K63-linked ubiquitin chains are a global signal for endocytosis and contribute to selective autophagy in plants.

In contrast to other eukaryotic model organisms, the closely related ubiquitin (Ub)-conjugating enzymes UBC35 and UBC36 are the main sources of K63-linked Ub chains in Arabidopsis.1 Although K63-linked chains have been associated with the regulation of vesicle trafficking, definitive proof for their role in endocytosis was missing. We show that the ubc35 ubc36 mutant has pleiotropic phenotypes related to hormone and immune signaling. Specifically, we reveal that ubc35-1 ubc36-1 plants have altered turnover of integral membrane proteins including FLS2, BRI1, and PIN1 at the plasma membrane. Our data indicates that K63-Ub chains are generally required for endocytic trafficking in plants. In addition, we show that in plants K63-Ub chains are involved in selective autophagy through NBR1, the second major pathway delivering cargoes to the vacuole for degradation. Similar to autophagy-defective mutants, ubc35-1 ubc36-1 plants display an accumulation of autophagy markers. Moreover, autophagy receptor NBR1 interacts with K63-Ub chains, which are required for its delivery to the lytic vacuole.2 Together, we show that K63-Ub chains act as a general signal required for the two main pathways delivering cargo to the vacuole and thus, to maintain proteostasis.

Biology of Two-Spotted Spider Mite (Tetranychus urticae): Ultrastructure, Photosynthesis, Guanine Transcriptomics, Carotenoids and Chlorophylls Metabolism, and Decoyinine as a Potential Acaricide.

Two-Spotted Spider Mites (TSSMs, Tetranychus urticae Koch 1836 (Acari: Tetranychidae)) is one of the most important pests in many crop plants, and their feeding activity is based on sucking leaf cell contents. The purpose of this study was to evaluate the interaction between TSSMs and their host Lima bean (Phaseolus lunatus) by analyzing the metabolomics of leaf pigments and the transcriptomics of TSSM guanine production. We also used epifluorescence, confocal laser scanning, and transmission electron microscopies to study the morphology and structure of TSSMs and their excreta. Finally, we evaluated the potential photosynthetic ability of TSSMs and the activity and content of Ribulose-1,5-bisphosphate Carboxylase/Oxigenase (RubisCO). We found that TSSMs express several genes involved in guanine production, including Guanosine Monophosphate Synthetase (GMPS) and decoyinine (DCY), a potential inhibitor of GMPS, was found to reduce TSSMs proliferation in infested Lima bean leaves. Despite the presence of intact chloroplasts and chlorophyll in TSSMs, we demonstrate that TSSMs do not retain any photosynthetic activity. Our results show for the first time the transcriptomics of guanine production in TSSMs and provide new insight into the catabolic activity of TSSMs on leaf chlorophyll and carotenoids. Finally, we preliminary demonstrate that DCY has an acaricidal potential against TSSMs.

Identification and Characterization of Physiological Pairing of E2 Ubiquitin-Conjugating Enzymes and E3 Ubiquitin Ligases.

The posttranslational attachment of the small protein modifier ubiquitin (Ub) is best known for its function in targeting proteins for degradation by the proteasome. However, ubiquitination also serves as a signal determining protein localization, activity, and interaction. Ubiquitination requires the sequential activity of E1 ubiquitin-activating enzyme (UBA), E2 ubiquitin-conjugating enzyme (UBC), and E3 ubiquitin ligase. Recognition of a target protein by an Ub-E2-E3 complex can result in its mono-ubiquitination (attachment of a single Ub moiety) or poly-ubiquitination, i.e., attachment of Ub chains. While the E3 ligase is important for the reaction specificity, the E2s catalyze the attachment of Ub to the target and to Ub itself to generate chains. In Arabidopsis thaliana, there are two E1s, 37 UBCs (and two ubiquitin-like conjugating enzymes) and more than 1400 E3 ligases, working in a combinatorial way. Therefore, in order to understand E3 ligase function, it is important to frame it within its possible E2s interactors. In this chapter, we propose a two-step identification and characterization of physiological E2-E3 pairs. In a first step, in vivo interacting E2s are identified through bimolecular fluorescence complementation (BiFC) using transient expression in Arabidopsis protoplast. In the second step, the activity of E2-E3 pairs is analyzed by a synthetic biology approach in which autoubiquitination is reconstituted in bacteria.

