Maya Bar

BAK1 is required for the attenuation of ethylene‐inducing xylanase (Eix)‐induced defense responses by the decoy receptor LeEix1

Elicitor recognition plays a key role in the reaction of plants to pathogens and the induction of plant defense responses. Furthermore, plant-microbe interactions involve numerous regulatory systems essential for plant defense against pathogens. Ethylene-inducing xylanase (Eix) is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). The Eix receptors (LeEix1 and LeEix2) belong to a superclade of leucine-rich repeat receptor-like proteins (RLP) with a signal for receptor-mediated endocytosis, which was shown to be essential for proper induction of defense responses. Both receptors are able to bind Eix, while only LeEix2 mediates defense responses. Here we demonstrate that LeEix1 heterodimerizes with LeEix2 upon application of the Eix elicitor. We show that LeEix1 attenuates Eix-induced internalization and signaling of the LeEix2 receptor. Furthermore, we demonstrate, using yeast two-hybrid and in planta bimolecular fluorescence complementation assays, that the brassinosteroid co-receptor, BAK1, binds LeEix1 but not LeEix2. In BAK1-silenced plants, LeEix1 was no longer able to attenuate plant responses to Eix, indicating that BAK1 is required for this attenuation. We suggest that LeEix1 functions as a decoy receptor for LeEix2, a function which requires BAK1.

EHD2 inhibits ligand-induced endocytosis and signaling of the leucine-rich repeat receptor-like protein LeEix2.

Plants are constantly being challenged by aspiring pathogens. In order to protect themselves, plants have developed numerous defense mechanisms that are either specific or non-specific to the pathogen. Pattern recognition receptors can trigger plant defense responses in response to specific ligands or patterns. EIX (ethylene-inducing xylanase) triggers a defense response via the LeEix2 receptor, while bacterial flagellin triggers plant innate immunity via the FLS2 receptor. Endocytosis has been suggested to be crucial for the process in both cases. Here we show that the EIX elicitor triggers internalization of the LeEix2 receptor. Treatment with endocytosis, actin or microtubule inhibitors greatly reduced the internalization of LeEix2. Additionally, we demonstrate that plant EHD2 binds to LeEix2 and is an important factor in its internalization and in regulation of the induction of defense responses such as the hypersensitive response, ethylene biosynthesis and induction of pathogenesis-related protein expression in the case of EIX/LeEix2 (an LRR receptor lacking a kinase domain), but does not appear to be involved in the FLS2 system (an LRR receptor possessing a kinase domain). Our results suggest that various endocytosis pathways are involved in the induction of plant defense responses.

Superheating of ice crystals in antifreeze protein solutions

It has been argued that for antifreeze proteins (AFPs) to stop ice crystal growth, they must irreversibly bind to the ice surface. Surface-adsorbed AFPs should also prevent ice from melting, but to date this has been demonstrated only in a qualitative manner. Here we present the first quantitative measurements of superheating of ice in AFP solutions. Superheated ice crystals were stable for hours above their equilibrium melting point, and the maximum superheating obtained was 0.44 °C. When melting commenced in this superheated regime, rapid melting of the crystals from a point on the surface was observed. This increase in melting temperature was more appreciable for hyperactive AFPs compared to the AFPs with moderate antifreeze activity. For each of the AFP solutions that exhibited superheating, the enhancement of the melting temperature was far smaller than the depression of the freezing temperature. The present findings clearly show that AFPs adsorb to ice surfaces as part of their mechanism of action, and this absorption leads to protection of ice against melting as well as freezing.

Endosomal signaling of the tomato leucine-rich repeat receptor-like protein LeEix2

Extracellular leucine-rich repeat (LRR) receptor-like proteins (RLPs) represent a unique class of cell-surface receptors, as they lack a functional cytoplasmic domain. Our knowledge of how RLPs that do not contain a kinase or Toll domain function is very limited. The tomato RLP receptor LeEix2 signals to induce defense responses mediated by the fungal protein ethylene-inducing xylanase (EIX). The movement of FYVE-positive endosomes before and after EIX application was examined using spinning disc confocal microscopy. We found that while FYVE-positive endosomes generally observe a random movement pattern, following EIX application a subpopulation of FYVE-positive endosomes follow a directional movement pattern. Further, cellular endosomes travel greater distances at higher speeds following EIX application. Time-course experiments conducted with specific inhibitors demonstrate the involvement of endosomal signaling in EIX-triggered defense responses. Abolishing the existence of endosomes or the endocytic event prevented EIX-induced signaling. Endocytosis/endosome inhibitors, such as Dynasore or 1-butanol, inhibit EIX-induced signaling. Moreover, treatment with Endosidin1, which inhibits an early step in plasma membrane/endosome trafficking, enhances the induction of defense responses by EIX. Our data indicate a distinct endosomal signaling mechanism for induction of defense responses in this RLP system.

