FUNCTIONAL CHARACTERIZATION OF TOMATO PROSYSTEMIN AND PROSYSTEMIN REGIONS: NOVEL TOOLS FOR PLANT DEFENSE

Prosystemin (ProSys) is a pro-hormone of 200 aminoacidic residues which releases a bioactive peptide hormone of 18 amino acids called Systemin (Sys) involved in the activation of a complex signaling cascade that leads to the production of defense compounds. The tomato genome contains only one copy of Prosys gene; it is composed of 4176 bp and is structured into 11 exons, of which the last one codes for Sys. Sys peptide was traditionally considered as the principal actor that confers protection against both biotic and abiotic environmental challenges observed in tomato plant overexpressing the ProSys. Thus, a single peptide hormone is capable of eliciting multiple defense pathways to counteract a wide range of unfavourable conditions for the plant. So far, it was unknown whether ProSys had any biological function other than being an intermediate in the synthesis of Sys. However, recent evidences suggest that Prosys devoid of the Sys sequence contributes to defense responses. This observation prompted us to investigate the biochemical and structural features of the ProSys protein. To this purpose ProSys has been expressed in BL21 (DE3) E. coli cells and purified. A detailed characterization of this pro-hormone by means of multidisciplinary approach revealed for the first time that this precursor behaves like an intrinsically disordered protein (IDP) possessing intrinsically disordered regions (IDRs) within the sequence. However, to find out an alternative delivery strategy not relying on transgenic plants, we decided to investigate the effects of exogenous application of the recombinant pro-hormone on the defense responses and its potential use as a plant protection tool in tomato. In particular, plant assays revealed that ProSys direct treatment of leaves is biologically active being very effective in the induction, both locally and systemically, of tomato defense-related genes, conferring protection against different pests. To our knowledge, this is the first biotic stress related IDP identified in plants, suggesting new interesting insights on the role of IDPs. into plant response against biotic stressors. IDPs are functionally important proteins lacking a stable or ordered three-dimensional structure. Despite being highly flexible, it has been demonstrated that IDPs have crucial roles in signal transduction process, cell-cycle regulation, gene expression and molecular recognition. The role of IDPs in these processes has been systematically studied in the animal kingdom. In contrast, less reports of these proteins from the plant kingdom are available in the scientific literature. In plant biology, IDPs play crucial roles among plant stress responses, signaling, and molecular recognition pathways, that resemble the functional roles of ProSys in the tomato defense pathways activated upon several biotic and abiotic stresses. These evidences aimed our study focused on the establishment of a relationship between ProSys structure and its biological activity. To this purpose different regions of ProSys have been expressed in BL21 (DE3) E. coli cells, purified and then characterized by a biophysical and biochemical point of view. Results showed that the recombinant fragments are disordered in agreement with what previously shown for the whole precursor. It was subsequently investigated whether the recombinant ProSys Fragments had any biological activity in activating defense responses upon biotic or abiotic attacks. In particular, by using a combination of gene expression analysis and bioassays, we proved that the exogenous supply of the recombinant ProSys Fragments to tomato plants promotes early and late plant defense genes, but only two fragments (namely Fragment I and III, encompassing the N-terminal part of the protein) were found to be the most promising. In addition, it was observed that the latter ones counteracted the development of Spodoptera littoralis larvae and the fungal leaf colonization. These results suggest that the direct application of these recombinant products, which are safe to humans and no-target organisms, may represent an exploitable tool for crop protection

Transcriptome and metabolome reprogramming in tomato plants by Trichoderma harzianum strain T22 primes and enhances defence responses against aphids

