A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants

Investigation of plant-bacteria interactions requires quantification of in planta bacterial titers by means of cumbersome and time-consuming colony-counting assays. Here, we devised a broadly applicable tool for bioluminescence-based quantitative and spatial detection of bacteria in plants. We developed vectors that enable Tn7 transposon-mediated integration of the luxCDABE luciferase operon into a specific genomic location found ubiquitously across bacterial phyla. These vectors allowed for the generation of bioluminescent transformants of various plant pathogenic bacteria from the genera Pseudomonas, Rhizobium (Agrobacterium), and Ralstonia. Direct luminescence measurements of plant tissues inoculated with bioluminescent Pseudomonas syringae pv. tomato DC3000 (Pto-lux) reported bacterial titers as accurately as conventional colony-counting assays in Arabidopsis thaliana, Solanum lycopersicum, Nicotiana benthamiana, and Marchantia polymorpha. We further showed the usefulness of our vectors in converting previously generated Pto derivatives to isogenic bioluminescent strains. Importantly, quantitative bioluminescence assays using these Pto-lux strains accurately reported the effects of plant immunity and bacterial effectors on bacterial growth, with a dynamic range of four orders of magnitude. Moreover, macroscopic bioluminescence imaging illuminated the spatial patterns of Pto-lux growth in/on inoculated plant tissues. In conclusion, our vectors offer untapped opportunities to develop bioluminescence-based assays for a variety of plant-bacteria interactions

Interactions between p27 and p88 replicase proteins of Red clover necrotic mosaic virus play an essential role in viral RNA replication and suppression of RNA silencing via the 480-kDa viral replicase complex assembly

AbstractRed clover necrotic mosaic virus (RCNMV), a positive-sense RNA virus with a bipartite genome, encodes p27 and p88 replicase proteins that are required for viral RNA replication and suppression of RNA silencing. In this study, we indentified domains in p27 and p88 responsible for their protein–protein interactions using in vitro pull-down assays with the purified recombinant proteins. Coimmunoprecipitation analysis in combination with blue-native polyacrylamide gel electrophoresis using mutated p27 proteins showed that both p27–p27 and p27–p88 interactions are essential for the formation of the 480-kDa complex, which has RCNMV-specific RNA-dependent RNA polymerase activity. Furthermore, we found a good correlation between the accumulated levels of the 480-kDa complex and replication levels and the suppression of RNA silencing activity. Our results indicate that interactions between RCNMV replicase proteins play an essential role in viral RNA replication and in suppressing RNA silencing via the 480-kDa replicase complex assembly

The rice OsERF101 transcription factor regulates the NLR Xa1-mediated immunity induced by perception of TAL effectors

イネが病原菌の感染力の源を検出して免疫を誘導する仕組みを解明 --病気に強い植物の開発に期待--. 京都大学プレスリリース. 2022-09-07.Plant nucleotide-binding leucine-rich repeat receptors (NLRs) initiate immune responses by recognizing pathogen effectors. The rice gene Xa1 encodes an NLR with an N-terminal BED domain, and recognizes transcription activator-like (TAL) effectors of Xanthomonas oryzae pv. oryzae (Xoo). Our goal is to elucidate the molecular mechanisms controlling the induction of immunity by Xa1. We used yeast two-hybrid assays to screen for host factors that interact with Xa1 and identified the AP2/ERF-type transcription factor OsERF101/OsRAP2.6. Molecular complementation assays were used to confirm the interactions among Xa1, OsERF101, and two TAL effectors. We created OsERF101-overexpressing and knockout mutant lines in rice and identified genes differentially regulated in these lines, many of which are predicted to be involved in regulation of response to stimulus. Xa1 interacts in the nucleus with the TAL effectors and OsERF101 via the BED domain. Unexpectedly, both the overexpression and knockout lines of OsERF101 displayed Xa1-dependent, enhanced resistance to an incompatible Xoo strain. Different sets of genes were up- or down-regulated in the overexpression and knockout lines. Our results indicate that OsERF101 regulates the recognition of TAL effectors by Xa1, and functions as a positive regulator of Xa1-mediated immunity. Further, an additional Xa1-mediated immune pathway is negatively regulated by OsERF101

GAPDH-A Facilitates Intercellular Movement of RCNMV

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process

Current structure observed in the central equatorial Pacific Ocean

東京水産大学環境システム学講座東京水産大学海鷹丸東京水産大学海鷹丸東京水産大学海鷹丸東京水産大学海鷹丸東京水産大学環境システム学講座東京水産大学海鷹

Long-Term Voyages and Bone Mass Among Seamen

東京水産大学海洋生産学専攻東京水産大学練習船海鷹丸東京水産大学練習船神鷹丸東京水産大学練習船海鷹丸東京水産大学練習船海鷹丸東京水産大学練習船神鷹丸東京水産大学練習船神鷹丸東京水産大学練習船海鷹丸東京水産大学練習船神鷹丸東京水産大学海洋生産学科東京水産大学海洋生産学

