Asymmetric subspecies-selection of an NLR-TF immune module and reconstruction of broad-spectrum disease resistance in rice

Asymmetric subspecies-selection of an NLR-TF immune module and reconstruction of broad-spectrum disease resistance in rice | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var 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Jiyun Liu , Jianjun Wang , Rongbai Li , Bizeng Mao , Jianlong Xu , Jong-Seong Jeon , Xuehui Huang , Bin Han , Yiwen Deng , Gongyou Chen , Zuhua He doi: https://doi.org/10.1101/2025.02.06.636770 Hui Lin 1 CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences , Shanghai 200032, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Fudan Chen 1 CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences , Shanghai 200032, China 2 College of Agriculture and Biotechnology, Zhejiang University , Hangzhou 310058, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Guanyun Cheng 3 Shanghai Collaborative Innovation Center of Agri-Seeds/State Key Laboratory of Microbial Metabolism, School of Agriculture and Biology, Shanghai Jiao Tong University , Shanghai 200240, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bingxiao Yan 1 CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences , Shanghai 200032, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Meng Yuan 4 National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University , Wuhan 430070, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jie Qiu 5 Shanghai Key Laboratory of Plant Molecular Sciences, Shanghai Collaborative Innovation Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University , Shanghai 200234, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ying Chen 1 CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences , Shanghai 200032, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yijie Wang 3 Shanghai Collaborative Innovation Center of Agri-Seeds/State Key Laboratory of Microbial Metabolism, School of Agriculture and Biology, Shanghai Jiao Tong University , Shanghai 200240, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kaixuan Cui 1 CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences , Shanghai 200032, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xiangyu Gong 1 CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences , Shanghai 200032, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shasha Liu 6 Key Laboratory of Plant Stress Biology, State Key Laboratory of Cotton Biology, School of Life Sciences, Henan University , Kaifeng 475004, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jiyun Liu 1 CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences , Shanghai 200032, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jianjun Wang 7 Zhejiang Academy of Agricultural Sciences , Hangzhou 310021, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rongbai Li 8 Agriculture College, Guangxi University , Nanning 530004, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bizeng Mao 2 College of Agriculture and Biotechnology, Zhejiang University , Hangzhou 310058, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jianlong Xu 9 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences , Beijing 100081, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jong-Seong Jeon 10 Graduate School of Green-Bio Science and Crop Biotech Institute, Kyung Hee University , Yongin 17104, Korea Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xuehui Huang 5 Shanghai Key Laboratory of Plant Molecular Sciences, Shanghai Collaborative Innovation Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University , Shanghai 200234, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bin Han 1 CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences , Shanghai 200032, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yiwen Deng 2 College of Agriculture and Biotechnology, Zhejiang University , Hangzhou 310058, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: zhhe{at}cemps.ac.cn gyouchen{at}sjtu.edu.cn ywdeng{at}cemps.ac.cn Gongyou Chen 3 Shanghai Collaborative Innovation Center of Agri-Seeds/State Key Laboratory of Microbial Metabolism, School of Agriculture and Biology, Shanghai Jiao Tong University , Shanghai 200240, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: zhhe{at}cemps.ac.cn gyouchen{at}sjtu.edu.cn ywdeng{at}cemps.ac.cn Zuhua He 1 CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences , Shanghai 200032, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: zhhe{at}cemps.ac.cn gyouchen{at}sjtu.edu.cn ywdeng{at}cemps.ac.cn Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Artificial selection has greatly shaped crop agronomic traits; however, the mechanistic basis of immunity selection has remained elusive. This study identifies a new rice NLR XA48 and its downstream transcription factor OsVOZ1, which confer bacterial blight resistance. XA48 perceives an ancient pathogen effector, XopG, to activates effector-triggered immunity (ETI). The XA48-OsVOZ1 module has undergone subspecies-specific selection. Xa48 is retained in indica but functionally lost in japonica rice. OsVOZ1 has also diverged into two haplotypes, indica kept both OsVOZ1 A/S alleles that match XA48; while japonica only inherited OsVOZ1 A that greatly decreases yield when Xa48 is reintroduced into japonica , mechanistically explaining the Xa48 loss in japonica . We resurrected wild rice broad-spectrum resistance by stacking XA48-mediated ETI with XA21-mediated pattern-triggered immunity (PTI). Thus, our study reveals that the asymmetric selection of an NLR-TF module shapes both disease resistance and reproduction, and provides a paradigm for breeding crops by harnessing the immunity of wild relatives. Plants have developed a sophisticated immune system to combat pathogens: PTI and ETI, which are governed by cell surface pattern-recognition receptors (PRRs) and intracellular NLR receptors, respecively 1 – 3 . Plant immunity has been shaped by crop domestication and breeding practices that aim to balance tradeoffs between growth and defense under diverse agricultural conditions 4 – 6 . It is unclear whether plant R genes and their associated immune components have been artificially selected during crop domestication 7 , 8 . Xanthomonas oryzae pv. oryzae ( Xoo ) causes bacterial leaf blight (BLB), one of the most devastating diseases of rice, and BLB outbreaks frequently occur after typhoons and heavy rainstorms 9 , 10 . The first cloned Xa gene, Xa21 , originated from the wild rice species Oryza longistaminata , which encodes a RLK that triggers race-specific PTI via perceiving the PAMP RaxX 11 , 12 . However, Xa21 does not impart disease resistance to Xoo strains from Northeast Asia due to the evolution of virulence in Xoo 13 , 14 . It is particularly important to isolate an Xa gene that can complement Xa21 for breeding modern rice with broad-spectrum resistance (BSR) to Xoo in face of the ever changing climate. Intriguingly, most functional Xa genes/loci have been isolated from wild relatives or represent recessive xa alleles of susceptibility genes 15 , suggesting negative selection of Xoo R genes during rice domestication. Given that extensive artificial selection has typically targeted high yielding and reliable disease resistance during breeding 6 , 16 , answering the question of whether and how R gene selection has occurred is particularly important for crop breeding, and we also need a practical breeding example to build BSR through integrating PTI and ETI in a real crop. Results Functional identification of the NLR gene Xa48 that confers lifetime Xoo resistance We combined map-based cloning and genome-wide association study (GWAS) to isolate an Xa gene that confers Xoo resistance and complements Xa21 . We first identified an indica variety Shuangkezao (SKZ) that exhibits strong resistance to the Korean Xoo strain J18 (DY89031), but high susceptibility to the Philippine Xoo strain PXO99A; while japonica TP309 line 106 harboring Xa21 is reverse 13 ( Extended Data Fig. 1a ), suggesting that SKZ has a R gene that confers race-specific resistance to Xoo and complements Xa21 . The gene, designated Xa48 , was identified by map-based cloning using a cross between SKZ and the japonica variety Nipponbare (NIPB). Xa48 was mapped to a 65-kb region on chromosome 3 using a bacterial artificial chromosome (BAC) of SKZ DNA ( Extended Data Fig. 1b ). Sequencing of the BAC clone revealed four candidates ( C1 to C4 ) harboring deletion/insertion polymorphisms in the NIPB and SKZ genomes ( Fig. 1a ). Download figure Open in new tab Extended Data Fig. 1 Map-based cloning identifies Xa48 . a , SKZ displayed strong resistance to J18 but high susceptible to PXO99A. Disease symptoms of rice variety SKZ, NIPB, TP309, and 106 at 14 dpi with Xoo strains J18 and PXO99A. Line 106 is transgenic TP309 expressing Xa21 . b , Xa48 was initially mapped to chromosome 3. c , d , Independent CRISPR/Cas9 knockout lines ( CR-C1 , CR-C2 , CR-C3 , CR-C4 ) of SKZ were generated ( c ), and representative lines’ lesion length of knockout mutants were measured ( d ). Note that only CR-C1 became susceptible to J18. e , Premature termination of XA48 translation in susceptible varieties caused by a single-base deletion and SNP. Schematic of the domain structure of XA48 is indicated. CC, coiled coil; NBS, nucleotide binding sequence; LRR, leucine-rich repeat. f , Transcriptional level of XA48 was detected in TP309- Xa48 ( f ) and NIPB- Xa48 ( g ) complement plants. g , h , NIPB- Xa48 transgenic plants are highly resistant to J18 as SKZ. The lesion length ( g ) and bacterial populations ( h ) were measured. Data were shown as mean ± SD, n ≥ 30 ( a, d, g ) and n = 3 ( f, h ), asterisks represented statistical significance (* P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed Student’s t-test). ns, not significant. Scale bars, 5 cm ( a, d, g ). Experiments were independently repeated three times with similar results ( a, d, g ). Download figure Open in new tab Fig. 1 Xa48 confers lifetime resistance against northeast Asian bacterial blight strains. a , Xa48 was preliminarily mapped to chromosome 3 between two insertion/deletion (InDel) markers InDel31 and InDel33, and was finely mapped to a 65-kb interval on a BAC of SKZ. Sequence analysis of the BAC revealed four candidates ( C1-C4 ) located in the mapping region. A deletion and SNPs were found within candidate gene C1 ( LOC_Os03g48370 ) in NIPB. b , Manhattan plot of GWAS for resistance