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Summary: The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate‐secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown.Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection.Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers in vitro with high affinity and specificity and that both proteins suppress the chitin‐triggered activation of two immune‐related plant mitogen‐activated protein kinases in the host Arabidopsis. Using RNAi‐mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium‐mediated penetration of both Arabidopsis epidermal cells and cellophane membranes in vitro.The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin‐triggered immunity and as proteins required for appressorium function. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the editors discuss various reports within the issue on topics including plant chemical biology, plant hormone research and agricultural biotechnology.

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases. Author summary: Chitin plays a central role in plant-fungi interactions, since it is a major component of the fungal cell wall that is targeted by host hydrolytic enzymes to inhibit the growth of fungal pathogens on the one hand, and release chitin fragments that are recognized by host immune receptors to activate further immune responses on the other hand. In turn, many fungal pathogens secrete chitin binding LysM effectors to which currently two functions have been assigned. Most LysM effectors that were functionally characterized to date function to prevent chitin recognition by host immune receptors through chitin sequestration. Additionally, some LysM effectors were shown to protect fungal hyphae against hydrolysis by host chitinases. The crystal structure of Mg1LysM from the Septoria blotch pathogen of wheat, Zymoseptoria tritici, revealed that chitin-induced dimerization of two Mg1LysM protomers through high affinity binding is required for hyphal protection against chitinases. Since Mg1LysM also forms ligand-independent homodimers, a supramolecular structure can be formed in which chitin-induced oligomerization of Mg1LysM ligand-independent homodimers form a contiguous Mg1LysM higher ordered structure that is anchored to the chitin in the fungal cell wall to prevent hydrolysis by host chitinases. [ABSTRACT FROM AUTHOR]

Summary: Fungi defend their ecological niche against antagonists by producing antibiosis molecules. Some of these molecules are only produced upon confrontation with the antagonist. The basidiomycete Coprinopsis cinerea induces the expression of the sesquiterpene synthase‐encoding gene cop6 and its two neighboring genes coding for cytochrome P450 monooxygenases in response to bacteria. We further investigated this regulation of cop6 and examined if the gene product is involved in the production of antibacterials. Cell‐free supernatants of axenic cultures of the Gram‐positive bacterium Bacillus subtilis were sufficient to induce cop6 transcription assessed using a fluorescent reporter strain. Use of this strain in a microfluidic device revealed that the cop6 gene was induced in all hyphae directly exposed to the supernatant and that induction occurred within less than one hour. Targeted replacement of the cop6 gene demonstrated the requirement of the encoded synthase for the biosynthesis of the sesquiterpene lagopodin B, a previously reported antibacterial compound from related species. Accordingly, lagopodin B from C. cinerea inhibited the growth of several Gram‐positive bacteria including B. subtilis but not Gram‐negative bacteria. Our results demonstrate that the C. cinerea vegetative mycelium responds to soluble compounds of a bacterial culture supernatant by local production of an antibacterial secondary metabolite. [ABSTRACT FROM AUTHOR]

Abstract: Bioactive natural products are important starting points for developing chemical tools for biological research. For elucidating their bioactivity profile, biological systems with concise complexity such as cell culture systems are frequently used, whereas unbiased investigations in more complex multicellular systems are only rarely explored. Here, we demonstrate with the natural product Rotihibin A and the plant research model system Arabidopsis thaliana that unbiased transcriptional profiling enables a rapid, label‐free, and compound economic evaluation of a natural product′s bioactivity profile in a complex multicellular organism. To this end, we established a chemical synthesis of Rotihibin A as well as that of structural analogues, followed by transcriptional profiling‐guided identification and validation of Rotihibin A as a TOR signaling inhibitor (TOR=target of rapamycin). These findings illustrate that a combined approach of transcriptional profiling and natural product research may represent a technically simple approach to streamline the development of chemical tools from natural products even for biologically complex multicellular biological systems. [ABSTRACT FROM AUTHOR]

