The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways. Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Detailed stratified GWAS analysis for severe COVID-19 in four European populations

Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended genome-wide association meta-analysis of a well-characterized cohort of 3255 COVID-19 patients with respiratory failure and 12 488 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a ~0.9-Mb inversion polymorphism that creates two highly differentiated haplotypes and characterized the potential effects of the inversion in detail. Our data, together with the 5th release of summary statistics from the COVID-19 Host Genetics Initiative including non-Caucasian individuals, also identified a new locus at 19q13.33, including NAPSA, a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung.S.E.H. and C.A.S. partially supported genotyping through a philanthropic donation. A.F. and D.E. were supported by a grant from the German Federal Ministry of Education and COVID-19 grant Research (BMBF; ID:01KI20197); A.F., D.E. and F.D. were supported by the Deutsche Forschungsgemeinschaft Cluster of Excellence ‘Precision Medicine in Chronic Inflammation’ (EXC2167). D.E. was supported by the German Federal Ministry of Education and Research (BMBF) within the framework of the Computational Life Sciences funding concept (CompLS grant 031L0165). D.E., K.B. and S.B. acknowledge the Novo Nordisk Foundation (NNF14CC0001 and NNF17OC0027594). T.L.L., A.T. and O.Ö. were funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project numbers 279645989; 433116033; 437857095. M.W. and H.E. are supported by the German Research Foundation (DFG) through the Research Training Group 1743, ‘Genes, Environment and Inflammation’. L.V. received funding from: Ricerca Finalizzata Ministero della Salute (RF-2016-02364358), Italian Ministry of Health ‘CV PREVITAL’—strategie di prevenzione primaria cardiovascolare primaria nella popolazione italiana; The European Union (EU) Programme Horizon 2020 (under grant agreement No. 777377) for the project LITMUS- and for the project ‘REVEAL’; Fondazione IRCCS Ca’ Granda ‘Ricerca corrente’, Fondazione Sviluppo Ca’ Granda ‘Liver-BIBLE’ (PR-0391), Fondazione IRCCS Ca’ Granda ‘5permille’ ‘COVID-19 Biobank’ (RC100017A). A.B. was supported by a grant from Fondazione Cariplo to Fondazione Tettamanti: ‘Bio-banking of Covid-19 patient samples to support national and international research (Covid-Bank). This research was partly funded by an MIUR grant to the Department of Medical Sciences, under the program ‘Dipartimenti di Eccellenza 2018–2022’. This study makes use of data generated by the GCAT-Genomes for Life. Cohort study of the Genomes of Catalonia, Fundació IGTP (The Institute for Health Science Research Germans Trias i Pujol) IGTP is part of the CERCA Program/Generalitat de Catalunya. GCAT is supported by Acción de Dinamización del ISCIII-MINECO and the Ministry of Health of the Generalitat of Catalunya (ADE 10/00026); the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (2017-SGR 529). M.M. received research funding from grant PI19/00335 Acción Estratégica en Salud, integrated in the Spanish National RDI Plan and financed by ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (European Regional Development Fund (FEDER)-Una manera de hacer Europa’). B.C. is supported by national grants PI18/01512. X.F. is supported by the VEIS project (001-P-001647) (co-funded by the European Regional Development Fund (ERDF), ‘A way to build Europe’). Additional data included in this study were obtained in part by the COVICAT Study Group (Cohort Covid de Catalunya) supported by IsGlobal and IGTP, European Institute of Innovation & Technology (EIT), a body of the European Union, COVID-19 Rapid Response activity 73A and SR20-01024 La Caixa Foundation. A.J. and S.M. were supported by the Spanish Ministry of Economy and Competitiveness (grant numbers: PSE-010000-2006-6 and IPT-010000-2010-36). A.J. was also supported by national grant PI17/00019 from the Acción Estratégica en Salud (ISCIII) and the European Regional Development Fund (FEDER). The Basque Biobank, a hospital-related platform that also involves all Osakidetza health centres, the Basque government’s Department of Health and Onkologikoa, is operated by the Basque Foundation for Health Innovation and Research-BIOEF. M.C. received Grants BFU2016-77244-R and PID2019-107836RB-I00 funded by the Agencia Estatal de Investigación (AEI, Spain) and the European Regional Development Fund (FEDER, EU). M.R.G., J.A.H., R.G.D. and D.M.M. are supported by the ‘Spanish Ministry of Economy, Innovation and Competition, the Instituto de Salud Carlos III’ (PI19/01404, PI16/01842, PI19/00589, PI17/00535 and GLD19/00100) and by the Andalussian government (Proyectos Estratégicos-Fondos Feder PE-0451-2018, COVID-Premed, COVID GWAs). The position held by Itziar de Rojas Salarich is funded by grant FI20/00215, PFIS Contratos Predoctorales de Formación en Investigación en Salud. Enrique Calderón’s team is supported by CIBER of Epidemiology and Public Health (CIBERESP), ‘Instituto de Salud Carlos III’. J.C.H. reports grants from Research Council of Norway grant no 312780 during the conduct of the study. E.S. reports grants from Research Council of Norway grant no. 312769. The BioMaterialBank Nord is supported by the German Center for Lung Research (DZL), Airway Research Center North (ARCN). The