Functional characterization of the first filamentous fungal tRNA-isopentenyltransferase and its role in the virulence of Claviceps purpurea.

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.

Detection of phytohormones in temperate forest fungi predicts consistent abscisic acid production and a common pathway for cytokinin biosynthesis.

The phytohormones, abscisic acid and cytokinin, once were thought to be present uniquely in plants, but increasing evidence suggests that these hormones are present in a wide variety of organisms. Few studies have examined fungi for the presence of these "plant" hormones or addressed whether their levels differ based on the nutrition mode of the fungus. This study examined 20 temperate forest fungi of differing nutritional modes (ectomycorrhizal, wood-rotting, saprotrophic). Abscisic acid and cytokinin were present in all fungi sampled; this indicated that the sampled fungi have the capacity to synthesize these two classes of phytohormones. Of the 27 cytokinins analyzed by HPLC-ESI MS/MS, seven were present in all fungi sampled. This suggested the existence of a common cytokinin metabolic pathway in fungi that does not vary among different nutritional modes. Predictions regarding the source of isopentenyl, cis-zeatin and methylthiol CK production stemming from the tRNA degradation pathway among fungi are discussed.

The Origins and Roles of Methylthiolated Cytokinins: Evidence From Among Life Kingdoms.

Cytokinins (CKs) are a group of adenine-derived, small signaling molecules of crucial importance for growth and multiple developmental processes in plants. Biological roles of classical CKs: isopentenyladenine (iP), trans and cis isomers of zeatin (tZ, cZ), and dihydrozeatin, have been studied extensively and their functions are well defined in many aspects of plant physiology. In parallel, extensive knowledge exists for genes involved in tRNA modifications that lead to the production of tRNA-bound methylthiolated CKs, especially in bacterial and mammalian systems. However, not much is known about the origins, fates, and possible functions of the unbound methylthiolated CKs (2MeS-CKs) in biological systems. 2MeS-CKs are the free base or riboside derivatives of iP or Z-type CKs, modified by the addition of a thiol group (-SH) at position 2 of the adenine ring that is subsequently methylated. Based on the evidence to date, these distinctive CK conjugates are derived exclusively via the tRNA degradation pathway. This review summarizes the knowledge on the probable steps involved in the biosynthesis of unbound 2MeS-CKs across diverse kingdoms of life. Furthermore, it provides examples of CK profiles of organisms from which the presence of 2MeS-CKs have been detected and confirms a close association and balance between the production of classical CKs and 2MeS-CKs. Finally, it discusses available reports regarding the possible physiological functions of 2MeS-CKs in different biological systems.

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase-coated magnetic beads: A key step toward codon emancipation.

Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate-based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase-coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real-time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1401-1407, 2017.

mTOR regulates the nucleoplasmic diffusion of Xrn2 under conditions of heat stress.

Stress induces various responses, including translational suppression and tRNA degradation in mammals. Previously, we showed that heat stress induces degradation of initiator tRNA(Met) (iMet) through 5'-3' exoribonuclease Xrn1 and Xrn2, respectively. In addition, we found that rapamycin inhibits the degradation of iMet under heat stress conditions. Here, we report that the mammalian target of rapamycin (mTOR) regulates the diffusion of Xrn2 from the nucleolus to the nucleoplasm, facilitating the degradation of iMet under conditions of heat stress. Our results suggest a mechanism of translational suppression through mTOR-regulated iMet degradation in mammalian cells.