Luminol-based Assay for Detection of Immunity Elicitor-induced Hydrogen Peroxide Production in Arabidopsis thaliana Leaves

In Arabidopsis thaliana, one of the very early immune-related responses induced after elicitor perception is the oxidative burst, i.e. reactive oxygen species (ROS) generation including superoxide anion and hydrogen peroxide (H2O2). ROS production plays different roles in a wide range of biotic and abiotic stress responses, including the closure of stomata and the regulation of cell expansion. In particular, elicitor-induced H2O2 is produced mainly by the membrane localized NAD(P)H oxidases RESPIRATORY BURST OXIDASE HOMOLOGUE D and F. In this protocol, we describe a simple and reproducible luminol/peroxidase-based assay to detect and evaluate immunity-related accumulation of H2O2 produced in Arabidopsis leaf discs treated with immunity elicitors, such as oligogalacturonides (OGs), flagellin (flg22) or the elongation factor-thermo-unstable (EF-Tu - elf18). This method is based on the detection of the luminescence released by excited-luminol molecules generated after the horseradish peroxidase (HRP)-catalyzed oxidation of luminol molecules in the presence of H2O2. Levels as well as duration of the luminescence are proportional to the amount of H2O2 produced by elicited leaf discs

Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea

Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced in response to pathogen invasion attempts at the infection site and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2′-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007). In this protocol the detection and quantification of camalexin produced in Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea is described

An aphid RNA transcript migrates systemically within plants and is a virulence factor

Aphids are sap-feeding insects that colonize a broad range of plant species and often cause feeding damage and transmit plant pathogens, including bacteria, viruses, and viroids. These insects feed from the plant vascular tissue, predominantly the phloem. However, it remains largely unknown how aphids, and other sap-feeding insects, establish intimate long-term interactions with plants. To identify aphid virulence factors, we took advantage of the ability of the green peach aphid Myzus persicae to colonize divergent plant species. We found that a M. persicae clone of near-identical females established stable colonies on nine plant species of five representative plant eudicot and monocot families that span the angiosperm phylogeny. Members of the novel aphid gene family Ya are differentially expressed in aphids on the nine plant species and are coregulated and organized as tandem repeats in aphid genomes. Aphids translocate Ya transcripts into plants, and some transcripts migrate to distal leaves within several plant species. RNAi-mediated knockdown of Ya genes reduces M. persicae fecundity, and M. persicae produces more progeny on transgenic plants that heterologously produce one of the systemically migrating Ya transcripts as a long noncoding (lnc) RNA. Taken together, our findings show that beyond a range of pathogens, M. persicae aphids translocate their own transcripts into plants, including a Ya lncRNA that migrates to distal locations within plants, promotes aphid fecundity, and is a member of a previously undescribed host-responsive aphid gene family that operate as virulence factors

The geography of technological innovation dynamics

Abstract Cities and metropolitan areas are major drivers of creativity and innovation in all possible sectors: scientific, technological, social, artistic, etc. The critical concentration and proximity of diverse mindsets and opportunities, supported by efficient infrastructures, enable new technologies and ideas to emerge, thrive, and trigger further innovation. Though this pattern seems well established, geography’s role in the emergence and diffusion of new technologies still needs to be clarified. An additional important question concerns the identification of the technological innovation pathways of metropolitan areas. Here, we explore the factors that influence the spread of technology among metropolitan areas worldwide and how geography and political borders impact this process. Our evidence suggests that political geography has been highly important for the diffusion of technological innovation till around two decades ago, slowly declining afterwards in favour of a more global patenting ecosystem. Further, the visualisation of the evolution of countries and metropolitan areas in a 2d space of competitiveness and diversification reveals the existence of two main technological innovation pathways, discriminating between different strategies towards progress. Our work provides insights for policymakers seeking to promote economic growth and technological advancement through tailored investments in prioritarian technological innovation areas

Ethylene production in Botrytis cinerea- and oligogalacturonide - induced immunity requires calcium-dependent protein kinases

Plant immunity against pathogens is achieved through rapid activation of defense responses that occur upon sensing of microbe- or damage-associated molecular pattern, respectively referred to as MAMPs and DAMPs. Oligogalacturonides (OGs), linear fragments derived from homogalacturonan hydrolysis by pathogen-secreted cell wall-degrading enzymes, and flg22, a 22-amino acid peptide derived from the bacterial flagellin, represent prototypical DAMPs and MAMPs, respectively. Both types of molecules induce protection against infections. In plants, like in animals, calcium is a second messenger that mediates responses to biotic stresses by activating calcium-binding proteins. Here we show that simultaneous loss of calcium-dependent protein kinases (CDPKs) CPK5, CPK6 and CPK11 affects Arabidopsis thaliana basal as well as elicitor- induced resistance to the necrotroph Botrytis cinerea, by affecting pathogen-induced ethylene production and accumulation of the ethylene biosynthetic enzymes 1-aminocyclopropane-1-carboxylic acid (ACC) synthase 2 (ACS2) and 6 (ACS6). Moreover, ethylene signaling contributes to OG-triggered immunity activation, and lack of CPK5, CPK6 and CPK11 affects the duration of OG- and flg22-induced gene expression, indicating that these kinases are shared elements of both DAMP and MAMP signaling pathways. This article is protected by copyright. All rights reserved

