Homologs of ToxB, a host-selective toxin gene from Pyrenophora tritici-repentis, are present in the genome of sister-species Pyrenophora bromi and other members of the Ascomycota.

Pyrenophora tritici-repentis requires the production of host-selective toxins (HSTs) to cause the disease tan spot of wheat, including Ptr ToxA, Ptr ToxB, and Ptr ToxC. Pyrenophora bromi, the species most closely related to P. tritici-repentis, is the causal agent of brown leaf spot of bromegrass. Because of the relatedness of P. bromi and P. tritici-repentis, we investigated the possibility that P. bromi contains sequences homologous to ToxA and/or ToxB, the products of which may be involved in its interaction with bromegrass. Multiplex polymerase chain reaction (PCR) revealed the presence of ToxB-like sequences in P. bromi and high-fidelity PCR was used to clone several of these loci, which were subsequently confirmed to be homologous to ToxB. Additionally, Southern analysis revealed ToxB from P. bromi to have a multicopy nature similar to ToxB from P. tritici-repentis. A combination of phylogenetic and Southern analyses revealed that the distribution of ToxB extends further into the Pleosporaceae, and a search of available fungal genomes identified a distant putative homolog in Magnaporthe grisea, causal agent of rice blast. Thus, unlike most described HSTs, ToxB homologs are present across a broad range of plant pathogenic ascomycetes, suggesting that it may have arose in an early ancestor of the Ascomycota.     

Homologs of ToxB, a host-selective toxin gene from Pyrenophora tritici-repentis, are present in the genome of sister-species Pyrenophora bromi and other members of the Ascomycota

Pyrenophora tritici-repentis requires the production of host-selective toxins (HSTs) to cause the disease tan spot of wheat, including Ptr ToxA, Ptr ToxB, and Ptr ToxC. Pyrenophora bromi, the species most closely related to P. tritici-repentis, is the causal agent of brown leaf spot of bromegrass. Because of the relatedness of P. bromi and P. tritici-repentis, we investigated the possibility that P. bromi contains sequences homologous to ToxA and/or ToxB, the products of which may be involved in its interaction with bromegrass. Multiplex polymerase chain reaction (PCR) revealed the presence of ToxB-like sequences in P. bromi and high-fidelity PCR was used to clone several of these loci, which were subsequently confirmed to be homologous to ToxB. Additionally, Southern analysis revealed ToxB from P. bromi to have a multicopy nature similar to ToxB from P. tritici-repentis. A combination of phylogenetic and Southern analyses revealed that the distribution of ToxB extends further into the Pleosporaceae, and a search of available fungal genomes identified a distant putative homolog in Magnaporthe grisea, causal agent of rice blast. Thus, unlike most described HSTs, ToxB homologs are present across a broad range of plant pathogenic ascomycetes, suggesting that it may have arose in an early ancestor of the Ascomycota.     

RNA-mediated gene silencing in the cereal fungal pathogen Cochliobolus sativus

A high-throughput RNA-mediated gene silencing system was developed for Cochliobolus sativus (anamorph: Bipolaris sorokiniana), the causal agent of spot blotch, common root rot and black point in barley and wheat. The green fluorescent protein gene (GFP) and the proteinaceous host-selective toxin gene (ToxA) were first introduced into C. sativus via the polyethylene glycol (PEG)-mediated transformation method. Transformants with a high level of expression of GFP or ToxA were generated. A silencing vector (pSGate1) based on the Gateway cloning system was developed and used to construct RNA interference (RNAi) vectors. Silencing of GFP and ToxA in the transformants was demonstrated by transformation with the RNAi construct expressing hairpin RNA (hpRNA) of the target gene. The polyketide synthase gene (CsPKS1), involved in melanin biosynthesis pathways in C. sativus, was also targeted by transformation with the RNAi vector (pSGate1-CsPKS1) encoding hpRNA of the CsPKS1 gene. The transformants with pSGate1-CsPKS1 exhibited an albino phenotype or reduced melanization, suggesting effective silencing of the endogenous CsPKS1 in C. sativus. Sectors exhibiting the wild-type phenotype of the fungus appeared in some of the CsPKS1-silenced transformants after subcultures as a result of inactivation or deletions of the RNAi transgene. The gene silencing system established provides a useful tool for functional genomics studies in C. sativus and other filamentous fungi.     

