Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

Precise spatial and temporal characterization of cytokinin (CK) responses reveals the function of the CK biosynthesis gene ISOPENTENYLTRANSFERASE 3 during nodule development in Medicago truncatula.     

The ecology of the banded civet (Hemigalus derbyanus) in Southeast Asia with implications for mesopredator release,zoonotic diseases,and conservation

Habitat loss and degradation threaten forest specialist wildlife species, but some generalist mesopredators exploit disturbed areas and human‐derived food, which brings them into closer contact with humans. Mesopredator release is also important for human health for known zoonotic disease reservoirs, such as Asian civets (Viverridae family), since this group includes the intermediator species for the SARS‐CoV‐1 outbreak. Here we use camera trapping to evaluate the habitat associations of the widespread banded civet (Hemigalus derbyanus) across its range in Southeast Asia. At the regional scale, banded civet detections among published studies were positively associated with forest cover and negatively associated with human population. At the local scale (within a landscape), hierarchical modeling of new camera trapping showed that abundance was negatively associated with forest loss and positively associated with distance to rivers. These results do not support mesopredator release and suggest a low likelihood overlap with humans in degraded habitats and, therefore, a low risk of zoonotic disease transmission from this species in the wild. We also estimate that banded civet distribution has contracted to under 21% of its currently recognized IUCN Red List range, only 12% of which falls within protected areas, and a precipitous recent decline in population size. Accordingly, we suggest the banded civet''s Red List status should be re‐evaluated in light of our findings.     

Phyllachora species infecting maize and other grass species in the Americas represents a complex of closely related species

The genus Phyllachora contains numerous obligate fungal parasites that produce raised, melanized structures called stromata on their plant hosts referred to as tar spot. Members of this genus are known to infect many grass species but generally do not cause significant damage or defoliation, with the exception of P. maydis which has emerged as an important pathogen of maize throughout the Americas, but the origin of this pathogen remains unknown. To date, species designations for Phyllachora have been based on host associations and morphology, and most species are assumed to be host specific. We assessed the sequence diversity of 186 single stroma isolates collected from 16 hosts representing 15 countries. Samples included both herbarium and contemporary strains that covered a temporal range from 1905 to 2019. These 186 isolates were grouped into five distinct species with strong bootstrap support. We found three closely related, but genetically distinct groups of Phyllachora are capable of infecting maize in the United States, we refer to these as the P. maydis species complex. Based on herbarium specimens, we hypothesize that these three groups in the P. maydis species complex originated from Central America, Mexico, and the Caribbean. Although two of these groups were only found on maize, the third and largest group contained contemporary strains found on maize and other grass hosts, as well as herbarium specimens from maize and other grasses that include 10 species of Phyllachora. The herbarium specimens were previously identified based on morphology and host association. This work represents the first attempt at molecular characterization of Phyllachora species infecting grass hosts and indicates some Phyllachora species can infect a broad range of host species and there may be significant synonymy in the Phyllachora genus.     

Insect immunity. Constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect

Two novel antimicrobial peptides, which we propose to name termicin and spinigerin, have been isolated from the fungus-growing termite Pseudacanthotermes spiniger (heterometabole insect, Isoptera). Termicin is a 36-amino acid residue antifungal peptide, with six cysteines arranged in a disulfide array similar to that of insect defensins. In contrast to most insect defensins, termicin is C-terminally amidated. Spinigerin consists of 25 amino acids and is devoid of cysteines. It is active against bacteria and fungi. Termicin and spinigerin show no obvious sequence similarities with other peptides. Termicin is constitutively present in hemocyte granules and in salivary glands. The presence of termicin and spinigerin in unchallenged termites contrasts with observations in evolutionary recent insects or insects undergoing complete metamorphosis, in which antimicrobial peptides are induced in the fat body and released into the hemolymph after septic injury.     

