Genome-Wide Expression Profiling of OsWRKY Superfamily Genes during Infection with Xanthomonas oryzae pv. oryzae Using Real-Time PCR.

WRKY transcription factors (TFs) are involved in regulating a range of biological processes such as growth, development, and the responses to biotic and abiotic stresses. Genome-wide expression profiling of OsWRKY TF superfamily genes in rice after infection with Xanthomonas oryzae pv. oryzae (Xoo) was performed to elucidate the function of OsWRKY TFs in the interaction between rice and Xoo. Of the 111 OsWRKY TF genes tested, the transcription of 94 genes changed after Xoo infection. The OsWRKY TF genes were classified into eight types according to their expression profiles. Eighty-two genes in Groups I, II, III, IV, VII were up-regulated after exposure to a compatible or an incompatible race of Xoo. Examination of salicylic acid (SA)-deficient rice lines revealed that SA was involved in Xa1-mediated resistance to Xoo infection. OsWRKY TF genes involved in Xa1-mediated resistance were classified according to their SA-dependent or -independent expression. In SA-deficient rice, the expression of 12 of 57 OsWRKY TF genes involved in Xa1-mediated resistance was compromised. Of these six OsWRKY TF genes were induced by SA. OsWRKY88, an example of a gene possibly involved in SA-dependent Xa1-mediated resistance, activated defense related genes and increased resistance to Xoo. Thus, expression profiling of OsWRKY TF genes may help predict the functions of OsWRKY TF genes involved in Xa1-mediated resistance.

A 4-Nitroquinoleneoxide-Induced Pleurotus eryngii Mutant Variety Increases Pin1 Expression in Rat Brain.

To develop Pleurotus eryngii varieties with improved medicinal qualities, protoplasts of P. eryngii were mutagenized using 4-nitroquinoleneoxide. The effects of the resulting variant mushrooms on a human cell were evaluated by applying their aqueous extracts to the human hepatoma cell line, HepG2, in vitro and examining any alteration in the proteomes of the treated HepG2. The P. eryngii mutant, NQ2A-12, was selected for its effects on increasing the expression level of Pin1 in HepG2. Pin1 is one of the peptidyl-prolyl cis-trans isomerases known to play an important role in repressing Alzheimer's disease pathogenesis. Validity of NQ2A-12 related to Alzheimer's disease was shown with an enhanced expression of Pin1 in a mouse brain tissue by injecting the NQ2A-12 extract. The mutant mushroom, NQ2A-12, could be developed as a new variety of P. eryngii with potential to protect against Alzheimer's disease.

Fusobacterium hwasookii sp. nov., Isolated from a Human Periodontitis Lesion.

In this study, we classified the five strains (ChDC F128(T), ChDC F145, ChDC F174, ChDC F206, and ChDC F300) as a novel species of genus Fusobacterium by DNA-DNA hybridization and multi-locus phylogenetic analysis (MLPA), based on a single sequence (24,715 bp) of 22 concatenated housekeeping genes, with morphological and chemotaxonomic characteristics. DNA-DNA hybridization data showed that the values of genomic relatedness between ChDC F128(T) and each of the other novel strains were ranged from 79.0 to 82.6 %, while those of genomic relatedness between ChDC F128(T) and type strain of each of subspecies of F. nucleatum or Fusobacterium periodonticum were ranged from 40.9 to 54.4 %. MLPA revealed that the 5 strains were clustered as one group and clearly discriminated with F. nucleatum and F. periodonticum with 100 % bootstrap value. The DNA G+C content of the five novel strains were ranged from 26.9 to 27.0 mol%. The cellular fatty acid analysis of clinical isolates and type strains revealed C14:0, C16:0, and cis-9 C16:1 as the major fatty acids. The cell wall peptidoglycan of the 5 strains was comprised of meso-lanthionine. These results show that the 5 strains are novel species and belong to the genus Fusobacterium. Strain ChDC F128(T) (=KCOM 1249(T) = KCTC 5108(T) = JCM 30218(T)) is suggested to be the type strain of a novel species of genus Fusobacterium, for which the name Fusobacterium hwasookii sp. nov. is proposed.

