PM1962) containing 100 μM acetosyringone for 30 min. Subsequently, the infected calli were co-cultured on NB medium (Coolaber PM1301) at 22 °C for 3 days. After thorough washing, the calli were transferred to NB selection medium containing 50 mg/L hygromycin. Following two rounds of selection (each lasting two weeks), resistant calli were transferred to NB regeneration medium supplemented with 2 mg/L 6-benzylaminopurine (6-BA) and 1 mg/L α-naphthaleneacetic acid (NAA). Regenerated plantlets (~3 weeks later) were transferred to 1/2 MS medium for 14 days and then to the soil. Part of the transgenic service was provided by Biorun Co., Ltd.

2.4. Mutation Detection

Transgene-free gene-editing plants were screened using a hygromycin painting assay after Transgenic T0 plants were obtained. Homozygously edited lines were identified from the T1 generation by Sanger sequencing and propagated. T2 generation plants from the selected homozygous lines were used for subsequent resistance evaluations and agronomic trait investigations.

To determine the transcript levels of target genes, total RNA was extracted from leaves of three-leaf-stage seedlings using the Ultrapure RNA Kit (DNase I) (Cwbio, Taizhou, Jiangsu, China; Cat. No. CW0597S). First-strand cDNA was synthesized with HiScript II Reverse Transcriptase (Vazyme, Nanjing, Jiangsu, China; Cat. No. R201). qRT-PCR was performed on a QuantStudio 1 Plus system (Applied Biosystems, Waltham, MA, USA) using ChamQ Blue Universal SYBR qPCR Master Mix (Vazyme; Cat. No. Q312). The rice Actin gene was used as an internal control, and the relative expression levels of target genes were calculated using the 2−ΔΔCt method. Three biological replicates were included for each sample.

Potential off-target effects of the designed sgRNAs were assessed in silico using Cas-OFFinder [31] allowing for up to three mismatches and an NGG PAM motif (Supplementary Table S2).

Primers used for sequencing and qPCR were listed in Supplementary Table S1.

2.5. Evaluation of Rice Resistance to Brown Planthopper

The brown planthopper population was maintained and propagated on the susceptible variety TN1. For the assay, 24 seedlings at the three-leaf stage for each tested line were planted together and confined within an insect-proof mesh cage. Approximately eight second-instar brown planthopper nymphs were placed on each plant. When the mortality rate of any line approach 90%, all plants were immediately photographed, and the mortality rate for each line was recorded. The experiment was performed with three biological replicates.

2.6. Evaluation of Rice Resistance to Blast

Dried filter papers containing mycelia of the M. oryzae strain Guy11 were placed on potato dextrose agar (PDA) medium (Coolaber, PM0520) and incubated at 26 °C for 7 days. Mycelia from the colony edge were then transferred to oatmeal agar plates and cultured for 14 days. Fungal spores were harvested and suspended in sterile water containing 0.02% (v/v) Tween 20 at a concentration of 2 × 105 spores/mL.

For the spray inoculation assay, 3-week-old rice seedlings were sprayed with the conidial suspension (105 conidia mL−1, 0.02% Tween 20) of M. oryzae. Inoculated plants were kept in darkness at high humidity for 24 h and then transferred to a growth chamber with a 16 h light/8 h dark cycle and 95% relative humidity for 10 days before photographing. Disease severity was scored for each plant using the scale described by Mackill [32]. To determine relative fungal biomass, genomic DNA was extracted from the second-top leaves of all individual plants and subjected to qPCR analysis using primers listed in Supplementary Table S1. The experiment included three biological replicates.

For the punch inoculation assay, the middle segments (7–9 cm long) of the second-top leaves from plants at the booting stage were detached. Three approximately equidistant spots were gently pressed (without breaking the epidermis) using a pipette tip on each leaf segment. A 5 μL droplet of the conidial suspension was applied to each spot. The leaf segments were placed in a culture dish containing a sterile solution of 6-BA (10 μg·mL−1) to maintain turgor pressure. Lesions were photographed, and their areas were measured 7 days post-inoculation using ImageJ 1.4.3.67. The inoculation experiments were repeated three times using leaves collected from two independent plants per line.