Leaf development and morphogenesis

The development of plant leaves follows a common basic program that is flexible and is adjusted according to species, developmental stage and environmental circumstances. Leaves initiate from the flanks of the shoot apical meristem and develop into flat structures of variable sizes and forms. This process is regulated by plant hormones, transcriptional regulators and mechanical properties of the tissue. Here, we review recent advances in the understanding of how these factors modulate leaf development to yield a substantial diversity of leaf forms. We discuss these issues in the context of leaf initiation, the balance between morphogenesis and differentiation, and patterning of the leaf margin.

The Coiled-Coil Domain of EHD2 Mediates Inhibition of LeEix2 Endocytosis and Signaling

Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis.

AtEHDs, novel Arabidopsis EH-domain-containing proteins involved in endocytosis

SUMMARY Endocytosis is an essential process by which the eukaryotic cell internalizes exogenous material. Studies in yeast and mammalian cells have revealed that endocytosis is a complex molecular process depending on regulated interactions between a variety of proteins and lipids through specific modules. One such module is the Eps15 homology (EH) domain, a conserved modular protein-interaction domain found in several endocytic proteins. The EH-domain-containing proteins function as regulators of endocytosis through their ability to interact with other proteins involved in this process. Here we describe the isolation and characterization of two Arabidopsis EH-domain-containing proteins (AtEHD1 and AtEHD2). We show that the two proteins are involved in endocytosis in plant systems and demonstrate that the Arabidopsis EHD proteins function similarly to mammalian EHDs. Similarly to hEHD2, over-expression of AtEHD2 has an inhibitory effect on endocytosis. While transgenic plants over-expressing AtEHD1 had no detectable phenotype, downregulation of AtEHD1 caused retardation of entry of endocytosed material into plant cells.

Compound leaf development in model plant species

Plant leaves develop in accordance with a common basic program, which is flexibly adjusted to the species, developmental stage and environment. Two key stages of leaf development are morphogenesis and differentiation. In the case of compound leaves, the morphogenesis stage is prolonged as compared to simple leaves, allowing for the initiation of leaflets. Here, we review recent advances in the understanding of how plant hormones and transcriptional regulators modulate compound leaf development, yielding a substantial diversity of leaf forms, focusing on four model compound leaf organisms: cardamine (Cardamine hirsuta), tomato (Solanum lycopersicum), medicago (Medicago truncatula) and pea (Pisum sativum).

A new crystal form of XT6 enables a significant improvement of its diffraction quality and resolution

Xylanases (1,4-beta-D-xylan xylanhydrolases; EC 3.2.1.8) hydrolyze the 1,4-beta-D-xylopyranosyl linkage of xylans. The detailed structural characterization of these enzymes is of interest for the elucidation of their catalytic mechanism and for their rational modification toward improved stability and specificity. An extracellular xylanase from Geobacillus stearothermophilus T-6 (XT6) has recently been cloned, overexpressed, purified and biochemically characterized. Previous crystallographic efforts resulted in a hexagonal crystal form, which subsequently proved to be of limited use for structural analysis, mainly because of its relatively poor diffraction quality and resolution. A systematic search for more suitable crystals of XT6 recently resulted in a new crystal form of this enzyme with significantly improved diffraction characteristics. The new crystals belong to a C-centred monoclinic crystal system (space group C2), with unit-cell parameters a = 121.5, b = 61.7, c = 89.1 A, beta = 119.7 degrees. These crystals diffract X-rays to better than 1.5 A resolution, showing a very clear diffraction pattern of relatively high quality. The crystals are mechanically strong and exhibit excellent radiation-stability when frozen under cold nitrogen gas. A full diffraction data set to 1.45 A resolution (94.1% completeness, R(merge) = 7.0%) has been collected from flash-frozen crystals of the native enzyme at 95 K using synchrotron radiation. Crystals of the E159A/E265A catalytic double mutant of XT6 were found to be isomorphous to those of native XT6. They were used for a full measurement of 1.8 A resolution diffraction data at 100 K (90.9% completeness; R(merge) = 5.0%). These data are currently being used for the high-resolution structure determination of XT6 and its mutant for mechanistic interpretations and rational introduction of thermostability.

EHD2 inhibits signaling of leucine rich repeat receptor-like proteins

Leucine-rich-repeat receptor protein (LRR-RLPs) and LRR-RLKs have been linked with signaling and defense responses in plants. EIX (ethylene-inducing xylanase) triggers a defense response via the LeEix2 receptor, while bacterial flagellin triggers plant innate immunity via the FLS2 receptor. Endocytosis has been suggested to be crucial for the process in both cases. Recently, we showed that the EIX elicitor triggers internalization of the LeEix2 receptor. Additionally, we demonstrate that plant EHD2 is an important factor in the internalization and regulation of the induction of plant immunity in the case of EIX/LeEix2 but does not appear to be involved in the flg/FLS2 system. Here we show that EHD2 is also involved in the signaling of the Cf4 and Cf9 receptors, causing inhibition of hypersensitive respond (HR) and ethylene biosynthesis upon over-expression of EHD2. Our results suggest that different endocytosis pathways are involved in the induction of plant defense responses.