Microbiological Research

Fungal innate immunity resembles mammalian innate immunity. It does not employ toll-like receptors (TLRs), but should employ endocytosis of non-fungal molecular patterns recognized by nuclear-localizing receptors (NLR). Downstream, both types of receptors are Mammalian Ste20 kinases (MSTs). We identified an MST3 ortholog in the plant pathogens Fusarium graminearum (FgMST3) and Magnaporthe oryzae (MoMST3). We knocked out both genes and investigated mutants using a standard panel of tests for growth, development, and pathogenicity for the respective fungi. Both{Delta} FgMST3 and{Delta} MoMST3 strains showed reduced pathogenicity. The deletions negatively affected conidia production and conidia germination but had little effect on growth rate. However, the two mutants reacted differently to various stress treatments, especially to Zn2+ and gentamicin. In addition, we constructed an innate immunity reporter system for F. graminearum to detect less than 4-hour responses to non-self-molecular patterns (NSMP) like bacterial outer membrane vesicles (OMVs) and trace levels of sucrose, indicating plant. The reporter gene responses to OMVs of MST3 mutant strains are severely reduced. Our results indicate that both MoMst3 and FgMst3 are involved in fungal innate immunity downstream of unknown NLR proteins, motivating studies to identify genes for the NLR receptors. Finding such and investigating how they work and vary between fungal species and strains should be essential for understanding fungal biotic interactions with viruses, bacteria, plants, and animals.

Wood-decaying fungi of the class Agaricomycetes (phylum Basidiomycota) are saprotrophs that break down lignocellulose and play an important role in the nutrient recycling. They secrete a wide range of extracellular plant cell wall degrading enzymes that break down cellulose, hemicellulose and lignin, the main building blocks of plant biomass. Although the production of these enzymes is regulated mainly at the transcriptional level, no activating regulators have been identified in any wood-decaying fungus in the class Agaricomycetes. We studied the regulation of cellulase expression in the wood-decaying fungus Schizophyllum commune. Comparative genomics and transcriptomics on two wild isolates revealed a Zn2Cys6-type transcription factor gene (roc1) that was highly up-regulated during growth on cellulose, when compared to glucose. It is only conserved in the class Agaricomycetes. A roc1 knockout strain showed an inability to grow on medium with cellulose as sole carbon source, and growth on cellobiose and xylan (other components of wood) was inhibited. Growth on non-wood-related carbon sources was not inhibited. Cellulase activity was reduced in the growth medium of the {Delta}roc1 strain. ChIP-Seq identified 1474 binding sites of the Roc1 transcription factor. Promoters of genes involved in lignocellulose degradation were enriched with these binding sites, especially those of LPMO (lytic polysaccharide monooxygenase) CAZymes, indicating that Roc1 directly regulates these genes. A GC-rich motif was identified as the binding site of Roc1, which was confirmed by a functional promoter analysis. Together, Roc1 is a key regulator of cellulose degradation and the first identified in wood-decaying fungi in the phylum Basidiomycota.

Ammonia opens trap formation in the nematode-trapping (NT) fungus Arthrobotrys oligospora, an intriguing morphological switch in NT fungi, where saprophytic mycelia are converted to pathogenic organs. Endocytosis plays a prominent role in nutrient uptake, signaling cascades, and maintenance of cellular homeostasis in higher eukaryotes. Here, we demonstrate that ammonia efficiently promotes endocytosis via the formation of 3D-adhesive mycelial nets in A. oligospora. Trap production is followed by the presence of massive multivesicular bodies (MVBs) and membrane rupture and repair. Additionally, both the ubiquitin-proteasome system and the endosomal sorting complex for transport (ESCRT) pathway are immediately linked to endocytosis regulation and MVB formation in ammonia-induced trap formation. Moreover, disruption of the ESCRT-1 complex subunit proteins AoHse and AoVps27 led to the complete loss of membrane endocytosis and trap formation. Finally, the deletion of the deubiquitinase AoSst2 caused a significant reduction in the number of trap structures produced in response to exposure to ammonia or nematodes. Overall, our results increase our knowledge of the molecular mechanisms underlying the phenotypic changes in the NT fungal group, demonstrating that the endocytosis-ESCRT-MVB pathway participates in the regulation of trapping organs. Author SummaryThe lifestyle switch of nematode-trapping (NT) fungi is a significant event that increases their pathogenicity to nematode prey, which has resulted in large losses to agricultural crops worldwide. Here, we describe the molecular mechanism underlying how this fungal group forms a NT structure in response to ammonia, a widely preferred nitrogen source in soil niches. Ammonia enhances the endocytosis process, ubiquitin-proteasome system, and endosomal sorting complex for transport (ESCRT) pathway of the model NT fungus A. oligospora, thereby generating enriched multivesicular bodies (MVBs) during trap formation. In this process, the cell membrane morphology is remarkably damaged and then repaired. We further found that disruption of the ESCRT-0 subcomplex or ubiquitinase severely blocked trap production and membrane reorganization. Our study provides a new understanding of endocytosis-ESCRT-MVB flux in the transition of fungal NT organs.

