FYVE1 Is Essential for Vacuole Biogenesis and Intracellular Trafficking in Arabidopsis

The plant vacuole is a central organelle that is involved in various biological processes throughout the plant life cycle. Elucidating the mechanism of vacuole biogenesis and maintenance is thus the basis for our understanding of these processes. Proper formation of the vacuole has been shown to depend on the intracellular membrane trafficking pathway. Although several mutants with altered vacuole morphology have been characterized in the past, the molecular basis for plant vacuole biogenesis has yet to be fully elucidated. With the aim to identify key factors that are essential for vacuole biogenesis, we performed a forward genetics screen in Arabidopsis (Arabidopsis thaliana) and isolated mutants with altered vacuole morphology. The vacuolar fusion defective1 (vfd1) mutant shows seedling lethality and defects in central vacuole formation. VFD1 encodes a Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing protein, FYVE1, that has been implicated in intracellular trafficking. FYVE1 localizes on late endosomes and interacts with Src homology-3 domain-containing proteins. Mutants of FYVE1 are defective in ubiquitin-mediated protein degradation, vacuolar transport, and autophagy. Altogether, our results show that FYVE1 is essential for plant growth and development and place FYVE1 as a key regulator of intracellular trafficking and vacuole biogenesis.The plant vacuole is the largest organelle in a plant cell in which proteins, metabolites, and ions can be stored or sequestered. The vacuole is essential for plant development and growth and is directly or indirectly involved in various biotic and abiotic stress responses (Zhang et al., 2014). The vacuole is also the central organelle for degradation of endocytic and autophagic protein substrates through the activity of vacuolar proteases. In both degradation pathways, substrates are transported to the vacuole by intracellular membrane trafficking. In endocytic degradation, plasma membrane-localized proteins are targeted to the vacuole for degradation by endosomes (Reyes et al., 2011). This process is important, among others, to control the abundance of plasma membrane receptors and thus downstream signaling events. Autophagic degradation is mainly involved in nutrient recycling. During this process, cytosolic proteins and organelles are either selectively or nonselectively transported by double membrane autophagosomes to the vacuole to be degraded (Liu and Bassham, 2012). Vacuolar transport defines an intracellular transport pathway by which de novo synthesized proteins or metabolic compounds are carried to the vacuole by vesicle transport (Drakakaki and Dandekar, 2013).In yeast (Saccharomyces cerevisiae), forward genetic screens aimed at finding mutants with defective vacuolar transport or vacuolar morphology have identified more than 30 VACUOLAR PROTEIN SORTING (VPS) and VACUOLAR MORPHOLOGY (VAM) genes (Banta et al., 1988; Raymond et al., 1992; Wada and Anraku, 1992). Closer analyses have shown that many of these mutants have defects both in protein sorting and in vacuole biogenesis, suggesting a close link between these processes. vps and vam mutants were classified into six mutant classes according to their phenotypes. The strategic success of these screens has been confirmed when later studies revealed that many of the genes categorized in the same mutant class were coding for subunits of the same protein complexes. Among them were complexes important for membrane transport and fusion events, such as the endosomal sorting complex required for transport (ESCRT)-I to ESCRT-III (Henne et al., 2011) or the homotypic fusion and vacuole protein sorting (HOPS) complex (Balderhaar and Ungermann, 2013).Sequence homologs of most yeast VPS genes can be found in the Arabidopsis (Arabidopsis thaliana) genome (Sanderfoot and Raikhel, 2003; Bassham et al., 2008), and some of them were reported to be involved in intracellular trafficking as well as vacuole biogenesis. For example, the Arabidopsis vacuoleless (vcl)/vps16 mutant is embryo lethal and lacks lytic vacuoles (Rojo et al., 2001). VPS16 is a subunit of the HOPS complex, suggesting that membrane fusion events mediated by VCL/VPS16 are also important for plant vacuole biogenesis. Several other Arabidopsis vps mutants were also shown to have altered vacuole morphology at the mature embryo stage (Shimada et al., 2006; Sanmartín et al., 2007; Ebine et al., 2008, 2014; Yamazaki et al., 2008; Zouhar et al., 2009; Shahriari et al., 2010), showing that there is a conserved mechanism regulating vacuolar transport and vacuole biogenesis. However, in contrast to yeast, in which mutants without vacuole or severe biogenesis defects are viable, plant vacuoles seem to be essential for plant development.We have previously shown that defects in the deubiquitinating enzyme (DUB) ASSOCIATED MOLECULE WITH THE Src homology-3 DOMAIN OF STAM3 (AMSH3) also lead to a severe vacuole biogenesis defect (Isono et al., 2010). AMSH homologs do not exist in budding yeast but are conserved in animals and plants. Our previous studies have shown that AMSH3 can directly interact with ESCRT-III subunits (Katsiarimpa et al., 2013). ESCRT-III is a multiprotein complex that is essential for multivesicular body (MVB) sorting (Winter and Hauser, 2006) and hence for plant growth and development (Haas et al., 2007; Spitzer et al., 2009; Katsiarimpa et al., 2011; Cai et al., 2014). AMSH proteins regulate intracellular trafficking events, including endocytic degradation, vacuolar transport, and autophagic degradation through its interaction with ESCRT-III (Isono et al., 2010; Katsiarimpa et al., 2011, 2013, 2014). Prior to our characterization of the amsh3 mutant, AMSH proteins had not been implicated in vacuole biogenesis. Thus, we reasoned that there might be additional, yet unidentified, factors important for regulating vacuole biogenesis in plants. Further, we reasoned that other mutants with a defect in vacuole biogenesis, analogous to amsh3, might also exhibit seedling lethality.Thus, with the goal to identify and characterize these factors, we carried out a two-step mutant screen. We first selected seedling lethal mutants from an ethyl methansulfonate (EMS)-mutagenized population and then examined the vacuole morphology in these mutants. The isolated mutants were designated vacuolar fusion defective (vfd). vfd1 is affected in the expression of a functional Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing FYVE1 protein. FYVE1 was originally identified in silico as one of 16 FYVE domain-containing proteins in Arabidopsis with no apparent homologs in yeast and mammals (van Leeuwen et al., 2004). FYVE domains bind phosphatidylinositol 3-P, a phospholipid that is a major constituent of endosomal membranes. Hence, FYVE domain-containing proteins are implicated in intracellular trafficking (van Leeuwen et al., 2004; Wywial and Singh, 2010). In a previous work, we have shown that a null mutant of FYVE1, fyve1-1, is defective in IRON-REGULATED TRANSPORTER1 (IRT1) polarization and that FYVE1 is essential for plant growth and development (Barberon et al., 2014). A very recent publication describing the same mutant has shown that FYVE1/FYVE domain protein required for endosomal sorting1 (FREE1) is also important for the early and late endosomal trafficking events (Gao et al., 2014). In this study, we show that FYVE1 is also regulating ubiquitin-dependent membrane protein degradation, vacuolar transport, autophagy, and vacuole biogenesis. Altogether, our results point toward FYVE1 being a key component of the intracellular trafficking machinery in plants.     

