Systemin is a small peptide with important functions in plant wound response signaling. Although the transcriptional responses of systemin action are well described, the signaling cascades involved in systemin perception and signal transduction at the protein level are poorly understood. Here we used a tomato cell suspension culture system to profile phosphoproteomic responses induced by systemin and its inactive Thr17Ala analog, allowing us to reconstruct a systemin-specific kinase/phosphatase signaling network. Our time-course analysis revealed early phosphorylation events at the plasma membrane, such as dephosphorylation of H⁺-ATPase, rapid phosphorylation of NADPH-oxidase and Ca²⁺-ATPase. Later responses involved transient phosphorylation of small GTPases, vesicle trafficking proteins and transcription factors. Based on a correlation analysis of systemin-induced phosphorylation profiles, we predicted substrate candidates for 44 early systemin-responsive kinases, which includes receptor kinases and downstream kinases such as MAP kinases, as well as nine phosphatases. We propose a regulatory module in which H⁺-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 mins of systemin treatment. We found the MAP kinase MPK2 to have increased phosphorylation level at its activating TEY-motif at 15 min post-treatment. The predicted interaction of MPK2 with LHA1 was confirmed by in vitro kinase assays, suggesting that the H⁺-ATPase LHA1 is re-activated by MPK2 later in the systemin response. Our data set provides a resource of proteomic events involved in systemin signaling that will be valuable for further in-depth functional studies in elucidation of systemin signaling cascades. Graphical Abstract Highlights Definition of early systemin-responsive phosphorylation time course. Reconstruction of kinase-substrate relationships from phosphorylation time profiles. Phosphatase PLL5 rapidly dephosphorylated H⁺-ATPase LHA1 inducing alkalinization of the medium. MAP-Kinase MPK2 re-phosphorylated LHA1 after 15 minutes.

In the early 2000s, two cucurbit-infecting begomoviruses were introduced into the eastern Mediterranean basin: the Old World Squash leaf curl virus (SLCV) and the New World Watermelon chlorotic stunt virus (WmCSV). These viruses have been emerging in parallel over the last decade in Egypt, Israel, Jordan, Lebanon and Palestine. We explored this unique situation by assessing the diversity and biogeography of the DNA-A component of SLCV and WmCSV in these five countries. There was fairly low sequence variation in both begomovirus species (SLCV π = 0.0077; WmCSV π = 0.0066). Both viruses may have been introduced only once into the eastern Mediterranean basin, but once established, these viruses readily moved across country boundaries. SLCV has been introduced at least twice into each of all five countries based on the absence of monophyletic clades. Similarly, WmCSV has been introduced multiple times into Jordan, Israel and Palestine. We predict that uncontrolled movement of whiteflies among countries in this region will continue to cause SLCV and WmCSV migration, preventing strong genetic differentiation of these viruses among these countries.

Systemin, the first signalling peptide identified in plants, mediates induced resistance against insect herbivores and necrotrophic pathogens in tomato^1–3. Initially, systemin was conceived as a hormone-like, long-distance messenger that triggers systemic defence responses far from the site of insect attack. It was later found to rather act as a phytocytokine, amplifying the local wound response for the production of downstream signals that activate defence gene expression in distant tissues⁴. Systemin perception and signalling rely on the systemin receptor SYR1⁵. However, the specifics of SYR1-dependent signalling and how systemin signalling differs from other immune signalling pathways remain largely unknown. Here we report that systemin activates the poltergeist-like phosphatase PLL2 in a SYR1-dependent manner. PLL2, in turn, regulates early systemin responses at the plasma membrane, including the rapid inhibition of proton pumps through dephosphorylation of their regulatory C-termini. PLL2 was found to be essential for downstream defence gene induction, ultimately contributing to insect resistance.