Abstract
Crops with broad-spectrum resistance loci are highly desirable in agricultural production because these loci often confer resistance to most races of a pathogen or multiple pathogen species. Here we discover a natural allele of proteasome maturation factor in rice, UMP1R2115, that confers broad-spectrum resistance to Magnaporthe oryzae, Rhizoctonia solani, Ustilaginoidea virens and Xanthomonas oryzae pv. oryzae. Mechanistically, this allele increases proteasome abundance and activity to promote the degradation of reactive oxygen species-scavenging enzymes including peroxidase and catalase upon pathogen infection, leading to elevation of H2O2 accumulation for defence. In contrast, inhibition of proteasome function or overexpression of peroxidase/catalase-encoding genes compromises UMP1R2115-mediated resistance. More importantly, introduction of UMP1R2115 into a disease-susceptible rice variety does not penalize grain yield while promoting disease resistance. Our work thus uncovers a broad-spectrum resistance pathway integrating de-repression of plant immunity and provides a valuable genetic resource for breeding high-yield rice with multi-disease resistance.
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Data availability
All data generated or analysed during this study are included in the main figures, extended data figures and supplementary information. Source data are also provided in supplementary information. Raw RNA-seq data are available at Genome Sequence Archive (http://ngdc.cncb.ac.cn: accession number CRA006650). Proteome data are available at OMIX database (https://ngdc.cncb.ac.cn/omix: accession number OMIX001081). The reference Nipponbare genome data were retrieved from EnsemblGenomes (ftp://ftp.ensemblgenomes.org/pub/release-31/plants/fasta/oryza_sativa/dna). Sequences of UMP1 homologues were retrieved from UniProt (https://www.uniprot.org/), and their accession numbers are as follows: Oryza punctata (A0A0E0KFV5), Oryza meridionalis (A0A0E0D398), Oryza glumipatula (A0A0D9YZR5), Leersia perrieri (A0A0D9VM90), Hordeum vulgare (F2CQM0), Triticum aestivum (A0A3B6QNA6), Brachypodium distachyon (I1IDJ7), Panicum hallii (A0A2S3GU64), Sorghum bicolor (A0A194YTW9), Zea mays (B4FLC7), Ananas comosus (A0A199VSL7), Arabidopsis thaliana (Q94B05), Punica granatum (A0A218XIA6), Cucumis sativus (A0A0A0KUK6), Homo sapiens (Q9Y244), Mus musculus (Q9CQT5) and Saccharomyces cerevisiae (P38293). Source data are provided with this paper.
References
Li, W., Deng, Y., Ning, Y., He, Z. & Wang, G. L. Exploiting broad-spectrum disease resistance in crops: from molecular dissection to breeding. Annu. Rev. Plant Biol. 71, 575–603 (2020).
Moore, J. W. et al. A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat. Nat. Genet. 47, 1494–1498 (2015).
Li, W. et al. A natural allele of a transcription factor in rice confers broad-spectrum blast resistance. Cell 170, 114–126 e115 (2017).
Gao, M. et al. Ca2+ sensor-mediated ROS scavenging suppresses rice immunity and is exploited by a fungal effector. Cell 184, 5391–5404 e5317 (2021).
Shi, J. et al. Identification of rice blast resistance genes in the elite hybrid rice restorer line Yahui2115. Genome 58, 91–97 (2015).
Liu, W. D., Liu, J. L., Triplett, L., Leach, J. E. & Wang, G. L. Novel insights into rice innate immunity against bacterial and fungal pathogens. Annu. Rev. Phytopathol. 52, 213–241 (2014).
Copeland, C. & Li, X. Regulation of plant immunity by the proteasome. Int. Rev. Cel. Mol. Bio. 343, 37–63 (2019).
Fang, W. W. et al. Selection of differential isolates of Magnaporthe oryzae for postulation of blast resistance genes. Phytopathology 108, 878–884 (2018).
Gemperline, D. C. et al. Proteomic analysis of affinity-purified 26S proteasomes identifies a suite of assembly chaperones in Arabidopsis. J. Biol. Chem. 294, 17570–17592 (2019).
Ramos, P. C., Hockendorff, J., Johnson, E. S., Varshavsky, A. & Dohmen, R. J. Ump1p is required for proper maturation of the 20S proteasome and becomes its substrate upon completion of the assembly. Cell 92, 489–499 (1998).
You, X. M. et al. Rice catalase OsCATC is degraded by E3 ligase APIP6 to negatively regulate immunity. Plant Physiol. 190, 1095–1099 (2022).
Park, C. H. et al. The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice. Plant Cell 24, 4748–4762 (2012).