BioMaterialBank Nord is member of popgen 2.0 network (P2N). P.K. Bergisch Gladbach, Germany and the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany. He is supported by the German Federal Ministry of Education and Research (BMBF). O.A.C. is supported by the German Federal Ministry of Research and Education and is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—CECAD, EXC 2030–390661388. The COMRI cohort is funded by Technical University of Munich, Munich, Germany. This work was supported by grants of the Rolf M. Schwiete Stiftung, the Saarland University, BMBF and The States of Saarland and Lower Saxony. K.U.L. is supported by the German Research Foundation (DFG, LU-1944/3-1). Genotyping for the BoSCO study is funded by the Institute of Human Genetics, University Hospital Bonn. F.H. was supported by the Bavarian State Ministry for Science and Arts. Part of the genotyping was supported by a grant to A.R. from the German Federal Ministry of Education and Research (BMBF, grant: 01ED1619A, European Alzheimer DNA BioBank, EADB) within the context of the EU Joint Programme—Neurodegenerative Disease Research (JPND). Additional funding was derived from the German Research Foundation (DFG) grant: RA 1971/6-1 to A.R. P.R. is supported by the DFG (CCGA Sequencing Centre and DFG ExC2167 PMI and by SH state funds for COVID19 research). F.T. is supported by the Clinician Scientist Program of the Deutsche Forschungsgemeinschaft Cluster of Excellence ‘Precision Medicine in Chronic Inflammation’ (EXC2167). C.L. and J.H. are supported by the German Center for Infection Research (DZIF). T.B., M.M.B., O.W. und A.H. are supported by the Stiftung Universitätsmedizin Essen. M.A.-H. was supported by Juan de la Cierva Incorporacion program, grant IJC2018-035131-I funded by MCIN/AEI/10.13039/501100011033. E.C.S. is supported by the Deutsche Forschungsgemeinschaft (DFG; SCHU 2419/2-1).Peer reviewe

Detailed stratified GWAS analysis for severe COVID-19 in four European populations

Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of a well-characterized cohort of 3,260 COVID-19 patients with respiratory failure and 12,483 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen (HLA) region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a highly pleiotropic ∼0.9-Mb inversion polymorphism and characterized the potential effects of the inversion in detail. Our data, together with the 5th release of summary statistics from the COVID-19 Host Genetics Initiative, also identified a new locus at 19q13.33, including NAPSA, a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung.Andre Franke and David Ellinghaus were supported by a grant from the German Federal Ministry of Education and Research (01KI20197), Andre Franke, David Ellinghaus and Frauke Degenhardt were supported by the Deutsche Forschungsgemeinschaft Cluster of Excellence “Precision Medicine in Chronic Inflammation” (EXC2167). David Ellinghaus was supported by the German Federal Ministry of Education and Research (BMBF) within the framework of the Computational Life Sciences funding concept (CompLS grant 031L0165). David Ellinghaus, Karina Banasik and Søren Brunak acknowledge the Novo Nordisk Foundation (grant NNF14CC0001 and NNF17OC0027594). Tobias L. Lenz, Ana Teles and Onur Özer were funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project numbers 279645989; 433116033; 437857095. Mareike Wendorff and Hesham ElAbd are supported by the German Research Foundation (DFG) through the Research Training Group 1743, "Genes, Environment and Inflammation". This project was supported by a Covid-19 grant from the German Federal Ministry of Education and Research (BMBF; ID: 01KI20197). Luca Valenti received funding from: Ricerca Finalizzata Ministero della Salute RF2016-02364358, Italian Ministry of Health ""CV PREVITAL – strategie di prevenzione primaria cardiovascolare primaria nella popolazione italiana; The European Union (EU) Programme Horizon 2020 (under grant agreement No. 777377) for the project LITMUS- and for the project ""REVEAL""; Fondazione IRCCS Ca' Granda ""Ricerca corrente"", Fondazione Sviluppo Ca' Granda ""Liver-BIBLE"" (PR-0391), Fondazione IRCCS Ca' Granda ""5permille"" ""COVID-19 Biobank"" (RC100017A). Andrea Biondi was supported by the grant from Fondazione Cariplo to Fondazione Tettamanti: "Biobanking of Covid-19 patient samples to support national and international research (Covid-Bank). This research was partly funded by a MIUR grant to the Department of Medical Sciences, under the program "Dipartimenti di Eccellenza 2018–2022". This study makes use of data generated by the GCAT-Genomes for Life. Cohort study of the Genomes of Catalonia, Fundació IGTP. IGTP is part of the CERCA Program / Generalitat de Catalunya. GCAT is supported by Acción de Dinamización del ISCIIIMINECO and the Ministry of Health of the Generalitat of Catalunya (ADE 10/00026); the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (2017-SGR 529). Marta Marquié received research funding from ant PI19/00335 Acción Estratégica en Salud, integrated in the Spanish National RDI Plan and financed by ISCIIISubdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER-Una manera de hacer Europa").Beatriz Cortes is supported by national grants PI18/01512. Xavier Farre is supported by VEIS project (001-P-001647) (cofunded by European Regional Development Fund (ERDF), “A way to build Europe”). Additional data included in this study