to Xoo strain J18 in rice accessions. The genome-wide significant value threshold (6.35E-8) is indicated by a horizontal dash-dot line. A zoomed-in plot for the GWAS peak was shown with two significantly associated nonsynonymous SNPs in the Xa48 region highlighted. c , d , Xa48 knockout CR-C1 plants completely lost resistance to strain J18, the lesion length was measured at 14 days post-inoculation (dpi) ( c ). Bacterial populations per leaf were measured at 0, 7 and 14 dpi ( d ). e , f , Complement transgenic plants (TP309- Xa48 ) exhibited high Xoo resistance. The lesion length at 14 dpi ( e ) and bacterial populations at 0, 7 and 14 dpi ( f ) were measured. g , Xa48 also confers resistance to other northeast Asian strains. Plants were inoculated with Xoo strain JL1, LN1, LN2 and LN3 collected from Northeast China, with J18 and PXO99A as avirulent and virulent control strain, respectively. Disease symptom was measured at 14 dpi. Data were shown as mean ± SD, n ≥ 25 ( c , e , g ) and n = 3 ( d , f ). Scale bars, 5 cm ( c , e , g ). For c to g , asterisks represented statistical significance (** P < 0.01, *** P < 0.001, two-tailed Student’s t-test). ns, not significant. Experiments were independently repeated three times with similar results ( c-g ). A GWAS analysis for resistance to strain J18 was executed across 1,945 rice accessions and revealed four associated loci on chromosomes 3, 4, 5 and 11 ( Fig. 1b ). Three of the four loci contained the Xa alleles Xa1 , xa5 and Xa47 , previously reported 17 – 19 . The other locus exhibits a substantial GWAS signature peak that overlaps with the mapped Xa48 locus. Next, we generated CRISPR/Cas9 knockout mutants ( CR-C1 , CR-C2 , CR-C3 , and CR-C4 ) in the four loci, and discovered that CR-C1 exhibited a susceptible phenotype to J18 ( Fig. 1c, d , Extended Data Fig. 1c, d ). Compared with SKZ, the C1 locus in NIPB has a single-base deletion ( Extended Data Fig. 1e ). These results strongly suggest that C1 is the candidate gene for Xa48 ( LOC_Os03g48370 ), which encodes an NLR receptor consisting of 1094 amino acids (aa) ( Extended Data Fig. 1e ). To further confirm Xa48 , a construct containing Xa48 genomic DNA and a 3,000-bp promoter region was transferred into japonica TP309 and NIPB ( Extended Data Fig. 1f ). The resulting TP309- Xa48 and NIPB- Xa48 lines were highly resistant to J18 ( Fig. 1e, f , Extended Data Fig. 1g, h ). Similarly, Xa48 confers resistance to Xoo strains from Northeast Asia, including LN2 13 ( Fig. 1g ). Therefore, we conclude that Xa48 is an NLR gene responsible for race-specific resistance to Xoo strains originating from Northeast Asia. In contrast to other Xa genes that generally confer resistance only in adult plants 15 , Xa48 also confers Xoo resistance in seedlings ( Extended Data Fig. 2a-d ), which supports its potential use in rice breeding. Xa48 is expressed at high levels in leaves and panicles, and it is not induced by pathogen infection ( Extended Data Fig. 2e-g ). Furthermore, RNA-seq analysis of wild type TP309 and TP309- Xa48 infected with Xoo revealed differentially expressed genes (DEGs) enriched in the biosynthesis of salicylic acid, ethylene and phytoalexin, implying the involvement of these signals in Xa48 -mediated immunity ( Extended Data Fig. 2h, i ). Download figure Open in new tab Extended Data Fig. 2 Xa48 confers lifetime Xoo resistance. a-d , Xa48 confers Xoo resistance in SKZ ( indica ) ( a , b ) and NIPB- Xa48 ( japonica ) ( c , d ) seedlings. Lesion lengths and bacterial populations of representative CR-Xa48 /SKZ mutants and NIPB- Xa48 were measured in seedlings inoculated with J18. e , GUS staining of pXa48::GUS reporter transgenic plants revealed Xa48 expression in leaf, stem, root, seed, and young panicle. The boxed sections were magnified for close views of GUS staining in the spikelet and anther. Scale bars, 1 cm. f , Expression patterns of Xa48 . Expression of Xa48 in different tissues was detected by qRT-PCR and normalized to expression in the root 1. Root1 was from 5-day-old seedling, leaf1, stem1 were from 25-day-old seedling, and root2, leaf2, stem2 were from 80-day-old plants. g , Xa48 was not induced by Xo o infection as revealed by qPCR. Four-week-old rice were inoculated with J18 and PXO99A, and leaf samples were collected during a time course of 0-72 hpi for RNA preparation. h , i , Hierarchical clustering and Gene Ontology (GO) analysis of differentially expressed genes in TP309 and TP309- Xa48 leaves inoculated with J18. Note that Xa48 enhanced gene induction in salicylic acid, ethylene, and diterpene phytoalexin biosynthesis processes, which play important roles in rice immunity. Data were shown as mean ± SD, n ≥ 30 ( a, c ) and n = 3 ( b, d, f, g ), asterisks represented statistical significance (** P < 0.01, *** P < 0.001, two-tailed Student’s t-test). ns, not significant. Experiments were independently repeated three times with similar results ( a, c ). XA48 perceives cognate effector AvrXa48 (XopG) and activates immunity Tn 5 -mediated mutagenesis of Xoo J18 was unsuccessful, so strain LN2 was used instead to search for avrXa48 . A LN2 mutant library was generated with Tn 5 and 20,000 insertion mutants were inoculated to Xa48 rice. Two Xoo mutants were obtained that exhibited virulence on Xa48 rice ( Fig. 2a , Extended Data Fig. 3a-c ). Plasmid rescue and complementation analysis of the Xoo mutants successfully identified avrXa48 , which is a predicted type III effector annotated as XopG without functional identification. For consistency, we use XopG hereafter. Production of XopG in Xoo PXO99A (PXO99A/XopG LN2 ) converted PXO99A into an avirulent strain on Xa48 rice ( Fig. 2b , Extended Data Fig. 3d ). Thus, XopG is the cognate effector to XA48. Download figure Open in new tab Extended Data Fig. 3 Identification and functional analysis of XopG through Tn 5 mutagenesis. a , b , Generation of avrXa48 / xopG insertion mutants. Tn 5 transposon-insertion shows two independent insert mutants (#1 and #2) in the same site ( a ), which cause loss-of-function of XopG. Southern blot confirms single insertion of Tn 5 in the two LN2 mutants ( b ). The non-TAL promoter of XopG is indicated with the featured sequence. c , Tn 5 transposon-inserted mutants of strain LN2 lost avirulence to NIPB- Xa48 . The lesion length was measured at 14 dpi. d , The introduction of XopG LN2 changed PXO99A into avirulence to NIPB- Xa48 . e , XopG directly interacts with the XA48 CC domain by SLC in N. benthamiana . XA21 and XA48-LRR domains, which do not interact with XopG were used as negative controls. f , XopG associates with the XA48 CC mainly in the cell periphery and nucleus as revealed by BiFC in N. benthamiana leaf cells. XopG and XA48-CC were fused to the N-terminal fragment of YFP (nYFP) and the C-terminal fragment of YFP (cYFP). Scale bars, 50 μm. g , XopG likely induces oligomerization of XA48 in rice cells as revealed by BN-PAGE assay. Total protein was detected by immunoblotting with anti-FLAG and-GFP antibodies. h , Schematic of the domain structure of XopG is indicated. Peptidase_M91 contains an HEXXH motif, characteristic of zinc metallopeptidases. i , j , XopG displayed self-cleavage activity ( i ) but did not cleavage XA48 ( j ). Immunoblots of XopG-MBP and MBP acts as control. XA48-FLAG with XopG-GFP detected by immunoblotting with anti-FLAG/GFP antibodies. k , Representative localization images of XopG-GFP transgenic rice, showing main localization to the PM and nucleus. Co-localization of XA48 with XopG was detected in the PM and nucleus transiently expressed in rice protoplasts. FM4-64 and DAPI staining indicate the PM and nucleus. Data were shown as mean ± SD, n ≥ 22 ( c, d ), scale bars, 5 cm, asterisks represented statistical significance (*** P < 0.001, two-tailed Student’s t-test). Experiments were independently repeated three times with similar results ( c, d, e, j ). Download figure Open in new tab Fig. 2 XA48 perceives the cognate effector XopG to activate immunity. a , Tn 5 transposon-inserted mutants of strain LN2 lost avirulence to TP309- Xa48 and SKZ. The lesion length was measured at 14 dpi. b , The introduction of XopG LN2 changed PXO99A virulence into avirulence to TP309- Xa48 and SKZ. c , Interaction of XA48-FLAG with XopG-GFP detected by co-IP in transgenic rice plants. Fusion proteins were immunodetected using anti-GFP or anti-FLAG antibody. Another Xoo effector protein XopV, which does not interact with XA48, was used as a negative control. d , Co-expression of XA48 and XopG induced hypersensitive response cell death in N. benthamiana leaves. Protein expression was detected by Western blot. Numbers in parentheses indicate the leaves that exhibited cell-death symptoms. e , XopG-like proteins from Xoo strain LN2, Pseudomonas syringae pv. tomato DC3000 ( Pst ), P. syringae pv. actinidae M228 ( Psa ), Xanthomonas translucens pv. translucens Km9 ( Xtt ), Salmonella enterica subsp. arizonae SA20100345 ( Sea ) and Escherichia coli O157, were individually introduced into strain PXO99A. Note that Clade I and II XopG-like proteins were avirulent to Xa48 . LN2 and PXO99A-XopG LN2 were used as avirulent controls. f , Mutants of XopG S155A and XopG A157D lost interaction with XA48-CC, as detected by SLC. g , h , XopG mutants (H142R, E143R, H146R) interact with XA48-CC, detected by split luciferase complementation (SLC) ( g ), but could not trigger XA48-mediaed cell death in N. benthamiana leaves ( h ). Protein expression was detected by Western blot. Numbers in parentheses indicate leaves exhibited cell-death symptoms. i , Disease resistance phenotype and lesion length of TP309- Xa48 inoculated with PXO99A/XopG LN2 and PXO99A expressing the three HEXXH motif mutant variants (PXO99A/XopG LN2 H142R , PXO99A/XopG LN2 E143R , and PXO99A/XopG LN2 H146R ). Note that the HEXXH motif is critical to its function in Xa48 -mediated resistance. LN2 and PXO99A-XopG LN2 were used as avirulent controls. j , XopG homologs among a total of 76 Xanthomonas are classified into five types based on protein sequences. Only Type I contains full-length XopG, whereas other types are truncated. TE, transposable element. The geographical distribution of the XopG homologs reveals that functional XopG in Type I, is mainly found in strains from northeast Asia. Data were shown as mean ± SD, n ≥ 20, asterisks represented statistical significance (*** P < 0.001, two-tailed Student’s t-test). ns, not significant. Scale bars, 5 cm ( a, b, e, i ). Experiments