Selective involvement of members of the eukaryotic initiation factor 4E family in the infection of Arabidopsis thaliana by potyviruses

Arabidopsis thaliana plants with mutations in the genes encoding eukaryotic initiation factor (eIF4E) or isoform of eIF4E (eIF(iso)4E) were tested for susceptibility to Clover yellow vein virus (ClYVV), a member of the genus Potyvirus. ClYVV accumulated in both inoculated and upper uninoculated leaves of mutant plants lacking eIF(iso)4E, but not in mutant plants lacking eIF4E. In contrast, Turnip mosaic virus (TuMV), another member of the genus Potyvirus, multiplied in mutant plants lacking eIF4E but not in mutant plants lacking eIF(iso)4E. These results suggest the selective involvement of members of the eIF4E family in infection by potyviruses.     

Local activation of Rap1 contributes to directional vascular endothelial cell migration accompanied by extension of microtubules on which RAPL, a Rap1-associating molecule, localizes

Endothelial cell migration is promoted by chemoattractants and is accompanied with microtubule extension toward the leading edge. Cytoskeletal microtubules polarize to function as rails for delivering a variety of molecules by motor proteins during cell migration. It remains, however, unclear how directional migration with polarized extension of microtubules is regulated. Here we report that Rap1 controls the migration of vascular endothelial cells. We found that Rap1-associating molecule, RAPL, which belongs to the Ras association domain family (Rassf), localized on microtubules and that activated Rap1 induced dissociation of RAPL from microtubules. A Rap1 activation-monitoring probe based on the fluorescence resonance energy transfer enabled us to demonstrate that local Rap1 activation occurs at the leading edge of the cells under the two types of cell migration, chemotaxis and wound healing. Time lapse imaging of microtubules marked by enhanced green fluorescent protein-RAPL showed the directional growth of microtubules toward the leading edge of the migrating cells. Using adenovirus, inactivation of Rap1 by expression of rap1GAPII inhibited wound healing. In addition, disconnection of Rap1 and RAPL by expression of a RAPL mutant also perturbed wound healing. Collectively, the locally activated Rap1 and its association with RAPL controls the directional migration of vascular endothelial cells.     

Diterpenoid Total Synthesis

A total synthesis of racemic 4-epipimaric and 4-episandaracopimaric acids is described.     

Assessment of Artificial MiRNA Architectures for Higher Knockdown Efficiencies without the Undesired Effects in Mice

RNAi-based strategies have been used for hypomorphic analyses. However, there are technical challenges to achieve robust, reproducible knockdown effect. Here we examined the artificial microRNA (amiRNA) architectures that could provide higher knockdown efficiencies. Using transient and stable transfection assays in cells, we found that simple amiRNA-expression cassettes, that did not contain a marker gene (−MG), displayed higher amiRNA expression and more efficient knockdown than those that contained a marker gene (+MG). Further, we tested this phenomenon in vivo, by analyzing amiRNA-expressing mice that were produced by the pronuclear injection-based targeted transgenesis (PITT) method. While we observed significant silencing of the target gene (eGFP) in +MG hemizygous mice, obtaining −MG amiRNA expression mice, even hemizygotes, was difficult and the animals died perinatally. We obtained only mosaic mice having both “−MG amiRNA” cells and “amiRNA low-expression” cells but they exhibited growth retardation and cataracts, and they could not transmit the –MG amiRNA allele to the next generation. Furthermore, +MG amiRNA homozygotes could not be obtained. These results suggested that excessive amiRNAs transcribed by −MG expression cassettes cause deleterious effects in mice, and the amiRNA expression level in hemizygous +MG amiRNA mice is near the upper limit, where mice can develop normally. In conclusion, the PITT-(+MG amiRNA) system demonstrated here can generate knockdown mouse models that reliably express highest and tolerable levels of amiRNAs.     

