In silico determination and mutational analysis of canidate effectors from phytopathogenic fungus Verticillium albo-atrum

Gliva Verticillium nonalfalfae, prej poimenovana V. albo-atrum, je fitopatogena askomiceta, ki povzroča bolezen prevodnega tkiva različnih rastlin, imenovano verticilijska uvelost. V Sloveniji največ škode povzroča V. nonalfalfae na hmelju (Humulus lupulus). Poleg fitosanitarnih ukrepov je najučinkovitejši pristop preprečevanja bolezni gojenje odpornih sort. Ključnega pomena pri razvoju novih ukrepov je poznavanje dejavnikov patogenosti glive, predvsem njihovih sekretornih proteinov, kot so efektorji, encimi in toksini. V doktorski disertaciji smo se osredotočili na efektorske proteine, ki smo jih želeli določiti z analizo razpoložljivih genomskih in proteomskih podatkov ter rezultatov RNA-Seq eksperimentov ter jih potrditi z mutacijsko analizo. Anotiranim genskim modelom glive V. nonalfafae (9269) smo z bioinformacijskimi orodji najprej določili in silico sekretom (944), ki je obogaten z encimi, ki so vpleteni v razgradnjo celičnih sten, s proteazami, lipazami, kutinazami in oksidoreduktazami, kar sovpada s patogenezo glive V. nonalfalfae kot hemibiotrofom. Nadalje smo identificirali genske modele izražene in planta (766), predvideli kandidatne sekretorne efektorske proteine (263) ter izbrali najboljše kandidate na podlagi razvrščanja po lastnostih že potrjenih glivnih efektorjev. V odpornih in neodpornih rastlinah hmelja po okužbi z V. nonalfalfae smo z RT-qPCR določili ekspresijo najbolj obetavnim (44) kandidatom. Z uporabo ATMT metode smo pripravili delecijske mutante petim izbranim efektorskim genom in umetno okužili neodporne sorte hmelja. Bolezenska znamenja smo ocenili z bolezenskim indeksom ter z izračunanimi vrednostmi rAUDPC. Bioinformacijski postopek za določanje efektorjev se je izkazal za relativno učinkovitega, saj smo z njim potrdili že prej določena efektorja VnaSSP4.2 in VnaUn.279 glive V. nonalfalfae. Delecijski mutant ΔVna1.565 je kazal zakasnelo virulenco, ΔVna8.691 pa povečano virulenco, medtem ko pri ostalih treh delecijskih mutantih nismo zaznali statistično značilnih razlik v primerjavi z divjim tipom glive, kljub njihovi visoki ekspresiji in planta. Njihova potencialna vloga v patogenezi glive je obravnavana v razpravi.Verticillium nonalfalfae, formerly V. albo-atrum, is a phytopathogenic ascomycete, which causes disease of the vascular tissue of various plants named verticillium wilt. In Slovenia, the most damage is done by Verticillium nonalfafae on hops (Humulus lupulus, L.). Apart from phytosanitary measures, the most effective approach for disease control is the cultivation of resistant varieties. The key to the development of new measures is knowledge of the factors of fungal pathogenicity, especially their secretory proteins, such as effectors, enzymes and toxins. In this doctoral study, we aimed to determine V. nonalfalfae candidate effectors, by analysing the available genomic and proteomic data, and the results of RNA-Seq experiments, and to confirm them with mutational analysis. From the annotated genetic models of V. nonalfafae (9269), we initially identified in silico secretome (944) enriched with enzymes involved in the degradation of cell walls, with proteases, lipases, cutinases and oxidoreductases, which corresponds to the hemibiotrophic life style of V. nonalfalfae. We further identified gene models expressed in planta (766), predicted candidate secretory effector proteins (263), and selected the best candidates based on the properties of already confirmed fungal effectors. We determined the expression of the most promising (44) candidates by RT-qPCR in resistant and susceptible varieties of hop after infection with V. nonalfalfae. Using the ATMT method, we prepared five deletion mutants of candidates with the highest expression, and artificially inoculated susceptible varieties of hop. Disease symptoms were assessed with a disease severity index and rAUDPC. The bioinformatic pipeline for determination of effectors proved to be relatively effective, since we determined previously verified V. nonalfalfae effectors VnaSSP4.2 and VnaUn.279. The deletion mutant ΔVna1.565 showed delayed disease symptoms and ΔVna8.691 showed increased virulence, while the remaining three deletion mutants did not show statistically significant differences compared to the wild type fungus, despite their high expression in planta. Their potential role in the pathogenesis of fungi is discussed

Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity

During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.</p

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus

Peer reviewe

COVID19 Disease Map, a computational knowledge repository of virus-host interaction mechanisms.

Funder: Bundesministerium für Bildung und ForschungFunder: Bundesministerium für Bildung und Forschung (BMBF)We need to effectively combine the knowledge from surging literature with complex datasets to propose mechanistic models of SARS-CoV-2 infection, improving data interpretation and predicting key targets of intervention. Here, we describe a large-scale community effort to build an open access, interoperable and computable repository of COVID-19 molecular mechanisms. The COVID-19 Disease Map (C19DMap) is a graphical, interactive representation of disease-relevant molecular mechanisms linking many knowledge sources. Notably, it is a computational resource for graph-based analyses and disease modelling. To this end, we established a framework of tools, platforms and guidelines necessary for a multifaceted community of biocurators, domain experts, bioinformaticians and computational biologists. The diagrams of the C19DMap, curated from the literature, are integrated with relevant interaction and text mining databases. We demonstrate the application of network analysis and modelling approaches by concrete examples to highlight new testable hypotheses. This framework helps to find signatures of SARS-CoV-2 predisposition, treatment response or prioritisation of drug candidates. Such an approach may help deal with new waves of COVID-19 or similar pandemics in the long-term perspective

The Role of Trust in Accounting Research

The purpose of this paper is to present a review of the knowledge about the trust concept and its application within the accounting research context. This is done by examining the different ontological and epistemological assumptions that accounting research are based on, by relating these assumptions to various conceptions of trust used in accounting research, and by revealing the explicit and implicit role that is given to trust in accounting research. The review is based on articles published between 1995 and 2002 in eleven influential accounting journals. The results show that few accounting articles are explicitly related to trust. Our review supports observations regarding the absence of empirical research made in previous research. Our classification of epistemological and ontological assumptions resulted in an even distribution between the mainstream and alternative approaches. The use of trust in the selected articles showed four different types reflecting an increasing role of trust in explaining accounting phenomena. To summarise, our review produces a dissociated impression of the role of trust in accounting research

Trust Research in Accounting – A Literature Review

Purpose: The purpose of the paper is to provide a structured overview of literature in the nexus of trust and accounting. This can serve as a basis for future research, and thus provide a framework for asking more precise and focused research questions. Design/methodology/approach: All papers published in prominent accounting journals during a 10-year period were scanned. Papers pertaining to the field of trust and accounting were categorized and analyzed in more detail, and qualitatively classified in accordance with selected dimensions. The review was focused on papers explicitly exploring the link between accounting and trust. Findings: The greater part of the papers is in the field of management accounting. The majority of published papers in the field are based on sociological theory, but there are some economics-based papers. Sociologically-based analysis seems to provide more structure, but is also less paradigmatic in nature than economic theory. Only a minority of papers has an explicit definition of the concept of trust. Our conclusion is that the state of research is clearly non-paradigmatic in nature. Origininality/value: This is the only literature review that provides a comprehensive overview of research on trust and accounting. Thus, it is an aid in future research in the area

Comprehensive analysis of Verticillium nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion.

The vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop. The secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed. Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease

Comprehensive analysis of Verticillium nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion

The vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop. The secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed. Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease