Use of the Plant Defense Protein Osmotin To Identify Fusarium oxysporum Genes That Control Cell Wall Properties

Fusarium oxysporum is the causative agent of fungal wilt disease in a variety of crops. The capacity of a fungal pathogen such as F. oxysporum f. sp. nicotianae to establish infection on its tobacco (Nicotiana tabacum) host depends in part on its capacity to evade the toxicity of tobacco defense proteins, such as osmotin. Fusarium genes that control resistance to osmotin would therefore reflect coevolutionary pressures and include genes that control mutual recognition, avoidance, and detoxification. We identified FOR (Fusarium Osmotin Resistance) genes on the basis of their ability to confer osmotin resistance to an osmotin-sensitive strain of Saccharomyces cerevisiae. FOR1 encodes a putative cell wall glycoprotein. FOR2 encodes the structural gene for glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting step in the biosynthesis of hexosamine and cell wall chitin. FOR3 encodes a homolog of SSD1, which controls cell wall composition, longevity, and virulence in S. cerevisiae. A for3 null mutation increased osmotin sensitivity of conidia and hyphae of F. oxysporum f. sp. nicotianae and also reduced cell wall β-1,3-glucan content. Together our findings show that conserved fungal genes that determine cell wall properties play a crucial role in regulating fungal susceptibility to the plant defense protein osmotin.Studies of plant-pathogen interactions strongly suggest that under the pressure to survive, plants and pathogens continuously react to one another''s defense arsenal and evolve to overcome these defenses (13). Plants recognize pathogen-associated molecular patterns, such as fungal cell wall fragments composed of chitin, glucans, oligosaccharides, or glycoprotein peptides (32). It has been established that pathogens evolved effector proteins to avoid plant surveillance mechanisms that recognize pathogen-associated molecular patterns and this in turn led to the evolution of plant surveillance mechanisms that recognize pathogen-specific effector proteins. All pathogen recognition mechanisms induce intracellular signaling that culminates in the synthesis of factors, such as antimicrobial plant proteins, that help in limiting the severity of infection (74). The antimicrobial proteins are therefore among the ultimate effectors of plant defense. There is evidence of recognition between plant antimicrobial proteins and pathogen-specific molecules (74). Therefore, pathogen mechanisms of resistance to the antimicrobial proteins and the antimicrobial proteins themselves must have coevolved. Consequently, we postulated that a screen for fungal genes that alter the sensitivity of a phytopathogen to an antifungal protein of the host plant (that is, a cognate plant defense effector) would lead to identification of genes involved in controlling pathogenicity, in controlling access of the antifungal protein to its target fungal molecules (such as genes controlling cell surface composition), and in controlling detoxification mechanisms.The plant antifungal protein selected to test this hypothesis was osmotin. Osmotin is an antifungal protein that is overexpressed in and secreted by salt-adapted cultured tobacco (Nicotiana tabacum) cells (63). It is a member of a family of ubiquitous plant proteins, referred to as plant pathogenesis-related proteins of family 5 (PR-5), that are implicated in defense against fungi (74). Osmotin gene and protein expression is induced by biotic stresses, and overexpression of osmotin delays development of disease symptoms in transgenic plants (41, 42, 43, 84). The genetic bases of the susceptibility and resistance of Saccharomyces cerevisiae to osmotin have been explored in our laboratory (49, 50). The results show that specific interactions of osmotin with the plasma membrane are responsible for cell death signaling. However, because the cell wall governs access of osmotin to the plasma membrane, differences in cell wall composition largely account for the differential osmotin sensitivity of various S. cerevisiae strains, and specific cell wall components play a significant role in modulating osmotin toxicity (30, 31, 49, 50, 81, 82). These studies in the model nonpathogenic fungus, S. cerevisiae, support our hypothesis that a screen for genes that alter the sensitivity of a phytopathogenic fungus to an antifungal defense effector protein of the host plant will uncover genes involved in controlling access of the antifungal protein to its target fungal molecules.Osmotin, like other plant defense antifungal proteins, has specific but broad-spectrum antifungal activity (74). One of the most osmotin-sensitive phytopathogenic fungi is Fusarium oxysporum. F. oxysporum is an ascomycete fungus, like S. cerevisiae, and has been touted as an appropriate multihost model for studying fungal virulence (53). It is a soilborne plant pathogen of economic significance, because it causes vascular wilt disease on a large variety of crop plants and produces toxic food contaminants (17, 58). In humans it also causes skin, nail, and eye disease that can become serious or life-threatening illnesses in immunocompromised patients (52, 69). F. oxysporum f. sp. lycopersici, F. oxysporum f. sp. nicotianae, and F. oxysporum f. sp. meloni, like S. cerevisiae, are quite sensitive to osmotin (1, 51; M. L. Narasimhan, unpublished data). Furthermore, it was recently shown that overexpression in F. oxysporum f. sp. nicotianae of an S. cerevisiae cell wall glycoprotein that increases the osmotin resistance of S. cerevisiae also increases the osmotin resistance of the plant pathogen and its virulence on tobacco, the osmotin-producing host plant (51). This suggested that osmotin resistance mechanisms may be conserved between S. cerevisiae and F. oxysporum and that S. cerevisiae could be used as a tool to uncover F. oxysporum genes that control osmotin sensitivity or resistance.In the current study, we expressed an F. oxysporum f. sp. nicotianae cDNA library in the osmotin-sensitive S. cerevisiae strain BWG1-7a and selected genes for their ability to increase osmotin tolerance. We report here the identification and characterization of three FOR (Fusarium Osmotin Resistance) genes that affect the cell wall in S. cerevisiae. The product of FOR1 has homology with a putative cell surface glycoprotein; FOR2 encodes glutamine:fructose-6-phosphate amidotransferase (GFAT), an enzyme that catalyzes the first step in the biosynthetic pathway leading to amino sugar-containing macromolecules, such as glycoproteins and chitin (64); and FOR3 has high homology with S. cerevisiae SSD1, a gene that controls cell wall composition and virulence (31, 78). FOR2 and FOR3 are the functional equivalents of the corresponding S. cerevisiae genes. Our parallel analysis using two model fungi verifies the notion that cell wall proteins play a critical role in determining the sensitivity/resistance of fungi to osmotin. In addition, these results implicate that the tobacco defense protein, osmotin, can serve as an effective/useful tool in identifying genes that control cell wall composition not only in a model fungus, such as S. cerevisiae, but also in phytopathogenic fungi, such as F. oxysporum.     

Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas

Background

Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.

Methodology/Principal Findings

In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.

Conclusions/Significance

In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.     

Suppression of uPA and uPAR Attenuates Angiogenin Mediated Angiogenesis in Endothelial and Glioblastoma Cell Lines

Background

In our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood.

Methodology/Principal Findings

In the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells.

Conclusions/Significance

Taken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway.     

Polo-like kinase 3 is Golgi localized and involved in regulating Golgi fragmentation during the cell cycle

The Golgi apparatus undergoes extensive fragmentation during mitosis in animal cells. Protein kinases play a critical role during fragmentation of the Golgi apparatus. We reported here that Polo-like kinase 3 (Plk3) may be an important mediator during Golgi breakdown. Specifically, Plk3 was concentrated at the Golgi apparatus in HeLa and A549 cells during interphase. At the onset of mitosis, Plk3 signals disintegrated and redistributed in a manner similar to those of Golgi stacks. Nocodazole activated Plk3 kinase activity, correlating with redistribution of Plk3 signals and Golgi fragmentation. In addition, treatment with brefeldin A (BFA), a Golgi-specific poison, also resulted in disappearance of concentrated Plk3 signals. Plk3 interacted with giantin, a Golgi-specific protein. Expression of Plk3, but not the kinase-defective Plk3 (Plk3(K52R)), resulted in significant Golgi breakdown. Given its role in cell cycle progression, Plk3 may be a protein kinase involved in regulation of Golgi fragmentation during the cell cycle.     

Three different pathological lesions in the <Emphasis Type="Italic">NF1</Emphasis> gene originating de novo in a family with neurofibromatosis type 1

Three members of a Portuguese family, who exhibited clinical evidence of neurofibromatosis type 1 (NF1), were found to possess different heritable and pathological mutations in their NF1 genes: a 1.5-Mb deletion spanning the entire NF1 gene, a truncating CGA-->TGA transition in exon 22 (R1241X), and a frameshift mutation in exon 29 (5406insT). All three lesions occurred de novo and are likely to have been generated by different mutational mechanisms. At least two of the mutations occurred on different chromosomal backgrounds. The probability of finding three non-identical NF1 gene lesions arising de novo in a family with NF1 is very remote, too low to be readily accepted as mere coincidence. A number of possible explanations for this unique finding were therefore explored, but none were found to be wholly convincing. This report nevertheless serves as a reminder that it is unwise, even in the case of an autosomal dominant condition, to extrapolate from the detection of a single mutation in a specific individual to assuming an identical molecular genetic aetiology in other clinically affected members of the same family.     