The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector.

Plant immunity is tightly controlled by a complex and dynamic regulatory network, which ensures optimal activation upon detection of potential pathogens. Accordingly, each component of this network is a potential target for manipulation by pathogens. Here, we report that RipAC, a type III-secreted effector from the bacterial pathogen Ralstonia solanacearum, targets the plant E3 ubiquitin ligase PUB4 to inhibit pattern-triggered immunity (PTI). PUB4 plays a positive role in PTI by regulating the homeostasis of the central immune kinase BIK1. Before PAMP perception, PUB4 promotes the degradation of non-activated BIK1, while after PAMP perception, PUB4 contributes to the accumulation of activated BIK1. RipAC leads to BIK1 degradation, which correlates with its PTI-inhibitory activity. RipAC causes a reduction in pathogen-associated molecular pattern (PAMP)-induced PUB4 accumulation and phosphorylation. Our results shed light on the role played by PUB4 in immune regulation, and illustrate an indirect targeting of the immune signalling hub BIK1 by a bacterial effector.

E2 ubiquitin-conjugating enzymes (UBCs): drivers of ubiquitin signalling in plants.

Most research in the field of ubiquitination has focused on E3 ubiquitin ligases because they are the specificity determinants of the ubiquitination process. Nevertheless, E2s are responsible for the catalysis during ubiquitin transfer, and are therefore, at the heart of the ubiquitination process. Arabidopsis has 37 ubiquitin E2s with additional ones mediating the attachment of ubiquitin-like proteins (e.g. SUMO, Nedd8 and ATG8). Importantly, E2s largely determine the type of ubiquitin chain built, and therefore, the type of signal that decides over the fate of the modified protein, such as degradation by the proteasome (Lys48-linked ubiquitin chains) or relocalization (Lys63-linked ubiquitin chains). Moreover, new regulatory layers impinging on E2s activity, including post-translational modifications or cofactors, are emerging that highlight the importance of E2s.

Exocyst subunit Exo70B2 is linked to immune signaling and autophagy.

During the immune response, activation of the secretory pathway is key to mounting an effective response, while gauging its output is important to maintain cellular homeostasis. The Exo70 subunit of the exocyst functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst regulation remains challenging. We show that, in Arabidopsis thaliana, Exo70B2 behaves as a bona fide exocyst subunit. Conversely, treatment with the salicylic acid (SA) defence hormone analog benzothiadiazole (BTH), or the immunogenic peptide flg22, induced Exo70B2 transport into the vacuole. We reveal that Exo70B2 interacts with AUTOPHAGY-RELATED PROTEIN 8 (ATG8) via two ATG8-interacting motives (AIMs) and its transport into the vacuole is dependent on autophagy. In line with its role in immunity, we discovered that Exo70B2 interacted with and was phosphorylated by the kinase MPK3. Mimicking phosphorylation had a dual impact on Exo70B2: first, by inhibiting localization at sites of active secretion, and second, it increased the interaction with ATG8. Phosphonull variants displayed higher effector-triggered immunity (ETI) and were hypersensitive to BTH, which induce secretion and autophagy. Our results suggest a molecular mechanism by which phosphorylation diverts Exo70B2 from the secretory into the autophagy pathway for its degradation, to dampen secretory activity.

Dissecting the plant exocyst.

The exocyst is an evolutionary conserved complex that mediates tethering of post-Golgi vesicles derived from the conventional secretory pathway to the plasma membrane (PM), before SNARE-mediated fusion. Through its tethering function, connecting secretory vesicles to the PM, it mediates spatiotemporal regulation of exocytosis. As an integral element of the secretory machinery, the exocyst has been implicated in a large variety of processes. However, emerging evidence suggests that it may also cater for unconventional secretory pathways, as well as autophagy. The exocyst entertains a multitude of interactions with proteins and membrane phospholipids, reflecting its highly dynamic nature and the complex regulatory processes that hardwire it with cellular signalling networks. However, our molecular understanding of this essential complex remains fragmentary. Here we review recent work focusing on the molecular features that have revealed both commonalities with yeast and animals, as well as unique characteristics of the plant exocyst.