Peng, H. et al. MBKbase for rice: an integrated omics knowledgebase for molecular breeding in rice. Nucleic Acids Res. 48, D1085–D1092 (2020).
Yin, X. et al. Rice copine genes OsBON1 and OsBON3 function as suppressors of broad-spectrum disease resistance. Plant Biotechnol. J. 16, 1476–1487 (2018).
Sun, W. et al. Ustilaginoidea virens: Insights into an emerging rice pathogen. Annu. Rev. Phytopathol. 58, 363–385 (2020).
Xie, K. B., Minkenberg, B. & Yang, Y. N. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc. Natl Acad. Sci. USA 112, 3570–3575 (2015).
Xie, X. et al. CRISPR-GE: a convenient software toolkit for CRISPR-based genome editing. Mol. Plant 10, 1246–1249 (2017).
Li, Y. et al. Multiple rice microRNAs are involved in immunity against the blast fungus Magnaporthe oryzae. Plant Physiol. 164, 1077–1092 (2014).
Li, Y. et al. Osa-miR169 negatively regulates rice immunity against the blast fungus Magnaporthe oryzae. Front. Plant. Sci. 8, 2 (2017).
Fan, J. et al. Infection of Ustilaginoidea virens intercepts rice seed formation but activates grain-filling-related genes. J. Integr. Plant Biol. 57, 577–590 (2015).
Jia, Y. et al. Rapid determination of rice cultivar responses to the sheath blight pathogen Rhizoctonia solani using a micro-chamber screening method. Plant Dis. 91, 485–489 (2007).
Han, X. et al. Quantitative trait loci mapping for bacterial blight resistance in rice using bulked segregant analysis. Int. J. Mol. Sci. 15, 11847–11861 (2014).
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).
Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for rna-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Li, Y. et al. Osa-miR398b boosts H2O2 production and rice blast disease-resistance via multiple superoxide dismutases. N. Phytol. 222, 1507–1522 (2019).
Fan, J. et al. Comparative iTRAQ proteome and transcriptome analyses of sweet orange infected by ‘Candidatus Liberibacter asiaticus’. Physiol. Plant. 143, 235–245 (2011).
Xie, C. et al. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 39, W316–W322 (2011).
Üstün, S. & Börnke, F. Ubiquitin proteasome activity measurement in total plant extracts. Bio-Protoc. 7, e2532 (2017).
Tamura, K. et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739 (2011).
Acknowledgements
We thank C. Lei (Chinese Academy of Agricultural Sciences) for providing rice lines IRBLz5-CA, IRBL9-W and IRBLkm-Ts; L. Zhu (Chinese Academy of Sciences) for providing the eGFP-tagged M. oryzae strain GZ8; W. Zhao (China Agricultural University) for providing M. oryzae CRB strains; Y. Chen (Zhejiang University) for providing the yeast strain BY4741 and its derived mutant Scump1; X. Chen and A.-P. Zheng (Sichuan Agricultural University) for providing Xoo strain P6 and R. solani strain AG1-IA, respectively; Y. Qian (Hangzhou Biogle Co., Ltd.) for assistance with rice genetic transformation; Z. Chen (Chengdu Tiancheng Weilai Technology) for help on natural variation analysis. This work was supported by the grants from the National Natural Science Foundation of China (32072503 to J.F., U19A2033 to W.-M.W., 32172417 to Y.L., 31901839 to Y.-Y.H. and 32101728 to Z.-X.Z.), the Sichuan Youth Science and Technology Innovation Research Team Foundation (2022JDTD0023 to J.F.), the Sichuan Natural Science Foundation (2022NSFSC1715 to X.-H.H. and 2022NSFSC0174 to H.W.), and the China Postdoctoral Science Foundation (2021M692335 to X.-H.H.).
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J.F., W.-M.W. and X.-H.H. designed the study and wrote the manuscript. X.-H.H., J.-L.W., S.S., J.L., J.F., J.-X.H., H.W., Z.-L.Y., Y.-F.B., X.Z., Y.Z., G.-B.L., J.-H.Z., Y.-P.J., D.-Q.L. M.P., Z.-X.Z., S.-X.Z., J.-W.Z., Y.-Y.H., Yan Li, X.Y., Y.N. and F.H. performed the experiments. J.F., W.-M.W., X.-H.H., J.X., Y.N., Yanli Lu and J.-L.W. analysed the data.
