MADS-Box Transcription Factor ZtRlm1 Is Responsible for Virulence and Development of the Fungal Wheat Pathogen Zymoseptoria tritici
Mohammadi, Naser ; Mehrabi, Rahim ; Mirzadi Gohari, Amir ; Roostaei, Mozaffar ; Mohammadi Goltapeh, Ebrahim ; Safaie, Naser ; Kema, Gert H.J. - \ 2020
Frontiers in Microbiology 11 (2020). - ISSN 1664-302X
Agrobacterium tumefaciens-mediated transformation - gene deletion - pathogenicity assay - phenotyping - Zymoseptoria tritici
Zymoseptoria tritici is one of the most economically destructive wheat diseases all over the world and is a model fungal plant pathogen within the ascomycetes. In this study, the instrumental role of the ZtRlm1 gene encoding a MADS-box transcription factor (TF) in the infection process of Z. tritici was functionally characterized as these proteins play critical roles in the global gene regulation required for various developmental and physiological processes. Our infection assays showed that ZtRlm1 mutants were attenuated in disease development as a 30 and 90% reduction in chloro-necrotic lesions and pycnidia formation, respectively, were observed in plants inoculated with ZtRlm1 mutant strains demonstrating that ZtRlm1 is a crucial factor playing a significant role in the late stage of infection corresponding with pycnidial formation. Our expression analysis demonstrated that the transcript level of ZtRlm1 is induced at 2 and 20 days post-inoculation, coinciding with pycnidial sporulation. In addition, microscopic analyses showed that branch intensity and biomass production were significantly reduced, indicating that impaired pycnidia formation is a result of impaired differentiation and biomass production in the ZtRlm1 mutants. Furthermore, melanization, a phenomenon required for fruiting body formation, was significantly hampered in ZtRlm1 mutants as they were not melanized under all tested temperature and media conditions. Overall, our data showed that impaired disease development of the ZtRlm1 mutants is mainly due to the significant impact of ZtRlm1 in different cellular processes, including differentiation, branching, fungal biomass production, and melanization, in which identification of downstream genes are of interest to increase our understanding of this pathosystem.
Repositioning of the global epicentre of non-optimal cholesterol
Taddei, Cristina ; Zhou, Bin ; Bixby, Honor ; Carrillo-Larco, Rodrigo M. ; Danaei, Goodarz ; Jackson, Rod T. ; Farzadfar, Farshad ; Sophiea, Marisa K. ; Cesare, Mariachiara Di; Iurilli, Maria Laura Caminia ; Martinez, Andrea Rodriguez ; Asghari, Golaleh ; Dhana, Klodian ; Gulayin, Pablo ; Kakarmath, Sujay ; Santero, Marilina ; Voortman, Trudy ; Riley, Leanne M. ; Cowan, Melanie J. ; Savin, Stefan ; Bennett, James E. ; Stevens, Gretchen A. ; Paciorek, Christopher J. ; Aekplakorn, Wichai ; Cifkova, Renata ; Giampaoli, Simona ; Kengne, Andre Pascal ; Khang, Young Ho ; Kuulasmaa, Kari ; Laxmaiah, Avula ; Margozzini, Paula ; Mathur, Prashant ; Nordestgaard, Børge G. ; Zhao, Dong ; Aadahl, Mette ; Abarca-Gómez, Leandra ; Rahim, Hanan Abdul ; Abu-Rmeileh, Niveen M. ; Acosta-Cazares, Benjamin ; Adams, Robert J. ; Ferrieres, Jean ; Geleijnse, Johanna M. ; He, Yuna ; Jacobs, Jeremy M. ; Kromhout, Daan ; Ma, Guansheng ; Dam, Rob M. van; Wang, Qian ; Wang, Ya Xing ; Wang, Ying Wei - \ 2020
Nature 582 (2020)7810. - ISSN 0028-0836 - p. 73 - 77.
