Characterization of a Hank’s Type Serine/Threonine Kinase and Serine/Threonine Phosphoprotein Phosphatase in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen P. aeruginosa PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the stp1 ORF, an ORF encoding Hank's type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in Escherichia coli. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn(2+)-, but not a Ca(2+)- or a Mg(2+)-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.     

Serine/Threonine Kinase 17A Is a Novel Candidate for Therapeutic Targeting in Glioblastoma

STK17A is a relatively uncharacterized member of the death-associated protein family of serine/threonine kinases which have previously been associated with cell death and apoptosis. Our prior work established that STK17A is a novel p53 target gene that is induced by a variety of DNA damaging agents in a p53-dependent manner. In this study we have uncovered an additional, unanticipated role for STK17A as a candidate promoter of cell proliferation and survival in glioblastoma (GBM). Unexpectedly, it was found that STK17A is highly overexpressed in a grade-dependent manner in gliomas compared to normal brain and other cancer cell types with the highest level of expression in GBM. Knockdown of STK17A in GBM cells results in a dramatic alteration in cell shape that is associated with decreased proliferation, clonogenicity, migration, invasion and anchorage independent colony formation. STK17A knockdown also sensitizes GBM cells to genotoxic stress. STK17A overexpression is associated with a significant survival disadvantage among patients with glioma which is independent of age, molecular phenotype, IDH1 mutation, PTEN loss, and alterations in the p53 pathway and partially independent of grade. In summary, we demonstrate that STK17A provides a proliferative and survival advantage to GBM cells and is a potential target to be exploited therapeutically in patients with glioma.     

Saccharomyces cerevisiae Env7 Is a Novel Serine/Threonine Kinase 16-Related Protein Kinase and Negatively Regulates Organelle Fusion at the Lysosomal Vacuole

Membrane fusion depends on conserved components and is responsible for organelle biogenesis and vesicular trafficking. Yeast vacuoles are dynamic structures analogous to mammalian lysosomes. We report here that yeast Env7 is a novel palmitoylated protein kinase ortholog that negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirmed the localization of tagged Env7 at the vacuolar membrane and implicated membrane association via the palmitoylation of its N-terminal Cys13 to -15. In vitro kinase assays established Env7 as a protein kinase. Site-directed mutagenesis of the Env7 alanine-proline-glutamic acid (APE) motif Glu269 to alanine results in an unstable kinase-dead allele that is stabilized and redistributed to the detergent-resistant fraction by interruption of the proteasome system in vivo. Palmitoylation-deficient Env7C13-15S is also kinase dead and mislocalizes to the cytoplasm. Microscopy studies established that env7Δ is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds. ENV7 function is not redundant with a similar role of vacuolar membrane kinase Yck3, as the two do not share a substrate, and ENV7 is not a suppressor of yck3Δ. Bayesian phylogenetic analyses strongly support ENV7 as an ortholog of the gene encoding human STK16, a Golgi apparatus protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system.     

The Aspergillus fumigatus Dihydroxyacid Dehydratase Ilv3A/IlvC Is Required for Full Virulence

Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.     

Assembly of XcpR in the Cytoplasmic Membrane Is Required for Extracellular Protein Secretion in Pseudomonas aeruginosa

A broad range of extracellular proteins secreted by Pseudomonas aeruginosa use the type II or general secretory pathway (GSP) to reach the medium. This pathway requires the expression of at least 12 xcp gene products. XcpR, a putative nucleotide-binding protein, is essential for the secretion process across the outer membrane even though the protein contains no hydrophobic sequence that could target or anchor it to the bacterial envelope. For a better understanding of the relationship between XcpR and the other Xcp proteins which are located in the envelope, we have studied its subcellular localization. In a wild-type P. aeruginosa strain, XcpR was found associated with the cytoplasmic membrane. This association depends on the presence of the XcpY protein, which also appears to be necessary for XcpR stability. Functional complementation of an xcpY mutant required the XcpY protein to be expressed at a low level. Higher expression precluded the complementing activity of XcpY, although membrane association of XcpR was restored. This behavior suggested that an excess of free XcpY might interfere with the secretion by formation of inactive XcpR-XcpY complexes which cannot properly interact with their natural partners in the secretion machinery. These data show that a precise stoichiometric ratio between several components may be crucial for the functioning of the GSP.     