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Nature Plants thanks Tsutomu Kawasaki, Shuai Huang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Comparative transcriptomic analysis of different rice accessions in response to M. oryzae infection.
a, Expression analysis of indicated defence-related genes at indicated time points of M. oryzae infection. OsUbi was used as the reference gene. The expression of indicated genes in LTH-0 hpi was set as the control. Data are mean ± s.d. (n = 3 biological replicates). b, Subclusters of differentially expressed genes. The gray lines in each subgraph represent the relative expression level of genes. The blue lines represent the average relative expression level of all genes in this cluster. c, Expression patterns of OsUMP1 homologs in rice responsive to M. oryzae infection. The fragments per kilobase of exon per million fragments mapped (FPKM) data were retrieved from the transcriptome data sets in this study. hpi, hours post inoculation. Values are mean ± s.d. (n = 2 biological replicates).
Extended Data Fig. 2 Disease assays of R2115 and its derived knockout mutants.
a, Schematic diagram of OsUMP1 locus and mutation types of CRISPR-Cas9-edited plants. b, Alignment of protein sequences between mutants and wild-type (WT). c, Disease phenotype of M. oryzae field isolates on R2115. Over 300 field isolates were separately punch-inoculated on to leaves of R2115 and LTH (set as the susceptible control). Disease phenotype was recorded at 5 days post inoculation (dpi). Representative images of leaves infected with 24 isolates are shown. d, Disease phenotype and relative fungal growth of ko-5 segregants infected with M. oryzae DZ108. Values are mean ± s.d. (n = 3 biological replicates). e, Disease phenotype of R2115 and ko-5 infected with incompatible M. oryzae strains CRB23 and CRB9. LTH was used as a control compatible to both CRB23 and CRB9. f, Disease assay with R. solani. Values are mean ± s.d. (n = 5 biological replicates). g, Disease assay with Xoo. Values are mean ± s.d. (n = 21, 24, 23 biological replicates, from left to right). P values were determined by two-sided unpaired t-test, compared to R2115.
Extended Data Fig. 3 The UMP1R2115 allele confers non-race-specific broad-spectrum resistance to M. oryzae.
a, Expression level of OsUMP1 in OsUMP1R2115 transgenic plants (T0 generation). The expression level of OsUMP1 was determined by RT-qPCR using OsUbi as the reference gene and TP309 as the control. Values are mean ± s.d. (n = 3 biological replicates). P values were determined by two-sided unpaired t-test, compared to TP309. b, Disease phenotype of indicated rice lines (T0 generation) at 7 days post inoculation (dpi) with M. oryzae GZ8. c and d, Disease phenotype and OsUMP1 expression of UMP1R2115-1 and UMP1R2115-13 segregants infected with M. oryzae GZ8. RT-qPCR analysis was performed with OsUbi as the reference gene and TP309 as the control. Values are mean ± s.d. (n = 3 biological replicates). e, Disease phenotype of TP309 and UMP1R2115 lines inoculated with a panel of M. oryzae strains. f, Quantification of lesion length in (e). Data are mean ± s.d. (n = 6 biological replicates). Significant difference was determined by two-sided unpaired ttest (* P < 0.05, ** P < 0.01).
Extended Data Fig. 4 OsUMP1 partially rescues heat sensitivity of S. cerevisiae ump1 mutant and is required for tolerance to canavanine.
a, Protein sequences of UMP1 homologs in different plant species were downloaded from the Uniprot database (https://www.uniprot.org), and aligned using DNAMAN software (version 5.2.2). Black, magenta, and turquoise shading sites indicate homology of 100%, ≥75%, and ≥50%, respectively. b, Phylogenetic tree was generated with MEGA5.1 using the Muscle method for sequence alignment and the Neighbor-Joining method for tree construction. The accession numbers for UMP1 homologs are as below: Oryza punctata (A0A0E0KFV5), Oryza meridionalis (A0A0E0D398), Oryza glumipatula (A0A0D9YZR5), Leersia perrieri (A0A0D9VM90), Hordeum vulgare (F2CQM0), Triticum aestivum (A0A3B6QNA6), Brachypodium distachyon (I1IDJ7), Panicum hallii (A0A2S3GU64), Sorghum bicolor (A0A194YTW9), Zea mays (B4FLC7), Ananas comosus (A0A199VSL7), Arabidopsis thaliana (Q94B05), Punica granatum (A0A218XIA6) and Cucumis sativus (A0A0A0KUK6). UMP1 homologs in Homo sapiens (Q9Y244), Mus musculus (Q9CQT5) and Saccharomyces cerevisiae (P38293) were included as outgroups for phylogenetic analysis. c, Yeast cells of wild-type (WT), Scump1 mutant, and Scump1 transformed with rice UMP1R2115 were incubated at 28 °C or 37 °C for 48 h. d, Seedlings of TP309, ko-11, and UMP1R2115 lines were grown in 50 μM canavanine for 14 days. e, Changes of shoot and root length of TP309, ko-11, and UMP1R2115 seedlings under 50 μM canavanine treatment. Values are mean ± s.d. The numbers of biological replicates (n) are indicated in the figure.
