SNARE protein FgVam7 controls growth,asexual and sexual development,and plant infection in Fusarium graminearum

Soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins play critical and conserved roles in membrane fusion and vesicle transport of eukaryotic cells. Previous studies have shown that various homologues of SNARE proteins are also important in the infection of host plants by pathogenic fungi. Here, we report the characterization of a SNARE homologue, FgVam7, from Fusarium graminearum that causes head blight in wheat and barley worldwide. Phylogenetic analysis and domain comparison reveal that FgVam7 is homologous to Vam7 proteins of Saccharomyces cerevisiae (ScVam7), Magnaporthe oryzae (MoVam7) and several additional fungi by containing a PhoX homology (PX) domain and a SNARE domain. We show that FgVam7 plays a regulatory role in cellular differentiation and virulence in F. graminearum. Deletion of FgVAM7 significantly reduces vegetative growth, conidiation and conidial germination, sexual reproduction and virulence. The ΔFgvam7 mutant also exhibits a defect in vacuolar maintenance and delayed endocytosis. Moreover, the ΔFgvam7 mutant is insensitive to salt and osmotic stresses, and hypersensitive to cell wall stressors. Further characterization of FgVam7 domains indicate that the PX and SNARE domains are conserved in controlling Vam7 protein localization and function, respectively. Finally, FgVam7 has been shown to positively regulate the expression of several deoxynivalenol (DON) biosynthesis genes TRI5, TRI6 and TRI101, and DON production. Our studies provide evidence for SNARE proteins as an additional means of regulatory mechanisms that govern growth, differentiation and virulence of pathogenic fungi.     

Septin 9 interacts with kinesin KIF17 and interferes with the mechanism of NMDA receptor cargo binding and transport

Intracellular transport involves the regulation of microtubule motor interactions with cargo, but the underlying mechanisms are not well understood. Septins are membrane- and microtubule-binding proteins that assemble into filamentous, scaffold-like structures. Septins are implicated in microtubule-dependent transport, but their roles are unknown. Here we describe a novel interaction between KIF17, a kinesin 2 family motor, and septin 9 (SEPT9). We show that SEPT9 associates directly with the C-terminal tail of KIF17 and interacts preferentially with the extended cargo-binding conformation of KIF17. In developing rat hippocampal neurons, SEPT9 partially colocalizes and comigrates with KIF17. We show that SEPT9 interacts with the KIF17 tail domain that associates with mLin-10/Mint1, a cargo adaptor/scaffold protein, which underlies the mechanism of KIF17 binding to the NMDA receptor subunit 2B (NR2B). Significantly, SEPT9 interferes with binding of the PDZ1 domain of mLin-10/Mint1 to KIF17 and thereby down-regulates NR2B transport into the dendrites of hippocampal neurons. Measurements of KIF17 motility in live neurons show that SEPT9 does not affect the microtubule-dependent motility of KIF17. These results provide the first evidence of an interaction between septins and a nonmitotic kinesin and suggest that SEPT9 modulates the interactions of KIF17 with membrane cargo.     

The Multivesicular Bodies (MVBs)-Localized AAA ATPase LRD6-6 Inhibits Immunity and Cell Death Likely through Regulating MVBs-Mediated Vesicular Trafficking in Rice

Previous studies have shown that multivesicular bodies (MVBs)/endosomes-mediated vesicular trafficking may play key roles in plant immunity and cell death. However, the molecular regulation is poorly understood in rice. Here we report the identification and characterization of a MVBs-localized AAA ATPase LRD6-6 in rice. Disruption of LRD6-6 leads to enhanced immunity and cell death in rice. The ATPase activity and homo-dimerization of LRD6-6 is essential for its regulation on plant immunity and cell death. An ATPase inactive mutation (LRD6-6^E315Q) leads to dominant-negative inhibition in plants. The LRD6-6 protein co-localizes with the MVBs marker protein RabF1/ARA6 and interacts with ESCRT-III components OsSNF7 and OsVPS2. Further analysis reveals that LRD6-6 is required for MVBs-mediated vesicular trafficking and inhibits the biosynthesis of antimicrobial compounds. Collectively, our study shows that the AAA ATPase LRD6-6 inhibits plant immunity and cell death most likely through modulating MVBs-mediated vesicular trafficking in rice.     

BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.     

