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The first line of plant defense occurs when a plant pattern recognition receptor (PRR) recognizes microbe‐associated molecular patterns. Plant PRRs are either receptor‐like kinases (RLKs), which have an extracellular domain for ligand binding, a single‐pass transmembrane domain, and an intracellular kinase domain for activating downstream signaling, or receptor‐like proteins (RLPs), which share the same overall structure but lack an intracellular kinase domain. The tomato (Solanum lycopersicum) LeEIX2 is an RLP that binds ethylene‐inducing xylanase (EIX), a fungal elicitor. To identify LeEIX2 receptor interactors, we conducted a yeast two‐hybrid screen and found a tomato protein that we termed SlRLK‐like. The interaction of LeEIX2 with SlRLK‐like was verified using co‐immunoprecipitation and bimolecular fluorescence complementation assays. The defense responses induced by EIX were markedly reduced when SlRLK‐like was overexpressed in Nicotiana benthamiana or Nicotiana tabacum, and knockout of SlRLK‐like using the CRISPR/Cas9 system increased EIX‐induced ethylene production and 1‐aminocyclopropane‐1‐carboxylate synthase (SlACS2) gene expression in tomato. Co‐expression of SlRLK‐like with LeEIX2 led to a reduction in its abundance, apparently through an endoplasmic reticulum‐associated degradation process. Notably, truncation of SlRLK‐like protein revealed that the malectin‐like domain is sufficient and essential for its function. Moreover, SlRLK‐like associated with the RLK FLS2, resulting in its degradation and concomitantly a reduction of the flagellin 22 (flg22)‐induced burst of reactive oxygen species. In addition, SlRLK‐like co‐expression with other RLPs, Ve1 and AtRLP23, also led to a reduction in their abundance. Our findings suggest that SlRLK‐like leads to a decreased stability of various PRRs, leading to a reduction in their abundance and resulting in attenuation of defense responses. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Challenging tomato or tobacco varieties with ethylene-inducing xylanase (EIX) from the fungus Trichoderma viride causes rapid induction of plant defence responses leading to programmed cell death. Using the yeast two-hybrid system, we isolated a novel protein, tomato small ubiquitin-related modifier protein (T-SUMO), which specifically interacts with EIX. T-SUMO, a cytoplasmic protein, is a member of the ubiquitin-like protein family. It shows homology to human protein sentrin/SUMO1, which suppresses tumour necrosis factor-induced cell death. Transgenic plants that express T-SUMO in the sense orientation suppress EIX induction of ethylene biosynthesis and cell death, while in the antisense orientation they enhance EIX-induced ethylene biosynthesis. These results indicate that T-SUMO is involved in mediating the signal generated by EIX that leads to induction of plant defence responses. [ABSTRACT FROM AUTHOR]

Summary Defense mechanisms of plants against pathogens often entail cell wall strengthening, ethylene biosynthesis, expression of pathogen-related proteins and hypersensitive responses (HR). Pathogen-derived elicitors trigger these defense responses. The Elicitor Ethylene-inducing Xylanase (EIX) elicits HR and other plant defense responses in some tobacco and tomato cultivars independently of its xylan degradation activity. The elicitation epitope on the EIX protein responsible for inducing the HR response has been elucidated. Through the generation of EIX-specific polyclonal antibodies and screening of combinatorial phage display peptide libraries an essential sequence of the EIX elicitation activity has been identified. This sequence consists of the pentapeptide TKLGE mapped to an exposed β-strand of the EIX protein. Substitution of the pentapeptide TKLGE to VKGT inhibited the elicitation activity but not the β-1-4-endoxylanase activity of the EIX protein further demonstrating that elicitation and enzyme activity are independent properties. Elucidation of a peptide sequence that is essential for elicitation of HR creates the opportunity to understand the control and signaling of plant defense. [ABSTRACT FROM AUTHOR]