Plastid methylerythritol phosphate pathway participates in the hypersensitive response-related cell death in Nicotiana benthamiana.

Programmed cell death (PCD), a characteristic feature of hypersensitive response (HR) in plants, is an important cellular process often associated with the defense response against pathogens. Here, the involvement of LytB, a gene encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase that participates in the final step of the plastid methylerythritol phosphate (MEP) pathway, in plant HR cell death was studied. In Nicotiana benthmiana plants, silencing of the NbLytB gene using virus-induced gene silencing (VIGS) caused plant growth retardation and albino leaves with severely malformed chloroplasts. In NbLytB-silenced plants, HR-related cell death mediated by the expression of either the human proapoptotic protein gene Bax or an R gene with its cognate Avr effector gene was inhibited, whereas that induced by the nonhost pathogen Pseudomonas syringae pv. syringae 61 was enhanced. To dissect the isoprenoid pathway and avoid the pleiotropic effects of VIGS, chemical inhibitors that specifically inhibit isoprenoid biosynthesis in plants were employed. Treatment of N. benthamiana plants with fosmidomycin, a specific inhibitor of the plastid MEP pathway, effectively inhibited HR-related PCD, whereas treatment with mevinolin (a cytoplasmic mevalonate pathway inhibitor) and fluridone (a carotenoid biosynthesis inhibitor) did not. Together, these results suggest that the MEP pathway as well as reactive oxygen species (ROS) generation in the chloroplast play an important role in HR-related PCD, which is not displaced by the cytosolic isoprenoid biosynthesis pathway.

Detection of Rhodococcus fascians, the Causative Agent of Lily Fasciation in South Korea.

Rhodococcus fascians is an important pathogen that infects various herbaceous perennials and reduces their economic value. In this study, we examined R. fascians isolates carrying a virulence gene from symptomatic lily plants grown in South Korea. Phylogenetic analysis using the nucleotide sequences of 16S rRNA, vicA, and fasD led to the classification of the isolates into four different strains of R. fascians. Inoculation of Nicotiana benthamiana with these isolates slowed root growth and resulted in symptoms of leafy gall. These findings elucidate the diversification of domestic pathogenic R. fascians and may lead to an accurate causal diagnosis to help reduce economic losses in the bulb market.

A human pathogenic bacterium Shigella proliferates in plants through adoption of type III effectors for shigellosis.

Shigella, which infects primates, can be transmitted via fresh vegetables; however, its molecular interactions with plants have not been elucidated. Here, we show that four Shigella strains, Shigella boydii, Shigella sonnei, Shigella flexneri 2a, and S. flexneri 5a, proliferate at different levels in Arabidopsis thaliana. Microscopic studies revealed that these bacteria were present inside leaves and damaged plant cells. Green fluorescent protein (GFP)-tagged S. boydii and S. flexneri 5a colonized leaves only, whereas S. flexneri 2a colonized both leaves and roots. Using Shigella mutants lacking type III secretion systems (T3SSs), we found that T3SSs that regulate the pathogenesis of shigellosis in humans also play a central role in bacterial proliferation in Arabidopsis. Strikingly, the immunosuppressive activity of two T3S effectors, OspF and OspG, was required for proliferation of Shigella in Arabidopsis. Of note, delivery of OspF or OspG effectors inside plant cells upon Shigella inoculation was confirmed using a split GFP system. These findings demonstrate that the human pathogen Shigella can proliferate in plants by adapting immunosuppressive machinery used in the original host human.

Smartphone operable centrifugal system (SOCS) for on-site DNA extraction from foodborne bacterial pathogen.

The on-site recovery of nucleic acid from foodborne bacteria is in high demand to further understand on-site molecular diagnosis, which is especially applicable in developing countries. Here, we first proposed a smartphone operable centrifugal system (SOCS) for nucleic acid extraction with the assistance of a low power consumable motor and hydrogel beads. The SOCS consists of a centrifugal motor, 3D-printed cartridge, a nucleic acid collection column, and a smartphone. The SOCS shows excellent DNA extraction performance within 6 min, and it can operate more than 100 times using a smartphone. The purified effluent DNA was accumulated in the nucleic acid collection column. The performance of the SOCS was confirmed by amplifying the recovered DNA from Escherichia coli O157:H7. Moreover, the artificially inoculated food and blood samples also confirmed the performance of SOCS. The proposed SOCS provides an on-site operable nucleic acid separation platform in terms of simplicity, easy usability, cost-effectiveness, and portability in pathogenic point-of-care diagnostics.

