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 fitness, 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 paper), we developed a 'TBF1-cassette' consisting of not only the immune-inducible promoter but also two pathogen-responsive upstream open reading frames (uORFs_TBF1) of the TBF1 gene. Here we demonstrate that inclusion of uORFs_TBF1-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.

Extended Data Figure 1. Conservation of uORF2_TBF1 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. b–d, 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 (Oryza sativa; Os09g28354).

Extended Data Figure 2. Characterization of uORFs_TBF1 and uORFs_bZIP11 in translational control, related to Fig. 1

a, Subcellular localization of the LUC-YFP fusion (a) and GFP_ER (b). SP, signal peptide from Arabidopsis basic chitinase; HDEL, ER retention signal. Representative of 8 images. Scale bar, 10 μm. c–e, mRNA levels of LUC in (Fig. 1b; n = 3), GFP_ER in (Fig. 1c; n = 4), and TBF1-YFP in (Fig. 1d; n = 3) 2 dpi before cell death was observed in plants expressing TBF1. f, Schematics of the 5′ leader sequences used in studying the translational activities of WT uORFs_bZIP11, mutant uorf2a_bZIP11 (ATG to CTG) or uorf2b_bZIP11 (ATG to TAG). g–i, uORFs_bZIP11-mediated translational control of cytosol-synthesized LUC (g; chemiluminescence with pseudo colour); ER-synthesized GFP_ER (h; fluorescence under UV); and cell death induced by overexpression of TBF1-YFP fusion (i; cleared using ethanol) after transient expression in N. benthamiana for 2 d (g, h) and 3 d (i), respectively. Representative of 4 images. j–l, mRNA levels of LUC in (g; n = 2 experiments with 3 technical replicates), GFP_ER in (h; n = 3 experiments with 3 technical replicates), and TBF1-YFP in (i; n = 3 experiments with 3 technical replicates). m, TE changes in LUC controlled by the 5′ leader sequence containing WT uORFs_bZIP11, mutant uorf2a_bZIP11 or uorf2b_bZIP11 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 2 experiments with 3 technical replicates. Bar with solid circles, mean with individual biological replicates.

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

The three developmental phenotypes observed in T1 (i.e., the first generation) Arabidopsis transgenic lines carrying 35S:uorfs_TBF1-snc1, 35S:uORFs_TBF1-snc1, TBF1p:uorfs_TBF1-snc1 and TBF1p:uORFs_TBF1-snc1 (above). Representative of 5 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 lines #1–4 after inoculation by spray (a) or infiltration (b). Mean ± s.e.m.. c, Hpa Noco2 growth as measured by spore counts 7 dpi. Mean ± s.e.m.. d–g, 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 Figure 1 for gel source data) and mRNA (i; mean from 2 experiments with 3 technical replicates). Solid circles, individual biological replicates. #1–4, four independent transgenic lines carrying TBF1p:uORFs_TBF1-snc1 with #1 and #2 shown in Fig. 2. hpi, hours after Psm ES4326 infection; CBB, Coomassie Brilliant Blue. See Source Data for sample size (n). Different letters above bar graphs indicate significant differences (P < 0.05).

Extended Data Figure 5. Functionality of uORFs_TBF1 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:uorfs_TBF1-LUC and 35S:uORFs_TBF1-LUC. Mean of LUC activities (RLU, relative light unit) of 3 biological replicates. Solid circles, individual biological replicates; and mean of LUC mRNA levels of 3 technical replicates after normalization to the 35S:uorfs_TBF1-LUC line #1. c, Representative lesion mimic disease (LMD) phenotypes (above) and percentage of AtNPR1-transgenic rice plants showing LMD in the second generation (T1) grown in the growth chamber (below).

Extended Data Figure 6. Effects of controlling transcription and translation of AtNPR1 on defence in T0 rice, related to Fig. 3

a–d, Lesion length measurements after infection by Xoo strain PXO347 in primary transformants (T0) for 35S:uorfs_TBF1-AtNPR1 (a), 35S:uORFs_TBF1-AtNPR1 (b), TBF1p:uorfs_TBF1-AtNPR1 (c) and TBF1p:uORFs_TBF1-AtNPR1 (d). Lines further analysed in T1 and T2 are circled. e, Average leaf lesion lengths. WT, recipient Oryza sativa cultivar ZH11. Mean ± s.e.m.. Different letters above indicate significant differences (P < 0.05). See Source Data for sample size (n).

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 4 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 indicate significant differences (P < 0.05). d, e, Relative levels of AtNPR1 mRNA (d) and protein (e; numbers below immunoblots; see Supplementary Figure 1 for gel source data) in response to Xoo infection. Mean of AtNPR1 mRNA levels of 3 technical replicates after normalization to 0 hpi (d). Solid circles, individual biological replicates.

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 indicate significant differences among constructs (P < 0.05).

Figure 1. uORFs_TBF1-mediated translational and TBF1 promoter-mediated transcriptional regulation

a, Schematics of WT uORFs_TBF1 or mutant uorfs_TBF1. b–d, LUC activity (b), GFP_ER fluorescence (c) and cell death induced by TBF1-YFP (d), representative of 6 images. e, Dual-luciferase system. f, Translational changes of the reporter to different treatments. Mean of the LUC/RLUC activity ratios normalized to Mock (n = 3). g, LUC/RLUC mRNA levels in (f). Mean ± s.d. of LUC/RLUC mRNA normalized to Mock (n = 6). h, Endogenous TBF1 mRNA levels (n = 3). UBQ5, internal control. Solid circles, individual biological replicates. See Extended Data Fig. 2.

Figure 2. Effects of controlling transcription and translation of snc1 in Arabidopsis

a, b, Effects on vegetative and reproductive growth. snc1, autoactivated mutant. #1 and #2, independent lines carrying TBF1p:uORFs_TBF1-snc1. Representative of 5 images. c, d, Psm ES4326 growth after inoculation by spray (c) or infiltration (d). e, f, Photos (representative of 6 images; scale bar, 0.5 cm) and quantification of Hpa Noco2. g–i, Rosette radius, fresh weight and total seed weight. Mean ± s.e.m.. Letters above indicate significant differences (P < 0.05). See Source Data for sample size (n) and Extended Data Fig. 4 for two additional lines.

Figure 3. Effects of controlling transcription and translation of AtNPR1 in rice

a, b, Symptoms and quantification after Xoo inoculation in field-grown T1 plants. c–f, Symptoms and quantification after Xoc (c, e, water-soaking) and M. oryzae (d, f) in T2 plants. g–i, Fitness under field conditions, including plant height (g), the number of grains per plant (h) and 1000-grain weight (i). Mean ± s.e.m.. Different letters above indicate significant differences (P < 0.05). See Source Data for sample size (n) and Extended Data Figs. 7, 8 for data from two additional lines and for more fitness parameters.