Fungi of the recently defined order Sebacinales (Basidiomycota) are involved in a wide spectrum of mutualistic symbioses (including mycorrhizae) with various plants, thereby exhibiting a unique potential for biocontrol strategies. The axenically cultivable root endophyte Piriformospora indica is a model organism of this fungal order. It is able to increase biomass and grain yield of crop plants. In barley, the endophyte induces local and systemic resistance to fungal diseases and to abiotic stress. To elucidate the lifestyle of P. indica, we analyzed its symbiotic interaction and endophytic development in barley roots. We found that fungal colonization increases with root tissue maturation. The root tip meristem showed no colonization, and the elongation zone showed mainly intercellular colonization. In contrast, the differentiation zone was heavily infested by inter-and intracellular hyphae and intracellular chlamydospores. The majority of hyphae were present in dead rhizodermal and cortical cells that became completely filled with chlamydospores. In some cases, hyphae penetrated cells and built a meshwork around plasmolyzed protoplasts, suggesting that the fungus either actively kills cells or senses cells undergoing endogenous programmed cell death. Seven days after inoculation, expression of barley BAX inhibitor-1 (HvBI-1), a gene capable of inhibiting plant cell death, was attenuated. Consistently, fungal proliferation was strongly inhibited in transgenic barley overexpressing GFP-tagged HvBI-1, which shows that P. indica requires host cell death for proliferation in differentiated barley roots. We suggest that the endophyte interferes with the host cell death program to form a mutualistic interaction with plants.biodiversity ͉ mycorrhiza ͉ rhizosphere ͉ Sebacinales ͉ systemic resistance

Cyclin-dependent protein kinase 5 (cdk5), a member of the cdk family, is active mainly in postmitotic cells and plays important roles in neuronal development and migration, neurite outgrowth, and synaptic transmission. In this study we investigated the relationship between cdk5 activity and regulation of the mitogen-activated protein (MAP) kinase pathway. We report that cdk5 phosphorylates the MAP kinase kinase-1 (MEK1) in vivo as well as the Ras-activated MEK1 in vitro. The phosphorylation of MEK1 by cdk5 resulted in inhibition of MEK1 catalytic activity and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In p35 (cdk5 activator) ؊/؊ mice, which lack appreciable cdk5 activity, we observed an increase in the phosphoryla- 1 is a member of the cyclin-dependent protein kinase family (cdc2, CDC28, and other generically cyclin-dependent CDKs). Although cdk5 binds to cyclin D, its activity is not regulated by cyclins and there is little evidence that cdk5 is involved in the progression of the cell cycle (for review see Ref.1; see also Refs. 2 and 3). cdk5 is active mainly in post-mitotic cells such as neurons (4, 5), retinal cells (6), and muscle cells (7), where its activators p35 (or its truncated form p25) (4, 5) and p39 (8 -11) are specifically expressed. cdk5 has been suggested to play important roles in neurite outgrowth (12, 13), neuronal migration (14 -16), dopamine signaling in the striatum (17), exocytosis (18 -21), differentiation of muscle cells (7), and organization of acetylcholine receptors at the neuromuscular junction (22). Although neuronal cytoskeletal proteins were initially identified as the major target substrates (4, 23, 24), the number of cdk5 substrates has expanded considerably (see Table I in Ref. 25). These include DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein involved in dopamine signaling (17), NUDEL (a murine homolog of the Aspergillus nidulans nuclear migration mutant NudE), a protein involved in neuronal migration and axon transport (26), and other proteins involved in cross-talk between protein kinases and phosphatases (27). cdk5 also modulates protein kinase reactions such as the small GTPase-Rac dependent phosphorylation of p21-activated kinase, which results in modification of the actin cytoskeleton (28). By virtue of phosphorylating these diverse substrates, cdk5 plays a multifunctional role in the nervous system. It has been demonstrated that the absence of cdk5 in cdk5 Ϫ/Ϫ mice results in embryonic lethality (16). Although the p35 knockout mice survive longer (14), both cdk5 Ϫ/Ϫ and p35 Ϫ/Ϫ mice exhibit similar defects in cortical neuronal migration and affect the development of the nervous system (14 -16). We observed that in cdk5 Ϫ/Ϫ mice brain stem neurons showed ballooning and hyperphosphorylation of cytoskeletal proteins as detected by the SMI31 antibody (see Fig. 1). Similar observations were obtained from p35 (Ϫ/Ϫ) mice. 2 The antibody cross-reacts with phosphorylated Lys-Ser-Pro (KSP) motifs in neurofilament proteins, tau, and MAPs ...

