Abstract
Rice is a staple crop for over half of the global population. However, blast disease caused by Magnaporthe orzae can result in more than a 30% loss in rice yield in epidemic years. Although some major resistance genes bolstering blast resistance have been identified in rice, their stacking in elite cultivars usually leads to yield penalties. Here we report that OsUBC45, a ubiquitin-conjugating enzyme functioning in the endoplasmic reticulum-associated protein degradation system, promotes broad-spectrum disease resistance and yield in rice. OsUBC45 is induced upon infection by M. oryzae, and its overexpression enhances resistance to blast disease and bacterial leaf blight by elevating pathogen-associated molecular pattern-triggered immunity (PTI) while nullifying the gene-attenuated PTI. The OsUBC45 overexpression also increases grain yield by over 10%. Further, OsUBC45 enhances the degradation of glycogen synthase kinase 3 OsGSK3 and aquaporin OsPIP2;1, which negatively regulate the grain size and PTI, respectively. The OsUBC45 reported in our study has the potential for improving yield and disease resistance for sustainable rice production.
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Data availability
Gene sequence information of rice from this study can be found at https://www.ricedata.cn/gene/, under the following accession numbers: OsUBC45, LOC_Os03g19500; OsPIP2;1, LOC_Os07g26690; OsGSK3, LOC_Os02g14130; OsBiP5, LOC_Os08g09770; OsCNX, LOC_Os04g32950; OsPDIL 2-1, LOC_Os05g06430; OsDOA10B, LOC_Os08g01040. Source data are provided with this paper.
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Acknowledgements
We thank L. Luo from Shanghai Agrobiological Gene Center, China, for providing the CRISPR/Cas9-knockout lines and overexpression lines of OsPIP2;1. We also thank J. Huang from Nanjing Agricultural University for providing the OsGSK3 antibody. This work is supported by grants from the National Natural Science Foundation of China (32293244 to Y.-L.P.; 32072368 to Q.C.), the National Rice Industry Program from the Ministry of Agriculture and Rural Affairs (CARS-01-36 to Y.-L.P.), the 111 Project (grant no. B13006 to Y.-L.P.) from the Ministry of Education, the Staple Crop Disease Resistance Breeding Program from China Agricultural University (Y.-L.P.) and Pinduoduo-China Agricultural University Research Fund (Y.-L.P.).
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Q.C. and Y.-L.P. designed the research. Y.W. performed most of the experiments. J. Yue, N.Y., Y.Z., C.Z., X.W., J. Yang and W.Z. contributed to the assays of rice protoplasts, western blots and plant inoculation. Q.C., Y.-L.P., V.B., Y.W. and Q.X. wrote the manuscript. Y.N., L.L. and H.Z. participated in the discussion of the results and modification of manuscript.
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Extended data
Extended Data Fig. 1 UPR is induced by chitin treatment.
The expression of UPR genes was induced by chitin treatment. Ten-day-old WT seedlings (ZH11) were treated with ddH2O or 10 μg ml−1 chitin for 6 h. The expression of the UPR genes was determined by qPCR. Data are presented as mean values +/− SEM, n = 3 replications. Asterisks indicate significant differences evaluated by two-tailed Student’s t test analysis, *p < 0.05, **p < 0.01. For exact p values, refer to Source Data.
Extended Data Fig. 2 Analysis of the evolutionary tree and tissue-specific expression of OsUBC45.
a, Schematic of OsUBC45. A UBCc domain (yellow) and a transmembrane domain (blue) are predicted in OsUBC45. The red bar is a ER membrane retention signal IEGK as predicted by the psort II program. b, The neighbor-joining phylogenetic tree of OsUBC45 and its orthologues from human and Arabidopsis. Bootstrap values from 1000 replicates are indicated at each node; the scale represents branch length. c, The expression level of OsUBC45 in different tissues was determined by qPCR. The individual tissues came from the rice in the filling stage. Data are presented as mean values +/− SEM, n = 3 replications. For exact p values, refer to Source Data. d, OsUBC45 interacted with OsDOA10B (LOC_Os08g01040) in a yeast two-hybrid assay. OsUBC45 was inserted into pPR3-N, while OsDOA10B was inserted into pBT3-STE. The transformants were grown on SD-Leu-Trp (SD-LW) and SD-Leu-Trp-His-Ade (SD-LWHA) plates containing 1 mM 3-AT. e, Ubiquitin-conjugating enzyme activity of His-MBP-OsUBC45. The OsUBC45-ubiquitin adduct (indicated by arrow) were detected using both anti-MBP and anti-ubiquitin antibodies.