The Deubiquitinating Enzyme AMSH1 and the ESCRT-III Subunit VPS2.1 Are Required for Autophagic Degradation in Arabidopsis

In eukaryotes, posttranslational modification by ubiquitin regulates the activity and stability of many proteins and thus influences a variety of developmental processes as well as environmental responses. Ubiquitination also plays a critical role in intracellular trafficking by serving as a signal for endocytosis. We have previously shown that the Arabidopsis thaliana ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM3 (AMSH3) is a deubiquitinating enzyme (DUB) that interacts with ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT-III (ESCRT-III) and is essential for intracellular transport and vacuole biogenesis. However, physiological functions of AMSH3 in the context of its ESCRT-III interaction are not well understood due to the severe seedling lethal phenotype of its null mutant. In this article, we show that Arabidopsis AMSH1, an AMSH3-related DUB, interacts with the ESCRT-III subunit VACUOLAR PROTEIN SORTING2.1 (VPS2.1) and that impairment of both AMSH1 and VPS2.1 causes early senescence and hypersensitivity to artificial carbon starvation in the dark similar to previously reported autophagy mutants. Consistent with this, both mutants accumulate autophagosome markers and accumulate less autophagic bodies in the vacuole. Taken together, our results demonstrate that AMSH1 and the ESCRT-III-subunit VPS2.1 are important for autophagic degradation and autophagy-mediated physiological processes.     

pH Regulation by NHX-Type Antiporters Is Required for Receptor-Mediated Protein Trafficking to the Vacuole in Arabidopsis

