Comparative sub-cellular proteome analyses reveals metabolic differentiation and production of effector-like molecules in the dieback phytopathogen Phytophthora cinnamomi.

Phytopathogenic oomycetes pose a significant threat to global biodiversity and food security. The proteomes of these oomycetes likely contain important factors that contribute to their pathogenic success, making their discovery crucial for elucidating pathogenicity. Phytophthora cinnamomi is a root pathogen that causes dieback in a wide variety of crops and native vegetation world-wide. Virulence proteins produced by P. cinnamomi are not well defined and a large-scale approach to understand the biochemistry of this pathogen has not been documented. Soluble mycelial, zoospore and secreted proteomes were obtained and label-free quantitative proteomics was used to compare the composition of the three sub-proteomes. A total of 4635 proteins were identified, validating 17.7% of the predicted gene set. The mycelia were abundant in transporters for nutrient acquisition, metabolism and cellular proliferation. The zoospores had less metabolic related ontologies but were abundant in energy generating, motility and signalling associated proteins. Virulence-associated proteins were identified in the secretome such as candidate effector and effector-like proteins, which interfere with the host immune system. These include hydrolases, cell wall degrading enzymes, putative necrosis-inducing proteins and elicitins. The secretome elicited a hypersensitive response on the roots of a model host and thus suggests evidence of effector activity

Protein methionine sulfoxide dynamics in Arabidopsis thaliana under oxidative stress

Reactive oxygen species such as hydrogen peroxide can modify proteins via direct oxidation of their sulfur-containing amino acids, cysteine and methionine. Methionine oxidation, studied here, is a reversible posttranslational modification that is emerging as a mechanism by which proteins perceive oxidative stress and function in redox signaling. Identification of proteins with oxidized methionines is the first prerequisite toward understanding the functional effect of methionine oxidation on proteins and the biological processes in which they are involved. Here, we describe a proteome-wide study of in vivo protein-bound methionine oxidation in plants upon oxidative stress using Arabidopsis thaliana catalase 2 knock-out plants as a model system. We identified over 500 sites of oxidation in about 400 proteins and quantified the differences in oxidation between wild-type and catalase 2 knock-out plants. We show that the activity of two plant-specific glutathione S-transferases, GSTF9 and GSTT23, is significantly reduced upon oxidation. And, by sampling over time, we mapped the dynamics of methionine oxidation and gained new insights into this complex and dynamic landscape of a part of the plant proteome that is sculpted by oxidative stress

Variability in an effector gene promoter of a necrotrophic fungal pathogen dictates epistasis and effector-triggered susceptibility in wheat

The fungus Parastagonospora nodorum uses proteinaceous necrotrophic effectors (NEs) to induce tissue necrosis on wheat leaves during infection, leading to the symptoms of septoria nodorum blotch (SNB). The NEs Tox1 and Tox3 induce necrosis on wheat possessing the dominant susceptibility genes Snn1 and Snn3B1/Snn3D1, respectively. We previously observed that Tox1 is epistatic to the expression of Tox3 and a quantitative trait locus (QTL) on chromosome 2A that contributes to SNB resistance/susceptibility. The expression of Tox1 is significantly higher in the Australian strain SN15 compared to the American strain SN4. Inspection of the Tox1 promoter region revealed a 401 bp promoter genetic element in SN4 positioned 267 bp upstream of the start codon that is absent in SN15, called PE401. Analysis of the world-wide P. nodorum population revealed that a high proportion of Northern Hemisphere isolates possess PE401 whereas the opposite was observed in representative P. nodorum isolates from Australia and South Africa. The presence of PE401 removed the epistatic effect of Tox1 on the contribution of the SNB 2A QTL but not Tox3. PE401 was introduced into the Tox1 promoter regulatory region in SN15 to test for direct regulatory roles. Tox1 expression was markedly reduced in the presence of PE401. This suggests a repressor molecule(s) binds PE401 and inhibits Tox1 transcription. Infection assays also demonstrated that P. nodorum which lacks PE401 is more pathogenic on Snn1 wheat varieties than P. nodorum carrying PE401. An infection competition assay between P. nodorum isogenic strains with and without PE401 indicated that the higher Tox1-expressing strain rescued the reduced virulence of the lower Tox1-expressing strain on Snn1 wheat. Our study demonstrated that Tox1 exhibits both ‘selfish’ and ‘altruistic’ characteristics. This offers an insight into a complex NE-NE interaction that is occurring within the P. nodorum population. The importance of PE401 in breeding for SNB resistance in wheat is discussed