Beneficial fungi in the genus Trichoderma are among the most widespread biocontrol agents of plant pathogens. Their role in triggering plant defences against pathogens has been intensely investigated, while, in contrast, very limited information is available on induced barriers active against insects. The growing experimental evidence on this latter topic looks promising, and paves the way towards the development of Trichoderma strains and/or consortia active against multiple targets. However, the predictability and reproducibility of the effects that these beneficial fungi is still somewhat limited by the lack of an in-depth understanding of the molecular mechanisms underlying the specificity of their interaction with different crop varieties, and on how the environmental factors modulate this interaction. To fill this research gap, here we studied the transcriptome changes in tomato plants (cultivar “Dwarf San Marzano”) induced by Trichoderma harzianum (strain T22) colonization and subsequent infestation by the aphid Macrosiphum euphorbiae. A wide transcriptome reprogramming, related to metabolic processes, regulation of gene expression and defence responses, was induced both by separate experimental treatments, which showed a synergistic interaction when concurrently applied. The most evident expression changes of defence genes were associated with the multitrophic interaction Trichoderma-tomato-aphid. Early and late genes involved in direct defence against insects wereinduced (i.e. peroxidase, GST, kinases and polyphenol oxidase, miraculin, chitinase), along with indirect defence genes, such as sesquiterpene synthase and geranylgeranyl phosphate synthase. Targeted and untargeted semi-polar metabolome analysis revealed a wide metabolome alteration showing an increased accumulation of isoprenoids in Trichodermatreated plants. The wide array of transcriptomic and metabolomics changes nicely fit with the higher mortality of aphids when feeding on Trichoderma treated plants,herein reported,and with the previously observed attractiveness of these latter towards the aphid parasitoid Aphidius ervi.Moreover, Trichoderma treated plants showed the over-expression of transcripts coding for several families of defence-related transcription factors (bZIP, MYB, NAC, AP2-ERF, WRKY), suggesting that the fungus contributes to the priming of plant responses against pest insects. Collectively, our data indicate that Trichoderma treatment of tomato plants induces transcriptomic and metabolomic changes, which underpin both direct and indirect defence responses

Temperature Differentially Influences the Capacity of Trichoderma Species to Induce Plant Defense Responses in Tomato Against Insect Pests

Species of the ecological opportunistic, avirulent fungus, Trichoderma are widely used in agriculture for their ability to protect crops from the attack of pathogenic fungi and for plant growth promotion activity. Recently, it has been shown that they may also have complementary properties that enhance plant defense barriers against insects. However, the use of these fungi is somewhat undermined by their variable level of biocontrol activity, which is influenced by environmental conditions. Understanding the source of this variability is essential for its profitable and wide use in plant protection. Here, we focus on the impact of temperature on Trichoderma afroharzianum T22, Trichoderma atroviride P1, and the defense response induced in tomato by insects. The in vitro development of these two strains was differentially influenced by temperature, and the observed pattern was consistent with temperature-dependent levels of resistance induced by them in tomato plants against the aphid, Macrosiphum euphorbiae, and the noctuid moth, Spodoptera littoralis. Tomato plants treated with T. afroharzianum T22 exhibited enhanced resistance toward both insect pests at 25°C, while T. atroviride P1 proved to be more effective at 20°C. The comparison of plant transcriptomic profiles generated by the two Trichoderma species allowed the identification of specific defense genes involved in the observed response, and a selected group was used to assess, by real-time quantitative reverse transcription PCR (qRT-PCR), the differential gene expression in Trichoderma-treated tomato plants subjected to the two temperature regimens that significantly affected fungal biological performance. These results will help pave the way toward a rational selection of the most suitable Trichoderma isolates for field applications, in order to best face the challenges imposed by local environmental conditions and by extreme climatic shifts due to global warming

Temperature Differentially Influences the Capacity of Trichoderma Species to Induce Plant Defense Responses in Tomato Against Insect Pests

Species of the ecological opportunistic, avirulent fungus, Trichoderma are widely used in agriculture for their ability to protect crops from the attack of pathogenic fungi and for plant growth promotion activity. Recently, it has been shown that they may also have complementary properties that enhance plant defense barriers against insects. However, the use of these fungi is somewhat undermined by their variable level of biocontrol activity, which is influenced by environmental conditions. Understanding the source of this variability is essential for its profitable and wide use in plant protection. Here, we focus on the impact of temperature on Trichoderma afroharzianum T22, Trichoderma atroviride P1, and the defense response induced in tomato by insects. The in vitro development of these two strains was differentially influenced by temperature, and the observed pattern was consistent with temperature-dependent levels of resistance induced by them in tomato plants against the aphid, Macrosiphum euphorbiae, and the noctuid moth, Spodoptera littoralis. Tomato plants treated with T. afroharzianum T22 exhibited enhanced resistance toward both insect pests at 25°C, while T. atroviride P1 proved to be more effective at 20°C. The comparison of plant transcriptomic profiles generated by the two Trichoderma species allowed the identification of specific defense genes involved in the observed response, and a selected group was used to assess, by real-time quantitative reverse transcription PCR (qRT-PCR), the differential gene expression in Trichoderma-treated tomato plants subjected to the two temperature regimens that significantly affected fungal biological performance. These results will help pave the way toward a rational selection of the most suitable Trichoderma isolates for field applications, in order to best face the challenges imposed by local environmental conditions and by extreme climatic shifts due to global warming