2.7. Evaluation of Rice Resistance to Bacterial Blight

The bacterial blight pathogen Xoo strain ZHE173 was cultured on nutrient agar (NA) medium (Coolaber, PM0670). Bacterial cells were resuspended in sterile water, and the concentration was adjusted to an optical density at 600 nm (OD600) of 0.5. For inoculation, scissors were dipped into the bacterial suspension and used to make a diagonal cut (~3 cm long) near the tip of the second-top leaf of healthy plants at the booting stage. Lesions were photographed and measured (length and area) approximately 3 weeks post-inoculation. The experiment included more than 15 biological replicates per line.

2.8. Investigation of Agronomic Traits

Nipponbare and the gene-edited lines were cultivated under controlled conditions in a smart greenhouse at Fujian Agriculture and Forestry University (Fuzhou, China). Plants were grown under controlled conditions: 28/25 °C (day/night), ~70% relative humidity, with a 13-h light/11-h dark photoperiod and a light intensity of 2000 μmol·m−2·s−1. At full maturity, plant height, panicle number, spikelets per panicle, filled grains per panicle, seed-setting rate, and 1000-grain weight were measured using eight biological replicates per line. Spikelets per panicle, filled grains per panicle, and seed-setting rate were determined based on the main panicle of each plant. For grain length and width measurements, seeds from all eight plants of a given line were pooled. Approximately 100 grains were randomly selected, scanned, and analyzed using the SmartGrain software [33].

3. Results

3.1. CRISPR/Cas9-Mediated Knockout of S Genes

The gRNA expression cassettes targeting these S genes were cloned and assembled into three binary vectors. The resulting constructs included pABX (targeting ACS2, Bsr-d1, and Xa5), pAEX (targeting ACS2, ERF922, and Xa5), and pAPX (targeting ACS2, Pi21, and Xa5) (Figure 2A). The vectors were named based on the initial letters of their respective target genes.

Figure 2.

Figure 2

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CRISPR/Cas9-mediated knockout of S genes in rice. (A) Schematic of the T-DNA structure in the gene-editing constructs. Pe35S and Pubi represent two strong constitutive promoters used in rice: the enhanced Cauliflower Mosaic Virus (CaMV) 35S promoter and the maize ubiquitin promoter, respectively. POsU6a, POsU6b, and POsU6c are three rice U6 small nuclear RNA (snRNA) promoters, each driving the transcription of a distinct sgRNA. T35S and TNOS are common terminators, derived from the CaMV 35S and A. tumefaciens nopaline synthase genes, respectively. The “Pe35S:HygR:T35S” cassette is shown with its text flipped 180°, denoting that it is oriented in the reverse direction relative to the other genetic elements. (B) Detailed genotypic analysis of the three gene-editing mutants. Nucleotides highlighted in red indicate the edited bases, while those on a grey background represent the Protospacer Adjacent Motif (PAM) sequence. For the Xa5 target in the apx mutant, only the flanking sequences are shown due to a large-fragment deletion (−214 bp). Frameshift mutations typically lead to premature termination. In contrast, the frameshift mutation in Bsr-d1 obtained in this study results in a protein of almost unchanged length compared to the wild-type.

A large number of transgenic lines were obtained via Agrobacterium-mediated transformation. From the T2 generation, three transgene-free lines with homozygous edits at all target loci were selected for further phenotypic analysis. These lines were designated abx, aex, and apx, corresponding to the triple-mutant acs2/bsr-d1/xa5, acs2/erf922/xa5, and acs2/pi21/xa5, respectively. The genotypes of these selected lines are shown in Figure 2B and Supplementary Figures S1–S3.

To further evaluate the impact of gene editing, we performed qPCR analysis of all target genes in each triple-mutant line. The results showed that the expression levels of the newly generated alleles were reduced to varying degrees, suggesting that even coding-region mutations can influence gene expression, potentially by affecting transcript stability. Overall, the transcriptional changes associated with Xa5 editing appeared relatively moderate across the mutant lines, even in the case of a large-fragment deletion—a more severe type of mutation (Supplementary Figure S4).

Sanger sequencing of the predicted off-target sites revealed no detectable editing events (Supplementary Figure S5), supporting the high specificity of the CRISPR/Cas9 system used in this study.

3.2. Resistance to Brown Planthopper

Following infestation with the brown planthopper, damage symptoms on wild-type (WT) Nipponbare and the three triple mutants (abx, aex, and apx) were photographed (Figure 3A). When the WT plants reached near-complete mortality, the survival rates of abx, aex, and apx were significantly higher, at 56.9%, 38.9%, and ~52.8%, respectively (Figure 3B). These results showed that all three gene-edited lines display significantly enhanced resistance to brown planthopper, with the improvements being particularly pronounced in the abx and apx mutants.

