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uORF-mediated translation allows engineered plant disease resistance without fitness costs

Abstract

Controlling plant disease has been a struggle for humankind since the advent of agriculture. Studies of plant immune mechanisms have led to strategies of engineering resistant crops through ectopic transcription of plants’ own defence genes, such as the master immune regulatory gene NPR1 (ref. 1). However, enhanced resistance obtained through such strategies is often associated with substantial penalties to fitness2, making the resulting products undesirable for agricultural applications. To remedy this problem, we sought more stringent mechanisms of expressing defence proteins. On the basis of our latest finding that translation of key immune regulators, such as TBF1 (ref. 3), is rapidly and transiently induced upon pathogen challenge (see accompanying paper4), we developed a ‘TBF1-cassette’ consisting of not only the immune-inducible promoter but also two pathogen-responsive upstream open reading frames (uORFsTBF1) of the TBF1 gene. Here we demonstrate that inclusion of uORFsTBF1-mediated translational control over the production of snc1-1 (an autoactivated immune receptor) in Arabidopsis thaliana and AtNPR1 in rice enables us to engineer broad-spectrum disease resistance without compromising plant fitness in the laboratory or in the field. This broadly applicable strategy may lead to decreased pesticide use and reduce the selective pressure for resistant pathogens.

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Figure 1: uORFsTBF1-mediated translational and TBF1 promoter-mediated transcriptional regulation.
Figure 2: Effects of controlling transcription and translation of snc1 in Arabidopsis.
Figure 3: Effects of controlling transcription and translation of AtNPR1 in rice.

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Acknowledgements

This study was supported by grants from National Institutes of Health 5R01 GM069594-11 and Howard Hughes Medical Institute and Gordon and Betty Moore Foundation (through grant GBMF3032) to X.D., National Natural Science Foundation of China (31371926) to M.Y., and the National Key Research and Development Program of China (2016YFD0100903) to S.W. We thank J. Motley, J. Marqués, P. Zwack and S. Zebell for comments on this manuscript.

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Authors

Contributions

G.X. and X.D. designed the research. G.X. performed the Arabidopsis-related experiments with help from E.Z. for fitness tests. L.L. isolated snc1 genomic DNA. S.K. maintained Hpa Noco2 strain in the laboratory and helped with inoculation; M.Y., C.A. and G.X. performed and S.W. supervised the rice-related experiments. G.X. and X.D. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Xinnian Dong.

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Competing interests

A patent based on this study has been filed by Duke University with G.X. and X.D. as inventors.

Additional information

Reviewer Information Nature thanks J. Bailey-Serres, P. Rushton and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Conservation of uORF2TBF1 nucleotide and peptide sequences in plant species.

a, Schematic of TBF1 mRNA structure. The 5′ leader sequence contains two uORFs, uORF1 and uORF2. CDS, coding sequence. bd, Alignment of uORF2 nucleotide sequences (b) and alignment (c) and phylogeny (d) of uORF2 peptide sequences in different plant species. The corresponding triplets encoding the conserved amino acids among these species are underlined. Identical residues (black background), similar residues (grey background), and missing residues (dashes) were identified using Clustlw2. At (Arabidopsis thaliana; AT4G36988), Pv (Phaseolus vulgaris; XP_007155927), Gm (Glycine max; XP_006600987), Gr (Gossypium raimondii; CO115325), Nb (Nicotiana benthamiana; CK286574), Ca (Cicer arietinum; XP_004509145), Pd (Phoenix dactylifera; XP_008797266), Ma (Musa acuminata subsp. Malaccensis; XP_009410098), Os (O. sativa; Os09g28354).

Extended Data Figure 2 Characterization of uORFsTBF1 and uORFsbZIP11 in translational control.

Related to Fig. 1. a, b, Subcellular localization of the LUC–YFP fusion (a) and GFPER (b). SP, signal peptide from Arabidopsis basic chitinase; HDEL, ER retention signal. Representative of eight images. Scale bar, 10 μm. ce, mRNA levels of LUC in (Fig. 1b; n = 3), GFPER in (Fig. 1c; n = 4), and TBF1–YFP in (Fig. 1d; n = 3) 2 d.p.i. before cell death was observed in plants expressing TBF1. f, Schematics of the 5′ leader sequences used in studying the translational activities of WT uORFsbZIP11, mutant uorf2abZIP11 (ATG to CTG), or uorf2bbZIP11 (ATG to TAG). gi, uORFsbZIP11-mediated translational control of cytosol-synthesized LUC (g; chemiluminescence with pseudo-colour); ER-synthesized GFPER (h; fluorescence under ultraviolet light); and cell death induced by overexpression of TBF1–YFP fusion (i; cleared using ethanol) after transient expression in N. benthamiana for 2 days (g, h) and 3 days (i), respectively. Representative of four images. jl, mRNA levels of LUC in (g; n = 2 experiments with three technical replicates), GFPER in (h; n = 3 experiments with three technical replicates), and TBF1–YFP in (i; n = 3 experiments with three technical replicates). m, Translational efficiency changes in LUC controlled by the 5′ leader sequence containing WT uORFsbZIP11, mutant uorf2abZIP11, or uorf2bbZIP11 in response to elf18 in N. benthamiana. Mean of the LUC/RLUC activity ratios (n = 12). n, LUC/RLUC mRNA changes in m. Mean of LUC/RLUC mRNA normalized to Mock from two experiments with three technical replicates. Bar with solid circles, mean with individual biological replicates.

