Direct association of Mg1 with a protease inhibitor confers resistance to rice root-knot nematode

doi:10.21203/rs.3.rs-1820368/v1

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Direct association of Mg1 with a protease inhibitor confers resistance to rice root-knot nematode

https://doi.org/10.21203/rs.3.rs-1820368/v1

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published 08 Jun, 2023

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The rice root-knot nematode (Meloidogyne graminicola) is one of the most destructive pests threatening rice (Oryza sativa L.) production in Asia; however, no rice resistance genes have been cloned. Here, we demonstrate that M. graminicola-resistance gene 1 (Mg1), an R gene highly expressed at the site of nematode invasion, determines resistance against the nematode in several rice varieties. Introgressing Mg1 into susceptible varieties dramatically increases resistance, for which the leucine-rich repeat domain is critical for recognizing root-knot nematode invasion. We also establish the transcriptome and cytological changes that allow a rapid and robust response during the incompatible interaction that occurs in resistant rice upon nematode invasion. Furthermore, we identified a putative protease inhibitor that directly interacts with Mg1 during Mg1-mediated resistance. Our findings provide insight into the molecular basis of nematode resistance as well as valuable resources for developing rice varieties with improved nematode resistance.

Rice (Oryza sativa L.) is a major staple crop, providing food for more than half of the world’s population¹. Plant-parasitic nematodes cause 10–25% annual yield losses in rice worldwide². Over 100 different nematode species attack rice³. Among them, root-knot nematodes (Meloidogyne spp.), one of the top 10 economically important plant-parasitic nematodes, have a remarkably wide host range and cause the most damage⁴. Rice root-knot nematode (M. graminicola) is the most prevalent root-knot nematode in rice cultivation systems and is a major threat to rice production, particularly in Asia, where approximately 90% of the world’s rice is produced and consumed^5-7.

M. graminicola is an obligate sedentary endoparasite and was first described by Golden and Birchfield (1965). It is widely distributed in South East Asia and other countries where rice is extensively cultivated (Mg distribution map: http://www.cabi.org/isc/datashe/33243)^8,9. M. graminicola infects upland, lowland, and deep-water rice, as well as rice grown in nurseries. Field populations of rice root-knot nematode are increasing dramatically due to changes in agricultural practices in response to water shortage and climate change, such as the development of aerobic rice⁵.

The infective second-stage juvenile (J2) of rice root-knot nematode enters the root in the elongation zone and migrates toward the vasculature, where it stimulates rice tissues to form a specialized feeding structure called giant cells, which provide the needed nourishment to complete their life cycle^6,10. After feeding, the J2s molt three more times to reach the reproductive adult stage, and the life cycle is completed within 2–3 weeks depending on the temperature and flooding conditions in the fields¹¹. Unlike the symptoms caused by other Meloidogyne species, M. graminicola induces hyperplasia and hypertrophy of surrounding rice root cells, resulting in hook-shaped galls at the root tips (Supplementary Fig. 1a). The adult females lay eggs inside the galls, where they are well-protected from flooded conditions, and J2s of the next generation will continue the infection cycle¹². Consequently, substantial damage to the rice root system leads to poor growth (Supplementary Fig. 1b) and severe rice yield losses⁷.

Control strategies such as crop rotation, continuous flooding, and nematicides, which are commonly applied in the field to mitigate yield losses caused by M. graminicola, are not efficient or feasible⁷. Breeding more resistant varieties offers an effective, economical, and environmentally friendly option to manage these nematodes. For example, one nematode resistance gene, Mi-1.2, found in the wild tomato species Lycopersicon peruvianum, is used commercially to enhance resistance to several root-knot nematode species in cultivated tomato (Solanum lycopersicum)^13,14. Ma from Myrobalan plum (Prunus cerasifera) confers broad-spectrum resistance to over 30 root-knot nematode species¹⁵ and is utilized for rootstock breeding in stone fruits¹⁶.

Rice germplasm constitutes natural sources of resistance against M. graminicola. Resistance to this nematode has been found in two rice species from Africa, the wild rice Oryza longistaminata and domesticated O. glaberrima¹⁷. However, introgressing resistance genes into Asian rice (O. sativa) is challenging because the interspecific progeny fails to express the same degree of resistance observed in African rice¹⁸. A new source of resistance has been identified recently in another wild rice species, O. glumaepatuma¹⁹. It has been reported that multiple quantitative trait loci (QTLs) are responsible for M. graminicola resistance traits using different mapping populations, but all of these loci need to be clarified. Six QTLs controlling partial nematode resistance were located on chromosomes 1, 2, 6, 7, 9, and 11 using recombinant inbred lines (RILs) derived from a cross between the O. sativa varieties Bala and Azucena²⁰. Jena et al.²¹ reported two QTLs on chromosomes 1 and 3 from the analysis of RILs derived from a cross between the varieties Annapurna and Ramakrishna. Likewise, more QTLs associated with M. graminicola resistance and tolerance have been detected in populations derived from a cross between the aerobic rice genotype IR78877–208-B-1-2 and the O. glaberrima genotype CG14^22,23. In addition, a genome-wide association study (GWAS) of a global rice panel revealed 11 genomic regions associated with nematode resistance, distributed on chromosomes 1, 3, 4, 5, 11, and 12²⁴. Similarly, 17 single nucleotide polymorphisms (SNPs) positively associated with nematode resistance were identified in Indian wild rice accessions via GWAS²⁵.

Although most Asian rice germplasm are susceptible to M. graminicola, a few highly resistant varieties have been reported in Asian rice, such as LD24 (an indica rice from Sri Lanka), Khao Pahk Maw (KPM, an aus rice from Thailand), and Zhonghua 11 (ZH11, a japonica rice from China)^24,26. Recent studies suggested that M. graminicola resistance is governed by a major locus, and the trait can be qualitative in certain varieties. In Asian rice (cv. Abhishek), a resistance locus was mapped to chromosome 10 through bulk segregant analysis²⁷. Phan et al.²⁶ reported that a potential dominant gene(s) confers incompatible nematode resistance in ZH11 with a hypersensitivity-like reaction (HR) following nematode infection. QTL sequencing (QTL-seq) of two rice populations generated from crosses between the resistant varieties LD24 or KPM and the susceptible variety Vialone Nano identified the same locus on chromosome 11, which was responsible for the resistance in LD24 or KPM²⁸. Despite these efforts, no candidate gene conferring complete resistance for breeding resistant rice cultivars has been cloned.

Here, we screened 207 Asian rice (O. sativa) varieties and identified three novel sources (SL 22-620, HKG 98, and Toga), along with the previously reported variety ZH11, as being fully resistant to M. graminicola infection. One allelic dominant gene, M. graminicola-resistance gene 1 (Mg1), determined the nematode resistance derived from SL 22-620, HKG 98, and ZH11. Map-based cloning and functional analysis revealed that Mg1 encodes a coiled-coil, nucleotide-binding, and leucine-rich repeat (CC-NB-LRR) protein, whose LRR domain plays a prominent role in its nematode resistance activity. Further, we identified a putative protease inhibitor involved in Mg1-mediated defense responses. Mg1-mediated resistance should protect rice from M. graminicola, offering a robust and completely dominant trait for rice root-knot nematode resistance breeding.