Figure 3.

Figure 3

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Evaluation of brown planthopper resistance of three triple-mutant lines (abx, aex, and apx). (A) Phenotypic comparison following brown planthopper infestation. The scale represents 5 cm. (B) Survival rates after brown planthopper infestation. Different lowercase letters (a–c) indicate significant differences among groups (p < 0.05).

3.3. Resistance to Blast

Blast resistance was evaluated using both spray and puncture inoculation assays. At 10 days post-spray-inoculation with M. oryzae, disease phenotypes of the WT and mutants (abx, aex, apx) were recorded (Figure 4A). Disease severity analysis indicated that 79.2% of WT plants were scored as susceptible (scale 3–5). In contrast, the edited lines exhibited a predominantly resistant phenotype, with 63.3% (abx), 65.1% (aex), and 66.7% (apx) of plants classified as scale 0–2 (Figure 4B). Consistent with this, quantification of relative fungal biomass by qPCR showed an average value of 1.96 in WT, which was significantly higher than the levels in abx (0.97), aex (0.40), and apx (1.33) (Figure 4C).

Figure 4.

Figure 4

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Evaluation of rice blast resistance of three triple-mutant lines (abx, aex, and apx). (A) Disease symptoms after spray-inoculation with M. oryzae. (B) Percentage classification of diseased leaves after spray inoculation. (C) Relative fungal biomass in leaves after spray inoculation. (D) Lesion development after puncture-inoculation with M. oryzae. (E) Lesion areas statistics. Different lowercase letters (a–d) indicate significant differences among groups (p < 0.05).

In the puncture inoculation assay, lesion development was assessed at 7 days post-inoculation (Figure 4D). The mean lesion area on WT was 11.04 cm2. All mutant lines displayed significantly reduced lesion areas of 4.00 cm2 (abx), 3.84 cm2 (aex), and 3.39 cm2 (apx) (Figure 4E).

Together, these results demonstrate that the gene-edited lines abx, aex, and apx show significantly enhanced resistance to rice blast compared to WT.

3.4. Resistance to Bacterial Blight

Disease symptoms on WT and the three triple mutants (abx, aex, and apx) were photographed at 21 days post-inoculation of Xoo (Figure 5A). The average lesion length was 14.47 cm in WT, but was significantly reduced to 8.20 cm, 7.02 cm, and 8.27 cm in abx, aex, and apx, respectively (Figure 5B). Similarly, the relative lesion length (lesion length/leaf length) was 41.51% in WT, while the mutants showed significantly lower values of 26.20%, 25.13%, and 26.94% (Figure 5C). The average lesion area followed the same trend, with WT at 11.18 cm2 compared to 6.45 cm2, 5.34 cm2, and 6.43 cm2 in the mutants (Figure 5D).

Figure 5.

Figure 5

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Evaluation of bacterial blight resistance of three triple-mutant lines (abx, aex, and apx). (A) Bacterial blight symptoms after inoculation with Xoo. (B) Lesion length on leaves induced by Xoo. (C) Relative lesion length (the rate of lesion length and leaf length). (D) Lesion area on leaves induced by Xoo. Different lowercase letters (a–b) indicate significant differences among groups (p < 0.05).

Collectively, these results demonstrate that all three gene-edited lines possess significantly improved resistance to bacterial blight.

3.5. Agronomic Traits

To assess whether the improved disease and pest resistance incurred any fitness cost, we evaluated the performance of major agronomic traits in the gene-edited lines under greenhouse conditions. As shown in Figure 6A, all mutant lines displayed normal growth and development comparable to WT at maturity. Comprehensive quantitative analysis revealed no significant alterations in key yield-related traits. Specifically, the plant height, heading date, and number of productive panicles per plant of the abx, aex, and apx mutants were similar to those of the WT (Figure 6B–D). Furthermore, yield components, including the number of spikelets per panicle, filled grains per panicle, seed-setting rate, grain length, grain width, and 1000-grain weight, were all maintained at levels comparable to the WT in all three mutant lines (Figure 6E–J). These results collectively demonstrate that the enhanced resistance in the abx, aex, and apx lines is achieved without compromising major agronomic traits.