Peptoniphilus mikwangii sp. nov., isolated from a clinical specimen of human origin.

A novel Gram-positive, anaerobic, and coccus-shaped bacterium, designated as strain ChDC B134(T), was isolated from a human postoperative infectious lesion in the right maxilla between the lateral incisor and canine and was characterized by polyphasic taxonomic analysis. 16S rRNA gene sequence analysis revealed that the strain ChDC B134(T) belonged to the genus Peptoniphilus, as it showed sequence similarities to Peptoniphilus indolicus KCTC 15023(T) (94.0 %) and Peptoniphilus asaccharolyticus KCTC 3321(T) (93.8 %). The prevalent fatty acids of of strain ChDC B134(T) were C16:0 (20.3 %), C18:1 cis 9 (34.3 %), and C18:0 (13.2 %). The DNA G+C content was 30.9 mol%. The cell wall diamino acid was D-ornithine, which is a property shared by other reference type strains of the genus Peptoniphilus. Based on the results of phenotypic, chemotaxonomic, and phylogenetic analysis, strain ChDC B134(T) (=KCOM 1628(T) = KCTC 15227(T) = JCM 30223(T)) should be classified as the type strain of a novel species of genus Peptoniphilus, for which the name Peptoniphilus mikwangii sp. nov. is proposed.

Nitrate inhibits soybean nodulation by regulating expression of CLE genes.

Nitrogen compounds such as nitrate act as a potential inhibitor for legume nodulation. In this study, we isolated a new CLE gene, GmNIC2, from nitrate-treated roots, which shares high sequence homology with nitrate-induced CLE gene GmNIC1. Similar to GmNIC1, the expression level of GmNIC2 was not significantly altered in roots by rhizobial inoculation and was much higher in young nodules than in roots. In addition, overexpression of GmNIC2 led to similar nodulation inhibition of transgenic hairy roots to that of GmNIC1, which occurred in GmNARK-dependent manner and at the local level. By analyzing GmNARK loss-of-function mutant, SS2-2, it was found that expression levels of GmNIC1 and GmNIC2 in the SS2-2 roots were lower than in the wild type (WT) roots in response to nitrate. In contrast to GmNIC1 and GmNIC2, expressions of GmRIC1 and GmRIC2 genes that are related to the autoregulation of nodulation (AON) were strongly suppressed both of the soybeans during all periods of nitrate treatment and even were not induced by additional inoculation with rhizobia. Taken together, the results of this study suggest that GmNIC2, as an active homologous gene located in chromosome 13, acts locally to suppress nodulation, like GmNIC1, and nitrate inhibition of nodulation is led by fine-tuned regulation of both nitrate-induced CLEs and rhizobia-induced CLEs.

Western blot membrane composed of electrospun polyvinylidene fluoride nanofiber membrane and polyethylene terephthalate sheet.

In a previous study, an electrospun polyvinylidene fluoride (PVDF) nanofiber membrane was developed for Western blotting. The membrane exhibited high sensitivity and high binding capacity for the detection of protein bands that was unlike that observed for conventional, microphase separation-based porous PVDF membranes. Nevertheless, the PVDF nanofiber membrane is quite expensive. The objective of this study was to develop an economical Western blot membrane using a hybrid electrospun PVDF nanofiber and polyethylene terephthalate (PET) sheet. The results showed that the detection sensitivity of the 4 gram per square meter (gsm) membrane was similar to those of the electrospun PVDF nanofiber membrane only, and the 7 gsm PVDF nanofiber membranes on a PET sheet and the electrospun PVDF nanofiber membrane. This means the protein detection sensitivity is not proportional to the thickness of the PVDF nanofiber membrane. The 4 gsm PVDF nanofiber membrane on a PET sheet can be used to detect proteins with high sensitivity and economic efficiency.

Surface modification of electrospun polyvinylidene fluoride nanofiber membrane by plasma treatment for protein detection.