Cre1 is an important transcription factor that regulates carbon catabolite repression (CCR) and is widely conserved across fungi. This gene has been extensively studied in several Ascomycota species, whereas its role in gene expression regulation in the Basidiomycota remains poorly understood. Here, we identified and investigated the role of cre1 in Coprinopsis cinerea, a basidiomycete model mushroom that can efficiently degrade lignocellulosic plant wastes. We used a rapid and efficient gene deletion approach based on PCR-amplified split-marker DNA cassettes together with in-vitro assembled Cas9-guide RNA ribonucleoproteins (Cas9-RNPs) to generate C. cinerea cre1 gene deletion strains. Gene expression profiling of two independent C. cinerea cre1 mutants showed significant deregulation of carbohydrate metabolism, plant cell wall degrading enzymes (PCWDEs), plasma membrane transporter-related and several transcription factor encoding genes, among others. Our results support the notion that, similarly to reports in the ascomycetes, Cre1 of C. cinerea orchestrates CCR through a combined regulation of diverse genes, including PCWDEs, transcription factors that positively regulate PCWDEs and membrane transporters which could import simple sugars that can induce the expression of PWCDEs. Somewhat paradoxically, though in accordance with other Agaricomycetes, genes related to lignin degradation were mostly downregulated in cre1 mutants, indicating they fall under different regulation than other PCWDEs. The gene deletion approach and the data presented in this paper expand our knowledge of CCR in the Basidiomycota and provide functional hypotheses on genes related to plant biomass degradation. ImportanceMushroom-forming fungi include some of the most efficient degraders of lignocellulosic plant biomass. They degrade dead plant materials by a battery of lignin-, cellulose-, hemicellulose- and pectin-degrading enzymes, the encoding genes of which are under tight transcriptional control. One of the highest-level regulation of these metabolic enzymes is known as carbon catabolite repression, which is orchestrates by the transcription factor Cre1, and ensures that costly lignocellulose-degrading enzyme genes are expressed only when simple carbon sources (e.g. glucose) are not available. Here, we identified the Cre1 ortholog in a litter-decomposer Agaricomycete, Coprinopsis cinerea, knocked it out and characterized transcriptional changes in the mutants. We identified several dozen lignocellulolytic enzyme genes as well as membrane transporters and other transcription factors as putative target genes. These results extend knowledge on carbon catabolite repression to litter decomposer Basidiomycota.

In this study, two distinct in vitro infection models of Aspergillus fumigatus, using murine macrophages (RAW264.7) and human lung epithelial cells (A549), were employed to identify the genes important for fungal adaptation during infection. Transcriptomic analyses of co-incubated Aspergillus uncovered 140 fungal genes up-regulated in common between both models that, when compared with a previously published in vivo transcriptomic study, allowed the identification of 13 genes consistently up-regulated in all three infection conditions. Among them, the maiA gene, responsible for a critical step in the L-phenylalanine degradation pathway, was identified. Disruption of maiA resulted in a mutant strain unable to complete the Phe degradation pathway, leading to an excessive production of pyomelanin when this amino acid served as the sole carbon source. Moreover, the disruption mutant exhibited noticeable cell wall abnormalities, with reduced levels of {beta}-glucans within the cell wall. the maiA-1 mutant strain induced reduced inflammation in primary macrophages and displayed significantly lower virulence in a neutropenic mouse model of infection. This is the first study linking the A. fumigatus maiA gene to fungal cell wall homeostasis and virulence.