were independently repeated twice with similar results ( a-i ). To explore the function of XopG in XA48-mediated ETI, we investigated whether XA48 directly interacts with XopG. An interaction between XopG and the coiled-coil (CC) domain of XA48 was observed in split luciferase complementation (SLC), bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) assays ( Fig. 2c , Extended Data Fig. 3e, f ). Furthermore, we established that the co-expression of XA48 and XopG induced cell death in Nicotiana benthamiana ( Fig. 2d ), and XopG induced the oligomerization of XA48 ( Extended Data Fig. 3g ). The effector-induced NLR oligomerization was also observed for the TNL receptor ROQ1 by another Xanthomonas effector XopQ in Arabidopsis 20 . Therefore, our findings show XA48 perceives XopG to activate immunity. XopG is a conserved effector in bacterial pathogens XopG contains a peptidase domain M91 with a conserved HEXXH motif ( Extended Data Fig. 3h ). This effector family includes the E. coli effector protein NleD, which cleaves and inactivates c-Jun N-terminal kinase 21 . To test the endopeptidase activity of XopG, self-cleavage of purified recombinant XopG fused to the maltose-binding protein (XopG-MBP) was observed in vitro ( Extended Data Fig. 3i ); however, there was no evidence that XopG can cleave XA48 in vivo ( Extended Data Fig. 3j ), which supported our observation that XopG promotes XA48 oligomerization ( Extended Data Fig. 3g ). Additionally, XopG was localized in the plasma membrane and nucleus, and it exhibited co-localization with XA48 ( Extended Data Fig. 3k ). We analyzed 38 proteins orthologous to XopG from other pathogenic bacteria and found they could be divided into three clades ( Extended Data Fig. 4a ). The xopG homolog genes were transformed into Xoo PXO99A, and the resulting PXO99A strains were inoculated to Xa48 rice. The PXO99A strains expressing XopG-like proteins from Clades I and II were avirulent on Xa48 plants, whereas those expressing Clade III proteins remained virulent ( Fig. 2e , Extended Data Fig. 4b ); this indicated that the XopG-like proteins from Clades I and II are likely to be recognized by XA48 to activate immunity. Next, an in-depth analysis revealed that the S and A aa residues at position 155 and 157 are conserved in XopG like proteins in Clades I and II but not in Clade III ( Extended Data Fig. 4a ). Further, site-directed mutagenesis was used to mutate S155 to A and A157 to D; the resulting XopG S155A and XopG A157D variants failed to interact with XA48-CC ( Fig. 2f ). When the two XopG variants were expressed in strain PXO99A (PXO99A/XopG S155A and PXO99A/XopG A157D ) and inoculated to Xa48 plants, the strains lost the ability to trigger XA48-mediated resistance ( Extended Data Fig. 4c ). We suspected that XopG belongs to an ancient effector family that impairs basal resistance to Xoo in rice ( Extended Data Fig. 5a ), and we speculated that the conserved HEXXH motif plays a critical role in XopG function. This hypothesis was tested by mutating 142H to R, 143E to A, and 146H to R. The H142R, E143A, and H146R mutants interacted with XA48-CC but could not induce XA48-mediated cell death ( Fig. 2g, h ). Furthermore, these mutants also lost the ability to trigger XA48 - mediated resistance in rice ( Fig. 2i , Extended Data Fig. 5b ). Download figure Open in new tab Extended Data Fig. 4 Functionality and specificity analysis suggest XopG is a conserved effector in bacterial pathogens . a , A phylogenetic tree and protein alignment of XopG and homologous proteins from different plant and animal bacterial pathogens were constructed using the Neighbor-Joining method, with 1,000 bootstrap replicates for statistical support, in MEGA11 software. Bootstrap values greater than 70% are indicated on the tree. XopG-like proteins are grouped into three clades. Note that the functional XopG LN2 belongs to Class I, labeled with green (left). The conserved HEXXH motif is indicated with red box. Amino acids in red (XopG S155 and XopG A157 ) are conserved in Clade I and Clade II, but changed in Clade III (right). b , Disease resistance phenotypes of NIPB- Xa48 inoculated with PXO99A/XopG LN2 , PXO99A/XopG Pst DC3000 , PXO99A/XopG Psa M228 , PXO99A/XopG Xtt KM9 , PXO99A/XopG Sea Serova , PXO99A/XopG E. coli O157 , which contain three clades of XopG-like proteins. c , Disease resistance phenotypes of NIPB- Xa48 inoculated with PXO99A expressing XopG S155A and XopG A157D . Note that the S155 and A157 of XopG from Clade I and II were critical to its functional in XA48-mediated resistance. Data were shown as mean ± SD, n ≥ 20 ( b , c ), scale bars, 5 cm, asterisks represented statistical significance (*** P < 0.001, two-tailed Student’s t-test). ns, not significant. Letters indicate significant differences ( P < 0.05) determined by two-way analysis of variance (ANOVA) with Tukey’s test. Experiments were independently repeated twice with similar results ( b , c ). Download figure Open in new tab Extended Data Fig. 5 HEXXH domain of XopG contributes to trigger XA48-mediated resistance in rice. a , Decreased basal Xoo resistance of xopG -OE in NIPB. Leaves were inoculated with PXO99A. The lesion length was measured at 14 dpi. b , Disease resistance phenotype and lesion length of NIPB- Xa48 inoculated with PXO99A/XopG LN2 , PXO99A/XopG LN2 H142R , PXO99A/XopG LN2 E143R , and PXO99A/XopG LN2 H146R . LN2 and PXO99A-XopG LN2 were used as avirulent controls. c , d , Avirulence and virulence detection of Type I and Type V of XopG variant strains on Xa48 . Data were shown as mean ± SD, n ≥ 22, scale bars, 5 cm, asterisks represented statistical significance (*** P < 0.001, two-tailed Student’s t-test). ns, not significant. Experiments were independently repeated twice with similar results. The sequence variability among XopG orthologs was investigated in 76 Xoo stains using available genome sequences (Supplementary Table 1), and the orthologs were classified into five structural types. Type I contained full-length XopG, whereas other types either lacked the HEXXH motif or were truncated ( Fig. 2j ). Consistent with the expected function of the HEXXH motif, Xoo strains LN2, LN4 and LN18 harbor a Type I XopG and are avirulent to Xa48 rice, whereas virulent strains PXO99A and JL28 harbor a Type V XopG ( Extended Data Fig. 5c, d ). The geographical distribution of Xoo strains revealed that Type I strains originated from Northeast Asia, whereas Xoo strains with mutated forms of XopG were mainly distributed in South Asia. These results support our previous finding that Xoo effectors exhibit a high level of divergence in both sequence and pathogenicity 13 . We propose that the prolonged arms race between XA48 and XopG has likely exerted a selective pressure on the evolution of Xa48 (see below for further details). XA48 interacts with OsVOZ transcription factors for immune signaling To elucidate the underlying molecular mechanism for XA48-mediated resistance, we conducted a yeast two-hybrid (Y2H) screen 22 to isolate XA48-interacting proteins. The transcription factors Vascular PLANT ONE-ZINC FINGER proteins, OsVOZ1 and OsVOZ2, were identified ( Fig. 3a, b , Extended Data Fig. 6a ). The XA48-OsVOZ1/2 interaction was validated in planta using SLC and co-IP assays ( Fig. 3c-f , Extended Data Fig. 6b ), and the results showed that OsVOZ1/2 interacted with full-length XA48 and its CC domain. We transiently co-expressed OsVOZ1/OsVOZ2-mCherry with XA48-YFP, and observed co-localization of XA48 and OsVOZ1/2 in the plasma membrane and nucleus ( Extended Data Fig. 6c, d ). This co-localization pattern is similar to that observed in the XA48-XopG association, hinting that XA48 might regulate OsVOZ1 and OsVOZ2. Download figure Open in new tab Extended Data Fig. 6 Identification of XA48-OsVOZ1/2 interaction. a , The candidate XA48-interacting proteins revealed by Y2H screen. Note that OsVOZ is the most hit protein. b , Western blots detected expression of XA48-CC-nLuc, cLuc-OsVOZ1 and cLuc-OsVOZ2 that were expressed in N. benthamiana for protein interaction. c , Representative images of XA48 subcellular localization. XA48-YFP was transiently expressed in rice protoplasts, which showed main localization to the PM/periphery and nucleus. OsRAC1-mCherry and NLS-mCherry served as a PM and nuclear marker, respectively. Scale bars, 10 µm. d , Co-localization of XA48 with OsVOZ1 and OsVOZ2 was detected in the PM and nucleus. Scale bars, 10 µm. e , Immunodetection of OsVOZs-GFP fusion protein in transgenic overexpression (OE) lines using anti-GFP antibody, with SKZ and NIPB- Xa48 as wild type controls. Actin was detected as a loading control. f , Schematic of OsVOZ1/2 knockout (KO) lines in NIPB background. Experiments were independently repeated twice with similar results ( b-e ). Download figure Open in new tab Fig. 3 XA48 interacts with and facilitates degradation of the transcription factors OsVOZ1 and OsVOZ2 for immune activation. a , b , The interaction between XA48 with OsVOZ1 ( a ) and OsVOZ2 ( b ) was detected by yeast two-hybrid (Y2H). Note that XA48 interacts with OsVOZ1/2 through the CC domain. SD/-L-T-H, synthetic dropout (SD) media lacking leucine, tryptophan, and histidine; SD/-L-T, SD media lacking leucine and tryptophan; 3AT, 3-aminotriazole. c , d , Co-IP assay confirms the interaction of XA48 and OsVOZ1/2. XA21 was expressed as negative control (Pruitt et al., 2015). e , f , SLC shows the interaction of XA48-CC and OsVOZ1/2 in planta . XA21 and PigmR-NBS (Zhai et al., 2022), which do not interact OsVOZs, were used as negative controls. g , h , OsVOZs negatively regulated XA48-mediated resistance against Xoo . Overexpression of OsVOZ1/2 resulted in decreased J18 resistance in SKZ ( g ) and NIPB- Xa48 transgenic plants ( h ). i , OsVOZs negatively regulate basal defense against Xoo . Knockout mutants of OsVOZ1/2 in NIPB increased resistance to J18. j , XA48-XopG promoted degradation of OsVOZ1 (left) and OsVOZ2 (right) in a cell-free assay. Extracts from OsVOZs-FLAG transgenic rice plants were incubated with cell extracts from transgenic rice XA48-GFP and XopG-GFP, and samples were collected over a time course of 0-20 min after mixing. GFP expression alone served as negative controls. k , Degradation promotion of OsVOZ1/2 by XopG-XA48 recognition was detected in planta. OsVOZ s-GFP/NIPB and OsVOZ s-GFP/NIPB- Xa48 plants were inoculated with PXO99A and PXO99A/XopG, respectively. proteins were collected over a time course of 0-72 hours post