Electrospun polyvinylidene fluoride (PVDF) nanofiber membranes have 3-dementional (3-D) open pore channel and hence have excellent application potential in Western blot. In this study we have modified electrospun PVDF nanofiber membrane by argon (Ar) plasma treatment to improve the surface hydrophilic and detection sensitivity. The results showed that the detection sensitivity of the Ar plasma-treated PVDF nanofiber membrane increased with increasing plasma treatment time without the need for a methanol pre-wet step. This suggests that the Ar plasma treated PVDF nanofiber membrane can be useful in Western blot with high sensitivity and without methanol pre-wet step.

Soybean nodule-enhanced CLE peptides in roots act as signals in GmNARK-mediated nodulation suppression.

The number of nodules formed in the roots of leguminous plants is systemically controlled by autoregulation of nodulation (AON). This study characterized two of the CLAVATA3/endosperm-surrounding region (CLE) genes involved in AON signal transduction. The GmRIC1 and GmRIC2 genes initiated expression solely in the roots at approximately 3 days after inoculation (DAI) with Nod factor-producing rhizobia, corresponding to the time point of AON, and the expression was up-regulated by cytokinins. Levels of GmRIC1 and GmRIC2 gene expression were much higher in the supernodulation mutant, SS2-2, than in wild-type (WT) soybeans during nodule development, even after initiation of nitrogen fixation. At 3 DAI, GmRIC2 was induced in the cells of the pericycle and the outer cortex, which undergo cell division to form nodule primordia and spreads from the central region to the whole nodule as it develops. Overexpression of GmRIC1 and GmRIC2 strongly suppressed the nodulation of WT roots as well as transgenic hairy roots in a GmNARK-dependent manner. This systemic suppression of nodulation was caused by the secretion of two CLE proteins into the extracellular space. Double grafting between WT and SS2-2 soybeans showed that signal Q is larger in SS2-2 than in WT roots during nodulation. The results of this study suggest that GmRIC1 and GmRIC2 are good candidates for root-derived signal Q in AON signal transduction.

Comparative proteomic analysis of soybean nodulation using a supernodulation mutant, SS2-2.

Legumes have the ability to form root nodules that fix atmospheric nitrogen through a symbiotic interaction with nitrogen-fixing bacteria. As a first step in dissecting the molecular process of nodulation, proteome reference maps of soybean roots and nodules were constructed. Time course analysis revealed that the transition from root to nodule was accompanied with downregulation of defense-response related proteins, including Mn-superoxide dismutase, peroxidase (Prx), PR10, and stress-induced protein, leading to the initiation of a symbiotic interaction between the two partners. Following nitrogenase biosynthesis, the host plant cooperated with the rhizobia to fix atmospheric nitrogen under microaerobic conditions via expression of leghemoglobins and antioxidant proteins. Comparative proteome analysis indicated lower expression of malate dehydrogenase (MDH), leghemoglobins and nitrogenase in the nodule development of the supernodulation mutant, SS2-2, as compared to the wild type, indicating that SS2-2 forms functionally immature nodules in higher numbers with the lower activity of nitrogen fixation.

Genetic analysis of genes controlling natural variation of seed coat and flower colors in soybean.

Soybean exhibits natural variation in flower and seed coat colors via the deposition of various anthocyanin pigments in the respective tissues. Although pigmentation in seeds or flowers has been well dissected at molecular level in several plant species, the genes controlling natural variation in anthocyanin traits in the soybean are not completely understood. To evaluate the genetic correlation between genetic loci and genes, 8 enzyme-encoding gene families and a transcription factor were localized in a soybean genome-wide genetic map. Among the seed coat color-controlling loci, the genetic location of the gene encoding for W1 was substantiated in the context of the current soybean molecular genetic map and O was postulated to correspond to anthocyanidin reductase. Among the genetic loci that regulate flower pigmentation, the genetic locations of the genes encoding for W1, W4, and Wp were identified, W3 was mapped on soybean linkage group B2 (chromosome 14), and W2 was postulated to correspond to an MYB transcription factor. Correlation studies between the developed markers and 3 color-controlling loci provided important empirical data that should prove useful in the design of marker-assisted breeding schemes as well as future association studies involving soybean.

Distinct roles of the pepper hypersensitive induced reaction protein gene CaHIR1 in disease and osmotic stress, as determined by comparative transcriptome and proteome analyses.

