Making Use of Plant uORFs to Control Transgene Translation in Response to Pathogen Attack

Reducing crop loss to diseases is urgently needed to meet increasing food production challenges caused by the expanding world population and the negative impact of climate change on crop productivity. Disease-resistant crops can be created by expressing endogenous or exogenous genes of interest through transgenic technology. Nevertheless, enhanced resistance by overexpressing resistance-produced genes often results in adverse developmental affects. Upstream open reading frames (uORFs) are translational control elements located in the 5′ untranslated region (UTR) of eukaryotic mRNAs and may repress the translation of downstream genes. To investigate the function of three uORFs from the 5′-UTR of ACCELERATED CELL 11 (uORFsACD11), we develop a fluorescent reporter system and find uORFsACD11 function in repressing downstream gene translation. Individual or simultaneous mutations of the three uORFsACD11 lead to repression of downstream translation efficiency at different levels. Importantly, uORFsACD11-mediated translational inhibition is impaired upon recognition of pathogen attack of plant leaves. When coupled with the PATHOGENESIS-RELATED GENE 1 (PR1) promoter, the uORFsACD11 cassettes can upregulate accumulation of Arabidopsis thaliana LECTIN RECEPTOR KINASE-VI.2 (AtLecRK-VI.2) during pathogen attack and enhance plant resistance to Phytophthora capsici. These findings indicate that the uORFsACD11 cassettes can be a useful toolkit that enables a high level of protein expression during pathogen attack, while for ensuring lower levels of protein expression at normal conditions.


Introduction
Food production demands increase with the expanding world population and the negative impact of global climate change [1][2][3].However, crop diseases become a major challenge to modern agriculture [4], with about 15% and 3% yield reduction caused by fungal/bacterial and viral pathogens, respectively [5,6].Microbial infection is a more severe threat to certain crops such as potato, in which it causes nearly 30% yield loss [5,6].The integration of enhanced resistance into new crop varieties by conventional breeding requires selection of desirable traits over several generations [7].In contrast, ectopic expression of resistance-conferring genes is a rapid and powerful approach for enhancing crop disease resistance [4,8].
The genetic engineering approach relies on our expanding knowledge of plant immune mechanisms [9].There are successful examples of specifically enhancing plant resistance to certain pathogens via ectopic expression of corresponding immunity-related genes, such as the legume-like lectin receptor kinase LecRK-I.9 which is recognizing RXLR effector protein IPI-O [10] and the bacterial Elongation Factor Thermo Unstable (EF-Tu) Receptor (EFR) [11,12].However, overexpressing resistance-conferring genes often lead to deleterious pleiotropic effects that antagonize normal plant growth [13][14][15].For example, overexpression of Arabidopsis thaliana NONEXPRESSER OF PR GENES 1 (AtNPR1) enhances plant disease resistance with conditional side effects [16][17][18].Thus, developing novel strategies to fine-tune transgene expression and translation is critical for balancing the trade-off between plant growth and the improved defense.A promising approach is the adoption of pathogen-inducible promoters.
Genes driven by pathogen-inducible promoters are specifically induced upon pathogen infection.Thus, pathogeninducible promoter-controlled expression of immunityrelated genes may be a rational solution to reduce unnecessary growth inhibition [19][20][21].For example, the promotor of Glycine max polyphenol oxidase gene, GmPPO12, is a pathogen-induced promoter that could be used in transgenic engineering [22].However, pathogen-inducible promoters often auto-activate transgenes in plants [23].For example, transgenic tobacco plants expressing the cryptogein or popA elicitor driven by the pathogen-inducible promoter hsr203J show broad-spectrum resistance, but some lines display runaway cell death due to hsr203J-induced gene auto-activation [24,25].Thus, regulatory elements with minimal side effects should be identified and used for fine-tuning transgene products at transcriptional and/or translational levels.A group of such candidates are from the translational control elements named upstream open reading frames (uORFs).
ACD11 is a ceramide-1-phosphate transfer protein that negatively regulates plant immunity [35,36].The protein stability of ACD11 is regulated by the E3 ligase XBA35.2 [37].We previously also showed that ACD11 can be stabilized via its physical interactions with BINDING PARTNER OF ACD11 1 (BPA1)-like (BPL) family proteins [36].However, whether there are other mechanisms regulating ACD11 gene products is still unknown.AtLecRK-VI.2 harbors an extracellular lectin motif [38,39] and positively regulates Arabidopsis resistance to bacterial pathogens [40].LecRK-VI.2also functions as a key component of systemic acquired resistance (SAR) by recognizing the putative SAR mobile signal extracellular nicotinamide adenine dinucleotide (eNAD+) [41].Heterologous expression of LecRK-VI.2 in N. benthamiana increases plant resistance to a broad range of bacteria pathogens [42].
To identify uORF's capacity of regulating downstream gene products, we have developed a fluorescence-based method to evaluate how a uORF might regulate protein production.Three predicted uORFs from the 5 ′ UTR of ACD11 were evaluated using this system.Individual or simultaneous mutations of the three uORFs ACD11 lead to repression of downstream translation variously.This translational inhibition was further impaired by pathogen inoculation.Therefore, we combined the uORFs ACD11 cassettes with the PATHOGENESIS-RELATED GENE 1 (PR1) promoter and demonstrated that they can fine-tune AtLecRK-VI.2-mediated resistance in transgenic N. benthamiana plants, indicating that the uORFs ACD11 cassettes can be a useful toolkit for engineering crop disease resistance with desired fitness cost.