Source data

Extended Data Figure 3 Three developmental phenotypes observed in primary Arabidopsis transformants expressing snc1.

The three developmental phenotypes observed in T1 (that is, the first generation) Arabidopsis transgenic lines carrying 35S:uorfsTBF1-snc1, 35S:uORFsTBF1-snc1, TBF1p:uorfsTBF1-snc1, and TBF1p:uORFsTBF1-snc1 (above). Representative of five images. Fisher’s exact test was used for the pairwise statistical analysis (below). Different letters in ‘Total’ indicate significant differences between type III versus type I + type II (P < 0.01).

Extended Data Figure 4 Effects of controlling transcription and translation of snc1 on defence and fitness in Arabidopsis.

Related to Fig. 2. a, b, Psm ES4326 growth in WT, snc1, transgenic line numbers 1–4 after inoculation by spray (a) or infiltration (b). Mean ± s.e.m. c, Hpa Noco2 growth as measured by spore counts 7 d.p.i. Mean ± s.e.m. dg, Analyses of plant radius (d), fresh weight (e), silique number (f), and total seed weight (g). Mean ± s.e.m. h, i, Relative levels of Psm ES4326-induced snc1 protein (h; numbers below immunoblots; see Supplementary Fig. 1 for gel source data) and mRNA (i; mean from two experiments with three technical replicates). Solid circles, individual biological replicates. Numbers 1–4, four independent transgenic lines carrying TBF1p:uORFsTBF1-snc1 with 1 and 2 shown in Fig. 2. h.p.i., hours after Psm ES4326 infection; CBB, Coomassie brilliant blue. See Source Data for sample size (n). Different letters above bars indicate significant differences (P < 0.05).

Source data

Extended Data Figure 5 Functionality of uORFsTBF1 in rice.

a, b, LUC activity (a) and mRNA levels (b) in three independent primary transgenic rice lines (called ‘T0’ in rice research) carrying 35S:uorfsTBF1-LUC and 35S:uORFsTBF1-LUC. Mean of LUC activities (RLU, relative light unit) of three biological replicates. Solid circles, individual biological replicates; and mean of LUC mRNA levels of three technical replicates after normalization to the 35S:uorfsTBF1-LUC line 1. c, Representative lesion mimic disease (LMD) phenotypes (above) and percentage of AtNPR1-transgenic rice plants showing lesion mimic disease in the second generation (T1) grown in the growth chamber (below).

Source data

Extended Data Figure 6 Effects of controlling transcription and translation of AtNPR1 on defence in T0 rice.

Related to Fig. 3. ad, Lesion length measurements after infection by Xoo strain PXO347 in primary transformants (T0) for 35S:uorfsTBF1-AtNPR1 (a), 35S:uORFsTBF1-AtNPR1 (b), TBF1p:uorfsTBF1-AtNPR1 (c), and TBF1p:uORFsTBF1-AtNPR1 (d). Lines further analysed in T1 and T2 are circled. e, Average leaf lesion lengths. WT, recipient O. sativa cultivar ZH11. Mean ± s.e.m. Different letters above bars indicate significant differences (P < 0.05). See Source Data for sample size (n).

Source data

Extended Data Figure 7 Effects of controlling transcription and translation of AtNPR1 on defence in T1 rice.

Related to Fig. 3. a, b, Representative symptoms observed in T1 AtNPR1-transgenic rice plants grown in the greenhouse (a) after Xoo inoculation and corresponding leaf lesion length measurements (b). PCR was performed to detect the presence (+) or the absence (−) of the transgene gene. c, Quantification of leaf lesion length of four lines for Xoo inoculation in field-grown T1 AtNPR1-transgenic rice plants. Mean ± s.e.m. See Source Data for sample size (n). Different letters above bars indicate significant differences (P < 0.05). d, e, Relative levels of AtNPR1 mRNA (d) and protein (e; numbers below immunoblots; see Supplementary Fig. 1 for gel source data) in response to Xoo infection. Mean of AtNPR1 mRNA levels of three technical replicates after normalization to 0 h.p.i. (d). Solid circles, individual biological replicates.

Source data

Extended Data Figure 8 Effects of controlling transcription and translation of AtNPR1 on fitness in T1 rice under field conditions.

Related to Fig. 3. Mean ± s.e.m. See Source Data for sample size (n). Different letters above bars indicate significant differences among constructs (P < 0.05).

Source data

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This file contains the uncropped immunoblots. (PDF 225 kb)

Supplementary Table 1

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Xu, G., Yuan, M., Ai, C. et al. uORF-mediated translation allows engineered plant disease resistance without fitness costs. Nature 545, 491–494 (2017). https://doi.org/10.1038/nature22372

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