A Capsicum annuum hypersensitive induced reaction protein1 (CaHIR1) was recently proposed as a positive regulator of hypersensitive cell death in plants. Overexpression of CaHIR1 in transgenic Arabidopsis plants conferred enhanced resistance against the hemi-biotrophic Pseudomonas syringae pv. tomato (Pst) and the biotrophic Hyaloperonospora parasitica. Infection by avirulent Pseudomonas strains carrying avrRpm1 or avrRpt2 caused enhanced resistance responses in transgenic plants, suggesting that CaHIR1 is involved in basal disease resistance in a race-nonspecific manner. H. parasitica exhibited low levels of asexual sporulation on CaHIR1 seedlings. In contrast, transgenic plants were susceptible not only to the necrotrophic fungal pathogen Botrytis cinerea but were also sensitive to osmotic stress caused by high salinity and drought. To identify proteins whose expression was altered by CaHIR1 overexpression in Arabidopsis leaves, a quantitative comparative proteome analysis using two-dimensional gel electrophoresis coupled with mass spectrometry was performed. Of about 400 soluble proteins, 11 proteins involved in several metabolic pathways were up- or down-regulated by CaHIR1 overexpression. Genes encoding glycine decarboxylase (At2g35370) and an unidentified protein (At2g03440), which were strongly upregulated in CaHIR1-overexpressing Arabidopsis, were also differentially induced at the transcriptional level by Pst infection. Arabidopsis carbonic anhydrase (At3g01500), highly similar to tobacco salicylic acid-binding protein 3, was up-regulated by CaHIR1 overexpression. The activity of an anti-oxidant enzyme, cooper/zinc superoxide dismutase (At2g28190), was also attenuated in transgenic Arabidopsis by CaHIR1 overexpression. Together, these results suggest that CaHIR1 overexpression in Arabidopsis mediates plant responses to biotrophic, hemi-biotrophic and necrotrophic pathogens, as well as to osmotic stress in different ways.

The glycine max xylem sap and apoplast proteome.

Molecular signaling interactions in the plant apoplast are important for defense and developmental responses. We examined the soybean proteome of the apoplastic conduit of root-to-shoot communication, the xylem stream, using gel electrophoresis combined with two types of tandem mass spectrometry. We examined soybeans for the presence of a Bradyrhizobium japonicum-induced, long distance developmental signal that controls autoregulation of nodulation (AON) to determine if xylem proteins (XPs) were involved directly or indirectly in AON. The xylem and apoplast fluids collected in hypocotyl, epicotyl, and stem tissue contained a highly similar set of secreted proteins. The XPs were different from those secreted from imbibing seed implying they play important basic roles in xylem function. The XPs of wild-type and nts1007 plants were indistinguishable irrespective of plant age, inoculation status, or time after inoculation suggesting that none was directly involved in AON. XPs were continuously loaded into the xylem stream, as they were present even 28 h after shoot decapitation. These results were consistent with semiquantitative RT-PCR studies that examined the expression of genes corresponding to the XPs under inoculated or uninoculated conditions. Monitoring the expression of XP genes by RT-PCR showed that four possessed root biased expression. This suggested that the corresponding protein products could be produced in roots and travel long distances to shoots. Of these, a species of lipid transfer protein is a candidate for a water-soluble, long-distance signal-carrier due to the presence of hydrophobic clefts that bind known plant signals in vitro. Two soybean XPs identified in this study, lipid transfer protein and Kunitz trypsin inhibitor (KTI), have known roles in plant signaling.

Cytokeratin-20 and seminal vesicle secretory protein VI as possible marker proteins in urinary bladder preneoplastic lesions induced by N-butyl-N-(4-hydroxybutyl) nitrosamine.

