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.
- Klíčová slova
- Claviceps purpurea, Cytokinin, Cytokinin oxidase/dehydrogenase, Phosphoribohydrolase, Virulence,
- MeSH
- Claviceps patogenita fyziologie MeSH
- cytokininy metabolismus MeSH
- interakce hostitele a patogenu fyziologie MeSH
- nemoci rostlin mikrobiologie MeSH
- žito * genetika metabolismus mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cytokininy MeSH
In plants, cytokinins (CKs) are synthesized de novo or by the degradation of modified tRNAs. Recently, the first fungal de novo pathway was identified within the plant pathogen Claviceps purpurea. As the deletion of the de novo pathway did not lead to a complete loss of CKs, this work focuses on the tRNA-modifying protein tRNA-isopentenyltransferase (CptRNA-IPT). The contribution of this enzyme to the CK pool of Claviceps and the role of CKs in the host-pathogen interaction are emphasized. The effects of the deletion of cptRNA-ipt and the double deletion of cptRNA-ipt and the key gene of de novo biosynthesis cpipt-log on growth, CK biosynthesis and virulence were analyzed. In addition, the sites of action of CptRNA-IPT were visualized using reporter gene fusions. In addition to CK-independent functions, CptRNA-IPT was essential for the biosynthesis of cis-zeatin (cZ) and contributed to the formation of isopentenyladenine (iP) and trans-zeatin (tZ). Although ΔcptRNA-ipt was reduced in virulence, the 'CK-free' double deletion mutant was nearly apathogenic. The results prove a redundancy of the CK biosynthesis pathway in C. purpurea for iP and tZ formation. Moreover, we show, for the first time, that CKs are required for the successful establishment of a host-fungus interaction.
- Klíčová slova
- Claviceps purpurea, cytokinin (CK) biosynthesis, host-pathogen interaction, tRNA degradation pathway, tRNA-isopentenyltransferase (tRNA-IPT),
- MeSH
- alkyltransferasy a aryltransferasy metabolismus MeSH
- biotest MeSH
- Claviceps enzymologie patogenita MeSH
- cytokininy metabolismus MeSH
- delece genu MeSH
- fungální léková rezistence účinky léků MeSH
- izoenzymy metabolismus MeSH
- mycelium metabolismus MeSH
- průmyslové fungicidy farmakologie MeSH
- RNA transferová metabolismus MeSH
- substrátová specifita účinky léků MeSH
- virulence účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- alkyltransferasy a aryltransferasy MeSH
- cytokininy MeSH
- izoenzymy MeSH
- průmyslové fungicidy MeSH
- RNA transferová MeSH
- tRNA isopentenyltransferase MeSH Prohlížeč
Oligosaccharides produced by submerged cultures of C. africana and C. sorghi were isolated by semipreparative HPLC. Structure of 6-O-beta-D-fructofuranosyl-D-glucopyranose (blastose), 1,6-bis-O-(beta-D-fructofuranosyl)-alpha-D-glucopyranoside (neokestose) and two sugar alcohols, 1-O-beta-D-fructofuranosyl-D-mannitol (fructosylmannitol) and 1,6-bis-O-(beta-D-fructofuranosyl)-D-mannitol (bisfructosylmannitol) was determined by NMR spectrometry. MALDI TOF MS analysis revealed molecular ions [M+Na]+ that indicate the presence of other tetra- and pentasaccharides (m/z = 689.4 and 851.5, respectively) and corresponding sugar alcohol (m/z = 691.4). Rapid conversion of sucrose into series of oligosaccharides and corresponding sugar alcohols was observed in all tested strains.
- MeSH
- Claviceps růst a vývoj izolace a purifikace metabolismus patogenita MeSH
- cukerné alkoholy chemie metabolismus MeSH
- fermentace MeSH
- molekulární struktura MeSH
- nemoci rostlin mikrobiologie MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- oligosacharidy biosyntéza chemie izolace a purifikace MeSH
- Sorghum mikrobiologie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cukerné alkoholy MeSH
- oligosacharidy MeSH