A Serine/Threonine Phosphorylation Site in the Ectodomain of a T Cell Receptor β Chain is Required for Activation by Superantigen

Abstract

The presence of consensus phosphorylation sites in the ectodomains of cell surface proteins suggests that such post‐translational modification may be important in regulation of surface receptor activity. To date, the only cell surface receptor for which such ectodomain phosphorylation has been conclusively demonstrated is the clonally expressed T cell antigen receptor (TCR). Attempts to conclusively identify individual phosphorylated residues in TCR α and β chains and determine their functional significance by biochemical approaches failed due to insufficient quantities of purified molecules. Here we present the results of an alternative approach where survey of phosphorylation sites in the TCR α and β chains was accomplished using site‐directed mutagenesis and retroviral vector expression, as well as in vitro phosphorylation of synthetic peptide substrates. All mutants studied directed the cell surface expression of normal amounts of TCR, and all transfectants could be stimulated to produce IL‐2 in response to substrate‐immobilized antibody to TCR. However, mutation of serine‐88 in the protein kinase A phosphorylation site of the TCR β chain resulted in a complete lack of response to the superantigen staphylococcal enterotoxin B (SEB). In addition, this mutation abolished TCR‐associated tyrosine phosphorylation, consistent with the impairment of cell signaling. Reversion of the serine‐88/alanine mutation with phosphorylatable threonine completely restored the SEB recognition by TCR. These results, interpreted in the context of the known three‐dimensional structure of the complex of SEB and TCR, are consistent with the view that serine‐88 is important for the contact of the TCR β chain with SEB.     

A Ferric Dicitrate Uptake System Is Required for the Full Virulence of Bacillus cereus

Bacillus cereus is an opportunistic human pathogen of increasing prevalence. Analysis of the Bacillus cereus genome sequence identified a potential ferric dicitrate uptake system. The three-gene operon was confirmed to be negatively regulated by the ferric uptake repressor (Fur). The Fec operon was genetically silenced using the integration suicide vector pMUTIN4. The mutant strain displayed no growth defect under iron-limited conditions but was unable to grow on ferric citrate as a sole iron source. The virulence of the mutant strain was attenuated in a lepidopteran infection model, highlighting the importance of iron uptake systems to the virulence of B. cereus and the potential of these systems to act as targets for novel antimicrobial agents.     

Rv2190c, an NlpC/P60 Family Protein, Is Required for Full Virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) possesses at least five genes predicted to encode proteins with NlpC/P60 hydrolase domains, including the relatively uncharacterized Rv2190c. As NlpC/P60 domain-containing proteins are associated with diverse roles in bacterial physiology, our objective was to characterize Rv2190c in M. tuberculosis growth and virulence. Our data indicate that lack of Rv2190c is associated with impaired growth, both in vitro and during an in vivo mouse model of TB. These growth defects are associated with altered colony morphology and phthiocerol dimycocerosate levels, indicating that Rv2190c is involved in cell wall maintenance and composition. In addition, we have demonstrated that Rv2190c is expressed during active growth phase and that its protein product is immunogenic during infection. Our findings have significant implications, both for better understanding the role of Rv2190c in M. tuberculosis biology and also for translational developments.     

The Extracytoplasmic Linker Peptide of the Sensor Protein SaeS Tunes the Kinase Activity Required for Staphylococcal Virulence in Response to Host Signals