Sporulation is the most widespread means of reproduction and dispersal in fungi. In the Basidiomycota, sexual spores are produced on specialised cells known as basidia, from which they are discharged forcibly by a powered process called ballistospory, the highest known acceleration in nature. However, the genetics of sporulation, in particular postmeiotic events related to spore morphogenesis and ballistospory, remain poorly known. Here, we explore the genetics of these processes, based on a new, highly conserved transcription factor, Sporulation-Related Regulator 1 (SRR1), and its putative downstream regulatory network. Reverse genetics of Srr1 in the model mushroom Coprinopsis cinerea and commercially produced oyster mushroom indicated a conserved role of Srr1 in sporulation across Agaricomycetes. RNA-Seq analysis and motif-based inference of a hypothetical SRR1 gene regulatory network allowed delimiting putative targets regulated by SRR1 in a direct and indirect manner. Using this network and comparative genomics, we identified genes associated with ballistospory, including a putative SRR1-target chitinase, which was found to be required for normal spore production and morphology. Overall, our study offers new insights into the genetic mechanisms governing postmeiotic spore morphogenesis and ballistospory in the Agaricomycetes.

Chitin is a critical structural component of fungal cell walls, yet our understanding of its synthesis across the kingdom Fungi remains limited. Here, we investigate chitin synthase diversity, transcription and localisation in the saprotrophic chytrid Rhizoclosmatium globosum (Rg), expanding insights into fungal cell wall biology beyond Dikaryan models. We identified 20 chitin synthase genes in the Rg genome, including canonical Division I and II types, and a distinctive chitin synthase gene containing a glycoside hydrolase domain linked to {beta}-glucan synthesis. Transcriptomic analysis through zoospore, germling and immature thallus developmental stages revealed stage-specific expression patterns, with active gene diversity correlating with increasing morphological complexity. Using electroporation-based transformation and fluorescent fusion constructs, we demonstrated successful expression and localisation of two chitin synthases during cell development. Localisation patterns showed dynamic redistribution from cytoplasmic dispersion in early encysted cells to concentrated signals at the sporangium wall. Expression in the apophysis and at the apophysis-sporangium junction indicates the importance of these structures in cell maintenance. Our findings highlight functional specialisation among chitin synthases and underscore the importance of cell wall integrity in chytrid development. This work establishes Rg as a genetically tractable model for studying chytrid cell biology and contributes to broader understanding of fungal evolution and cell wall dynamics.

Aspergillus fungi are opportunistic pathogens that affect a large number of people worldwide. Many aspects of Aspergillus spp. pathogenesis toward humans are known, but their ability to enhance their infectious potential by manipulating the environmental pH of its host has not been considered yet. In this study, we tested the hypothesis that by producing oxalic acid, Aspergillus niger can manipulate pH during lung infection and thus, interfering with this process could limit pathogenicity. To test this hypothesis, we co-cultured A. niger with oxalotrophic bacteria in increasingly complex testing systems (Petri dishes and 3D-cell cultures systems). In in vitro tests, oxalotrophic bacteria limit oxalic acid production and suppressed the pH shift induced by A. niger. In 3D-cell cultures (Transwells(R) and Bronchioles-on-a-chip), A. niger also modified pH, Ca2+ and oxalic acid concentrations. Co-inoculation with as little as 10 cells of the oxalatrophic bacterium strongly inhibited the germination and development of A. niger and returned each of the three parameters to the baseline physiological values of uninfected cells. This biocontrol interaction between oxalotrophic bacteria and oxalate-producing A. niger could represent a paradigm shift in the fight against opportunistic fungal pathogens, where the host environment is rendered less permissive to fungal development.

Burkholderia gladioli pv. alliicola, B.cepacia, and B. orbicola are common bacterial pathogens of onion. Onions produce organosulfur thiosulfinate defensive compounds after cellular decompartmentalization. Using whole genome sequencing and in silico analysis, we identified putative thiosulfinate tolerance gene (TTG) clusters in multiple onion-associated Burkholderia species similar to those characterized in other Allium-associated bacterial endophytes and pathogens. Sequence analysis revealed the presence of three Burkholderia TTG cluster types with both Type A and Type B being broadly distributed in B. gladioli, B. cepacia, and B. orbicola in both the chromosome and plasmids. Based on isolate natural variation and generation of isogenic strains, we determined the in vitro and in vivo contribution of TTG clusters in B. gladioli, B. cepacia, and B. orbicola. The Burkholderia TTG clusters contributed to enhanced allicin tolerance and improved growth in filtered onion extract by all three species. TTG clusters also made clear contributions to B gladioli foliar necrosis symptoms and bacterial populations. Surprisingly, the TTG cluster did not contribute to bacterial populations in onion bulb scales by these three species. Based on our findings, we hypothesize onion-associated Burkholderia may evade or inhibit the production of thiosulfinates in onion bulb tissues.