Localization of Ptr ToxA Produced by Pyrenophora tritici-repentis Reveals Protein Import into Wheat Mesophyll Cells

The plant pathogenic fungus Pyrenophora tritici-repentis secretes host-selective toxins (HSTs) that function as pathogenicity factors. Unlike most HSTs that are products of enzymatic pathways, at least two toxins produced by P. tritici-repentis are proteins and, thus, products of single genes. Sensitivity to these toxins in the host is conferred by a single gene for each toxin. To study the site of action of Ptr ToxA (ToxA), toxin-sensitive and -insensitive wheat (Triticum aestivum) cultivars were treated with ToxA followed by proteinase K. ToxA was resistant to protease, but only in sensitive leaves, suggesting that ToxA is either protected from the protease by association with a receptor or internalized. Immunolocalization and green fluorescent protein tagged ToxA localization demonstrate that ToxA is internalized in sensitive wheat cultivars only. Once internalized, ToxA localizes to cytoplasmic compartments and to chloroplasts. Intracellular expression of ToxA by biolistic bombardment into both toxin-sensitive and -insensitive cells results in cell death, suggesting that the ToxA internal site of action is present in both cell types. However, because ToxA is internalized only in sensitive cultivars, toxin sensitivity, and therefore the ToxA sensitivity gene, are most likely related to protein import. The results of this study show that the ToxA protein is capable of crossing the plant plasma membrane from the apoplastic space to the interior of the plant cell in the absence of a pathogen.     

Chromosome-based molecular characterization of pathogenic and non-pathogenic wheat isolates of Pyrenophora tritici-repentis

The ToxA gene of Pyrenophora tritici-repentis encodes a host-selective toxin (Ptr ToxA) that has been shown to confer pathogenicity when used to transform a non-pathogenic wheat isolate. Major karyotype polymorphisms between pathogenic and non-pathogenic strains, and to a lesser extent among pathogenic strains, and among non-pathogenic strains were identified. ToxA was localized to a 3.0 Mb chromosome. PCR-based subtraction was carried out with the ToxA chromosome as tester DNA and genomic DNA from a non-pathogenic isolate as driver DNA. Seven of 8 single-copy probes that originated from the 3.0 Mb chromosome could be assigned to a 2.75 Mb chromosome of a non-pathogenic isolate. Nine different repetitive DNA probes originated from the 3.0 Mb chromosome, including sequences that correspond to known fungal transposable elements. Two additional single-copy probes that originated from a 3.4 Mb chromosome were unique to the pathogens and they correspond to a peptide synthetase gene. Our findings suggest substantial differences between pathogenic and non-pathogenic isolates of P. tritici-repentis.     

Ptr ToxA interacts with a chloroplast-localized protein

Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces host-selective toxins that are determinants of pathogenicity or virulence. Ptr ToxA (ToxA), a proteinaceous toxin produced by P. tritici-repentis, is a necrotizing toxin produced by the most common races isolated from infected wheat. Recent studies have shown that ToxA is internalized into the mesophyll cells and localizes to chloroplasts of sensitive wheat cultivars only. We employed a yeast two-hybrid screen in an effort to determine plant proteins that interact with ToxA and found that ToxA interacts with a chloroplast protein, designated ToxA binding protein 1 (ToxABP1). ToxABP1 contains a lysine-rich region within a coiled-coil domain that is similar to phosphotidyl-inositol binding sites present in animal proteins involved in endocytosis. In both ToxA-sensitive and -insensitive cultivars, ToxABP1 is expressed at similar levels and encodes an identical protein. ToxABP1 protein is present in both chloroplast membranes and chloroplast stroma. ToxA appears to interact primarily with a multimeric complex of ToxABP1 protein associated with the chloroplast membrane.     