Extended Data Fig. 3 Rice yield and grain size were reduced in the osubc45 mutants.
a, Morphology of the WT and osubc45 mutant plants at the mature stage. Bar = 5 cm. b, Plant height of the WT and osubc45 mutant plants. Data are presented as mean values +/− SEM, n = 5 independent plants. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test analysis, **p < 0.01. c, Number of panicles per WT and osubc45 mutant plants. Data are presented as mean values +/− SEM, n = 5 independent hills. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test analysis, *p < 0.05, **p < 0.01. d, Panicle length of WT and osubc45-9 and osubc45-23. Data are presented as mean values +/− SEM, n = 10 independent panicles. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test, *p < 0.05 and **p < 0.01. e-f, Primary branches per panicle (e) and secondary branches per panicle (f) of WT and osubc45 mutants. Data are presented as mean values +/− SEM, n = 10 independent panicles. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test, **p < 0.01. g, Grain number per panicle of WT, osubc45-9 and osubc45-23. Data are presented as mean values +/− SEM, n = 10 independent panicles. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test, **p < 0.01. h-i, Grain length (h) and Grain width (i) of WT and osubc45 mutant plants. Data are presented as mean values +/− SEM, n = 10 grains, 3 replications were performed. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test (**p < 0.01). For exact p values, refer to Source Data.
Extended Data Fig. 4 The osubc45 mutants were sensitive to rice blast by spray inoculation.
a, The osubc45 mutants increased the susceptibility to rice blast by spray inoculation. The WT and osubc45 mutant plants were spray-inoculated with the virulent isolate RB22. Leaves were photographed at 7 dpi. b, Lesion number of the leaves was measured at 7 dpi. Data are presented as mean values +/− SEM, n = 3 replications. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test, **p < 0.01. For exact p values, refer to Source Data.
Extended Data Fig. 5 Effects of overexpression of OsUBC45 on rice growth and yield.
a, Morphology of WT and OsUBC45 overexpression lines at maturity. Bar = 5 cm. b, Plant height of the WT and OsUBC45 overexpression plants. Data are presented as mean values +/− SEM, n = 5 independent plants. c, Panicle number per plant of (WT and OsUBC45 transgenic lines). Data are presented as mean values +/− SEM, n = 5 independent plants. d, Primary branches per panicle of WT and OsUBC45 transgenic lines. e, Secondary branches per panicle of WT and OsUBC45 transgenic plants. (d)-(e) Data are presented as mean values +/− SEM, n = 10 independent panicles (*p < 0.05, **p < 0.01). For exact p values, refer to Source Data.
Extended Data Fig. 6 OsUBC45 enhanced resistance to rice blast by spray inoculation.
a, OsUBC45 transgenic plants increased resistance to rice blast by spray inoculation. WT and OsUBC45 transgenic plants were sprayed with virulent isolate RB22. Leaves were photographed at 7 dpi. b, Lesion number of the inoculated leaves was measured at 7 dpi. Data are presented as mean values +/− SEM, n = 3 replications. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test, **p < 0.01. c, Lesion length of the drop inoculation in Fig. 4d. Data are presented as mean values +/− SEM, n = 6 independent lesions. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test, **p < 0.01. For exact p values, refer to Source Data.
Extended Data Fig. 7 DGS1 interacted with OsGSK3 and promoted its degradation.
a, Proteins that were identified to interact with OsUBC45 by yeast two-hybrid screening. b, OsUBC45 interacts with OsGSK3 by LCI assay. c, The expression of OsGSK3 in WT, osubc45 mutants and OsUBC45 overexpression lines were determined by qPCR. Data are presented as mean values +/− SEM (n = 3 replications). d, Chitin treatment did not affect the degradation of OsGSK3. Protein levels of OsGSK3 in WT, osubc45 mutants and OsUBC45-OE plants were detected using anti-OsGSK3 antibody with/without chitin treatment. e, DGS1 interacted with OsGSK3 in yeast. DGS1 was inserted into pBT3-STE, while OsGSK3 was inserted into pPR3-N. The transformants were grown on SD-LW and SD-LWHA plates containing 2 mM 3-AT. f, DGS1 interacted with OsGSK3 by LCI assay. g, DGS1 promoted the degradation of OsGSK3-HA-Nluc in rice protoplasts. Different combinations of plasmids were transformed into rice protoplasts of WT. Proteins from the rice protoplasts were used for protein gel blot analysis.