Studies on the immunogenic potential of plant-expressed cholera toxin B subunit

Nicotiana tabacum var. Samsun was transformed via Agrobacterium-mediated transformation with a gene encoding the cholera toxin B subunit (CTB) of Vibrio cholerae, modified to contain a sequence coding for an endoplasmic reticulum retention signal (SEKDEL), under the control of the cauliflower mosaic virus 35S promoter. Total protein from the transgenic leaf tissue was isolated and an aliquot containing 5 g recombinant CTB was injected intradermally into Balb/c (H2K^d) mice. CTB-specific serum IgG was detected in animals that had been administered plant-expressed or native purified CTB. A T-cell proliferation study using splenocytes and cytokine estimations in supernatants generated by in vitro stimulation of macrophages isolated from the immuno-primed animals was carried out. Inhibition of proliferation of T lymphocytes was observed in splenic T lymphocytes isolated from animals injected with either native or plant-expressed CTB. Macrophages isolated from mice immunised with native or plant-expressed CTB showed enhanced secretion of interleukin-10 but secretion of lipopolysaccharide-induced interleukin-12 and tumor necrosis factor alpha was inhibited. These studies suggest that plant-expressed protein behaved like native CTB with regards to effects on T-cell proliferation and cytokine levels, indicating the suitability of plant expression systems for the production of bacterial antigens, which could be used as edible vaccine. The transgene was found to be inherited in the progeny and was expressed to yield a pentameric form of CTB as evident by its interaction with G_M1 ganglioside.Abbreviations BAP 6-Benzylaminopurine - Con A Concanavilin A - CTB Cholera toxin B subunit - ctxB Gene encoding cholera toxin B subunit - ELISA Enzyme-linked immunosorbent assay - HRP Horseradish peroxidase - IL-10 Interleukin-10 - IL-12 Interleukin-12 - LPS Lipopolysaccharide - NAA Naphthaleneacetic acid - PBS Phosphate-buffered saline - TNF Tumour necrosis factor alphaCommunicated by H. Uchimiya     

NSD1 mutations are the major cause of Sotos syndrome and occur in some cases of Weaver syndrome but are rare in other overgrowth phenotypes

Sotos syndrome is a childhood overgrowth syndrome characterized by a distinctive facial appearance, height and head circumference >97th percentile, advanced bone age, and developmental delay. Weaver syndrome is characterized by the same criteria but has its own distinctive facial gestalt. Recently, a 2.2-Mb chromosome 5q35 microdeletion, encompassing NSD1, was reported as the major cause of Sotos syndrome, with intragenic NSD1 mutations identified in a minority of cases. We evaluated 75 patients with childhood overgrowth, for intragenic mutations and large deletions of NSD1. The series was phenotypically scored into four groups, prior to the molecular analyses: the phenotype in group 1 (n=37) was typical of Sotos syndrome; the phenotype in group 2 (n=13) was Sotos-like but with some atypical features; patients in group 3 (n=7) had Weaver syndrome, and patients in group 4 (n=18) had an overgrowth condition that was neither Sotos nor Weaver syndrome. We detected three deletions and 32 mutations (13 frameshift, 8 nonsense, 2 splice-site, and 9 missense) that are likely to impair NSD1 functions. The truncating mutations were spread throughout NSD1, but there was evidence of clustering of missense mutations in highly conserved functional domains between exons 13 and 23. There was a strong correlation between presence of an NSD1 alteration and clinical phenotype, in that 28 of 37 (76%) patients in group 1 had NSD1 mutations or deletions, whereas none of the patients in group 4 had abnormalities of NSD1. Three patients with Weaver syndrome had NSD1 mutations, all between amino acids 2142 and 2184. We conclude that intragenic mutations of NSD1 are the major cause of Sotos syndrome and account for some Weaver syndrome cases but rarely occur in other childhood overgrowth phenotypes.     

Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB

The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis.