The KEEP ON GOING Protein of Arabidopsis Regulates Intracellular Protein Trafficking and Is Degraded during Fungal Infection

In plants, the trans-Golgi network and early endosomes (TGN/EE) function as the central junction for major endomembrane trafficking events, including endocytosis and secretion. Here, we demonstrate that the KEEP ON GOING (KEG) protein of Arabidopsis thaliana localizes to the TGN/EE and plays an essential role in multiple intracellular trafficking processes. Loss-of-function keg mutants exhibited severe defects in cell expansion, which correlated with defects in vacuole morphology. Confocal microscopy revealed that KEG is required for targeting of plasma membrane proteins to the vacuole. This targeting process appeared to be blocked at the step of multivesicular body (MVB) fusion with the vacuolar membrane as the MVB-associated small GTPase ARA6 was also blocked in vacuolar delivery. In addition, loss of KEG function blocked secretion of apoplastic defense proteins, indicating that KEG plays a role in plant immunity. Significantly, KEG was degraded specifically in cells infected by the fungus Golovinomyces cichoracearum, suggesting that this pathogen may target KEG to manipulate the host secretory system as a virulence strategy. Taking these results together, we conclude that KEG is a key component of TGN/EE that regulates multiple post-Golgi trafficking events in plants, including vacuole biogenesis, targeting of membrane-associated proteins to the vacuole, and secretion of apoplastic proteins.     

Essential Role of the E3 Ubiquitin Ligase NOPPERABO1 in Schizogenous Intercellular Space Formation in the Liverwort Marchantia polymorpha

The vast majority of land plants develop gas-exchange tissues with intercellular spaces (ICSs) connected directly to the air. Although the developmental processes of ICS have been described in detail at the morphological and ultrastructural level in diverse land plants, little is known about the molecular mechanism responsible for ICS formation. The liverwort Marchantia polymorpha develops a multilayered tissue with a large ICS (air chamber), whose formation is initiated at selected positions of epidermal cells. We isolated a mutant of M. polymorpha showing impaired air-chamber formation, nopperabo1 (nop1), from T-DNA–tagged lines. In nop1 plants, no ICS was formed; consequently, a single-layered epidermis developed on the dorsal side of the thallus. The causal gene NOP1 encodes a Plant U-box (PUB) E3 ubiquitin ligase carrying tandem ARMADILLO (ARM) repeats in the C terminus. An in vitro ubiquitination assay indicated that the NOP1 protein possesses E3 ubiquitin ligase activity in a U-box–dependent manner. Confocal microscopy and biochemical analysis showed that NOP1 was localized to the plasma membrane. Our investigation demonstrated the essential role of the PUB-ARM–type ubiquitin ligase in ICS formation in M. polymorpha, which sheds light on the molecular mechanism of schizogenous ICS formation in land plants.     

Arabidopsis RZFP34/CHYR1, a Ubiquitin E3 Ligase,Regulates Stomatal Movement and Drought Tolerance via SnRK2.6-Mediated Phosphorylation

Abscisic acid (ABA) is a phytohormone that plays a fundamental role in plant development and stress response, especially in the regulation of stomatal closure in response to water deficit stress. The signal transduction that occurs in response to ABA and drought stress is mediated by protein phosphorylation and ubiquitination. This research identified Arabidopsis thaliana RING ZINC-FINGER PROTEIN34 (RZP34; renamed here as CHY ZINC-FINGER AND RING PROTEIN1 [CHYR1]) as an ubiquitin E3 ligase. CHYR1 expression was significantly induced by ABA and drought, and along with its corresponding protein, was expressed mainly in vascular tissues and stomata. Analysis of CHYR1 gain-of-function and loss-of-function plants revealed that CHYR1 promotes ABA-induced stomatal closure, reactive oxygen species production, and plant drought tolerance. Furthermore, CHYR1 interacted with SNF1-RELATED PROTEIN KINASE2 (SnRK2) kinases and could be phosphorylated by SnRK2.6 on the Thr-178 residue. Overexpression of CHYR1^T178A, a phosphorylation-deficient mutant, interfered with the proper function of CHYR1, whereas CHYR1^T178D phenocopied the gain of function of CHYR1. Thus, this study identified a RING-type ubiquitin E3 ligase that functions positively in ABA and drought responses and detailed how its ubiquitin E3 ligase activity is regulated by SnRK2.6-mediated protein phosphorylation.     

