How independent are TSE agents from their hosts?

Central to understanding the nature TSE agents (or prions) is how their genetic information is distinguished from the host. Are TSEs truly infectious diseases with host-independent genomes, or are they aberrations of a host component derived from the host genome? Recent experiments tested whether glycosylation of host PrP affects TSE strain characteristics. Wild-type mice were infected with 3 TSE strains passaged through transgenic mice with PrP devoid of glycans at 1 or both N-glycosylation sites. Strain-specific characteristics of 1 TSE strain changed but did not change for 2 others. Changes resulted from the selection of mutant TSE strains in a novel replicative environment. In general the properties of established TSEs support the genetic independence of TSE agents from the host, and specifically the primary structure of PrP does not directly encode TSE agent properties. However sporadic TSEs, challenge this independency. The prion hypothesis explains emerging TSEs relatively successfully but poorly accounts for the diversity and mutability of established TSE strains, or how many different infectious conformations are sustained thermodynamically. Research on early changes in RNA expression and events at the ribosome may inform the debate on TSE agent properties and their interaction with host cell machinery.     

Differences in early callose deposition during adapted and non-adapted powdery mildew infection of resistant Arabidopsis lines

The deposition of callose, a (1,3)-β-glucan cell wall polymer, can play an essential role in the defense response to invading pathogens. We could recently show that Arabidopsis thaliana lines with an overexpression of the callose synthase gene PMR4 gained complete penetration resistance to the adapted powdery mildew Golovinomyces cichoracearum and the non-adapted powdery mildew Blumeria graminis f. sp hordei. The penetration resistance is based on the transport of the callose synthase PMR4 to the site of attempted fungal penetration and the subsequent formation of enlarged callose deposits. The deposits differed in their total diameter comparing both types of powdery mildew infection. In this study, further characterization of these callose deposits revealed that size differences were especially pronounced in the core region of the deposits. This suggests that specific, pathogen-dependent factors exist, which might regulate callose synthase transport to the core region of forming deposits.     

PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens

Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall without activating the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant 5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene that confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [¹⁴C]‐acetyl‐CoA to oligogalacturonides. Through site‐directed mutagenesis, we show that three amino acids within a highly conserved esterase domain in putative PMR5 orthologs are necessary for PMR5 function. A suppressor screen of mutagenized pmr5 seed selecting for increased powdery mildew susceptibility identified two previously characterized genes affecting the acetylation of plant cell wall polysaccharides, RWA2 and TBR. The rwa2 and tbr mutants also suppress powdery mildew disease resistance in pmr6, a mutant defective in a putative pectate lyase gene. Cell wall analysis of pmr5 and pmr6, and their rwa2 and tbr suppressor mutants, demonstrates minor shifts in cellulose and pectin composition. In direct contrast to their increased powdery mildew resistance, both pmr5 and pmr6 plants are highly susceptibile to multiple strains of the generalist necrotroph Botrytis cinerea, and have decreased camalexin production upon infection with B. cinerea. These results illustrate that cell wall composition is intimately connected to fungal disease resistance and outline a potential route for engineering powdery mildew resistance into susceptible crop species.     

Foraminifers of the Viséan–Serpukhovian boundary interval in Western Palaeotethys: a review

A detailed revision of foraminiferal zonal schemes in sections throughout Europe and North Africa for the Viséan–Serpukhovian boundary interval suggests that several foraminiferal taxa might have the potential to form reliable markers throughout the Palaeotethys. This would support the currently investigated boundary definition based on the First Appearance Datum of the conodont Lochriea ziegleri. However, correlation of these foraminiferal markers in the Western Palaeotethys region has encountered several problems, partly arising from taxonomic issues, but mainly because of apparent discrepancies between the First Occurrence Data (FOD). Analysis of the available foraminiferal data has revealed that some taxa show marked delays in their FODs, due to the timing of westward dispersal within the Palaeotethys, emanating from a probable source in eastern Russia. As a result of this investigation, two dispersal routes have been identified, a northern branch and a southern branch. In general, the displacements within the southern branch occurred more rapidly than in the northern branch. In addition to different dispersal routes, separation of the main foraminiferal markers in stratigraphical sections from different regions can result from isolation of shallow‐water facies of the inner platform from those of relatively deeper‐water settings in the outer platform, the latter showing more consistent foraminiferal FODs. The differences in palaeobathymetry and associated energy levels have enabled two foraminiferal zonal schemes to be distinguished for the Viséan–Serpukhovian boundary interval in the Western Palaeotethys, one for the inner platform and a second one for the outer platform.     