Plant root systems are indispensable for water uptake, nutrient acquisition, and anchoring plants in the soil. Previous studies using auxin inhibitors definitively established that auxin plays a central role regulating root growth and development. Most auxin inhibitors affect all auxin signaling at the same time, which obscures an understanding of individual events. Here, we report that jasmonic acid (JA) functions as a lateral root (LR)-preferential auxin inhibitor in Arabidopsis (Arabidopsis thaliana) in a manner that is independent of the JA receptor, CORONATINE INSENSITIVE1 (COI1). Treatment of wild-type Arabidopsis with either (-)-JA or (+)-JA reduced primary root length and LR number; the reduction of LR number was also observed in coi1 mutants. Treatment of seedlings with (-)-JA or (+)-JA suppressed auxin-inducible genes related to LR formation, diminished accumulation of the auxin reporter DR5::GUS, and inhibited auxin-dependent DII-VENUS degradation. A structural mimic of (-)-JA and (+)-coronafacic acid also inhibited LR formation and stabilized DII-VENUS protein. COI1-independent activity was retained in the double mutant of transport inhibitor response1 and auxin signaling f-box protein2 (tir1 afb2) but reduced in the afb5 single mutant. These results reveal JAs and (+)-coronafacic acid to be selective counter-auxins, a finding that could lead to new approaches for studying the mechanisms of LR formation. [ABSTRACT FROM AUTHOR]

Jasmonates are lipid-derived plant hormones that regulate plant defenses and numerous developmental processes. Although the biosynthesis and molecular function of the most active form of the hormone, (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), have been unraveled, it remains poorly understood how the diversity of bioactive jasmonates regulates such a multitude of plant responses. Bioactive analogs have been used as chemical tools to interrogate the diverse and dynamic processes of jasmonate action. By contrast, small molecules impairing jasmonate functions are currently unknown. Here, we report on jarin-1 as what is to our knowledge the first small-molecule inhibitor of jasmonate responses that was identified in a chemical screen using Arabidopsis thaliana. Jarin-1 impairs the activity of JA-Ile synthetase, thereby preventing the synthesis of the active hormone, JA-Ile, whereas closely related enzymes are not affected. Thus, jarin-1 may serve as a useful chemical tool in search for missing regulatory components and further dissection of the complex jasmonate signaling networks. [ABSTRACT FROM AUTHOR]

Rapid transient elevation of cytoplasmic calcium (Ca2+) levels in plant cells is an early signaling event triggered by many environmental cues including abiotic and biotic stresses. Cellular Ca2+ levels and their alterations can be monitored by genetically encoded reporter systems such as the bioluminescent protein, aequorin. Employment of proteinaceous Ca2+ sensors is usually performed in transgenic lines that constitutively express the reporter construct. Such settings limit the usage of these Ca2+ biosensors to particular reporter variants and plant genetic backgrounds, which can be a severe constraint in genetic pathway analysis. Here we systematically explored the potential of Arabidopsis thaliana leaf mesophyll protoplasts, either derived from a transgenic apoaequorin-expressing line or transfected with apoaequorin reporter constructs, as a complementary biological resource to monitor cytoplasmic changes of Ca2+ levels in response to various biotic stress elicitors. We tested a range of endogenous and pathogen-derived elicitors in seedlings and protoplasts of the corresponding apoaequorin-expressing reporter line. We found that the protoplast system largely reflects the Ca2+ signatures seen in intact transgenic seedlings. Results of inhibitor experiments including the calculation of IC50 values indicated that the protoplast system is also suitable for pharmacological studies. Moreover, analyses of Ca2+signatures in mutant backgrounds, genetic complementation of the mutant phenotypes and expression of sensor variants targeted to different subcellular localizations can be readily performed. Thus, in addition to the prevalent use of seedlings, the leaf mesophyll protoplast setup represents a versatile and convenient tool for the analysis of Ca2+ signaling pathways in plant cells. [ABSTRACT FROM AUTHOR]

Sexual recombination drives genetic diversity in eukaryotic genomes and fosters adaptation to novel environmental challenges. Although strictly asexual microorganisms are often considered as evolutionary dead ends, they comprise many devastating plant pathogens. Presently, it remains unknown how such asexual pathogens generate the genetic variation that is required for quick adaptation and evolution in the arms race with their hosts. Here, we show that extensive chromosomal rearrangements in the strictly asexual plant pathogenic fungus Verticillium dahliae establish highly dynamic lineage-specific (LS) genomic regions that act as a source for genetic variation to mediate aggressiveness. We show that such LS regions are greatly enriched for in planta-expressed effector genes encoding secreted proteins that enable host colonization. The LS regions occur at the flanks of chromosomal breakpoints and are enriched for retrotransposons and other repetitive sequence elements. Our results suggest that asexual pathogens may evolve by prompting chromosomal rearrangements, enabling rapid development of novel effector genes. Likely, chromosomal reshuffling can act as a general mechanism for adaptation in asexually propagating organisms. [ABSTRACT FROM AUTHOR]