Quantitative variation in effector activity of ToxA isoforms from Stagonospora nodorum and Pyrenophora tritici-repentis

ToxA is a proteinaceous necrotrophic effector produced by Stagonospora nodorum and Pyrenophora tritici-repentis. In this study, all eight mature isoforms of the ToxA protein were purified and compared. Circular dichroism spectra indicated that all isoforms were structurally intact and had indistinguishable secondary structural features. ToxA isoforms were infiltrated into wheat lines that carry the sensitivity gene Tsn1. It was observed that different wheat lines carrying identical Tsn1 alleles varied in sensitivity to ToxA. All ToxA isoforms induced necrosis when introduced into any Tsn1 wheat line but we observed quantitative variation in effector activity, with the least-active version found in isolates of P. tritici-repentis. Pathogen sporulation increased with higher doses of ToxA. The isoforms that induced the most rapid necrosis also induced the most sporulation, indicating that pathogen fitness is affected by differences in ToxA activity. We show that differences in toxin activity encoded by a single gene can contribute to the quantitative inheritance of necrotrophic virulence. Our findings support the hypothesis that the variation at ToxA results from selection that favors increased toxin activity.     

Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants

We made a series of improved Gateway binary vectors (pGWBs) for plant transformation. Fifteen different reporters and tags, sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7, and TAP, were employed. Some vectors carry the 2x35S-Omega promoter for higher-level expression. The kanamycin- and hygromycin-resistant markers are independently available for each of the 43 types of vectors, thus an additional transformation of once-transformed plants can be carried out easily. Their small size and high-copy number in Escherichia coli make possible easier handling at plasmid preparation and sequencing. Improved pGWBs should be a powerful tool for transgenic research in plants.     

Structure of Ptr ToxA: an RGD-containing host-selective toxin from Pyrenophora tritici-repentis

Tan spot of wheat (Triticum aestivum), caused by the fungus Pyrenophora tritici-repentis, has significant agricultural and economic impact. Ptr ToxA (ToxA), the first discovered proteinaceous host-selective toxin, is produced by certain P. tritici-repentis races and is necessary and sufficient to cause cell death in sensitive wheat cultivars. We present here the high-resolution crystal structure of ToxA in two different crystal forms, providing four independent views of the protein. ToxA adopts a single-domain, beta-sandwich fold of novel topology. Mapping of the existing mutation data onto the structure supports the hypothesized importance of an Arg-Gly-Asp (RGD) and surrounding sequence. Its occurrence in a single, solvent-exposed loop in the protein suggests that it is directly involved in recognition events required for ToxA action. Furthermore, the ToxA structure reveals a surprising similarity with the classic mammalian RGD-containing domain, the fibronectin type III (FnIII) domain: the two topologies are related by circular permutation. The similar topologies and the positional conservation of the RGD-containing loop raises the possibility that ToxA is distantly related to mammalian FnIII proteins and that to gain entry it binds to an integrin-like receptor in the plant host.     

Investigating the secretory pathway of the baculovirus-insect cell system using a secretory green fluorescent protein

