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Natural variation in ZmFBL41 confers banded leaf and sheath blight resistance in maize

Abstract

Rhizoctonia solani is a widely distributed phytopathogen that causes banded leaf and sheath blight in maize and sheath blight in rice. Here, we identified an F-box protein (ZmFBL41) that confers resistance to banded leaf and sheath blight through a genome-wide association study in maize. Rice overexpressing ZmFBL41 showed elevated susceptibility to R. solani. Two amino acid substitutions in this allele prevent its interaction with ZmCAD, which encodes the final enzyme in the monolignol biosynthetic pathway, resulting in the inhibition of ZmCAD degradation and, consequently, the accumulation of lignin and restriction of lesion expansion. Knocking out the ZmCAD-homologous gene OsCAD8B in rice enhanced susceptibility to R. solani. The results reveal a susceptibility mechanism in which R. solani targets the host proteasome to modify the secondary metabolism of the plant cell wall for its invasion. More importantly, it provides an opportunity to generate R. solani–resistant varieties of different plant species.

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Fig. 1: GWAS for BLSB resistance in maize.
Fig. 2: Natural variations in ZmFBL41 were significantly associated with maize resistance to R. solani.
Fig. 3: Function of ZmFBL41 in BLSB resistance.
Fig. 4: ZmFBL41B73 targets and triggers degradation of ZmCAD.
Fig. 5: Function of CAD in R. solani resistance.
Fig. 6: Two amino acid substitutions in the LRR domain of ZmFBL41Chang7-2 stabilize ZmCAD by blocking protein–protein interactions.
Fig. 7: A model for ZmFBL41-mediated BLSB resistance.

Data availability

Data supporting the findings of this work are available within the paper and its Supplementary Information. All materials used in this study have been described in previous studies8,33,34. The RNA-sequencing data for all 368 inbred lines have been deposited in the NCBI Sequence Read Archive under accession code SRP026161. The genotype after imputation can be downloaded from http://www.maizego.org/Resources.html. The widely used mapping population, although not owned by us, can—to our knowledge—be accessed by any researcher through appropriate application. Most of the data are from the GenBank of CIMMYT (details in Supplementary Table 1), which are public available, others can be obtained from the National GenBank, Institute of Crop Germplasm Resources of Chinese Academy of Agricultural Sciences (CAAS, http://www.cgris.net) with an MTA. The researchers can also contact the founders of lines of the mapping population (J.Y., yjianbing@mail.hzau.edu.cn). Source data for Figs. 3a,c, 5,a,c,e and Supplementary Figs. 1a, 2c, 6e,f have been provided in Supplementary Table 8.

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Acknowledgements

We thank B. Liu and Y. Zhang from Shandong Agricultural University for seed propagation of maize transposon-insertion lines. This study was supported by the National Key Research and Development Program of China (2016YFD0101003 and 2016YFD0100903), the National Natural Science Foundation of China (31601279), the Key Research and Development Program of Shandong Province (2017GNC10104 and 2018GNC110018) and the Shandong Modern Agricultural Technology and Industry system (SDAIT-17-06).

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N.L. inoculated the maize population, resequenced ZmFBL41, carried out the functional analysis of ZmFBL41 and ZmCAD, screened ZmFBL41-interacting proteins, carried out the expression analysis of ZmFBL41 and PR genes, measured the lignin content and wrote the paper. B.L. studied the degradation of ZmCAD and measured the lignin content. H.W. analyzed the GWAS data. X.L. inoculated the maize population. F.Y. studied the degradation of ZmCAD and constructed the OsCAD8B knockout line. X.D. analyzed the raw data. J.Y. provided the maize inbred lines and SNP data platform. Z.C. designed the experiments and wrote the paper.

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Correspondence to Zhaohui Chu.

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Integrated supplementary information

Supplementary Fig. 1 Phenotypic variation in maize BLSB resistance in the natural-variation population.

(a) Distribution of lesion length in different maize inbred lines inoculated with R. solani. (b) Box-plot of lesion lengths from different origins. Center values are medians, solid lines indicate variability outside the upper and lower quartiles, and dots denote outliers. (c) Quantile-quantile plot for the GWAS under a general linear model (GLM). Statistical significance was determined by a two-sided t-test. TST, tropical or subtropical lines; NSS, non-stiff stalk; SS, stiff stalk.

Supplementary Fig. 2 Correlation analysis between lesion length and disease index from 10 maize lines inoculated with R. solani.

(a) Disease symptoms of SH and RH lines after inoculation with R. solani in the field. Scale bars, 10 cm. (b) Lesion length measured at 7 dpi in the field. Five SH and five RH lines were used and 5 plants of each line were measured. Independent experiments were repeated three times. (c) Disease index counted at 14 dpi in the field. Five SH and five RH lines were used and 10 plants of each line were measured. Independent experiments were repeated five times. In box plots, center values are medians, solid lines indicate variability outside the upper and lower quartiles, and dots denote outliers.

