A petal‐specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal‐specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal‐specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal‐specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical β‐glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal‐specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA‐like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal‐specific promoter in molecular breeding of floricultural crops.     

An oral Salmonella vaccine promotes the down-regulation of cell surface Toll-like receptor 4 (TLR4) and TLR2 expression in mice

A single oral immunization with the Lon-protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) induced protective immunity in mice against a subcutaneous challenge with virulent Listeria monocytogenes as well as virulent Salmonella serovar Typhimurium. The populations of cell surface Toll-like receptor 4 (TLR4) and TLR2 on peritoneal macrophages decreased at week 6 after immunization. This population decrease was not reversed after a challenge with either Salmonella or Listeria. These results suggest that oral immunization with CS2022 induced immune tolerance correlated with the down-regulation of cell surface TLR expression. This down-regulation may in part account for the development of cross-protection against a Listeria challenge by immunization with CS2022.     

Participation of caspase-3-like protease in oxidation-induced impairment of erythrocyte membrane properties

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.     

Repression of sigK intervening (skin) element gene expression by the CI-like protein SknR and effect of SknR depletion on growth of Bacillus subtilis cells

The Bacillus subtilis phage DNA-like sigK intervening (skin) element (48 kb) is excised from the chromosome by DNA rearrangement, and a composite gene, sigK (spoIIIC and spoIVCB), is created on the chromosome during sporulation. In this study, we first focused on the role of sknR (skin repressor), which has homology with the gene encoding the Xre repressor of defective phage PBSX. The depletion of SknR caused overexpression of the region between yqaF and yqaN (the yqaF-yqaN operon) and a growth defect in B. subtilis. Point mutation analysis and an electrophoretic mobility shift assay (EMSA) suggested that SknR functions as a negative regulator of gene expression in the yqaF-yqaN operon of the skin element through direct interaction with operators of 2-fold symmetry located in the intergenic region between sknR and yqaF. Deletion analysis revealed that the lethal effect of depletion of SknR was related to overexpression of yqaH and yqaM, whose products were previously reported to associate with DnaA and DnaC, respectively. Furthermore, overexpression of either yqaH or yqaM caused cell filamentation and abnormal chromosome segregation, which suggested that overproduction of these proteins inhibits DNA replication. Moreover, overexpression of yqaM inhibited the initiation of replication. Taken together, these data demonstrate that the B. subtilis skin element carries lethal genes, which are induced by the depletion of sknR.     

Identification of the putative protein phosphatase gene PTC1 as a virulence-related gene using a silkworm model of Candida albicans infection

Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1Δ, yvh1Δ, sit4Δ, and ptc1Δ) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1Δ, yvh1Δ, and sit4Δ mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1Δ revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.     

PLANT U-BOX PROTEIN10 Regulates MYC2 Stability in Arabidopsis

MYC2 is an important regulator for jasmonic acid (JA) signaling, but little is known about its posttranslational regulation. Here, we show that the MYC2 C-terminal region interacted with the PLANT U-BOX PROTEIN10 (PUB10) armadillo repeats in vitro. MYC2 was efficiently polyubiquitinated by PUB10 with UBC8 as an E2 enzyme and the conserved C249 in PUB10 was required for activity. The inactive PUB10(C249A) mutant protein retained its ability to heterodimerize with PUB10, thus blocking PUB10 E3 activity as a dominant-negative mutant. Both MYC2 and PUB10 were nucleus localized and coimmunoprecipitation experiments confirmed their interaction in vivo. Although unstable in the wild type, MYC2 stability was enhanced in pub10, suggesting destabilization by PUB10. Moreover, MYC2 half-life was shortened or prolonged by induced expression of PUB10 or the dominant-negative PUB10(C249A) mutant, respectively. Root growth of pub10 seedlings phenocopied 35S:MYC2 seedlings and was hypersensitive to methyl jasmonate, whereas 35S:PUB10 and jin1-9 (myc2) seedlings were hyposensitive. In addition, the root phenotype conferred by MYC2 overexpression in double transgenic plants was reversed or enhanced by induced expression of PUB10 or PUB10(C249A), respectively. Similar results were obtained with three other JA-regulated genes, TAT, JR2, and PDF1.2. Collectively, our results show that MYC2 is targeted by PUB10 for degradation during JA responses.     

An insect cell line discrimination method by RAPD-PCR

Summary RAPD-PCR with a tenmar single primer for discrimination of insect cell lines was devised. The base sequence of the primers used were TTCGAGCCAG, CCGCATCTAC, GAACGGACTC, and TGAGTGGGTG (GC contents were 60%). Genome DNA was extracted by modified Landry et al. (1993) method. The reaction mixture consisted of 10 μl buffer, 8 μl dNTP mixture (2.5 mM each), 4 μl primer (50 μM), Taq DNA polymerase (2.5 units), 1 μl template DNA; and the reaction was run at 94° C for 2 min (denaturation), followed by 31 cycles of 94° C for 1 min, 42° C for 1 min (annealing), and 72° C for 2 min (extension) and terminated with 72° C for 7 min. By developing the reaction products with agarose gel electrophoresis, it became evident that DNA fragments were amplified with all the primers used. Among four primers, the second primer was selected as a suitable primer for distinguishing cell lines. With this method, cell lines derived from different species were clearly distinguished.     

Role of internalization in the pathogenicity of Shiga toxin-producing Escherichia coli infection in a gnotobiotic murine model

We investigated the role of bacterial internalization in the killing caused by Shiga toxin-producing Escherichia coli (STEC) infection using a gnotobiotic murine model. A high number of internalized STEC was found in the colonic epithelial cells of STEC-infected mice by both an ex vivo assay and transmission electron microscopy. Most of these mice were killed within 10 days after infection. However, the implantation of lactic acid bacteria in such mice before infection markedly decreased the number of internalized STECs and also completely protected these hosts from killing by a STEC infection. The inhibition of such internalization by immunoglobulin also prevented the hosts from being killed. The Shiga toxin levels in these hosts indicated an inhibition of the penetration of Shiga toxins produced in the colon to the underlying tissue. These results suggested that the internalization plays an important role in the pathogenicity caused by STEC infection in a gnotobiotic murine model.