was obtained in part by the COVICAT Study Group (Cohort Covid de Catalunya) supported by IsGlobal and IGTP, EIT COVID-19 Rapid Response activity 73A and SR20-01024 La Caixa Foundation. Antonio Julià and Sara Marsal were supported by the Spanish Ministry of Economy and Competitiveness (grant numbers: PSE-010000-2006-6 and IPT-010000-2010-36). Antonio Julià was also supported the by national grant PI17/00019 from the Acción Estratégica en Salud (ISCIII) and the FEDER. The Basque Biobank is a hospitalrelated platform that also involves all Osakidetza health centres, the Basque government's Department of Health and Onkologikoa, is operated by the Basque Foundation for Health Innovation and Research-BIOEF. Mario Cáceres received Grants BFU2016-77244-R and PID2019-107836RB-I00 funded by the Agencia Estatal de Investigación (AEI, Spain) and the European Regional Development Fund (FEDER, EU). Manuel Romero Gómez, Javier Ampuero Herrojo, Rocío Gallego Durán and Douglas Maya Miles are supported by the “Spanish Ministry of Economy, Innovation and Competition, the Instituto de Salud Carlos III” (PI19/01404, PI16/01842, PI19/00589, PI17/00535 and GLD19/00100), and by the Andalussian government (Proyectos Estratégicos-Fondos Feder PE-0451-2018, COVID-Premed, COVID GWAs). The position held by Itziar de Rojas Salarich is funded by grant FI20/00215, PFIS Contratos Predoctorales de Formación en Investigación en Salud. Enrique Calderón's team is supported by CIBER of Epidemiology and Public Health (CIBERESP), "Instituto de Salud Carlos III". Jan Cato Holter reports grants from Research Council of Norway grant no 312780 during the conduct of the study. Dr. Solligård: reports grants from Research Council of Norway grant no 312769. The BioMaterialBank Nord is supported by the German Center for Lung Research (DZL), Airway Research Center North (ARCN). The BioMaterialBank Nord is member of popgen 2.0 network (P2N). Philipp Koehler has received non-financial scientific grants from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, and the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany. He is supported by the German Federal Ministry of Education and Research (BMBF).Oliver A. Cornely is supported by the German Federal Ministry of Research and Education and is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy – CECAD, EXC 2030 – 390661388. The COMRI cohort is funded by Technical University of Munich, Munich, Germany. Genotyping was performed by the Genotyping laboratory of Institute for Molecular Medicine Finland FIMM Technology Centre, University of Helsinki. This work was supported by grants of the Rolf M. Schwiete Stiftung, the Saarland University, BMBF and The States of Saarland and Lower Saxony. Kerstin U. Ludwig is supported by the German Research Foundation (DFG, LU-1944/3-1). Genotyping for the BoSCO study is funded by the Institute of Human Genetics, University Hospital Bonn. Frank Hanses was supported by the Bavarian State Ministry for Science and Arts. Part of the genotyping was supported by a grant to Alfredo Ramirez from the German Federal Ministry of Education and Research (BMBF, grant: 01ED1619A, European Alzheimer DNA BioBank, EADB) within the context of the EU Joint Programme – Neurodegenerative Disease Research (JPND). Additional funding was derived from the German Research Foundation (DFG) grant: RA 1971/6-1 to Alfredo Ramirez. Philip Rosenstiel is supported by the DFG (CCGA Sequencing Centre and DFG ExC2167 PMI and by SH state funds for COVID19 research). Florian Tran is supported by the Clinician Scientist Program of the Deutsche Forschungsgemeinschaft Cluster of Excellence “Precision Medicine in Chronic Inflammation” (EXC2167). Christoph Lange and Jan Heyckendorf are supported by the German Center for Infection Research (DZIF). Thorsen Brenner, Marc M Berger, Oliver Witzke und Anke Hinney are supported by the Stiftung Universitätsmedizin Essen. Marialbert Acosta-Herrera was supported by Juan de la Cierva Incorporacion program, grant IJC2018-035131-I funded by MCIN/AEI/10.13039/501100011033. Eva C Schulte is supported by the Deutsche Forschungsgemeinschaft (DFG; SCHU 2419/2-1).N

Plant growth promotion and biocontrol traits of Vitis vinifera "Glera" bacterial endophytes in the sustainable management of viticulture

This thesis describes the results obtained during the three years PhD course which was focused on plant-bacteria interactions, in particular Vitis vinifera cultivar Glera and endophytes, previously isolated from the same plant species. The project aimed to investigate the possible beneficial effects that four selected endophyte strains, belonging to Pantoea and Bacillus genera, can give to Glera rooting cuttings and their potential utility as environmental-friendly alternative approach in plant growth promotion, stress alleviation, and biocontrol. The project was prompted to reduce the extensive use of agro-chemicals in agriculture and to promote a sustainable management of viticulture. As a first step, P. agglomerans (GL83) and B. licheniformis (GL174) selected for their valuable plant growth promoting traits were GFP-tagged and used to inoculate in vitro Glera apical cuttings in order to confirm their endophytic nature. Exploiting laser scanning confocal microscopy and a cultivation dependent method the colonization of stem endosphere of Glera apical cuttings after 20 and 30 days post inoculation (dpi) was demonstrated. The analysis was