Localization of the reb operon expression is inconsistent with that of the R-body production in the stem nodules formed by Azorhizobium caulinodans mutants having a deletion of praR.

Azorhizobium caulinodans, a kind of rhizobia, has a reb operon encoding pathogenic R-body components, whose expression is usually repressed by a transcription factor PraR. Mutation on praR induced a high expression of reb operon and the formation of aberrant nodules, in which both morphologically normal and shrunken host cells were observed. Histochemical GUS analyses of praR mutant expressing reb operon-uidA fusion revealed that the bacterial cells within the normal host cells highly expressed the reb operon, but rarely produced R-bodies. On the other hand, the bacterial cells within the shrunken host cells frequently produced R-bodies but rarely expressed the reb operon. This suggests that R-body production is not only regulated at the transcriptional level, but by other regulatory mechanisms as well.

Physiological characterization of poly-ß-hydroxybutyrate accumulation in the moderately thermophilic hydrogen-oxidizing bacterium Hydrogenophilus thermoluteolus TH-1.

Hydrogenophilus thermoluteolus strain TH-1 is a thermophilic hydrogen-oxidizing microorganism that has the highest growth rate among autotrophs. Genomic analysis revealed that this strain comprises the complete gene set for poly-ß-hydroxybutyrate (PHB) synthesis, i.e., three copies of acetyl-CoA acetyltransferase and polyhydroxyalkanoate synthase and one copy of acetoacetyl-CoA reductase and 3-hydroxyacyl-CoA dehydrogenase/3-hydroxybutyryl-CoA epimerase. An investigation on PHB accumulation in strain TH-1 demonstrated that PHB accumulation was induced by nitrogen limitation under autotrophic as well as heterotrophic conditions. This strain accumulated up to 430.4 ± 14.3 mg L-1 PHB during a 3-h incubation under nitrogen-limited heterotrophic conditions. The highest PHB accumulation rates under autotrophic and heterotrophic conditions were 38.6% (w/w) of the dry cells after a 6-h induction and 53.8% after 3 h, respectively. Although PHB granules started to accumulate after 15 min of nitrogen limitation under heterotrophic conditions, a drastic decrease of PHB was observed after 9 h of induction.

Transcriptional switch for programmed cell death in pith parenchyma of sorghum stems.

Pith parenchyma cells store water in various plant organs. These cells are especially important for producing sugar and ethanol from the sugar juice of grass stems. In many plants, the death of pith parenchyma cells reduces their stem water content. Previous studies proposed that a hypothetical D gene might be responsible for the death of stem pith parenchyma cells in Sorghum bicolor, a promising energy grass, although its identity and molecular function are unknown. Here, we identify the D gene and note that it is located on chromosome 6 in agreement with previous predictions. Sorghum varieties with a functional D allele had stems enriched with dry, dead pith parenchyma cells, whereas those with each of six independent nonfunctional D alleles had stems enriched with juicy, living pith parenchyma cells. D expression was spatiotemporally coupled with the appearance of dead, air-filled pith parenchyma cells in sorghum stems. Among D homologs that are present in flowering plants, Arabidopsis ANAC074 also is required for the death of stem pith parenchyma cells. D and ANAC074 encode previously uncharacterized NAC transcription factors and are sufficient to ectopically induce programmed death of Arabidopsis culture cells via the activation of autolytic enzymes. Taken together, these results indicate that D and its Arabidopsis ortholog, ANAC074, are master transcriptional switches that induce programmed death of stem pith parenchyma cells. Thus, targeting the D gene will provide an approach to breeding crops for sugar and ethanol production.

Morphological and electrophysiological differences in tarsal chemosensilla between the wild silkmoth Bombyx mandarina and the domesticated species Bombyx mori.

Gustatory and olfactory senses of phytophagous insects play important roles in the recognition of host plants. In the domestic silkmoth Bombyx mori and its wild species Bombyx mandarina, the morphologies and responses of adult olfactory organs (antennae) have been intensely investigated. However, little is known about these features of adult gustatory organs and the influence of domestication on the gustatory sense. Here we revealed that both species have two types of sensilla (thick [T] and slim [S] types) on the fifth tarsomeres of the adult legs. In both species, females have 3.6-6.9 times more T-sensilla than males. Therefore, T-sensilla seem to play more important roles in females than in males. Moreover, gustatory cells of T-sensilla of B. mandarina females responded intensely to mulberry leaf extract in electrophysiological experiments, while T-sensilla of B. mori females (N4 strain) hardly responded to mulberry leaf extract. These results suggest that T-sensilla of B. mandarina females are involved in the recognition of oviposition sites. We also observed that, in three B. mori strains (N4, p50T, and Kinshu × Showa), the densities of sensilla on the fifth tarsomeres were much lower than in B. mandarina. These results indicate that domestication has influenced the tarsal gustatory system of B. mori.