Screening rice resistance to M. graminicola. To identify resistant sources against M. graminicola, we evaluated 207 rice varieties, comprising 197 lines of the United States Department of Agriculture (USDA) Rice Mini-Core Collection²⁹ representing the genetic diversity found in 18,709 rice cultivars collected worldwide and 10 lines collected in our laboratory, by assessing gall numbers in nematode-infected plants under controlled conditions. Most rice varieties were susceptible to nematode infection, with substantial variation observed across varieties 2 weeks after inoculation (Supplementary Fig. 2, Supplementary Table 1). However, we identified three resistant varieties, Toga (MC79, an indica rice from India), SL 22-620 (MC162, an aus rice from Sierra Leone), and HKG 98 (MC174, an aus rice from Mali), and the previously identified resistant japonica variety ZH11. Of the four varieties, Toga exhibited the lowest gall number. Plants from these resistant varieties bore at most two galls, compared to 27 galls in the susceptible japonica variety Nipponbare (Fig. 1a). Plant growth was barely affected in the resistant varieties after M. graminicola infection, in contrast to the stunted growth observed for Nipponbare (Fig. 1b). We performed a nematode attraction assay by counting the number of J2s touching the root tip at 2, 4, and 6 h after inoculation and observed no significant difference between Nipponbare and the four resistant varieties (Fig. 1c, Supplementary Fig. 3). We also assessed nematode penetration and development after inoculation. In the root system of the resistant rice varieties, the number of nematodes was significantly lower and nematode development was delayed compared to Nipponbare (Fig. 1d-e; Supplementary Fig. 4). At 15 days post inoculation (dpi), Nipponbare galls were filled with eggs and females, whereas the resistant varieties contained few females or eggs.

Molecular cloning of Mg1. To investigate whether the nematode resistance of ZH11, Toga, SL 22-620, and HKG 98 is governed by the same locus, we crossed the resistant varieties to obtain a set of progeny for an allelism test. All F₁ and F₂ offspring derived from SL 22-620×ZH11 and HKG 98×ZH11 crosses exhibited the same resistant phenotype as their parents (Supplementary Table 2). However, the F₁ plants derived from ZH11×Toga and HKG 98×Toga crosses were resistant, but the F₂ offspring segregated for resistant:susceptible phenotypes in a 15:1 ratio (160:16 or 182:15; χ2 = 2.42 or 0.63 < 3.84, P > 0.05) (Supplementary Table 2). This ratio indicated that the genes responsible for the resistance of SL 22-620, HKG 98, and ZH11 are allelic, while Toga carries a non-allelic resistance locus.

To map the common gene governing nematode resistance in SL 22-620, HKG 98, and ZH11, we developed three F₂ populations segregating for nematode resistance using two resistant varieties, HKG 98 and ZH11, and three susceptible varieties, Nipponbare, MH63, and Lehui 188, as parents. The F₁ plants derived from the crosses ZH11×MH63 and HKG 98×Nipponbare were highly resistant to M. graminicola (Supplementary Fig. 5a-b). Gall number formed on individual plants for each F₂population showed a continuous distribution with an apparent valley in the distribution curve (Supplementary Fig. 5c-e), and the segregation of the resistant to susceptible plants fitted a 3:1 ratio (245:85, 249:75, or 155:36; χ2 = 0.003, 0.498, or 0.591 < 3.84, P > 0.05) (Supplementary Fig. 5f). These results suggested that a single dominant gene, hereafter designated as M. graminicola-resistance gene 1 (Mg1), likely confers the nematode resistance observed in HKG 98 and ZH11.

Next, we carried out a bulk segregant analysis (BSA) using bulked resistant (R) and susceptible (S) F₂ plants. We identified two polymorphic markers (RM11-20 and RM11-25) and three polymorphic markers (CR2, CR8, and FJ5) from a contiguous region on chromosome 11 that distinguish the R and S pools from the ZH11×Lehui 188 and HKG 98×Nipponbare populations, respectively (Supplementary Fig 5g, h). We also used the bulked DNA samples for QTL-seq analysis, which highlighted a genomic region on chromosome 11 from 20.39 to 29.02 Mb with the most significant QTL peak (Supplementary Fig 5i).

Using 6,244 F₂ plants derived from the cross between ZH11 and Lehui 188, we delimited Mg1 to a 26.86–28.23-Mb interval flanked by markers WXM35 and 11-27M (Fig. 2a, b). High-resolution mapping using an additional 10,836 F₂ and F_2:3plants narrowed the candidate region down to 52.2 kb between markers MH15 and CR28, based on the Nipponbare reference genome (Fig. 2c). In the ZH11 reference genome, the corresponding interval flanked by markers MH15 and CR28 covered 75.4 kb (Fig. 2d). Likewise, using 7,929 F₂ and F_2:3 individuals derived from the cross between HKG 98 and Nipponbare, we further delimited Mg1 to a 38.3-kb interval between markers CR20 and CR28 in Nipponbare, which further narrowed the target region to 47.5 kb in ZH11 (Supplementary Fig 6).

Sequence analysis located Mg1 within a gene cluster encoding nucleotide-binding (NB) and leucine-rich repeat (LRR) receptors (NLRs). When we compared the genome sequences of ZH11 and Nipponbare, we identified a large structural variation within our target region (Fig. 2d). The 52.2-kb region of Nipponbare contained four annotated NLR-like genes (LOC_Os11g44960, LOC_Os11g44970, LOC_Os11g44990, and LOC_Os11g45050), two genes encoding expressed proteins, and three retrotransposon genes. We preliminarily annotated the corresponding 75.4-kb region of ZH11 using open reading frame (ORF) (http://hollywood.mit.edu/GENSCAN.html) and protein structure (http://smart.embl-heidelberg.de/) predictions. Among the 16 genes predicted in this region, six genes encoded NLR-like proteins, which we designated C3, C4, C5, C61, C62, and C9; the remaining 10 genes were predicted to encode transposable elements or expressed proteins. Because NLR-like proteins are implicated in disease resistance, we selected the six NLR genes for further investigation. C3 is a truncated NLR protein with only the LRR domain, but was located outside of the 47.5-kb target interval deduced from mapping results of the HKG 98×Nipponbare population. Therefore, we considered C4, C5, C61, C62, and C9 as the most likely candidates for Mg1.

We obtained full-length cDNAs for each gene by rapid amplification of cDNA ends (RACE)-PCR (Fig. 2e, Supplementary Fig. 7a). None of the five remaining NLR genes contained introns in their coding region, and only the untranslated region (UTR) of C61 contained four introns (Fig. 2e). Next, we performed reverse transcription quantitative PCR (RT-qPCR) to determine if the expression of these genes is induced by nematode infection. Only C9 showed a nematode-induced expression pattern at 24–48 hours post inoculation (hpi) (Fig. 2f). By contrast, C61 expression did not significantly change from 24 to 48 hpi, but decreased at 3 and 4 dpi (Supplementary Fig. 8). C3 and C5 had the lowest expression of all tested genes, below the limit of detection by RT-qPCR. We later confirmed these expression patterns by transcriptome deep sequencing (RNA-seq) (Fig. 3b).

Functional validation of Mg1. We introduced targeted mutations in each candidate gene in ZH11 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing. We designed two sequence-specific single guide RNAs (sgRNAs) to disrupt their coding sequences (CDSs) (Fig. 2e, Supplementary Fig. 7a). We sequenced the target sites in all transgenic plants and used homozygous progeny carrying insertions/deletions (InDels) or a deletion between the two target sites for nematode inoculation (Fig. 2e, Supplementary Fig. 7b). Compared to wild-type ZH11, all independent C61-knockout plants were highly susceptible to M. graminicola, similar to Nipponbare, and bore significantly more galls and nematodes (Fig. 2g, h,). By contrast, CRISPR-edited plants for C3, C4, C5, C9, and C62 retained the full resistance phenotype characteristic of ZH11 (Supplementary Fig. 7c, d). We concluded that C61 is the M. graminicola resistance gene Mg1.