Successful reproduction of flowering plants requires constant communication between female tissues and growing pollen tubes. Female cells secrete molecules and peptides as nutrients or guidance cues for fast and directional tube growth, which is executed by dynamic changes of intracellular activities within pollen tubes. Compared with the extensive interest in female cues and intracellular activities of pollen tubes, how female cues are sensed and interpreted intracellularly in pollen is poorly understood. We show here that COBL10, a glycosylphosphatidylinositol ( GPI)-anchored protein, is one component of this pollen tube internal machinery. Mutations in COBL10 caused gametophytic male sterility due to reduced pollen tube growth and compromised directional sensing in the female transmitting tract. Deposition of the apical pectin cap and cellulose microfibrils was disrupted in cobl10 pollen tubes. Pollen tube localization of COBL10 at the apical plasma membrane is critical for its function and relies on proper GPI processing and its C-terminal hydrophobic residues. GPI-anchored proteins are widespread cell sensors in mammals, especially during egg-sperm communication. Our results that COBL10 is critical for directional growth of pollen tubes suggest that they play critical roles in cell-cell communications in plants. [ABSTRACT FROM AUTHOR]

Recognition of microbe-associated molecular patterns (MAMPs) leads to the generation of MAMP-triggered immunity (MTI), which restricts the invasion and propagation of potentially infectious microbes. It has been described that the perception of different bacterial and fungal MAMPs causes the repression of flavonoid induction upon light stress or sucrose application. However, the functional significance of this MTI-associated signaling output remains unknown. In Arabidopsis (Ambidopsis thaliana), FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR act as the pattern recognition receptors for the bacterial MAMP epitopes flg22 (of flagellin) and elfl8 (of elongation factor [EF]-Tu), respectively. Here, we reveal that reactive oxygen species spiking and callose deposition are dispensable for the repression of flavonoid accumulation by both pattern recognition receptors. Importantly, FLS2-triggered activation of PATHOGENESIS-RELATED (PR) genes and bacterial basal defenses are enhanced in transparent testa4 plants that are devoid of flavonoids, providing evidence for a functional contribution of flavonoid repression to MTI. Moreover, we identify nine small molecules, of which eight are structurally unrelated, that derepress flavonoid accumulation in the presence of flg22. These compounds allowed us to dissect the FLS2 pathway. Remarkably, one of the identified compounds uncouples flavonoid repression and PR gene activation from the activation of reactive oxygen species, mitogen-activated protein kinases, and callose deposition, corroborating a close link between the former two outputs. Together, our data imply a model in which MAMP-induced repression of flavonoid accumulation serves a role in removing the inherent inhibitory action of flavonoids on an MTI signaling branch. [ABSTRACT FROM AUTHOR]

Chitin is a homopolymer of β-(1,4)-linked N-acetyl-D-glucosamine (GlcNAc) and a major structural component of fungal cell walls. In plants, chitin acts as a microbe-associated molecular pattern (MAMP) that is recognized by lysin motif (LysM)-containing plant cell surface-localized pattern recognition receptors (PRRs) that activate a plethora of downstream immune responses. To deregulate chitin-induced plant immunity and successfully establish infection, many fungal pathogens secrete LysM domain-containing effector proteins during host colonization. The LysM effector Ecp6 from the tomato (Solanum lycopersicum) leaf mold fungus Cladosporium fulvum can outcompete plant PRRs for chitin binding because two of its three LysM domains cooperate to form a composite groove with ultra-high (pM) chitin-binding affinity. However, most functionally characterized LysM effectors contain only two LysMs, including Magnaporthe oryzae MoSlp1, Verticillium dahliae Vd2LysM, and Colletotrichum higginsianum ChElp1 and ChElp2. Here, we performed modeling, structural, and functional analyses to investigate whether such dual-domain LysM effectors can also form ultra-high chitin-binding affinity grooves through intramolecular LysM dimerization. However, our study suggests that intramolecular LysM dimerization does not occur. Rather, our data support the occurrence of intermolecular LysM dimerization for these effectors, associated with a substantially lower chitin binding affinity than monitored for Ecp6. Interestingly, the intermolecular LysM dimerization allows for the formation of polymeric complexes in the presence of chitin. Possibly, such polymers may precipitate at infection sites to eliminate chitin oligomers, and thus suppress the activation of chitin-induced plant immunity., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αβ-defensins (Csαβ-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.