inoculation (hpi) for Western blotting. l , OsVOZs negatively regulates Xa21 -mediated resistance. Overexpression of OsVOZ1/2 resulted into decreased resistance to PXO99A and PXO61 in OsVOZs -OE/ Xa21 transgenic plants. m , Binding specificity of OsVOZ1 towards the CCCAC motif of the JAZ promoter. Biotin-labeled probes were incubated with MBP or OsVOZ1-MBP, with unlabeled competitor fragments co-incubated. The bands representing the DNA-protein complexes (shift) and the free probes are indicated by arrows. n , OsJAZs showed decreased expression in CR-Osvoz1 plants compared with wildtype NIPB, as detected by qRT-PCR. o , OsVOZ1 enhances promoter activity OsJAZs . OsVOZ1-GFP activates the expression of the pro OsJAZs -LUC fusion reporter in N. benthamiana . The LUC activity was measured by normalizing to REN signal. p , OsJAZs were down-regulated by XA48-XopG. NIPB- Xa48 were inoculated with PXO99A and PXO99A/XopG, respectively, and leaf samples were collected at 24hpi for RNA preparation. Data were shown as mean ± SD, n ≥ 15 ( g - i , l ), n = 3 ( n - p ), asterisks represented statistical significance (*P < 0.05, **P < 0.01, *** P < 0.001, two-tailed Student’s t-test). Scale bars, 5 cm ( g - i , l ). Experiments were independently repeated three times with similar results. OsVOZ2 was reported to affect Xoo virulence 23 . To determine the roles of OsVOZs in XA48-mediated resistance, we generated transgenic plants overexpressing OsVOZ1/2 ( OsVOZ1 -OE and OsVOZ2 -OE) in SKZ ( Xa48 , indica ), NIPB- Xa48 ( japonica ) and the knockout mutants ( CR-Osvoz1 / 2 ) by CRISPR/Cas9 editing ( Extended Data Fig. 6e, f ). A rice osvoz1/osvoz2 double mutant was not obtained due to its lethality 24 , and the OsVOZ1/2 -OE line showed attenuated XA48-mediated resistance ( Fig. 3g, h ). In contrast, CR-Osvoz mutants displayed increased basal resistance when compared with wild type NIPB ( Fig. 3i ). Therefore, OsVOZs function as negative regulators in both basal and XA48-triggered immunity against Xoo . The biological importance of the XA48-OsVOZs interaction in XA48-mediated resistance was then investigated. When the accumulation of OsVOZ1-FLAG and OsVOZ2-FLAG was measured in cell-free protein degradation assays, the presence of XA48 and XopG promoted the degradation of OsVOZ1 and OsVOZ2 ( Fig. 3j ). The XopG-XA48 promoting degradation of OsVOZ1/2 was also investigated in planta . Inoculation with PXO99A/XopG but not PXO99A promoted degradation of OsVOZ1/2 in OsVOZs -OE/NIPB- Xa48 rice as compared to OsVOZs -OE/NIPB ( Fig. 3k ). These results confirmed that OsVOZs undergo degradation to release the suppression of immunity during ETI activation. OsVOZ1 and OsVOZ2 were previously shown to interact with Piz-t, an NLR that confers resistance to the rice blast fungus, M. oryzae . OsVOZ1/2 negatively regulated basal defense and positively regulated Piz-t-mediated ETI, presumably by modulating Piz-t expression and protein levels 24 . To evaluate this possibility in the rice- Xoo interaction, plants overexpressing OsVOZs were generated in the Xa21 line 106. When these plants were inoculated with the virulent Xoo strains PXO99A and PXO61, OsVOZs were shown to negatively regulate XA21-mediated PTI against Xoo ( Fig. 3l ). Therefore, OsVOZs function in both PTI and ETI against Xoo , which may adopt differential signaling mechanisms to cope with pathogens with diverse infection lifestyles. To dissect the immune suppression function of OsVOZs, Cleavage Under Targets & Tagmentation (CUT&Tag) assay was executed in OsVOZ1-GFP/NIPB transgenic plants. DNA motif (CCCAC) had a high binding affinity for OsVOZ1-MBP ( Fig. 3m ). Next, RNA-seq analysis of CR-Osvoz1 and wild type NIPB revealed a set of DEGs involved in defense response, such as amino acid biosynthetic, metabolic process, and jasmonic acid (JA) signaling ( Extended Data Fig. 7a ). Expression level of JASMONATE-ZIM-DOMAINs ( OsJAZs ), encoding JA signaling and defense repressors in rice 25 , 26 , were downregulated in CR-Osvoz1 rice as compared to NIPB ( Fig. 3n ) and enriched the CCCAC motif in their promoters, indicating that JAZ-mediated immune suppression was alleviated. EMSA assays indicated that OsVOZ1-MBP bound specifically to CCCAC motifs in promoters of OsJAZ5 , OsJAZ9 , OsJAZ11 , OsJAZ12 , OsJAZ14 , and OsJAZ15 tested ( Extended Data Fig. 7b ), suggesting that OsVOZ1-regulates expression of these genes. Binding of OsVOZ1 to OsJAZ promoters induced OsJAZ expression based on luciferase-to-Renilla (LUC/REN) ratio ( Fig. 3o ); this suggested that OsVOZ1 activated OsJAZ expression by directly binding to their promoters. Furthermore, qRT-PCR analysis revealed that OsJAZs expression decreased when NIPB- Xa48 rice was inoculated with Xoo PXO99A/XopG as compared to the wild type PXO99A ( Fig. 3p ). Therefore, JA signaling at least plays a role in the XA48-OsVOZ1 module-mediated Xoo resistance. Collectively, these data establish an immune signaling pathway where the XA48-XopG immune complex triggers ETI by interacting with and promoting degradation of OsVOZs thereby inhibiting expression of the JAZ immune suppressors. Download figure Open in new tab Extended Data Fig. 7 OsVOZ1 specifically binds to the promoter of OsJAZ family, which contain the conserved CCCAC motif. a , Gene Ontology (GO) analysis of differentially expressed genes in CR-Osvoz1 and NIPB. Note that loss of OsVOZ1 function activated genes involved in JA-related defense. b , EMSA was performed to investigate binding affinity of OsVOZ1 to the cis-elements in OsJAZs promoter. Biotin-labeled probes were incubated with MBP or OsVOZ1-MBP. Unlabeled competitor fragments were added to evaluate binding specificity. Experiments were independently repeated twice with similar results ( b ). Xa48 was retained in indica but functionally lost in japonica The Xa48 locus present in japonica lines NIPB and TP309 was examined, and both genomes contained a nonfunctional allele due to loss-of-function mutations ( Extended Data Fig.1e ). Based on this finding, we analyzed the divergence of Xa48 alleles in the genomes of 3K rice germplasm 27 . A total of 1,945 available accessions were inoculated with Xoo strain J18 ( Fig. 4a ), and 405 accessions (20.8%) were identified with resistance to strain J18. Of these 405 accessions, 145 (35.8%) carried a functional Xa48 ; the others harbored loss-of-function mutations in Xa48 , suggesting that other R genes were active against Xoo J18. Intriguingly, rice lines with a functional Xa48 allele are exclusively indica subspecies ( Fig. 4a , Supplementary Table 2). This suggests that Xa48 might have undergo differential selection in the two subspecies, leading to its functional retention in indica but loss in japonica . Download figure Open in new tab Fig. 4 Subspecies-species selection of OsVOZ1 contribute to Xa48 -induced reproductive penalty in japonica . a , The frequency of functional Xa48 was assessed in sequenced rice accessions. Xa48 was detected in resistant accessions by PCR-based sequencing. Note that near all Xa48 -containing accessions are indica rice, and no japonica rice was detected to contain functional but loss-of-function Xa48 mutants. b , Dynamic frequency changes of functional Xa48 during indica and japonica domestication and improvement. c , The introduction of Xa48 led to a significantly decrease in grain yield in the japonica rice TP309 by reducing seed setting in multiple-locations and seasons field trials. d , The introduction of Xa48 did not affect grain productivity in the indica variety Kasalath in multiple-locations and seasons field trials. e , OsVOZ1 exists a major non-synonymous polymorphism SNP G and SNP T , resulting in an amino acid change from alanine (OsVOZ1 A ) to serine (OsVOZ1 S ). f , Dynamic frequency changes of the OsVOZ1 A and OsVOZ1 S alleles during indica and japonica domestication and improvement. Note that nearly all japonica accessions carry the OsVOZ1 A allele, whereas indica rice harbors both alleles. g , Seed setting rates of 70 indica rice accessions that contain either OsVOZ1 S or OsVOZ1 A haplotype with or without ( Xa48 + or Xa48 - ) from the 3K rice genome project, showing no effect of OsVOZ1 A/S alleles on reproductivity in indica rice bringing native Xa48 . h , i , Overexpression of OsVOZ1 A and OsVOZ1 S alleles significantly decreased resistance to J18 ( h ) without effect on seed setting in NIPB ( i ). Scale bars, 5 cm. j , OsVOZ1 A and OsVOZ1 S overexpression compromised Xa48- mediated resistance . OsVOZ1 A -OE/NIPB- Xa48 and OsVOZ1 S -OE/NIPB- Xa48 were generated by crossing OsVOZ1 A -OE and OsVOZ1 S -OE with NIPB- Xa48 , respectively. Homozygous plants were selected from the F 2 populations. Scale bars, 5 cm. k , l , Overexpression of OsVOZ1 S but not OsVOZ1 A restored normal seed setting in NIPB -Xa48 ( japonic a) ( k ). The Field trail of two seasons and locations revealed that the seed setting of OsVOZ1 S -OE/NIPB- Xa48 was stored as the wild type NIPB ( l ). Scale bars, 1 cm. Data were shown as mean ± SD, n ≥ 12 ( c, d, g, i, l ), asterisks represented statistical significance (* P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed Student’s t-test). ns, not significant. Letters indicate significant differences ( P < 0.05) determined by two-way analysis of variance (ANOVA) with Tukey’s test ( g , l ). Experiments were independently repeated three times with similar results ( c, d, g, I, l ). Next, we determined the sequence of Xa48 in other genomic projects with 2,451 Asian rice accessions including wild rice with available genome sequences 28 – 30 (Supplementary Table 3). Based on GWAS peak SNPs ( P =1.47E - 16 ), we examined the dynamic frequency change of functional Xa48 allele during rice domestication and improvement. In the wild rice species, O. rufipogon , the functional allele frequency was moderate (9.8%). In indica , the allele frequency increased from 7.8% to 24.9% during breeding improvements that occurred from landraces to cultivars. In contrast, the functional allele of temperate japonica decreased from a rare frequency in landraces (1.0%) and improved populations (0.3%) (Supplementary Table 4). These results suggest positive and negative selection for Xa48 in indica and japonica , respectively. Considering the agroecological-specific distribution of XopG-positive Xoo strains in Northeast Asia ( Fig. 2j ) where japonica rice is cultivated, we suggest that a negative selection pressure occurred for XopG in Xoo to deal with XA48-mediated resistance. Xa48 