We also sequenced the genomic region corresponding to C61 from the resistant cultivars SL 22-620 and HKG 98 and determined that the nucleotide and deduced amino acid sequences of C61 are identical to ZH11, suggestive of the same origin for this locus. We then created C61-knockout lines in the SL 22-620 and HKG 98 backgrounds. Plants homozygous for the C61-knockouts in SL 22-620 and HKG 98 were highly susceptible to M. graminicola (Fig. 2i, Supplementary Fig. 9a, b), further validating that Mg1 is required for M. graminicola resistance in ZH11, SL 22-620, and HKG 98.

Mg1 is a unique CC-NB-LRR protein. Mg1 encodes a protein of 1,033 amino acids with a coiled-coil (CC) domain, an NB-ARC (APAF1, R gene products, and CED-4) domain, and an LRR domain (Supplementary Fig. 10a). The NB-ARC domain contained the conserved motifs P-loop, Resistance Nucleotide Binding Site (RNBS)-A, Kinase-2/Walker B, RNBS-B, RNBS-C, GLPL, RNBS-D, and MHD (Supplementary Fig. 10b). Mg1 was located in a complex NLR gene cluster with large structural variation between rice genotypes. Therefore, we compared the chromosomal organization of NLR genes within this locus among the four rice varieties ZH11, Nipponbare, ZS97, and R498 with high-quality reference genomes (Fig. 3a). Ten NLRs were dispersed in Nipponbare (over a 210-kb interval), R498 (240 kb), and ZH11 (360 kb), but only eight in the same region of ZS97 (220 kb). Gene colinearity analysis illustrated the highly diversified NLR gene family between rice varieties (Fig. 3b), making it difficult to distinguish orthologous relationships among homologs within this locus. Phylogenetic analysis using amino acid sequences encoded by all NLR genes mentioned above identified three major groups of resistance (R) genes (A, B, and C), further demonstrating the high diversity among homologs (Fig. 3c).

Mg1 confers resistance to M. graminicola. To confirm the resistance function of Mg1, we transformed a 7.4-kb genomic fragment of C61 with its promoter into the susceptible cultivar Nipponbare (Fig. 4a). Homozygous T₂ transgenic plants derived from independent T₀ transformants were highly resistant to M. graminicola, similar to ZH11, compared to wild-type Nipponbare (Fig. 4b-d). We also overexpressed the C61 coding sequence in Nipponbare under the control of the rice Actin1 promoter (Fig. 4a, b). The resulting independent homozygous T₂ transgenic lines displayed enhanced resistance to M. graminicola compared to the wild-type Nipponbare (Fig. 4c, d). Thus, we concluded that C61 confers nematode resistance and is Mg1.

To facilitate breeding of new resistant cultivars, we designed an InDel marker, CR24, and a cleaved amplified polymorphic sequences (CAPS) marker, WXM1, flanking or within the Mg1 locus, that co-segregates with Mg1, as allele-specific molecular markers (Supplementary Fig. 11a). In addition to ZH11, SL 22-620, and HKG 98, two previously identified resistant varieties (LD24 and KPM) harbored an Mg1 resistance allele, while Toga and the other susceptible varieties did not (Supplementary Fig. 11b, c). Toga showed no correlation between its resistance phenotype and the Mg1-associated genotypes, consistent with our previous finding that Toga contains a resistant locus distinct from Mg1. To evaluate the potential utility of Mg1 in rice breeding, we introgressed the Mg1 locus into the susceptible variety Huazhan, which is commonly used as a restorer line in rice breeding programs, through successive backcrossing. The resulting near-isogenic line, NIL-Mg1, was highly resistant to nematode infection, similar to ZH11 (Supplementary Fig. 11d, e). Moreover, Mg1 had no adverse effect on plant growth or yield traits (thousand-grain weight) under field conditions in NIL-Mg1, suggesting the potential for Mg1 in rice breeding (Supplementary Fig. 11f, g).

A phylogenetic analysis showed that Mg1 clusters with LOC_Os11g44960 and LOC_Os11g45050 in Nipponbare, sharing 93.9% and 93.8% sequence identity, respectively (Fig. 3c, Supplementary Fig. 12). Amino acid alignment revealed 31 and 63 amino acid substitutions between Mg1 and LOC_Os11g44960 or LOC_Os11g45050, respectively (Supplementary Fig. 12). When comparing Mg1 with both susceptible alleles in Nipponbare, we identified SNPs causing 15 amino acid substitutions, all of which occurred within the LRR domains, especially the last two LRRs. To determine which of the Mg1 domains are responsible for nematode resistance, we generated a series of chimeric constructs by replacing the sequence for different Mg1 domains with the corresponding domains from LOC_Os11g44960 and LOC_Os11g45050 (indicated as #1-#6 in Fig. 4e) and expressed them in Nipponbare under the Mg1 promoter of ZH11 (Fig. 4e). We then evaluated nematode resistance in homozygous transgenic lines for each chimeric construct. All chimeric genes were expressed to similar levels (Supplementary Fig. 13). Neither LOC_Os11g44960 nor LOC_Os11g45050 (#6) conferred nematode resistance (Fig. 4f). Only two chimeric constructs (#2 and #5) encoding the LRR or NB-ARC-LRR domain of Mg1, but none of the other domain combinations tested, enhanced nematode resistance, indicating that the specific recognition of nematode effectors through LRR domain is responsible for the activation of Mg1 and nematode resistance.

Effector-triggered immunity mediated by R proteins is often accompanied by cell death. Isolated CC and NB domains, or the full length of some NLR proteins, activate cell death in the absence of the corresponding avirulence factors when their encoding genes are transiently expressed in Nicotiana benthamiana^30,31. To examine whether Mg1 similarly induces cell death, we individually expressed full-length Mg1 or the sequence encoding the CC, NB, and LRR domains in Agrobacterium tumefaciens–infiltrated N. benthamiana leaves. Although all proteins accumulated in the infiltrated leaves, none induced cell death (Supplementary Fig. 14a, b). Mutations in the MHD motif of the NB-ARC domain result in autoactivation of R proteins³². We therefore changed an aspartate to valine in the MHD motif of Mg1 (Mg1^D493V) and expressed the corresponding construct in N. benthamiana leaves, which clearly induced HR-like cell death at 3 dpi (Supplementary Fig. 14a). Taken together, these results indicate that the activation of Mg1 by nematode effectors may be sufficient to trigger defense responses accompanied by cell death.

Expression analysis of Mg1. Since typical hook-shaped galls form at the root tips of M. graminicola–infected rice plants, we investigated the Mg1 expression pattern in ZH11 with a Mg1 promoter-β-glucuronidase (GUS) reporter construct. We used an ~3-kb promoter fragment upstream of the Mg1 start codon to drive GUS expression and transformed the resulting construct into ZH11 to generate stable transgenic lines. We predominantly detected GUS staining in leaf mesophyll cells, root tips, and vascular tissues (Fig. 5a). Consistent with its role in recognizing nematode attack, we observed high GUS expression in galls upon M. graminicola infection. In addition, RT-PCR analysis revealed that Mg1 is constitutively expressed in roots, stems, and leaves, with the highest expression levels reached in root tips (Fig. 5b).

