Abstract
Among phytohormones, cytokinins (CKs) play an important role in controlling crucial aspects of plant development. Not only plants but also diverse microorganisms are able to produce phytohormones, including CKs, though knowledge concerning their biosynthesis and metabolism is still limited. In this work we demonstrate that the fungus Leptosphaeria maculans, a hemi-biotrophic pathogen of oilseed rape (Brassica napus), causing one of the most damaging diseases of this crop, is able to modify the CK profile in infected B. napus tissues, as well as produce a wide range of CKs in vitro, with the cis-zeatin derivatives predominating. The endogenous CK spectrum of L. maculans in vitro consists mainly of free CK bases, as opposed to plants, where other CK forms are mostly more abundant. Using functional genomics, enzymatic and feeding assays with CK bases supplied to culture media, we show that L. maculans contains a functional: (i) isopentenyltransferase (IPT) involved in cZ production; (ii) adenosine kinase (AK) involved in phosphorylation of CK ribosides to nucleotides; and (iii) CK-degradation enzyme cytokinin oxidase/dehydrogenase (CKX). Our data further indicate the presence of cis–trans isomerase, zeatin O-glucosyltransferase(s) and N6-(Δ2-isopentenyl)adenine hydroxylating enzyme. Besides, we report on a crucial role of LmAK for L. maculans fitness and virulence. Altogether, in this study we characterize in detail the CK metabolism of the filamentous fungi L. maculans and report its two novel components, the CKX and CK-related AK activities, according to our knowledge for the first time in the fungal kingdom. Based on these findings, we propose a model illustrating CK metabolism pathways in L. maculans.
Highlights
Phytohormones play essential roles in coordination of plant growth, development and stress responses
Using functional genomics and feeding assays, we show that L. maculans contains functional IPT and adenosine kinase (AK) genes, encoding enzymes that catalyze the formation of cZ and the phosphorylation of CK ribosides into nucleotides, respectively
We have shown that L. maculans produces a wide spectrum of CKs in vitro, present both in the mycelium (Figure 1) and culture medium (Supplementary Figure 1), as well as in planta upon infection in B. napus (Table 1)
Summary
Phytohormones play essential roles in coordination of plant growth, development and stress responses. CKs primarily control cell growth and differentiation; regulate apical dominance, axillary bud growth, leaf senescence, seed dormancy or nutritional signaling. They are involved in responses to different environmental stimuli, such as biotic or abiotic stresses (Sakakibara, 2006). The first and rate-limiting step in biosynthesis of isoprenoid CKs consists of the attachment of an isoprenoid side chain to the adenine nucleotide catalyzed by isopentenyltransferases (IPTs). Plants use two different types of IPTs, depending on the adenine-containing substrate. Adenylate IPTs exist in multiple copies in all flowering plants and are mainly responsible for the synthesis of iP- and tZtype CKs (Miyawaki et al, 2006). The activation of nucleotides to free CK bases is achieved by a one-step reaction catalyzed by LONELY GUY (LOG) (Kurakawa et al, 2007), or by a two-step reaction catalyzed by 5 -ribonucleotide phosphohydrolase and adenosine nucleosidase (Sakakibara, 2006)
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