An efficient isolation of foodborne pathogen using surface-modified porous sponge.

Rapid and efficient detection of pathogenic bacteria from food is critical to prevent epidemic food poisoning. However, the isolation of pathogenic bacteria from spoiled food is hampered by the lack of proper cell cultivation and/or isolation methods. Most of currently used methods suffer from complex, time-consuming culturing steps, low scalability, and high operation cost. Herein, we developed an alternative approach for the isolation of pathogenic bacteria directly from food using a surface-modified, highly porous sponge via initiated chemical vapor deposition (iCVD) process. A hydrophobic polymer, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetra-siloxane) (pV4D4), was deposited conformally on amphiphilic 3-dimensional (3D) melamine sponge to incorporate hydrophobicity as well as oleophilicity to the porous sponge surface, which is appropriate for absorbing oil component selectively from food extracts. Furthermore, the surface-modified sponge was capable of the isolation of Escherichia coli O157:H7 (E. coli O157:H7) from heterogeneous mixture with oil/water/food particles with undistinguisible efficiency compare to artificial model system. The surface-modified sponge developed in this study will be a novel platform for oil/water separation and isolation of foodborne pathogens directly from heterogeneous mixture to enhance the efficiency of molecular diagnostics.

Calreticulin modulates the intracellular survival of mycobacteria by regulating ER-stress-mediated apoptosis.

Endoplasmic reticulum (ER)-stress-mediated apoptosis is a host defense mechanism against Mycobacterium tuberculosis (Mtb) infection. Calreticulin (CRT) is the major calcium-binding chaperone protein. Previous reports have suggested a close relationship between the cell-surface expression of CRT and apoptosis. In this study, the role of CRT during Mtb infection was examined. The results showed that Mtb infection induces CRT production by macrophages and that CRT levels are correlated with the degree of apoptotic cell death. The enhanced production of CRT was associated with the ER stress induced by Mtb infection. A significant increase in CRT translocation from the cytosol to the plasma membrane after 24 h of infection suggested the importance of CRT localization in the induction of apoptosis during Mtb infection. An investigation of the factors associated with CRT translocation and the ability of ectopically expressed CRT to induce apoptosis showed that pretreatment with a reactive oxygen species scavenger decreased Mtb-induced CRT expression, leading to the reduction of CHOP and caspase-3 activation. The intracellular survival of Mtb was significantly higher in macrophages transfected with a CRT-specific small interfering RNA than in control cells. The key role of CRT in inducing apoptosis included its interaction with CXCR1 and TNFR1 in Mtb-infected macrophages. The CRT/CXCR1/TNFR1 complex was shown to induce the extrinsic apoptotic pathway during Mtb infection. Together, these results demonstrate that CRT is critical for the intracellular survival of Mtb, via ER-stress-induced apoptosis, as well as the importance of ER stress-mediated CRT localization in the pathogenesis of tuberculosis.

Draft Genome Sequence of the Endophytic Bacterium Variovorax paradoxus KB5, Which Has Antagonistic Activity against a Phytopathogen, Pseudomonas syringae pv. tomato DC3000.

Variovorax paradoxus KB5, isolated from the inside of Arabidopsis thaliana leaves, showed antibacterial activity against the phytopathogen Pseudomonas syringae pv. tomato DC3000. Here, we report a draft genome sequence of V. paradoxus KB5, which contains a delftibactin-like nonribosomal peptide biosynthetic gene cluster.

Roles of endoplasmic reticulum stress-mediated apoptosis in M1-polarized macrophages during mycobacterial infections.