To determine Mg1 subcellular localization, we transiently expressed a construct encoding an Mg1-green fluorescent protein (GFP) fusion protein in rice protoplasts and N. benthamiana. We mainly observed fluorescence signals in the cytoplasm (Fig. 5c). The CC-NB-LRR protein ZAR1 (HOPZ-ACTIVATED RESISTANCE 1) forms a resistosome in the plasma membrane to trigger immunity and cell death^33,34. Therefore, to further explore Mg1 localization, we searched for potential modifications in Mg1 that promote association with the plasma membrane. The online software CSS-Palm predicted the cysteine 12 residue in the Mg1 N-terminal CC domain as a potential site for palmitoylation (Supplementary Table 3). To assess the importance of this residue in Mg1-mediated nematode defense responses, we introduced a C12S mutation into the autoactivated D493V mutant of Mg1 (Mg1^C12S,D493V) and tested its effect on cell death. The C12S mutation completely abolished the cell death–inducing capacity of Mg1^D493V (Fig. 5d). Consistent with this result, treating N. benthamiana leaves infiltrated with the Mg1^D493V construct with the palmitoylation inhibitor 2-bromopalmitate (2-BP) suppressed the cell death phenotype (Fig. 5e). These results indicated that the C12 residue in the Mg1 N-terminal CC domain mediates palmitoylation and membrane localization and is required for the cell death activity of Mg1 (Fig. 5e).

Rice resistance responses to M. graminicola infection. To understand the underlying resistance mechanism mediated by Mg1, we performed a global comparative transcriptome profiling between the resistant (ZH11 and HKG 98) and susceptible (Nipponbare) varieties at the early stage (1 dpi) of nematode infection. Compared to control conditions (no infection), we detected 547 differentially expressed genes (DEGs) (Log₂FC ≥ 1, P-adjust < 0.05) upon nematode infection in Nipponbare (Fig. 6a); notably, we identified more DEGs for ZH11 (838 genes) and HKG 98 (1,329 genes) following nematode treatment (Fig. 6a). Hierarchical clustering analysis of all DEGs suggested that the resistant varieties display an overall enhanced nematode response compared to Nipponbare (Supplementary Fig. 15a). Most of the DEGs were upregulated upon nematode invasion, with 258 genes induced in all three genotypes and 339 common genes specifically induced in ZH11 and HKG 98 (Fig. 6b). We investigated the 339 genes co-upregulated in the resistant varieties using Gene Ontology (GO) enrichment analysis and observed a significant overrepresentation (P-adjust < 0.05) for genes involved in defense response, protein phosphorylation, kinase activity, and response to stress (Supplementary Fig. 15b). Based on a similar pathway enrichment analysis via Kyoto Encyclopedia of Genes and Genomes (KEGG), we determined that plant–pathogen interaction, biosynthesis of secondary metabolites, plant hormone signal transduction, and MAPK signaling pathway are enriched in the resistant varieties (Fig. 6c). We also investigated the expression patterns of the defense-related genes PATHOGENESIS-RELATED GENE 1a (OsPR1a), OsPR10, CDPK-RELATED PROTEIN KINASE 5 (OsCRK5), and OsWRKY45 in ZH11 and Nipponbare at different time points following nematode inoculation by RT-qPCR. We detected a more rapid and much stronger induction of gene expression in ZH11 compared to Nipponbare (Fig. 6d).

The importance of jasmonate (JA) and salicylic acid (SA) hormone pathways for nematode resistance is well established in compatible plant–nematode interactions^35,36. We thus explored the roles of JA and SA in Mg1-mediated nematode resistance. Genes involved in SA and JA biosynthesis and signaling were differentially activated upon nematode infection, with much stronger induction was observed in resistant genotypes (Fig. 6e). Transcript levels of the SA pathway–related genes PHENYLALANINE AMMONIA-LYASE (PAL), NONEXPRESSOR OF PR 1 (NPR1), PR1b, and TGACG-Binding (TGA) transcription factor genes were higher in ZH11 and HKG 98 than in Nipponbare. Similarly, the JA-related genes 12-OXOPHYTODIENOATE REDUCTASE (OPR), LIPOXYGENASE (LOX), and ALLENE OXIDE SYNTHASE 2 (AOS2) were expressed to higher levels in the resistant varieties after nematode infection (Fig. 6e). Notably, the SA-deficient transgenic NahG line and the SA-signaling mutant npr1 in the ZH11 background had more galls and lower resistance against M. graminicola. Chemical inhibition of JA biosynthesis by exogenously applying diethyldithiocarbamic acid (DIECA) also compromised the nematode resistance of wild-type ZH11, but we observed no additive effect for the SA-defective mutants (Fig. 6f). These data indicate that SA and JA are required for Mg1-mediated resistance.

Lignin and callose accumulate as physical barriers following nematode infection³⁷. To investigate the defense responses in the resistant and susceptible lines, we assessed lignin and callose staining in nematode-infected root galls at 3 dpi. We observed stronger red staining surrounding the feeding cells in the resistant varieties compared to Nipponbare, suggesting increased lignin accumulation in the resistant varieties (Fig. 6g). Callose deposition, seen as many bright speckles, also increased in the resistant varieties (Supplementary Fig. 15c). In agreement, RNA-seq revealed that genes related to lignin and callose biosynthesis are differentially regulated by nematode invasion between resistant and susceptible genotypes (Supplementary Fig. 15d). Taken together, global gene expression profiles and histological analyses indicated that ZH11 and HKG 98 undergo a series of resistance responses to prevent nematode parasitism at the early stage of infection.

MgBP1 is required for Mg1-mediated nematode resistance. To dissect the signaling components involved in Mg1-mediated resistance, we performed a yeast two-hybrid (Y2H) screen using the Mg1 CC or LRR domains as bait and identified the candidate MG1-BINDING PROTEIN 1 (MgBP1, LOC_Os12g25090) for further characterization. MgBP1 is a small protein of 100 amino acids in the potato type I serine protease inhibitor family. MgBP1 expression was dramatically upregulated upon nematode infection, with higher expression seen in the resistant varieties (Fig. 7a). Yeast colonies co-transformed with constructs encoding MgBP1 and the LRR, CC-NB, or CC domain grew well on selective medium (Fig. 7b). A luciferase complementation imaging (LCI) assay in N. benthamiana validated the interaction between MgBP1 and the Mg1 CC domain, but not with the other Mg1 domains tested (Fig. 7c). A co-immunoprecipitation (Co-IP) assay also supported this specific interaction. Following immunoprecipitation with an anti-GFP antibody, we detected MgBP1-FLAG with an anti-FLAG antibody in protein extracts from N. benthamiana leaves co-infiltrated with CC-GFP and MgBP1-FLAG constructs but not in the control (Fig. 7d). We detected no interaction between MgBP1 and the Mg1 LRR domain in this assay (Fig. 7e).

Since protease inhibitors are involved in protein turnover, we tested the influence of MgBP1 on Mg1 accumulation. Co-expressing Mg1 and MgBP1 constructs in N. benthamiana leaves raised Mg1 abundance (Fig. 7f). In line with this observation, Mg1^D493V-triggered cell death was stronger when Mg1^D493V was co-expressed with MgBP1 relative to Mg1^D493V alone (Fig. 7g). Thus, MgBP1 might protect Mg1 from degradation to achieve effective nematode resistance. To explore whether Mg1-mediated resistance depends on MgBP1, we used CRISPR/Cas9-mediated editing to mutate MgBP1 in the ZH11 background (Supplementary Fig. 16) and obtained two independent T₂ homozygous mutants for nematode resistance. Compared to ZH11, the edited mgbp1 mutants had significantly reduced resistance to M. graminicola, although they were not as susceptible as Nipponbare (Fig. 7h, i). These results suggest that MgBP1 contributes to Mg1-mediated defense responses by directly protecting Mg1 from degradation.