Plasmid Constructs.
To build fluorescenceand luminescence-based reporter system, full length of GFP coding sequence (CDS), terminator of nopaline synthase gene (tNOS), CaMV 35S promoter (35S), and Luciferase CDS were amplified and inserted sequentially into the pSuper vector which contains a MAS promoter.5 ′ -UTR TBF1 , 5 ′ -UTR ACD11 , and AtLecRK-VI.2 with a C-terminal-fused FLAG tag were amplified from WT Arabidopsis (Col-0).Site-directed mutagenesis of uORFs ACD11 was performed to change ATG to CTG.To generate transgenic Arabidopsis expressing NAT-GFP, 5′UTR ACD11 , GFP, and tNOS were inserted sequentially into pSuper.To generate transgenic N. benthamiana, different combinations of uORFs ACD11 cassettes and AtLecRK-VI.2 with C-terminal fused FLAG tag and tNOS were inserted sequentially into pSuper.Primers used in this study are listed in Table S1.

Plant Materials and Transgenic Plants. N. benthamiana
plants used in this study were grown in the glasshouse at 26 °C under a 16-hours light/8-hours dark photoperiod for 6 weeks.Arabidopsis were grown at 24 °C under a 12-hours light/12-hours dark photoperiod and 60% relative humidity for one month.
To generate transgenic Arabidopsis plants, the indicated plasmid was introduced into Agrobacterium strain GV3101.Arabidopsis WT (Col-0) plants were transformed using the standard Agrobacterium-mediated floral dip protocol.Transgenic plants were screened on 1/2 MS medium containing 50 mg/l hygromycin.
To generate transgenic N. benthamiana, the indicated plasmid was transformed into Agrobacterium strain GV3101.Agrobacterium-mediated N. benthamiana transformation was performed as previously described [43].Transgenic plants were screened on 1/2 MS medium containing 50 mg/l Kanamycin.

Phytophthora capsici Culture Conditions, Culture
Filtrate Acquisition and Inoculation Assay.The P. capsici strain LT263 used in this study was cultured and maintained at 25 °C in 10% (v/v) V8 juice medium (containing 0.1 M CaCO 3 ) in the dark.To produce CF, mycelium was cultured in liquid V8 medium at 25 °C for 3 days.The culture was then passed through a 22 μm sterile filter unit (Merck Millipore, https://www.merckmillipore.com) to generate CF.For plant infiltration, a 1/10 CF solution was used.