Alteration of macrophage function has an important regulatory impact on the survival of intracellular mycobacteria. We found that macrophages infected with attenuated Mycobacterium tuberculosis (Mtb) strain H37Ra had elevated expression of M1-related molecules, whereas the M2 phenotype was dominant in macrophages infected with virulent Mtb H37Rv. Further, the TLR signalling pathway played an important role in modulating macrophage polarization against Mtb infection. Interestingly, endoplasmic reticulum (ER) stress was significantly increased in M1 polarized macrophages and these macrophages effectively removed intracellular Mtb, indicating that ER stress may be an important component of the host immune response to Mtb in M1 macrophages. This improved understanding of the mechanisms that regulate macrophage polarization could provide new therapeutic strategies for tuberculosis.

The Role of Prostate Apoptosis Response-4 (Par-4) in Mycobacterium tuberculosis Infected Macrophages.

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that forms a complex with glucose-regulated protein 78 (GRP78) to induce apoptosis. Previously, we reported that ER stress-induced apoptosis is a critical host defense mechanism against Mycobacterium tuberculosis (Mtb). We sought to understand the role of Par-4 during ER stress-induced apoptosis in response to mycobacterial infection. Par-4 and GRP78 protein levels increased in response Mtb (strain: H37Ra) infection. Furthermore, Par-4 and GRP78 translocate to the surface of Mtb H37Ra-infected macrophages and induce apoptosis via caspase activation. NF-κB activation, Mtb-mediated ER stress, and Par-4 production were significantly diminished in macrophages with inhibited ROS production. To test Par-4 function during mycobacterial infection, we analyzed intracellular survival of Mtb H37Ra in macrophages with Par-4 overexpression or knockdown. Mtb H37Ra growth was significantly reduced in Par-4 overexpressing macrophages and increased in knockdown macrophages. We also observed increased Par-4, GRP78, and caspases activation in Bacillus Calmette-Guérin (BCG)-infected prostate cancer cells. Our data demonstrate that Par-4 is associated with ER stress-induced apoptosis resulting in reduced intracellular survival of mycobacteria. BCG treatment increases Par-4-dependent caspase activation in prostate cancer cells. These results suggest ER stress-induced Par-4 acts as an important defense mechanism against mycobacterial infection and regulates cancer.

Draft Genome Sequence of the Endophytic Strain Rhodococcus kyotonensis KB10, a Potential Biodegrading and Antibacterial Bacterium Isolated from Arabidopsis thaliana.

Rhodococcus kyotonensis KB10 is an endophytic bacterium isolated from Arabidopsis thaliana The organism showed mild antibacterial activity against the phytopathogen Pseudomonas syringae pv. tomato DC3000. This study reports the genome sequence of R. kyotonensis KB10. This bacterium contains an ectoine biosynthesis gene cluster and has the potential to degrade nitroaromatic compounds. The identified bacterium may be a suitable biocontrol agent and degrader of environmental pollutants.

Genome Sequence of the Endophytic Bacterium Bacillus thuringiensis Strain KB1, a Potential Biocontrol Agent against Phytopathogens.

ITALIC! Bacillus thuringiensisis the most widely known microbial pesticide used in agricultural applications. Herein, we report a draft genome sequence of the endophytic bacterium ITALIC! Bacillus thuringiensisstrain KB1, which exhibits antagonism against phytopathogens.

A Leaf-Inhabiting Endophytic Bacterium, Rhodococcus sp. KB6, Enhances Sweet Potato Resistance to Black Rot Disease Caused by Ceratocystis fimbriata.

Rhodococcus species have become increasingly important owing to their ability to degrade a wide range of toxic chemicals and produce bioactive compounds. Here, we report isolation of the Rhodococcus sp. KB6, which is a new leaf-inhabiting endophytic bacterium that suppresses black rot disease in sweet potato leaves. We determined the 7.0 Mb draft genome sequence of KB6 and have predicted 19 biosynthetic gene clusters for secondary metabolites, including heterobactins, which are a new class of siderophores. Notably, we showed the first internal colonization of host plants with Rhodococcus sp. KB6 and discuss its potential as a biocontrol agent for sustainable agriculture.