Prediction of q-values and conformations of gadolinium chelates for magnetic resonance imaging

For gadolinium chelates, we determined that there is a linear correlation between calculated solvent-accessible surface area and q-value, the number of rapidly exchanging water molecules directly bound to the gadolinium ion. A calibration curve was developed to predict q-value based on the solvent-accessible surface area of gadolinium. This predictive method was validated with the following gadolinium crystal structures: (ethylenediaminetetraacetic acid)-gadolinium(III) [Gd(EDTA)] [Templeton, L. K., Templeton, D. H., Zalkin, A., and Ruben, H. W. (1982) Anomalous Scattering by Praseodymium, Samarium, and Gadolinium and Structures of their Thylenediaminetetraacetate (EDTA) Salts. Acta Crystallogr., Sect. B 38, 2155], (1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid)-gadolinium(III) [Gd(DOTA)] [Dubost, J.-P., Leger, J.-M., Langlois, M.-H., Meyer, D., and Schaefer, M. (1991) Structure of a Magnetic Resonance Imaging Agent - The Gadolinium-DOTA Complex C(16)H(24)N(4)O(8)NaGd, 5H(2)O. C. R. Acad. Sci., Ser. 2 312, 349], (diethylenetriaminepentaacetic acid)-gadolinium(III) [Gd(DTPA)] [Stezowski, J. J., and Hoard, J. L. (1984) Heavy Metal Ionophores - Correlations Among Structural Parameters of Complexed Nonpeptide Polyamino Acids. Isr. J. Chem. 24, 323], (diethylenepenta-acetato)-gadolinium(III) [Gd(DTPA-BEA)] [Smith, P. H., Brainard, J. R., Morris, D. E., Jarvinen, G. D., and Ryan, R. R. (1989) Solution and Solid-State Characterization of Europium and Gadolinium Schiff-Base Complexes and Assessment of their Potential as Contrast Agents in Magnetic Resonance Imaging. J. Am. Chem. Soc. 111, 7437], and (1,7,13-triaza-4,10, 16-trioxacyclo-octadecane-N,N',N' '-triacetato)-gadolinium(III) [Gd(TTTA)] [Chen, D., Squattrito, P. J., Martell, A. E., and Clearfield, A. (1990) Synthesis and Crystal Structure of a 9-Coordinate Gadolinium(III) Complex of 1,7,13-Triaza-4,10, 16-Trioxacyclooctadecane-N,N',N' '-Tri-Acetic Acid. Inorg. Chem. 29, 4366]. Predicted q-values were in complete agreement with experimentally determined q-values. A genetic algorithm-based conformational search method was developed to generate valid 3D models for gadolinium chelates. The method was successfully tested on the following gadolinium chelates: Gd(EDTA) (Templeton et al., 1982), Gd(DOTA) (Dubost et al., 1991), Gd(DTPA-BEA) (Smith et al., 1989), Gd(TTTA) (Chen et al., 1990), Gd(triethylene glycol) [Rogers, R. D., Voss, E. J., and Etzenhouser, R. D. (1988) F-Element Crown Ether Complexes. 17. Synthetic and Structural Survey of Lanthanide Chloride Tiethylene Glycol Complexes. Inorg. Chem. 27, 533], and Gd(tetraethylene glycol) [Rogers, R. D., Etzenhouser, R. D., Murdoch, J. S., and Reyes, E. (1991) Macrocycle Complexation Chemistry. 35. Survey of the Complexation of the Open-Chain 15-Crown-5 Analogue tetraethylene Glycol with the Lanthanide Chlorides. Inorg. Chem. 30, 1445].     

Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets

Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.     

Endothelial cell confluence regulates Weibel-Palade body formation

Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (VWF) are characteristic of the mammalian endothelium. We hypothesized that vascular-specific antigens such as VWF are linked to endothelial identity and proliferation in vitro. To test this idea, the cellular accumulation of VWF in WPBs was monitored as a function of cell proliferation, confluence and passage number in human umbilical vein endothelial cells (HUVECs). We found that as passage number increased the percentage of cells containing VWF in WPBs was reduced significantly, whilst the protein was still detected within the secretory pathway at all times. However, the endothelial-specific marker protein, PECAM-1, is present on all cells even when WPBs are absent, indicating partial maintenance of endothelial identity. Biochemical studies show that a significant pool of immature pro-VWF can be detected in sub-confluent HUVECs; however, a larger pool of mature, processed VWF is detected in confluent cells. Newly synthesized VWF must thus be differentially sorted and packaged along the secretory pathway in semi-confluent versus confluent endothelial cells. Our studies thus show that WPB formation is linked to the formation of a confluent endothelial monolayer.     

NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells

Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22^phox and gp91^phox, as well as the Ca²⁺ concentration-responsive gp91^phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22^phox was not detectable, both gp91^phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22^phox or gp91^phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death. superoxide anion; diphenylene iodonium; p22^phox; gp91^phox; adenosine 3',5'-cyclic monophosphate response element; caspases