The Magnaporthe oryzae Effector AvrPiz-t Targets the RING E3 Ubiquitin Ligase APIP6 to Suppress Pathogen-Associated Molecular Pattern–Triggered Immunity in Rice

Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.     

Arabidopsis Phytochrome B Promotes SPA1 Nuclear Accumulation to Repress Photomorphogenesis under Far-Red Light

Phytochrome A (phyA) is the primary photoreceptor mediating deetiolation under far-red (FR) light, whereas phyB predominantly regulates light responses in red light. SUPPRESSOR OF PHYA-105 (SPA1) forms an E3 ubiquitin ligase complex with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), which is responsible for the degradation of various photomorphogenesis-promoting factors, resulting in desensitization to light signaling. However, the role of phyB in FR light signaling and the regulatory pathway from light-activated phytochromes to the COP1-SPA1 complex are largely unknown. Here, we confirm that PHYB overexpression causes an etiolation response with reduced ELONGATED HYPOCOTYL5 (HY5) accumulation under FR light. Notably, phyB exerts its nuclear activities and promotes seedling etiolation in both the presence and absence of phyA in response to FR light. PhyB acts upstream of SPA1 and is functionally dependent on it in FR light signaling. PhyB interacts and forms a protein complex with SPA1, enhancing its nuclear accumulation under FR light. During the dark-to-FR transition, phyB is rapidly imported into the nucleus and facilitates nuclear SPA1 accumulation. These findings support the notion that phyB plays a role in repressing FR light signaling. Activity modulation of the COP1-SPA E3 complex by light-activated phytochromes is an effective and pivotal regulatory step in light signaling.     

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.     

An E4 Ligase Facilitates Polyubiquitination of Plant Immune Receptor Resistance Proteins in Arabidopsis

Proteins with nucleotide binding and leucine-rich repeat domains (NLRs) serve as immune receptors in animals and plants that recognize pathogens and activate downstream defense responses. As high accumulation of NLRs can result in unwarranted autoimmune responses, their cellular concentrations must be tightly regulated. However, the molecular mechanisms of this process are poorly detailed. The F-box protein Constitutive expressor of PR genes 1 (CPR1) was previously identified as a component of a Skp1, Cullin1, F-box protein E3 complex that targets NLRs, including Suppressor of NPR1, Constitutive 1 (SNC1) and Resistance to Pseudomonas syringae 2 (RPS2), for ubiquitination and further protein degradation. From a forward genetic screen, we identified Mutant, snc1-enhancing 3 (MUSE3), an E4 ubiquitin ligase involved in polyubiquitination of its protein targets. Knocking out MUSE3 in Arabidopsis thaliana results in increased levels of NLRs, including SNC1 and RPS2, whereas overexpressing MUSE3 together with CPR1 enhances polyubiquitination and protein degradation of these immune receptors. This report on the functional role of an E4 ligase in plants provides insight into the scarcely understood NLR degradation pathway.     

Cytoplastic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in Nicotiana benthamiana

Autophagy as a conserved catabolic pathway can respond to reactive oxygen species (ROS) and plays an important role in degrading oxidized proteins in plants under various stress conditions. However, how ROS regulates autophagy in response to oxidative stresses is largely unknown. Here, we show that autophagy-related protein 3 (ATG3) interacts with the cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs) to regulate autophagy in Nicotiana benthamiana plants. We found that oxidative stress inhibits the interaction of ATG3 with GAPCs. Silencing of GAPCs significantly activates ATG3-dependent autophagy, while overexpression of GAPCs suppresses autophagy in N. benthamiana plants. Moreover, silencing of GAPCs enhances N gene-mediated cell death and plant resistance against both incompatible pathogens Tobacco mosaic virus and Pseudomonas syringae pv tomato DC3000, as well as compatible pathogen P. syringae pv tabaci. These results indicate that GAPCs have multiple functions in the regulation of autophagy, hypersensitive response, and plant innate immunity.     