Gene validation and remodelling using proteogenomics of Phytophthora cinnamomi, the causal agent of Dieback

Phytophthora cinnamomi is a pathogenic oomycete that causes plant dieback disease across a range of natural ecosystems and in many agriculturally important crops on a global scale. An annotated draft genome sequence is publicly available (JGI Mycocosm) and suggests 26,131 gene models. In this study, soluble mycelial, extracellular (secretome), and zoospore proteins of P. cinnamomi were exploited to refine the genome by correcting gene annotations and discovering novel genes. By implementing the diverse set of sub-proteomes into a generated proteogenomics pipeline, we were able to improve the P. cinnamomi genome annotation. Liquid chromatography mass spectrometry was used to obtain high confidence peptides with spectral matching to both the annotated genome and a generated 6-frame translation. Two thousand seven hundred sixty-four annotations from the draft genome were confirmed by spectral matching. Using a proteogenomic pipeline, mass spectra were used to edit the P. cinnamomi genome and allowed identification of 23 new gene models and 60 edited gene features using high confidence peptides obtained by mass spectrometry, suggesting a rate of incorrect annotations of 3% of the detectable proteome. The novel features were further validated by total peptide support, alongside functional analysis including the use of Gene Ontology and functional domain identification. We demonstrated the use of spectral data in combination with our proteogenomics pipeline can be used to improve the genome annotation of important plant diseases and identify missed genes. This study presents the first use of spectral data to edit and manually annotate an oomycete pathogen

MOL#8268 1 Title Page. The natural mutation encoding a C-terminus truncated 5-HT 2B receptor is a gain of proliferative functions

Number of tables, 1 Number of references, 31 Number of words in the abstract, 230 Number of words in the Introduction, 744 Number of words in the Discussion. 1172 List of non-standard abbreviations receptor was identified in one patient diagnosed with pulmonary hypertension after intake of the anorexigen dexfenfluramine. Although reported to generate a lack of function, this C-terminus truncated 5-HT 2B receptor should somehow affect transduction pathways relevant to pulmonary hypertension. In our study, we investigated putative modifications in transduction of the R393X mutated 5-HT 2B receptor. In stably transfected cells, we confirmed the loss of IP 3 stimulation due to the G αq uncoupling, despite conserved ligand affinity between the normal and mutated receptors. We also observed a partial loss of nitric oxide synthase stimulation. However, the truncated R393X receptor presented (i) a strong gain of efficacy in cell proliferation as assessed by mitogen-activated protein kinase activity and thymidine incorporation, (ii) a preferential coupling to G α13 as shown by blocking antiserum, and (iii) an apparent lack of internalization upon agonist stimulation as observed by confocal microscopy. This work demonstrates that, in the 5-HT 2B receptor, the C-terminus including the palmitoylation and phosphorylation sites is absolutely required for proper transduction and internalization. For the first time, we show that the lack of C-terminus can generate a switch of coupling to G α13 , a reduced NO synthase activation and an increase in cell proliferation. All these modifications are relevant in pathophysiological vasoconstriction. MOL#8268 4 Introduction

Genes that determine immunology and inflammation modify the basic defect of impaired ion conductance in cystic fibrosis epithelia

