
An erad-related ubiquitin-conjugating enzyme boosts broad-spectrum disease resistance and yield in rice
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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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access
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Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A NATURAL ALLELE OF PROTEASOME MATURATION FACTOR IMPROVES RICE RESISTANCE TO MULTIPLE PATHOGENS Article 16 January 2023
PHYTOALEXIN SAKURANETIN ATTENUATES ENDOCYTOSIS AND ENHANCES RESISTANCE TO RICE BLAST Article Open access 23 April 2024 GENETICALLY-ENCODED TARGETED PROTEIN DEGRADATION TECHNOLOGY TO REMOVE
ENDOGENOUS CONDENSATION-PRONE PROTEINS AND IMPROVE CROP PERFORMANCE Article Open access 29 January 2025 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. REFERENCES * Dean, R. et al. The top 10 fungal pathogens in molecular
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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.). AUTHOR
INFORMATION AUTHORS AND AFFILIATIONS * MOA Key Lab of Pest Monitoring and Green Management and Frontiers Science Center for Molecular Design Breeding, China Agricultural University, Beijing,
China Yu Wang, Jiaolin Yue, Nan Yang, Chuan Zheng, Yunna Zheng, Xi Wu, Jun Yang, Vijai Bhadauria, Wensheng Zhao, You-Liang Peng & Qian Chen * Peking University Institute of Advanced
Agricultural Sciences, Weifang, China Huawei Zhang * School of Life Sciences, Shandong University, Qingdao, China Lijing Liu * State Key Laboratory for Biology of Plant Diseases and Insect
Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China Yuese Ning * State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental
Biology, the Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, China Qi Xie * University of Chinese Academy of Sciences, Beijing, China Qi Xie Authors * Yu Wang View
author publications You can also search for this author inPubMed Google Scholar * Jiaolin Yue View author publications You can also search for this author inPubMed Google Scholar * Nan Yang
View author publications You can also search for this author inPubMed Google Scholar * Chuan Zheng View author publications You can also search for this author inPubMed Google Scholar *
Yunna Zheng View author publications You can also search for this author inPubMed Google Scholar * Xi Wu View author publications You can also search for this author inPubMed Google Scholar
* Jun Yang View author publications You can also search for this author inPubMed Google Scholar * Huawei Zhang View author publications You can also search for this author inPubMed Google
Scholar * Lijing Liu View author publications You can also search for this author inPubMed Google Scholar * Yuese Ning View author publications You can also search for this author inPubMed
Google Scholar * Vijai Bhadauria View author publications You can also search for this author inPubMed Google Scholar * Wensheng Zhao View author publications You can also search for this
author inPubMed Google Scholar * Qi Xie View author publications You can also search for this author inPubMed Google Scholar * You-Liang Peng View author publications You can also search for
this author inPubMed Google Scholar * Qian Chen View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS 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. CORRESPONDING AUTHORS Correspondence to Qi Xie,
You-Liang Peng or Qian Chen. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Food_ thanks Tsutomu Kawasaki and
the anonymous reviewers for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations. 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 REPORTING SUMMARY SUPPLEMENTARY TABLE List of primer sequences (5′→3′)
used in this study. SOURCE DATA STATISTICAL SOURCE DATA All statistical source data. SOURCE DATA OF GELS OR BLOTS All unprocessed western blots and/or gels. RIGHTS AND PERMISSIONS Springer
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CITE THIS ARTICLE 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).
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