The brassinosteroid-responsive protein OCTOPUS is a novel regulator of Arabidopsis thaliana immune signaling.

Phloem is a critical tissue for transport of photosynthates and extracellular signals in vascular plants. However, it also represents an ideal environment for pathogens seeking access to valuable host nutrients. Although many vascular pathogens induce economically relevant crop damage, there is still little known about the mechanisms by which immune signaling operates through the phloem. An existing phosphoproteomic dataset was mined to identify proteins that were both phosphorylated in response to the defense-elicitor flagellin (flg22) and expressed in vascular cells. A single candidate, OCTOPUS (OPS), is polarly associated with the plasma membrane of sieve element cells and has been characterized as an inhibitor of brassinosteroid insensitive-2 in promotion of brassinosteroid-related phytohormone signaling. The observation that OPS is differentially phosphorylated in response to flg22 led us to the examine whether OPS may also regulate flg22-induced immune signaling. Two independent alleles of ops exhibited enhanced immunity outputs across multiple signaling branches of PAMP-triggered immunity (PTI), constitutively and in response to flg22 treatment. Together with our observation that interactions between OPS and brassinosteroid insensitive-2 were disrupted by induction of salicylic acid and depletion of brassinosteriod, these data support a model whereby OPS modulates brassinolide and immune signaling to control downstream responses. We present OPS as a novel addition to the list of proteins with documented roles in PAMP-PTI signaling. These results further indicate that immune signaling in the phloem may be a significant and unique component of the host detection and response to pathogens in vascular plants.

Class I TCP transcription factor AtTCP8 modulates key brassinosteroid-responsive genes.

The plant-specific TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factor family is most closely associated with regulating plant developmental programs. Recently, TCPs were also shown to mediate host immune signaling, both as targets of pathogen virulence factors and as regulators of plant defense genes. However, comprehensive characterization of TCP gene targets is still lacking. Loss of function of the class I TCP gene AtTCP8 attenuates early immune signaling and, when combined with mutations in AtTCP14 and AtTCP15, additional layers of defense signaling in Arabidopsis (Arabidopsis thaliana). Here, we focus on TCP8, the most poorly characterized of the three to date. We used chromatin immunoprecipitation and RNA sequencing to identify TCP8-bound gene promoters and differentially regulated genes in the tcp8 mutant; these datasets were heavily enriched in signaling components for multiple phytohormone pathways, including brassinosteroids (BRs), auxin, and jasmonic acid. Using BR signaling as a representative example, we showed that TCP8 directly binds and activates the promoters of the key BR transcriptional regulatory genes BRASSINAZOLE-RESISTANT1 (BZR1) and BRASSINAZOLE-RESISTANT2 (BZR2/BES1). Furthermore, tcp8 mutant seedlings exhibited altered BR-responsive growth patterns and complementary reductions in BZR2 transcript levels, while TCP8 protein demonstrated BR-responsive changes in subnuclear localization and transcriptional activity. We conclude that one explanation for the substantial targeting of TCP8 alongside other TCP family members by pathogen effectors may lie in its role as a modulator of BR and other plant hormone signaling pathways.

EPSIN1 Modulates the Plasma Membrane Abundance of FLAGELLIN SENSING2 for Effective Immune Responses.