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For mycelium inoculation, N. benthamiana leaves were inoculated by 5 mm disks of 4-day growth mycelium at 24 hours postinfiltration.Lesion areas were measured and photographed under UV light at 48 hpi.For zoospore inoculation on leaves of Arabidopsis, P. capsici mycelium was incubated in liquid medium for 3 days and then washed 3 times with distilled water.Washed mycelium was incubated in sterilized water at 25 °C in darkness for 12 hours.The cultures were cold shocked at 4 °C for 20 min and incubated at 25 °C for 2 hours to release zoospore.Leaves were soaked in 100 spores/μl P. capsici zoospores for 30 minutes and then held under moist conditions for subsequent analysis by confocal microscopy.
Stock solution for trypan blue staining was produced by mixing trypan blue (0.02 g), glycerol (10 ml), phenol (10 g), lactic acid (10 ml), and sterilized water (10 ml).N. benthamiana leaves were soaked in trypan stock solution for 24 hours at 24 °C.Leaves were then destained using ethanol for 5 days.Samples were put in ethanol for taking pictures under white light.
2.5.Transient Expression in N. benthamiana.Transient expression in N. benthamiana was conducted as previous reported [44].Briefly, Agrobacterium strains with mentioned constructs were cultured for 48 hours, collected, washed, and then resuspended in 10 mM MgCl 2 to an optical density (OD) at 600 nm (0.4) and infiltrated into five-week-old N. benthamiana leaves.

qRT-PCR Analysis.
To perform real-time PCR, total RNA was extracted by a Total RNA Kit (Tiangen Biotech Co., Ltd., Beijing, China).The cDNAs were synthesized using the HiScript II Q RT SuperMix for qPCR (Vazyme Biotech Co., Ltd., Nanjing, China).Real-time PCR was performed by using an AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) on an ABI Prism Q5 system.AtUBQ10 or NbELF18 were used as internal references.Primers used for in real-time PCR are listed in Table S1.The qRT-PCR results were concluded from three biological replicates.

Western Blotting and Confocal
Microscopy.Plant leaves for protein extraction were ground in liquid nitrogen.Extraction buffer (0.1% Triton X-100, 150 mM KCL, 50 mM HEPES and 1 mM EDTA, protease inhibitor cocktail; pH 7.5) and 1 mM DTT was used for protein extraction.For Western blot assays, Flag, GFP (Abmart), and LUC (Sigma) antibodies were used.
To detect GFP accumulation after different treatments, confocal images were obtained at 12 hours after treatment by a confocal microscope (Zeiss LSM980, Germany).The average GFP florescence densities were quantified per 100 pixels of 20 randomly selected cells (relative unit) using Ima-geJ (https://imagej.en.softonic.com/).

A Fluorescence-and Luminescence-Based Reporter
System for the Function Investigation of uORFs.To investigate the function of uORFs, we designed a fluorescenceand luminescence-based reporter system to visibly measure the regulatory effect of uORFs (Figure 1(a)).In this system, the expression of green fluorescent protein (GFP) is under the control of mannopine synthase (MAS) promoter and the 5′ UTR of indicated gene.The luciferase (LUC) gene driven by CaMV 35S promoter is used as an internal reference.GFP fluorescence and LUC luminescence intensities were quantified using a microplate reader.The relative fluorescence ratio (GFP/LUC) was calculated to remove perturbation resulted from agro-infiltration (Figure 1(a)).Notably, no significant interference was detected between green fluorescence and luminescence (Figure S1).
To check the reliability of our system, we used the previously reported cis translational repressor 5 ′ -UTR TBF1 as a positive control [31,34].5 ′ -UTR TBF1 significantly repressed GFP protein accumulation in our system (Figures 1(b)-1(d)), indicating the effectiveness of this reporter system.