Immune responses induced by oligogalacturonides are differentially affected by AvrPto and loss of BAK1/BKK1 and PEPR1/PEPR2

Plants possess an innate immune system capable of restricting invasion by most potential pathogens. At the cell surface, the recognition of microbe-associated molecular patterns (MAMPs) and/or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) represents the first event for the prompt mounting of an effective immune response. Pathogens have evolved effectors that block MAMP-triggered immunity. The Pseudomonas syringae effector AvrPto abolishes immunity triggered by the peptide MAMPs flg22 and elf18, derived from the bacterial flagellin and elongation factor Tu, respectively, by inhibiting the kinase function of the corresponding receptors FLS2 and EFR, as well as their co-receptors BAK1 and BKK1. Oligogalacturonides (OGs), a well-known class of DAMPs, are oligomers of α-1,4-linked galacturonosyl residues, released on partial degradation of the plant cell wall homogalacturonan. We show here that AvrPto affects only a subset of the OG-triggered immune responses and that, among these responses, only a subset is affected by the concomitant loss of BAK1 and BKK1. However, the antagonistic effect on auxin-related responses is not affected by either AvrPto or the loss of BAK1/BKK1. These observations reveal an unprecedented complexity among the MAMP/DAMP response cascades. We also show that the signalling system mediated by Peps, another class of DAMPs, and their receptors PEPRs, contributes to OG-activated immunity. We hypothesize that OGs are sensed through multiple and partially redundant perception/transduction complexes, some targeted by AvrPto, but not necessarily comprising BAK1 and BKK1.n

Aphids curtail the impact of feeding damage by limiting oligogalacturonide release and suppressing cell wall associated immunity

Aphids are insect pests that use piercing-sucking mouthparts to navigate into leaf tissues and feed from phloem sap. During feeding and probing, aphids penetrate the rigid plant cell walls and deliver effector proteins mixed with saliva into the host plant cells. Cell wall-damage usually leads to the release of damage-associated molecular patterns (DAMPs), including oligogalacturonides (OGs), which promote PRR-triggered immunity (PTI) against pathogens. Whether OG-induced plant defence responses play a role in aphid-plant interactions is not fully understood. Here we show that OGs are released in aphid-exposed leaves but less so than in non-treated plants indicating that these insects may suppress the accumulation of OGs during feeding. Prior exposure of plants to OGs induces plant defense responses that reduce the ability of M. persicae to colonize plants. This plant defense is suppressed by the aphid effector Mp10 (also known as chemosensory protein 4 or CSP4) that is delivered by aphids into the cytoplasm of plant cells. Mp10 suppresses PTI responses to OGs and a variety of other elicitors. Prolonged leaf exposure to Mp10 leads to chlorosis that is dependent on SGT1 and EDS1, central regulators of intracellular NLR receptor signaling and effector-triggered immunity (ETI). However, we identified other aphid effectors that suppress ETI. Therefore, aphids actively manage the impact of feeding damage by limiting OG release and suppressing cell wall associated immunity

The aphid effector Mp10/CSP4 is conserved among hemipteran sap-feeders and targets cell surface receptor trafficking processes in plants.

<p>Sap-feeding insects of the order Hemiptera, such as aphids, whiteflies and leafhoppers, use stylets to navigate to the plant vascular system for long-term feeding. We previously demonstrated that aphids deposit the effector Mp10 into the cytoplasm of mesophyll cells. Mp10 belongs to a family of chemosensory proteins (CSPs) commonly present in arthropods. In aphids Mp10 is also known as CSP4. How CSPs regulate cellular processes in plants, and in eukaryotic cells in general, is largely unknown. We found that Mp10 suppresses PTI in response to a variety of elicitor molecules, including aphid-derived elicitors. Mp10 interacts with conserved plant target proteins that have key roles in the trafficking and stabilization of membrane proteins, in agreement with our data showing that Mp10 inhibits ROS and Ca2+ bursts to flg22 and promotes the degradation and cellular trafficking of the pattern recognition receptor FLS2. Mining of available genome-sequence data revealed that Mp10 belongs to a clade shared with homologues from other plant-feeding hemipterans, and Mp10 orthologues from other sap-feeders share PTI-suppressive functions and interactions with plant targets. Thus, CSPs in the Mp10 clade may have evolved to suppress PTI in plants early in the evolution of plant-feeding hemipterans. Given that aphids deposit Mp10 in cells upon probing, this effector appears to act as a local anesthetic to inhibit plant perception of aphid feeding.</p&gt