Figure 6.

Figure 6

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Agronomic traits of three triple-mutant lines (abx, aex, and apx). (A) Plant type at maturity stage. (B–J) Comparison of four genotypes across nine key agronomic traits: (B) Plant height, the scale represents 10 cm. (C) Heading date, (D) Panicle number, (E) Spikelets per panicle, (F) Filled grains per panicle, (G) Seed-setting rate, (H) Grain length, (I) Grain width, and (J) 1000-grain weight. Bars sharing the same letter (a) are not significantly different.

4. Discussion

This study employed multiplex CRISPR/Cas9 technology to simultaneously disrupt key S genes conferring vulnerability to brown planthopper, rice blast, and bacterial leaf blight. We successfully generated triple mutant rice lines exhibiting significantly enhanced resistance to these three major biotic stresses. Under controlled greenhouse conditions, the improved resistance was achieved without significant compromise in major agronomic traits. This work offers a strategy and valuable genetic material for breeding multi-resistant and high-yielding crops through multiplex gene editing.

The successful stacking of resistance, however, also revealed complex phenotypic outcomes associated with editing ethylene signaling genes. Previous studies showed that RNAi-mediated suppression of ACS2 (involved in ethylene biosynthesis) enhanced brown planthopper resistance but reduced blast resistance, revealing a “dual role” for ethylene in stress responses [24]. Here, we generated an aex line by knocking out ERF922 in the acs2 mutant background. ERF922 functions as a negative regulator of blast resistance [26], and is annotated as an ethylene response factor, suggesting it may act downstream of ACS2. Phenotypic assessment revealed that while the aex line showed weaker brown planthopper resistance compared to the abx (bsr-d1 in acs2 background) and apx (pi21 in acs2 background) lines, its resistance was still significantly stronger than that of WT. This indicates that the aex line retained a substantial portion of the enhanced brown planthopper resistance of the acs2. Concurrently, the aex line also exhibited stronger blast resistance than the WT. This suggests two non-exclusive possibilities. First, functional redundancy within the ACS gene family may allow residual ethylene synthesis in the acs2 mutant, insufficient to fully abolish blast susceptibility. Second, beyond its classical role in ethylene response, the ERF transcription factor ERF922 may integrate multiple hormonal signals such as ABA, JA, or SA [34]. Mechanistically, given that insect resistance is often mediated by JA and ethylene pathways, whereas pathogen resistance is primarily governed by the SA pathway, ERF922 likely acts as a convergence point that modulates the physiological antagonism between these signaling pathways. It potentially functions by repressing SA-dependent defense genes to prioritize ethylene-driven stress responses. The depletion of ERF922 may relieve this repression, thereby reactivating SA-mediated immunity against blast fungus even when upstream ethylene biosynthesis is compromised in the acs2 background. If this model is correct, then the defense response activated by its loss could be partially independent of upstream ethylene synthesis. However, this hypothesis remains to be further validated by measuring hormone dynamics and the expression of defense marker genes in the mutants. Thus, within complex hormonal networks regulating susceptibility, targeting downstream “signaling hub” genes like ERF922 can sometimes be more effective in coordinating broad-spectrum resistance than targeting upstream biosynthetic genes like ACS2. This offers important insights for designing future strategies for multi-resistance gene editing.

We also found that knocking out Xa5 significantly enhanced bacterial leaf blight resistance without observable growth or yield penalties. This result aligns with the use of its natural weak-function alleles in breeding [29] and with prior work employing the same target site [35]. qPCR analysis indicated that Xa5 transcript levels in the mutants were only slightly decreased compared to those in the WT (Supplementary Figure S4), which may reflect persistent transcription despite knockout. We speculate on two potential explanations for the low penalty of xa5: First, Xa5 may function primarily as a susceptibility target for pathogen effectors and be non-essential for normal development. Second, CRISPR/Cas9-induced edits near the start codon that cause premature termination may not always result in complete loss-of-function, as they can trigger translational reinitiation. Notably, both edit types generated here (a single-nucleotide insertion and a 214-bp deletion) possess downstream in-frame start codons (ATG), as illustrated in Supplementary Figures S1–S3. A similar phenomenon in wheat RHT1 was shown to produce partially functional truncated proteins via reinitiation [36]. However, whether the observed absence of fitness costs is due to the dispensability of Xa5 in normal development or to residual function of a truncated isoform remains to be experimentally clarified. Future studies involving protein-level assays (e.g., Western blot) or genetic complementation will be essential to resolve this question, which has important implications for the long-term stability and breeding utility of Xa5-edited lines.