uORFs ACD11
Are Cis-Acting Elements That Repress Downstream Translation.Three uORFs were identified in the 5 ′ -UTR ACD11 of Arabidopsis Col-0 ecotype and were named as uORF1, uORF2, and uORF3 (Figure 2(a), Figure S2a).The Col-0 uORF1 and uORF3 were found to be conserved in 96.6% and 100% of the 1,135 accessions in the uORFlight database, respectively (Figure S3a) [30].Two major types of uORF2 could be identified in 1,135 accessions.Except Col-0 type, Ws-2 type uORF2 contained a synonymous nucleotide substitution (G to C in the 60th base) (Figure S3a, S3b).All three uORFs ACD11 are highly conserved across Arabidopsis accessions which suggests that they may be functional.
A null mutant of uORFs ACD11 was created by mutating the start codon of all three ACD11 uORFs to CTG (hereafter "uorf1/2/3") and constructed into our vector.5 ′ -UTR ACD11 with native uORFs ACD11 (hereafter "NAT") was used as a control (Figure 2(b)).When expressed in N. benthamiana leaves, uorf1/2/3 exhibited much stronger GFP fluorescence than NAT.In contrast, they generated similar intensities of luminescence (Figure 2(b)).Similarly, GFP/LUC values showed that leaves expressing the uorf1/2/3 construct displayed significantly higher GFP fluorescence than those expressing NAT (Figure 2(c)).qRT-PCR and Western blot assays demonstrated that uorf1/2/3 and NAT constructs generate similar levels of GFP mRNA in N. benthamiana leaves (Figure 2(d)), but uorf1/2/3 expression leads to higher GFP protein accumulation (Figure 2(e)).These results indicate that uORFs ACD11 negatively regulate downstream protein accumulation at the translation level.Notably, the absence of 5 ′ UTR ACD11 does not change GFP transcript, protein or fluorescence level as compared to uorf1/2/3 (Figures 2(b)-2(e)), indicating that the non-uORF regions in 5 ′ -UTR ACD11 may not affect the expression of downstream protein.

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To test whether uORFs function as trans-or cis-elements, uorf1/2/3 construct was coinfiltrated with the constructs harboring coding cassettes of uORF1, uORF2, uORF3, or all of them.The NAT construct was used as control.GFP translation cannot be blocked via coexpression peptides of any individuals or all three uORFs (Figure 2(f )), demonstrating that uORFs act in cis.

uORFs ACD11 Have Variable Contributions to
Translational Repression.We next investigated the individual contributions of uORFs ACD11 to translational repression.A series of uORFs ACD11 mutants were created by individually or simultaneously mutating the start codon of uORF1, uORF2, and/or uORF3 to CTG (Figure 3(a)).These mutants exhibited comparable luminescence signals but highly variable GFP fluorescence (Figure 3(a)).Despite their relatively weak Kozak strength (Figure S2b), all three uORFs ACD11 are involved in the translational repression of downstream gene (Figures 3(a)-3(d)).The presence of any single uORF ACD11 was able to sustain the inhibition phenotype (Figures 3(a)-3(d)).Among them, uORF1 tends to be the dominant inhibitory element.Disruption of uORF1 alone could significantly increase GFP signal, which was not observed on uORF2 or uORF3 disruption (Figures 3(a)-3(d)).Nevertheless, a mutant with intact uORF3 and the disrupted uORF1 and uORF2 leads to the lowest GFP   The result is consistent with the report that uORF length is not correlated with the suppression capacity [45].Taken together, these observations indicate that uORFs ACD11 have complex genetic interactions, and they are not simply additive in repressing downstream protein translation.

uORFs ACD11 -Mediated Translational Repression Is
Attenuated upon Pathogen Infection.The 5′-UTR ACD11 -GFP transgenic Arabidopsis plants were produced to test the response of uORFs ACD11 to pathogen infection (Figure S4a).GFP fluorescence generated in these transgenic plants was too weak to detect using in vivo fluorescence imaging (Figure S4b), but visible under confocal microscope (Figure 4(a)).Transgenic Arabidopsis leaves were challenged with the oomycete pathogen Phytophthora capsici (Figure S4c).GFP fluorescence was enhanced in leaves inoculated with P. capsici zoospores or culture filter (CF) (Figures 4(a) and 4(b)) [46].qRT-PCR analysis showed that the transcript accumulation level of GFP was unchanged after inoculation (Figure 4(c)).Western blot assay confirmed that the inoculation of either P. capsici zoospores or CF promoted GFP accumulation at the protein level (Figure 4 .Interestingly, Pst DC3000 AvrRPT2 , an avirulent strain that induces hypersensitive response in Arabidopsis [47,48], showed higher GFP-induction efficiency than the wild-type (WT) Pst DC3000 (Figures 4(a)-4(d)).This observation indicates that the translational repression ability of uORFs ACD11 can be attenuated after plant recognizing different pathogens.

