Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have ...become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.
• Arbuscular mycorrhizal (AM) fungi greatly improve mineral uptake by host plants in nutrient-depleted soil and can intracellularly colonize root cortex cells in the vast majority of higher plants. ...However, AM fungi possess common fungal cell wall components such as chitin that can be recognized by plant chitin receptors to trigger immune responses, raising the question as to how AM fungi effectively evade chitin-triggered immune responses during symbiosis.
• In this study, we characterize a secreted lysin motif (LysM) effector identified from the model AM fungal species Rhizophagus irregularis, called RiSLM.
• RiSLM is one of the highest expressed effector proteins in intraradical mycelium during the symbiosis. In vitro binding assays show that RiSLM binds chitin-oligosaccharides and can protect fungal cell walls from chitinases. Moreover, RiSLM efficiently interferes with chitin-triggered immune responses, such as defence gene induction and reactive oxygen species production in Medicago truncatula. Although RiSLM also binds to symbiotic (lipo)chitooligosaccharides it does not interfere significantly with symbiotic signalling in Medicago. Host-induced gene silencing of RiSLM greatly reduces fungal colonization levels.
• Taken together, our results reveal a key role for AM fungal LysM effectors to subvert chitin-triggered immunity in symbiosis, pointing to a common role for LysM effectors in both symbiotic and pathogenic fungi.
Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). ...Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi.
Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships ...with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development.
Strigolactones (SLs) influence the ability of legumes to associate with nitrogen-fixing bacteria. In this study, we determine the precise stage at which SLs influence nodulation. We show that SLs ...promote infection thread formation, as a null SL-deficient pea (Pisum sativum) mutant forms significantly fewer infection threads than wild-type plants, and this reduction can be overcome by the application of the synthetic SL GR24. We found no evidence that SLs influence physical events in the plant before or after infection thread formation, since SL-deficient plants displayed a similar ability to induce root hair curling in response to rhizobia or Nod lipochitooligosaccharides (LCOs) and SL-deficient nodules appear to fix nitrogen at a similar rate to those of wild-type plants. In contrast, an SL receptor mutant displayed no decrease in infection thread formation or nodule number, suggesting that SL deficiency may influence the bacterial partner. We found that this influence of SL deficiency was not due to altered flavonoid exudation or the ability of root exudates to stimulate bacterial growth. The influence of SL deficiency on infection thread formation was accompanied by reduced expression of some early nodulation genes. Importantly, SL synthesis is down-regulated bymutations in genes of the Nod LCO signaling pathway, and this requires the downstream transcription factor NSP2 but not NIN. This, together with the fact that the expression of certain SL biosynthesis genes can be elevated in response to rhizobia/Nod LCOs, suggests that Nod LCOs may induce SL biosynthesis. SLs appear to influence nodulation independently of ethylene action, as SL-deficient and ethylene-insensitive double mutant plants display essentially additive phenotypes, and we found no evidence that SLs influence ethylene synthesis or vice versa.
Most land plants live symbiotically with arbuscular mycorrhizal fungi. Establishment of this symbiosis requires signals produced by both partners: strigolactones in root exudates stimulate ...pre‐symbiotic growth of the fungus, which releases lipochito‐oligosaccharides (Myc‐LCOs) that prepare the plant for symbiosis. Here, we have investigated the events downstream of this early signaling in the roots. We report that expression of miR171h, a microRNA that targets NSP2, is up‐regulated in the elongation zone of the root during colonization by Rhizophagus irregularis (formerly Glomus intraradices) and in response to Myc‐LCOs. Fungal colonization was much reduced by over‐expressing miR171h in roots, mimicking the phenotype of nsp2 mutants. Conversely, in plants expressing an NSP2 mRNA resistant to miR171h cleavage, fungal colonization was much increased and extended into the elongation zone of the roots. Finally, phylogenetic analyses revealed that miR171h regulation of NSP2 is probably conserved among mycotrophic plants. Our findings suggest a regulatory mechanism, triggered by Myc‐LCOs, that prevents over‐colonization of roots by arbuscular mycorrhizal fungi by a mechanism involving miRNA‐mediated negative regulation of NSP2.
Chitin oligosaccharides (COs) hold high promise as organic fertilizers in the ongoing agro-ecological transition. Short- and long-chain COs can contribute to the establishment of symbiotic ...associations between plants and microorganisms, facilitating the uptake of soil nutrients by host plants. Long-chain COs trigger plant innate immunity. A fine investigation of these different signaling pathways requires improving the access to high-purity COs. Here, we used the response surface methodology to optimize the production of COs by enzymatic hydrolysis of water-soluble chitin (WSC) with hen egg-white lysozyme. The influence of WSC concentration, its acetylation degree, and the reaction time course were modelled using a Box–Behnken design. Under optimized conditions, water-soluble COs up to the nonasaccharide were formed in 51% yield and purified to homogeneity. This straightforward approach opens new avenues to determine the complex roles of COs in plants.
