Complementary peptides represent a credible alternative to agrochemicals by activating translation of targeted proteins

The study addresses how to safely and specifically enhance plant performance without genetic modification or conventional agrochemicals, amid rising pesticide resistance, environmental and health concerns, and climate-induced yield pressures. The authors hypothesize that short, hydrophilic complementary peptides (cPEPs) designed from coding sequences can bind their cognate mRNAs in planta and selectively enhance translation of targeted proteins, thereby modulating corresponding phenotypes (growth, stress tolerance, defense), and offering a potential alternative tool for agronomy and weed control.

• Design and synthesis of complementary peptides (cPEPs): Short hydrophilic peptides (5–60 amino acids; ≥33% D, N, E, Q, K, R) were designed to be complementary to coding sequences of target genes (including luciferase, NSP1, SKL/EIN2, RH10, DCL1, ABI5, SGR1, HSP101, CPK3, and others). Peptides and scrambled controls were synthesized (Smart Biosciences), dissolved at 2–10 mM, aliquoted, and stored at −80 °C.
• Plant materials and growth: Arabidopsis thaliana (Col-0 and transgenic LUC lines), Medicago truncatula (A17), Nicotiana benthamiana, Solanum lycopersicum (tomato), Glycine max (soybean), Barbarea vulgaris, Amaranthus hypochondriacus. Plants were grown in controlled chambers under specified photoperiod, temperature, humidity, and media conditions. Rhizobium inoculations and pathogen challenges were performed as described.
• Peptide application regimens: Spraying, watering, or media addition. Typical concentrations were 10–100 µM; for Arabidopsis protein quantification, 100 µM daily for 5 days; for flowering/chlorophyll, 10 µM thrice weekly until bolting; for growth/weed assays, mixes at 20 µM each peptide; for luciferase assays, 50 µM cPEP was optimal and 24 h post-treatment yielded maximal effect.
• RNA–peptide interaction assays: RNA co-immunoprecipitation (RNA-IP) using HA-tagged peptides and anti-HA magnetic beads, followed by RT-PCR to detect target mRNAs (e.g., luciferase) in Arabidopsis.
• FRET-FLIM: In N. benthamiana leaves expressing NSP1 mRNA (WT, ΔcPEP deletion, or ΔcPEP replaced with irrelevant sequence), SYTOX-labeled mRNAs and FAM-tagged cPEPnsp1 were used to assess proximity/interaction via fluorescence lifetime imaging microscopy.
• Gene expression and protein assays: qRT-PCR for mRNA levels; luciferase activity assays in planta and in vitro; GUS activity from ProNSP1-NSP1-GUS translational fusions; western blots using target-specific antibodies; chlorophyll content via SPAD meter; root length and leaf area quantified with ImageJ.
• Pathogenicity assays: Botrytis cinerea infection on A. thaliana and tomato leaves with peptide treatments before/after inoculation; Aphanomyces euteiches infection in Medicago with peptide treatments; pathogen load quantified via α-tubulin qPCR.
• Heat stress assays: Arabidopsis seedlings exposed to 45 °C for 45 min with pre/post peptide treatments; soybean seedlings exposed to 45 °C for 24 h with peptide pre-treatments; survival assessed post-recovery.
• Translation mechanism tests: Cycloheximide (200 µg/mL) co-treatment with cPEPluc in Arabidopsis luciferase lines to test dependence on translation; Wheat germ (TnT SP6) in vitro transcription/translation with LUC ± cPEPluc or scrambled control.
• Ribosome engagement profiling: 5′P sequencing (GMUCT-based) in Arabidopsis treated with cPEPcpk3-HA vs scrambled to quantify 5′P read accumulations in defined windows around start/stop codons, indicative of ribosome occupancy.
• cPEP interactome: Co-immunoprecipitation using HA-tagged cPEPcpk3 or scrambled in Arabidopsis, followed by quantitative MS (LC-MS/MS; MaxQuant/Perseus analysis) to identify enriched protein interactors.
• Polysome association: Sucrose gradient fractionation of polysomes from Arabidopsis treated with cPEPluc-HA or scrambled; HA western blot of fractions to detect cPEP presence in polysomal fractions.
• Phenotype assays: Lateral root counts in Medicago WT, nsp1 mutant, and 35S-NSP1 lines after cPEPnsp1 vs scrambled; Medicago nodulation counts after cPEPskl vs irrelevant peptide; Arabidopsis flowering time shifts with individual cPEPs and combinations; additive effects assessed by flowering time and leaf area.
• Statistics: Wilcoxon or Student’s t-tests as appropriate; replicate numbers specified per assay; proteomics volcano plots with FDR control in Perseus.