Different uORFs ACD11 Cassettes Lead to Variable
Accumulation of AtLecRK-VI.2 for Acquired Resistance.
P. capsici resistance levels were tested for N. benthamiana plants transiently expressing AtLecRK-VI.2 under the control of different uORFs ACD11 cassettes.Compared to 7 BioDesign Research empty vector, all cassettes significantly enhanced plant immunity to P. capsici at 48 hours postinoculation (hpi), with uorf1/2/3-LecRK-VI.2delivering the highest resistance (Figure 5(c)).No increased resistance could be found in leaves expressing uORF1, uORF2, or uORF3 (Figure 5(d)), indicating that none of the peptides encoded by the three uORFs are directly involved in P. capsici resistance.
LecRK-VI.2 functions as an extracellular pyridine nucleotide receptor involved in SAR [41].To test whether the observed alteration protein levels in the three transgenic line affected SAR response, we assessed the ability of DC3000 hopq 1-1 to colonize upper leaves three days after the inoculation of lower leaves.Systemic resistance was observed in wildtype and the three transgenic lines (Figure 7(h)).BioDesign Research However, none of the three transgenic lines exhibited a proportionally stronger SAR response from controls (Figure 7(h)).

Discussion
Fine-tuning of quantitative traits is highly valued by breeders as it affords a sound approach to harness useful characteristics for breeding without serious field impairment [49,50].Naturally occurred weak alleles affecting important traits have contributed to great advances in domestication, evolution, and breeding [51,52], but their utilization is restricted by low availability.Ectopic expression of foreign genes is an alternative approach to introduce the desired traits.However, overexpression can lead to undesirable phenotypes.For example, ectopic expression of AtNPR1 in rice using the maize ubiquitin promoter resulted in abnormal plant development and a reduction in seed size under some conditions [17].Constitutive expression of the coding region of tobacco tbz17 (Nttbz17) results in plants with thicker leaves [53].Here, we successfully controlled the transgene products by using uORFs ACD11 and PR1 promoter to achieve a fine-tuned resistance level without growth retardation in transgenic plants (Figure 7(i)).
A uORF is a small ORF containing a start codon located upstream of their regulated gene.The three uORFs in 5 ′ -UTR ACD11 are conserved among Arabidopsis accessions, indicating their important regulatory roles.Their variable translational repression efficiencies may at least partially depend on the Kozak sequence context around their start codons [54].Despite their relatively low Kozak strength, the three uORFs ACD11 effectively repress the translation of downstream gene in a redundant manner.Among them, uORF3 has the shortest length of 9 base pairs but the most favorable Kozak strength, which leads to its strongest translational repression efficiency.This observation explains why the uorf1/2 cassette has strong repression capacity and is suitable for fine-tuning transgene expression and balancing plant growth and immunity.
Natural uORFs has been successfully used in engineering plant defense.Ectopic expression of AtNPR1 driven by 35S:uORFs AtTBF1 renders broad-spectrum disease resistance to rice plants with no apparent growth retardation [34].The potential value of edited uORFs is also emerging gradually.For example, uORF engineering of an important enzyme in vitamin C synthesis, LsGGP2, improves the tolerance of lettuce to oxidation stress and ascorbate content [55].Here, we have shown that a modified uORF, uORFs ACD11 , can be used to down regulate gene translation under normal growth conditions while enabling the activation of translation when a pathogen is detected.
