Trichoderma spp. are among the most widely used plant beneficial fungi in agriculture. Its interaction with the plant triggers resistance responses by the activation of Induced Systemic Resistance mediated by Jasmonic acid and Ethylene and/or Systemic Acquired Resistance, which involves Salicylic acid, with the consequent control of a wide range of plant parasites. However, the benefit they can confer to plants may be reduced or nullified by environmental conditions or fungal ecological fitness. A novel approach to enhance their effectiveness in plant defense is to combine them with bioactive molecules including plant-derived compounds. Here, we show that plant treatment with Trichoderma afroharzianum (strain T22) and Systemin, a tomato peptide active in triggering plant defense, confers protection against the fungal pathogens Fusarium oxysporum, Botrytis cinerea and the insect pest Tuta absoluta. The observed defensive response was associated with an increase of Jasmonic acid and related metabolites and a decrease of Salicylic acid.

Tomato Prosystemin (ProSys), the precursor of Systemin, a small peptidic hormone, is produced at very low concentration in unchallenged plants, while its expression greatly increases in response to several different stressors triggering an array of defence responses. The molecular mechanisms that underpin such a wide array of defence barriers are not fully understood and are likely correlated with the intrinsically disordered (ID) structure of the protein. ID proteins interact with different protein partners forming complexes involved in the modulation of different biological mechanisms. Here we describe the ProSys-protein network that shed light on the molecular mechanisms underpinning ProSys associated defence responses. Three different approaches were used. In silico prediction resulted in 98 direct interactors, most clustering in phytohormone biosynthesis, transcription factors and signal transduction gene classes. The network shows the central role of ProSys during defence responses, that reflects its role as central hub. In vitro ProSys interactors, identified by Affinity Purification-Mass Spectrometry (AP-MS), revealed over three hundred protein partners, while Bimolecular Fluorescent Complementation (BiFC) experiments validated in vivo some interactors predicted in silico and in vitro. Our results demonstrate that ProSys interacts with several proteins and reveal new key molecular events in the ProSys-dependent defence response of tomato plant.

The in-depth studies over the years on the defence barriers by tomato plants have shown that the Systemin peptide controls the response to a wealth of environmental stress agents. This multifaceted stress reaction seems to be related to the intrinsic disorder of its precursor protein, Prosystemin (ProSys). Since latest findings show that ProSys has biological functions besides Systemin sequence, here we wanted to assess if this precursor includes peptide motifs able to trigger stress-related pathways. Candidate peptides were identified in silico and synthesized to test their capacity to trigger defence responses in tomato plants against different biotic stressors. Our results demonstrated that ProSys harbours several repeat motifs which triggered plant immune reactions against pathogens and pest insects. Three of these peptides were detected by mass spectrometry in plants expressing ProSys, demonstrating their effective presence in vivo. These experimental data shed light on unrecognized functions of ProSys, mediated by multiple biologically active sequences which may partly account for the capacity of ProSys to induce defense responses to different stress agents.

Background: Plant-derived peptides can act as resistance inducers and represent promising tools for sustainable crop protection. Despite growing interest and application, their broader effects on plant-associated microbiomes remain insufficiently characterized. Here, we investigated the impact of an immunomodulatory peptide derived from the tomato defense protein Prosystemin on the tomato phyllosphere microbiome and leaf volatilome. Results: The peptide was applied as a foliar spray at biweekly intervals from planting to two months post-germination to approximate common agricultural practices. Shotgun metagenomic sequencing combined with qPCR revealed abundant bacterial communities (up to 4.6 log ₁₀ bacterial 16S rRNA gene copies) dominated by Actino-, Alphaproteo- and Gammaproteobacteria across all samples. Peptide treatment was associated with a significant shift in community structure, characterized by reduced alpha diversity and increased microbial associations. Several genera, including Acinetobacter , Sphingobium , Sphingomonas , Brevundimonas , and Massilia , increased in relative abundance following treatment. Functional profiling indicated rearrangements in gene categories related to stress response and metabolic adaptation. Notably, volatilome analysis further revealed elevated monoterpene emissions in peptide treated plants, consistent with activation of defense-associated metabolism. Members of the Sphingomonadaceae family, particularly Sphingobium yanoikuyae , appear well suited to persist under peptide-associated conditions and may therefore contribute to the observed community restructuring, although causal mechanisms remain to be tested. Conclusion: Beyond its established role in protecting tomato against pests and necrotrophic fungi, the Prosystemin-derived peptide provides an opportunity to investigate peptide-triggered plant responses and their interactions with the plant microbiota.

In an increasingly challenging agricultural environment, the identification of novel tools for protecting crops from stress agents while securing marketable production is a key objective. Here we investigated the effects of three previously characterized Prosystemin-derived functional peptide fragments as protective agents against salt stress and as biostimulants modulating tomato yield and quality traits. The treatments of tomato plants with femtomolar amounts of the peptides alleviated salt stress symptoms, likely due to an increase in root biomass up to 18% and the upregulation of key antioxidant genes such as APX2 and HSP90. In addition, the peptides exhibited biostimulant activity, significantly improving root area (up to 10%) and shoot growth (up to 9%). We validated such activities through two-year field trials carried out on industrial tomato crops. Peptide treatments confirmed their biostimulant effects, leading to a nearly 50% increase in marketable production compared to a commonly used commercial product and consistently enhancing fruit °Brix values.

The use of plant-derived peptides as resistance inducers provides innovative and potentially environmentally friendly methods to safeguard crop health. Although their use is increasing rapidly, their broader impact, particularly on plant-associated microbiomes, remains underexplored. This study investigated the influence of a promising immunomodulatory peptide derived from the tomato defense protein prosystemin on the tomato phyllosphere microbiome. We applied the peptide via foliar spray biweekly to simulate common agricultural practices from the planting stage to two months post-germination. Using a shotgun metagenomics approach combined with qPCR, we identified bacterial communities of high abundance (up to 4.6 log₁₀ bacterial 16S rRNA copies) and high diversity, mainly comprising Actino-, Alphaproteo– and Gammaproteobacteria, on all tomato leaves. The peptide treatment led to a significant and targeted shift in the bacterial community, characterized by reduced diversity and network complexity and species loss, i.e., Streptomyces. The enrichment was predominantly observed in bacterial genera such as Acinetobacter, Sphingobium, Sphingomonas, Brevundimonas, and Massilia, which are typically associated with improved plant growth and stress resilience. Intriguingly, shifts in both taxonomic and functional profile upon peptide application aligned with patterns typically observed during plant defense activation, involving jasmonic acid and related secondary metabolites. Members of the Sphingomonadaceae family, particularly Sphingobium yanoikuyae, have emerged as potential drivers of such microbial dynamics, likely adapting to plant upregulated defenses and potentially supporting its resilient phenotype. Overall, in addition to its well-established role in combating tomato pests and necrotrophic fungi, the prosystemin-derived peptide paves the way for disentangling peptide-induced resistance and its interplay with the plant microbiota.