then extended at 45, 52 and 60 dpi in Glera cuttings which were in the meantime transferred in hydroponic non-sterile medium. Both strains were mostly visualized inside the xylem vessels of the stem until 60 dpi confirming the strains endophytic attitude and persistence. Then, the four strains were directly applied in the field by inoculating rootstocks (Kober 5BB) and Glera buds with each bacteria suspension (GL83, GL174, GL189, GL452 and the consortium of these four) both during the hydration step of rooting cuttings production line and by administering the bacterial suspensions at the root level, after plants have developed roots. Then, both the morphological and physiological plant parameters were recorded sampling plants at different time-point of their development. GL83 and GL174 were the most promising grapevine bio-fertilizer strains, especially when rootstocks and buds were inoculated during the hydration step of rooting cuttings production line. Inoculated plants presented improved leaf physiological parameters such as photosynthesis rate and transpiration flux linked to an increase of root and shoot fresh biomasses compared to the control plants. Moreover, an experiment aimed to simulate a fertilization procedure using 1-year-old Glera grapevine plants, which were bio-fertilized once a week for seven consecutive weeks with GL83 and GL174 confirmed these strains as promising bio-fertilizer candidates. Furthermore, GL83 was able to relieve the effects of drought stress in inoculated Glera rooting cuttings. In fact, stressed-inoculated plants presented enhanced physiological parameters compared to stressed-control plants and, in addition, GL83 plant growth promoting traits seemed to be water stress dependent. Another issue investigated during these three PhD years was the role of Bacillus cyclic lipopeptides (LPs) families as elicitors of plant defence responses, through induced systemic resistance (ISR) pathway. LPs are amphiphilic compounds produced by some beneficial bacterial strains freely living in the soil or undergoing mutualistic interactions with plants. The natural functions of LPs suggest their role in antagonism toward other microorganisms, like plant pathogens, and their involvement in motility and attachment to surfaces encouraging bacteria biofilm formation and development. So far, the mechanisms underlying the induced physiological responses in host plants, upon the exposure to these compounds remain unclear. Challenging Arabidopsis cell suspension cultures with the commercial Bacillus LPs families (surfactin, fengycin and iturin) both the early cell response and the later events like defence-gene expression were evaluated. Through Evans blue test no cell death increase was detected challenging Arabidopsis cell culture with three different concentrations of the LPs families. The administration of the highest concentration of surfactin (50 µg/ml) to A. thaliana cell cultures expressing the Ca2+-sensitive photoprotein aequorin in the cytosol evoked a cytosolic Ca2+ transient, suggesting that the perception of surfactin could be mediated by Ca2+, which is one of the main important intracellular messenger that generate a wide range of different spatial and temporal signals depending on the stimulus perceived. Then, to evaluate the possible long-term effects generated upon the perception of LPs, a semi-quantitative PCR defence-gene expression analysis was performed at 2 and 6 hours after LPs cell culture-exposure. Lipoxygenase 1 (LOX1), Pathogen-related protein 1 (PR1), Phenylalanine ammonia lyase (PAL1), Non-expressor of Pathogenesis-Related genes 1 (NPR1) and Mitogen-Activated Protein Kinase 3 (MAPK3) were the main genes investigated which are considered reliable markers of ISR and systemic acquired resistance (SAR). The results showed that ~3-fold increase of LOX1 and PAL1 expression was recorded in treated samples respect to controls. A ~2-fold increase in the NPR1 gene expression was observed comparing samples at 2 h with control. No differences in PR1 and MAPK3 gene expression were detected between samples and controls. The up-regulation of the LOX1 and NPR1 could suggest that surfactin elicits the activation of jasmonate-mediated pathway that could culminate in ISR. Instead, up-regulation of PAL1 without PR1 expression may indicate an initial SAR triggering, afterwards inhibited by some steps of ISR pathway. A UPLC-MS analysis performed at the Gembloux Agro-Bio Tech (University of Liège, Belgium) revealed that some selected Glera Bacillus endophytes produce surfactin and fengycin. Moreover, a quite uncommon type of surfactin called pumilacidin was detected and semi-purified. An in vitro antagonism assays against some plant pathogen bacteria showed that pumilacidin had a quite strong antibacterial effect. This is a preliminary result and other tests against, for example, infectious fungi such as B. cinerea need to be performed. Nevertheless, this is an important starting point for developing new sustainable practices with potential application in disease control. In conclusion, among grapevine cultivable endophytic strains, GL83 and GL174 showed to be promising candidates as bio-fertilizer enhancing both the grapevine growth and health: GL83 was able to promote growth and to confer an increased tolerance to drought stress whereas GL174 seemed to have a protecting action against some fungal grapevine pathogens. The possibility of endophyte