ACD11 encodes a phingosine transfer protein.Its knockout causes activation of defense response and programmed cell death (PCD), indicating that ACD11 negatively regulates plant immunity [35].The protein stability of ACD11 is also regulated by the E3 ligase XBA35.2 and its binding partners of BPL family proteins [36,37].The three uORFs characterized in this study may be an additional control layer of ACD11 protein accumulation.In plants, the protein levels of such negative regulators are strictly controlled by multiple layers of mechanisms.Other examples of this multilayered regulation include the pentatricopeptide repeats proteinlike (PPRL) protein negatively regulates RESISTANT TO P. SYRINGAE 2-(RPS2-) mediated resistance pathway and is downregulated by a natural antisense short interfering RNA (nat-siRNA) derived from Arabidopsis GTP-BINDING 2 (ATGB2) gene (nat-siRNAATGB2) at the transcription level [56].EDS1-interacting J protein 1 (EIJ1) plays a negative role in plant defense response and its protein accumulation is restricted during pathogen infection process [57].
Based on our data, uORFs ACD11 -mediated translational repression was attenuated upon either PAMP treatment or pathogen inoculation.Similar phenomenon has been reported in multiple other genes such as uORF TBF1 [31,58,59].The R-motif, a highly enriched consensus sequence consisting of mostly purines, can be found in the 5 ′ -UTR of these genes.However, no R-motif can be identified from 5 ′ UTR ACD11 , indicating that ACD11 and R-motif containing genes like TBF1 may respond to pathogen infection via distinct mechanisms.Additionally, the translational repression ability of uORFs ACD11 become weaker after the avirulent strain treatment.Avirulent strains lead to effector-triggered immunity (ETI) in host cells.Compared with basal resistance, ETI induces a stronger and faster defense response against pathogens.We speculated that the inhibition of uORFs ACD11 -mediated translational repression ability was a part of defense response, and thus, uORFs ACD11 -mediated translational repression was weaker upon avirulent strains.
The integration of pPR1 is an improvement to the uORFs ACD11 cassettes.The adoption of pathogen-inducible promoters is a logical solution to reduce growth distortion triggered by defense-related transgenes [19,20], but they should be carefully selected for genetic engineering to avoid those with unfavorable auto-activation effect [24,25].In this study, no obvious auto-activation is detected for pPR1-uORFs ACD11 cassettes and the strategy successfully avoids potential growth retardation induced by AtLecRK-VI.2.
In this study, we fine-tune AtLecRK-VI.2 expression via the combinations of pPR1 and uORFs ACD11 cassettes.In this way, AtLecRK-VI.2 expression is strictly induced by pathogen infection and optimized to provide satisfactory resistance protection with no apparent impair on plant growth.These cassettes can be used for fine-tuning other genes of interest.They would be a useful toolkit for precisely engineering crop disease resistance and other important agronomic traits.

Figure 1 :
Figure 1: Establishment of a fluorescenceand luminescence-based reporter system to study the functions of uORFs.(a) Diagram of the fluorescenceand luminescence-based reporter system.Illustration of the constructed plasmids used in this study is shown at the top.Workflow of our system is shown below.(b) Repression of GFP production by the 5′-UTR of TBF1.Photos were taken 48 hours post agro-infiltration.Illustration of the indicated plasmids was shown below.(c) Relative fluorescence of leaves expressing indicated plasmids.Relative fluorescence was calculated based on the formula shown in (c) (mean ± SD; n = 8, Student's t-test, P < 0:01).(d) Decreased GFP protein level confirmed by Western blot assay.The GFP protein accumulation was detected by α-GFP antibody.Equal loading of each sample is indicated using the luciferase protein.