With climate change, water shortages, and changes in rice cultivation systems, the incidence of M. graminicola is increasing, especially in Asia. Mining resistance genes for crop disease improvement is an environmentally friendly and practical solution to mitigate this concern. Here, we evaluated the response of 207 rice varieties to this nematode under controlled conditions, and four varieties (Toga, SL 22-620, HKG 98, and ZH11) displayed high resistance. Mg1, a dominant resistance gene on chromosome 11, is the common resistance gene in SL 22-620, HKG 98, and ZH11. Mg1 introgressed into the susceptible rice cultivar Huazhan significantly enhanced its resistance to M. graminicola, with no adverse effects on thousand-grain weight. Thus, Mg1 and the Mg1-linked molecular markers developed here will have great value in marker-assisted breeding. Furthermore, Toga, which carries a distinct resistance gene and does not carry the resistant allele of the Mg1 locus, provides another resource for breeding rice with durable nematode resistance through resistance-gene pyramiding or diversification.

NLR genes are enriched on rice chromosome 11 (Rice Chromosome 11 and 12 Sequencing Consortia 2005), and many of them are clustered. We located Mg1 in a complex NLR gene cluster on chromosome 11 and observed large structural variations between rice cultivars. This locus, previously referred to as AC134922, is one of the most diversified R gene loci in rice genomes³⁸. R genes undergo adaptive evolution in response to pathogens, and their clustering may increase the opportunity for genetic exchange, acting as potential resistance reservoirs against future pathogen strains³⁹. Retrotransposons, which are abundant at this locus, may be an important driving force in R gene evolution and diversication⁴⁰. Comprehensive sequence comparison across diverse rice cultivars will help to better understand the evolutionary mechanisms acting on R genes. Whether other Mg1 homologs are responsible for disease resistance remains to be studied. We established, through bioinformatic sequence analysis and marker-assisted screening, that few rice varieties belonging to japonica, indica, and aus subspecies carry Mg1, suggesting that Mg1 has not been widely selected for during rice breeding. We propose that the Mg1 locus from the resistant varieties shares the same origin, but how this specific Mg1 locus was introgressed into rice varieties distributed in different regions of the world is still elusive.

Plants rely on membrane-associated pattern recognition receptors (PRRs) and intracellular NLRs to activate the two tiers of plant immune responses, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), to defend against various pathogens^41,42 . By directly or indirectly recognizing pathogen effector proteins, NLRs initiate robust disease resistance responses that typically result in localized host cell death^43,44. The LRR domain of NLRs mainly mediates direct or indirect pathogen effector sensing^45,46. Mg1 encodes a CC-NB-LRR-type protein, and the expression of its susceptible alleles failed to trigger nematode resistance. Domain swap analysis between Mg1 and its susceptible orthologs demonstrated the pivotal role of the Mg1 C-terminal LRR domain in conferring nematode resistance. The Mg1 LRR domain contains many amino acid substitutions when compared to its susceptible alleles in Nipponbare, indicating that potential adaptive divergence in the LRR domain may alter pathogen recognition specificity. Taken together, we hypothesize that the Mg1 LRR domain determines the specificity of nematode recognition and is largely implicated in Mg1 activation.

Plant parasitic nematodes deliver effectors into the host cells through their stylet to support parasitism. Nematodes are attracted to and penetrate roots of resistant plants; however, the development of the feeding site is substantially hindered, and the nematodes starve to death or leave the roots^47,48. Resistant plants initiate a localized HR near the feeding site in roots. However, how resistance genes are activated is not well understood. The root-knot nematode effectors Map-1 and Cg-1 may be involved in resistance mediated by the resistance gene Mi-1, but further validation is needed^49,50. Direct evidence of nematode effectors implicated in ETI comes from potato cyst nematode (Globodera rostochiensis and Globodera pallida)-secreted effectors RAN-BINDING PROTEIN LIKE-1 (GpRBP1) and VENOM-ALLERGEN PROTEIN 1 (GrVAP1), which are directly or indirectly recognized by the R proteins Gpa2 and Cf-2, respectively^51,52. To develop rice varieties with durable nematode resistance, it will be crucial to identify the corresponding avirulence effectors of Mg1 and to elucidate the Mg1 mechanisms for perceiving nematode presence.

In the absence of pathogens, NLR proteins are repressed through self-inhibitory intramolecular interactions. It is crucial for plants to tightly control the NLR protein state to avoid fitness penalties. The presence of effectors induces a conformational change, which releases the intramolecular interactions to activate NLR signaling^53,54. We provided evidence that Mg1-mediated resistance is highly dependent on the presence of nematode. First, we detected high Mg1 expression at the nematode invasion site, although this was not influenced by nematode invasion, suggesting that Mg1 may be inactive prior to nematode infection. The perception of the cognate effectors secreted from nematodes may therefore be a prerequisite for Mg1 activation. Second, neither Mg1 nor its individual domains triggered cell death in N. benthamiana leaves, in contrast to the cell death phenotype observed for NLRs like Brown Planthopper Resistance Gene 9 (BPH9), RESISTANT TO P. SYRINGAE 5 (RPS5), MILDEW A (MLA), Stem rust 33 (Sr33), and Sr50^31,55. The autoactivated form of Mg1 (Mg1^D493V) mimicked effector-mediated NLR activation. Furthermore, the Mg1 CC domain self-associated, similar to other NLRs (Supplementary Fig 17a, b), whose self-association or oligomerization is key for NLR activation^33,56. Recent breakthroughs on the CNL (CC-containing NLR) protein ZAR1 demonstrated that rearranging the CC domain leads to oligomeric resistosomes at the plasma membrane, serving as calcium-permeable cation-selective channels that trigger immune signaling and cell death^33,34,53. In addition, a cysteine at position 12 of Mg1, which was predicted as a palmitoylation site, was required for cell death activity. Palmitoylation-dependent membrane localization of Pit, a rice blast resistance protein, may activate rice immunity⁵⁷. Our data suggest that palmitoylation-mediated membrane localization is also required for Mg1 function. Finally, our results indicate that similar defense responses are activated in resistant plants during incompatible interactions with pathogens, nematodes, and insects⁵⁸. Indeed, SA and JA pathways were required for Mg1-mediated resistance, and lignin and callose deposition was highly induced at the nematode feeding site in resistant rice varieties. These transcriptome and cytological changes likely facilitate a robust response to nematode threats. Our results corroborate previous reports that phytohormone pathways and secondary metabolites contribute to nematode resistance in compatible rice–nematode interactions³⁵.

NLRs are a class of resistance genes widely used in crop disease resistance breeding. Our current understanding of NLR signaling mechanisms in crops is very limited. We revealed that MgBP1, a member of the potato type I serine protease inhibitor family, contributes to Mg1-mediated nematode defense responses by directly interacting with Mg1. MgBP1 was highly expressed in resistant plants compared to susceptible Nipponbare. Protease inhibitors inactivate proteases to regulate protein degradation or turnover and combat insect and nematode infection by inhibiting their digestive enzymes⁵⁹. Enhanced resistance to several nematode species by expressing protease inhibitor genes has been described in various host plants^60,61. Whether MgBP1 has nematocidal activity deserves further investigation. Protease inhibitors are also involved in endogenous physiological processes, such as plant growth and development, seed germination, senescence, and programmed cell death, by counteracting the activities of specific proteases^62,63. Here, we observed the higher accumulation of Mg1 when co-expressed with a construct encoding MgBP1, although we cannot rule out the possibility that MgBP1 similarly affects the levels of other defense components in response to nematode infection. Further characterization using a proteomics approach might help address this question. In addition, whether nematode-secreted peptidases target Mg1 to evade immunity is unknown. Thus, the underlying molecular mechanism of how MgBP1 contributes to the defense response remains to be elucidated.