Among the invaluable benefits of basidiomycete genomics is the dramatically enhanced insight into the potential capacity to biosynthesize natural products. This study focuses on adenylate-forming reductases, which is a group of natural product biosynthesis enzymes that resembles non-ribosomal peptide synthetases, yet serves to modify one substrate, rather than to condense two or more building blocks. Phylogenetically, these reductases fall in four classes. The phylogeny of Heterobasidion annosum (Russulales) and Serpula lacrymans (Boletales) adenylate-forming reductases was investigated. We identified a previously unrecognized phylogenetic branch within class III adenylate-forming reductases. Three representatives were heterologously produced and their substrate preferences determined in vitro: NPS9 and NPS11 of S. lacrymans preferred l-threonine and benzoic acid, respectively, while NPS10 of H. annosum accepted phenylpyruvic acid best. We also investigated two class IV adenylate-forming reductases of Coprinopsis cinerea, which each were active with l-alanine, l-valine, and l-serine as substrates. Our results show that adenylate-forming reductases are functionally more diverse than previously recognized. As none of the natural products known from the species investigated in this study includes the identified substrates of their respective reductases, our findings may help further explore the diversity of these basidiomycete secondary metabolomes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Chitin-binding lysin motif (LysM) effectors contribute to the virulence of various plant-pathogenic fungi that are causal agents of foliar diseases. Here, we report the LysM effectors of the soil-borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are conserved in a collection of V. dahliae strains. Remarkably, and in contrast with the previously studied LysM effectors of other plant pathogens, no expression of core LysM effectors was monitored in planta in a taxonomically diverse panel of host plants. Moreover, targeted deletion of the individual LysM effector genes in V. dahliae strain JR2 did not compromise virulence in infections on Arabidopsis, tomato or Nicotiana benthamiana. Interestingly, an additional lineage-specific LysM effector is encoded in the genome of V. dahliae strain VdLs17, but not in any other V. dahliae strain sequenced to date. Remarkably, this lineage-specific effector is expressed in planta and contributes to the virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or N. benthamiana. Functional analysis revealed that this LysM effector binds chitin, is able to suppress chitin-induced immune responses and protects fungal hyphae against hydrolysis by plant hydrolytic enzymes. Thus, in contrast with the core LysM effectors of V. dahliae, this lineage-specific LysM effector of strain VdLs17 contributes to virulence in planta., (© 2016 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

While host immune receptors detect pathogen-associated molecular patterns to activate immunity, pathogens attempt to deregulate host immunity through secreted effectors. Fungi employ LysM effectors to prevent recognition of cell wall-derived chitin by host immune receptors, although the mechanism to compete for chitin binding remained unclear. Structural analysis of the LysM effector Ecp6 of the fungal tomato pathogen Cladosporium fulvum reveals a novel mechanism for chitin binding, mediated by intrachain LysM dimerization, leading to a chitin-binding groove that is deeply buried in the effector protein. This composite binding site involves two of the three LysMs of Ecp6 and mediates chitin binding with ultra-high (pM) affinity. Intriguingly, the remaining singular LysM domain of Ecp6 binds chitin with low micromolar affinity but can nevertheless still perturb chitin-triggered immunity. Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex. DOI:http://dx.doi.org/10.7554/eLife.00790.001.

In this chapter a method for the heterologous production of fungal proteins in the yeast Pichia pastoris is described. Starting with cloning of the sequence encoding the gene of interest into the expression vector, this protocol describes P. pastoris transformation, production of the protein in a fermentor, and purification of the protein. This method has successfully been used for the production of a number of fungal effector proteins.