High blood cholesterol is typically considered a feature of wealthy western countries1,2. However, dietary and behavioural determinants of blood cholesterol are changing rapidly throughout the world3 and countries are using lipid-lowering medications at varying rates. These changes can have distinct effects on the levels of high-density lipoprotein (HDL) cholesterol and non-HDL cholesterol, which have different effects on human health4,5. However, the trends of HDL and non-HDL cholesterol levels over time have not been previously reported in a global analysis. Here we pooled 1,127 population-based studies that measured blood lipids in 102.6 million individuals aged 18 years and older to estimate trends from 1980 to 2018 in mean total, non-HDL and HDL cholesterol levels for 200 countries. Globally, there was little change in total or non-HDL cholesterol from 1980 to 2018. This was a net effect of increases in low- and middle-income countries, especially in east and southeast Asia, and decreases in high-income western countries, especially those in northwestern Europe, and in central and eastern Europe. As a result, countries with the highest level of non-HDL cholesterol—which is a marker of cardiovascular risk—changed from those in western Europe such as Belgium, Finland, Greenland, Iceland, Norway, Sweden, Switzerland and Malta in 1980 to those in Asia and the Pacific, such as Tokelau, Malaysia, The Philippines and Thailand. In 2017, high non-HDL cholesterol was responsible for an estimated 3.9 million (95% credible interval 3.7 million–4.2 million) worldwide deaths, half of which occurred in east, southeast and south Asia. The global repositioning of lipid-related risk, with non-optimal cholesterol shifting from a distinct feature of high-income countries in northwestern Europe, north America and Australasia to one that affects countries in east and southeast Asia and Oceania should motivate the use of population-based policies and personal interventions to improve nutrition and enhance access to treatment throughout the world.
Effects of an artificial hay aroma and compound feed formulation on feed intake pattern, rumen function and milk production in lactating dairy cows
Binti Abd Rahim, Sholeha ; Laar, Harmen van; Dijkstra, J. ; Navarro-Villa, A. ; Fowers, R. ; Hendriks, W.H. ; Pellikaan, W.F. ; Leen, F. ; Martín-Tereso, J. - \ 2020
Animal 14 (2020)3. - ISSN 1751-7311 - p. 529 - 537.
The Kempen system is a dairy feeding system in which diet is provided in the form of a compound feed (CF) and hay offered ad libitum. Ad libitum access to CF and hay allows cows in this system to achieve a high DM intake (DMI). Out of physiological concerns, the voluntary hay intake could be increased and the consumption pattern of CF could be manipulated to maintain proper rumen functioning and health. This study investigated the effects of an artificial hay aroma and CF formulation on feed intake pattern, rumen function and milk production in mid- to late-lactating dairy cows. Twenty Holstein–Friesian cows were assigned to four treatments in a 4 × 4 Latin square design. Diet consisted of CF and grass hay (GH), fed separately, and both offered ad libitum, although CF supply was restricted in maximum meal size and speed of supply by an electronic system. Treatments were the combination of two CF formulations – high in starch (CHS) and fibre (CHF); and two GH – untreated (UGH) and the same hay treated with an artificial aroma (TGH). Meal criteria were determined using three-population Gaussian–Gaussian–Weibull density functions. No GH × CF interaction effects on feed intake pattern characteristics were found. Total DMI and CF intake, but not GH intake, were greater (P < 0.01) in TGH treatment, and feed intake was not affected by type of CF. Total visits to feeders per day, visits to the GH feeder, visits to the CF feeder and CF eating time (all P < 0.01) were significantly greater in cows fed with TGH. Meal frequency, meal size and meal duration were unaffected by treatments. Cows fed CHF had a greater milk fat (P = 0.02), milk urea content (P < 0.01) and a greater milk fat yield (P < 0.01). Cows fed TGH had a greater milk lactose content and lactose yield (P < 0.05), and milk urea content (P < 0.01). Cows fed TGH had smaller molar proportions of acetic acid and greater molar proportions of propionic acid compared with UGH. In conclusion, treatment of GH with an artificial aroma increased CF intake and total DMI, but did not affect hay intake. Additionally, GH treatment increased the frequency of visits to both feeders, and affected rumen volatile fatty acid profile. Type of CF did not affect meal patterns, ruminal pH, nor fermentation profiles.
Transcriptome and proteome analyses of proteases in biotroph fungal pathogen Cladosporium fulvum
Karimi Jashni, Mansoor ; Burgt, Ate van der; Battaglia, Evy ; Mehrabi, Rahim ; Collemare, Jérôme ; Wit, Pierre J.G.M. de - \ 2020
Journal of Plant Pathology: rivista di patologia vegetale 102 (2020). - ISSN 1125-4653 - p. 377 - 386.