Bacterial pathogens often employ two-component systems (TCSs), typically consisting of a sensor kinase and a response regulator, to control expression of a set of virulence genes in response to changing host environments. In Staphylococcus aureus, the SaeRS TCS is essential for in vivo survival of the bacterium. The intramembrane-sensing histidine kinase SaeS contains, along with a C-terminal kinase domain, a simple N-terminal domain composed of two transmembrane helices and a nine amino acid-long extracytoplasmic linker peptide. As a molecular switch, SaeS maintains low but significant basal kinase activity and increases its kinase activity in response to inducing signals such as human neutrophil peptide 1 (HNP1). Here we show that the linker peptide of SaeS controls SaeS’s basal kinase activity and that the amino acid sequence of the linker peptide is highly optimized for its function. Without the linker peptide, SaeS displays aberrantly elevated kinase activity even in the absence of the inducing signal, and does not respond to HNP1. Moreover, SaeS variants with alanine substitution of the linker peptide amino acids exhibit altered basal kinase activity and/or irresponsiveness to HNP1. Biochemical assays reveal that those SaeS variants have altered autokinase and phosphotransferase activities. Finally, animal experiments demonstrate that the linker peptide-mediated fine tuning of SaeS kinase activity is critical for survival of the pathogen. Our results indicate that the function of the linker peptide in SaeS is a highly evolved feature with very optimized amino acid sequences, and we propose that, in other SaeS-like intramembrane sensing histidine kinases, the extracytoplasmic linker peptides actively fine-control their kinases.     

Membrane Topology of Outer Membrane Protein AlgE,Which Is Required for Alginate Production in Pseudomonas aeruginosa

The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa secretes a viscous extracellular polysaccharide, called alginate, as a virulence factor during chronic infection of patients with cystic fibrosis. In the present study, it was demonstrated that the outer membrane protein AlgE is required for the production of alginate in P. aeruginosa. An isogenic marker-free algE deletion mutant was constructed. This strain was incapable of producing alginate but did secrete alginate degradation products, indicating that polymerization occurs but that the alginate chain is subsequently degraded during transit through the periplasm. Alginate production was restored by introducing the algE gene. The membrane topology of the outer membrane protein AlgE was assessed by site-specific insertions of FLAG epitopes into predicted extracellular loop regions.Pseudomonas aeruginosa is an ubiquitous opportunistic human pathogen responsible for chronic infections of the lungs of patients with cystic fibrosis (CF), in whom it is the leading cause of mortality and morbidity (9). The establishment of a chronic infection in the lungs of patients with CF coincides with the switch of P. aeruginosa to a stable mucoid variant, producing copious amounts of the exopolysaccharide alginate; this is typically a poor prognostic indicator for these patients (24, 31). Alginate is a linear unbranched exopolysaccharide consisting of 1,4-linked monomers of β-d-mannuronic acid and its C-5 epimer, α-l-guluronic acid, which is known to be produced by only two bacterial genera, Pseudomonas and Azotobacter (34). The switch to a mucoid phenotype coincides with the appearance of a 54-kDa protein in the outer membrane; this protein has been identified and has been designated AlgE (13, 31).The genes encoding the alginate biosynthesis machinery are located within a 12-gene operon (algD-alg8-alg44-algK-algE-algG-algX-algL-algI-algJ-algF-algA). AlgA and AlgD, along with AlgC (not encoded in the operon), are involved in precursor synthesis (34). Alg8 is the catalytic subunit of the alginate polymerase located at the inner membrane (35). AlgG is a C-5 mannuronan epimerase (19). AlgK contains four putative Sel1-like repeats, similar to the tetratricopeptide repeat motif often found in adaptor proteins involved in the assembly of multiprotein complexes (3, 10). AlgX shows little homology to any known protein, and its role is unclear (14). Knockout mutants of AlgK, AlgG, and AlgX have nonmucoid phenotypes, although they produce short alginate fragments, due to the activity of the alginate lyase (AlgL), which degrades the nascent alginate (1, 14, 19-21, 36). AlgF, AlgI, and AlgJ are involved in acetylation of alginate, but they are not ultimately required for its production (12). The membrane-anchored protein, Alg44, is required for polymerization and has a PilZ domain for the binding of c-di-GMP, a secondary messenger essential for alginate production (16, 25, 33). The periplasmic C terminus of Alg44 shares homology with the membrane fusion proteins involved in the bridging of the periplasm in multidrug efflux pumps (11, 43). The periplasmic alginate lyase, AlgL, appears to be required for the translocation of intact alginate across the periplasm (1, 26). AlgE is an outer membrane, anion-selective channel protein through which alginate is presumably secreted (30). A protein complex or scaffold through which the alginate chain can pass and be modified and which spans the periplasm bridging the polymerase located (Alg8) at the outer membrane pore (AlgE) has been proposed (21). Indeed, it has been demonstrated that both the inner and the outer membranes are required for the in vitro polymerization of alginate (35).The requirement of AlgE for the biosynthesis of alginate in P. aeruginosa was first observed by complementation of an alginate-negative mutant derived by chemical mutagenesis with a DNA fragment containing algE (8) Secondary structure predictions suggested that AlgE forms an 18-stranded β barrel with extended extracellular loops. Several of these loops show high densities of charged amino acids, suggesting a functional role in the translocation of the anionic alginate polymer (29, 30). Preliminary analysis of AlgE crystals has been reported (48).In this study, the role of AlgE in alginate biosynthesis was investigated and the membrane topology of AlgE was assessed by site-directed insertion mutagenesis.     