mTOR activity is essential for retinal pigment epithelium regeneration in zebrafish

The retinal pigment epithelium (RPE) plays numerous critical roles in maintaining vision and this is underscored by the prevalence of degenerative blinding diseases like age-related macular degeneration (AMD), in which visual impairment is caused by progressive loss of RPE cells. In contrast to mammals, zebrafish possess the ability to intrinsically regenerate a functional RPE layer after severe injury. The molecular underpinnings of this regenerative process remain largely unknown yet hold tremendous potential for developing treatment strategies to stimulate endogenous regeneration in the human eye. In this study, we demonstrate that the mTOR pathway is activated in RPE cells post-genetic ablation. Pharmacological and genetic inhibition of mTOR activity impaired RPE regeneration, while mTOR activation enhanced RPE recovery post-injury, demonstrating that mTOR activity is essential for RPE regeneration in zebrafish. RNA-seq of RPE isolated from mTOR-inhibited larvae identified a number of genes and pathways dependent on mTOR activity at early and late stages of regeneration; amongst these were components of the immune system, which is emerging as a key regulator of regenerative responses across various tissue and model systems. Our results identify crosstalk between macrophages/microglia and the RPE, wherein mTOR activity is required for recruitment of macrophages/microglia to the RPE injury site. Macrophages/microglia then reinforce mTOR activity in regenerating RPE cells. Interestingly, the function of macrophages/microglia in maintaining mTOR activity in the RPE appeared to be inflammation-independent. Taken together, these data identify mTOR activity as a key regulator of RPE regeneration and link the mTOR pathway to immune responses in facilitating RPE regeneration.     

Comparison of Erysiphe cichoracearum and E. cruciferarum and a survey of 360 Arabidopsis thaliana accessions for resistance to these two powdery mildew pathogens

In previous work, UEA1 and UCSC1, two geographically distinct, powdery mildew isolates, were recognized for their ability to infect Arabidopsis thaliana. We have clarified the identity of these isolates by determining their host ranges, reexamining their morphology, and comparing their DNA sequences for the 5.8S ribosomal RNA and two flanking internal transcribed spacer sequences. These experiments confirm that UEA1 is a member of Erysiphe cruciferarum and that UCSC1 belongs to E. cichoracearum. Interactions of the two Erysiphe isolates with 360 A. thaliana accessions were examined to provide a comprehensive profile of naturally occurring powdery mildew resistance in this weedy species. The majority of A. thaliana accessions (213) were susceptible to both isolates. Among the accessions exhibiting some degree of resistance, most (84) responded differentially to UEA1 and UCSC1 and the remainder were resistant to both isolates. Notably, resistance to UCSC1 cosegregated with RPW7, a locus previously demonstrated to confer resistance to UEA1 in Ms-0 x Landsberg (erecta) crosses. With this large collection of resistant accessions, questions about species specificity, genetic diversity and the evolution of resistance to powdery mildews can be addressed.     

The Arabidopsis sku6/spiral1 gene encodes a plus end-localized microtubule-interacting protein involved in directional cell expansion

The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1). However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic spindle. SPR1:GFP was concentrated at the growing ends of cortical microtubules and was dependent on polymer growth state; the microtubule-related fluorescence dissipated upon polymer shortening. The protein has a repeated motif at both ends, separated by a predicted rod-like domain, suggesting that it may act as an intermolecular linker. These observations suggest that SPR1 is involved in microtubule polymerization dynamics and/or guidance, which in turn influences touch-induced directional cell expansion and axial twisting.     