Rutaecarpine suppresses atherosclerosis in ApoE−/− mice through upregulating ABCA1 and SR-BI within RCT

ABCA1 and scavenger receptor class B type I (SR-BI)/CD36 and lysosomal integral membrane protein II analogous 1 (CLA-1) are the key transporter and receptor in reverse cholesterol transport (RCT). Increasing the expression level of ABCA1 and SR-BI/CLA-1 is antiatherogenic. The aim of the study was to find novel antiatherosclerotic agents upregulating expression of ABCA1 and SR-BI/CLA-1 from natural compounds. Using the ABCA1p-LUC and CLA-1p-LUC HepG2 cell lines, we found that rutaecarpine (RUT) triggered promoters of ABCA1 and CLA-1 genes. RUT increased ABCA1 and SR-BI/CLA-1 expression in vitro related to liver X receptor alpha and liver X receptor beta. RUT induced cholesterol efflux in RAW264.7 cells. ApoE-deficient (ApoE^−/−) mice treated with RUT for 8 weeks showed ∼68.43, 70.23, and 85.56% less en face lesions for RUT (L), RUT (M), and RUT (H) groups, respectively, compared with the model group. Mouse macrophage-specific antibody and filipin staining indicated that RUT attenuated macrophages and cholesterol accumulations in atherosclerotic lesions, respectively. Additionally, ABCA1 and SR-BI expression was highly induced by RUT in livers of ApoE^−/− mice. Meanwhile, RUT treatment significantly increased the fecal ³H-cholesterol excretion, which demonstrated that RUT could promote RCT in vivo. RUT was identified to be a candidate that protected ApoE^−/− mice from developing atherosclerosis through preferentially promoting activities of ABCA1 and SR-BI within RCT.     

Human sodium/inositol cotransporter 2 (SMIT2) transports inositols but not glucose in L6 cells

To characterize the function of the sodium/inositol symporter SMIT2 in skeletal muscle, human SMIT2 cDNA was transfected into L6 myoblasts using pcDNA3.1 expression vector. Compared with the pcDNA3.1 vector only transfection, this overexpression increased the uptake of [³H]d-chiro-inositol (DCI) by 159-fold. [³H]myo-Inositol uptake increased by 37-fold. In contrast, [¹⁴C]d-glucose, [¹⁴C]2-deoxy-d-glucose, or [¹⁴C]3-O-methyl-d-glucose uptake remained unchanged in the presence of either 0, 5.5, or 25 mM unlabeled glucose. The K_m of DCI and myo-inositol for DCI uptake was 111.0 and 158.0 μM, respectively, whereas glucose competed for DCI uptake with a K_i of 6.1 mM. Insulin treatment of non-transfected L6 cells (2 μM for 24 h) increased [³H]DCI specific uptake 18-fold. DCI transport is up regulated by insulin and competitively inhibited by millimolar levels of glucose. Therefore, expression and/or function of SMIT2, a high affinity transporter specific for DCI and myo-inositol, may be reduced in diabetes mellitus, insulin resistance and polycystic ovary syndrome causing the abnormal DCI metabolism observed in these conditions.     

Inhibition of RelB by 1,25‐dihydroxyvitamin D3 promotes sensitivity of breast cancer cells to radiation

Aberrant constitutive expression of the NF‐κB c‐Rel and RelA subunits in breast cancer cells was shown to promote their survival. Recently, we demonstrated that aggressive breast cancers constitutively express high levels of the RelB subunit, which promotes their more invasive phenotype via induction of the BCL2 gene. As these cancers are frequently resistant to therapy, here we tested the hypothesis that RelB promotes their survival. High RelB expressing Hs578T and MDA‐MB‐231 breast cancer cells were more resistant to γ‐radiation than MCF7 and ZR‐75 cells, which express lower RelB levels. Knockdown of RelB in Hs578T led to decreased survival in response to γ‐irradiation, while conversely ectopic expression of RelB in MCF7 cells protected these cells from radiation. Similar data were obtained upon treatment of Hs578T or MCF7 cells with the chemotherapeutic agent doxorubicin. High serum levels of 25‐hydroxyvitamin D are associated with decreased breast cancer risk and mortality, although, the mechanisms of its protective actions have not been fully elucidated. Treatment of Hs578T and Her‐2/neu‐driven NF639 cells with 1,25‐dihydroxyvitamin D₃ decreased RelB/RELB gene expression and levels of pro‐survival targets Survivin, MnSOD and Bcl‐2, while increasing their sensitivity to γ‐irradiation. Thus, RelB, which promotes survival and a more highly invasive phenotype of breast cancer cells, is a target of 1,25‐dihydroxyvitamin D₃, providing one mechanism for the observed protective role of 25‐hydroxyvitamin D in patients with breast cancer. J. Cell. Physiol. 220: 593–599, 2009. © 2009 Wiley‐Liss, Inc.