Figure 2 :
Figure 2: uORFs of ACD11 are cis-acting elements that repress downstream translation.(a) Diagram of three uORFs identified in the 5′-UTR of ACD11.The main ORF (mORF) indicates ACD11.(b) Repression of GFP production by the 5′-UTR of ACD11.Photos were taken 48 hours post agro-infiltration.Illustration of the indicated plasmids was shown below.(c) Relative fluorescence of leaves expressing indicated plasmids.Relative fluorescence was calculated based on the formula shown in Figure 1(c) (mean ± SD; n = 8, Student's t-test, P < 0:01).(d) Relative transcript accumulation levels of GFP.Transcript accumulation levels of GFP were analyzed by qRT-PCR with NbELF18 as internal reference.Bars represent standard errors from three biological replicates (mean ± SD; n = 3; * * , P < 0:01; Student's t -test).(e) Decreased GFP protein expression confirmed by Western blot assay.The GFP protein accumulation was detected by α-GFP antibody.Equal loading of each sample is indicated by the luciferase protein.(f) ACD11 uORFs act in cis.uORFs was expressed in trans in leaves expressing uorf1/2/3 plasmid.Relative fluorescence was calculated based on the formula shown in Figure 1(c) (mean ± SD; n ≥ 8 , Student's t-test, P < 0:01).

Figure 3 :
Figure 3: Various uORFs combinations show different downstream repressing efficiencies.(a) Repression of GFP production by ACD11 uORF mutants.Photos were taken 48 hours post agro-infiltration.Illustration of the indicated plasmids was shown below.(b) Relative fluorescence in leaves expressing indicated plasmids.Relative fluorescence was calculated based on the formula shown in Figure 1(c) (mean ± SD; n = 8, Student's t-test, P < 0:01).(c) Relative transcript accumulation levels of GFP.Transcript accumulation levels of GFP were analyzed by qRT-PCR with NbELF18 as internal reference.Bars represent standard errors from three biological replicates (mean ± SD; n = 3; * * , P < 0:01; Student's t-test).(d) Decreased GFP protein expression confirmed by Western blot assay.The GFP protein accumulation was detected by α-GFP antibody.Equal loading of each sample is indicated by the luciferase protein.

Figure 4 :
Figure 4: uORF ACD11 -mediated translational repression is attenuated upon pathogen infection.(a) GFP accumulation after pathogenrelated treatments.Confocal microscope images were taken 12 hours posttreatment.Bar = 50 μm.(b) Mean fluorescence value of indicated leaves.The average GFP florescence densities were quantified per 100 pixels of 20 randomly selected cells (relative unit) using ImageJ.(c) Relative transcript accumulation levels of GFP.Transcript accumulation levels of GFP were analyzed by qRT-PCR with AtUBQ10 as internal reference.Bars represent standard errors from three biological replicates (mean ± SD; n = 3; * * , P < 0:01; Student's t -test).(d) GFP protein accumulation detected by Western blot assay.The α-GFP antibody was used to detect the expression of GFP protein.Equal loading of each sample is indicated by Ponceau staining.(e) GFP protein accumulation changes after P. capsici inoculation.The GFP protein accumulation was detected by α-GFP antibody.Equal loading of each sample is indicated by Ponceau staining.

Figure 5 :
Figure 5: Different uORFs ACD11 cassettes lead to variable AtLecRK-VI.2 protein accumulation and pathogen resistance levels.(a) AtLecRK-VI.2 protein accumulation levels detected by Western blot assay.The α-FLAG antibody was used to detect the expression of AtLecRK-VI.2 protein.Equal loading of each sample is indicated by Ponceau staining.(b) Relative transcript accumulation levels of AtLecRK-VI.2.Transcript accumulation levels of AtLecRK-VI.2 were analyzed by qRT-PCR with NbELF18 as internal reference.Bars represent standard errors from three independent biological replicates (mean ± SD; n = 3; * * , P < 0:01; Student's t-test).(c) Expression of AtLecRK-VI.2 driven by ACD11 uORF-mutants provides variable levels of resistance to P. capsici infection in N. benthamiana.Lesion areas were calculated at 48 hpi.The result was calculated from three independent replicates with at least five leaves per replicate (mean ± SD; n ≥ 18; * * , P < 0:01, Student's t-test).(d) No pathogen resistance is induced by uORF1, uORF2, or uORF3.Leaves were stained by trypan blue and destained with ethanol.Photos were taken in white light and shown in (a).Bar = 2 cm.The lesion areas were calculated from three independent experiments were shown below (mean ± SD; n ≥ 18; * * , P < 0:01, Student's t-test).