The secretory pathway is important in actively transporting proteins into the extracellular environment of eucaryotic cells. In this study a green fluorescent protein (GFP) mutant engineered to contain a secretion signal was used as a model protein in order to visualize the secretion process inside insect cells. Fluorescent microscopy indicated that significant amounts of secreted green fluorescent protein (sGFP) accumulated in High-Five, Trichoplusia ni, cells following infection with a baculovirus vector containing the gene under the polyhedrin promoter. Laser scanning confocal microscopy was used to reconstruct whole cell images of the infected High-Five cells at multiple days postinfection. While the protein was widely distributed at 2 days postinfection, certain intracellular regions appeared to contain higher or lower concentrations of the sGFP. A layer by layer examination indicated pockets in which sGFP was absent, and these appear to be vesicles that have recently released the sGFP or are not yet accumulating sGFP. By 3 days postinfection, the sGFP in some cells was concentrated in a number of widely dispersed globules, which may represent the vesicle remnants of a deteriorating secretory pathway. In contrast, nonsecreted GFP was more uniformly distributed in the cells than sGFP and did not accumulate in vesicles. In addition to GFP, the lectins wheat germ agglutinin (WGA) and concanavalin A (ConA), which have affinities for sugar residues, were used to examine the secretory pathway. The WGA, which is a Golgi marker, was distributed around the nucleus prior to infection but then was found to be polarized in one region of the cell following the baculovirus infection. The expansion of other cellular compartments following the baculovirus infection may have caused a change in intracellular distribution of the Golgi. While some of the sGFP was found to colocalize with the WGA label, much of the sGFP was outside this Golgi region. In contrast, ConA labeling, which was not as specific as WGA, was found throughout the cell both before and after infection similar to the sGFP distribution. These studies demonstrate that confocal visualization of fluorescent proteins can be used as an in vivo tool for examining secretory processing in insect cells.     

Protein expression and subcellular localization of the general purine transporter UapC from Aspergillus nidulans

The uapC gene of Aspergillus nidulans belongs to a family of nucleobase-specific transporters conserved in prokaryotic and eucaryotic organisms. We report the use of immunological and green fluorescent protein based strategies to study protein expression and subcellular distribution of UapC. A chimeric protein containing a plant-adapted green fluorescent protein (sGFP) fused to the C-terminus of UapC was shown to be functional in vivo, as it complements a triple mutant (i.e., uapC(-) uapA(-) azgA(-)) unable to grow on uric acid as the sole nitrogen source. UapC-GFP is located in the plasma membrane and, secondarily, in internal structures observed as fluorescent dots. A strong correlation was found between cellular levels of UapC-GFP fluorescence and known patterns of uapC gene expression. This work represents the first in vivo study of protein expression and subcellular localization of a filamentous fungal nucleobase transporter.     

Construction of two Gateway vectors for gene expression in fungi

We report the construction of two Gateway fungal expression vectors pCBGW and pGWBF. The pCBGW was generated by introducing an expression cassette, which consists of a Gateway recombinant cassette (attR1-Cmr-ccdB-attR2) under the control of fungal promoter PgpdA and a terminator TtrpC, into the multiple cloning site of fungal vector pCB1004. The pGWBF is a binary vector, which was generated from the plant expression vector pGWB2 by replacing the CaMV35S promoter with PgpdA. The pGWBF can be transformed into fungi efficiently with Agrobacterium-mediated transformation. The applicability of two newly constructed vectors was tested by generating the destination vectors pGWBF-GFP and pCBGW-GFP and examining the expression of GFP gene in Trichoderma viride and Gibberella fujikuroi, respectively. Combining with the advantage of Gateway cloning technology, pCBGW and pGWBF will be useful in fungi for large-scale investigation of gene functions by constructing the interested gene destination/expression vectors in a high-throughput way.     

A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells

Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.     

Construction of a native promoter-containing transposon vector for the stable monitoring of the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp

Green fluorescent protein (GFP) is the most potential useful marker for the in situ monitoring of biofilm microbes. The objective of this study was to construct and compare the efficacy of transposon vectors containing native and foreign promoters in monitoring the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp. The promoter of nitrite reductase (Pnir) was cloned from LYS-86 and utilized to construct the transposon vector pUT/mini-Tn5-km2-Pnir-gfp. Another transposon vector, pUT/mini-Tn5-km2-Plac-gfp, containing the lactose promoter Plac was also constructed. These two transposon vectors and pUT-luxAB-gfp containing the promoter PpsbA were individually inserted into the chromosome of P. stutzeri LYS-86 by conjugation. Three GFP-tagged recombinant strains, LYS-Plac-gfp, LYS-Pnir-gfp, and LYS-PpsbA-gfp, were selected from the conjugants. Green fluorescence was observed only in LYS-Pnir-gfp, suggesting that the native promoter Pnir may be more suitable for GFP expression in P. stutzeri than the foreign promoters Plac and PpsbA. Indeed, LYS-Pnir-gfp maintained stable GFP fluorescence over 16 subcultures without significant changes in the denitrifying capacity.     