Extended Data Fig. 8 DGS1 interacted with OsPIP2;1 and promoted its degradation.
a, OsUBC45 interacted with OsPIP2;1 according to a yeast two-hybrid assay. OsUBC45 was inserted into pBT3-STE, while OsPIP2;1 was inserted into pPR3-N. The transforms were grown on SD-LW and SD-LWHA plates containing 3 mM 3-AT. b, The expression of OsPIP2;1 in WT, osubc45 mutants and OsUBC45 overexpression lines were determined by qPCR. Data are presented as mean values +/− SEM, n = 3 replications. c, DGS1 interacted with OsPIP2;1 in yeast two-hybrid assays. DGS1 was inserted into pBT3-STE, while OsPIP2;1 was inserted into pPR3-N. The transformants were grown on SD-LW and SD-LWHA plates containing 2 mM 3-AT. d, DGS1 associated with OsPIP2;1 in the LCI assay. e, DGS1 negatively regulated the stability of OsPIP2;1-Myc in rice protoplasts. Different combinations of plasmids were used to transform rice protoplasts of WT. Proteins isolated from the rice protoplasts were used for western blot analysis.
Extended Data Fig. 9 OsPIP2;1 increased susceptibility to bacterial blight.
a, The expression of OsPIP2;1 is inhibited by M. oryzae treatment. The WT plants were inoculated with the M. oryzae virulent isolate RB22 for the corresponding times. Data are presented as mean values +/− SEM, n = 3 replications. Asterisks indicate significant differences evaluated by two-sided Student’s t-tests, **p < 0.01. b, OsPIP2;1 negatively regulated bacterial blight resistance. Leaves were photographed at 14 dpi. c, Lesion length of bacterial blight in leaves (b). Data are presented as mean values +/− SEM, n = 3 independent lesions. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test (*p < 0.05, **p < 0.01). For exact p values, refer to Source Data.
Extended Data Fig. 10 OsUBC45 positively regulates disease resistance by accumulating more ROS under M. oryzae treatment.
a, H2O2 contents of WT, osubc45-9 and OsUBC45-OE2 seedlings after inoculation with M. oryzae for 24 h. Seedlings treated with 0.02% Tween-20 served as mock controls. Data are presented as mean values +/− SEM, n = 3 replications. Asterisks indicate significant differences evaluated by two-sided Student’s t-tests, **p < 0.01. FW, fresh weight. b, DAB staining of different rice leaves infected with M. oryzae for 48 h. H2O2 accumulation was shown as dark-brown spots. c, Relative DAB staining intensity was measured by Image J. Data are presented as mean values +/− SEM, n = 3 independent leaves. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test, **p < 0.01. d, AUR, AR and H2DCF-DA probing of H2O2 in leaf sheath cells of WT, osubc45-9 and OsUBC45-OE2 plants infected with M. oryzae for 48 h. Bars = 50 μm. e–g, Statistical analysis of H2O2 accumulation in (d). Fluorescence intensity of AUR (e). Fluorescence intensity of AR (f). Fluorescence intensity of H2DCF-DA (g). Data are presented as mean values +/− SEM, n = 5 independent views. Asterisks indicate significant differences evaluated by two-tailed Student’s t-test, **p < 0.01. For exact p values, refer to Source Data.
Supplementary information
Supplementary Table
List of primer sequences (5′→3′) used in this study.
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Wang, Y., Yue, J., Yang, N. et al. An ERAD-related ubiquitin-conjugating enzyme boosts broad-spectrum disease resistance and yield in rice. Nat Food 4, 774–787 (2023). https://doi.org/10.1038/s43016-023-00820-y
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DOI: https://doi.org/10.1038/s43016-023-00820-y
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