decreases grain yield in japonica Our next objective was to determine the underlying mechanism leading to the elimination of Xa48 in japonica but not indica . Considering the growth penalty associated with disease resistance in rice 31 , 32 , agronomic traits were compared in the following rice lines: SKZ vs CR-Xa48/ SKZ (CRISPR/Cas9 knockout line), Kasalath (without Xa48 ) vs transgenic Kasalath- Xa48 ( indica ) ( Extended Data Fig. 8a ), NIPB vs NIPB- Xa48 , and TP309 vs TP309- Xa48 ( japonica ). Morphological differences in these transgenic lines were not observed when compared to their respective wild types; however, one exception was the lower plant height observed in NIPB- Xa48 and TP309- Xa48 ( Extended Data Fig. 8b, c ). Interestingly, seed setting rate decreased significantly in japonica NIPB- Xa48 and TP309- Xa48 but remained unchanged in indica CR-Xa48/ SKZ and Kasalath- Xa48 plants based on field trials over multiple seasons and locations, resulting in lower grain yield in NIPB- Xa48 and TP309- Xa48 as compared to wild type NIPB and TP309 ( Fig. 4c, d , Extended Data Fig. 8d-f ). These findings indicated the reintroduction of Xa48 greatly decreased reproductivity in japonica rice. Download figure Open in new tab Extended Data Fig. 8 Xa48 decreases grain yield in japonica but not in indica . a , Lesion length of Xa48 complement lines in indica Kasalath that contains a truncated Xa48 mutant (shown above) and harbors the OsVOZ1 A allele. Note that wild type Kasalath is also resistant to J18, mediated by an additional unrecognized Xa genes. Scale bars, 5cm. b-d , Mature plants ( b ), plant height ( c ) and panicles ( d ) of NIPB- Xa48 , TP309- Xa48 , Kasalath- Xa48 , and CR-Xa48 /SKZ. Note that panicle size was not affected in these plants with or without Xa48 , whereas Xa48 led to a decrease in plant height in both japonica varieties NIPB and TP309, but not in indica rice SKZ and Kasalath. Scale bars, 10 cm ( b ) and 5 cm ( d ). e , The introduction of Xa48 significantly decreased grain yield in japonica rice NIPB by reducing seed setting in multiple-location and season field trials during 2021, 2022 and 2023. f , Knockout of Xa48 (CR- Xa48 ) did not affect grain productivity in indica rice SKZ in multiple-location and-season field trials during 2021, 2022 and 2023. g , KEGG analysis of differentially expressed genes in TP309- Xa48 vs TP309 young panicle. Note that the Xa48 introduction induced differential expression of many genes including those involved in sugar and amino acid metabolism, which may contribute to the seed development penalty in japonica . h , Xa48 does not cause much difference of gene expression in indica CR-Xa48 / SKZ vs SKZ . Data were shown as mean ± SD, n ≥ 15 ( a, c, e, f ), asterisks represented statistical significance (*** P < 0.001, two-tailed Student’s t-test). ns, not significant. Experiments were independently repeated three times with similar results ( a, e, f ). We performed RNA-seq analysis in the young panicles of the following rice pairs: TP309- Xa48 vs wild type TP309, and CR-Xa48 /SKZ vs SKZ. The DEGs were enriched for glucose and amino acid metabolism pathways in japonica ( Extended Data Fig. 8g, h ), which may explain the defective grain development observed in in japonica . Taken together, these results suggest that the presence of Xa48 decreases agronomic value with respect to reproduction and has been removed through artificial selection and mutation in japonica . A natural OsVOZ1 variant contributes to Xa48 -induced yield penalty in japonica rice The genetic mechanism underlying Xa48 subspecies-specific selection was further explored by focusing on OsVOZ1, which contains a non-synonymous SNP, SNP G/T . This SNP resulted in an amino acid change from A to S ( Fig. 4e ). The allele frequency of OsVOZ1 S was highly differentiated between indica and japonica rice and was examined during rice domestication and modern improvement. The allele frequency in wild rice was 5.0%, and increased to 38.6% during indica rice domestication, and continued to rise in modern indica cultivars (85.3%) ( Fig. 4f , Supplementary Table 5). However, OsVOZ1 S was rarely found in the japonica group (0.3%), which instead harbors the OsVOZ1 A allele. In summary, the allele frequency changes of Xa48 and OsVOZ1 S suggested that the XA48-OsVOZ1 immune module may be favored and has been continuously co-selected during indica rice domestication and improvement. In contrast, Xa48 and OsVOZ1 S alleles in japonica rice have almost been eliminated possibly driven by purifying selection. The SLC and Y2H assays demonstrated that both OsVOZ1 A and OsVOZ1 S interacted with XA48, indicating that this SNP does not impact the interaction of either OsVOZ1 variant with XA48 ( Extended Data Fig. 9a, b ). To determine the effect of OsVOZ1 alleles on seed development, we randomly selected 70 indica accessions and divided them into four categories, namely OsVOZ1 S with or without Xa48 , and OsVOZ1 A with or without Xa48 . Seed setting rate of indica accessions were then assessed in a natural rice paddy. No significant difference was observed among these categories ( Fig. 4g ), suggesting that SNP G/T has no effect on seed setting in indica rice. To further support this conclusion, the indica variety SKZ containing OsVOZ1 S was crossed with indica variety Kasalath containing OsVOZ1 A ( Extended Data Fig. 9c ); this indica × indica cross was not anticipated to impart significant changes in seed setting rate of the progeny, which generally occur in indica × japonica crosses. We identified four distinct groups of progeny populations by genotyping ( OsVOZ1 S Xa48 - , OsVOZ1 A Xa48 - , OsVOZ1 S Xa48 + , and OsVOZ1 A Xa48 + ); none showed difference in seed setting rate ( Extended Data Fig. 9d ). Moreover, we crossed SKZ with the indica varieties 9311 and TN1 (both containing OsVOZ1 S and a mutated Xa48 , Extended Data Fig. 9c ) to obtain two OsVOZ1 S - Xa48 - and OsVOZ1 S - Xa48 + progenies and obtained similar results ( Extended Data Fig. 9e ). Additionally, when the pXa48:Xa48 transgene was introduced into the indica varieties TN1 and ZS97 containing OsVOZ1 S and OsVOZ1 A , respectively, the resulting transgenic lines, TN1- Xa48 and ZS97- Xa48 , exhibited resistance to Xoo J18 but showed no difference in seed setting rate ( Extended Data Fig. 9f-i ). These results indicate that the XA48 - mediated immunity module exhibits greater versatility in coping with different alleles of OsVOZ1 in indica rice, while the XA48-OsVOZ1 A module specifically disturbs seed setting in japonica . Download figure Open in new tab Extended Data Fig. 9 XA48-OsVOZ1 A/S immune modules shape different seed setting between japonica and indica . a , b , OsVOZ1 S interacts with XA48, determined by SLC ( a ) and Y2H ( b ) assays. c , The allelic variation of Xa48 and OsVOZ1 in different varieties was shown in the indica ( i ) and japonica ( j ) varieties sued in the study. d , Development of indica inbreed lines with four combinations of OsVOZ1 A/S and Xa48 , OsVOZ1 S - Xa48 - , OsVOZ1 S - Xa48 + , OsVOZ1 A - Xa48 - and OsVOZ1 A - Xa48 + , derived from SKZ (OsVOZ1 S ) crossing to Kasalath (OsVOZ1 A ) (F 5 ), which showed no difference in seed setting rate. e , Premature mutation of Xa48 in indica TN1 and 9311 at codon position 226. SKZ crossing to 9311 and TN1, which brings the same OsVOZ1 S allele, to generate inbreed lines OsVOZ1 S - Xa48 - and OsVOZ1 S - Xa48 + (F 5 ) respectively, which showed the same seed setting rate. f-i , Lesion lengths and seed setting rates of complement line TN1- Xa48 ( f, g ) and ZS97- Xa48 ( h, i ) ( indica ). The results indicated that the complement indica lines exhibited J18-resisatnce and no difference on seed setting rates. Scale bars, 5cm. Data were shown as mean ± SD, n ≥ 15 ( d-i ). For d , letters indicate significant differences ( P < 0.05) determined by two-way analysis of variance (ANOVA) with Tukey’s test. For e-i , asterisks represented statistical significance (*** P < 0.001, two-tailed Student’s t-test). ns, not significant. Experiments were independently repeated three times with similar results. Our many attempts to edit the G/T SNP were unsuccessful, so we examined the function of this SNP in the seed setting rate of japonica using a transformation strategy. First, we generated lines overexpressing either allele ( OsVOZ1 A -OE or OsVOZ1 S -OE) in NIPB using the maize Ubiquitin promoter, which showed no differences in seed setting relative to NIPB, but were significantly less resistant to Xoo ( Fig. 4h, i ), supporting the above notion that OsVOZ1 negatively regulates basal Xoo resistance ( Fig. 3i ). Next, we crossed the OsVOZ1 A /S overexpression lines with NIPB- Xa48 to generate OsVOZ1 A -OE/NIPB- Xa48 and OsVOZ1 S -OE/NIPB- Xa48 plants, which displayed attenuated XA48-mediated resistance ( Fig. 4j ), in a manner consistent with results described above ( Fig. 3g, h ). The seed setting rate of OsVOZ1 A -OE/NIPB- Xa48 was lower than that in wild type NIPB. Interestingly, OsVOZ1 S -OE/NIPB- Xa48 restored the normal wild type seed setting rate ( Fig. 4k, l ). These results collectively demonstrate that the XA48-OsVOZ1 A module inhibits reproduction in japonica , and explain the asymmetric selection of the XA48-OsVOZ1 immune module between the two subspecies and the functional loss of Xa48 in japonica . Breeding for BSR by integrating XA21-mediated PTI and XA48-mediated ETI Our results demonstrated that Xa48 confers a resistance spectrum that is complementary to that of Xa21 , which originates from wild relative 11 , 12 . We proposed that the combination of Xa48 and Xa21 , which trigger ETI and PTI, respectively, may be a good approach that has practical implication for providing BSR in rice breeding programs. This strategy was investigated by traditional cross breeding or transgenic approach ( Extended Data Fig. 10a ). The resulting Xa21 Xa48 plants conferred BSR to 28 Xoo strains that were previously known to be avirulent on either Xa21 or Xa48 but not both ( Fig. 5a, b , Extended Data Fig. 10b ). Importantly, we established that stacking Xa21 and Xa48 conferred a high level of resistance to four Xoo strains (FJ-1∼4) that were virulent to both Xa21 and Xa48 ( Fig. 5c ); furthermore, this resistance was associated with increased expression of several pathogenesis-related ( PR ) genes during Xoo infection ( Extended Data Fig. 10c ). These results document that PTI and ETI pathways can be deployed together to enhance basal