• Targeted RNA interaction and specificity: HA-tagged cPEPluc specifically co-immunoprecipitated luciferase mRNA, unlike scrambled controls. FRET-FLIM showed close proximity between cPEPnsp1-FAM and NSP1 mRNA in cytoplasm; deleting the corresponding RNA region (NSP1 ΔcPEP) abolished interaction; replacing it with an irrelevant sequence conferred responsiveness to the corresponding irrelevant peptide.
• Protein upregulation without mRNA increase: qPCR showed no change in mRNA abundance upon cPEP treatment, yet target protein activity/levels increased (e.g., luciferase activity increased vs water, scrambled, or irrelevant peptides; NSP1-GUS protein increased with cPEPnsp1).
• Design breadth and parameters: Ten additional luciferase-targeting 10-aa cPEPs across all three frames were all active in increasing luciferase. Peptide length window of 5–40 aa enhanced activity; longer peptides (>40 aa) were ineffective. Optimal concentration was ~50 µM; maximal effect at ~24 h post-treatment.
• Proteome specificity: Global proteomics of Arabidopsis treated with cPEPluc-HA vs scrambled detected no significant proteome-wide changes at p<0.01, supporting target specificity.
• Broad applicability across targets/species: 11 cPEPs validated against 11 proteins in three plant species. In Medicago, cPEPnsp1 reduced lateral root number (WT responsive; nsp1 mutant and NSP1 overexpressor insensitive), cPEPskl reduced nodule numbers, and cPEPrh10 enhanced resistance to Aphanomyces (increased root growth; decreased pathogen α-tubulin expression in roots).
• Phenotypic modulation in Arabidopsis: cPEPdcl1 reduced primary root length; cPEPabi5 decreased chlorophyll content; cPEPsgr1 increased chlorophyll; cPEPhsp101 improved survival after heat shock; defense-targeting cPEPs reduced Botrytis lesion size; cpk3 mutant was unresponsive to cPEPcpk3. Flowering time: SHY2 and MRB1 cPEPs accelerated flowering; BRI1, BAK1, TAP46, SPT, EIN2, GA2OX7, PHYB, HAGS, SHR, WUS cPEPs delayed development. Combining EIN2, BRI1, BAK1, WUS cPEPs produced additive delay: individual peptides up to ~17% reduction in development vs mix ~23% (assessed by flowering day and leaf growth).
• Mechanism—translation activation: Cycloheximide blocked cPEPluc-induced luciferase increase, indicating dependence on translation rather than protein stability. In wheat germ in vitro translation, cPEPluc increased luciferase activity. 5′P-Seq revealed increased ribosome occupancy around the start codon on CPK3 transcripts upon cPEPcpk3 treatment, but not on close homologs (CPK6/9/32), consistent with enhanced initiation on-target. Co-IP/MS identified ribosomal protein RPL19 and BAM3 enriched with cPEPcpk3-HA; rpl19 mutants were insensitive to cPEPcpk3 and cPEPdcl1, demonstrating RPL19 requirement. cPEPluc-HA localized to polysomal fractions (8–12), supporting interaction with ribosomal machinery.
• Agronomic outcomes: In tomato, cPEPjar1 decreased Botrytis lesion area by 23%. In soybean, cPEPhsp101 increased heat stress survival by 67%, and a growth-promoting mix (MRB1, SHY2, SGR1) increased plant height by 24%. Weed control: a mix targeting EIN2, BRI1, BAK1, WUS reduced Barbarea vulgaris leaf surface by 30% and Amaranthus hypochondriacus leaf surface by 15%.

The work demonstrates that exogenously applied complementary peptides can selectively bind their cognate mRNAs in planta and enhance translation, increasing targeted protein abundance without elevating mRNA levels. This targeted protein upregulation translates into predictable phenotypic outcomes consistent with known gene functions, spanning growth regulation, stress tolerance (heat), chlorophyll modulation, and pathogen resistance. Mechanistically, cPEPs promote ribosome recruitment/initiation on target transcripts, involve ribosomal component RPL19, and associate with polysomes, supporting a direct role in translational control. The approach offers a non-transgenic, externally applicable tool, potentially species-selective by sequence design, that could complement or replace certain agrochemicals, mitigate resistance issues, and address traits not easily modulated by chemicals. The findings also suggest the intriguing possibility of endogenous natural cPEPs arising from lncRNAs, UTRs, alternative ORFs, or proteolytic fragments, though their existence and roles in plants remain to be established.

This study introduces complementary peptides as a generalizable, designable modality to enhance translation of specific plant proteins via sequence-directed interaction with target mRNAs and the ribosomal machinery. cPEPs reliably increase target protein abundance, elicit expected phenotypes across multiple species, show additive effects when combined, and deliver agronomically relevant benefits (enhanced disease resistance, heat tolerance, growth promotion, and weed suppression). Future research should identify and characterize potential natural cPEPs in plant genomes, refine design rules and delivery strategies, comprehensively assess off-targets and species specificity, elucidate detailed molecular mechanisms (including roles of ribosomal subunits such as RPL19), and evaluate efficacy, durability, and safety in field conditions.

• Experiments were conducted under controlled laboratory/growth chamber conditions; field performance and durability of effects were not assessed.
• Proteome-wide analyses showed no significant changes at p<0.01 for one model system, but broader off-target assessments across diverse tissues and species remain limited.
• The effective peptide length window (5–40 aa) and hydrophilicity criteria were empirically defined; comprehensive design rules are not yet established.
• While mechanistic data implicate translation initiation and RPL19, the full molecular pathway and the role of other ribosomal or auxiliary factors require further clarification.
• The existence and identity of endogenous natural cPEPs (natcPEPs) were hypothesized; their detection is technically challenging due to small size and current MS sensitivity limits.