The Major Site of Phosphorylation within the Rous Sarcoma Virus MA Protein Is Not Required for Replication

About one-third of the MA protein in Rous sarcoma virus (RSV) is phosphorylated. Previous analyses of this fraction have suggested that serine residues 68 and 106 are the major sites of phosphorylation. As a follow-up to that study, we have characterized mutants which have these putative phosphorylation sites changed to alanine, either separately or together. None of the substitutions (S68A, S106A, or S68/106A) had an effect on the budding efficiency or infectivity of the virus. Upon examination of the ³²P-labeled viral proteins, we found that the S68A substitution did not affect phosphorylation in vivo at all. In contrast, the S106A substitution prevented all detectable phosphorylation of MA, suggesting that there is only one major site of phosphorylation in MA. We also found that the RSV MA protein is phosphorylated on tyrosine, but the amount was low and detectable only with large numbers of virions and an antibody specific for phosphotyrosine.     

Dyrk1A, a Serine/Threonine Kinase, is Involved in ERK and Akt Activation in the Brain of Hyperhomocysteinemic Mice

Hyperhomocysteinemia due to cystathionine beta synthase (CBS) deficiency is associated with diverse brain disease. Whereas the biological actions linking hyperhomocysteinemia to the cognitive dysfunction are not well understood, we tried to establish relationships between hyperhomocysteinemia and alterations of signaling pathways. In the brain of CBS-deficient mice, a murine model of hyperhomocysteinemia, we previously found an activation of extracellular signal-regulated kinase (ERK) pathway and an increase of Dyrk1A, a serine/threonine kinase involved in diverse functions ranging from development and growth to apoptosis. We then investigated the relationship between Dyrk1A and the signaling pathways initiated by receptor tyrosine kinases (RTK), the ERK and PI3K/Akt pathways. We found a significant increase of phospho-ERK, phospho-MEK, and phospho-Akt in the brain of CBS-deficient and Dyrk1a-overexpressing mice. This increase was abolished when CBS-deficient and Dyrk1A-transgenic mice were treated with harmine, an inhibitor of Dyrk1A kinase activity, which emphasizes the role of Dyrk1A activity on ERK and Akt activation. Sprouty 2 protein level, a negative feedback loop modulator that limits the intensity and duration of RTK activation, is decreased in the brain of CBS-deficient mice, but not in the brain of Dyrk1A transgenic mice. Furthermore, a reduced Dyrk1A and Grb2 binding on sprouty 2 and an increased interaction of Dyrk1A with Grb2 were found in the brain of Dyrk1A transgenic mice. The consequence of Dyrk1A overexpression on RTK activation seems to be a decreased interaction of sprouty 2/Grb2. These observations demonstrate ERK and Akt activation induced by Dyrk1A in the brain of hyperhomocysteinemic mice and open new perspectives to understand the basis of the cognitive defects in hyperhomocysteinemia.     