defense in rice. It is also noteworthy that the Xa21 Xa48 rice lines retained a high level of Xoo resistance in the field trail after a flooding ( Fig. 5d ). Download figure Open in new tab Extended Data Fig. 10 Xa48 - Xa21 stacking rice for broad-spectrum Xoo resistance was developed. a , Development of transgenic TP309 ( japonica ) with integrating Xa21Xa48 through crossing and selfing selection (F 5 generation). b , Broad-spectrum disease resistance of Xa21Xa48 plants against northeast Asian Xoo strain J18, LN1, LN2, LN3 and JL1. Scale bars, 5 cm. c , The defense genes PR4 , PR5 , and PR10 expression were significantly enhanced in Xa21Xa48 plants during Xoo infection. d , Development of indica rice combining endogenous Xa48 and Xa21 through crossing SKZ and BG139 ( Xa21 ) and selfing (F 4 generation). e , De novo development of japonica regaining Xa48 and OsVOZ1 S by crossing CR-Osvoz1 /NIPB and NIPB- Xa48 . f , Mature plant and panicle of CR-Osvoz1 , which showed defective growth and development phenotypes. Scale bars, 10 cm. Data were shown as mean ± SD, n = 3 ( c ), asterisks represented statistical significance (* P < 0.05, ** P < 0.01, and *** P < 0.001, two-tailed Student’s t-test). ns, not significant. Experiments were independently repeated twice with similar results ( c ). Download figure Open in new tab Fig. 5 Broad-spectrum bacterial blight resistance is reconstructed by stacking Xa48 -mediated ETI and Xa21 -mediated PTI in modern rice. a , b , Lesion lengths of TP309, Xa21 (106), Xa48 , and Xa21Xa48 plants inoculated with 28 Xoo strains that are either virulent to Xa48 or Xa21 . Note that Xa21Xa48 plants conferred broad-spectrum resistance in comparison to plants with the single R gene triggering PTI ( a ) and ETI ( b ) respectively. c , Improved resistance to strains FJ-1, FJ-2, FJ-3, and FJ-4 in Xa21Xa48 plants. The strains were isolated from the infected leaves of rice plants grown in Fujian province (Southeast China), which bring a mutated XopG (Type V in Fig 2j ) and are moderately virulent to respective Xa21 and Xa48 . d , Xoo nursery test of Xa21Xa48 lines. Plants were kept under natural flooding after heavy rainstorms associated with a typhoon, leading natural Xoo infection in the Shanghai station. Disease symptom was recorded one and a half months after floodwaters. Note that Xa21Xa48 rice conferred high filed resistance against Xoo infection. e , Representative leaves of SKZ and BG139 ( Xa21 ) inoculated with PXO99A and J18. f , Lesion lengths of japonica regaining Xa48 and OsVOZ1 S by crossing CR-Osvoz1 /NIPB with NIPB- Xa48 . g , De novo design of japonica rice restoring Xa48 and OsVOZ1 S . The Xa48 - OsVOZ1 S japonica plants ( OsVOZ1 S -OE/ Xa48 /CR- Osvoz1 ) in NIPB background were grown in the paddy field for agronomic trait measurement with multiple-locations and seasons. Notably, the Xa48 - OsVOZ1 S japonica plants restored normal seed setting. Data were shown as mean ± SD, n ≥ 15, asterisks represented statistical significance (*** P < 0.001, two-tailed Student’s t-test). ns, not significant. Letters indicate significant differences ( P < 0.05) determined by two-way analysis of variance (ANOVA) with Tukey’s test ( f , g ). Scale bars, 5 cm ( c , e , f ). Experiments were independently repeated three times with similar results ( a-c, e-g ). Rice line BG139, an indica variety containing Xa21 , was crossed with SKZ to generate Xa21 Xa48 inbred indica lines; these contained intact Xa21 and Xa48 immune modules from wild rice and exhibited BSR to Xoo strains ( Fig. 5e , Extended Data Fig. 10d ). Because of the conventional approach for introducing the two genes, the utilization of these Xa21 Xa48 lines in rice breeding programs is underway at several seed companies. In parallel, we de novo developed japonica lines expressing the Xa48 -OsVOZ1 S immune module: we first knocked out OsVOZ1 A in NIPB, the resulting CR-Osvoz1 NIPB plants were then crossed with NIPB- Xa48 to generate NIPB- Xa48 / CR-Osvoz1 . The later line was crossed again with OsVOZ1 S -OE/NIPB to generate OsVOZ1 S -OE/ Xa48 NIPB plants, which retained the XA48-OsVOZ1 S module and conferred resistance to Xoo J18 without a yield penalty ( Fig. 5f, g , Extended Data Fig. 10e ). It is important to mention that Osvoz1 knockout plants were attenuated in growth and reproduction ( Extended Data Fig. 10f ), suggesting a critical function in plant growth and development. Collectively, our results provide a foundation for exploiting application of R genes from wild rice relatives with different immune modules and provides a breeding paradigm that high field disease resistance can be achieved through combining PTI and ETI in modern crop breeding. Discussion Our study presents holistic approaches with Xa48 for cultivating modern crops that exhibit both high disease resistance and optimal yield performance. First, leveraging Xa48 and Xa21 to reconstruct BSR to bacterial blight in rice, providing a success breeding example of how synchronized PTI and ETI indeed improves disease resistance in a real crop, as proposed 33 – 36 . This approach could also extend the shelf life of single R genes that have been defeated by virulent pathogen strains. Second, this study describes the use of genome editing, transgenes, and hybridization methods to created novel BLB-resistant, penalty-free japonica lines that recruit the XA48-OsVOZ1 S immune module. This breeding concept has the potential to be applied to other crop pathosystems, including rice for resistance to blast disease and wheat for resistance to stripe rust, utilizing reliable PTI and ETI modules. XA48 and its associated transcription factor OsVOZ1 constitute an immune module that underwent differential artificial selection, leading to the loss of XA48 in japonica rice. This differential selection was driven by two main forces: one from the pathogen-host interaction, as presented with the XopG natural variation and distribution, and the other coming from artificial selection through selective breeding for high yield ( Fig. 6a, b ). Notably, the arms race of XA48 and XopG led to XopG enriched in strains from Northeast Asia where japonica rice is cultivated; In contrast, XopG had undergone massive loss-of-function events when Xa48 is retained within the indica lineage, which has been widely cultivated in tropical and subtropical regions, encompassing the southern reaches of the Yangtze River and the Mekong River valley 37 , where typhoons and heavy rainstorms are common occurrences, as we observed in the natural Xoo nursery ( Fig. 6a ). This specific agroecological scenario, coupled with high pathogen pressure, is likely a contributing factor to the retention of Xa48 in indica rice. Download figure Open in new tab Fig. 6 Working model of subspecies-specific selection of the XA48-OsVOZ1 module and design in japonica . a , Rice leaf bacterial blight spread after floodwaters associated the typhoon in the Shaoxing experimental station (Southeast China) in the 2021 summer, showing paddy flooding (top, July) and bacterial blight symptom in the field (bottom, September). b , A proposal model for subspecies-specific selection of the XA48-OsVOZ1 immune module between indica and japonica . XA48 perceives the cognate type III bacterial effector XopG to activate immunity and promote degradation of the immunosuppressive transcription factor OsVOZ1, which activates OsJAZs , the suppressors of immunity. OsVOZ1 evolved two alleles, OsVOZ1 A and OsVOZ1 S , japonica rice only inherits OsVOZ1 A while indica rice keeps both. The XA48-OsVOZ1 A combination poses a negative effect on reproductivity in japonica , leading to Xa48 functional loss in japonica . Xa48 is kept and does not affect reproduction in indica with either OsVOZ1 A or OsVOZ1 S allele, which is historically cultivated in Southeast Asia where Xoo frequently outbreaks due to typhoons and floodwaters. The reintroduction of XA48 in japonica lines resulted in reduced seed production due to combination of Xa48 and the japonica allele OsVOZ1 A , while the Xa48 immune module is much more versatile in coping with different alleles of OsVOZ1 A/S in indica ( Fig. 6b ). The immune system incompatibility is reminiscent of hybrid necrosis or weakness seen in plants, where genetic incompatibility triggers autoimmunity in hybrids. This autoimmunity primarily arises from a conflict involving NLRs, which are crucial for achieving a compatible immune response and better yield performance in hybrids but become problematic when their interactions lead to incompatibility 38 – 41 . Therefore, our study documents that the incompatible immune system has led to the artificial selection for an ETI network that shapes both disease resistance and reproduction in a crop. Methods Plant materials and growth conditions The japonica model varieties NIPB, TP309 and transgenic TP309 line 106 expressing Xa21 , the indica varieties, SKZ, Kasalath, ZS97, 9311 and TN1, and 1,945 available rice accessions used in the 3K genome project 27 , were used and grown in the experimental paddy fields at Shanghai for the summer season and Hainan Island for the winter season under natural field conditions. Nicotiana benthamiana grown at a growth chamber under long-day conditions (16-h day/8-h night, 22°C) for 3-4 weeks, was used for transient expression experiments, protein-protein interaction, and cell death assays. Pathogens Xoo strains (listed in Supplementary Table 6) from Asia and elsewhere were stored and used in this study. Xtt Km9, Pst DC3000, Psa M228, E. coli O157 and Sea SA20100345 were also used in this study. Map-based cloning and screening of rice BAC library SKZ was crossed to NIPB to generate an F 2 mapping population and inoculated with Xoo strain J18. Xa48 was delimited to a 65-kb genomic region between InDel33 and InDel31 on chromosome 3. The genomic DNA of SKZ was partially digested with Hin dIII and inserted into the BAC cloning vector plndigoBAC-5 for library construction. A BAC clone was selected that spanned the Xa48 locus. Sequence analysis of the BAC clone revealed four ORFs within the 65-kb region, which showed several insertion, deletion and SNPs between NIPB and SKZ. GWAS analysis of Xoo resistance The genotyping matrix for GWAS was obtained from the 3K Rice Genomes Project 27 and other rice genome projects 37 , 42 . The panel includes 4,591 rice accessions with 5.23 million SNPs. 1,945 accessions were phenotyped for J18 resistance, a total of 3,605,578 SNPs with minor allele frequency above 0.05 