Pathogen-derived cytokinins (CKs) have been recognized as important virulence factor in several host-pathogen interactions and it was demonstrated multiple times that phytopathogenic fungi form CKs via the tRNA degradation pathway. In contrast to previous studies, the focus of this study is on the second step of CK formation and CK degradation to improve our understanding of the biosynthesis in fungi on the one hand, and to understand CK contribution to the infection process of Claviceps purpurea on the other hand. The ergot fungus Claviceps purpurea is a biotrophic phytopathogen with a broad host range including economically important crops causing harvest intoxication upon infection. Its infection process is restricted to unfertilized ovaries without causing macroscopic defense symptoms. Thus, sophisticated host manipulation strategies are implicated. The cytokinin (CK) plant hormones are known to regulate diverse plant cell processes, and several plant pathogens alter CK levels during infection. C. purpurea synthesizes CKs via two mechanisms, and fungus-derived CKs influence the host-pathogen interaction but not fungus itself. CK deficiency in fungi with impact on virulence has only been achieved to date by deletion of a tRNA-ipt gene that is also involved in a process of translation regulation. To obtain a better understanding of CK biosynthesis and CKs' contribution to the plant-fungus interaction, we applied multiple approaches to generate strains with altered or depleted CK content. The first approach is based on deletion of the two CK phosphoribohydrolase (LOG)-encoding genes, which are believed to be essential for the release of active CKs. Single and double deletion strains were able to produce all types of CKs. Apparently, log gene products are dispensable for the formation of CKs and so alternative activation pathways must be present. The CK biosynthesis pathway remains unaffected in the second approach, because it is based on heterologous overexpression of CK-degrading enzymes from maize (ZmCKX1). Zmckx1 overexpressing C. purpurea strains shows strong CKX activity and drastically reduced CK levels. The strains are impaired in virulence, which reinforces the assumption that fungal-derived CKs are crucial for full virulence. Taken together, this study comprises the first analysis of a log depletion mutant that proved the presence of alternative cytokinin activation pathways in fungi and showed that heterologous CKX expression is a suitable approach for CK level reduction.
Disease symptoms of some phytopathogenic fungi are associated with changes in cytokinin (CK) levels. Here, we show that the CK profile of ergot-infected rye plants is also altered, although no pronounced changes occur in the expression of the host plant's CK biosynthesis genes. Instead, we demonstrate a clearly different mechanism: we report on the first fungal de novo CK biosynthesis genes, prove their functions and constitute a biosynthetic pathway. The ergot fungus Claviceps purpurea produces substantial quantities of CKs in culture and, like plants, expresses enzymes containing the isopentenyltransferase and lonely guy domains necessary for de novo isopentenyladenine production. Uniquely, two of these domains are combined in one bifunctional enzyme, CpIPT-LOG, depicting a novel and potent mechanism for CK production. The fungus also forms trans-zeatin, a reaction catalysed by a CK-specific cytochrome P450 monooxygenase, which is encoded by cpp450 forming a small cluster with cpipt-log. Deletion of cpipt-log and cpp450 did not affect virulence of the fungus, but Δcpp450 mutants exhibit a hyper-sporulating phenotype, implying that CKs are environmental factors influencing fungal development.
- MeSH
- alkyltransferasy a aryltransferasy metabolismus MeSH
- Claviceps genetika růst a vývoj metabolismus MeSH
- cytokininy biosyntéza MeSH
- delece genu MeSH
- geny hub genetika MeSH
- isopentenyladenosin biosyntéza MeSH
- rostlinné geny genetika MeSH
- systém (enzymů) cytochromů P-450 genetika MeSH
- žito mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ergot alkaloids produced by the fungus Claviceps parasitizing on cereals, include three major groups: clavine alkaloids, d-lysergic acid and its derivatives and ergopeptines. These alkaloids are important substances for the pharmatech industry, where they are used for production of anti-migraine drugs, uterotonics, prolactin inhibitors, anti-Parkinson agents, etc. Production of ergot alkaloids is based either on traditional field cultivation of ergot-infected rye or on submerged cultures of the fungus in industrial fermentation plants. In 2010, the total production of these alkaloids in the world was about 20,000 kg, of which field cultivation contributed about 50%. This review covers the recent advances in understanding of the genetics and regulation of biosynthesis of ergot alkaloids, focusing on possible applications of the new knowledge to improve the production yield.
- MeSH
- Claviceps genetika metabolismus MeSH
- enzymy metabolismus MeSH
- fermentace MeSH
- genetické inženýrství metody MeSH
- molekulární struktura MeSH
- námelové alkaloidy biosyntéza chemie genetika farmakologie MeSH
- průmyslová mikrobiologie metody MeSH
- žito mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