Protein trafficking requires proper ion and pH homeostasis of the endomembrane system. The NHX-type Na⁺/H⁺ antiporters NHX5 and NHX6 localize to the Golgi, trans-Golgi network, and prevacuolar compartments and are required for growth and trafficking to the vacuole. In the nhx5 nhx6 T-DNA insertional knockouts, the precursors of the 2S albumin and 12S globulin storage proteins accumulated and were missorted to the apoplast. Immunoelectron microscopy revealed the presence of vesicle clusters containing storage protein precursors and vacuolar sorting receptors (VSRs). Isolation and identification of complexes of VSRs with unprocessed 12S globulin by 2D blue-native PAGE/SDS-PAGE indicated that the nhx5 nhx6 knockouts showed compromised receptor-cargo association. In vivo interaction studies using bimolecular fluorescence complementation between VSR2;1, aleurain, and 12S globulin suggested that nhx5 nhx6 knockouts showed a significant reduction of VSR binding to both cargoes. In vivo pH measurements indicated that the lumens of VSR compartments containing aleurain, as well as the trans-Golgi network and prevacuolar compartments, were significantly more acidic in nhx5 nhx6 knockouts. This work demonstrates the importance of NHX5 and NHX6 in maintaining endomembrane luminal pH and supports the notion that proper vacuolar trafficking and proteolytic processing of storage proteins require endomembrane pH homeostasis.     

The Endoplasmic Reticulum Is the Main Membrane Source for Biogenesis of the Lytic Vacuole in Arabidopsis

Vacuoles are multifunctional organelles essential for the sessile lifestyle of plants. Despite their central functions in cell growth, storage, and detoxification, knowledge about mechanisms underlying their biogenesis and associated protein trafficking pathways remains limited. Here, we show that in meristematic cells of the Arabidopsis thaliana root, biogenesis of vacuoles as well as the trafficking of sterols and of two major tonoplast proteins, the vacuolar H⁺-pyrophosphatase and the vacuolar H⁺-adenosinetriphosphatase, occurs independently of endoplasmic reticulum (ER)–Golgi and post-Golgi trafficking. Instead, both pumps are found in provacuoles that structurally resemble autophagosomes but are not formed by the core autophagy machinery. Taken together, our results suggest that vacuole biogenesis and trafficking of tonoplast proteins and lipids can occur directly from the ER independent of Golgi function.     

miRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival

miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha ^loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.     

Gibberellin Is Required for Flowering in Arabidopsis thaliana under Short Days

Mutants of Arabidopsis thaliana deficient in gibberellin synthesis (ga1-3 and ga1-6), and a gibberellin-insensitive mutant (gai) were compared to the wild-type (WT) Landsberg erecta line for flowering time and leaf number when grown in either short days (SD) or continuous light (CL). The ga1-3 mutant, which is severely defective in ent-kaurene synthesis because it lacks most of the GA1 gene, never flowered in SD unless treated with exogenous gibberellin. After a prolonged period of vegetative growth, this mutant eventually underwent senescence without having produced flower buds. The gai mutant and the “leaky” ga1-6 mutant did flower in SD, but took somewhat longer than WT. All the mutants flowered readily in CL, although the ga1-3 mutant showed some delay. Unlike WT and ga1-3, the gai mutant failed to respond to gibberellin treatment by accelerating flowering in SD. A cold treatment promoted flowering in the WT and gai, but failed to induce flowering in ga1-3. From these results, it appears that gibberellin normally plays a role in initiating flowering of Arabidopsis.     

Auxin-Mediated Ribosomal Biogenesis Regulates Vacuolar Trafficking in Arabidopsis

In plants, the mechanisms that regulate the transit of vacuolar soluble proteins containing C-terminal and N-terminal vacuolar sorting determinants (VSDs) to the vacuole are largely unknown. In a screen for Arabidopsis thaliana mutants affected in the trafficking of C-terminal VSD containing proteins, we isolated the ribosomal biogenesis mutant rpl4a characterized by its partial secretion of vacuolar targeted proteins and a plethora of developmental phenotypes derived from its aberrant auxin responses. In this study, we show that ribosomal biogenesis can be directly regulated by auxins and that the exogenous application of auxins to wild-type plants results in vacuolar trafficking defects similar to those observed in rpl4a mutants. We propose that the influence of auxin on ribosomal biogenesis acts as a regulatory mechanism for auxin-mediated developmental processes, and we demonstrate the involvement of this regulatory mechanism in the sorting of vacuolar targeted proteins in Arabidopsis.     

STARCH-EXCESS4 Is a Laforin-Like Phosphoglucan Phosphatase Required for Starch Degradation in Arabidopsis thaliana

Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear α-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases α-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism.     

Phospholipase A2 Is Required for PIN-FORMED Protein Trafficking to the Plasma Membrane in the Arabidopsis Root

Phospholipase A₂ (PLA₂), which hydrolyzes a fatty acyl chain of membrane phospholipids, has been implicated in several biological processes in plants. However, its role in intracellular trafficking in plants has yet to be studied. Here, using pharmacological and genetic approaches, the root hair bioassay system, and PIN-FORMED (PIN) auxin efflux transporters as molecular markers, we demonstrate that plant PLA₂s are required for PIN protein trafficking to the plasma membrane (PM) in the Arabidopsis thaliana root. PLA₂α, a PLA₂ isoform, colocalized with the Golgi marker. Impairments of PLA₂ function by PLA₂α mutation, PLA₂-RNA interference (RNAi), or PLA₂ inhibitor treatments significantly disrupted the PM localization of PINs, causing internal PIN compartments to form. Conversely, supplementation with lysophosphatidylethanolamine (the PLA₂ hydrolytic product) restored the PM localization of PINs in the pla₂α mutant and the ONO-RS-082–treated seedling. Suppression of PLA₂ activity by the inhibitor promoted accumulation of trans-Golgi network vesicles. Root hair–specific PIN overexpression (PINox) lines grew very short root hairs, most likely due to reduced auxin levels in root hair cells, but PLA₂ inhibitor treatments, PLA₂α mutation, or PLA₂-RNAi restored the root hair growth of PINox lines by disrupting the PM localization of PINs, thus reducing auxin efflux. These results suggest that PLA₂, likely acting in Golgi-related compartments, modulates the trafficking of PIN proteins.     

SORTING NEXIN1 Is Required for Modulating the Trafficking and Stability of the Arabidopsis IRON-REGULATED TRANSPORTER1

Dicotyledonous plants growing under limited iron availability initiate a response resulting in the solubilization, reduction, and uptake of soil iron. The protein factors responsible for these steps are transmembrane proteins, suggesting that the intracellular trafficking machinery may be involved in iron acquisition. In search for components involved in the regulation of Arabidopsis thaliana iron deficiency responses, we identified the members of the SORTING NEXIN (SNX) protein family. SNX loss-of-function plants display enhanced susceptibility to iron deficiency in comparison to the wild type. The absence of SNX led to reduced iron import efficiency into the root. SNX1 showed partial colocalization with the principal root iron importer IRON-REGULATED TRANSPORTER1 (IRT1). In SNX loss-of-function plants, IRT1 protein levels were decreased compared with the wild type due to enhanced IRT1 degradation. This resulted in diminished amounts of the IRT1 protein at the plasma membrane. snx mutants exhibited enhanced iron deficiency responses compared with the wild type, presumably due to the lower iron uptake through IRT1. Our results reveal a role of SNX1 for the correct trafficking of IRT1 and, thus, for modulating the activity of the iron uptake machinery.     

Arabidopsis ATG8-INTERACTING PROTEIN1 Is Involved in Autophagy-Dependent Vesicular Trafficking of Plastid Proteins to the Vacuole

Selective autophagy has been extensively studied in various organisms, but knowledge regarding its functions in plants, particularly in organelle turnover, is limited. We have recently discovered ATG8-INTERACTING PROTEIN1 (ATI1) from Arabidopsis thaliana and showed that following carbon starvation it is localized on endoplasmic reticulum (ER)-associated bodies that are subsequently transported to the vacuole. Here, we show that following carbon starvation ATI1 is also located on bodies associating with plastids, which are distinct from the ER ATI bodies and are detected mainly in senescing cells that exhibit plastid degradation. Additionally, these plastid-localized bodies contain a stroma protein marker as cargo and were observed budding and detaching from plastids. ATI1 interacts with plastid-localized proteins and was further shown to be required for the turnover of one of them, as a representative. ATI1 on the plastid bodies also interacts with ATG8f, which apparently leads to the targeting of the plastid bodies to the vacuole by a process that requires functional autophagy. Finally, we show that ATI1 is involved in Arabidopsis salt stress tolerance. Taken together, our results implicate ATI1 in autophagic plastid-to-vacuole trafficking through its ability to interact with both plastid proteins and ATG8 of the core autophagy machinery.     