Glycerol-3-phosphate acquisition in spirochetes: distribution and biological activity of glycerophosphodiester phosphodiesterase (GlpQ) among Borrelia species

Relapsing-fever spirochetes achieve high cell densities (>10(8)/ml) in their host's blood, while Lyme disease spirochetes do not (<10(5)/ml). This striking contrast in pathogenicity of these two groups of bacteria suggests a fundamental difference in their ability to either exploit or survive in blood. Borrelia hermsii, a tick-borne relapsing-fever spirochete, contains orthologs to glpQ and glpT, genes that encode glycerophosphodiester phosphodiesterase (GlpQ) and glycerol-3-phosphate transporter (GlpT), respectively. In other bacteria, GlpQ hydrolyzes deacylated phospholipids to glycerol-3-phosphate (G3P) while GlpT transports G3P into the cytoplasm. Enzyme assays on 17 isolates of borreliae demonstrated GlpQ activity in relapsing-fever spirochetes but not in Lyme disease spirochetes. Southern blots demonstrated glpQ and glpT in all relapsing-fever spirochetes but not in the Lyme disease group. A Lyme disease spirochete, Borrelia burgdorferi, that was transformed with a shuttle vector containing glpTQ from B. hermsii produced active enzyme, which demonstrated the association of glpQ with the hydrolysis of phospholipids. Sequence analysis of B. hermsii identified glpF, glpK, and glpA, which encode the glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase, respectively, all of which are present in B. burgdorferi. All spirochetes examined had gpsA, which encodes the enzyme that reduces dihydroxyacetone phosphate (DHAP) to G3P. Consequently, three pathways for the acquisition of G3P exist among borreliae: (i) hydrolysis of deacylated phospholipids, (ii) reduction of DHAP, and (iii) uptake and phosphorylation of glycerol. The unique ability of relapsing-fever spirochetes to hydrolyze phospholipids may contribute to their higher cell densities in blood than those of Lyme disease spirochetes.     

Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans GON-1

We demonstrate that in humans, two metalloproteases, ADAMTS-9 (1935 amino acids) and ADAMTS-20 (1911 amino acids) are orthologs of GON-1, an ADAMTS protease required for gonadal morphogenesis in Caenorhabditis elegans. ADAMTS-9 and ADAMTS-20 have an identical modular structure, are distinct in possessing 15 TSRs and a unique C-terminal domain, and have a similar gene structure, suggesting that they comprise a new subfamily of human ADAMTS proteases. ADAMTS20 is very sparingly expressed, although it is detectable in epithelial cells of the breast and lung. However, ADAMTS9 is highly expressed in embryonic and adult tissues, and therefore we characterized the ADAMTS-9 protein further. Although the ADAMTS-9 zymogen has many proprotein convertase processing sites, pulse-chase analysis, site-directed mutagenesis, and amino acid sequencing demonstrated that maturation to the active form occurs by selective proprotein convertase (e.g. furin) cleavage of the Arg(287)-Phe(288) bond. Although lacking a transmembrane sequence, ADAMTS-9 is retained near the cell surface as well as in the ECM of transiently transfected COS-1 and 293 cells. COS-1 cells transfected with ADAMTS9 (but not vector-transfected cells) proteolytically cleaved bovine versican and aggrecan core proteins at the Glu(441)-Ala(442) bond of versican V1 and the Glu(1771)-Ala(1772) bond of aggrecan, respectively. In contrast, the ADAMTS-9 catalytic domain alone was neither localized to the cell surface nor able to confer these proteolytic activities on cells, demonstrating that the ancillary domains of ADAMTS-9, including the TSRs, are required both for specific extracellular localization and for its versicanase and aggrecanase activities.