This study further confirms that disrupting S genes, which act as negative regulators of immunity, represents an effective breeding strategy for combating multiple biotic stresses. However, the uneven distribution of S gene resources poses a significant constraint: while pathogen-related S genes are relatively well-characterized, well-defined insect susceptibility genes remain scarce. This likely reflects fundamental differences in infestation strategies. Insect herbivory involves complex physical and chemical elicitation, and plant defense relies on induced, multi-layered barriers—such as cell wall fortification and secondary metabolite accumulation—governed by intricate networks, making single major S genes less probable. Insect resistance-related S genes may instead focus more on auxiliary components affecting insect nutrition, digestion, or oviposition recognition. Consequently, loss-of-function alleles of such genes are less likely to achieve immune effects comparable to those in disease resistance, thereby imposing greater demands on phenotypic screening and functional gene discovery strategies.

Validating the stability of agronomic traits across diverse environments is undoubtedly the gold standard for evaluating the breeding potential of gene-edited crops, particularly when manipulating resistance genes associated with growth-defense trade-offs. However, comprehensive multi-location field trials were not conducted in this study due to strict biosafety regulations regarding gene-edited crops in China, which currently restrict open-field experimentation. Therefore, our assessment focused on establishing a robust phenotypic baseline under controlled greenhouse conditions. The mutants obtained in this study enhanced resistance and maintained WT-level yield components in this optimized environment. It is a necessary and promising prerequisite for future field evaluations once regulatory policies permit. It should also be noted that the present study focused on the combined effect of triple editing; systematic comparison with single- and double-gene mutants was not performed, which limits our ability to precisely delineate the individual contribution of each edited gene. Future work involving such comparisons will help clarify potential synergies or trade-offs among these susceptibility genes. Moreover, while we have demonstrated significantly enhanced resistance using representative strains/populations (e.g., the blast isolate Guy11), future studies should include a wider range of pathogen isolates and insect populations to further evaluate the resistance spectrum and stability.

In summary, this study used multiplex CRISPR/Cas9 editing to pyramid disruptive mutations of S genes conferring rice susceptibility to brown planthopper, blast, and bacterial leaf blight, respectively, into a single rice background, creating novel lines with triple resistance and no significant agronomic trade-offs. Our work provides directly valuable genetic resources for multi-resistant rice breeding and demonstrates the feasibility and advantages of a modular, designable S gene editing strategy. With ongoing discovery of S genes and advances in editing technology, designing and cultivating “smart crops” capable of withstanding multiple biotic stresses is becoming an increasingly attainable breeding goal.

Acknowledgments

We thank Yaoguang Liu of South China Agricultural University for providing with vector construction plasmids, and Guodong Lu of Fujian Agriculture and Forestry University for providing with M. oryzae isolate Guy11.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants15040601/s1, Supplementary Table S1: Primers used in this study. Supplementary Table S2: Potential off-target sites for the sgRNAs used in this study. Supplementary Figures S1–S3: Sanger sequencing chromatograms of the edited sites in the triple-mutant lines abx, aex, and apx. The PAM site is highlighted in red text. For Xa5 edits that are located extremely close to the start codon, the newly predicted start codon is highlighted in blue. Supplementary Figure S4: Expression analysis of the targeted S genes. Supplementary Figure S5: Sanger sequencing validation of predicted off-target sites.

plants-15-00601-s001.zip (6.2MB, zip)

Author Contributions

G.Q., R.Z. and Y.S. conceived the study, designed the experiments, and wrote the manuscript. Q.S., C.X. and J.J. performed vector construction and genetic transformation. J.P., Q.S., L.L. and T.C. conducted the resistance evaluation and agronomic trait investigation. L.L. carried out genotypic identification of the mutants. H.D. and C.L. assisted in revising the manuscript. R.Z. and Y.S. provided financial support and supervised the project. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author(s).

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding this study.

Funding Statement

This work is supported by the National Natural Science Foundation of China (32302465, 32371588, 32271617, 32401402, 32471656).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

plants-15-00601-s001.zip (6.2MB, zip)

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author(s).

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