Streptococcus pneumoniae is a Gram-positive opportunistic pathogen that can colonize the upper respiratory tract. It is a leading cause of a wide range of infectious diseases, including community-acquired pneumonia, meningitis, otitis media and bacteraemia. Pneumococcal infections cause 1-2 million deaths per year, most of which occur in developing countries, where this bacterial species is probably the most important pathogen during early infancy. Here, we focused on choline-binding proteins (CBPs), i.e., PspC, PspA and LytA, and their integration into and interaction with the cell wall of S. pneumoniae. The three pneumococcal proteins have different surface-exposed regions but share related choline-binding anchors. These surface-exposed pneumococcal proteins are in direct contact with host cells and have diverse functions. PspC and PspA bind several host plasma proteins, whereas LytA plays a role in cell division and the lytic phase. We explored the role of the three CBPs on adhesion and pathogenicity in a human host by performing relevant imaging and functional analyses, such as electron microscopy, confocal laser scanning microscopy and functional quantitative assays targeting biofilm formation and the haemolytic capacity of S. pneumoniae. In vitro biofilm formation assays and electron microscopy experiments were used to examine the ability of knockout mutant strains lacking the lytA, pspC or pspA genes to adhere to surfaces. The mutant strains were compared with the S. pneumoniae D39 reference strain. We found that LytA plays an important role in robust synthesis of the biofilm matrix. PspA and PspC appeared crucial for the haemolytic effects of S. pneumoniae on human red blood cells. Furthermore, all knockout mutants caused less damage to endothelial cells than wild-type bacteria, highlighting the significance of CPBs for the overall pathogenicity of S. pneumoniae. Hence, in addition to their structural function within the cell wall of S. pneumoniae, each of these three surface-exposed CBPs controls or mediates multiple steps during bacterial pathogenesis.

Proteins with internal repeats (PIRs) are the second most abundant class of fungal cell wall resident proteins. In yeasts, PIRs preserve the wall stability under stressful conditions. They are characterized by conserved N-terminal amino acid sequences repeated in tandem (PIR domains), and a Cys-rich C-terminal domain. Despite PIRs have been inferred in several filamentous fungi genomes, they have not been studied beyond yeasts. In this work, PIRs diversity, evolution and biological role, focused on a new PIRs class, were addressed. Bioinformatic inference of PIRs in fungi indicated they were an innovation in Ascomycota. Predicted PIRs clustered in two main groups: classical yeasts PIRs (N-terminal PIR domains; C-terminal Cys-rich domain), and PIRs from filamentous fungi with an inverted architecture (N-terminal Cys-rich domain; C-terminal PIR domains), which could harbor additional GPI-signals. As representatives of the second group, Neurospora crassa (Nc) PIR-1 (NCU04033) and PIR-2 (NCU07569) were studied. Confocal microscopy of eGFP-labeled PIR-1 and PIR-2 revealed they accumulate in apical plugs; additionally, PIR-1 requires the Kex2 processing site for correct maturation, and its predicted C-terminal GPI modification signal resulted functional. Moreover, Nc {Delta}pir-1 and {Delta}pir-2 single mutants showed a growth rate similar to that of Nc WT, but the double mutant Nc {Delta}pir-1/{Delta}pir-2 grew significatively slower. Similarly, Nc {Delta}pir-1 and Nc {Delta}pir-2 were mildly sensitive to calcofluor white, although Nc {Delta}pir-1/{Delta}pir-2 double mutant was severely impaired. Despite the inverted architecture of PIR-1 and PIR-2, they resulted in cell wall stabilizers as classical yeast PIRs.

