JAZ8 Interacts With VirE3 Attenuating Agrobacterium Mediated Root Tumorigenesis

Agrobacterium tumefaciens can cause crown gall tumors by transferring both an oncogenic piece of DNA (T-DNA) and several effector proteins into a wide range of host plants. For the translocated effector VirE3 multiple functions have been reported. It acts as a transcription factor in the nucleus binding to the Arabidopsis thaliana pBrp TFIIB-like protein to activate the expression of VBF, an F-box protein involved in degradation of the VirE2 and VIP1 proteins, facilitating Agrobacterium-mediated transformation. Also VirE3 has been found at the plasma membrane, where it could interact with VirE2. Here, we identified AtJAZ8 in a yeast two-hybrid screening with VirE3 as a bait and confirmed the interaction by pull-down and bimolecular fluorescence complementation assays. We also found that the deletion of virE3 reduced Agrobacterium virulence in a root tumor assay. Overexpression of virE3 in Arabidopsis enhanced tumorigenesis, whereas overexpression of AtJAZ8 in Arabidopsis significantly decreased the numbers of tumors formed. Further experiments demonstrated that AtJAZ8 inhibited the activity of VirE3 as a plant transcriptional regulator, and overexpression of AtJAZ8 in Arabidopsis activated AtPR1 gene expression while it repressed the expression of AtPDF1.2. Conversely, overexpression of virE3 in Arabidopsis suppressed the expression of AtPR1 whereas activated the expression of AtPDF1.2. Our results proposed a novel mechanism of counter defense signaling pathways used by Agrobacterium, suggesting that VirE3 and JAZ8 may antagonistically modulate the salicylic acid/jasmonic acid (SA/JA)-mediated plant defense signaling response during Agrobacterium infection.

Deciphering the Role of Ion Channels in Early Defense Signaling against Herbivorous Insects

Plants and insect herbivores are in a relentless battle to outwit each other. Plants have evolved various strategies to detect herbivores and mount an effective defense system against them. These defenses include physical and structural barriers such as spines, trichomes, cuticle, or chemical compounds, including secondary metabolites such as phenolics and terpenes. Plants perceive herbivory by both mechanical and chemical means. Mechanical sensing can occur through the perception of insect biting, piercing, or chewing, while chemical signaling occurs through the perception of various herbivore-derived compounds such as oral secretions (OS) or regurgitant, insect excreta (frass), or oviposition fluids. Interestingly, ion channels or transporters are the first responders for the perception of these mechanical and chemical cues. These transmembrane pore proteins can play an important role in plant defense through the induction of early signaling components such as plasma transmembrane potential (Vm) fluctuation, intracellular calcium (Ca2+), and reactive oxygen species (ROS) generation, followed by defense gene expression, and, ultimately, plant defense responses. In recent years, studies on early plant defense signaling in response to herbivory have been gaining momentum with the application of genetically encoded GFP-based sensors for real-time monitoring of early signaling events and genetic tools to manipulate ion channels involved in plant-herbivore interactions. In this review, we provide an update on recent developments and advances on early signaling events in plant-herbivore interactions, with an emphasis on the role of ion channels in early plant defense signaling.

Brassica napus Mediator Subunit16 Induces BnMED25- and BnWRKY33-Activated Defense Signaling to Confer Sclerotinia sclerotiorum Resistance

The plant mediator is a highly conserved protein complex that interacts with transcription factors (TFs) and RNA polymerase II (RNAP II) to relay regulatory information during transcription. Plant immune response is one of the biological processes that is orchestrated by this regulatory mechanism. Brassica napus, an important oil crop, is severely attacked by a devastating disease Sclerotinia stem rot. Here, we explored broad-spectrum disease resistant roles of B. napus mediator subunit 16 (BnMED16) and its host defense mechanism against fugal pathogen Sclerotinia sclerotiorum. We found that BnMED16 expression was significantly increased by S. sclerotiorum infection, and its homologous overexpression resulted in rapid and comprehensive defense responses from the beginning to the end. This affected signal transduction with multiple channels including pathogen recognition, intracellular Ca2+ concentration, reactive oxygen species (ROS) accumulation and clearance, and activation of mitogen-activated protein kinase (MAPK) signaling cascades initially. Subsequently, pathogen-/defense-related genes and hormone-responsive pathways were highly activated, which resulted in enhanced cell wall and secretion of defense proteases. Furthermore, the biochemical analysis showed that BnMED16 interacts with BnMED25 and BnWRKY33. Additionally, BnMED25 also interacts with TFs BnMYC2, BnCOI1, and BnEIN3 of the JA/ET signal transduction pathway. Taken together, we proposed a hypothetical model that BnMED16 confers S. sclerotiorum resistance by enhancing BnMED25-mediated JA/ET defense pathways and BnWRKY33-activated defense signaling in B. napus. The BnMED16 overexpressing lines with enhanced broad-spectrum disease resistance could be useful for breeding Sclerotinia-resistant oilseed rape varieties, as well as serving as basis for further strategy development in resistance breeding.