Tomato Prosystemin Is Much More than a Simple Systemin Precursor

Systemin (Sys) is an octadecapeptide, which upon wounding, is released from the carboxy terminus of its precursor, Prosystemin (ProSys), to promote plant defenses. Recent findings on the disordered structure of ProSys prompted us to investigate a putative biological role of the whole precursor deprived of the Sys peptide. We produced transgenic tomato plants expressing a truncated ProSys gene in which the exon coding for Sys was removed and compared their defense response with that induced by the exogenous application of the recombinant truncated ProSys (ProSys(1-178), the Prosystemin sequence devoid of Sys region). By combining protein structure analyses, transcriptomic analysis, gene expression profiling and bioassays with different pests, we demonstrate that truncated ProSys promotes defense barriers in tomato plants through a hormone-independent defense pathway, likely associated with the production of oligogalacturonides (OGs). Both transgenic and plants treated with the recombinant protein showed the modulation of the expression of genes linked with defense responses and resulted in protection against the lepidopteran pest Spodoptera littoralis and the fungus Botrytis cinerea. Our results suggest that the overall function of the wild-type ProSys is more complex than previously shown, as it might activate at least two tomato defense pathways: the well-known Sys-dependent pathway connected with the induction of jasmonic acid biosynthesis and the successive activation of a set of defense-related genes, and the ProSys(1-178)-dependent pathway associated with OGs production leading to the OGs mediate plant immunity

Plant peptides and Arbuscular mycorrhizal fungi colonization as natural enhancer of crop resistance against biotic stresses

L'obiettivo del progetto è l’identificazione di nuovi strumenti per la difesa sostenibile del pomodoro, basati sul trattamento delle piante con peptidi di origine vegetale (usati singolarmente o in varie combinazioni) e funghi micorrizici arbuscolari

Colonization of Solanum melongena and Vitis vinifera Plants by Botrytis cinerea Is Strongly Reduced by the Exogenous Application of Tomato Systemin

Plant defense peptides are able to control immune barriers and represent a potential novel resource for crop protection. One of the best-characterized plant peptides is tomato Systemin (Sys) an octadecapeptide synthesized as part of a larger precursor protein. Upon pest attack, Sys interacts with a leucine-rich repeat receptor kinase, systemin receptor SYR, activating a complex intracellular signaling pathway that leads to the wound response. Here, we demonstrated, for the first time, that the direct delivery of the peptide to Solanum melongena and Vitis vinifera plants protects from the agent of Grey mould (Botrytis cinerea). The observed disease tolerance is associated with the increase of total soluble phenolic content, the activation of antioxidant enzymes, and the up-regulation of defense-related genes in plants treated with the peptide. Our results suggest that in treated plants, the biotic defense system is triggered by the Sys signaling pathway as a consequence of Sys interaction with a SYR-like receptor recently found in several plant species, including those under investigation. We propose that this biotechnological use of Sys, promoting defense responses against invaders, represents a useful tool to integrate into pest management programs for the development of novel strategies of crop protection

Peptidi biostimolanti e bioprotettivi e loro impiego in agricoltura

La presente invenzione si riferisce a nuovi peptidi isolati aventi attività biostimolante e bioprotettiva contro lo stress abiotico e biotico nelle piante, nonché a composizioni comprendenti detti peptidi. Preferibilmente, i peptidi secondo l'invenzione provengono da piante di Solanum lycopersicum (pomodoro) e sono prodotti in modo ricombinante o sintetico. Viene anche descritto l'uso di detti peptidi e/o di detta composizione per aumentare la resistenza a stress biotici e/o abiotici in una pianta