exploitation to help plants to cope with abiotic as well as biotic environmental stresses, interfering with plant morphology and physiology or priming their systemic responses against pathogens, opens new scenarios for an environmentally friendly shift in agricultural practices with much lower impact of synthetic agro-chemicals. Results obtained from this work are aligned with the huge amount of studies, grown exponentially in the last years, in which the endophytes abilities and properties are often assayed in a single plant species or within groups of closely related plant genotypes. In addition, investigations are usually performed using microbial species that are relatively easy to cultivate leaving out that plant biome is always characterized by wide and complex interactions. The plant phenotype is determined not only by the plant responses to the environment but it is also orchestrated by the associated microbiota, the responses of the microbiota to the environment, and the complex interactions between individuals in the endosphere microbial plant community. Future exciting challenges, based on classical approaches and new valuable technologies (i.e. next-generation sequencing) applied to greenhouse and field conditions would allow to explore and characterize the contributions of genetic and metabolic elements involved in the interactions between host plants and endophytes, providing new ecological and evolutionary insights and a better knowledge of the plant-microbiome-environment relationships.Questa tesi presenta e discute i risultati ottenuti durante i tre anni del corso in Evoluzione, Ecologia e Conservazione della scuola di dottorato in Bioscienze presso l’Università degli studi di Padova. Il progetto di dottorato si è focalizzato sulle interazioni piante-microrganismi, in particolare tra Vitis vinifera cultivar Glera e batteri endofiti, precedentemente isolati dalla stessa specie vegetale. Il progetto mira a studiare i possibili effetti benefici che alcuni batteri endofiti, appartenenti ai generi Pantoea e Bacillus, possono conferire alle barbatelle di Glera in termini di promozione della crescita, riduzione di stress ambientali e protezione contro i patogeni. Il progetto ha come scopo la riduzione dell’utilizzo di sostanze inquinanti, come fertilizzanti chimici e pesticidi, in agricoltura e di promuovere una gestione più sostenibile della viticoltura. Innanzitutto sono stati selezionati due ceppi, Pantoea agglomerans (GL83) e Bacillus licheniformis (GL174), sulla base delle loro attività di promozione della crescita precedentemente definite; quindi entrambi sono stati trasformati con il gene che codifica la proteina fluorescente GFP e utilizzati per inoculare, in vitro, talee apicali di Glera per confermare la loro natura endofitica. Sfruttando tecniche di microscopia confocale a fluorescenza e un metodo di coltivazione diretta in piastra, è stato possibile dimostrare la capacità di tali ceppi di colonizzare i tessuti interni del fusto dopo 20 e 30 giorni dall'inoculo. L'analisi è stata poi estesa a 45, 52 e 60 giorni dall’inoculo, durante i quali le talee di vite sono state trasferite in mezzo idroponico non sterile. Entrambi i ceppi sono stati visualizzati all'interno del sistema vascolare del fusto e nei tessuti interni della radice fino a 60 giorni dall’inoculo. Il passo successivo ha previsto la messa a punto di esperimenti in campo utilizzando quattro ceppi endofiti (GL83, GL174, GL189, GL452 e il consorzio di questi quattro) i quali sono stati inoculati in due diversi momenti nella linea di produzione delle barbatelle. Nel primo test i batteri sono stati somministrati a portainnesti (Kober 5BB) e gemme di Glera durante la fase di idratazione, mentre nel secondo dopo che le barbatelle avevano sviluppato le radici. Sono stati quindi valutati sia parametri di crescita sia parametri fisiologici campionando le barbatelle in diversi stadi del loro sviluppo. I ceppi più promettenti sono stati GL83 e GL174 specialmente quando somministrati a portainnesti e gemme durante la fase di idratazione. Le piante inoculate presentavano parametri fisiologici più elevati rispetto a quelli di piante di controllo, nonché un aumento della massa radicale e della parte aerea. E’ stato anche condotto un esperimento mirato a simulare la fertilizzazione in vigneto; questo ha previsto l’utilizzo di piante di vite di 1 anno, le quali sono state irrigate una volta alla settimana per sette settimane consecutive con GL83 e GL174. I risultati emersi confermano che questi due ceppi sono due ottimi candidati per un futuro utilizzo in vigneto come bio-fertilizzanti. Inoltre, GL83 è stato in grado di alleviare gli effetti del deficit idrico in barbatelle di Glera. Infatti, le piante inoculate soggette a stress idrico presentavano parametri morfologici e fisiologici più elevati rispetto a quelli delle relative piante di controllo. Un altro tema trattato durante questo lavoro di dottorato è stato il ruolo delle tre famiglie di lipopeptidi prodotti dal genere Bacillus (surfactine, fengicine e iturine) nell’indurre risposte di difesa nelle piante. I lipopeptidi sono molecole anfipatiche prodotte come metaboliti secondari da alcuni generi batterici come Bacillus e Pseudomonas; la loro struttura li rende composti biologicamente attivi contro microrganismi potenzialmente patogeni quali batteri, funghi e micoplasmi. E’ stato inoltre riportato che queste sostanze sono in grado di