Cladosoprium fulvum - Fungal proteases - Gene expression - Plant-microbe interaction - Tomato pathogen
Proteases are key components of the hydrolytic enzyme arsenal employed by fungal pathogens to invade their host plants. The recent advances in -omics era have facilitated identification of functional proteases involved in plant-fungus interactions. By comparison of the publically available sequences of fungal genomes we found that the number of protease genes present in the genome of Cladosporium fulvum, a biotrophic tomato pathogen, is comparable with that of hemibiotrophs. To identify host plant inducible protease genes and their products, we performed transcriptome and proteome analyses of C. fulvumin vitro and in planta by means of RNA-Seq/RT-qPCR and mass spectrometry. Transcriptome data showed that 14 out of the 59 predicted proteases are expressed during in vitro and in planta growth of C. fulvum, of which nine belong to serine proteases S8 and S10 and the rest belong to metallo- and aspartic proteases. Mass spectrometry confirmed the presence of six proteases at proteome level during plant infection. Expression of limited number of proteases by C. fulvum might sustain biotrophic growth and benefits its stealth pathogenesis.
|The impact of feed intake patterns on ruminal fermentation and milk production of dairy cattle
Binti Abd Rahim, Sholeha ; Laar, Harmen van; Martin-Tereso, J. ; Hendriks, W.H. ; Dijkstra, J. - \ 2019
In: Proceedings of the 44th Animal Nutrition Research Forum. - - p. 8 - 9.
|Potential underlying factors of subacute rumen acidosis: Effects of frequency and duration of feed availability on performance and milk fatty acid profile in dairy cows
Binti Abd Rahim, Sholeha ; Laar, Harmen van; Martín-Tereso, Javier ; Hendriks, W.H. ; Dijkstra, J. - \ 2019
In: Proceedings of the 17th International Conference on Production Disease in Farm Animals. - - p. 165 - 165.
|Potential underlying factors of subacute rumen acidosis: Effects of frequency and duration of feed availability on performance and milk fatty acid profile in dairy cows
Binti Abd Rahim, Sholeha - \ 2019
|The impact of feed intake patterns on ruminal fermentation and milk production of dairy cattle
Binti Abd Rahim, Sholeha - \ 2019
Author Correction: Phosphopantetheinyl transferase (Ppt)-mediated biosynthesis of lysine, but not siderophores or DHN melanin, is required for virulence of Zymoseptoria tritici on wheat
Derbyshire, Mark C. ; Gohari, Amir Mirzadi ; Mehrabi, Rahim ; Kilaru, Sreedhar ; Steinberg, Gero ; Ali, Solaf ; Bailey, Andy ; Hammond-Kosack, Kim ; Kema, Gert H.J. ; Rudd, Jason J. - \ 2019
Scientific Reports 9 (2019)1. - ISSN 2045-2322
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Phosphopantetheinyl transferase (Ppt)-mediated biosynthesis of lysine, but not siderophores or DHN melanin, is required for virulence of Zymoseptoria tritici on wheat
Derbyshire, Mark C. ; Gohari, Amir Mirzadi ; Mehrabi, Rahim ; Kilaru, Sreedhar ; Steinberg, Gero ; Ali, Solaf ; Bailey, Andy ; Hammond-Kosack, Kim ; Kema, Gert H.J. ; Rudd, Jason J. - \ 2018
Scientific Reports 8 (2018)1. - ISSN 2045-2322
Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB) disease of wheat. Z. tritici is an apoplastic fungal pathogen, which does not penetrate plant cells at any stage of infection, and has a long initial period of symptomless leaf colonisation. During this phase it is unclear to what extent the fungus can access host plant nutrients or communicate with plant cells. Several important primary and secondary metabolite pathways in fungi are regulated by the post-translational activator phosphopantetheinyl transferase (Ppt) which provides an essential co-factor for lysine biosynthesis and the activities of non-ribosomal peptide synthases (NRPS) and polyketide synthases (PKS). To investigate the relative importance of lysine biosynthesis, NRPS-based siderophore production and PKS-based DHN melanin biosynthesis, we generated deletion mutants of ZtPpt. The ∆ZtPpt strains were auxotrophic for lysine and iron, non-melanised and non-pathogenic on wheat. Deletion of the three target genes likely affected by ZtPpt loss of function (Aar- lysine; Nrps1-siderophore and Pks1- melanin), highlighted that lysine auxotrophy was the main contributing factor for loss of virulence, with no reduction caused by loss of siderophore production or melanisation. This reveals Ppt, and the lysine biosynthesis pathway, as potential targets for fungicides effective against Z. tritici.