Plant and nematode materials. The rice (Oryza sativa L.) lines used in this experiment are listed in Supplementary Table 1 and include 197 varieties from the United States Department of Agriculture (USDA) Rice Mini-Core Collection and 10 rice varieties maintained in our laboratory. All rice materials were originally provided by Dr. Chengcai Chu at the Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences (CAS). The SA-deficient NahG transgenic line and the npr1 mutant in the ZH11 background were kindly provided by Dr. Jiuyou Tang (IGDB, CAS). The Meloidogyne graminicola culture was provided by Dr. Gaofeng Wang at Huazhong Agricultural University (HZAU) and was originally isolated from a rice field from Haikou in Hainan Province, China. A pure nematode culture was maintained on Nipponbare (O. sativa) growing in a sand:potting soil mixture (3:1) at 26–28°C under a 16-h-light/8-h-dark regime with 250 μmol m^–2 s^–1 light intensity and 70–75% relative humidity. Four weeks after inoculation, infected roots were cut into 1-cm pieces and ground with a blender. The slurry was washed through a stack of sieves (mesh number: #60, #200, and #500). Eggs collected from the bottom sieve were incubated in a hatching chamber with distilled water at 28℃ for 1 day. Freshly hatched second-stage juveniles (J2s) were used for the experiments.

Nematode infection experiments. Rice seeds were germinated at 28°C for 2–3 days and then transferred to a sand:potting soil mixture for growth. The seedlings were watered daily and fertilized twice a week with Hoagland solution. Fourteen-day-old rice plants were inoculated with 150 M. graminicola J2s per plant or mock-inoculated with 0.1% (w/v) agarose. The infection level of the plants was evaluated at 15 dpi by counting the number of galls per plant. For the large-scale screening, 12 plants in two separate pots (six plants per pot) were assessed for each cultivar. Acid fuchsin staining was performed to visualize the galls as described previously³⁵. Roots were boiled in 0.8% (v/v) acetic acid and 0.013% (w/v) acid fuchsin for 3 min and then destained in acidified glycerol. Nematode development was assessed under a stereomicroscope by counting the number of nematodes of various stages at the times indicated in the text. The nematode infection assay was performed three times with at least 15 plants for each genotype.

Nematode attraction assay. The nematode attraction assay was performed as previously described⁶⁴. In brief, 1 mL of 23% (w/v) pluronic F-127 (Sigma-Aldrich) containing 1,000 J2s was aliquoted into 12-well tissue culture plates at 4℃. Then, a 1-cm-long root tip was placed into each well. The number of nematodes touching the terminal 1.5 mm of the root tip was scored at the indicated time points (2, 4, and 6 hpi). The experiment was performed three times with at least 12 roots for each treatment.

RNA extraction and RT-qPCR analysis. Total RNA was isolated from rice roots using TRIpure reagent (Aidlab, Beijing, China), and the RNA concentration was quantified with a NanoDrop-1000 spectrophotometer (Thermo Scientific, USA). First-strand cDNA was synthesized using HiScriptII QRT SuperMix (Vazyme, Nanjing, China) following the manufacturer’s instructions. Gene-specific primers were designed and quantitative PCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) with an iCycler iQ 5 thermal cycler (Bio-Rad, California, USA) real-time PCR system. OsEXPNAR (LOC_Os07g02340) and OsActin (LOC_Os03g50885) were used as internal controls; the expression of reference genes was not influenced by various stress conditions, including nematode infection⁶⁵. A dissociation curve was generated to verify amplicon specificity. The experiment was performed three times, and relative gene expression was determined using the 2^–ΔΔCt method compared to the internal control⁶⁶. The primers used for qPCR are listed in Supplementary Table 4.

RNA-seq and data analysis. Three rice cultivars, Nipponbare, HKG 98, and ZH11, were used for RNA-seq analysis. Six-day-old rice seedlings were inoculated individually with 150 M. graminicola J2s suspended with 0.1% (w/v) agarose solution. Controls were mock-inoculated with agarose solution alone. At 1 dpi, root tips (2 mm in length) or visible galls were collected and frozen in liquid nitrogen for further use. Forty root samples were pooled for each treatment, and two biological replicates were performed. Total RNA was extracted using the Ultrapure RNA Kit (CWBIO, Beijing, China). RNA integrity was assessed using an RNA Nano 6000 assay kit on a Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Library construction for mRNA sequencing was performed using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations. The libraries were sequenced using an Illumina Hiseq platform as 100-bp single-end reads (Majorbio, Shanghai, China). Raw reads were filtered by removing adapter sequences, reads containing poly-N, and low-quality reads (quality score < 20). The resulting clean reads were aligned and assembled to the Nipponbare reference genome (MSU7 release from the http://rice.uga.edu) using TopHat v2.0.12 and Cufflinks. Then, transcripts per million reads (TPM) values for each gene were calculated based on the length of the gene and read counts mapped to this gene using RSEM software. Differential gene expression was determined using the DESeq2 R package (1.18.0) with Log₂FC ≥ 1 and a false discovery rate (FDR) < 0.05 as the threshold. Values of Log₁₀(TPM+1) were used for hierarchical clustering analysis. The expression heatmap and KEGG enrichment analyses were generated with values of Log₂FC using TBtools v1.0⁶⁷. GO enrichment was determined using Goatools. The cutoff for significant enrichment was P-adjust < 0.05.

Mapping and cloning Mg1. To map Mg1, the mapping F₂ populations derived from the crosses ZH11×Lehui 188, ZH11×MH63, and HKG 98×Nipponbare were generated. Moreover, four F₂ populations derived from the crosses SL 22-620×ZH11, HKG 98×ZH11, ZH11×Toga, and HKG 98×Toga were generated for allelism tests. Nematode resistance was scored by counting the number of galls per F₂ individual. To confirm the phenotypes of the F₂ individuals, their F_2:3 progeny were evaluated. The CTAB method was used to extract DNA from fresh leaves of individual rice seedlings.

Mg1 was initially localized to a region on chromosome 11 by BSA. Briefly, resistant (R) and susceptible (S) DNA samples, extracted from 30 F₂ individuals each with extremely resistant or susceptible phenotypes, were pooled and used as R and S bulk samples. A total of 134 simple sequence repeat (SSR) markers distributed evenly on all 12 rice chromosomes were used to screen polymorphisms between R and S pools as well as both parents. QTL-seq was conducted to detect resistance loci. Each of the DNA samples pooled above was used for whole-genome resequencing with >30× genome coverage. The resulting short reads were aligned to the Nipponbare reference sequence using BWA software. The GATK toolkit was used for SNP calling. The SNP-index and derived ∆(SNP-index) were calculated according to the pipeline described by Takagi et al.⁶⁸. Smoothed G′ value analysis was conducted according to Magwene et al.³⁰. A QTL was identified as a peak or valley of the ∆(SNP-index) plot or G′ value plot. The x-axis corresponds to the chromosomal position. The ∆(SNP-index) plot (the 2^nd top) is shown with statistical confidence intervals under the null hypothesis of no QTL (blue, P < 0.1; green, P < 0.05; red, P < 0.01). For G′ value and its corresponding P-value (–Log₁₀ value) plot (bottom two), the red threshold line represents q < 0.01.

