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.

Enzyme‐enzyme interactions can be discovered by affinity purification mass spectrometry (AP‐MS) under in vivo conditions. Tagged enzymes can either be transiently transformed into plant leaves or stably transformed into plant cells prior to AP‐MS. The success of AP‐MS depends on the levels and stability of the bait protein, the stability of the protein‐protein interactions, and the efficiency of trypsin digestion and recovery of tryptic peptides for MS analysis. Unlike in‐gel‐digestion AP‐MS, in which the gel is cut into pieces for several independent trypsin digestions, we uses a proteomics‐based in‐solution digestion method to directly digest the proteins on the beads following affinity purification. Thus, a single replicate within an AP‐MS experiment constitutes a single sample for LC‐MS measurement. In subsequent data analysis, normalized signal intensities can be processed to determine fold‐change abundance (FC‐A) scores by use of the SAINT algorithm embedded within the CRAPome software. Following analysis of co‐sublocalization of “bait” and “prey,” we suggest considering only the protein pairs for which the intensities were more than 2% compared with the bait, corresponding to FC‐A values of at least four within‐biological replicates, which we recommend as minimum. If the procedure is faithfully followed, experimental assessment of enzyme‐enzyme interactions can be carried out in Arabidopsis within 3 weeks (transient expression) or 5 weeks (stable expression). © 2019 The Authors.Basic Protocol 1: Gene cloning to the destination vectorsAlternate Protocol: In‐Fusion or Gibson gene cloning protocolBasic Protocol 2: Transformation of baits into the plant cell culture or plant leafBasic Protocol 3: Affinity purification of protein complexesBasic Protocol 4: On‐bead trypsin/LysC digestion and C18 column peptide desalting and concentrationBasic Protocol 5: Data analysis and quality control

The study of protein–protein interactions (PPIs) is fundamental in understanding the unique role of proteins within cells and their contribution to complex biological systems. While the toolkit to study PPIs has grown immensely in mammalian and unicellular eukaryote systems over recent years, application of these techniques in plants remains under-utilized. Affinity purification coupled to mass spectrometry (AP-MS) and proximity labeling coupled to mass spectrometry (PL-MS) are two powerful techniques that have significantly enhanced our understanding of PPIs. Relying on the specific binding properties of a protein to an immobilized ligand, AP is a fast, sensitive and targeted approach used to detect interactions between bait (protein of interest) and prey (interacting partners) under near-physiological conditions. Similarly, PL, which utilizes the close proximity of proteins to identify potential interacting partners, has the ability to detect transient or hydrophobic interactions under native conditions. Combined, these techniques have the potential to reveal an unprecedented spatial and temporal protein interaction network that better understands biological processes relevant to many fields of interest. In this review, we summarize the advantages and disadvantages of two increasingly common PPI determination techniques: AP-MS and PL-MS and discuss their important application to plant systems.

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.

Feijoa sellowiana (O. Berg) is a broadly widespread fruit tree species at a very early stage of domestication. Although appreciated for its flavored berries rich in nutrients and nutraceuticals, and as an ornamental plant, feijoa is still considered an underutilized species and little information is available about its genetic background, cultivar traceability and divergence. This study aimed to investigate, for the first time, the genetic diversity of feijoa through the application of AFLPs. Specifically, twenty cultivars from different countries and six wild types (WTs) from their area of origin (Misiones, Argentina) were analyzed. The AFLPs proved to be informative, revealing the values of the percentage of polymorphic loci (PPB), Nei’s genetic diversity (h), and the Shannon index (I) at 69.36%, 0.27, and 0.43, respectively, consistent with the average of long-lived perennial and outcrossing species. However, despite the limited number of WTs examined, the genetic variability (h) was higher (approximately 37%) within the six samples compared to cultivars. The population structure analysis identified three clusters, with WTs forming a separated cluster (III) as expected. Cultivars were divided into two clusters (I and II), with cluster I exhibiting a closer genetic proximity to WTs compared to cluster II. This finding was further confirmed using the UPGMA dendrogram based on Provesti distances. This work raised awareness of the genetic variability among the feijoa’s widespread cultivars and demonstrated that the limited genetic breeding programs over the last decades resulted in low diversity among them. Moreover, these results confirm the hypothesis that all varieties are derived from a single narrow ancestral population. The potential of this species is considerable and needs to be further investigated to exploit its peculiarities.

Castanea sativa Mill. is a valuable species with historical and economic importance in Europe, particularly in the Mediterranean area. In Italy, chestnut cultivation has been developed for centuries, leading to the recognition of more than 300 varieties. Nevertheless, a profusion of local names has been assigned by growers, causing the occurrence of synonyms and homonyms across the country. This research focused on genetic characterization and identification using 21 single sequence repeats (SSRs) for four chestnut varieties (i.e., Pastinese, Nerattino, Carpinese, and Rossola) commonly used for flour production in the Tuscan Apennine Mountains (Pistoia Province). A comprehensive number of 55 accessions identified by local growers as belonging to the four varieties were analyzed, in addition to a few “Marrone” accessions as outgroups. The 21 microsatellites were highly informative, detecting 98 alleles and displaying an average polymorphism information content (PIC) equal to 0.582. In addition, a considerable amount of genetic diversity was revealed, as shown by the heterozygosity levels (He = 0.634 and Ho = 0.475). The STRUCTURE analysis provided clear distinctions among the different varieties, splitting them into four separate groups. This result was also confirmed by UPGMA dendrogram and principal co-ordinates analysis (PCoA). However, one accession (Carp_5), previously identified as Carpinese, showed an allelic profile attributable to Pastinese, suggesting that farmers might have performed mislabeling or grafting propagation errors. Thus, our results confirm the use of SSRs to allocate the accessions of different varieties, uncovering possible synonyms and homonyms. Specifically, in the context of the Pistoiese mountain region, this tool can favor the traceability of processed products, such as flour, enhancing the quality and economic value of the local market. Lastly, our findings have revealed a considerable genetic variability within the Tuscan chestnut varieties whose preservation is mandatory to face climate change challenges through sustainable forest management practices.