Breaking Bad News: Dynamic Molecular Mechanisms of Wound Response in Plants.

Recognition and repair of damaged tissue are an integral part of life. The failure of cells and tissues to appropriately respond to damage can lead to severe dysfunction and disease. Therefore, it is essential that we understand the molecular pathways of wound recognition and response. In this review, we aim to provide a broad overview of the molecular mechanisms underlying the fate of damaged cells and damage recognition in plants. Damaged cells release the so-called damage associated molecular patterns to warn the surrounding tissue. Local signaling through calcium (Ca2+), reactive oxygen species (ROS), and hormones, such as jasmonic acid, activates defense gene expression and local reinforcement of cell walls to seal off the wound and prevent evaporation and pathogen colonization. Depending on the severity of damage, Ca2+, ROS, and electrical signals can also spread throughout the plant to elicit a systemic defense response. Special emphasis is placed on the spatiotemporal dimension in order to obtain a mechanistic understanding of wound signaling in plants.

Extracellular self-DNA as a damage-associated molecular pattern (DAMP) that triggers self-specific immunity induction in plants.

Mammals sense self or non-self extracellular or extranuclear DNA fragments (hereinafter collectively termed eDNA) as indicators of injury or infection and respond with immunity. We hypothesised that eDNA acts as a damage-associated molecular pattern (DAMP) also in plants and that it contributes to self versus non-self discrimination. Treating plants and suspension-cultured cells of common bean (Phaseolus vulgaris) with fragmented self eDNA (obtained from other plants of the same species) induced early, immunity-related signalling responses such as H2O2 generation and MAPK activation, decreased the infection by a bacterial pathogen (Pseudomonas syringae) and increased an indirect defence to herbivores (extrafloral nectar secretion). By contrast, non-self DNA (obtained from lima bean, Phaseolus lunatus, and Acacia farnesiana) had significantly lower or no detectable effects. Only fragments below a size of 700 bp were active, and treating the eDNA preparation DNAse abolished its inducing effects, whereas treatment with RNAse or proteinase had no detectable effect. These findings indicate that DNA fragments, rather than small RNAs, single nucleotides or proteins, accounted for the observed effects. We suggest that eDNA functions a DAMP in plants and that plants discriminate self from non-self at a species-specific level. The immune systems of plants and mammals share multiple central elements, but further work will be required to understand the mechanisms and the selective benefits of an immunity response that is triggered by eDNA in a species-specific manner.

Sources of specificity in plant damaged-self recognition.

Plants perceive injury and herbivore attack via the recognition of damage-associated molecular patterns (DAMPs) and herbivore-associated molecular patterns (HAMPs). Although HAMPs in particular are cues that can indicate the presence of a specific enemy, the application of pure DAMPs or HAMPs frequently activates general downstream responses: membrane depolarization, Ca(2+) influxes, oxidative stress, MAPKinase activation and octadecanoid signaling at the molecular level, and the expression of digestion inhibitors, cell wall modifications and other general defenses at the phenotypic level. We discuss the relative benefits of perceiving the non-self versus the damaged-self and of specific versus non-specific responses and suggest that the perception of a complex mixture of DAMPs and HAMPs triggers fine-tuned plant responses. DAMPs such as extracellular ATP (eATP), cell wall fragments, signaling peptides, herbivore-induced volatile organic compounds (HI-VOCs) and eDNA hold the key for a more complete understanding of how plants perceive that and by whom they are attacked.

A New Approach to Produce HIV-1 Envelope Trimers: BOTH CLEAVAGE AND PROPER GLYCOSYLATION ARE ESSENTIAL TO GENERATE AUTHENTIC TRIMERS.

The trimeric envelope spike of HIV-1 mediates virus entry into human cells. The exposed part of the trimer, gp140, consists of two noncovalently associated subunits, gp120 and gp41 ectodomain. A recombinant vaccine that mimics the native trimer might elicit entry-blocking antibodies and prevent virus infection. However, preparation of authentic HIV-1 trimers has been challenging. Recently, an affinity column containing the broadly neutralizing antibody 2G12 has been used to capture recombinant gp140 and prepare trimers from clade A BG505 that naturally produces stable trimers. However, this antibody-based approach may not be as effective for the diverse HIV-1 strains with different epitope signatures. Here, we report a new and simple approach to produce HIV-1 envelope trimers. The C terminus of gp140 was attached to Strep-tag II with a long linker separating the tag from the massive trimer base and glycan shield. This allowed capture of nearly homogeneous gp140 directly from the culture medium. Cleaved, uncleaved, and fully or partially glycosylated trimers from different clade viruses were produced. Extensive biochemical characterizations showed that cleavage of gp140 was not essential for trimerization, but it triggered a conformational change that channels trimers into correct glycosylation pathways, generating compact three-blade propeller-shaped trimers. Uncleaved trimers entered aberrant pathways, resulting in hyperglycosylation, nonspecific cross-linking, and conformational heterogeneity. Even the cleaved trimers showed microheterogeneity in gp41 glycosylation. These studies established a broadly applicable HIV-1 trimer production system as well as generating new insights into their assembly and maturation that collectively bear on the HIV-1 vaccine design.

Damaged-self recognition in common bean (Phaseolus vulgaris) shows taxonomic specificity and triggers signaling via reactive oxygen species (ROS).

Plants require reliable mechanisms to detect injury. Danger signals or "damage-associated molecular patterns" (DAMPs) are released from stressed host cells and allow injury detection independently of enemy-derived molecules. We studied the response of common bean (Phaseolus vulgaris) to the application of leaf homogenate as a source of DAMPs and measured the production of reactive oxygen species (ROS) as an early response and the secretion of extrafloral nectar (EFN) as a jasmonic acid (JA)-dependent late response. We observed a strong taxonomic signal in the response to different leaf homogenates. ROS formation and EFN secretion were highly correlated and responded most strongly to leaf homogenates produced using the same cultivar or closely related accessions, less to a distantly related cultivar of common bean or each of the two congeneric species, P. lunatus and P. coccineus, and not at all to homogenates prepared from species in different genera, not even when using other Fabaceae. Interestingly, leaf homogenates also reduced the infection by the bacterial pathogen, Pseudomonas syringae, when they were applied directly before challenging, although the same homogenates exhibited no direct in vitro inhibitory effect in the bacterium. We conclude that ROS signaling is associated to the induction of EFN secretion and that the specific blend of DAMPs that are released from damaged cells allows the plant to distinguish the "damaged-self" from the damaged "non-self." The very early responses of plants to DAMPs can trigger resistance to both, herbivores and pathogens, which should be adaptive because injury facilitates infection, independently of its causal reason.