Stress and sexual reproduction affect the dynamics of the wheat pathogen effector AvrStb6 and strobilurin resistance
Kema, Gerrit H.J. ; Mirzadi Gohari, Amir ; Aouini, Lamia ; Gibriel, Hesham A.Y. ; Ware, Sarah B. ; Den Bosch, Frank van; Manning-Smith, Robbie ; Alonso-Chavez, Vasthi ; Helps, Joe ; M’Barek, Sarrah Ben; Mehrabi, Rahim ; Diaz-Trujillo, Caucasella ; Zamani, Elham ; Schouten, Henk J. ; Lee, Theo A.J. van der; Waalwijk, Cees ; Waard, Maarten A. de; Wit, Pierre J.G.M. de; Verstappen, Els C.P. ; Thomma, Bart P.H.J. ; Meijer, Harold J.G. ; Seidl, Michael F. - \ 2018
Nature Genetics 50 (2018). - ISSN 1061-4036 - p. 375 - 380.
Host resistance and fungicide treatments are cornerstones of plant-disease control. Here, we show that these treatments allow sex and modulate parenthood in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that the Z. tritici–wheat interaction complies with the gene-for-gene model by identifying the effector AvrStb6, which is recognized by the wheat resistance protein Stb6. Recognition triggers host resistance, thus implying removal of avirulent strains from pathogen populations. However, Z. tritici crosses on wheat show that sex occurs even with an avirulent parent, and avirulence alleles are thereby retained in subsequent populations. Crossing fungicide-sensitive and fungicide-resistant isolates under fungicide pressure results in a rapid increase in resistance-allele frequency. Isolates under selection always act as male donors, and thus disease control modulates parenthood. Modeling these observations for agricultural and natural environments reveals extended durability of host resistance and rapid emergence of fungicide resistance. Therefore, fungal sex has major implications for disease control.
The ZtVf1 transcription factor regulates development and virulence in the foliar wheat pathogen Zymoseptoria tritici
Mohammadi, Naser ; Mehrabi, Rahim ; Mirzadi Gohari, Amir ; Mohammadi Goltapeh, Ebrahim ; Safaie, Naser ; Kema, Gert H.J. - \ 2017
Fungal Genetics and Biology 109 (2017). - ISSN 1087-1845 - p. 26 - 35.
Functional analysis - Fungal transformation - Gene deletion - Mycosphaerella graminicola - USER-friendly cloning
The dimorphic fungal pathogen, Zymoseptoria tritici undergoes discrete developmental changes to complete its life cycle on wheat. Molecular mechanisms underlying morphogenesis during infection process of Z. tritici are poorly understood. In this study, we have investigated the role of ZtVf1 gene encoding a transcription factor belonging to C2-H2 subfamily. In planta assays revealed that ZtVf1 is required for virulence. Reduced necrotic lesions and low pycnidia density within the lesions resulted in significantly reduced virulence of ZtVf1 mutants. Cytological analysis showed that the impaired virulence of ZtVf1 mutants attributed to reduced penetration and colonization along with hampered pycnidia differentiation. In vitro phenotyping showed that ZtVf1 deletion affects hyphal branching and biomass production suggesting that the reduced tissue colonization by the ZtVf1 mutant might be due to lower hyphal branching and less fungal biomass production. In addition, the majority of infected substomatal cavities by the ZtVf1 mutant filled with compacted mycelia mat that did not differentiate to mature pycnidia indicating that the impaired melanization negatively affected pycnidia formation and maturation. The ZtVf1 might target multiple genes belonging to different cellular processes whose identification is of eminent interest to increase our understanding of this pathosystem. Overall, the data provided in this study indicates that attenuated pathogenicity of ZtVf1 mutant is due to involvement of this gene in the regulation of both early and late stages of infection.
Karyotype Variability in Plant-Pathogenic Fungi
Mehrabi, Rahim ; Mirzadi Gohari, Amir ; Kema, Gert H.J. - \ 2017
Annual Review of Phytopathology 55 (2017). - ISSN 0066-4286 - p. 483 - 503.
Chromosomal rearrangement - Cytological karyotyping - Electrophoretic - Evolutionary adaptation - Genome evolution - Horizontal gene transfer - Polymorphisms
Recent advances in genetic and molecular technologies gradually paved the way for the transition from traditional fungal karyotyping to more comprehensive chromosome biology studies. Extensive chromosomal polymorphisms largely resulting from chromosomal rearrangements (CRs) are widely documented in fungal genomes. These extraordinary CRs in fungi generate substantial genome plasticity compared to other eukaryotic organisms. Here, we review the most recent findings on fungal CRs and their underlying mechanisms and discuss the functional consequences of CRs for adaptation, fungal evolution, host range, and pathogenicity of fungal plant pathogens in the context of chromosome biology. In addition to a complement of permanent chromosomes called core chromosomes, the genomes of many fungal pathogens comprise distinct unstable chromosomes called dispensable chromosomes (DCs) that also contribute to chromosome polymorphisms. Compared to the core chromosomes, the structural features of DCs usually differ for gene density, GC content, housekeeping genes, and recombination frequency. Despite their dispensability for normal growth and development, DCs have important biological roles with respect to pathogenicity in some fungi but not in others. Therefore, their evolutionary origin is also reviewed in relation to overall fungal physiology and pathogenicity.