Phosphoinositides control the localization of HOPS subunit VPS41, which together with VPS33 mediates vacuole fusion in plants.

The vacuole is an essential organelle in plant cells, and its dynamic nature is important for plant growth and development. Homotypic membrane fusion is required for vacuole biogenesis, pollen germination, stomata opening, and gravity perception. Known components of the vacuole fusion machinery in eukaryotes include SNARE proteins, Rab GTPases, phosphoinositides, and the homotypic fusion and vacuolar protein sorting (HOPS) tethering complex. HOPS function is not well characterized in plants, but roles in embryogenesis and pollen tube elongation have been reported. Here, we show that Arabidopsis HOPS subunits VPS33 and VPS41 accumulate in late endosomes and that VPS41, but not VPS33, accumulates in the tonoplast via a wortmannin-sensitive process. VPS41 and VPS33 proteins bind to liposomes, but this binding is inhibited by phosphatidylinosiltol-3-phosphate [PtdIns(3)P] and PtdIns(3,5)P2, which implicates a nonconserved mechanism for HOPS recruitment in plants. Inducible knockdown of VPS41 resulted in dramatic vacuole fragmentation phenotypes and demonstrated a critical role for HOPS in vacuole fusion. Furthermore, we provide evidence for genetic interactions between VPS41 and VTI11 SNARE that regulate vacuole fusion, and the requirement of a functional SNARE complex for normal VPS41 and VPS33 localization. Finally, we provide evidence to support VPS33 and SYP22 at the initial stage for HOPS-SNARE interactions, which is similar to other eukaryotes. These results highlight both conserved and specific mechanisms for HOPS recruitment and function during vacuole fusion in plants.

Vacuolar trafficking and biogenesis: a maturation in the field.

The vacuole is a prominent organelle that is essential for plant viability. The vacuole size, and its role in ion homeostasis, protein degradation and storage, place significant demands for trafficking of vacuolar cargo along the endomembrane system. Recent studies indicate that sorting of vacuolar cargo initiates at the ER and Golgi, but not the trans-Golgi network/early endosome, as previously thought. Furthermore, maturation of the trans-Golgi network into pre-vacuolar compartments seems to contribute to a major route for plant vacuolar traffic that works by bulk flow and ends with membrane fusion between the pre-vacuolar compartment and the tonoplast. Here we summarize recent evidence that indicates conserved and plant-specific mechanisms involved in sorting and trafficking of proteins to this major organelle.

Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsis zigzag1 hypocotyls.

Gravitropism in Arabidopsis shoots depends on the sedimentation of amyloplasts in the endodermis, and a complex interplay between the vacuole and F-actin. Gravity response is inhibited in zigzag-1 (zig-1), a mutant allele of VTI11, which encodes a SNARE protein involved in vacuole fusion. zig-1 seedlings have fragmented vacuoles that fuse after treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and underscore a role of phosphoinositides in vacuole fusion. Using live-cell imaging with a vertical stage microscope, we determined that young endodermal cells below the apical hook that are smaller than 70 µm in length are the graviperceptive cells in dark-grown hypocotyls. This result was confirmed by local wortmannin application to the top of zig-1 hypocotyls, which enhanced shoot gravitropism in zig-1 mutants. Live-cell imaging of zig-1 hypocotyl endodermal cells indicated that amyloplasts are trapped between juxtaposed vacuoles and their movement is severely restricted. Wortmannin-induced fusion of vacuoles in zig-1 seedlings increased the formation of transvacuolar strands, enhanced amyloplast sedimentation and partially suppressed the agravitropic phenotype of zig-1 seedlings. Hypergravity conditions at 10 g were not sufficient to displace amyloplasts in zig-1, suggesting the existence of a physical tether between the vacuole and amyloplasts. Our results overall suggest that vacuole membrane remodeling may be involved in regulating the association of vacuoles and amyloplasts during graviperception.

