SUMMARYElicitor 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.
). † These authors contributed equally to this work. SUMMARYExtracellular 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.
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.
Plant-microbe interactions involve numerous regulatory systems essential for plant defense against pathogens. An ethylene-inducing xylanase (Eix) of Trichoderma viride is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). We demonstrate that tomato cyclopropyl isomerase (SlCPI), an enzyme involved in sterol biosynthesis, interacts with the LeEix2 receptor. Moreover, we examined the role of SlCPI in signaling during the LeEix/Eix defense response. We found that SlCPI is an important factor in the regulation of the induction of defense responses such as the hypersensitive response, ethylene biosynthesis, and the induction of pathogenesis-related protein expression in the case of LeEix/Eix. Our results also suggest that changes in the sterol composition reduce LeEix internalization, thereby attenuating the induction of plant defense responses.
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