Clostridioides difficile is a Gram-positive and obligate anaerobic spore-former pathogen. C. difficile spores are essential for the transmission and recurrence of C. difficile infections (CDI). A major challenge in sporulation studies in C. difficile is the low yield and asynchronous nature of this process. In this work, a hypersporulating strain, derivative of R20291, with an early sporulation onset and enhanced sporulation efficiency was isolated by serendipity. This strain had 1000-fold higher sporulation efficiency than the parental R20291 strain in sessile culture conditions. Electron micrographs revealed that spores of both strains have similar hair-like projections, electron-dense outer exosporium layer features. Whole genome sequencing and genomic analyses revealed that the hypersporulating strain had a 2356 bp-deletion spanning three ORF, including a non-essential proC1 involved in proline metabolism, and a missense mutation in rsbV, an anti-anti-SigB factor of RsbW. These observations suggest that this RsbV-variant might contribute to constitutive repression of the SigB-dependent general stress response, and therefore, derepressing sporulation in this hypersporulating strain.

The biosynthesis of antioxidant pigments, namely, betalains, has predominantly been found in Caryophyllales. The potential betalains biosynthesis was firstly explored in Aspergillus sydowii H-1 under controlled culture conditions. This study identified, knocked out, and overexpressed genes involved in the betanin biosynthesis and assessed the activities of tyrosinase, 45-DOPA dioxygenase and LigB. The results indicated these betanin biosynthesis pathway was crucial for colony morphology, conidiogenesis, stress response, and violet pigment synthesis. Moreover, AsDODA and AsLigB catalyzed the conversion of L-DOPA into 45-seco-DOPA, a key intermediate in the formation of betalamic acid in vitro. Additionally, transcription factors such as AsbHLH, AsMYB1R, and AsWD40 positively regulated the expression of betalain biosynthesis genes. This research provides new insights into the evolutionary origins of betalain-producing species, expanding the scope of betalain biosynthesis to include Aspergillus species. ImportanceTo date, betalains were restricted to plants of the order Caryophyllales, fungi of Basidiomycota and several types of bacteria. This study is the first to demonstrate the potential of Ascomycetes A. sydowii H-1 to synthesize betalains under controlled culture conditions, providing a detailed genetic and biological characterization of the associated genes and metabolic pathways. This finding demonstrates that betalain biosynthesis can be expanded to other Aspergillus.

Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.

Currently, the fungal class Archaeosporomycetes consists of one order, Archaeosporales with four families: Archaeosporaceae, Ambisporaceae, Geosiphonaceae, and Polonosporaceae. In the present study, the objective was to re-analyze the phylogeny and morphology of the Archaeosporomycetes from order to genus level. The different ecological strategies and, consequently, distinct evolutionary patterns of these taxa, as well as their morphological characters and other data updated here, suggest the need to divide Archaeosporales into four orders: (i) the type order Archaeosporales, (ii) Ambisporales ord. nov., both with four genera, (iii) Geosiphonales and (iv) Polonosporales ord. nov., both with single families and genera. Remarkably, the order Geosiphonales was described in the past, but was not considered in the Archaeosporomycetes until now. Phylogenetically, the four main clades (orders here proposed) of Archaeosporomycetes are well supported, with bootstrap values higher than 95% in all analyses, except Ambisporales/Ambisporaceae for RAxML-NG FBP analysis in the SSU tree (75%). Ecologically, this class includes three orders of arbuscular mycorrhizal fungi (AMF) forming symbiotic associations with plants, while Geosiphonales form an endocytobiosis with the cyanobacterium Nostoc. Morphologically, there are at least two AMF orders with spore bimorphism, which has not (yet) been described for Polonosporales. The only known species of Polonosporales, Polonospora polonica, forms spores directly on the neck of sporiferous saccules and the spores can morphologically be differentiated from all other taxa in Archaeosporomycetes by the formation of three permanent, rather thick spore walls, of which two form de novo during spore formation. The outer spore wall of Archaeosporales and Ambisporales are semi-permanent, evanescent or even short-lived, or show multiple fissures during aging, when it is more resistant. Ambisporales can easily be differentiated from Archaeosporales for instance by larger spores of the acaulosporoid morph and thicker spore walls. Our phylogenetic analyses suggested that Archaeosporales can be divided into two families: Antiquisporaceae that was described to form intraradical hyphae, vesicles and spores, staining darkly in Trypan blue, and Archaeosporaceae whose hyphae generally do not or only faintly stain in this reagent, and vesicles and intraradical spores have been rarely, if ever reported.