Figure 6 :
Figure 6: Pathogen-inducible protein accumulation of AtLecRK-VI.2 is achieved by using the combinations of different uORFs and the NbPR1 promoter.(a) Cell death induced by indicated gene products.Leaves infiltrated with Agrobacterium harboring indicated constructs.Photos were taken at 5 dpi in white light.(b) CF-induced AtLecRK-VI.2 transcript accumulation confirmed by qRT-PCR.Transcript accumulation levels of AtLecRK-VI.2 were analyzed by qRT-PCR.P. capsici CF was infiltrated into leaves expressing the indicated plasmid.Total RNA was extracted 1 hour posttreatment.The NbELF18 gene was used as an internal reference.Bars represent standard errors from three biological replicates (mean ± SD; n = 3; * * , P < 0:01; Student's t-test).(c) Induced AtLecRK-VI.2 protein accumulation confirmed by Western blot assay.CF was infiltrated into leaves expressing the indicated plasmid.Proteins was extracted 2 hours posttreatment.The α-FLAG antibody was used to detect the expression of AtLecRK-VI.2 protein.Equal loading of each sample is indicated by Ponceau staining.(d) Enhanced resistance to P. capsici infection provided by ACD11 uORF-mutants coupled with the PR1 promoter and AtLecRK-VI.2 in N. benthamiana.Lesion areas were calculated from three independent experiments with at least five leaves per replicate (mean ± SD; n ≥ 18; * * , P < 0:01, Student's t-test).

Figure 7 :
Figure 7: pPR1-uORFs ACD11 cassettes fine-tune AtLecRK-VI.2-mediated resistance to pathogens in transgenic N. benthamiana.(a, b) The growth phenotypes of indicated transgenic N. benthamiana plants.Photos were taken at 8 weeks after sowing (a).Plant heights (from soil to the top of plants) were calculated and showed in (b).(c) AtLecRK-VI.2 transcript accumulation confirmed by qRT-PCR.Transcript accumulation levels of AtLecRK-VI.2 were analyzed by qRT-PCR.CF was infiltrated into the indicated leaves.Total RNA was extracted 1 hour posttreatment.The NbELF18 gene was used as an internal reference.Bars represent standard errors from three biological replicates (mean ± SD; n = 3; * * , P < 0:01; Student's t-test).(d, e) Induced AtLecRK-VI.2 protein accumulation upon P. capsici infection.P. capsici mycelium was inoculated on the indicated leaves.Proteins was extracted 12 hours postinoculation.The α-FLAG antibody was used to detect the expression of AtLecRK-VI.2 protein.Equal loading of each sample is indicated by Ponceau staining.(f) Enhanced resistance to P. capsici infection in transgenic N. benthamiana.Lesion areas were calculated from three independent experiments with at least five leaves per replicate (mean ± SD; n ≥ 18; * * , P < 0:01, Student's t-test).(g)Fine-tuning resistance to P. syringae hopq 1-1 in transgenic plants.Error bars represent the mean ± SD; n = 3; lowercase letters indicate statistical significance tested between multiple groups by oneway ANOVA at P < 0:05.(h) Fine-tuning systemic acquired resistance to P. syringae hopq 1-1 in transgenic plants.Three lower leaves on each 4-week-old soil-grown plant were infiltrated with H 2 O or P. syringae hopq 1-1 suspension (OD = 0:0001).Two systemic leaves were challenge-inoculated with a P. syringae hopq 1-1 suspension (OD = 0:0001) at 24 hours after the last infiltration.Three days later, eight leaves were collected to examine pathogen growth.Error bars represent the mean ± SD; n = 3; lowercase letters indicate statistical significance tested between multiple groups by one-way ANOVA at P < 0:05.(i) A model for the usage of uORFs ACD11 in precisely engineering crop disease resistance at required levels to minimize negative impacts on plant growth.uORFs ACD11 inhibits downstream gene translation in normal conditions and releases it upon pathogens.uORFs ACD11 coupled with PR1 promoter can confer fine-tuned resistance in transgenic plants specifically when plants are facing pathogen threats.