Practical whole-tooth restoration utilizing autologous bioengineered tooth germ transplantation in a postnatal canine model

Whole-organ regeneration has great potential for the replacement of dysfunctional organs through the reconstruction of a fully functional bioengineered organ using three-dimensional cell manipulation in vitro. Recently, many basic studies of whole-tooth replacement using three-dimensional cell manipulation have been conducted in a mouse model. Further evidence of the practical application to human medicine is required to demonstrate tooth restoration by reconstructing bioengineered tooth germ using a postnatal large-animal model. Herein, we demonstrate functional tooth restoration through the autologous transplantation of bioengineered tooth germ in a postnatal canine model. The bioengineered tooth, which was reconstructed using permanent tooth germ cells, erupted into the jawbone after autologous transplantation and achieved physiological function equivalent to that of a natural tooth. This study represents a substantial advancement in whole-organ replacement therapy through the transplantation of bioengineered organ germ as a practical model for future clinical regenerative medicine

Bronchoalveolar lavage galactomannan for the diagnosis of chronic pulmonary aspergillosis

Diagnosing chronic pulmonary aspergillosis (CPA) is complicated, and there are limited data available regarding the identification of galactomannan (GM) in clinical specimens to assist the detection of this infection. The purpose of this study was to evaluate the detection of GM in bronchoalveolar lavage fluid (BALF) and serum and to assess its utility for diagnosing CPA. We retrospectively reviewed the diagnostic and clinical characteristics of 144 patients, with and without CPA, in Nagasaki University Hospital, Japan, whose BAL and serum specimens were examined for the presence of GM. The Platelia Aspergillus enzyme immunoassay (PA EIA) was performed according to the manufacturer\u27s instructions. The mean values of BALF GM antigen were 4.535 (range, 0.06214.120) and 0.430 (range, 0.0629.285) in CPA (18) and non-CPA (126) patients, respectively. The mean values of serum GM antigen were 1.557 (range, 0.2325.397) and 0.864 (range, 0.0288.956) in CPA and non-CPA patients, respectively. PA EIA of BALF is superior to the test with serum, with the optimal cut-off values for BALF and serum of 0.4 and 0.7, respectively. The sensitivity and specificity of PA EIA in BALF at a cut-off of 0.4 were 77.2% and 77.0%, respectively, whereas with serum at a cut-off of 0.7, they were 66.7% and 63.5%, respectively. GM testing using BALF showed reasonable sensitivity and specificity as compared to that using serum. Thus, assessing GM levels in BALF may enhance the accuracy of diagnosing CPA

Clinical features of pulmonary cryptococcosis in non-HIV patients in Japan

OBJECTIVE: To clarify the clinical features of pulmonary cryptococcosis in Japanese non-HIV population.METHODS: Retrospective investigation of 151 pulmonary cryptococcosis cases between 1977 and 2012 was executed. The underlying disease (UDs), aggravating factors, radiological characteristics, and treatment were examined.RESULTS: Sixty-seven patients (44.4%) had no UDs. The common UDs were diabetes (32.1%) followed by hematologic disease (22.6%), and collagen disease (22.6%). Peripherally distributed pulmonary nodules/masses were most commonly seen. Lesions in the right middle lobe (p = 0.01) and air bronchogram (P = 0.05) were significantly more frequent, respectively, in patients with UDs than patients without them. Azoles were mainly selected for the patients without meningoencephalitis. Mean treatment duration for patients with and without UDs was 6.64 and 2.87 months, respectively. Patients whose pulmonary nodules improved after treatment continued to experience gradual reduction of cryptococcosis antigen titers, even if antigen titers were positive at the time of treatment cessation. The average time for antigen titers to become negative after treatment cessation was 13.1 and 10.7 months for patients with and without UDs, respectively. When groups were compared according to the presence of meningoencephalitis complications, deaths, and survivals, factors contributing to cryptococcosis prognosis included higher age, hypoproteinemia, hypoalbuminemia, steroid use, high C-reactive protein levels, and meningoencephalitis complications.CONCLUSIONS: It is crucial to consider the presence of UDs and meningoencephalitis for the choice of antifungals and treatment duration for cryptococcosis in non-HIV patients. Three- and six months-administration of azoles for pulmonary cryptococcosis with or without UDs, respectively is reasonable

A study on health control for cadets in sea training 1. : body composition and blood circulation among cadets in sea training

東京水産大学海洋システム工学講座東京水産大学水産資源経営講座東京水産大学練習船海鷹丸東京水産大学練習船海鷹丸東京水産大学練習船海鷹丸東京水産大学練習船海鷹丸東京水産大学練習船海鷹丸東京水産大学練習船青鷹丸東京水産大学練習船青鷹丸東京水産大学名誉教