Because of their beneficial impact on plants, the highly diverse mycorrhizal fungi grouped in the order Sebacinales lay claim to high ecological and agricultural significance. Here, we describe for the first time associations of Sebacinoid members with bacteria. Using quantitative PCR, denaturating gradient gel electrophoresis and fluorescence in situ hybridization, we detected an intimate association between Piriformospora indica and Rhizobium radiobacter, an alpha-Proteobacterium. The stability of the association, vertical transmission of the bacteria during asexual fungal reproduction and fungal plant colonization was monitored using R. radiobacter-specific primers. Treatment of mycelium or fungal protoplasts with antibiotics highly efficient against the free bacteria failed to cure the fungus. Barley seedlings dip-inoculated with R. radiobacter showed growth promotion and systemic resistance to the powdery mildew fungus Blumeria graminis comparable to P. indica inoculation. By screening additional isolates of the Sebacina vermifera complex, three species-specific associations with bacteria from the genera Paenibacillus, Acinetobacter and Rhodococcus were found. These findings suggest that Sebacinales species regularly undergo complex interactions involving host plants and bacteria reminiscent of other ectomycorrhizal and endomycorrhizal associations.

SummaryBeneficial effects elicited by the root endophyte Piriformospora indica are widely known, but the mechanism by which these are achieved is still unclear. It is proposed that phytohormones produced by the fungal symbiont play a crucial role in the interaction with the plant roots.Biochemical analyses of the underlying biosynthetic pathways for auxin production have shown that, on tryptophan feeding, P. indica can produce the phytohormones indole-3-acetic acid (IAA) and indole-3-lactate (ILA) through the intermediate indole-3-pyruvic acid (IPA). Time course transcriptional analyses after exposure to tryptophan designated the piTam1 gene as a key player. A green fluorescence protein (GFP) reporter study and transcriptional analysis of colonized barley roots showed that piTam1 is induced during the biotrophic phase.Piriformospora indica strains in which the piTam1 gene was silenced via an RNA interference (RNAi) approach were compromised in IAA and ILA production and displayed reduced colonization of barley (Hordeum vulgare) roots in the biotrophic phase, but the elicitation of growth promotion was not affected compared with the wild-type situation.Our results suggest that IAA is involved in the establishment of biotrophy in P. indica-barley symbiosis and might represent a compatibility factor in this system.

Cyclin-dependent kinase 5 (cdk5) is found in an active form only in neuronal cells. Activation by virtue of association with the cyclin-like neuronal proteins p35 (or its truncated form p25) and p39 is the only mechanism currently shown to regulate cdk5 catalytic activity. In addition to cyclin binding, other members of the cdk family require for maximal activation phosphorylation of a Ser͞Thr residue (Thr 160 in the case of cdk-2) that is conserved in all cdks except cdk8. This site is phosphorylated by cdk-activating kinases, which, however, do not phosphorylate cdk5. To examine the possible existence of a phosphorylation-dependent regulatory mechanism in the case of cdk5, we have metabolically labeled PC12 cells with 32 P i and shown that the endogenous cdk5 is phosphorylated. Bacterially expressed cdk5 also can be phosphorylated by PC12 cell lysates. Phosphorylation of cdk5 by a PC12 cell lysate results in a significant increase in cdk5͞p25 catalytic activity. Ser 159 in cdk5 is homologous to the regulatory Thr 160 in cdk2. A Ser 159 -to-Ala (S159A) cdk5 mutant did not show similar activation, which suggests that cdk5 is also regulated by phosphorylation at this site. Like other members of the cdk family, cdk5 catalytic activity is inf luenced by both p25 binding and phosphorylation. We show that the cdk5-activating kinase (cdk5AK) is distinct from the cdk-activating kinase (cyclin H͞cdk7) that was reported previously to neither phosphorylate cdk5 nor affect its activity. We also show that casein kinase I, but not casein kinase II, can phosphorylate and activate cdk5 in vitro.Cyclin-dependent kinase 5 (cdk5) is a serine͞threonine kinase present at relatively high levels in neurons (1, 2). On the basis of sequence similarity, it is a member of the cyclin-dependent kinase (cdk) family. Full activation of cyclin-dependent kinases characteristically requires combination with one of the cyclins and also phosphorylation of a threonine or serine residue located on the cdk T loop by a cdk-activating kinase. These two events are considered to act cooperatively to open the catalytic site and relieve its obstruction by the T loop (3-6). However, cdk5 is not activated by cyclins, and activation by phosphorylation of its T loop serine has not been demonstrated previously (3). Activation of cdk5 results instead from combination with cdk5-specific activators that include p35 or its truncated form, p25 (7,8), and p39 (9). Although cdk5 is expressed in many mammalian tissues, the cdk5 activators are largely restricted to neurons. The mechanism of cdk5 interaction with its activators is thought to be analogous to that of cyclindependent kinases and forms highly stable and active complexes after prolonged incubation (7-10). Substrates of cdk5 include the neuronal cytoskeletal proteins neurofilament-H (1, 11), tau (12), and MAP (13). However, the embryonic lethality of cdk-5 ''knockout'' mice suggests additional important functions for this enzyme (14).Because cdk-activating kinase (cyclin H͞cdk7) has been found not to ...