were maintained for the GWAS study. GWAS was performed by GEMMA with the linear mixed model 43 . Kinship matrix was incorporated in the GWAS models to control false-positives. The effective number of SNPs was calculated as 786,858 by GEC software 44 , and therefore by applying genome-wide type I error rate at α□=□0.05, we determined the significance threshold as 6.35E-8 (0.05/786,858). The GWAS plot was visualized using the CMplot R package 45 . Allele frequency inference during rice domestication and improvement A total of 2,451 Asian rice accessions were used for inference of the allele frequency changes of Os VOZ1 and Xa48 . These accessions include 169 wild rice (88 and 81 genetically close to indica and japonica , respectively), 1,158 temperate japonica , 135 tropical japonica , 22 basmati , 79 aus and 888 indica rice 29 , 30 . Detailed information is listed in the Supplementary Table 3. Landraces and cultivar accessions for indica and temperate japonica were classified based on Wei et al. (2021). Variants in the Os VOZ1 and Xa48 genes were called by GATK v3.7 46 . The allele frequency was calculated for variants in Xa48 and Os VOZ1 for all rice groups. Plasmid construction and rice transformation For CRISPR/Cas9-mediated knockout mutation in rice, 20-bp gene-specific guide RNA sequences were constructed targeting the candidates C1 , C2 , C3 , C4 of the mapping region and OsVOZs were PCR-based cloned and subcloned into pYLCRISPR/Cas9-MH vector 47 . To generate the constructs for OsVOZ1 and OsVOZ2 fusions driven by the maize Ubiquitin 1 promoter (Ubi), coding regions (CDS) were inserted into PUN1301-pUBI-GFP or PUN1301-pUBI-FLAG vectors. For complementation, DNA fragments containing the whole Xa48 promoter and coding region were inserted into binary vector pCAMBIA1300 to generate plasmid pXa48::Xa48 . All constructs were introduced into Agrobacterium strain EHA105, and then transformed into different rice backgrounds to generate more than 15 independent lines; an exception was the CRISPR/Cas9 knockout mutants that usually generated more than 100 transformants for mutation screening. All transgenic plants were selected by PCR-based sequencing, gene expression assays or western blot. For transient expression in N. benthamiana , the constructs were introduced into Agrobacterium strain GV3101. The primer sequences used for cloning are listed in Supplementary Table 7. The following transgenic lines/mutants were developed in this study: CR-C1 ( CR-Xa48 /SKZ), CR-C2 , CR-C3 , CR-C4 , NIPB- Xa48 , TP309- Xa48 , Kasalath- Xa48 , TN1- Xa48 , ZS97- Xa48 , OsVOZ1 -OE/SKZ, OsVOZ2 -OE/SKZ, OsVOZ1 -OE/NIPB- Xa48 , OsVOZ2 -OE/NIPB- Xa48 , OsVOZ1 A -OE, OsVOZ1 S -OE, OsVOZ1 A -OE/NIPB- Xa48 , OsVOZ1 S -OE/NIPB- Xa48 , xopG -OE, XA48 -OE, CR-Osvoz1/ NIPB, CR-Osvoz2/ NIPB, NIPB- Xa48/CR-Osvoz1 , OsVOZ1 S -OE/NIPB- Xa48/CR-Osvoz1 , OsVOZ1 A -OE/NIPB- Xa48/CR-Osvoz1 and Xa21 / Xa48 . All transformants were grown in experimental paddy fields located at Shanghai and Hainan Island. Pathogen inoculation The Xoo strains used in this study included strains from Northeast Asia, South Asia and other regions, and Xoo transformants expressing xopG homologs from other pathogenic bacteria (listed in Supplementary Table 6). For inoculation experiments, Xoo strains grown on PSA medium (10 g/L tryptone, 10 g/L sucrose, 1g/L glutamate, 15 mg/L cephalexin, pH 7.0) at 28□ for 3 days were suspended in sterile water to OD 600 = 1.0. Two-month-old plants were inoculated by leaf-clipping and syringe infiltration methods. Lesion length was measured at 14 days post inoculation (dpi). Xoo growth curve were generated by suspending inoculated leaf tissue (20 cm) in 10 ml sterile water to collect bacteria; suspensions were diluted to count colony-forming units. Inoculation experiments were repeated independently at least three times. Rice Xoo nursery test To investigate Xoo infection and potential epidemic after flooding, rice was grown at the Shaoxing (Zhejiang province) and Shanghai experimental station in June, 2021. Plants were maintained for 5 d under natural flooding due to August rainstorms. Plants were maintained without bactericide application to allow Xoo to infect naturally. Disease symptoms were recorded 45 d after flooding. Tn 5 insertion mutagenesis of Xoo Competent Xoo LN2 cells were mixed with 1 μL of the EZ-Tn 5 Tnp Transposome and subjected to electroporation as recommended by the manufacturer (Epicentre, Madison, USA). The transformed Xoo cells were transferred to 1.5 mL Eppendorf tubes, incubated at 180 rpm at 28°C for 1.5 h, and then cultured on PSA containing the kanamycin (20 μg/mL) at 28°C for 3 d. Over 20,000 Km-resistance colonies were individually numbered and then transferred to new PSA plates with Km for further investigation. Plasmid rescue and XopG identification Xoo genomic DNA was extracted by the CTAB method and digested. The digested DNA was separated in 1.3% agarose gels at 80V for 16-20 h and then transferred to Immobilon-Ny+ Transfer Membranes (Merck, USA). The DNA was labeled, hybridized, and detected using the DIG-High Prime DNA Labeling and Detection Starter Kit. Plasmid rescue was performed with the EZ-Tn 5 TM Tnp Transposome TM kit following the manufacturer’s instructions. For XopG identification, the genomic DNA was isolated from two Tn 5 mutants of Xoo strain LN2, which were compatible with NIPB- Xa48 . The genomic DNA was extracted, digested with Pst I enzyme, and hybridized in Southern blots where Tn 5 was used as a probe. Detection was conducted with the Dig-Labeling Kit (Roche) as recommended by the manufacturer. The Pst I-digested gDNA fragments were also self-ligated using T 4 ligase and transformed into competent cells of E. coli EC100D (Lucigen, USA). The plasmids of single clone were isolated and fragments flanking the Tn 5 insertion site were sequenced using the primers included in the Tnp Transposome kit. The sequenced fragments were analyzed using BLAST programs available at the National Center for Biotechnology Information (NCBI). Expression of xopG homologues from other bacterial pathogens Both xopG and xopG homologues were PCR-amplified (see primers in Supplementary Table 7) and ligated into pHM1 vector at the Sal I- Hin dIII site. Competent cells of Xoo PXO99A were electroporated with 1 μL purified plasmid DNA and transferred to NA containing spectinomycin. The transformed Xoo PXO99A strains were verified by immunoblotting using FLAG as the antibody. Yeast two-hybrid screening and protein interaction analysis Y2H screening was conducted to identify XA48-interacting proteins with a rice pathogen-induced cDNA library as previously described 22 . For the Y2H assay, the coding sequences of target genes, including xopG , were amplified with gene specific primers and cloned into yeast expression vectors pDEST22 (AD)/32 (BD) (Invitrogen). Constructs were co-transformed into yeast strain AH109, grown on selective medium (lacking Trp and Leu or Trp, Leu and His) containing 3-aminotriazole (Sigma-Aldrich, A8056) for 3 d at 30□. Split luciferase complementation assays For SLC assays, coding sequences of the target genes were cloned into pCAMBIA-35S-nLuc or pCAMBIA-35S-cLuc, which were transformed into Agrobacterium strain GV3101. Agrobacterium cells were collected and resuspended in infiltration buffer (10 mM MgCl 2 , 10 mM MES, 150 mM acetosyringone, pH 5.6), and incubated for 2-3h at 30°C before infiltration into N. benthamiana leaves. Two days after transformation, luciferase activity was measured as recommended by the manufacturer (Promega), images were captured using the Tanon-5200 Chemiluminescent imaging system (Tanon). Bimolecular fluorescence complementation assay For BiFC assays, XopG and XA48 CC were fused to the N-terminal fragment of YFP (nYFP) and the C-terminal fragment of YFP (cYFP), which were transformed into Agrobacterium strain GV3101. Agrobacterium cells were collected, resuspended in infiltration buffer, and incubated for 2-3 h at 30°C before infiltration into N. benthamiana leaves. Fluorescence images were harvested at 48 post-infiltration (hpi) using a Leica TCS SP5 II confocal microscope. Protein co-immunoprecipitation Protein co-IP assays were performed to verify protein-protein interactions in planta . Xa48-CC was cloned to pCAMBIA1300-35S-eGFP(C)-rbcsE9 and OsVOZ1 , OsVOZ2 and xopG were cloned to pCAMBIA1300-35S-FLAG(C)-rbcsE9. Protein extracts were prepared from N. benthamiana or transgenic rice leaves in IP buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 1mM PMSF and protease inhibitor cocktail (11836153001, Sigma)]. Supernatants were incubated with anti-GFP beads for 2 h at 4°C and then washed four times with washing buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM PMSF and protease inhibitor cocktail). The bound proteins were released from the beads by boiling for 5 min in SDS loading buffer. The reagents used included the antibodies, FLAG (Sigma, F1804), GFP (Abcam, ab290), goat anti-rabbit IgG secondary antibody (Thermo Fisher, 31460) and goat anti-mouse IgG secondary antibody (CWBIO, CW0102). Subcellular localization For protein subcellular localization assay, coding sequences of the genes were cloned into PA7-35S-YFP or PA7-35S-mCherry, which were transformed into rice protoplasts prepared from leaf sheaths of 10-day-old seedlings. NLS (Nuclear Localization Signal) was used as a nuclear marker and OsRAC1 as a plasma membrane marker 22 . After incubation in the dark for 16-20□h at 26°C, fluorescence was observed with a confocal microscope (Zeiss LSM 880). Cell death assay in N. benthamiana The N. benthamiana cell death assay was used to determine the function of XA48-XopG interaction and XA48 activation. XopG-GFP and XA48-GFP constructs were transformed into Agrobacterium GV3101 and expressed in N. benthamiana . The expression of each protein was confirmed by immunoblotting using anti-GFP and cell death on infiltrated leaves was photographed at 36-48 hpi. Cell-free degradation assay Total proteins were extracted from N. benthamiana leaves or transgenic rice leaves with the extraction buffer (50mM Tris-HCL, pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 5 mM dithiothreitol, 5 mM adenosine 5‘-triphosphate, and 1x protease inhibitor cocktail). The extracts were clarified by two sequential centrifugations at 12,000 g for 5 min. Samples were incubated at 25 °C at the indicated time points. The reaction was terminated by boiling the reaction in SDS sample buffer and then analyzed