Combating a Global Threat to a Clonal Crop : Banana Black Sigatoka Pathogen Pseudocercospora fijiensis (Synonym Mycosphaerella fijiensis) Genomes Reveal Clues for Disease Control
Arango Isaza, Rafael E. ; Diaz-Trujillo, Caucasella ; Dhillon, Braham ; Aerts, Andrea ; Carlier, Jean ; Crane, Charles F. ; V. de Jong, Tristan ; Vries, Ineke de; Dietrich, Robert ; Farmer, Andrew D. ; Fortes Fereira, Claudia ; Garcia, Suzana ; Guzman, Mauricio ; Hamelin, Richard C. ; Lindquist, Erika A. ; Mehrabi, Rahim ; Quiros, Olman ; Schmutz, Jeremy ; Shapiro, Harris ; Reynolds, Elizabeth ; Scalliet, Gabriel ; Souza, Manoel ; Stergiopoulos, Ioannis ; Lee, Theo A.J. van der; Wit, Pierre J.G.M. de; Zapater, Marie Françoise ; Zwiers, Lute Harm ; Grigoriev, Igor V. ; Goodwin, Stephen B. ; Kema, Gert H.J. - \ 2016
Plos Genetics 12 (2016)8. - ISSN 1553-7390
Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.
Flexible gateway constructs for functional analyses of genes in plant pathogenic fungi
Mehrabi, Rahim ; Mirzadi Gohari, Amir ; Silva, Gilvan Ferreira da; Steinberg, Gero ; Kema, Gert H.J. ; Wit, Pierre J.G.M. de - \ 2015
Fungal Genetics and Biology 79 (2015). - ISSN 1087-1845 - p. 186 - 192.
Binary vectors - Gateway technology - Gene deletion - GFP - RFP - Zymoseptoria tritici
Genetic manipulation of fungi requires quick, low-cost, efficient, high-throughput and molecular tools. In this paper, we report 22 entry constructs as new molecular tools based on the Gateway technology facilitating rapid construction of binary vectors that can be used for functional analysis of genes in fungi. The entry vectors for single, double or triple gene-deletion mutants were developed using hygromycin, geneticin and nourseothricin resistance genes as selection markers. Furthermore, entry vectors containing green fluorescent (GFP) or red fluorescent (RFP) in combination with hygromycin, geneticin or nourseothricin selection markers were generated. The latter vectors provide the possibility of gene deletion and simultaneous labelling of the fungal transformants with GFP or RFP reporter genes. The applicability of a number of entry vectors was validated in Zymoseptoria tritici, an important fungal wheat pathogen.
Corrigendum to "Proteome catalog of Zymoseptoria tritici captured during pathogenesis in wheat". [Fung. Genet. Biol. 79 (2015) 42-53]
Mbarek, Sarrah Ben; Cordewener, J.H.G. ; Lee, T.A.J. Van der; America, A.H.P. ; Gohari, Amir Mirzadi ; Mehrabi, Rahim ; Hamza, Sonia ; Wit, P.J.G.M. de; Kema, Gert - \ 2015
Fungal Genetics and Biology 82 (2015). - ISSN 1087-1845 - p. 291 - 291.