Extended Data Fig. 5 OsUMP1 regulates proteasome function and affects protein ubiquitination and accumulation in rice.
a and b, Relative proteasome activity normalized to proteasome amount (related to Fig. 2b,d) in leaves of UMP1R2115 (a) and ko-11 (b) lines upon infection with M. oryzae GZ8. Values are mean ± s.d. (n = 3 biological replicates). P values were determined by two-sided unpaired t-test, compared to TP309 at 0 hpi. c, Detection of ubiquitination of total proteins in TP309, UMP1R2115, and ko-11 plants. Seedlings of indicated lines were treated with/without MG132 (50 μM) for 12 h prior to M. oryzae infection. Samples were collected at 12 h post M. oryzae infection for western blot analysis with a ubiquitin antibody. Detection with a HSP antibody was used for loading normalization. d and e, GO enrichment analysis of differentially accumulated proteins (DAPs) between TP309 and UMP1R2115-1. Leaves from four-leaf-stage seedlings were used for iTRAQ proteomic analysis. Top ten enriched GO terms were presented for up-regulated (d) or down-regulated (e) DAPs. Rich factor refers to the ratio of DAP number against annotated protein number in background genome for a certain term. Term of interest was indicated by a red rectangle. Significance was determined by Fisher’s exact test (P < 0.05).
Extended Data Fig. 6 UMP1R2115 enhances defence responses against M. oryzae, which is inhibited by MG132.
a and b, Expression analysis of defence-related genes in leaves of TP309, UMP1R2115 (a), and mutant ko-11 (b) lines inoculated with M. oryzae strain GZ8. OsUbi was used as the reference gene. The expression of indicated genes in TP309-0 hpi (hours post inoculation) was set as the control. Data are mean ± s.d. (n = 3 biological replicates). P values were determined by two-sided unpaired t-test, compared to TP309 at corresponding time points. c and i, RT-qPCR analysis of OsUMP1 in leaves of R2115 (c) and LTH (i) inoculated with GZ8. OsUbi was used as the reference gene. d and j, Measurements of 26S proteasome amount in R2115 (d) and LTH (j). e and k, Measurements of 26S proteasome activity in R2115 (e) and LTH (k). f and l, Measurements of POD amount in R2115 (f) and LTH (l). g and m, Measurements of CAT amount in R2115 (g) and LTH (m). h and n, Quantification of H2O2 in R2115 (h) or LTH (n). Data are mean ± s.d. (n = 3 biological replicates for c–n). o, Measurements of 26S proteasome activity in leaves of UMP1R2115 transgenic lines infected with M. oryzae strain GZ8 at 5 dpi. MG132 treatment was conducted at 1 day before M. oryzae inoculation. p and q, Measurements of the abundance of POD (p) and CAT (q). r, Quantification of H2O2. Data are mean ± s.d. (n = 3 biological replicates for o–r). P values were determined by two-sided unpaired t-test, compared to corresponding water-treated controls (o–r).
Extended Data Fig. 7 Rice E3 ligase APIP6 promotes the degradation of OsCatB but not OsAPX8.
a, Sequence alignment of three rice catalases OsCatA, OsCatB, and OsCatC using DNAMAN software (version 5.2.2). Black shading sites indicate 100% homology, while turquoise shading sites indicate ≥50% homology. Note that the overall homology between OsCatB and OsCatC is 83.9%. b, Yeast-two-hybrid assay for detecting interaction of APIP6 with OsCatB/OsAPX8. DDO, SD/-Leu-Trp. QDO, SD/-Leu-Trp-His-Ade. c and d, Protein degradation assay in rice protoplasts. OsCatB-HA (c) or OsAPX8-HA (d) were co-expressed with APIP6-GFP or APIP6H58Y-GFP in rice protoplasts. The proteasome inhibitor MG132 (50 μM) was added at 12 h prior to sampling. LUC-Myc was used as an internal control.