To fine-map Mg1, more DNA markers primarily based on InDel polymorphisms and derived cleaved amplified polymorphic sequences (dCAPSs) were designed according to the published sequences of the japonica Nipponbare and ZH11 (http://www.mbkbase.org/) and the indica R498 and Minghui63 (MH63) varieties (https://rice.hzau.edu.cn/rice_rs2/). We used the flanking markers Tj146-3 and Tj146-7 to screen ZH11×Lehui 188 F₂ plants, and the 214 obtained recombinants delineated Mg1 to a 1.37-Mb interval between markers WXM35 and 11-27M. Similarly, Mg1 was delineated to a 640-kb interval between markers WXM37 and CR16 using 5,792 F₂ plants generated from the cross between HKG 98 and Nipponbare. The F₂ and F_2:3 populations consisting of 10,836 and 2,137 individuals for the crosses of ZH11×Lehui 188 (or MH63) and HKG 98×Nipponbare were used for linkage analysis and identification of recombinants.

To identify all candidate genes, the coding regions (http://hollywood.mit.edu/GENSCAN.html) and protein structure (http://smart.embl-heidelberg.de/) of the target region were predicted. To determine the expression levels of these candidates, reads obtained from the RNA-seq were aligned and mapped to the target locus of ZH11 using TopHat and Cufflinks.

The near-isogenic lines carrying Mg1 (NIL-Mg1) were generated by repeated backcrossing of the resistant parent ZH11 to the recurrent parent Huazhan and were selected from the BC₃F₄ population through marker-assisted selection. The tightly linked Mg1 markers and additional SSR markers were used to screen the resistance locus and genetic background. Huazhan and NIL-Mg1 plants were examined for nematode resistance and agronomic traits. The agronomic performance experiment was conducted in a standard paddy field of Wuhan, China. The plants were grown in a randomized block design with three replicates. At harvest, thousand-grain weight was measured.

All PCR primers and markers are listed in Supplementary Table 4.

Sequence analysis. The J. Craig Venter Institute (JCVI) toolkit was used for genome assembly, annotation, and comparative genomics⁶⁹. We used the default parameters of this toolkit to draw a colinearity plot to compare the distribution of the Mg1-related NLR gene cluster on chromosome 11 between four rice varieties (ZH11, Nipponbare, ZS97, and R498). The coding sequences of the NLR genes within the cluster from ZH11 were used to identify the colinear orthologs. Protein sequences within the NLR gene cluster were obtained for different rice varieties. ClustalX v2.1 was used to align the amino acid sequences. Conserved domains were predicted by BlastP, SMART (http://smart.embl-heidelberg.de/), and LRR search (https://lrrsearch.com/) online tools. The phylogenetic tree was generated with the predicted full-length amino acid sequences of NLR proteins using the MUSCLE algorithm of the phylogenetic analysis software MEGA7⁷⁰. A bootstrap consensus tree was constructed inferred from 1,000 replicates using the neighbor-joining method, and the P-distance model with complete gap deletion was used to calculate the evolutionary distances.

Vector construction and transformation. To identify the full-length sequences of candidate genes, 5′ RACE and 3′ RACE were conducted. Total RNA was isolated from M. graminicola–infected ZH11 roots using the RNAprep pure Plant Kit (TIANGEN, China). 5′ RACE cDNA and 3′ RACE cDNA were synthesized according to Scotto-Lavino et al.⁷¹. Two rounds of amplification were performed using gene-specific primers and adaptor primers listed in Supplementary Table 4. The largest PCR products were cloned, and three individual clones were sequenced.

Targeted mutagenesis of candidate genes was performed using CRISPR/Cas9 as previously described⁷². Two specific sgRNAs were designed for each gene. The polycistronic tRNA-gRNA (PTG) fragments were assembled using the Golden Gate assembly method and subsequently inserted into the pRGEB32 binary vector (courtesy of Kabin Xie at HZAU). The resulting constructs were introduced into rice variety ZH11 via Agrobacterium tumefaciens–mediated transformation according to a conventional protocol. The construct targeting Mg1 was also transformed into rice varieties HKG 98 and SL 22-620. Plants regenerated from hygromycin-resistant calli and their progeny were genotyped by PCR using primers flanking the target sites, and mutations were confirmed by sequencing. T₁ or T₂ homozygous transgenic lines derived from each independent T₀ plant were used for the resistance test.

To construct the Mg1 complementation vector, a 7,381-bp genomic fragment containing 3 kb of the promoter region, the entire coding region, and 3 kb of 3′-flanking sequence of Mg1 was amplified from ZH11 using gene-specific primers, and the PCR product was inserted into the binary vector pCAMBIA2300 (courtesy of Chengcai Chu at CAS) digested by SalI and XbaI. The overexpression vector was constructed by sub-cloning the Mg1 coding sequence into the binary vector pCAMBIA2301 under the control of the rice Actin1 promoter with SalI and PstI digestion. The constructs were introduced into susceptible variety Nipponbare via A. tumefaciens–mediated transformation to generate independent lines for functional verification of Mg1. RT-qPCR was performed to evaluate gene expression levels.

For the domain swapping experiment, the sequences encoding various domains (CC, CC-NB-ARC, NB-ARC, NB-ARC-LRR, LRR, and the full-length protein) of Mg1 or its two orthologs from Nipponbare (LOC_Os11g44960 and LOC_Os11g45050) were amplified. A series of chimeric fragments recombined at different positions between Mg1 and its orthologs were generated by overlapping PCR. The resulting products and the Mg1 promoter were ligated into the pCAMBIA2300 binary vector with XbaI and SalI restriction sites. The constructs encoding the Mg1^D493V and Mg1^C12S,D493V variants were generated using overlap extension PCR primers carrying point mutations, and the amplified products were cloned into the pCAMBIA1300-35S vector with XbaI and SalI sites and verified by sequencing. These constructs were tested for cell death activity after transient infiltration in Nicotiana benthamiana leaves or transformed into Nipponbare for resistance function analysis.

A 3-kb promoter fragment upstream of the ATG start codon of Mg1 was amplified from ZH11 genomic DNA and cloned into the pCAMBIA2391Z vector with SalI and EcoRI to build the GUS reporter construct. This Mg1pro:GUS vector was introduced into ZH11, and T₂ transgenic lines were used for subsequent analysis.

For subcellular localization, the Mg1 coding sequence was digested with XbaI and SalI and inserted into the pCAMBIA2300-35S-GFP vector in-frame and upstream of GFP.

To generate constructs for Y2H assays, various fragments of Mg1 were cloned into the pGADT7 (AD) and pGBKT7 (BD) vectors. The full-length coding sequence of MgBP1 was cloned in-frame and downstream of the sequence encoding the GAL4 DNA activation domain in the pGADT7 vector with NdeI and XhoI digestion.

For LCI assays, the full-length coding sequence of MgBP1 was cloned into the pCAMBIA-35S-CLuc vector to form the CLuc-MgBP1 fusion construct. Different fragments of Mg1 were amplified and inserted into the pCAMBIA-35S-NLuc and pCAMBIA-35S-CLuc vectors to express C-terminal NLuc and N-terminal CLuc fusion constructs, respectively.

For Co-IP assays, the coding sequences being tested were separately cloned into pCAMBIA1300-35S-3×Flag and pCAMBIA2300-35S-GFP vectors using XbaI and SalI sites to produce C-terminal-tagged fusion constructs.

All primers used to construct vectors are listed in Supplementary Table 4. All constructs were verified by DNA sequencing.