by western blotting. All protein immunodetection experiments were performed independently three times with similar results. GUS reporter assay For the β-glucuronidase (GUS) reporter gene constructs, the 3-kb promoter region of Xa48 was inserted upstream of the GUS coding sequence in expression vector pCAMBIA1300-GUS and then introduced into Agrobacterium strain EHA105 to generate GUS reporter transgenic plants. For GUS staining, plant tissues were incubation-stained in buffer (50 mM NaPO 4 , pH 7.0, 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe 6 , 0.1% Triton X-100, and 1 mM X-Gluc) overnight at 37°C, and then dehydrated with a graded ethanol series. RNA preparation and RNA-seq analysis Total RNAs were extracted using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). For quantification of gene expression, 1□μg RNA was reverse-transcribed into cDNA using oligo (dT) primers and SuperScript III. qRT-PCR was performed using SYBR Premix Ex Taq (TaKaRa) and gene-specific primers were listed in Supplementary Table 7, rice ACTIN served as an internal control to normalize expression levels. The 2 -△△CT method was used to calculate the relative expression levels with three biological repeats. For RNA-seq analysis, RNA samples were prepared from Xoo -infected rice leaves at 24 hpi and from panicles of different rice genotypes at the booting stage. RNA-seq was performed by Shanghai Biotechnology Corporation. Three biological replicates were used for each genotype/treatment. The entire RNA-seq dataset was deposited in the NCBI Gene Sequence Read Archive (SRA) under accession number: PRJNA1028151. CUT& Tag assay Cleavage Under Targets and Tagmentation (CUT&Tag) assays were conducted using the Hyperactive Universal CUT&Tag Assay Kit (Vazyme, Cat# TD903). Briefly, two-week-old seedling leaves of OsVOZ1-OE/NIPB and OsVOZ1-OE/SKZ were cross-linked with 1% formaldehyde and stopped by glycine solution. The tissue was ground into fine powder and nuclei were prepared in NE1 buffer (20□mM HEPES, pH 7.5, 10□mM NaCl, 0.5□mM spermidine, 0.1% Triton X-100, 20% glycerol) for 10□min on ice. Samples were resuspended in NE2 buffer (20□mM HEPES, pH 7.5, 150□mM NaCl, 0.5□mM spermidine, Protease Inhibitor) and centrifuged at 3000 g for 5□min. Subsequently, the anti-GFP antibody (Abcam, Cat#ab290) was added and incubated overnight at 4°C, accompanied by gentle rotation. Secondary antibody was added and incubated for 1□h at room temperature. DNA library was constructed using TruePrep Index Kit V2 for Illumina (Vazyme, Cat# TD202) with proper primers. Electrophoretic mobility shift assay OsVOZ1-MBP recombinant protein was developed and purified in E. coli . DNA fragments were end-labeled with Cy5, which was incubated with 0.2 ug protein in 20 ul binding buffer (100 mM Tris-HCl, pH 7.5, 250 mM KCl, 25 mM MgCl 2 , 10 mM EDTA, 10 mM DTT and 10% glycerol) at room temperature for 30 min. 50-200-fold non-labeled competitor DNA was added for competition assays. The reaction mixture was electrophoresed on a native polyacrylamide gel, and then was imaged by autoradiography (Fujifilm FLA 9000 plus DAGE) according to the manufacturer. Dual-luciferase transcriptional activity assay A dual-Luc reporter system in N. benthamiana leaves was performed following a previously reported protocol 22 . Briefly, pCAMBIA1300-35s-GFP and pCAMBIA1300-35s-OsVOZ1-GFP serve as effectors. 3 kb promoter region of OsJAZs were cloned into pGreenII-0800-Luc vector to generate Pro OsJAZ ::Luc reporter. The effector and reporter plasmids were introduced into Agrobacterium strain GV3101 and co-expressed in N. benthamiana leaves. The LUC and REN luciferase activities were measured using Dual-Luciferase Reporter Assay Kit (Promega) following the manufacturer’s instructions. Phylogenetic analysis For phylogenetic analysis of XopG, we utilized the amino acid sequence of XopG as a query to identify homolog proteins in other Gram-negative bacteria via the Domain Enhanced Lookup Time Accelerated BLAST (BLASTp) program available at NCBI. Subsequently, XopG and its identified homologs were employed to construct a phylogenetic tree using the Neighbor-Joining method, with 1,000 bootstrap replicates for statistical support, in MEGA11 software. Breeding approaches for stacking Xa 21 and Xa 48 We used two approaches to stacking Xa21 and Xa48 . First, transgenic TP309- Xa21 (106) was crossed to TP309- Xa48 , and the progeny were self-crossed continually to generate homozygous polymeric Xa21Xa48 plants (F 5 ). In a second approach, the traditional indica variety BG139 (containing Xa21 ) was crossed to SKZ (containing Xa48 ), and the progeny self-crossed to generate homozygous breeding material Xa21 Xa48 (F 4 ). De novo development of japonica rice expressing Xa 48 and OsVOZ 1 S To re-introduce Xa48 and OsVOZ1 S into japonica rice, we first generated OsVOZ1- KO lines in NIPB plants ( CR-Osvoz1 ). We then crossed CR-Osvoz1 to NIPB- Xa48 to develop NIPB- Xa48/CR-Osvoz1 , and crossed to OsVOZ1 S -OE to develop OsVOZ1 S -OE/NIPB- Xa48/CR-Osvoz1 plants. The Xa48-OsVOZ1 S japonica plants ( OsVOZ1 S -OE/NIPB- Xa48/CR-Osvoz1 NIPB ) were grown in the paddy field for measurement of agronomic traits. Field trails and agronomic trait measurements One-month-old seedlings were transplanted into paddy fields located at the Shanghai and Hainan Experiment Stations during 2021∼2023 for multiple-season and-location field test. At least 12 independent lines of each genotype were grown over multiple seasons at both locations. Upon maturity, agronomic traits including plant height, tiller number, seed setting rate, grain number per panicle, grain weight per plant, and 1,000-grain weight were analyzed. Average values of 15 plants or 12 representative progeny lines of each genotype (T3) were measured and compared. Statistical analysis Quantification analysis on lesion length, bacterial growth, plant growth, seed setting rate, grain productivity and other measurements were conducted in GraphPad Prism. All values were presented as mean ± SD and the number (n) of samples or replicates were indicated in the corresponding figure legends. Significance of difference was analyzed using Student’s t test for pairwise comparisons and one-way or two-way ANOVA followed by Tukey’s test for multiple groups’ comparison. Detailed information about quantifications and statistical analysis values is provided in figure legends, or within specific sections of Methods. No methods were used for sample randomization or sample size estimation and no data were excluded from analysis. Data availability The RNA-seq data generated in this study have been deposited in the SRA database under accession PRJNA1028151. The full genomic sequence of the Xa48 can be found in GenBank 2754159. All data are available in the main text or the supplementary materials. Source data are provided with this paper. Author contributions H.L., F.C., Y.D., G.C., and Z.H. conceived and designed the experiments. H.L., F.C., B.Y., G-Y.C., M.Y., and Y.C. performed experiments, and Y.W. and G-Y.C. performed Tn5 mutagenesis. J.Q., X.H. and B.H. performed population genomics and artificial selection analysis. K.C., X.G., S.L., J.L., and J-S.J. assisted with pathogen inoculation and field trials. B.M. assisted in rice transformation experiments. R.L. J.W. and J.X. grew and provided wild rice and rice germplasm. Z.H., Y.D. and G.C. supervised the project. Z.H. and Y.D. provided theoretical contributions to the project. H.L., F.C., Y.D., G.C. and Z.H. analyzed the data and wrote the paper. All authors discussed and commented on the manuscript. Competing interests The authors declare no competing interests. Acknowledgements We would like to thank Dr. Qifa Zhang for helpful discussion, Dr. Pamela Ronald for providing the Xa21 transgenic seeds, Ms. Xin Wang for rice transformation, Ziyao Lei for field inoculation, and Mr. Jianyao Shou for helping with Xoo nursery. This work was supported by Biological Breeding-National Science and Technology Major Projects (2023ZD04070 to Y.D.), the National Natural Science Foundation of China (32088102, 31930090 to Z.H.; 32361143515, 31830072 to G.C.; U20A2021 to Y.D.; 32402392 to H.L), the Chinese Academy of Sciences (XDB27040201 to Z.H. and XDA24010304 to Y.D.), Shanghai Science and Technology Development Funds (24YF2751900 to H.L), Shanghai Agricultural Science and Technology Innovation Program (Grant No. K2024-02-08-00-12-F00050 to H.L), and the National Key Research and Development Program of China (2022YFE0198100 to Y.D.). References 1. ↵ Jones , J. D. & Dangl , J. L . The plant immune system . Nature 444 , 323 – 329 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 2. Couto , D. & Zipfel , C . Regulation of pattern recognition receptor signalling in plants . Nat. Rev. Immunol . 16 , 537 – 552 ( 2016 ). OpenUrl CrossRef PubMed 3. ↵ Xin , X. F. , Kvitko , B. & He , S. Y . 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Share Asymmetric subspecies-selection of an NLR-TF immune module and reconstruction of broad-spectrum disease resistance in rice Hui Lin , Fudan Chen , Guanyun Cheng , Bingxiao Yan , Meng Yuan , Jie Qiu , Ying Chen , Yijie Wang , Kaixuan Cui , Xiangyu Gong , Shasha Liu , Jiyun Liu , Jianjun Wang , Rongbai Li , Bizeng Mao , Jianlong Xu , Jong-Seong Jeon , Xuehui Huang , Bin Han , Yiwen Deng , Gongyou Chen , Zuhua He bioRxiv 2025.02.06.636770; doi: https://doi.org/10.1101/2025.02.06.636770 Share This Article: Copy Citation Tools Asymmetric subspecies-selection of an NLR-TF immune module and reconstruction of broad-spectrum disease resistance in rice Hui Lin , Fudan Chen , Guanyun Cheng , Bingxiao Yan , Meng Yuan , Jie Qiu , Ying Chen , Yijie Wang , Kaixuan Cui , Xiangyu Gong , Shasha Liu , Jiyun Liu , Jianjun Wang , Rongbai Li , Bizeng Mao , Jianlong Xu , Jong-Seong Jeon , Xuehui Huang , Bin Han , Yiwen Deng , Gongyou Chen , Zuhua He bioRxiv 2025.02.06.636770; doi: https://doi.org/10.1101/2025.02.06.636770 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Genetics Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17648) Bioengineering (13871) Bioinformatics (41880) Biophysics (21423) Cancer Biology (18561) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13364) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22475) Immunology (17713) Microbiology (40328) Molecular Biology (17148) Neuroscience (88473) Paleontology (666) Pathology (2827) Pharmacology and Toxicology (4816) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)