O_LILysM effectors are suppressors of chitin-triggered plant immunity in biotrophic and hemibiotrophic fungi. Their role in necrotrophic fungi is unclear as these last are known to activate plant defenses and induce cell death. C_LIO_LITo characterize the role of the BcLysM1 gene encoding a putative LysM effector in the necrotrophic fungus Botrytis cinerea, its expression was followed by transcriptional fusion and by RT-qPCR in planta. Two tagged-recombinant proteins were produced, and two independent deletion strains were constructed and characterized. C_LIO_LIBcLysM1 is induced in the early phase of infection, and more specifically in multicellular appressoria called infection cushions. The BcLysM1 protein binds the chitin in the fungus cell wall and protects hyphae against degradation by external chitinases. It is also able to sequester chitooligosaccharides and to prevent them from inducing ROS production in A. thaliana. Using mycelium as inoculum, deletion strains show a delay in infection initiation and a default in adhesion to bean leaf surfaces. C_LIO_LIThis study demonstrates for the first time a dual role for a LysM effector in mycelium adhesion on the plant and in host defenses suppression, both of them occurring during the asymptomatic phase of infection by a necrotrophic fungus. C_LI

Slippery skin of onion caused by Burkholderia gladioli pv. alliicola (Bga) is a common bacterial disease reported from onion growing regions around the world. Despite the increasing attention in recent years, our understanding of the virulence mechanisms of this pathogen remains limited. In this study, we characterized the predicted genetic determinants of virulence in Bga strain 20GA0385 using reverse genetics approach. Using the closely related rice pathogen, B. glumae as a reference, comparative genomics analysis was performed to identify Bga candidate virulence factors and regulators. Marked and unmarked deletion mutants were generated using allelic exchange and the mutants were functionally validated using in vitro and in vivo assays. The role of mutants in pathogenic phenotypes was analyzed using onion foliar/seedling necrosis assays, the Red Scale Necrosis (RSN) assay and in planta bacterial population counts. The phytotoxin toxoflavin was a major contributor to foliar necrosis and bacterial populations whereas the type II and type III secretion system (T2SS/T3SS) were dispensable for foliar symptoms. In onion scale tissue, the T2SS single mutant gspC and its double and triple mutant derivatives all contributed to scale lesion area. Neither the lipocyclopeptide icosalide, toxoflavin, nor T3SS were required for scale symptoms. Our results suggest the quorum sensing tofIMR system in Bga regulates, toxoflavin, T2SS, and T3SS, contributing to onion symptom production. We show different virulence factors contribute to onion tissue-specific virulence patterns in Bga and that decreases in scale symptoms often do not result in decreased Bga populations in onion tissue.

Highlights- Xoo produces filamentous morphology, which is transient and yields pleomorphic progenies in vitro - In planta, initial attachment of rod-shaped Xoo is detected at xylem pits - The Xoo infection front migrates basipetally in the vascular bundle and progresses laterally from major to minor veins via transverse veins - Xoo breaks out of the xylem vessels and enter the neighboring xylem parenchyma - Xoo assumes filamentous morphology that can traverse from the xylem across the bundle sheath into mesophyll tissue - Mobility in xylem vessels depends predominantly on rod-shaped Xoo, while infection of mesophyll tissue at later stages appears to be linked to filamentous morphology SummaryXanthomonas oryzae pv. oryzae (Xoo) is classified as a xylem pathogen responsible for bacterial blight of rice causing substantial yield losses in Asia and Africa. Xoo virulence depends on the ability to trigger SWEET sucrose efflux transporters in the xylem parenchyma (XP) by injection of transcription activation like effectors (TALe) into host cells, likely to access host-derived sucrose. To establish infection, Xoo must overcome physical barriers, immune responses and the hydraulic xylem flow. To gain insights into the colonization process, we used translational SWEET11a-GUS reporter lines, scanning electron microscopy, and confocal laser scanning microscopy of Xoo tagged with a fluorescent protein. We found that Xoo can differentiate in vitro into filamentous forms. We mapped the infection route of Xoo along the vasculature, identified distinct spatiotemporal phases of Xoo colonization marked by rod-shaped and, notably, filamentous Xoo cells. Rod-shaped Xoo were found to attach to xylem pits during basipetal progression of the infection. Notably, we found that at later infection stages, Xoo could enter the XP. Strikingly, Xoo adopted a filamentous phenotype that traversed bundle sheath cells and entered mesophyll cells. Chlorosis and necrosis of leaves is thus likely not just due to blockage of xylem flow, but to direct tissue damage. Filamentation had been reported as important for virulence of human pathogens e.g. Yersinia pestis, uropathogenic E. coli and Shigella and had been associated to sugar utilization in Bacillus subtilis. We thus hypothesize that Xoo differentiation during host colonization is critical for virulence. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/680524v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@f66d8borg.highwire.dtl.DTLVardef@91ca66org.highwire.dtl.DTLVardef@17f2846org.highwire.dtl.DTLVardef@12d46e7_HPS_FORMAT_FIGEXP M_FIG C_FIG