Supplementary Fig. 3 Structure and subcellular localization of ZmFBL41.

(a) A schematic diagram of the ZmFBL41 protein. The F-box and LRR domains are indicated. (b) ZmFBL41 is localized to the cytoplasm of N. benthamiana leaf cells. Scale bars, 50 μm. Localization is representative of three independent experiments.

Supplementary Fig. 4 Evaluation of ZmFBL41 overexpression lines and transposon-insertion line.

(a) Schematic of Mutator insertion relative to ZmFBL41 in maize line W22. Protein-coding regions are indicated as red bars. UTRs are indicated as white bars. The Mutator insertion is indicated by a blue triangle. The primers (F1, R1 and TIR-F) used for PCR of ZmFBL41 are indicated. (b) Identification of the zmfbl41 transposon-insertion line by PCR. See also Supplementary Fig. 8h. (c) Detection of ZmFBL41 gene expression in the zmfbl41 insertion line by qRT-PCR. In box plots, center values are medians and solid lines indicate variability outside the upper and lower quartiles. Statistical significance was determined by a two-sided t-test. Independent experiments were repeated five times. (d) Identification of ZmFBL41-overexpressing lines by RT-PCR. Gel is representative of three independent experiments. See also Supplementary Fig. 8i.

Supplementary Fig. 5 ZmFBL41 interacts with ZmSKP1 protein.

(a) A schematic diagram of ZmFBL41B73 and the truncations used for constructing bait vectors. (b) Y2H assay indicates that the interaction of ZmFBL41B73 and ZmSKP1-1 depends on the F-box domain. -WL, medium lacking Trp and Leu, -WLHA, medium lacking Trp, Leu, His and adenine. The images are representative of three independent experiments. (c) ZmFBL41B73 interacts with ZmSKP1-1 in a Co-IP assay. The ZmFBL41B73-HA or ZmFBL41B73ΔLRR-HA and ZmSKP1-1-Myc proteins were co-expressed in N. benthamiana leaves. Co-IP was carried out with an antibody to Myc, and the proteins were analysed by using Western blotting with the anti-HA (for ZmFBL41B73 and ZmFBL41B73ΔLRR), and anti-Myc (for ZmSKP1-1) antibodies. Blot is representative of three independent experiments. See also Supplementary Fig. 8j. (d) BiFC assay shows the ZmFBL41B73-ZmSKP1-1 interaction in N. benthamiana leaf cells. Scale bars, 50 μm. The images are representative of three independent experiments.

Supplementary Fig. 6 Identification of zmcad transposon-insertion line and OsCAD8B knockout transgenic lines.

(a) Schematic of Mutator insertion relative to ZmCAD in maize line W22. Protein-coding regions are indicated by red bars. The Mutator insertion is indicated by a blue triangle. The primers (F2, R2 and TIR-F) used for PCR of ZmCAD are indicated. (b) Identification of the zmcad transposon-insertion line by PCR. See also Supplementary Fig. 8k. (c) The target site designed to knock out the OsCAD8B gene by the CRISPR/Cas9 system. (d) Verification of knockout lines by PCR-based sequencing. A transgenic line (abbreviated as OsCAD8BKO) for OsCAD8B knockout was generated from the Zhonghua 11 genetic background. (e) Comparison of the plant height between the wild-type and the OsCAD8BKO line. 10 single 10-day-old seedlings of WT and OsCAD8BKO were measured. Scale bars, 4 cm. Independent experiments were repeated three times. (f) Determination of lignin content in the OsCAD8BKO line. 10 single plants of WT and OsCAD8BKO were measured. Independent experiments were repeated three times. In box plots, center values are medians and solid lines indicate variability outside the upper and lower quartiles. Statistical significance was determined by a two-sided t-test.

Supplementary Fig. 7 Sequence comparison of the LRR domains of ZmFBL41 between B73 and Chang7-2.

(a) Comparison of the B73 and Chang7-2 LRR domain CDS sequences. (b) Amino acid sequences of the LRR domains of B73 and Chang7-2. Identical bases are highlighted in black.

Supplementary Fig. 8 Raw blot and gel images related to main figures and Supplementary Figs.

(a) Western blots related to Fig. 4b. (b) Western blots related to Fig. 4d. (c) Western blots related to Fig. 4e. (d) Western blots related to Fig. 4f. (e) Western blots related to Fig. 6b. (f) Western blots related to Fig. 6c. (g) Western blots related to Fig. 6d. (h) Gel images related to Supplementary Fig. 4b. (i) Gel images related to Supplementary Fig. 4d. (j) Western blots related to Supplementary Fig. 5c. (k) Gel images related to Supplementary Fig. 6b.

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Li, N., Lin, B., Wang, H. et al. Natural variation in ZmFBL41 confers banded leaf and sheath blight resistance in maize. Nat Genet 51, 1540–1548 (2019). https://doi.org/10.1038/s41588-019-0503-y

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