Dual Regulation of Gene Expression Mediated by Extended MAPK Activation and Salicylic Acid Contributes to Robust Innate Immunity in Arabidopsis thaliana

Network robustness is a crucial property of the plant immune signaling network because pathogens are under a strong selection pressure to perturb plant network components to dampen plant immune responses. Nevertheless, modulation of network robustness is an area of network biology that has rarely been explored. While two modes of plant immunity, Effector-Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), extensively share signaling machinery, the network output is much more robust against perturbations during ETI than PTI, suggesting modulation of network robustness. Here, we report a molecular mechanism underlying the modulation of the network robustness in Arabidopsis thaliana. The salicylic acid (SA) signaling sector regulates a major portion of the plant immune response and is important in immunity against biotrophic and hemibiotrophic pathogens. In Arabidopsis, SA signaling was required for the proper regulation of the vast majority of SA-responsive genes during PTI. However, during ETI, regulation of most SA-responsive genes, including the canonical SA marker gene PR1, could be controlled by SA-independent mechanisms as well as by SA. The activation of the two immune-related MAPKs, MPK3 and MPK6, persisted for several hours during ETI but less than one hour during PTI. Sustained MAPK activation was sufficient to confer SA-independent regulation of most SA-responsive genes. Furthermore, the MPK3 and SA signaling sectors were compensatory to each other for inhibition of bacterial growth as well as for PR1 expression during ETI. These results indicate that the duration of the MAPK activation is a critical determinant for modulation of robustness of the immune signaling network. Our findings with the plant immune signaling network imply that the robustness level of a biological network can be modulated by the activities of network components.     

Amphidinol 3 preferentially binds to cholesterol in disordered domains and disrupts membrane phase separation

Amphidinol 3 (AM3), a polyhydroxy-polyene metabolite from the dinoflagellate Amphidinium klebsii, possesses potent antifungal activity. AM3 is known to interact directly with membrane sterols and permeabilize membranes by forming pores. Because AM3 binds to sterols such as cholesterol and ergosterol, it can be assumed that AM3 has some impact on lipid rafts, which are membrane domains rich in sphingolipids and cholesterol. Hence, we first examined the effect of AM3 on phase-separated liposomes, in which raft-like ordered and non-raft-like disordered domains are segregated. Consequently, AM3 disrupted the phase separation at 22 μM, as in the case of methyl-β-cyclodextrin, a well-known raft-disrupter that extracts sterol from membranes. The surface plasmon resonance measurements and dye leakage assays show that AM3 preferentially recognizes cholesterol in the disordered membrane, which may reflect a weaker lipid-cholesterol interaction in disordered membrane than in ordered membrane. Finally, to gain insight into the AM3-induced coalescence of membrane phases, we measured membrane fluidity using fluorescence correlation spectroscopy, demonstrating that AM3 significantly increases the order of disordered phase. Together, AM3 preferentially binds to the disordered phase rather than the ordered phase, and enhances the order of the disordered phase, consequently blending the separated phases.     

Microsatellite scanning of the immunogenome associates MAPK14 and ELTD1 with graft-versus-host disease in hematopoietic stem cell transplantation

Graft-versus-host disease (GVHD) is the main complication after hematopoietic stem cell transplantation (HSCT). Evidence for non-HLA gene polymorphisms as a cause of GVHD lacks consistency, which is, in part, due to methodological issues of previous candidate gene association studies and small effect size of their results, demanding for larger scale and more robust approaches. Here, non-HLA gene polymorphisms were studied on a large population (922 HSCT pairs) from a homogeneous ethnic background with selection/correction for important clinical confounders. A methodology was applied exploiting the strength of confirmatory typing in an independent study cohort. Targeting an immunogenome of 2,909 genes, an approach of pooled DNA typing of 4,321 microsatellite (MS) markers in two independent screening steps and confirmation of associated markers by further individual genotyping on combined screening cohorts was used to identify genetic susceptibility loci for moderate to severe GVHD (grades 2–4). Ten MS loci (D5S424, D6S0035i, D1S0818i, DXS0151i, D17S0219i, DXS0629i, DXS0324i, D17S0271i, D6S0330i, and D1S1335i) passed the two pooled DNA typing steps and confirmation by individual sample genotyping; two of these (D1S0818i–ELTD1 and D6S0035i–MAPK14) remain associated following application of Bonferroni’s correction and multivariate analysis. The MAPK14 locus was exemplarily explored by typing of haplotype single nucleotide polymorphisms (SNP) confirming this association. This study identified several new MS susceptibility loci for GVHD that warrant further investigation. Immunogenome scanning using MS markers is a useful method for the identification of non-HLA gene loci associating with HSCT outcomes.