Identification and functional characterization of putative (a)virulence factors in the fungal wheat pathogen Zymoseptoria tritici
Mirzadi Gohari, A. - \ 2015
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Gert Kema; Rahim Mehrabi. - Wageningen : Wageningen University - ISBN 9789462575912 - 159
triticum aestivum - wheat - plant pathogenic fungi - mycosphaerella graminicola - virulence factors - genetic analysis - pathogenesis - bioinformatics - triticum aestivum - tarwe - plantenziekteverwekkende schimmels - mycosphaerella graminicola - virulente factoren - genetische analyse - pathogenese - bio-informatica
Zymoseptoria tritici (Desm.) Quaedvlieg & Crous (previously known as Mycosphaerella graminicola) is the causal agent of septoria tritici blotch (STB), which is a devastating foliar wheat disease worldwide. It is responsible for significant yield losses occurring annually in all major wheat-growing areas and threatens global food security. Z. tritici is a hemi-biotrophic fungal pathogen that, after stomatal penetration, establishes a stealthy biotrophic and symptomless relation with its host plant that is followed by a sudden switch to a necrotrophic growth phase coinciding with chlorosis that eventually develops in large necrotic blotches containing many pycnidia producing asexual splash-borne conidia. Under natural conditions - once competent mating partners are present and conditions are conducive- pseudothecia are formed producing airborne ascospores. Disease management of STB is primarily achieved through fungicide applications and growing commercial cultivars carrying Stb resistance genes. However, the efficacy of both strategies is limited as strains resistant to fungicides frequently develop and progressively dominate natural populations, which hampers disease management; also the deployed Stb genes are often overcome by existing or newly developed isolates of the fungus. Hence, there is a need for discovery research to better understand the molecular basis of the host-pathogen interaction that enables breeders to identify and deploy new Stb genes, which will eventually contribute to more sustainable disease control.
Chapter 1 introduces the subject of the thesis and describes various aspects of the lifestyle of Z. tritici with emphasis on dissecting the various stages and physiological processes during pathogenesis on wheat. In addition, it includes a short summary and discussion of the current understanding of the role of (a)virulence factors in the Z. tritici–wheat pathosystem.
Chapter 2 describes new gateway technology-driven molecular tools comprising 22 entry constructs facilitating rapid construction of binary vectors for functional analyses of fungal genes. The entry vectors for single, double or triple gene deletion mutants were developed using hygromycin, geneticin and nourseothricin resistance genes as selection markers. Furthermore, these entry vectors contain the genes encoding green fluorescent (GFP) or red fluorescent (RFP) protein in combination with the three selection markers, which enables simultaneous tagging of gene deletion mutants for microscopic analyses. The functionality of these entry vectors was validated in Z. tritici and described in Chapters 3, 4 and 5.
Chapter 3 describes the functional characterization of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans. ZtWor1 is up-regulated during initiation of colonization and fructification, and regulates expression of candidate effector genes, including one that was discovered after comparative proteome analysis of Z. tritici wild-type and ΔZtWor1 strains. Cell fusion and anastomosis occurred frequently in ΔZtWor1 strains, which is reminiscent of mutants of MgGpb1, the β-subunit of the heterotrimeric G protein. Comparative expression profiling of ΔZtWor1, ΔMgGpb1 and ΔMgTpk2 (the catalytic subunit of protein kinase A) strains, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenicity of many fungal plant pathogens.
Chapter 4 describes combined bioinformatics and expression profiling studies during pathogenesis in order to discover candidate effectors of Z. tritici important for virulence. In addition, a genetic approach was followed to map quantitative trait loci (QTLs) in Z. tritici carrying putative effectors. Functional analysis of two top effector candidates, small-secreted proteins SSP15 and SSP18, which were selected based on their expression profile in planta, showed that they are dispensable for virulence of Z. tritici. These analyses suggest that generally adopted criteria for effector discovery, such as protein size, number of cysteine residues and up-regulated expression during pathogenesis, should be taken with caution and cannot be applied to every pathosystem, as they likely represent only a subset of effector genes.
Chapter 5 describes the functional characterization of ZtCpx1 and ZtCpx2 encoding a secreted and a cytoplasmic catalase-peroxidase (CP) in Z. tritici, respectively. Gene replacement of ZtCpx1 resulted in mutant strains that were sensitive to exogenously added H2O2 and in planta phenotyping showed they are significantly less virulent compared to wild-type. All mutant phenotypes could be restored to wild-type by complementation with the wild-type allele of ZtCpx1 driven by its native promoter. Additionally, functional analysis of ZtCpx2 confirmed that this gene encodes a secreted CP and is, however, dispensable for virulence of Z. tritici on wheat. However, we showed that both genes act synergistically, as the generated double knock-out strain showed a significantly stronger reduction in virulence than the individual single knock-out strains. Hence, both genes are required by Z. tritici for successful infection and colonization of wheat.
In Chapter 6 I discuss and summarize the genetic approaches used in this study, reflect on the major findings and bottlenecks encountered, and propose new strategies to identify effectors of Z. tritici in the future.
Identification and functional characterization of proteases and protease inhibitors involved in virulence of fungal tomato pathogens
Karimi Jashni, M. - \ 2015
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jerome Collemare; Rahim Mehrabi. - Wageningen : Wageningen University - ISBN 9789462574571 - 183
passalora fulva - plantenziekteverwekkende schimmels - virulentie - proteïnasen - proteïnaseremmers - plant-microbe interacties - genomica - solanum lycopersicum - tomaten - eiwitexpressieanalyse - passalora fulva - plant pathogenic fungi - virulence - proteinases - proteinase inhibitors - plant-microbe interactions - genomics - solanum lycopersicum - tomatoes - proteomics
Pathogens cause disease on both animal and plant hosts. For successful infection and establishment of disease, pathogens need proper weaponry to protect themselves against host defenses and to promote host colonization to facilitate uptake of nutrients for growth and reproduction. Indeed, plant pathogens secrete various types of effector molecules (proteins and secondary metabolites) to manipulate host responses for their own needs. Secreted proteases and protease inhibitors (PIs) are such effector molecules. Proteases can hydrolyze plant defense proteins and PIs can inhibit plant proteases that are part of the host surveillance system. Despite the importance of proteases and PIs secreted by fungal pathogens, little information about their role in virulence is available. The recent advances in genomics, bioinformatics, transcriptomics and proteomics have facilitated identification and functional analysis of proteases and PIs relevant to plant-fungus interactions.
Chapter 1 is an introduction to the thesis outlining the general concept of plant-microbe interactions. It briefly describes the current knowledge of pathogenicity mechanisms employed by fungal plant pathogens and defense mechanisms employed by their host plants. It further introduces proteases and PIs and their potential role in modifying pathogenesis-related (PR) proteins to facilitate fungal virulence. It completes with an outline of the PhD research project.
In chapter 2, we analyzed and compared the number of putatively secreted proteases present in the genomes of 30 fungi with different lifestyles. The analysis showed that fungi with a saprotrophic and hemibiotrophic lifestyle contain more secreted protease genes than biotrophs. Surprisingly, the number of protease genes present in the genome of Cladosporium fulvum, a biotrophic tomato pathogen, is comparable with that of hemibiotrophs and saprotrophs. We analyzed all C. fulvum protease genes both at the transcriptome and proteome level by means of RNA-Seq/RT-qrtPCR and mass spectrometry analyses, respectively. Results showed that many proteases of C. fulvum are not expressed during growth in planta, likely sustaining the biotrophic growth pattern of this fungus.
In chapter 3, using an alignment-based gene prediction tool, we identified pseudogenes containing disruptive mutations (DMs) that likely lead to the production of nonfunctional proteins, including a group of putatively secreted proteases from C. fulvum. Fewer DMs were observed in other fungi including Dothistroma septosporum, a hemibiotrophic pine needle pathogen and close relative of C. fulvum, and suggested that the difference in pseudogenization of proteases between these two pathogens might in part explain their different lifestyle.
In chapter 4, we analyzed the tomato genome and identified 30 candidate chitinases genes, of which six encoded chitin binding domain (CBD)-containing chitinases. Transcriptome and proteome data were collected after inoculation of tomato with several fungal pathogens and allowed the identification of two CBD-chitinases (SlChi2 and SlChi13) with a putative role in protecting tomato against C. fulvum and F. oxysporum f. sp. lycopersici (F. oxysporum), respectively. Purified CBD-chitinases SlChi1, SlChi2, SlChi4 and SlChi13 were incubated with secreted protein extracts (SPEs) from seven fungal tomato pathogens and we could show that SPEs from F. oxysporum, Verticillium dahliae, and Botrytis cinerea modified SlChi1 and SlChi13. LC-MS/MS analysis revealed that incubation with SPE from F. oxysporum removed the N-terminal 37 and 49 amino acids, comprising part and complete CBD domain from SlChi1 and SlChi13, respectively. Removal of the CBD of SlChi1 and SlChi13 by SPE of F. oxysporum reduced the antifungal activity of the two chitinases. We identified a fungal metalloprotease (FoMep1) and a subtilisin serine protease (FoSep1) that synergistically cleaved both SlChi1 and SlChi13. Transgenic F. oxysporum in which the genes encoding these two proteases were knocked out by homologous recombination lost the ability to cleave the two chitinases and were compromised in virulence on tomato compared to the parental wild type. These results suggest an important role of the two chitinases in defense of tomato against this pathogen.