scatenare risposte di difesa nelle piante, ma i meccanismi che sottendono all’attivazione di tali risposte e il percorso specifico che seguono, sono tuttora poco noti. Tramite l’utilizzo di un sistema modello semplificato come le colture cellulari in sospensione di Arabidopsis thaliana, È stato possibile studiare sia la fase di percezione dei lipopeptidi sia eventi a valle come l’espressione di alcuni geni marker di difesa. Prima di tutto è stata valutata la possibile citotossicità di questi composti utilizzando l’Evans blue test; sono state somministrate tutte le tre famiglie di lipopeptidi in commercio del genere Bacillus in tre differenti concentrazioni e in alcun caso È stata riscontrata citotossicità . Utilizzando poi una linea cellulare transgenica di Arabidopsis esprimente stabilmente la foto-proteina luminescente Ca2+-sensibile equorina nel citosol è stato possibile studiare l’eventuale coinvolgimento dello ione Ca2+ come trasduttore del segnale dopo la somministrazione di lipopeptidi. Solo la concentrazione più elevata di surfactine (50 µg/ml) ha indotto un transiente citosolico di Ca2+, suggerendo il coinvolgimento di questo ione nella percezione delle stesse. Successivamente, per studiare i possibili effetti a lungo termine delle surfactine nell’indurre le risposte di difesa della pianta sono state effettuate delle analisi di espressione genica tramite PCR semi-quantitativa dopo 2 e 6 ore dal trattamento delle colture cellulari con surfactine 50 µg/ml. I geni studiati sono stati LOX1, PR1, PAL1, NPR1 e MAPK3 considerati geni marker per risposte di difesa sistemiche indotte o acquisite, ISR e SAR. I risultati ottenuti dimostrano un’attivazione delle risposte di difesa della pianta; in particolare LOX1 e PAL1 sono risultati essere 3 volte più espressi nei campioni trattati rispetto al controllo, mentre NPR1 è risultato essere 2 volte più espresso dopo 2 ore dalla somministrazione del trattamento. Invece PR1 e MAPK3 sono risultati invariati nel livello di espressione tra cellule trattate e di controllo. L’up-regolazione di geni come LOX1 e NPR1 suggerisce l’attivazione della via dipendente dall’acido jasmonico la quale conferisce alla pianta uno stato di allerta (priming) contro successivi attacchi da patogeno. PAL1 invece è uno dei geni up-regolati in seguito all’attivazione della SAR. E’ comunque risaputo che ISR e SAR possono agire in modo sinergico aumentando le difese indotte della pianta. Analisi attraverso UPLC-MS condotte nel Lab. del Dott. Marc Ongena presso Gembloux Agro-Bio Tech (University of Liège, Gembloux, Belgio) hanno rivelato la capacità di altri ceppi endofiti di Glera del genere Bacillus di produrre lipopeptidi ciclici, surfactine e fengicine. Una sostanza in particolare, chiamata pumilacidina, è stata semi-purificata e testata in vitro tramite test di antagonismo contro alcuni batteri patogeni per le piante. I risultati hanno mostrato che questa sostanza ha una capacità anti microbica piuttosto marcata. Questo risultato preliminare deve essere confermato da ulteriori studi condotti con funghi patogeni come B. cinerea, ma è un punto di partenza importante per lo sviluppo di nuove pratiche sostenibili per il bio-controllo. In conclusione, GL83 e GL174, tra i ceppi di endofiti di vite coltivabili, si sono dimostrati essere piuttosto promettenti come bio-fertilizzanti, in quanto capaci di migliorare sia la crescita che lo stato di salute della vite. In particolare, GL83 è stato capace di promuovere la crescita e di conferire maggiore tolleranza in situazioni di deficit idrico, mentre GL174 sembra avere un’azione protettiva nei confronti di alcuni patogeni della vite. La possibilità di sfruttare gli endofiti per supportare la pianta in situazioni di stress biotici ed abiotici, in grado di interferire sulla morfologia e la fisiologia della pianta stessa e su risposte di difese sistemiche contro i patogeni, apre nuovi scenari nella gestione sostenibile della viticoltura che faccia meno uso di input chimici. I risultati di questo lavoro confermano quelli ottenuti negli ultimi anni, in cui le proprietà degli endofiti sono state studiate in una singola specie vegetale o in gruppi di genotipi strettamente correlati. Inoltre, spesso gli studi vengono condotti con specie microbiche relativamente facili da coltivare, tralasciando che il bioma della pianta È caratterizzato da ampie e complesse interazioni. Il fenotipo vegetale infatti non è determinato unicamente dalla risposta della pianta all’ambiente, ma è anche orchestrato dalla risposta dell’intero microbiota all’ambiente e dalle complesse interazioni tra gli individui e la comunità microbica della rizosfera. Le prospettive future, basate allo stesso tempo su approcci classici e tecnologie all’avanguardia (NGS), e applicate in differenti condizioni colturali, permetteranno di valutare e caratterizzare in termini genetici e metabolici gli effetti delle interazioni tra pianta ospite ed endofiti. Questo apporterà nuovi spunti e conoscenze sia dal punto di vista ecologico che evoluzionistico in merito alle interazioni pianta-microbioma-ambiente

Plant growth promotion and biocontrol traits of Vitis vinifera "Glera" bacterial endophytes in the sustainable management of viticulture