Arbuscular Mycorrhiza and Root Nodule Symbiosis are symbiotic interactions with a high benefit for plant growth and crop production. Thus, it is of great interest to understand the developmental ...process of these symbioses in detail. We analysed very early symbiotic responses of Medicago truncatula root hair cells, by stimulation with lipochitinoligosaccharides specific for the induction of nodules (Nod-LCOs), or the interaction with mycorrhiza (Myc-LCOs). Intracellular micro electrodes were used, in combination with Ca2+ sensitive reporter dyes, to study the relations between cytosolic Ca2+ signals and membrane potential changes. We found that sulfated Myc- as well as Nod-LCOs initiate a membrane depolarization, which depends on the chemical composition of these signaling molecules, as well as the genotype of the plants that were studied. A successive application of sulfated Myc-LCOs and Nod-LCOs resulted only in a single transient depolarization, indicating that Myc-LCOs can repress plasma membrane responses to Nod-LCOs. In contrast to current models, the Nod-LCO-induced depolarization precedes changes in the cytosolic Ca2+ level of root hair cells. The Nod-LCO induced membrane depolarization thus is most likely independent of cytosolic Ca2+ signals and nuclear Ca2+ spiking.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Lipo-chitooligosaccharides (LCOs) are historically known for their role as microbial-derived signaling molecules that shape plant symbiosis with beneficial rhizobia or mycorrhizal fungi. Recent ...studies showing that LCOs are widespread across the fungal kingdom have raised questions about the ecological function of these compounds in organisms that do not form symbiotic relationships with plants. To elucidate the ecological function of these compounds, we investigate the metabolomic response of the ubiquitous human pathogen Aspergillus fumigatus to LCOs. Our metabolomics data revealed that exogenous application of various types of LCOs to A. fumigatus resulted in significant shifts in the fungal metabolic profile, with marked changes in the production of specialized metabolites known to mediate ecological interactions. Using network analyses, we identify specific types of LCOs with the most significant effect on the abundance of known metabolites. Extracts of several LCO-induced metabolic profiles significantly impact the growth rates of diverse bacterial species. These findings suggest that LCOs may play an important role in the competitive dynamics of non-plant-symbiotic fungi and bacteria. This study identifies specific metabolomic profiles induced by these ubiquitously produced chemicals and creates a foundation for future studies into the potential roles of LCOs as modulators of interkingdom competition.
The activation of silent biosynthetic gene clusters (BGC) for the identification and characterization of novel fungal secondary metabolites is a perpetual motion in natural product discoveries. Here, we demonstrated that one of the best-studied symbiosis signaling compounds, lipo-chitooligosaccharides (LCOs), play a role in activating some of these BGCs, resulting in the production of known, putative, and unknown metabolites with biological activities. This collection of metabolites induced by LCOs differentially modulate bacterial growth, while the LCO standards do not convey the same effect. These findings create a paradigm shift showing that LCOs have a more prominent role outside of host recognition of symbiotic microbes. Importantly, our work demonstrates that fungi use LCOs to produce a variety of metabolites with biological activity, which can be a potential source of bio-stimulants, pesticides, or pharmaceuticals.
Myrosinase, an S-glycosidase, hydrolyzes plant anionic 1-thio-β-d-glucosides (glucosinolates) considered part of the plant defense system. AlthoughO-glycosidases are ubiquitous, myrosinase is the ...only knownS-glycosidase. Its active site is very similar to that of retaining O-glycosidases, but one of the catalytic residues in O-glycosidases, a carboxylate residue functioning as the general base, is replaced by a glutamine residue. Myrosinase is strongly activated by ascorbic acid. Several binary and ternary complexes of myrosinase with different transition state analogues and ascorbic acid have been analyzed at high resolution by x-ray crystallography along with a 2-deoxy-2-fluoro-glucosyl enzyme intermediate. One of the inhibitors, d-gluconhydroximo-1,5-lactam, binds simultaneously with a sulfate ion to form a mimic of the enzyme-substrate complex. Ascorbate binds to a site distinct from the glucose binding site but overlapping with the aglycon binding site, suggesting that activation occurs at the second step of catalysis,i.e. hydrolysis of the glycosyl enzyme. A water molecule is placed perfectly for activation by ascorbate and for nucleophilic attack on the covalently trapped 2-fluoro-glucosyl-moiety. Activation of the hydrolysis of the glucosyl enzyme intermediate is further evidenced by the observation that ascorbate enhances the rate of reactivation of the 2-fluoro-glycosyl enzyme, leading to the conclusion that ascorbic acid substitutes for the catalytic base in myrosinase.
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