Novel Expression System for Combined Vaccine Production in <Emphasis Type="Italic">Edwardsiella tarda</Emphasis> Ghost and Cadaver Cells

To develop combined vaccine systems, we have generated Edwardsiella tarda ghosts (ETG) displaying a foreign protein on the outer membrane and also Ed. tarda cadaver (ETC) expressing a heterologous protein in the cytoplasm. Green fluorescent protein (GFP) was used as a model foreign protein. A constitutive promoter (EtPR C28-1) cloned newly from Ed. tarda was used as a promoter for the expression of foreign protein. Comparison of the strength of the new promoter with a commercially available constitutive promoter (P_HCE) showed higher expression levels of the novel expression system. The N-terminal domain of ice nucleation protein (InaN), an outer membrane protein of Pseudomonas syringae, was used as an anchor motif for surface display of GFP. By transformation of Ed. tarda with the constructed vectors, GFP was successfully expressed on the surface of ETG and in the cytoplasm of ETC. When compared to P_HCE driven expression, approximately more than 2 times of GFP was expressed on ETG and in ETC by EtPR C28-1 promoter when judged by fluorescent spectrophotometry. Furthermore, significantly higher expression of GFP on the surface of ETG by EtPR C28-1 than by P_HCE was demonstrated by serum agglutination assay. These results suggest that the newly cloned Ed. tarda constitutive promoter is capable to express foreign proteins not only on the surface of Ed. tarda ghosts but also in the cytoplasm of Ed. tarda cadavers, and can be used as an efficient promoter for the expression of heterologous antigens of the ETG and ETC-based combined vaccines.     

GFP工程酵母菌的构建及其对Cu2+的荧光响应

从酿酒酵母基因组DNA中克隆到金属硫蛋白启动子(PCUP1)片段,将绿色荧光蛋白(GFP)基因置于PCUP1的调控下,构建重组质粒pCUP9K-GFP,并通过氯化锂法转化毕赤酵母,获得工程菌株。工程菌细胞及其发酵液中可检出GFP荧光,表明PCUP1能启动外源基因GFP转录,使工程菌表达并分泌GFP。研究发现,工程菌培养液中分别加入10μmol/L的铜、铬、镉和砷离子后,铜处理组GFP荧光强度明显增加,其余三种离子对工程菌荧光强度影响不大;用铜离子诱导后,工程菌发酵上清液的荧光强度明显增强,并与铜离子浓度(0～1mmol/L)呈正相关。研究表明,该工程菌中启动子PCUP1受铜离子诱导,GFP的表达对铜离子具有剂量依赖性,在一定浓度范围内,GFP荧光强度与铜离子浓度呈正相关。     

Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters

It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation.     

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class.     

Agrobacterium tumefaciens-mediated transformation of Leptosphaeria spp. and Oculimacula spp. with the reef coral gene DsRed and the jellyfish gene gfp

Four filamentous ascomycetes, Leptosphaeria maculans, L. biglobosa, Oculimacula yallundae and O. acuformis, were transformed via Agrobacterium tumefaciens-mediated transformation with the genes encoding DsRed and GFP. Using vectors pCAMDsRed and pCAMBgfp, either germinated conidia of Leptosphaeria spp. and O. yallundae or physically fragmented cultures of Oculimacula spp. were transformed. In vitro, the expression of the two reporter proteins in mycelium of both Oculimacula and both Leptosphaeria species was sufficient to distinguish each species in co-inoculated cultures. In planta, transformants of L. maculans or L. biglobosa expressing DsRed or GFP could be observed together in leaves of Brassica napus. Either reporter protein could be used to view the colonization of leaf petioles by both Leptosphaeria spp. and growth in the xylem vessels could be clearly observed. With the generation of these transformants, further studies on interactions between pathogen species involved in disease complexes on various host species and between opposite mating types of the same species are now possible.