The Catalytic Activity of the Ubp3 Deubiquitinating Protease Is Required for Efficient Stress Granule Assembly in Saccharomyces cerevisiae

The interior of the eukaryotic cell is a highly compartmentalized space containing both membrane-bound organelles and the recently identified nonmembranous ribonucleoprotein (RNP) granules. This study examines in Saccharomyces cerevisiae the assembly of one conserved type of the latter compartment, known as the stress granule. Stress granules form in response to particular environmental cues and have been linked to a variety of human diseases, including amyotrophic lateral sclerosis. To further our understanding of these structures, a candidate genetic screen was employed to identify regulators of stress granule assembly in quiescent cells. These studies identified a ubiquitin-specific protease, Ubp3, as having an essential role in the assembly of these RNP granules. This function was not shared by other members of the Ubp protease family and required Ubp3 catalytic activity as well as its interaction with the cofactor Bre5. Interestingly, the loss of stress granules was correlated with a decrease in the long-term survival of stationary-phase cells. This phenotype is similar to that observed in mutants defective for the formation of a related RNP complex, the Processing body. Altogether, these observations raise the interesting possibility of a general role for these types of cytoplasmic RNP granules in the survival of G₀-like resting cells.     

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.     

Interaction of the Deubiquitinating Enzyme Ubp2 and the E3 Ligase Rsp5 Is Required for Transporter/Receptor Sorting in the Multivesicular Body Pathway

Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.     

Fast-Suppressor Screening for New Components in Protein Trafficking,Organelle Biogenesis and Silencing Pathway in Arabidopsis thaliana Using DEX-Inducible FREE1-RNAi Plants

Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex(endosomal sorting complex required for transport).FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body(MVB),vacuolar protein transport and vacuole biogenesis,and autophagic degradation.FREE1 knockout plants show defective MVB formation,abnormal vacuolar transport,fragmented vacuoles,accumulated autophagosomes,and seedling lethality.To further uncover the underlying mechanisms of FREE1 function in plants,we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants.The obtained mutants are termed as suppressors of free1(sof).To date,229 putative sof mutants have been identified.Barely detecting of FREE1 protein with M3plants further identified 84 FREE1-related suppressors.Also145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants.Through next-generation sequencing(NGS)of bulked DNA from F2mapping population of two RNAi-related sof mutants,FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly.These FREE1-and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis,as well as for uncovering the new components involved in the regulation of silencing pathways in plants.     

Mqo,a Tricarboxylic Acid Cycle Enzyme,Is Required for Virulence of Pseudomonas syringae pv. tomato Strain DC3000 on Arabidopsis thaliana