Stringent Expression Control of Pathogenic R-body Production in Legume Symbiont Azorhizobium caulinodans.

R bodies are insoluble large polymers consisting of small proteins encoded by reb genes and are coiled into cylindrical structures in bacterial cells. They were first discovered in Caedibacter species, which are obligate endosymbionts of paramecia. Caedibacter confers a killer trait on the host paramecia. R-body-producing symbionts are released from their host paramecia and kill symbiont-free paramecia after ingestion. The roles of R bodies have not been explained in bacteria other than CaedibacterAzorhizobium caulinodans ORS571, a microsymbiont of the legume Sesbania rostrata, carries a reb operon containing four reb genes that are regulated by the repressor PraR. Herein, deletion of the praR gene resulted in R-body formation and death of host plant cells. The rebR gene in the reb operon encodes an activator. Three PraR binding sites and a RebR binding site are present in the promoter region of the reb operon. Expression analyses using strains with mutations within the PraR binding site and/or the RebR binding site revealed that PraR and RebR directly control the expression of the reb operon and that PraR dominantly represses reb expression. Furthermore, we found that the reb operon is highly expressed at low temperatures and that 2-oxoglutarate induces the expression of the reb operon by inhibiting PraR binding to the reb promoter. We conclude that R bodies are toxic not only in paramecium symbiosis but also in relationships between other bacteria and eukaryotic cells and that R-body formation is controlled by environmental factors.IMPORTANCECaedibacter species, which are obligate endosymbiotic bacteria of paramecia, produce R bodies, and R-body-producing endosymbionts that are released from their hosts are pathogenic to symbiont-free paramecia. Besides Caedibacter species, R bodies have also been observed in a few free-living bacteria, but the significance of R-body production in these bacteria is still unknown. Recent advances in genome sequencing technologies revealed that many Gram-negative bacteria possess reb genes encoding R-body components, and interestingly, many of them are animal and plant pathogens. Azorhizobium caulinodans, a microsymbiont of the tropical legume Sesbania rostrata, also possesses reb genes. In this study, we demonstrate that A. caulinodans has ability to kill the host plant cells by producing R bodies, suggesting that pathogenicity conferred by an R body might be universal in bacteria possessing reb genes. Furthermore, we provide the first insight into the molecular mechanism underlying the expression of R-body production in response to environmental factors, such as temperature and 2-oxoglutarate.

DRP3 and ELM1 are required for mitochondrial fission in the liverwort Marchantia polymorpha.

Mitochondria increase in number by the fission of existing mitochondria. Mitochondrial fission is needed to provide mitochondria to daughter cells during cell division. In Arabidopsis thaliana, four kinds of genes have been reported to be involved in mitochondrial fission. Two of them, DRP3 (dynamin-related protein3) and FIS1 (FISSION1), are well conserved in eukaryotes. The other two are plant-specific ELM1 (elongated mitochondria1) and PMD (peroxisomal and mitochondrial division). To better understand the commonality and diversity of mitochondrial fission factors in land plants, we examined mitochondrial fission-related genes in a liverwort, Marchantia polymorpha. As a bryophyte, M. polymorpha has features distinct from those of the other land plant lineages. We found that M. polymorpha has single copies of homologues for DRP3, FIS1 and ELM1, but does not appear to have a homologue of PMD. Citrine-fusion proteins with MpDRP3, MpFIS1 and MpELM1 were localized to mitochondria in M. polymorpha. MpDRP3- and MpELM1-defective mutants grew slowly and had networked mitochondria, indicating that mitochondrial fission was blocked in the mutants, as expected. However, knockout of MpFIS1 did not affect growth or mitochondrial morphology. These results suggest that MpDRP3 and MpELM1 but neither MpFIS1 nor PMD are needed for mitochondrial fission in M. polymorpha.