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This work presents an in vivo stem-mounted sensor for Nicotiana tabacum plants and an in situ cell suspension sensor for Solanum lycopersicum cells. Stem-mounted sensors are mechanically stable and less sensitive to plant and air movements than the previously demonstrated leaf-mounted sensors. Interdigitated-electrode-arrays with a dual working electrode configuration were used with an auxiliary electrode and an Ag/AgCl quasi-reference electrode. Signal amplification by redox cycling is demonstrated for a plant-based sensor responding to enzyme expression induced by different cues in the plants. Functional biosensing is demonstrated, first for constitutive enzyme expression and later, for heat-shock-induced enzyme expression in plants. In the cell suspension with redox cycling, positive detection of the enzyme β-glucuronidase (GUS) was observed within a few minutes after applying the substrate (pNPG, 4-Nitrophenyl β-D-glucopyranoside), following redox reactions of the product (p-nitrophenol (pNP)). It is assumed that the initial reaction is the irreversible reduction of pNP to p-hydroxylaminophenol. Next, it can be either oxidized to p-nitrosophenol or dehydrated and oxidized to aminophenol. Both last reactions are reversible and can be used for redox cycling. The dual-electrode redox-cycling electrochemical signal was an order of magnitude larger than that of conventional single-working electrode transducers. A simple model for the gain is presented, predicting that an even larger gain is possible for sub-micron electrodes. In summary, this work demonstrates, for the first time, a redox cycling-based in vivo plant sensor, where diffusion-based amplification occurs inside a tobacco plant's tissue. The technique can be applied to other plants as well as to medical and environmental monitoring systems.

The ability to interact with plants, both to sense and to actuate, would open new opportunities for precision agriculture. These interactions can be achieved by using the plant as part of the sensing system. The present work demonstrates real-time monitoring of β-glucuronidase (GUS) expression in transgenic tobacco plants using its activity as a biomarker for functional sensing. As "proof of concept", we demonstrated GUS enzyme biosensing under constitutive expression in Msk8 tomato cells and transgenic tobacco plants and in heat shock inducible BY2 tobacco cells and tobacco plants. The sensing was done using a three-electrode microchip in Msk8 or BY2 cell culture or in tobacco plant leaves. The electrode microchip was used to transduce the expression of the GUS enzyme by chronoamperometry to a measurable electrical current signal. For the constitutive expression of GUS in Msk8 cells, the system sensitivity was 0.076 mA/mM-cm 2 and the limit of detection was 0.1 mM. For the heat shock inducible BY2 cells the GUS enzyme activity was detected 12-26 h after the heat shock was applied (40 °C for 2 h) using two different substrates: p-nitrophenyl-β-glucuronide (with sensitivity of 0.051 mA/mM-cm 2 ) and phenolphthalein-β-glucuronide (with sensitivity of 0.029 mA/mM-cm 2 )., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Plant-pathogen interactions involve a large number of wide regulatory systems, necessary for plant defense responses against pathogen attack. The fungal protein ethylene-inducing xylanase (EIX) elicits defense responses in specific cultivars of tobacco and tomato. The response to EIX is controlled by a single locus encoding for LeEIX2, a leucine-rich-repeat receptor-like-protein (LRR-RLP). As an RLP, LeEIX2 does not possess an obvious cytoplasmic signaling moiety such as a kinase domain. To study LeEIX2 mode of action, it is essential to identify the potential interactors involved after EIX perception. Here, we describe the in vivo co-IP methodology used for protein interaction verification and ethylene and ROS (reactive oxygen species) measurements used for physiological effects assessment.

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.

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., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

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.

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.

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 beta-strand of the EIX protein. Substitution of the pentapeptide TKLGE to VKGT inhibited the elicitation activity but not the beta-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.