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.

• Keywords
• Claviceps purpurea, Cytokinin, Cytokinin oxidase/dehydrogenase, Phosphoribohydrolase, Virulence,
• MeSH
• Claviceps pathogenicity   physiology   MeSH
• Cytokinins metabolism   MeSH
• Host-Pathogen Interactions physiology   MeSH
• Plant Diseases microbiology   MeSH
• Secale * genetics   metabolism   microbiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytokinins MeSH

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

• MeSH
• Biological Evolution MeSH
• Cytokinins metabolism   MeSH
• Escherichia coli enzymology   growth & development   MeSH
• Phylogeny MeSH
• Plants, Genetically Modified genetics   growth & development   metabolism   MeSH
• Molecular Sequence Data MeSH
• Mutation genetics   MeSH
• Mutagenesis, Site-Directed MeSH
• Nostoc enzymology   genetics   MeSH
• Oxidoreductases genetics   metabolism   MeSH
• Dimethylallyltranstransferase genetics   metabolism   MeSH
• Gene Expression Regulation, Enzymologic MeSH
• Recombinant Proteins metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Nicotiana enzymology   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• cytokinin oxidase MeSH Browser
• Cytokinins MeSH
• Oxidoreductases MeSH
• Dimethylallyltranstransferase MeSH
• Recombinant Proteins MeSH