A Novel Protein Kinase Localized to Lipid Droplets Is Required for Droplet Biogenesis in Trypanosomes

Ubiquitous among eukaryotes, lipid droplets are organelles that function to coordinate intracellular lipid homeostasis. Their morphology and abundance is affected by numerous genes, many of which are involved in lipid metabolism. In this report we identify a Trypanosoma brucei protein kinase, LDK, and demonstrate its localization to the periphery of lipid droplets. Association with lipid droplets was abrogated when the hydrophobic domain of LDK was deleted, supporting a model in which the hydrophobic domain is associated with or inserted into the membrane monolayer of the organelle. RNA interference knockdown of LDK modestly affected the growth of mammalian bloodstream-stage parasites but did not affect the growth of insect (procyclic)-stage parasites. However, the abundance of lipid droplets dramatically decreased in both cases. This loss was dominant over treatment with myriocin or growth in delipidated serum, both of which induce lipid body biogenesis. Growth in delipidated serum also increased LDK autophosphorylation activity. Thus, LDK is required for the biogenesis or maintenance of lipid droplets and is one of the few protein kinases specifically and predominantly associated with an intracellular organelle.Trypanosoma brucei is a single-celled eukaryotic pathogen responsible for human African trypanosomiasis (also known as African sleeping sickness) and nagana in domestic animals. More than 50,000 cases of human disease occur yearly, with over 70 million people at risk. No vaccine exists, and chemotherapy is difficult to administer and prone to pathogen resistance. As T. brucei transits between the mammalian bloodstream and the tsetse fly vector during its life cycle, the organism encounters and adapts to profoundly different environmental conditions. The parasite undergoes dramatic changes in both energy (7, 51) and lipid biosynthesis and metabolism (39, 47, 49) as it shifts between these environments.Protein kinases function in numerous regulatory aspects of the cell, including control of the cell cycle and morphology, responses to stress, and transmission of signals from the extracellular environment or between compartments of the cell. As is the case in other eukaryotes, protein kinases, particularly those associated with membranes, are expected to play pivotal roles in the cell''s ability to sense and appropriately respond to its environment. Trypanosoma brucei possesses over 170 protein kinases (16, 44). Most of these can be assigned to the standard groups of protein kinases based on sequence similarity within the kinase domain. However, sequence similarities with kinases from more well-studied organisms are rarely strong enough to allow one-to-one orthologous relationships to be determined (44), and even those which appear orthologous by sequence have sometimes shown functional divergence (46). Hence, an understanding of the roles of specific protein kinases of trypanosomatids requires an individualized assessment. The initial genome analysis of the trypanosomatids (16) showed a lack of receptor tyrosine kinases, but nine T. brucei predicted serine/threonine kinases were annotated as possessing transmembrane domains. One of these was recently shown to be strategically located at a key interface between the host and parasite: the flagellar pocket (38). This eukaryotic translation initiation factor 2α (eIF2α) family kinase was postulated to play a sensory role in monitoring protein transport.Only a very small number of protein kinases of various organisms have been observed to localize to the membranes of intracellular organelles, most of them to the endoplasmic reticulum (ER) (14, 27, 50). Lipid droplets (also known as lipid bodies, adiposomes, or oil bodies in plants) are thought to arise from the ER, although the routes of protein localization to them are not well understood. They are increasingly recognized as legitimate organelles due to their dynamic roles in energy metabolism (40), lipid trafficking (41), and protection against toxic effects of nonesterified lipids and sterols (18). Studies also suggest that they function as potential protein storage depots (12) and in antigen presentation (10). Although recent efforts to expand the lipid droplet proteome have resulted in a vastly increased and in many cases surprising catalogue of potentially associated proteins (3, 5, 11, 12, 23, 37), relatively little is known as to how these structures form and are regulated within the cell.We examine here a novel T. brucei protein kinase with a predicted transmembrane domain. Surprisingly, this protein is localized intracellularly in association with lipid droplets. RNAi-mediated knockdown of this newly identified kinase, dubbed LDK (for lipid droplet kinase), reveals a role in the formation or maintenance of lipid droplets in both mammalian bloodstream-form (BF) and insect procyclic-form (PF) stages of the parasite life cycle.     

