Immune‐enhancing <scp>miPEPs</scp> reduce plant diseases and offer new solutions in agriculture

Abstract

The biggest challenge for global agriculture today is to combat pathogens, especially fungi, with fewer and fewer active molecules available. In fact, fungi are responsible for 10%–20% of crop losses, equivalent to a loss of up to $200 billion per year (www.ars.usda.gov). Moreover, the use of current fungicides (mainly chemicals) is often associated with health and environmental issues (Fungicide_Uses_and_Risks). Finally, fungi are becoming increasingly resistant to the fungicides currently used (Fungicide_Resistance_Management). In this context, the identification of new compound families controlling fungi in agriculture is of critical importance to maintain or increase global crop yields. Diseases can be managed in different ways: by direct toxicity to fungi to control fungal growth and reproduction (Fungicides) or by enhancing the plant defence response using elicitors. MiPEPs are short peptides encoded by primary transcripts of miRNAs and activate their transcription (Lauressergues et al., 2015). Due to their straightforward application through irrigation or spraying of plants with synthetic peptides, miPEPs appear as a credible alternative to chemicals in agronomy by improving crop growth and reducing weed growth (Ormancey et al., 2021). Here, we used the full set of miPEPs corresponding to conserved miRNAs (from miR156 to miR399) to identify those that can enhance PR1 expression, a gene induced in response to several pathogens, and commonly used as a marker of plant defence responses (Venegas-Molina et al., 2020). We treated Arabidopsis thaliana seedlings expressing PR1 promoter fused to the β-glucuronidase (GUS) with each of the 89 miPEPs (Lauressergues et al., 2022), in the absence of any elicitor or pathogen, and quantified GUS activity. Out of the 89 miPEPs tested, 17 miPEPs were able to significantly decrease PR1 expression, and three, miPEP169c, miPEP169h and miPEP396b, were able to increase PR1 expression (Figure 1a; Figure S1a). In order to know whether this ability to induce PR1 expression was correlated with a better resistance to pathogens, we inoculated A. thaliana plants with the necrotrophic fungus Botrytis cinerea and treated them in parallel with the three active miPEPs. Interestingly, the different miPEPs were able to decrease fungal colonization, without any direct toxicity of the peptides on fungal spores (Figure 1b; Figures S1b and S2a). To go further, we validated that each miPEP was able to up-regulate the expression of their pri-miRNA (Figures S1c–e). Then, we measured the expression of several plant defence marker genes in response to each of the miPEPs using qRT-PCR (Table S1). The expression of four marker genes was significantly induced by the three miPEPs (Figure 1c; Figure S2): PDF1.1 and PDF1.2 for miPEP169c, RD20 for miPEP396b and OSM34 for miPEP169h. Interestingly, these four genes are known to be involved in antifungal responses. PDF1.1 and PDF1.2 are defensins involved in resistance to pathogens (De Coninck et al., 2010), RD20 is a peroxygenase controlling cell death (Hanano et al., 2015) and OSM34 belongs to the osmotin family involved in cell death (Hakim et al., 2018). While the direct target genes of miR169 and miR396 are well known, the signalling pathways controlled by them are not well-characterized (Figure S3). To know whether the functions of these miPEPs are conserved among plants, we identified the entire miR169 and miR396 families in three crops: strawberry, bean and tomato, and identified their miPEPs (Table S2). Treatment of plants with these peptides allowed us to identify miPEPs reducing the incidence of B. cinerea infection in the three plant species (Figure 1d; Figures S4 and S5). Our analysis reveals very distinct roles of the different miR169 and miR396 members in these plant species (Figure S4). While some members reduce fungal infection, some others increase plant susceptibility. This explains the conflicting roles of miR169 across different species, promoting (Song et al., 2018), or decreasing plant resistance (Soto-Suárez et al., 2017). Hence, our analysis highlights the requirement to study each member of miRNA families to fully understand miRNA biological roles. We then focussed on the most active miPEP in tomato, that is SlmiPEP169d, which is potentially the closest functional homologue of AtmiPEP169c-h. We tested whether this miPEP could protect tomato against other pathogens: Alternaria solani, a fungus responsible for early blight, and Pseudomonas syringae pv. tomato, a bacterium responsible for bacterial speck. We performed infection assays on tomato plants, treated independently with the active miPEP. In each case, a decrease in disease severity was observed when plants were treated with SlmiPEP169d, correlated with a decreased quantity of bacteria in leaves (Figures 1e,f; Figure S6), confirming its activity at promoting the general resistance against pathogens. Plant diseases are responsible for huge yield losses in crop productions. To know whether the protective effect of the tomato SlmiPEP169d against different pathogens impinge on the final yield, we carried out field trials with this peptide. Interestingly, the plants treated with SlmiPEP169d showed less necrotrophic spots on leaves than untreated plants (Figure 1g). We then measured the total plant weight and the total number of fruits obtained at a time point of the crop cycle. Interestingly, SlmiPEP169d was able to improve tomato crop yields by 35% (Figures 1h,i), confirming the relevance of our approach to identify miPEPs active in crop protection through appropriate molecular screening. This work was funded by the ANR project BiomiPEP (ANR-16-CE12-0018-01). We thank FERA Science (www.fera.co.uk) for their help in phenotyping. We thank Dr Aldon (LRSV) for providing the bacterial strain. The authors declare no conflict of interest. JPC and MB designed the research; MO, BG, CB, CM and NJ performed the molecular biology and plant experiments; HSC performed bioinformatics; JPC, MB, PT and SP wrote the paper. Data S1 Material and Methods. Figure S1 Three A. thaliana miPEPs increase plant defense. Figure S2 miPEPs modulate plant defense and are not toxic agaisnt B. cinerea. Figure S3 Discussion. Figure S4 Infection assays of miPEPs on strawberry, bean and tomato. Figure S5 Pictures of B. cinerea infection assays on strawberry, bean and tomato. Figure S6 Tomato infection assays with P. syringae pv. tomato. Table S1 List of primers used. Table S2 Sequences of peptides. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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