Plant pathogenic bacteria, such as Pseudomonas syringae pv. tomato strain DC3000, the causative agent of tomato bacterial speck disease, grow to high levels in the apoplastic space between plant cells. Colonization of plant tissue requires expression of virulence factors that modify the apoplast to make it more suitable for pathogen growth or facilitate adaptation of the bacteria to the apoplastic environment. To identify new virulence factors involved in these processes, DC3000 Tn5 transposon insertion mutants with reduced virulence on Arabidopsis thaliana were identified. In one of these mutants, the Tn5 insertion disrupted the malate:quinone oxidoreductase gene (mqo), which encodes an enzyme of the tricarboxylic acid cycle. mqo mutants do not grow to wild-type levels in plant tissue at early time points during infection. Further, plants infected with mqo mutants develop significantly reduced disease symptoms, even when the growth of the mqo mutant reaches wild-type levels at late stages of infection. Mutants lacking mqo function grow more slowly in culture than wild-type bacteria when dicarboxylates are the only available carbon source. To explore whether dicarboxylates are important for growth of DC3000 in the apoplast, we disrupted the dctA1 dicarboxylate transporter gene. DC3000 mutants lacking dctA1 do not grow to wild-type levels in planta, indicating that transport and utilization of dicarboxylates are important for virulence of DC3000. Thus, mqo may be required by DC3000 to meet nutritional requirements in the apoplast and may provide insight into the mechanisms underlying the important, but poorly understood process of adaptation to the host environment.One important aspect of interactions between plant pathogens and their hosts is the ability of the pathogen to obtain nutrients within the plant tissue. Nutrient acquisition is essential for growth within the host, since both cell division and DNA replication can be influenced by nutrient availability. Bacterial plant pathogens differ in the strategies they use to get necessary nutrients during infection. Some pathogens, such as Agrobacterium tumefaciens, elicit production of specific carbon and nitrogen sources by the plant (1). Other pathogens may rely on metabolites that are readily available in the plant apoplast or may stimulate the release of water or nutrients from surrounding plant cells (27).Little is known about how pathogenic Pseudomonas syringae strains acquire nutrients when growing in their hosts. P. syringae strains are gram-negative gammaproteobacteria, which as a group cause disease on many agriculturally important plants. For example, P. syringae pv. tomato strain DC3000 causes disease on tomato, A. thaliana, and several agriculturally important Brassicas, such as turnip, mustard, collard, and cauliflower (8, 51). Initially, DC3000 colonizes plant surfaces and then enters the plant tissue through natural openings (such as stomata) or wounds (34, 38). DC3000 then establishes itself in the plant apoplast, the intercellular space between plant cells (38). Once in the apoplast of susceptible hosts, DC3000 multiplies to high levels, and the infected plants develop disease symptoms, including chlorosis (yellowing) of the leaf tissue and necrotic spots or patches called lesions (38, 49). Pseudomonads, such as P. aeruginosa and P. fluorescens, preferentially utilize tricarboxylic acid (TCA) cycle intermediates (20, 29, 33, 44), and DC3000 utilizes these carbon sources in culture (19). Some studies to investigate nutrient acquisition of DC3000 have been carried out (3, 7, 40); however, it is not clear what carbon sources DC3000 utilizes when growing in plant tissue.Several virulence factors are necessary for DC3000 to enter, grow inside the plant, and cause disease. Like many other bacterial pathogens, DC3000 uses a type III secretion system (TTSS) (15), which is encoded by the hrp/hrc genes, to inject effector proteins into plant cells (16, 27). Many of these effectors suppress host defenses, and it is likely that some may be involved in modulating the apoplastic environment or nutrient acquisition (16). DC3000 also produces the phytotoxin coronatine, which promotes entry of the bacteria into the plant apoplast by stimulating the opening of stomata (34) and is required for bacterial growth in the apoplast by suppressing salicylic acid (SA)-dependent host defenses (4, 45). Coronatine also promotes disease symptom development via an SA-independent mechanism (4). While much emphasis has been placed on exploring how type III-secreted effectors and coronatine promote DC3000 virulence, other factors are also likely to be important during pathogenesis.To identify additional factors involved in pathogenesis, we undertook a genetic screen to identify novel virulence factors (5, 24). DC3000 mutants with reduced virulence were identified by assaying for their ability to elicit disease symptoms on A. thaliana and tomato plants (24, 37). One of these mutants, AK4C9, had reduced virulence on both hosts. The gene disrupted in this mutant is the malate:quinone oxidoreductase gene (mqo), which encodes an enzyme of the TCA cycle. mqo mutants grow more slowly than wild-type DC3000 in planta and in culture when dicarboxylates are the only carbon source, suggesting that dicarboxylates are important for the growth of DC3000 in the apoplast. In the present study, we explore the role of Mqo and a dicarboxylate transporter, DctA1, in DC3000 pathogenesis.     

The Shigella flexneri Type 3 Secretion System Is Required for Tyrosine Kinase-Dependent Protrusion Resolution,and Vacuole Escape during Bacterial Dissemination

Shigella flexneri is a human pathogen that triggers its own entry into intestinal cells and escapes primary vacuoles to gain access to the cytosolic compartment. As cytosolic and motile bacteria encounter the cell cortex, they spread from cell to cell through formation of membrane protrusions that resolve into secondary vacuoles in adjacent cells. Here, we examined the roles of the Type 3 Secretion System (T3SS) in S. flexneri dissemination in HT-29 intestinal cells infected with the serotype 2a strain 2457T. We generated a 2457T strain defective in the expression of MxiG, a central component of the T3SS needle apparatus. As expected, the ΔmxiG strain was severely affected in its ability to invade HT-29 cells, and expression of mxiG under the control of an arabinose inducible expression system (ΔmxiG/pmxiG) restored full infectivity. In this experimental system, removal of the inducer after the invasion steps (ΔmxiG/pmxiG (Ara withdrawal)) led to normal actin-based motility in the cytosol of HT-29 cells. However, the time spent in protrusions until vacuole formation was significantly increased. Moreover, the number of formed protrusions that failed to resolve into vacuoles was also increased. Accordingly, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to trigger tyrosine phosphorylation in membrane protrusions, a signaling event that is required for the resolution of protrusions into vacuoles. Finally, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to escape from the formed secondary vacuoles, as previously reported in non-intestinal cells. Thus, the T3SS system displays multiple roles in S. flexneri dissemination in intestinal cells, including the tyrosine kinase signaling-dependent resolution of membrane protrusions into secondary vacuoles, and the escape from the formed secondary vacuoles.