Imaging-based resistance assay using enhanced luminescence-tagged Pseudomonas syringae reveals a complex epigenetic network in plant defense signaling pathways

High-throughput resistance assays in plants have a limited selection of suitable pathogens. In this study, we developed a Pseudomonas syringae strain chromosomally tagged with the NanoLuc (NL) luciferase from the deep-sea shrimp Oplophorus gracilirostris, a bioluminescent marker significantly brighter than the conventional firefly luciferase. Our reporter strain tagged with NL was over 100 times brighter than P. syringae tagged with the LuxCDABE operon from Photorhabdus luminescens, one of the existing luciferase-based strains. In planta-imaging was improved by using the surfactant Silwet L-77, particularly at a lower reporter concentration. Using this imaging system, over 30 epigenetic mutants were analyzed for their resistance traits since the defense signaling pathway is known to be epigenetically regulated. SWC1, a defense-related chromatin remodeling complex, was found to be a positive defense regulator, which supported one of two earlier conflicting reports. Compromises in DNA methylation in the CG context led to enhanced resistance against virulent Pst. DCLs and AGOs, important in the biogenesis and exerting the effector function of small RNAs, respectively, showed modest but distinct requirements for ETI and basal resistance to Pst. In addition, the transcriptional expression of an epigenetic component was found to be a significant predictor of its immunity contribution. In summary, this study showcased how a high-throughput resistance assay enabled by a pathogen strain with an improved luminescent reporter could provide insightful knowledge on complex defense signaling pathways.

Toll/interleukin-1 Receptor Domain-Only Protein of Arabidopsis Thaliana AtTX14 2IR, Produced By Alternative Splicing, Can Induce Defense Responses

Toll/interleukin -1 receptor (TIR) domains, which have NAD+ cleavage activity, are used as signaling modules in NOD-like receptors for defense responses. It has been shown that TIR domains not only form homo- or heterodimers with TIR domain-containing proteins but also interact with various proteins. A previous study showed that overexpression of Arabidopsis thaliana (Arabidopsis) AtTX14, encoding an N-terminal TIR domain and a C-terminal domain with unknown function, resulted in dwarfism and constitutive defense signaling or autoimmunity. Transgenic Arabidopsis overexpressing AtTX14 displays enhanced defense responses and associated dwarf phenotypes at 28 °C compared with those at 22 °C, which differs from other mutant or transgenic Arabidopsis with constitutive defense responses. We found that AtTX14 is alternatively spliced to encode three different proteins, and the TIR domain itself can induce autoimmunity and elevated defense responses to the bacterial pathogen Pseudomonas syringae pv. tomato. In addition, we revealed that the transcription of AtTX14 is regulated by a positive feedback mechanism. With transient overexpression of three AtTX14 protein forms in tobacco leaves, providing a heterologous system free from the positive feedback of AtTX14 in Arabidopsis, we demonstrated that expression of a splicing variant encoding the TIR domain-only protein is sufficient to activate defense signaling. A deeper understanding of interaction networks involving AtTX14 will broaden our knowledge on how plant defense signaling is regulated in response to pathogen infection and ambient temperature changes.

Negative regulation of plastidial isoprenoid pathway by herbivore-induced β-cyclocitral in Arabidopsis thaliana

Insect damage to plants is known to up-regulate defense and down-regulate growth processes. While there are frequent reports about up-regulation of defense signaling and production of defense metabolites in response to herbivory, much less is understood about the mechanisms by which growth and carbon assimilation are down-regulated. Here we demonstrate that insect herbivory down-regulates the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in Arabidopsis (Arabidopsis thaliana), a pathway making primarily metabolites for use in photosynthesis. Simulated feeding by the generalist herbivore Spodoptera littoralis suppressed flux through the MEP pathway and decreased steady-state levels of the intermediate 1-deoxy-D-xylulose 5-phosphate (DXP). Simulated herbivory also increased reactive oxygen species content which caused the conversion of β-carotene to β-cyclocitral (βCC). This volatile oxidation product affected the MEP pathway by directly inhibiting DXP synthase (DXS), the rate-controlling enzyme of the MEP pathway in Arabidopsis and inducing plant resistance against S. littoralis. βCC inhibited both DXS transcript accumulation and DXS activity. Molecular models suggested that βCC binds to DXS at the binding site for the thymine pyrophosphate cofactor and blocks catalysis, which was confirmed by direct assays of βCC with the purified DXS protein in vitro. Another intermediate of the MEP pathway, 2-C-methyl-D-erythritol-2, 4-cyclodiphosphate, which is known to stimulate salicylate defense signaling, showed greater accumulation and enhanced export out of the plastid in response to simulated herbivory. Together, our work implicates βCC as a signal of herbivore damage in Arabidopsis that increases defense and decreases flux through the MEP pathway, a pathway involved in growth and carbon assimilation.