This thesis describes the results obtained during the three years PhD course which was focused on plant-bacteria interactions, in particular Vitis vinifera cultivar Glera and endophytes, previously isolated from the same plant species. The project aimed to investigate the possible beneficial effects that four selected endophyte strains, belonging to Pantoea and Bacillus genera, can give to Glera rooting cuttings and their potential utility as environmental-friendly alternative approach in plant growth promotion, stress alleviation, and biocontrol. The project was prompted to reduce the extensive use of agro-chemicals in agriculture and to promote a sustainable management of viticulture. As a first step, P. agglomerans (GL83) and B. licheniformis (GL174) selected for their valuable plant growth promoting traits were GFP-tagged and used to inoculate in vitro Glera apical cuttings in order to confirm their endophytic nature. Exploiting laser scanning confocal microscopy and a cultivation dependent method the colonization of stem endosphere of Glera apical cuttings after 20 and 30 days post inoculation (dpi) was demonstrated. The analysis was then extended at 45, 52 and 60 dpi in Glera cuttings which were in the meantime transferred in hydroponic non-sterile medium. Both strains were mostly visualized inside the xylem vessels of the stem until 60 dpi confirming the strains endophytic attitude and persistence. Then, the four strains were directly applied in the field by inoculating rootstocks (Kober 5BB) and Glera buds with each bacteria suspension (GL83, GL174, GL189, GL452 and the consortium of these four) both during the hydration step of rooting cuttings production line and by administering the bacterial suspensions at the root level, after plants have developed roots. Then, both the morphological and physiological plant parameters were recorded sampling plants at different time-point of their development. GL83 and GL174 were the most promising grapevine bio-fertilizer strains, especially when rootstocks and buds were inoculated during the hydration step of rooting cuttings production line. Inoculated plants presented improved leaf physiological parameters such as photosynthesis rate and transpiration flux linked to an increase of root and shoot fresh biomasses compared to the control plants. Moreover, an experiment aimed to simulate a fertilization procedure using 1-year-old Glera grapevine plants, which were bio-fertilized once a week for seven consecutive weeks with GL83 and GL174 confirmed these strains as promising bio-fertilizer candidates. Furthermore, GL83 was able to relieve the effects of drought stress in inoculated Glera rooting cuttings. In fact, stressed-inoculated plants presented enhanced physiological parameters compared to stressed-control plants and, in addition, GL83 plant growth promoting traits seemed to be water stress dependent. Another issue investigated during these three PhD years was the role of Bacillus cyclic lipopeptides (LPs) families as elicitors of plant defence responses, through induced systemic resistance (ISR) pathway. LPs are amphiphilic compounds produced by some beneficial bacterial strains freely living in the soil or undergoing mutualistic interactions with plants. The natural functions of LPs suggest their role in antagonism toward other microorganisms, like plant pathogens, and their involvement in motility and attachment to surfaces encouraging bacteria biofilm formation and development. So far, the mechanisms underlying the induced physiological responses in host plants, upon the exposure to these compounds remain unclear. Challenging Arabidopsis cell suspension cultures with the commercial Bacillus LPs families (surfactin, fengycin and iturin) both the early cell response and the later events like defence-gene expression were evaluated. Through Evans blue test no cell death increase was detected challenging Arabidopsis cell culture with three different concentrations of the LPs families. The administration of the highest concentration of surfactin (50 µg/ml) to A. thaliana cell cultures expressing the Ca2+-sensitive photoprotein aequorin in the cytosol evoked a cytosolic Ca2+ transient, suggesting that the perception of surfactin could be mediated by Ca2+, which is one of the main important intracellular messenger that generate a wide range of different spatial and temporal signals depending on the stimulus perceived. Then, to evaluate the possible long-term effects generated upon the perception of LPs, a semi-quantitative PCR defence-gene expression analysis was performed at 2 and 6 hours after LPs cell culture-exposure. Lipoxygenase 1 (LOX1), Pathogen-related protein 1 (PR1), Phenylalanine ammonia lyase (PAL1), Non-expressor of Pathogenesis-Related genes 1 (NPR1) and Mitogen-Activated Protein Kinase 3 (MAPK3) were the main genes investigated which are considered reliable markers of ISR and systemic acquired resistance (SAR). The results showed that ~3-fold increase of LOX1 and PAL1 expression was recorded in treated samples respect to controls. A ~2-fold increase in the NPR1 gene expression was observed comparing samples at 2 h with control. No differences in PR1 and MAPK3 gene expression were detected between samples and controls. The up-regulation of the LOX1 and NPR1 could suggest that surfactin elicits the activation of jasmonate-mediated pathway that could culminate in ISR. Instead, up-regulation of PAL1 without PR1 expression may indicate an initial SAR triggering, afterwards inhibited by some steps of ISR pathway. A UPLC-MS analysis performed at the Gembloux Agro-Bio Tech (University of Liège, Belgium) revealed that