In chapter 5, we searched for host target(s) of the apoplastic effector Avr9 secreted by C. fulvum during infection of tomato. Based on the structural homology of Avr9 with carboxy peptidase inhibitors, we hypothesized that the host target of Avr9 might be apoplastic proteases. To isolate and identify Avr9 targets in apoplastic fluids, we used synthetic biotinylated Avr9, and performed pull-down and far-western blotting assays with apoplastic fluids from tomato inoculated with a C. fulvum race lacking the Avr9 gene. However, we found no specific Avr9-interacting proteins from pull-down complexes analyzed by mass spectrometry or by far-western blotting. Then, we hypothesized that glycosylation of Avr9 might be required for its biological function. The results of mass spectrometry analysis revealed that Avr9 is N-glycosylated when secreted by C. fulvum, containing at least two GlcNac and six mannose residues. The necrosis-inducing activity of glycosylated and non-glycosylated Avr9 was assayed but appeared not significantly different; however, we could not produce sufficient amounts of (biotinylated)-glycosylated Avr9 to perform pull-down assays for identification of potential glycosylated Arv9-interacting proteins by mass spectrometry.
Previous studies as well as the results present in this PhD thesis showed that fungal pathogens secrete a plethora of effectors including proteases and PIs. Many of identified proteases and PIs mediate effector-triggered immunity in host plants. In chapter 6, we reviewed the recent advances on the various roles of proteases and PIs in compromising basal defense responses induced by microbe-associated molecular patterns.
Chapter 7 is a summarizing discussion of the PhD thesis. We showed determinative roles of proteases and PIs in shaping plant-pathogen interactions. The expression and pseudogenization studies on proteases of C. fulvum showed that the genome content does not necessarily reflect the lifestyle of this fungus. This is true for many classes of fungal genes, including proteases. Fungi contain many different types of proteases whose functions may partly overlap. This hampers the discovery of their biological functions. We could demonstrate that two different types of proteases (metalloprotease (FoMep1) and subtilisin serine protease (FoSep1)) of F. oxysporum act synergistically to modify and reduce antifungal activity of two plant CBD-chitinases. Identifying additional proteases is achievable by a targeted proteomics approach using known targets as we did in chapter 4. However, identification of biological functions of proteases is a technical challenge when targets are not known. Multi-gene targeting of protease and PI genes is required to reveal their function in plant-pathogen interactions, which can only be addressed by using advanced genetic tools in future research.
Schimmels zijn genetische wildebrassen (interview met Gert Kema)
Mehrabi, R. ; Wit, P. de; Kema, G.H.J. - \ 2011
Gewasbescherming 42 (2011)2 (april 2011). - ISSN 0166-6495 - p. Nieuws - 95.
Pathogene schimmels doen spontaan waar organisaties als Greenpeace van gruwen: ze wisselen genen of zelfs hele chromosomen uit met andere schimmelsoorten. Dat blijkt uit een artikel in FEMS Microbiology Reviews van de Wageningse genetici Rahim Mehrabi, Pierre de Wit en Gert Kema van Wageningen UR. Jarenlang leidde de schimmelsoort Pyrenophora triticirepentis een voor tarwe onschuldig bestaan. Maar rond de Tweede Wereldoorlog tastte de schimmel opeens tarwe aan, net als de pathogene schimmelsoort Stagonospora nodorum. Toen genetici onlangs de genoomsequenties van beide schimmels gingen vergelijken, bleek er een ‘eilandje’ van Stagonospora-DNA in het genoom van Pyrenophora te zitten. Ook bleek dat dit pakketje genen in schimmels rond 1940 aanwezig was, maar daarvoor niet. Het is dus erg aannemelijk dat P. tritici-repentis een pathogene tarweschimmel werd na DNA-uitwisseling met S. nodorum.
|Pathogenen wisselen genen uit. Uitwisseling genen kan leiden tot agressievere schimmels (interview met Gert Kema)
Mehrabi, R. ; Wit, P. de; Kema, G.H.J. - \ 2011
Harderwijk : ProGrass (Professional Grass Service)
Pathogene schimmels wisselen genen of zelfs hele chromosomen uit met andere schimmelsoorten. Dat blijkt uit een artikel in FEMS Microbiology Reviews van de Wageningse genetici Rahim Mehrabi, Pierre de Wit en Gert Kema. Het kan leiden tot nieuwe of veel agressievere schimmels. "Dit soort processen zijn niet te voorkomen', zegt onderzoeker Kema. "Het is belangrijk alert te blijven en te onderzoeken op welke manier zo'n nieuwe schimmel aangepakt kan worden en de verspreiding ervan kan worden voorkomen."