BACKGROUND: The cystic fibrosis (CF) basic defect, caused by dysfunction of the apical chloride channel CFTR in the gastrointestinal and respiratory tract epithelia, has not been employed so far to support the role of CF modifier genes. METHODS: Patients were selected from 101 families with a total of 171 F508del-CFTR homozygous CF patients to identify CF modifying genes. A candidate gene based association study of 52 genes on 16 different chromosomes with a total of 182 genetic markers was performed. Differences in haplotype and/or diplotype distribution between case and reference CF subpopulations were analysed. RESULTS: Variants at immunologically relevant genes were associated with the manifestation of the CF basic defect (0.01<Praw<0.0001 at IL1B, TLR9, TNFalpha, CD95, STAT3 and TNFR). The intragenic background of F508del-CFTR chromosomes determined disease severity and manifestation of the basic defect (Praw=0.0009). Allele distributions comparing transmitted and non-transmitted alleles were distorted at several loci unlinked to CFTR. CONCLUSIONS: The inherited capabilities of the innate and adaptive immune system determine the manifestation of the CF basic defect. Variants on F508del-CFTR chromosomes contribute to the observed patient-to-patient variability among F508del-CFTR homozygotes. A survivor effect, manifesting as a transmission disequilibrium at many loci, is consistent with the improvement of clinical care over the last decades, resulting in a depletion of risk alleles at modifier genes. Awareness of non-genetic factors such as improvement of patient care over time is crucial for the interpretation of CF modifier studies

Bacillus cereus Biovar Anthracis Causing Anthrax in Sub-Saharan Africa—Chromosomal Monophyly and Broad Geographic Distribution

Through full genome analyses of four atypical Bacillus cereus isolates, designated B. cereus biovar anthracis, we describe a distinct clade within the B. cereus group that presents with anthrax-like disease, carrying virulence plasmids similar to those of classic Bacillus anthracis. We have isolated members of this clade from different mammals (wild chimpanzees, gorillas, an elephant and goats) in West and Central Africa (Côte d’Ivoire, Cameroon, Central African Republic and Democratic Republic of Congo). The isolates shared several phenotypic features of both B. anthracis and B. cereus, but differed amongst each other in motility and their resistance or sensitivity to penicillin. They all possessed the same mutation in the regulator gene plcR, different from the one found in B. anthracis, and in addition, carry genes which enable them to produce a second capsule composed of hyaluronic acid. Our findings show the existence of a discrete clade of the B. cereus group capable of causing anthrax-like disease, found in areas of high biodiversity, which are possibly also the origin of the worldwide distributed B. anthracis. Establishing the impact of these pathogenic bacteria on threatened wildlife species will require systematic investigation. Furthermore, the consumption of wildlife found dead by the local population and presence in a domestic animal reveal potential sources of exposure to humans

Disruption of tuftelin 1, a desmosome associated protein, causes skin fragility, woolly hair and palmoplantar keratoderma

Desmosomes are dynamic complex protein structures involved in cellular adhesion. Disruption of these structures by loss of function variants in desmosomal genes lead to a variety of skin and heart related phenotypes. Here, we report tuftelin 1 as a desmosome-associated protein, implicated in epidermal integrity. In two siblings with mild skin fragility, woolly hair and mild palmoplantar keratoderma, but without a cardiac phenotype, we identified a homozygous splice site variant in the TUFT1 gene, leading to aberrant mRNA splicing and loss of tuftelin 1 protein. Patients' skin and keratinocytes showed acantholysis, perinuclear retraction of intermediate filaments, and reduced mechanical stress resistance. Immunolabeling and transfection studies showed that tuftelin 1 is positioned within the desmosome and its location dependent on the presence of the desmoplakin carboxy-terminal tail. A Tuft1 knock-out mouse model mimicked the patients' phenotypes. Altogether, this study reveals tuftelin 1 as a desmosome-associated protein, whose absence causes skin fragility, woolly hair and palmoplantar keratoderma