some selected Glera Bacillus endophytes produce surfactin and fengycin. Moreover, a quite uncommon type of surfactin called pumilacidin was detected and semi-purified. An in vitro antagonism assays against some plant pathogen bacteria showed that pumilacidin had a quite strong antibacterial effect. This is a preliminary result and other tests against, for example, infectious fungi such as B. cinerea need to be performed. Nevertheless, this is an important starting point for developing new sustainable practices with potential application in disease control. In conclusion, among grapevine cultivable endophytic strains, GL83 and GL174 showed to be promising candidates as bio-fertilizer enhancing both the grapevine growth and health: GL83 was able to promote growth and to confer an increased tolerance to drought stress whereas GL174 seemed to have a protecting action against some fungal grapevine pathogens. The possibility of endophyte exploitation to help plants to cope with abiotic as well as biotic environmental stresses, interfering with plant morphology and physiology or priming their systemic responses against pathogens, opens new scenarios for an environmentally friendly shift in agricultural practices with much lower impact of synthetic agro-chemicals. Results obtained from this work are aligned with the huge amount of studies, grown exponentially in the last years, in which the endophytes abilities and properties are often assayed in a single plant species or within groups of closely related plant genotypes. In addition, investigations are usually performed using microbial species that are relatively easy to cultivate leaving out that plant biome is always characterized by wide and complex interactions. The plant phenotype is determined not only by the plant responses to the environment but it is also orchestrated by the associated microbiota, the responses of the microbiota to the environment, and the complex interactions between individuals in the endosphere microbial plant community. Future exciting challenges, based on classical approaches and new valuable technologies (i.e. next-generation sequencing) applied to greenhouse and field conditions would allow to explore and characterize the contributions of genetic and metabolic elements involved in the interactions between host plants and endophytes, providing new ecological and evolutionary insights and a better knowledge of the plant-microbiome-environment relationships

Clustering consistency with Dirichlet process mixtures

Dirichlet process mixtures are flexible non-parametric models, particularly suited to density estimation and probabilistic clustering. In this work we study the posterior distribution induced by Dirichlet process mixtures as the sample size increases, and more specifically focus on consistency for the unknown number of clusters when the observed data are generated from a finite mixture. Crucially, we consider the situation where a prior is placed on the concentration parameter of the underlying Dirichlet process. Previous findings in the literature suggest that Dirichlet process mixtures are typically not consistent for the number of clusters if the concentration parameter is held fixed and data come from a finite mixture. Here we show that consistency for the number of clusters can be achieved if the concentration parameter is adapted in a fully Bayesian way, as commonly done in practice. Our results are derived for data coming from a class of finite mixtures, with mild assumptions on the prior for the concentration parameter and for a variety of choices of likelihood kernels for the mixture

Biocontrol traits of Bacillus licheniformis GL174, a culturable endophyte of Vitis vinifera cv. Glera 06 Biological Sciences 0604 Genetics 06 Biological Sciences 0607 Plant Biology 06 Biological Sciences 0605 Microbiology

Abstract Background Bacillus licheniformis GL174 is a culturable endophytic strain isolated from Vitis vinifera cultivar Glera, the grapevine mainly cultivated for the Prosecco wine production. This strain was previously demonstrated to possess some specific plant growth promoting traits but its endophytic attitude and its role in biocontrol was only partially explored. In this study, the potential biocontrol action of the strain was investigated in vitro and in vivo and, by genome sequence analyses, putative functions involved in biocontrol and plant-bacteria interaction were assessed. Results Firstly, to confirm the endophytic behavior of the strain, its ability to colonize grapevine tissues was demonstrated and its biocontrol properties were analyzed. Antagonism test results showed that the strain could reduce and inhibit the mycelium growth of diverse plant pathogens in vitro and in vivo. The strain was demonstrated to produce different molecules of the lipopeptide class; moreover, its genome was sequenced, and analysis of the sequences revealed the presence of many protein-coding genes involved in the biocontrol process, such as transporters, plant-cell lytic enzymes, siderophores and other secondary metabolites. Conclusions This step-by-step analysis shows that Bacillus licheniformis GL174 may be a good biocontrol agent candidate, and describes some distinguished traits and possible key elements involved in this process. The use of this strain could potentially help grapevine plants to cope with pathogen attacks and reduce the amount of chemicals used in the vineyard