A Serine/Threonine Protein Kinase Gene Isolated by an in vivo Binding Procedure Using the Arabidopsis Floral Homeotic Gene Product, AGAMOUS

During the course of characterizing fragments bound to an Arabidopsisfloral homeotic protein AGAMOUS in vivo, a gene encoding a putativeserine/threonine protein kinase was found on one of the fragments.The deduced 426 amino acid residues of the gene, named APK2a,are 65% identical to a previously reported Arabidopsisserine/threonineprotein kinase, APKla. The gene is composed of 6 exons and mapsat 10 cM from the upper end of chromosome 1. Northern hybridizationexperiments indicated that the gene is strongly expressed inleaves, moderately in roots, and very weakly in flowers. Furtherin situ analysis of the expression in floral buds showed thatthe APK2a gene is expressed at pedicels, is not expressed atthe floral organ primordia of wild type floral buds, but ismoderately expressed in the floral organ primordia of the agamousmutant. In vitro binding assay suggests that the AGAMOUS proteinbinds to a sequence similar to, but different from, the knownMADS-binding consensus sequences, the CArG box, located 3' downstreamof the APK2a gene. These results suggest that APK2a gene expressionis negatively regulated by the AG protein. A close homologue of the APK2a gene, named APK2b, was also isolatedfrom the Arabidopsis cDNA library. The expression pattern ofthe APK2b gene differs from that of APK2a. It is strongly expressedin leaves, moderately in flowers, and weakly in roots. ⁴Present address: Biomolecular Engineering Research Institute,6-2-3, Fruedai, Suita, Osaka, 565 Japan.     

Recruitment of the Host Plant Heat Shock Protein 70 by Tomato Yellow Leaf Curl Virus Coat Protein Is Required for Virus Infection

A functional capsid protein (CP) is essential for host plant infection and insect transmission of Tomato yellow leaf curl virus (TYLCV) and other monopartite begomoviruses. We have previously shown that TYLCV CP specifically interacts with the heat shock protein 70 (HSP70) of the virus insect vector, Bemisia tabaci. Here we demonstrate that during the development of tomato plant infection with TYLCV, a significant amount of HSP70 shifts from a soluble form into insoluble aggregates. CP and HSP70 co-localize in these aggregates, first in the cytoplasm, then in the nucleus of cells associated with the vascular system. CP-HSP70 interaction was demonstrated by co-immunopreciptation in cytoplasmic - but not in nuclear extracts from leaf and stem. Inhibition of HSP70 expression by quercetin caused a decrease in the amount of nuclear CP aggregates and a re-localization of a GFP-CP fusion protein from the nucleus to the cytoplasm. HSP70 inactivation resulted in a decrease of TYLCV DNA levels, demonstrating the role of HSP70 in TYLCV multiplication in planta. The current study reveals for the first time the involvement of plant HSP70 in TYLCV CP intracellular movement. As described earlier, nuclear aggregates contained TYLCV DNA-CP complexes and infectious virions. Showing that HSP70 localizes in these large nuclear aggregates infers that these structures operate as nuclear virus factories.     

A Kelch Motif-Containing Serine/Threonine Protein Phosphatase Determines the Large Grain QTL Trait in Rice

A thorough understanding of the genetic basis of rice grain traits is critical for the improvement of rice (Oryza sativa L.) varieties. In this study, we generated an F₂ population by crossing the large‐grain japonica cultivar CW23 with Peiai 64 (PA64), an elite indica small‐grain cultivar. Using QTL analysis, 17 QTLs for five grain traits were detected on four different chromosomes. Eight of the QTLs were newly‐identified in this study. In particular, qGL3‐1, a newly‐identified grain length QTL with the highest LOD value and largest phenotypic variation, was fine‐mapped to the 17 kb region of chromosome 3. A serine/threonine protein phosphatase gene encoding a repeat domain containing two Kelch motifs was identified as the unique candidate gene corresponding to this QTL. A comparison of PA64 and CW23 sequences revealed a single nucleotide substitution (C→A) at position 1092 in exon 10, resulting in replacement of Asp (D) in PA64 with Glu (E) in CW23 for the 364^th amino acid. This variation is located at the D position of the conserved sequence motif AVLDT of the Kelch repeat. Genetic analysis of a near‐isogenic line (NIL) for qGL3‐1 revealed that the allele qGL3‐1 from CW23 has an additive or partly dominant effect, and is suitable for use in molecular marker‐assisted selection.     