BACKGROUND: A simple and non-invasive methods for the diagnosis of transitional cell carcinoma of the bladder are needed for the prevention of invasive tumor. A proteomic technology has recently been developed to facilitate protein profiling of biological mixtures. We tried to detect the marker proteins by proteomic approach during the initiation stages on N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder carcinogenesis in rats. METHODS: Ten rats in group A were given 0.05% BBN in drinking water for 12 weeks. Other 10 rats in group B with 10 rats were designated as a control group and were not given BBN. Whole urinary bladders of all rats were excised at 12 weeks from the beginning of the experiment. Conventional proteomics was performed with high resolution 2-dimension gel electrophoresis followed by computational image analysis and protein identification using mass spectrometry. RESULTS: A comparison of urinary bladder hyperplasia tissue with control tissue showed that five proteins; actin gamma2 propeptide, cytokeratin-20 (CK-20), proapolipoprotein, alpha2 actin (alpha-cardiac actin) and heat shock 27 kDa protein-1 were over-expressed in hyperplastic tissues. Three proteins; transcription factor myocardin, seminal vesicle secretory protein VI (SVS-VI) precursor and hypothetical protein RMT-7 were under-expressed in hyperplastic tissues. CONCLUSION: In our animal mode, BBN-induced urinary bladder mucosal hyperplasia resulted in an increase in the expression of five proteins and a decrease in the expression of three proteins. Of these, CK-20 and SVS-VI seem to be of particular interest. However other method such as Western blotting seems to be needed for confirmation of these proteins and more information on human bladder tissue is needed for clinical application.

Differentially expressed genes related to symbiotic association in a supernodulating soybean mutant and its wild-type.

SUMMARY To understand the molecular basis of symbiotic association, a cDNA-AFLP technique was used to identify differentially expressed transcripts between a supernodulating soybean mutant, SS2-2, and its wild-type, Sinpaldalkong 2. As sources of cDNA-AFLP templates, trifoliates of 2-week-old plants were collected 1 week after Bradyrhizobium japonicum inoculation. A total of 147 bands out of 4000 amplicons were recognized as differentially expressed fragments, with 40 transcript-derived fragments (TDFs) in SS2-2 and 65 TDFs in Sinpaldalkong 2. Qualitative and quantitative real-time RT-PCR assays suggested that the expression patterns of genes in both genotypes were clearly differentiated. TDFs homologous to nodulin (65S2) and a putative senescence-associated protein (9S1) were up-regulated in SS2-2, whereas Sinpaldalkong 2 showed up-regulation of a receptor-like kinase (48sin1) and a kinase-like protein (17sin1). This indicates that different genes may be involved in regulation of the symbiotic programme that distinguishes SS2-2 from its wild-type. A TDF showing a change in a single base from A (Sinpaldalkong 2) to T (SS2-2) in this study was identified as a Glycine max nodule autoregulation receptor-like protein kinase precursor, previously identified by map-based cloning. These results demonstrate that cDNA-AFLP is a powerful technique to detect interesting genes without prior assumptions about the nature of the genes. The differentially expressed genes between Sinpaldalkong 2 and SS2-2 suggest that different signal transduction pathways for symbiosis may be involved in the two soybean genotypes.

Investigation of early protein changes in the urinary bladder following partial bladder outlet obstruction by proteomic approach.

We investigated the pathophysiological mechanism by proteomic approach as a possible tool to detect the marker proteins to develop lower urinary tract symptoms following bladder outlet obstruction (BOO). Rats were randomized into 3 groups; control, sham operation and BOO groups. BOO group was divided into 1, 3, and 5 day-group. Conventional proteomics was performed with high resolution 2-D gel electrophoresis followed by computational image analysis and protein identification using mass spectrometry using rat urinary bladders. A comparison of bladder of BOO group with control bladder showed that three proteins of optineurin, thioredoxin and preprohaptoglobin were over-expressed in the bladder of BOO group. In addition, four proteins, such as peroxiredoxin 2, transgelin, hippocampal cholinergic neurostimulating peptide (HCNP) and beta-galactoside-binding lectin, were under-expressed in the bladder of BOO group. These data supported that downregulation of HCNP might make detrusor muscle be supersensitive to acetylcholine, up-regulation of optineurin means the protection of nerve injury, and down-regulation of transgelin means the decreased contractility of detrusor muscle. Beside these proteins, other proteins are related to oxidative stress or have a nonspecific function in this study. However more information is needed in human bladder tissue for clinical usage.