The plasma membrane (PM) provides a critical interface between plant cells and their environment to control cellular responses. To perceive the bacterial flagellin peptide flg22 for effective defense signaling, the immune receptor FLAGELLIN SENSING2 (FLS2) needs to be at its site of function, the PM, in the correct abundance. However, the intracellular machinery that controls PM accumulation of FLS2 remains largely undefined. The Arabidopsis (Arabidopsis thaliana) clathrin adaptor EPSIN1 (EPS1) is implicated in clathrin-coated vesicle formation at the trans-Golgi network (TGN), likely aiding the transport of cargo proteins from the TGN for proper location; but EPS1's impact on physiological responses remains elusive. Here, we identify EPS1 as a positive regulator of flg22 signaling and pattern-triggered immunity against Pseudomonas syringae pv tomato DC3000. We provide evidence that EPS1 contributes to modulating the PM abundance of defense proteins for effective immune signaling because in eps1, impaired flg22 signaling correlated with reduced PM accumulation of FLS2 and its coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1). The eps1 mutant also exhibited reduced responses to the pathogen/damage-associated molecular patterns elf26 and AtPep1, which are perceived by the coreceptor BAK1 and cognate PM receptors. Furthermore, quantitative proteomics of enriched PM fractions revealed that EPS1 was required for proper PM abundance of a discrete subset of proteins with different cellular functions. In conclusion, our study expands the limited understanding of the physiological roles of EPSIN family members in plants and provides novel insight into the TGN-associated clathrin-coated vesicle trafficking machinery that impacts plant PM-derived defense processes.

Simplified Enrichment of Plasma Membrane Proteins from Arabidopsis thaliana Seedlings Using Differential Centrifugation and Brij-58 Treatment.

The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.

Isolation of Microsomal Membrane Proteins from Arabidopsis thaliana.

Cellular membranes define the boundaries between organelles and the cytosol or the extracellular environment, thus providing functional separation between subcellular compartments. In addition, membranes assist in a diverse range of cellular functions, including serving as signaling platforms, mediating transport of molecules, and facilitating trafficking of cargo between cellular compartments. Because membrane functionality is largely defined by protein composition, exploring the roles of membrane proteins is of interest to many researchers. This article focuses on the subcellular fractionation of microsomes, which are membrane-derived vesicles formed during cell lysis. In plants, microsomes mainly consist of the plasma membrane and membranes derived from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and tonoplast. The article describes the different steps involved in enriching for and solubilizing microsomal membrane proteins from Arabidopsis thaliana seedlings and cultured cells by differential centrifugation. Solubilized microsomal proteins can be used for subsequent immunoblot analysis, co-immunoprecipitation, or proteomic studies. © 2016 by John Wiley & Sons, Inc.

Sensitivity to Flg22 is modulated by ligand-induced degradation and de novo synthesis of the endogenous flagellin-receptor FLAGELLIN-SENSING2.

FLAGELLIN-SENSING2 (FLS2) is the plant cell surface receptor that perceives bacterial flagellin or flg22 peptide, initiates flg22-signaling responses, and contributes to bacterial growth restriction. Flg22 elicitation also leads to ligand-induced endocytosis and degradation of FLS2 within 1 h. Why plant cells remove this receptor precisely at the time during which its function is required remains mainly unknown. Here, we assessed in planta flg22-signaling competency in the context of ligand-induced degradation of endogenous FLS2 and chemical interference known to impede flg22-dependent internalization of FLS2 into endocytic vesicles. Within 1 h after an initial flg22 treatment, Arabidopsis (Arabidopsis thaliana) leaf tissue was unable to reelicit flg22 signaling in a ligand-, time-, and dose-dependent manner. These results indicate that flg22-induced degradation of endogenous FLS2 may serve to desensitize cells to the same stimulus (homologous desensitization), likely to prevent continuous signal output upon repetitive flg22 stimulation. In addition to impeding ligand-induced FLS2 degradation, pretreatment with the vesicular trafficking inhibitors Wortmannin or Tyrphostin A23 impaired flg22-elicited reactive oxygen species production that was partially independent of BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1. Interestingly, these inhibitors did not affect flg22-induced mitogen-activated protein kinase phosphorylation, indicating the ability to utilize vesicular trafficking inhibitors to target different flg22-signaling responses. For Tyrphostin A23, reduced flg22-induced reactive oxygen species could be separated from the defect in FLS2 degradation. At later times (>2 h) after the initial flg22 elicitation, recovery of FLS2 protein levels positively correlated with resensitization to flg22, indicating that flg22-induced new synthesis of FLS2 may prepare cells for a new round of monitoring the environment for flg22.