Signaling pathways involved in pathogenicity and development of the fungal wheat pathogen Mycosphaerella graminicola
Mycosphaerella graminicola(Fuckel) J. Schröt is the causal agent of septoria tritici leaf blotch, which is the major foliar wheat disease in most temperate areas.Direct losses and the cost of control strategies contribute to the huge economical importance of this disease. M. graminicolais a dimorphic fungal pathogen that grows in yeast-like or filamentous fashion depending on the environmental conditions. Filamentation is required for pathogenicity of this non-appressorium forming pathogen, which has a hemibiotrophic lifestyle characterized by an initial biotrophic phase (about 10 days) that is followed by a necrotrophic phase during which numerous asexual and eventually sexual fructifications are produced. This thesis is part of an ongoing research program aimed at understanding the genetic and molecular control of pathogenicity. The research was initiated with the generation of EST and genomic sequence data of the pathogen. This thesis describes the functional analyses of 10 genes encoding proteins involved in signal transduction pathways operating in M. graminicola when grown in vitro and in planta . Chapter one gives an introduction to the research topic. In chapter two, the role of the mitogen-activated protein kinase (MAPK)-encoding gene, MgFus3 , the ortholog of Fus3 controlling mating in Saccharomyces cerevisiae , is described. Disruption of this gene prevented melanization of mycelia and formation of pycnidia in vitro. MgFus3 mutants are non-pathogenic. This is ascribed to impaired penetration of stomata, possibly due to inability of the mutants to recognize stomata. Chapter threedescribes the MAPK-encoding gene, MgSlt2 . This is the ortholog of Slt2 in S. cerevisiae that regulates cell wall biosynthesis. In M. graminicola , MgSlt2 plays a role in cell wall integrity since MgSlt2 mutants were affected in polarized growth and showedprogressive autolysis during aging. They were also hypersensitive to glucanase and several fungicides and did not produce aerial mycelium or melanin on potato dextrose agar (PDA). Pathogenicity assays revealed that virulence of MgSlt2 mutants was severely reduced. Electron microscopy and histopathological analyses showed that MgSlt2 mutants penetrated wheat stomata regularly, but were unable to establish invasive growth and did not produce asexual fructifications. BecauseMgSlt2 is involved in cell wall integrity, MgSlt2 mutants are probably more sensitive to hitherto unknown plant defense compounds, which might explain the compromised colonization of mesophyll tissue.Chapter fourdescribes characterization of MAPK-encoding gene, MgHog1 , and the p21-activated kinase (PAK)-encoding gene, MgSte20 .MgHog1 mutants were osmosensitive, highly resistant to the fungicides fludioxonil, fenpiclonil and iprodione did not melanize and were unable to switch from yeast-like to filamentous growth. As a result of the impaired dimorphic switch, MgHog1 mutants were unable to establish infectious germ tubes and therefore failed to penetrate wheat leaves. This demonstrates that dimorphic transition is a key factor in pathogenicity of M. graminicola. Phenotypes of MgSte20 mutants were identical to the wild-type isolate in all tested conditions.Fructification of M. graminicola is a complex process requiring differentiation of the infectious hyphae in the substomatal cavities during the latter stages of infection. In Chapter five, functional analyses of genes encoding the catalytic ( MgTpk2 ) and the regulatory ( MgBcy1 ) subunits of PKA showed that these genes are essential for asexual fructification. MgTpk2 and MgBcy1 mutants were able to germinate, penetrate and colonize mesophyll tissue, but were unable to differentiate pycnidia. Our data provide evidence that the cAMP pathway regulates filamentation through MgTpk2 and MgBcy1 . Disruption of MgTpk2 impaired filamentation. In addition, the MgTpk2 mutants became melanized faster and secreted a dark-brown pigment into yeast glucose broth medium (YGB), whereas MgBcy1 mutants showed delayed melanization on PDA and were osmosensitive. Overall, the divergent functions of the regulatory and the catalytic subunits of PKA indicate that proper regulation of PKA activity is required for various physiological processes including differentiation, filamentation, osmoregulation and melanization. Chapter sixdescribes the characterization of three Gaprotein-encoding genes ( MgGpa1, MgGpa2 and MgGpa3 ) and one Gbprotein-encoding gene ( MgGpb1 ) in M. graminicola . Phylogenetic comparisons and sequence analyses of Gaproteins of M. graminicola revealed that MgGPA1 and MgGPA3 can be categorized as homologs of the mammalian Gaiand Gasfamilies, respectively, whereas MgGPA2 is unrelated to mammalian Gaproteins. MgGpa1 , MgGpa3 and MgGpb1 mutants exhibited different phenotypes when grown on PDA at 20 °C that were not observed when the temperature was raised to 28 °C. Melanization did not occur in the MgGpa1 and MgGpb1 mutants, and the former formed fluffy mycelia in YGB and hardly produced spores. MgGpb1 mutants showed a nested type of growth on PDA that resulted from hampered filamentation, numerous cell fusions and increased anastomosis. Therefore, we concluded that MgGpa1 negatively regulates filamentation, which is positively regulated by MgGpa3 and MgGpb1 . Interestingly, unlike the response of the MgGpa1 mutants, exogenous cAMP restored the phenotype of the wild-type in the MgGpb1 and MgGpa3 mutants, indicating a stimulating function for MgGPB1 and MgGPA3 and an inhibitory function for MgGPA1 in the regulation of the cAMP pathway. Pathogenicity assays revealed that MgGpa1 , MgGpa3 and MgGpb1 are required for virulence of M. graminicola whereas MgGpa2 is dispensable.Finally, in chapterseven the results described in this thesis are discussed in a broader perspective. The roles of the various kinases as well as G proteins during development in vitro and in planta for fungal pathogenesis in general and for M. graminicola in particular are highlighted. Also, the potential of M. graminicola as a new model fungus is addressed. The power of comparative genomics can be fully exploited once the genomic sequence of the fungus is available.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Subjects: | gene expression, genetic analysis, mycosphaerella graminicola, pathogenicity, regulatory genes, septoria, signal transduction, triticum aestivum, wheat, genetische analyse, genexpressie, pathogeniteit, regulator-genen, signaaltransductie, tarwe, |
| Online Access: | https://research.wur.nl/en/publications/signaling-pathways-involved-in-pathogenicity-and-development-of-t |
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