Metabolic engineering of the C16 homoterpene TMTT in Lotus japonicus through overexpression of (E,E)-geranyllinalool synthase attracts generalist and specialist predators in different manners.

Plant defenses against herbivores include the emission of specific blends of volatiles, which enable plants to attract natural enemies of herbivores. We characterized a plastidial terpene synthase gene, PlTPS2, from lima bean (Phaseolus lunatus). The recombinant PlTPS2 protein was multifunctional, producing linalool, (E)-nerolidol and (E,E)-geranyllinalool, precursors of (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene [TMTT]. Transgenic Lotus japonicus and Nicotiana tabacum plants, expressing PlTPS2 or its homolog Medicago truncatula TPS3 (MtTPS3), were produced and used for bioassays with herbivorous and predatory mites. Transgenic L. japonicus plants expressing PlTPS2 produced (E,E)-geranyllinalool and TMTT, whereas wild-type plants and transgenic plants expressing MtTPS3 did not. Transgenic N. tabacum expressing PlTPS2 produced (E,E)-geranyllinalool but not TMTT. Moreover, in olfactory assays, the generalist predatory mite Neoseiulus californicus but not the specialist Phytoseiulus persimilis was attracted to uninfested, transgenic L. japonicus plants expressing PlTPS2 over wild-type plants. The specialist P. persimilis was more strongly attracted by the transgenic plants infested with spider mites than by infested wild-type plants. Predator responses to transgenic plant volatile TMTT depend on various background volatiles endogenously produced by the transgenic plants. Therefore, the manipulation of TMTT is an ideal platform for pest control via the attraction of generalist and specialist predators in different manners.

Separation of early and late responses to herbivory in Arabidopsis by changing plasmodesmal function.

Herbivory results in an array of physiological changes in the host that are separable from the associated physical damage. We have made the surprising observation that an Arabidopsis line (pdko3) mutated in genes encoding plasmodesmal proteins is defective in some, but not all, of the typical plant responses to herbivory. We tested the responses of plasma transmembrane potential (Vm) depolarization, voltage gated K(+) channel activity, cytosolic calcium [Ca2+]cyt and reactive oxygen species (ROS) (H2 O2 and NO) release, shoot-to-root signaling, biosynthesis of the phytohormone jasmonic acid (JA) and the elicitation of volatile organic compounds (VOCs). Following herbivory and the release of factors present in insect oral secretions (including a putative ß-galactofuranose polysaccharide), both the pdko3 and wild type (WT) plants showed a increased accumulation of [Ca2+]cyt , NO and H2 O2 . In contrast, unlike WT plants, the mutant line showed an almost complete loss of voltage gated K(+) channel activity and Vm depolarization, a loss of shoot-induced root-Vm depolarization, a loss of activation and regulation of gene expression of the JA defense pathway, and a much diminished release and altered profile of VOCs. The mutations in genes for plasmodesmal proteins have provided valuable genetic tools for the dissection of the complex spectrum of responses to herbivory and shown us that the responses to herbivory can be separated into a calcium-activated oxidative response and a K(+) -dependent Vm-activated jasmonate response associated with the release of VOCs.

Transcriptome pyrosequencing of the parasitoid wasp Cotesia vestalis: genes involved in the antennal odorant-sensory system.

Cotesia vestalis is an endoparasitic wasp that attacks larvae of the diamondback moth (Plutella xylostella), a herbivore of cruciferous plants. Females of C. vestalis use herbivore-induced plant odorants released from plants infested by P. xylostella as a host-searching cue. Transcriptome pyrosequencing was used to identify genes in the antennae of C. vestalis adult females coding for odorant receptors (ORs) and odorant binding proteins (OBPs) involved in insect olfactory perception. Quantitative gene expression analyses showed that a few OR and OBP genes were expressed exclusively in the antenna of C. vestalis adult females whereas most other classes of genes were expressed in the antennae of both males and females, indicating their diversity in importance for the olfactory sensory system. Together, transcriptome profiling of C. vestalis genes involved in the antennal odorant-sensory system helps in detecting genes involved in host- and food-search behaviors through infochemically-mediated interactions.