Intrinsically disordered Prosystemin discloses biologically active repeat motifs.

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.

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.

Not Only Systemin: Prosystemin Harbors Other Active Regions Able to Protect Tomato Plants.

Prosystemin is a 200-amino acid precursor expressed in Solanaceae plants which releases at the C-terminal part a peptidic hormone called Systemin in response to wounding and herbivore attack. We recently showed that Prosystemin is not only a mere scaffold of Systemin but, even when deprived of Systemin, is biologically active. These results, combined with recent discoveries that Prosystemin is an intrinsically disordered protein containing disordered regions within its sequence, prompted us to investigate the N-terminal portions of the precursor, which contribute to the greatest disorder within the sequence. To this aim, PS1-70 and PS1-120 were designed, produced, and structurally and functionally characterized. Both the fragments, which maintained their intrinsic disorder, were able to induce defense-related genes and to protect tomato plants against Botrytis cinerea and Spodoptera littoralis larvae. Intriguingly, the biological activity of each of the two N-terminal fragments and of Systemin is similar but not quite the same and does not show any toxicity on experimental non-targets considered. These regions account for different anti-stress activities conferred to tomato plants by their overexpression. The two N-terminal fragments identified in this study may represent new promising tools for sustainable crop protection.

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.

Selection of Endophytic Beauveria bassiana as a Dual Biocontrol Agent of Tomato Pathogens and Pests.

Endophytic fungi (EF) can enhance both plant growth and defense barriers against pests and pathogens, contributing to the reduction of chemical pesticides and fertilizers use in agriculture. Beauveria bassiana is an entomopathogenic fungus showing endophytism in several crops, often associated with a good capacity to limit the development of pests and disease agents. However, the diversity of the protective efficacy and plant response to different strains can be remarkable and needs to be carefully assessed for the successful and predictable use of these beneficial microorganisms. This study aims to select B. bassiana strains able to colonize tomato plants as endophytes as well as to control two important disease agents, Botrytis cinerea and Alternaria alternata, and the pest aphid, Macrosiphum euphorbiae. Nine wild-type isolates and one commercial strain were screened for endophytism, then further characterized for plant-growth promotion plus inhibition of disease development and pest infestation. Four isolates proved to have a good control activity against the biotic stressors tested, but only Bb716 was also able to promote plant growth. This work provides a simple workflow for the selection of beneficial EF, paving the way towards more effective use of B. bassiana in Integrate Pest Management (IPM) of tomato.

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.

Zucchini Plants Alter Gene Expression and Emission of (E)-ß-Caryophyllene Following Aphis gossypii Infestation.

Zucchini (Cucurbita pepo L.) is widely cultivated in temperate regions. One of the major production challenges is the damage caused by Aphis gossypii (Homoptera: Aphididae), a polyphagous aphid, which can negatively affect its host plant, both directly by feeding and indirectly by vectoring viruses. To gain insights into the transcriptome events that occur during the zucchini-aphid interaction and to understand the early-to-late defense response through gene expression profiles, we performed RNA-sequencing (RNA-Seq) on zucchini leaves challenged by A. gossypii (24, 48, and 96 h post-infestation; hpi). Data analysis indicated a complex and dynamic pattern of gene expression and a transient transcriptional reconfiguration that involved more than 700 differentially expressed genes (DEGs), including a large number of defense-related genes. The down-regulation of key genes of plant immunity, such as leucine-rich repeat (LRR) protein kinases, transcription factors, and genes associated with direct (i.e., protease inhibitors, cysteine peptidases, etc.) and indirect (i.e., terpene synthase) defense responses, suggests the aphid ability to manipulate plant immune responses. We also investigated the emission of volatile organic compounds (VOCs) from infested plants and observed a reduced emission of (E)-ß-caryophyllene at 48 hpi, likely the result of aphid effectors, which reflects the down-regulation of two genes involved in the biosynthesis of terpenoids. We showed that (E)-ß-caryophyllene emission was modified by the duration of plant infestation and by aphid density and that this molecule highly attracts Aphidius colemani, a parasitic wasp of A. gossypii. With our results we contributed to the identification of genes involved in cucurbit plant interactions with phloem feeders. Our findings may also help pave the way toward developing tolerant zucchini varieties and to identify molecules for sustainable management of harmful insect populations.

Transcriptome and Metabolome Reprogramming in Tomato Plants by Trichoderma harzianum strain T22 Primes and Enhances Defense Responses Against Aphids.

Beneficial fungi in the genus Trichoderma are among the most widespread biocontrol agents of plant pathogens. Their role in triggering plant defenses 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 toward 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 defense responses, was induced both by separate experimental treatments, which showed a synergistic interaction when concurrently applied. The most evident expression changes of defense genes were associated with the multitrophic interaction Trichoderma-tomato-aphid. Early and late genes involved in direct defense against insects were induced (i.e., peroxidase, GST, kinases and polyphenol oxidase, miraculin, chitinase), along with indirect defense 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 Trichoderma treated 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 toward the aphid parasitoid Aphidius ervi. Moreover, Trichoderma treated plants showed the over-expression of transcripts coding for several families of defense-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 defense responses.

Tomato Plants Treated with Systemin Peptide Show Enhanced Levels of Direct and Indirect Defense Associated with Increased Expression of Defense-Related Genes.

Plant defense peptides represent an important class of compounds active against pathogens and insects. These molecules controlling immune barriers can potentially be used as novel tools for plant protection, which mimic natural defense mechanisms against invaders. The constitutive expression in tomato plants of the precursor of the defense peptide systemin was previously demonstrated to increase tolerance against moth larvae and aphids and to hamper the colonization by phytopathogenic fungi, through the expression of a wealth of defense-related genes. In this work we studied the impact of the exogenous supply of systemin to tomato plants on pests to evaluate the use of the peptide as a tool for crop protection in non-transgenic approaches. By combining gene expression studies and bioassays with different pests we demonstrate that the exogenous supply of systemin to tomato plants enhances both direct and indirect defense barriers. Experimental plants, exposed to this peptide by foliar spotting or root uptake through hydroponic culture, impaired larval growth and development of the noctuid moth Spodoptera littoralis, even across generations, reduced the leaf colonization by the fungal pathogen Botrytis cinerea and were more attractive towards natural herbivore antagonists. The induction of these defense responses was found to be associated with molecular and biochemical changes under control of the systemin signalling cascade. Our results indicate that the direct delivery of systemin, likely characterized by a null effect on non-target organisms, represents an interesting tool for the sustainable protection of tomato plants.

Prosystemin, a prohormone that modulates plant defense barriers, is an intrinsically disordered protein.

Prosystemin, originally isolated from Lycopersicon esculentum, is a tomato pro-hormone of 200 aminoacid residues which releases a bioactive peptide of 18 aminoacids called Systemin. This signaling peptide is involved in the activation of defense genes in solanaceous plants in response to herbivore feeding damage. Using biochemical, biophysical and bioinformatics approaches we characterized Prosystemin, showing that it is an intrinsically disordered protein possessing a few secondary structure elements within the sequence. Plant treatment with recombinant Prosystemin promotes early and late plant defense genes, which limit the development and survival of Spodoptera littoralis larvae fed with treated plants.