Protein secretome of moss plants (Physcomitrella patens) with emphasis on changes induced by a fungal elicitor.

Studies on extracellular proteins (ECPs) contribute to understanding of the multifunctional nature of apoplast. Unlike vascular plants (tracheophytes), little information about ECPs is available from nonvascular plants, such as mosses (bryophytes). In this study, moss plants (Physcomitrella patens) were grown in liquid culture and treated with chitosan, a water-soluble form of chitin that occurs in cell walls of fungi and insects and elicits pathogen defense in plants. ECPs released to the culture medium were compared between chitosan-treated and nontreated control cultures using quantitative mass spectrometry (Orbitrap) and 2-DE-LC-MS/MS. Over 400 secreted proteins were detected, of which 70% were homologous to ECPs reported in tracheophyte secretomes. Bioinformatics analyses using SignalP and SecretomeP predicted classical signal peptides for secretion (37%) or leaderless secretion (27%) for most ECPs of P. patens, but secretion of the remaining proteins (36%) could not be predicted using bioinformatics. Cultures treated with chitosan contained 72 proteins not found in untreated controls, whereas 27 proteins found in controls were not detected in chitosan-treated cultures. Pathogen defense-related proteins dominated in the secretome of P. patens, as reported in tracheophytes. These results advance knowledge on protein secretomes of plants by providing a comprehensive account of ECPs of a bryophyte.

Characterization of the genome, proteome, and structure of yersiniophage ϕR1-37.

The bacteriophage vB_YecM-ϕR1-37 (ϕR1-37) is a lytic yersiniophage that can propagate naturally in different Yersinia species carrying the correct lipopolysaccharide receptor. This large-tailed phage has deoxyuridine (dU) instead of thymidine in its DNA. In this study, we determined the genomic sequence of phage ϕR1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. The 262,391-bp genome of ϕR1-37 is one of the largest sequenced phage genomes, and it contains 367 putative open reading frames (ORFs) and 5 tRNA genes. Mass-spectrometric analysis identified 69 phage particle structural proteins with the genes scattered throughout the genome. A total of 269 of the ORFs (73%) lack homologues in sequence databases. Based on terminator and promoter sequences identified from the intergenic regions, the phage genome was predicted to consist of 40 to 60 transcriptional units. Image reconstruction revealed that the ϕR1-37 capsid consists of hexameric capsomers arranged on a T=27 lattice similar to the bacteriophage ϕKZ. The tail of ϕR1-37 has a contractile sheath. We conclude that phage ϕR1-37 is a representative of a novel phage type that carries the dU-containing genome in a ϕKZ-like head.

Human L-selectin preferentially binds synthetic glycosulfopeptides modeled after endoglycan and containing tyrosine sulfate residues and sialyl Lewis x in core 2 O-glycans.

Endoglycan is a mucin-like glycoprotein expressed by endothelial cells and some leukocytes and is recognized by L-selectin, a C-type lectin important in leukocyte trafficking and extravasation during inflammation. Here, we show that recombinant L-selectin and human T lymphocytes expressing L-selectin bind to synthetic glycosulfopeptides (GSPs). These synthetic glycosulfopeptides contain 37 amino acid residues modeled after the N-terminus of human endoglycan and contain one or two tyrosine sulfates (TyrSO(3)) along with a nearby core-2-based Thr-linked O-glycan with sialyl Lewis x (C2-SLe(x)). TyrSO(3) at position Y118 was more critical for binding than at Y97. C2-SLe(x) at T124 was required for L-selectin recognition. Interestingly, under similar conditions, neither L-selectin nor T lymphocytes showed appreciable binding to the sulfated carbohydrate epitope 6-sulfo-SLe(x). P-selectin also bound to endoglycan-based GSPs but with lower affinity than toward GSPs modeled after PSGL-1, the physiological ligand for P- and L-selectin that is expressed on leukocytes. These results demonstrate that TyrSO(3) residues in association with a C2-SLe(x) moiety within endoglycan and PSGL-1 are preferentially recognized by L-selectin.

The closest relatives of icosahedral viruses of thermophilic bacteria are among viruses and plasmids of the halophilic archaea.

We have sequenced the genome and identified the structural proteins and lipids of the novel membrane-containing, icosahedral virus P23-77 of Thermus thermophilus. P23-77 has an approximately 17-kb circular double-stranded DNA genome, which was annotated to contain 37 putative genes. Virions were subjected to dissociation analysis, and five protein species were shown to associate with the internal viral membrane, while three were constituents of the protein capsid. Analysis of the bacteriophage genome revealed it to be evolutionarily related to another Thermus phage (IN93), archaeal Halobacterium plasmid (pHH205), a genetic element integrated into Haloarcula genome (designated here as IHP for integrated Haloarcula provirus), and the Haloarcula virus SH1. These genetic elements share two major capsid proteins and a putative packaging ATPase. The ATPase is similar with the ATPases found in the PRD1-type viruses, thus providing an evolutionary link to these viruses and furthering our knowledge on the origin of viruses.

Quickly-released peroxidase of moss in defense against fungal invaders.

Mosses (Bryophyta) are nonvascular plants that constitute a large part of the photosynthesizing biomass and carbon storage on Earth. Little is known about how this important portion of flora maintains its health status. This study assessed whether the moss, Physcomitrella patens, responds to treatment with chitosan, a fungal cell wall-derived compound inducing defense against fungal pathogens in vascular plants. Application of chitosan to liquid culture of P. patens caused a rapid increase in peroxidase activity in the medium. For identification of the peroxidase(s), matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/MS, other methods and the whole-genome sequence of P. patens were utilized. Peroxidase gene knock-out mutants were made and inoculated with fungi. The peroxidase activity resulted from a single secreted class III peroxidase (Prx34) which belonged to a P. patens specific phylogenetic cluster in analysis of the 45 putative class III peroxidases of P. patens and those of Arabidopsis and rice. Saprophytic and pathogenic fungi isolated from another moss killed the Prx34 knockout mutants but did not damage wild-type P. patens. The data point out the first specific host factor that is pivotal for pathogen defense in a nonvascular plant. Furthermore, results provide conclusive evidence that class III peroxidases in plants are needed in defense against hostile invasion by fungi.