Histochemical staining. Lignin was detected using Wiesner reagent (3% [v/v] phloroglucinol-HCl) according to Mitra and Loqué⁷³. Five days after nematode inoculation, galls and root tips were harvested, embedded in 7% (w/v) agarose, and cut into 50-μm sections with a vibratome (Leica VT 1000S, Germany). Sections were incubated for 5 min in Wiesner reagent, which was subsequently replaced by sterile water. A light microscope (Leica DM2500, Germany) was used for the imaging. Callose staining was performed according to Millet et al.⁷⁴. Three days after nematode inoculation, approximately 2-cm-long roots tips were cut and immediately fixed in a 3:1 (v/v) ethanol:acetic acid solution for 24 h. After rehydration and water washes, roots were treated with 10% (w/v) NaOH at 37℃ for 2 h to clear the tissues. Then, the roots were incubated in 0.01% (w/v) aniline blue in 150 mM K₂HPO₄solution (pH 9.5) for 25 min. The roots, including galls, were mounted onto slides, and callose deposition was examined immediately using a microscope under UV light (excitation, 390 nm; emission, 460 nm). For Mg1 promoter-GUS analysis, root fragments with or without nematode infection were vacuum-infiltrated with GUS staining solution (100 mM Tris-HCl, pH 7.0, 50 mM NaCl, 1 mM 5-bromo-4-chloro-3-indolyl-β-glucuronic acid, 1 mM potassium ferricyanide, pH 7.0, and 0.06% [v/v] Triton X-100) twice for 10 min each and incubated overnight at 37℃. The reaction was stopped with 70% (v/v) ethanol. Stained roots were visualized with a stereoscope.

Agroinfiltration assays in N. benthamiana. A. tumefaciens strain EHA105 harboring different binary vectors was syringe-infiltrated into fully expanded leaves of 5-week-old N. benthamiana. Briefly, the A. tumefaciens cultures were pelleted and resuspended in infiltration buffer (10 mM MES, pH 5.6, 10 mM MgCl₂, and 200 μM acetosyringone) to a final concentration of OD₆₀₀ = 1.0. To enhance transient gene expression, an A. tumefaciens strain carrying the p19 silencing suppressor was included with all infiltrations. The suspensions were kept at 25°C for at least 3 h without shaking. For co-infiltration, equal volumes of suspensions carrying the indicated constructs were mixed and infiltrated into N. benthamiana leaves. After infiltration, plants were cultured at 25℃ for 36–48 h in a 16-h-light/8-h-dark photoperiod with a light intensity around 100 μmol m⁻² s⁻¹. Images presented in the figures are representative of at least five leaves. For cell death analysis, leaves were exposed under UV light (365 nm) using an ultraviolet imager.

Subcellular localization assay. Mg1 was transiently transfected in rice protoplasts to investigate Mg1 subcellular localization. Rice protoplasts from 10-day-old ZH11 seedlings were isolated and transfected as previously described⁷⁵. Transfected protoplasts were incubated in W5 solution for 16 h and used for fluorescence observation. To confirm Mg1 subcellular localization, the Mg1-GFP construct was transiently infiltrated in N. benthamiana leaves as described above. Fluorescence signals were captured with a Leica TCS SP8 confocal microscope system using an excitation laser wavelength of 488 nm with an emission filter of 500–550 nm.

Y2H assay. The Y2H screening was conducted using the LRR or CC domain of Mg1 as the bait following the manufacturer’s instructions (Matchmaker Gold Yeast Two-Hybrid Library Screening System, Clontech, Dalian, China). A root cDNA library of rice cultivar ZH11 generated with Make Your Own ‘Mate & Plate’ Library System (Clontech, Dalian, China) was used for screening. Positive protein–protein interactions were selected by growing yeast colonies on synthetic defined (SD) medium lacking leucine, tryptophan, and histidine (SD–L–T–H, TDO). Combinations of AD/SV40 with BD/p53 or BD/Lam served as positive or negative controls, respectively. To test Mg1 self-interaction and to confirm the protein–protein interactions, different combinations of Mg1 fragments and their interacting candidates, as indicated in the figures, were co-transformed into yeast strain AH109. Colonies grown on SD–L–T (DDO) medium were cultured, diluted, and plated on TDO medium. Plates were kept at 30°C for 3 d before being photographed. All Y2H experiments were performed three times independently.

Firefly LCI assay. To investigate the protein interactions, the LCI assays were carried out as previously described⁷⁶. A. tumefaciens strain EHA105 carrying the indicated constructs was co-infiltrated into N. benthamiana leaves as described above. Two days after infiltration, the infiltrated leaves were sprayed with 1 mM luciferin (Sigma-Aldrich, L9504) supplemented with 0.01% (v/v) Triton X-100 in darkness for 3 min. Luminescence images were captured using a low-light cooled CCD imaging apparatus (NightOWL II LB983).

Co-IP assay. A. tumefaciens–infiltrated leaves were collected, and total protein was extracted by incubating the homogenized samples in protein extraction buffer (10% [v/v] glycerol, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.5% [v/v] Triton X-100, and 1× protease inhibitor cocktail from Roche, USA) for 30 min. After centrifugation at 16,000 g for 10 min, the supernatant was incubated with anti-GFP agarose beads (Chromotek, gta-20) for 3 h at 4°C. The beads were washed four times with extraction buffer. The proteins were then eluted by boiling with 2× SDS loading buffer for 5 min. Co-immunoprecipitated proteins were analyzed by immunoblot with 1:10,000 diluted anti-FLAG and anti-GFP antibodies (Sigma-Aldrich). The Clarity Western ECl Substrate (Bio-Rad) and Odyssey LI-COR Imaging System were used to detect the blot signals.

Chemical treatments. To inhibit JA biosynthesis, 100 μM diethyldithiocarbamic acid (DIECA), purchased from Sigma, was exogenously applied to rice leaves 24 h before nematode inoculation. DIECA was prepared in water containing 0.02% (v/v) Tween 20 as previously described³⁵. Distilled water containing 0.02% (v/v) Tween 20 was used as a control. For the cell death assay, the palmitoylation inhibitor 2-bromopalmitate (2-BP) was infiltrated at a concentration of 100 μM into N. benthamiana leaves 6 h before Mg1 agro-infiltration. To determine Mg1 protein stability, 50 μM cycloheximide (CHX) was infiltrated into N. benthamiana leaves 4 h before immunoblot analysis.

Statistical analysis. The Chi-square test for goodness-of-fit was performed with MS Excel. The nematode infection and gene expression data were analyzed by GraphPad PRISM software. Significant differences between the treatments were evaluated using the least significant difference (LSD) test at a 5% probability level using the Statistix 9 software package.

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Acknowledgments

We thank Dr. Kabin Xie and Dr. Gaofeng Wang (HZAU, China) for kindly providing vectors and materials for analysis, Dr. Guanzhu Han (Nanjing Normal University, China) and Dr. Hongru Wang (University of California, USA) for assisting on sequence analysis and critical discussions, Gupo Li (CAS, China) for field management of rice materials. The work was supported by grants from Huazhong Agricultural University Scientific & Technological Self-Innovation Foundation (2016RC004, 2021ZKPY015) to XG.

Author contributions

X.G. supervised the work. X.G. and X.W. designed the experiments and wrote the manuscript with input from all authors. X.W., R.C., D.X. and R.H. performed majority of the experiments and data analysis. L.J. helped with the mapping analysis. H.L. and Y.W. contributed to bioinformatics analysis. D.P. contributed to disease assays. C.C., J.T. and C.S. provided germplasm for screening and developed some of the mapping populations used in this study.

Conflict of interest

The authors declare that they have no conflict of interest in relation to this work.

Data availability

The data supporting the findings of the study are available within the paper and its supplementary data published online.

There is NO Competing Interest.

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Direct association of Mg1 with a protease inhibitor confers resistance to rice root-knot nematode

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