Extended Data Fig. 8 Overexpressing OsAPX8 or OsCatB in UMP1R2115 plants and expression analysis of defence-related genes in UMP1R2115 and TP309 plants treated with chitin or flg22.
a and b, Expression level of OsAPX8 (a) or OsCatB (b) in UMP1R2115 plants overexpressing OsAPX8 or OsCatB. The expression level was determined by RT-qPCR using OsUbi as the reference gene and null segregant as the control. Values are mean ± s.d. (n = 3 biological replicates). P values were determined by two-sided unpaired t-test, compared to the null segregant. c and d, Expression analysis of defence-related genes in TP309 and UMP1R2115-1 plants treated with chitin (40 μg ml−1; c) or flg22 (20 nM; d). OsUbi was used as the reference gene. TP309-0 hpt (hours post treatment) was set as the control. Values are mean ± s.d. (n = 3 biological replicates).
Extended Data Fig. 9 UMP1R2115 enhances rice resistance to multiple pathogens and H2O2 accumulation, which is compromised by overexpression of OsAPX8 or OsCatB.
a-f, Disease phenotype, lesion length, and H2O2 quantification of leaves from TP309, UMP1R2115-1/13, and ko-11 plants infected with R. solani (a-c) or Xoo (d-f). g-l, Disease phenotype, lesion length, and H2O2 quantification of leaves from OsAPX8-overexpressing lines in UMP1R2115-1 background infected with R. solani (g-i) or Xoo (j-l). m-r, Disease phenotype, lesion length, and H2O2 quantification of leaves from OsCatB-overexpressing lines in UMP1R2115-1 background infected with R. solani (m-o) or Xoo (p-r). For R. solani assay, disease phenotype and lesion length were recorded at 2 days post inoculation (dpi), while H2O2 quantification was performed at 1 dpi. For Xoo assay, disease phenotype and lesion length were recorded at 14 dpi, while H2O2 quantification was performed at 2 dpi. Values are mean ± s.d. (n = 5 biological replicates for b, h, n and 3 biological replicates for c, f, i, l, o, r). P values were determined by two-sided unpaired t-test, compared to TP309 (b, c, e, f) or null segregants (h, i, k, l, n, o, q, r).
Extended Data Fig. 10 Natural variation of OsUMP1 and SNPs/InDels affecting OsUMP1 expression.
a, Natural variation analysis of OsUMP1 using DNA polymorphism data among over 5000 rice accessions in MBKbase (http://mbkbase.org/rice). Using default parameters, we identified 157 major haplotypes for 2.5-kb promoter plus the gene region of OsUMP1 (Supplementary Table 8). There were four SNPs/InDels in the coding region and 42 SNPs/InDels in the promoter. Note that R2115-specific SNPs and InDels were marked in red. Sequences around the eleven specific SNPs/InDels (S1~S11) were analyzed for cis-elements, which are presented in Supplementary Table 9. To identify SNPs/InDels responsible for up-regulation of OsUMP1 in R2115, a series mutations were generated in the promoter of UMP1R2115 (m1~m7). b, Dual Firefly/Renilla luciferase reporter assay. Different versions of the OsUMP1 promoter were ligated with the Firefly luciferase-encoding region and separately co-expressed with 35S-Renilla luciferase in Nicotiana benthamiana leaves. Firefly and Renilla luciferases activities were measured from leaves treated with/without chitin (50 μg ml−1). Values are mean ± s.d. (n = 3 biological replicates). P values were determined by two-sided unpaired t-test, compared to R2115.
Supplementary information
Supplementary Tables 1–10
Supplementary Table 1. Mapping results of RNA-seq reads. Supplementary Table 2. The complete list of DEGs from rice in response to Magnaporthe oryzae infection (the data represent normalized fragments per kilobase of exon per million fragments mapped). Supplementary Table 3. Gene Ontology enrichment analysis for genes in subcluster 3. Supplementary Table 4. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for genes in subcluster 3. Supplementary Table 5. Expression data of subcluster 3 genes involved in programmed cell death and proteasome. Supplementary Table 6. Proteomic analysis of UMP1R2115-1 transgenic plants compared with wild-type TP309. Supplementary Table 7. GO enrichment analysis for differentially accumulated proteins in UMP1R2115-1 compared with TP309. Supplementary Table 8. Natural variation of OsUMP1 in rice populations. Supplementary Table 9. Cis-elements located around 11 R2115-specific SNPs/InDels in the OsUMP1 promoter. Supplementary Table 10. Primers used in this study.
Source data
Source Data Extended Data Fig. 5
Unprocessed western blots.
Source Data Extended Data Fig. 7
Unprocessed western blots.
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Hu, XH., Shen, S., Wu, JL. et al. A natural allele of proteasome maturation factor improves rice resistance to multiple pathogens. Nat. Plants 9, 228–237 (2023). https://doi.org/10.1038/s41477-022-01327-3
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DOI: https://doi.org/10.1038/s41477-022-01327-3