A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis

Plant pathogenic fungi cause massive yield losses and affect both quality and safety of food and feed produced from infected plants. The main objective of plant pathogenic fungi is to get access to the organic carbon sources of their carbon-autotrophic hosts. However, the chemical nature of the carbon source(s) and the mode of uptake are largely unknown. Here, we present a novel, plasma membrane-localized sucrose transporter (Srt1) from the corn smut fungus Ustilago maydis and its characterization as a fungal virulence factor. Srt1 has an unusually high substrate affinity, is absolutely sucrose specific, and allows the direct utilization of sucrose at the plant/fungal interface without extracellular hydrolysis and, thus, without the production of extracellular monosaccharides known to elicit plant immune responses. srt1 is expressed exclusively during infection, and its deletion strongly reduces fungal virulence. This emphasizes the central role of this protein both for efficient carbon supply and for avoidance of apoplastic signals potentially recognized by the host.     

A Novel Basal Body Protein That Is a Polo-like Kinase Substrate Is Required for Basal Body Segregation and Flagellum Adhesion in Trypanosoma brucei

The Polo-like kinase (PLK) in Trypanosoma brucei plays multiple roles in basal body segregation, flagellum attachment, and cytokinesis. However, the mechanistic role of TbPLK remains elusive, mainly because most of its substrates are not known. Here, we report a new substrate of TbPLK, SPBB1, and its essential roles in T. brucei. SPBB1 was identified through yeast two-hybrid screening with the kinase-dead TbPLK as the bait. It interacts with TbPLK in vitro and in vivo, and is phosphorylated by TbPLK in vitro. SPBB1 localizes to both the mature basal body and the probasal body throughout the cell cycle, and co-localizes with TbPLK at the basal body during early cell cycle stages. RNAi against SPBB1 in procyclic trypanosomes inhibited basal body segregation, disrupted the new flagellum attachment zone filament, detached the new flagellum, and caused defective cytokinesis. Moreover, RNAi of SPBB1 confined TbPLK at the basal body and the bilobe structure, resulting in constitutive phosphorylation of TbCentrin2 at the bilobe. Altogether, these results identified a basal body protein as a TbPLK substrate and its essential role in promoting basal body segregation and flagellum attachment zone filament assembly for flagellum adhesion and cytokinesis initiation.     

A SIX1 Homolog in Fusarium oxysporum f. sp. conglutinans Is Required for Full Virulence on Cabbage

Fusarium oxysporum is a soil-born fungus that induces wilt and root rot on a variety of plants. F. oxysporum f. sp. conglutinans (Foc) can cause wilt disease on cabbage. This study showed that a homolog of SIX1 protein in the Arabidopsis infecting isolate Fo5176 (Fo5176-SIX1) had four isoforms in the conidia of Foc by proteomic analysis. Thus, we analyzed the roles of protein Foc-SIX1. Gene expression analysis showed that, compared to the expression in mycelia, dramatically altered expression of Foc-SIX1 could be detected after infecting cabbages, and Foc-SIX1 was highly expressed in conidia under axenic culture condition. Furthermore, we knocked out the Foc-SIX1 gene and found that Foc-ΔSIX1 mutants had significantly reduced virulence compared with wild type isolate, and full virulence was restored by complementation of Foc-ΔSIX1 mutants with Foc-SIX1. Thus, we concluded that SIX1 in Foc was required for full virulence on cabbage. We also complemented Foc-ΔSIX1 with SIX1 gene in F. oxysporum f. sp. lycopersici (Fol) and found Foc-ΔSIX1::Fol-SIX1 mutants did not affect the virulence of Foc-ΔSIX1. The results confirmed that Fol-SIX1 was not capable of replacing the role of Foc-SIX1 in Foc on the disease symptom development of cabbage. The roles of Fol-SIX1 on virulence might rely on host specificity.