Identification of a Sphingolipid α-Glucuronosyltransferase That Is Essential for Pollen Function in Arabidopsis

Glycosyl inositol phosphorylceramide (GIPC) sphingolipids are a major class of lipids in fungi, protozoans, and plants. GIPCs are abundant in the plasma membrane in plants, comprising around a quarter of the total lipids in these membranes. Plant GIPCs contain unique glycan decorations that include a conserved glucuronic acid (GlcA) residue and various additional sugars; however, no proteins responsible for glycosylating GIPCs have been identified to date. Here, we show that the Arabidopsis thaliana protein INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE1 (IPUT1) transfers GlcA from UDP-GlcA to GIPCs. To demonstrate IPUT1 activity, we introduced the IPUT1 gene together with genes for a UDP-glucose dehydrogenase from Arabidopsis and a human UDP-GlcA transporter into a yeast mutant deficient in the endogenous inositol phosphorylceramide (IPC) mannosyltransferase. In this engineered yeast strain, IPUT1 transferred GlcA to IPC. Overexpression or silencing of IPUT1 in Nicotiana benthamiana resulted in an increase or a decrease, respectively, in IPC glucuronosyltransferase activity in vitro. Plants in which IPUT1 was silenced accumulated IPC, the immediate precursor, as well as ceramides and glucosylceramides. Plants overexpressing IPUT1 showed an increased content of GIPCs. Mutations in IPUT1 are not transmitted through pollen, indicating that these sphingolipids are essential in plants.     

Clathrin Light Chains Regulate Clathrin-Mediated Trafficking,Auxin Signaling,and Development in Arabidopsis

Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development.     

Involvement of Arabidopsis Hexokinase1 in Cell Death Mediated by Myo-Inositol Accumulation

Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. We recently identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalyzing the limiting step of myo-inositol (MI) synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD. Here, we identified a suppressor of PCD by screening for mutations that abolish the mips1 cell death phenotype. Our screen identified the hxk1 mutant, mutated in the gene encoding the hexokinase1 (HXK1) enzyme that catalyzes sugar phosphorylation and acts as a genuine glucose sensor. We show that HXK1 is required for lesion formation in mips1 due to alterations in MI content, via SA-dependant signaling. Using two catalytically inactive HXK1 mutants, we also show that hexokinase catalytic activity is necessary for the establishment of lesions in mips1. Gas chromatography-mass spectrometry analyses revealed a restoration of the MI content in mips1 hxk1 that it is due to the activity of the MIPS2 isoform, while MIPS3 is not involved. Our work defines a pathway of HXK1-mediated cell death in plants and demonstrates that two MIPS enzymes act cooperatively under a particular metabolic status, highlighting a novel checkpoint of MI homeostasis in plants.     

Cytochrome P450 CYP89A9 Is Involved in the Formation of Major Chlorophyll Catabolites during Leaf Senescence in Arabidopsis

Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis.     

Insights into the Localization and Function of the Membrane Trafficking Regulator GNOM ARF-GEF at the Golgi Apparatus in Arabidopsis

GNOM is one of the most characterized membrane trafficking regulators in plants, with crucial roles in development. GNOM encodes an ARF-guanine nucleotide exchange factor (ARF-GEF) that activates small GTPases of the ARF (ADP ribosylation factor) class to mediate vesicle budding at endomembranes. The crucial role of GNOM in recycling of PIN auxin transporters and other proteins to the plasma membrane was identified in studies using the ARF-GEF inhibitor brefeldin A (BFA). GNOM, the most prominent regulator of recycling in plants, has been proposed to act and localize at so far elusive recycling endosomes. Here, we report the GNOM localization in context of its cellular function in Arabidopsis thaliana. State-of-the-art imaging, pharmacological interference, and ultrastructure analysis show that GNOM predominantly localizes to Golgi apparatus. Super-resolution confocal live imaging microscopy identified GNOM and its closest homolog GNOM-like 1 at distinct subdomains on Golgi cisternae. Short-term BFA treatment stabilizes GNOM at the Golgi apparatus, whereas prolonged exposures results in GNOM translocation to trans-Golgi network (TGN)/early endosomes (EEs). Malformed TGN/EE in gnom mutants suggests a role for GNOM in maintaining TGN/EE function. Our results redefine the subcellular action of GNOM and reevaluate the identity and function of recycling endosomes in plants.     

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle–dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).     

Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration

Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)–mediated double-strand DNA break (DSB) repair. Here, we report a negative role of X-RAY CROSS COMPLEMENTATION GROUP4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate.