Adi Avni

The Receptor for the Fungal Elicitor Ethylene-Inducing Xylanase Is a Member of a Resistance-Like Gene Family in Tomato

An ethylene-inducing xylanase (EIX) is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum). The LeEix locus in tomatoes was characterized by map-based cloning, which led to the identification of a novel gene cluster from which two members (LeEix1 and LeEix2) were isolated. Similar to the tomato Ve resistance genes in tomato plants, the deduced amino acid sequences encoded by LeEix1 and LeEix2 contain a Leu zipper, an extracellular Leu-rich repeat domain with glycosylation signals, a transmembrane domain, and a C-terminal domain with a mammalian endocytosis signal. Silencing expression of the LeEix genes prevented the binding of EIX to cells of an EIX-responsive plant and thus inhibited the hypersensitive response. Overexpression of either LeEix1 or LeEix2 genes in EIX-nonresponsive tobacco plants enabled the binding of EIX, although only LeEix2 could transmit the signal that induced the hypersensitive response. Overexpressing LeEix2 in mammalian COS-7 cells enables binding of EIX, indicating physical interaction between the EIX elicitor and LeEix2 gene product. Structural analysis of the LeEix proteins suggests that they belong to a class of cell-surface glycoproteins with a signal for receptor-mediated endocytosis. Mutating the endocytosis signal in LeEix2 (Tyr 993 to Ala) abolished its ability to induce the hypersensitive response, suggesting that endocytosis plays a key role in the signal transduction pathway.

Induction of Ethylene Biosynthesis in Nicotiana tabacum by a Trichoderma viride Xylanase Is Correlated to the Accumulation of 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Synthase and ACC Oxidase Transcripts

Xylanase (EIX) from the fungus Trichoderma viride elicits ethylene biosynthesis in leaf tissues of Nicotiana tabacum cv Xanthi but not in cv Hicks. The increase in ethylene biosynthesis is accompanied by an accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC), an increase in extractable ACC synthase activity, and increases in ACC synthase and ACC oxidase transcripts. Priming of leaves with ethylene (120 [mu]L/L, 14 h) sensitizes the tissue, resulting in an enhanced response to EIX and increases in both the in vivo ACC oxidase activity and ACC oxidase transcript level. EIX and ethylene independently induce ACC oxidase. Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine is not accompanied by a reduction in ACC oxidase transcript level, indicating that ethylene biosynthesis is not required. In contrast to the differential induction of ethylene biosynthesis by EIX in Xanthi versus Hicks cultivars, both cultivars respond to a chemical stress (induced by CuSO4) by enhancing ethylene production. This induction is accompanied by an increase in ACC synthase transcript but not in that of ACC oxidase.

A Novel Plant Cysteine Protease Has a Dual Function as a Regulator of 1-Aminocyclopropane-1-Carboxylic Acid Synthase Gene Expression

The hormone ethylene influences plant growth, development, and some defense responses. The fungal elicitor Ethylene-Inducing Xylanase (EIX) elicits ethylene biosynthesis in tomato (Lycopersicon esculentum) and tobacco (Nicotiana tabacum) leaves by induction of 1-aminocyclopropane-1-caboxylic acid synthase (Acs) gene expression. A minimal promoter element in the LeAcs2 gene required for EIX responsiveness was defined by deletion analysis in transgenic tomato plants. The sequence between −715 and −675 of the tomato Acs2 gene was found to be essential for induction by EIX. A Cys protease (LeCp) was isolated that specifically binds to this cis element in vitro. Ectopic expression of LeCp in tomato leaves induced the expression of Acs2. Moreover, chromatin immunoprecipitation showed that LeCp binds in vivo to the Acs promoter. We propose a mechanism for the dual function of the LeCp protein. The protease acts enzymatically in the cytoplasm. Then, upon signaling, a small ubiquitin-related modifier protein binds to it, enabling entrance into the nucleus, where it acts as a transcription factor. Thus, LeCp can be considered a dual-function protein, having enzymatic activity and, upon elicitor signaling, exhibiting transcriptional factor activity that induces LeAcs2 expression.

16S rRNA Phylogeny of Sponge-Associated Cyanobacteria

ABSTRACT Phylogenetic analyses of 16S rRNA sequences of sponge-associated cyanobacteria showed them to be polyphyletic, implying that they derived from multiple independent symbiotic events. Most of the symbiont sequences were affiliated to a group of Synechococcus and Prochlorococcus species. However, other symbionts were related to different groups, such as the Oscillatoriales.

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.

Involvement of Arabidopsis ROF2 (FKBP65) in thermotolerance

The ROF2 (FKBP65) is a heat stress protein which belongs to the FK506 Binding Protein (FKBP) family. It is homologous to ROF1 (FKBP62) which was recently shown to be involved in long term acquired thermotolerance by its interaction with HSP90.1 and modulation of the heat shock transcription factor HsfA2. In this study, we have demonstrated that ROF2 participates in long term acquired thermolerance, its mode of action being different from ROF1. In the absence of ROF2, the small heat shock proteins were highly expressed and the plants were resistant to heat stress, opposite to the effect observed in the absence of ROF1. It was further demonstrated that ROF2 transcription is modulated by HsfA2 which is also essential for keeping high levels of ROF2 during recovery from heat stress. ROF2 localization to the nucleus was observed several hours after heat stress exposure and its translocation to the nucleus was independent from the presence of HSP90.1 or HsfA2. ROF2 has been shown to interact with ROF1, to form heterodimers and it is suggested that via this interaction it can join the complex ROF1-HSP90.1- HsfA2. Transient expression of ROF2 together with ROF1 repressed transcription of small HSPs. A model describing the mode of action of ROF2 as a heat stress modulator which functions in negative feedback regulation of HsfA2 is proposed.

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.

Sumoylation of Arabidopsis heat shock factor A2 (HsfA2) modifies its activity during acquired thermotholerance.

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulate many cellular processes. In this work we show SUMOylation of the heat shock transcription factor, AtHsfA2, in connection with the plant’s response to heat stress and acquired thermotolerance. Using the Yeast two hybrid and the bimolecular fluorescence complementation system, we have found that AtSUMO1 physically interacts with AtHsfA2. Further investigation allowed us to determine that Lys 315 of AtHsfA2 is the main SUMOylation site. Overexpression of AtSUMO1 led to a decrease in AtHsfA2 transcriptional activation of heat shock promoters. We have examined the effect of AtSUMO1 on AtHsfA2 during heat shock treatments. The phenotype of seedlings overexpressing AtSUMO1 resembled the phenotype of AtHsfA2 knock out seedlings, which were more sensitive than wild type seedlings to repeated heat treatment. Furthermore, AtSUMO1 overexpressing seedlings exhibited lower expression levels of small heat shock proteins as compared with wild type seedlings after heat treatment. Based on our findings, we suggest that AtSUMO1 is involved in the regulation of AtHsfA2 in acquired thermotolerance.

Enhancing plant growth and fiber production by silencing GA 2-oxidase

Enhancing plant height and growth rates is a principal objective of the fiber, pulp, wood and biomass product industries. Many biotechnological systems have been established to advance that task with emphasis on increasing the concentration of the plant hormone gibberellin, or on its signalling. In this respect, the most studied gibberellin biosynthesis enzyme is the GA 20-oxidase which catalyses the rate limiting step of the pathway. Overexpression of the gene resulted in an excessively high activity of the gibberellin deactivating enzyme, GA 2-oxidase. Consequently, this feedback regulation limits the intended outcome. We assume that silencing GA 2-oxidase transcription would abolish this antithetical effect, thereby allowing greater gibberellin accumulation. Here, we show that silencing the gibberellin deactivating enzyme in tobacco model plants results in a dramatic improvement of their growth characteristics, compared with the wild type and GA 20-oxidase over-expressing plants. Moreover, the number of xylem fiber cells in the silenced lines exceeded that of GA 20-oxidase over-expressing plants, potentially, making GA 2-oxidase silencing more profitable for the wood and fiber industries. Interestingly, crossing GA 20-oxidase over-expressing plants with GA 2-oxidase silenced plants did not yield consequential additive effects. Our findings unveil the benefits of silencing GA 2-oxidase to substantially increase tobacco growth and fiber production, which suggest using this approach in cultivated forest plantations and industrial herbaceous plants, worldwide.