The human pathogen Aspergillus fumigatus produces fusarinine-type (FusTS) and ferrichrome-type siderophores (FchTS), both of which have been shown to be crucial for virulence of this mold. After the common first siderophore biosynthetic step, SidA-catalyzed hydroxylation of ornithine, the pathway splits. For FusTS biosynthesis, SidF incorporates an anhydromevalonyl group, while for FchTS biosynthesis, SidL and an as yet unknown enzyme incorporate an acetyl group. The transacylases SidF and SidL share only limited similarity in their C-terminal GNAT (Gcn5-related N-acetyltransferases) motif-containing domains. SidF is transcriptionally induced by iron limitation and localizes to peroxisomes, whereas SidL is a cytosolic enzyme with largely iron-independent expression. Here, we discovered that simultaneous inactivation of both SidF and SidL abolished the biosynthesis of both FusTS and FchTS and caused a growth defect under iron limitation, similar to the inactivation of SidA. Biosynthesis of both FusTS and FchTS depended on both the unique N-terminal and the GNAT motif-containing C-terminal SidF domains. In conclusion, SidF is the hitherto unknown FchTS biosynthetic enzyme: in contrast to SidL, SidF is a bifunctional enzyme accepting acetyl-CoA and anhydromevalonyl-CoA as substrates for biosynthesis of both FusTS and FchTS. Furthermore, this study revealed interdependence of FusTS and FchTS production, and that the peroxisomal localization of FusTS enzymes is important for optimizing FusTS production at the expense of FchTS. Phylogenetic analyses supported the relevance of these findings to other fungal species and revealed overlapping but distinct consensus sequences for the GNAT motifs of SidL and SidF, most likely reflecting their different substrate specificities. IMPORTANCEAdaptation to the host niche is key for any pathogenic organism. Aspergillus fumigatus is a major fungal pathogen causing 90% of invasive aspergillosis cases, which is associated with a high mortality rate. Siderophore-mediated iron acquisition has been shown to be essential for virulence of A. fumigatus and other fungal pathogens. In recent years, the hyphal siderophore biosynthetic pathway has been largely elucidated with exception of a single unknown enzyme, which we identified here as SidF. In contrast to another siderophore biosynthetic acyltransferase, SidL, SidF is a bifunctional enzyme accepting different substrates. As simultaneous inactivation of SidF and SidL, which share a common protein domain and a common substrate, blocks the biosynthesis of all siderophores, simultaneous targeting of SidF and SidL may allow development of new antifungal drugs. Phylogenetic analyses supported the relevance of these findings to other fungal species Moreover, this study clarified the rational for partial peroxisomal localization of siderophore biosynthesis and their metabolic interdependence. The human pathogen Aspergillus fumigatus produces fusarinine-type (FusTS) and ferrichrome-type siderophores (FchTS), both of which have been shown to be crucial for virulence of this mold. After the common first siderophore biosynthetic step, SidA-catalyzed hydroxylation of ornithine, the pathway splits. For FusTS biosynthesis, SidF incorporates an anhydromevalonyl group, while for FchTS biosynthesis, SidL and an as yet unknown enzyme incorporate an acetyl group. The transacylases SidF and SidL share only limited similarity in their C-terminal GNAT (Gcn5-related N-acetyltransferases) motif-containing domains. SidF is transcriptionally induced by iron limitation and localizes to peroxisomes, whereas SidL is a cytosolic enzyme with largely iron-independent expression. Here, we discovered that simultaneous inactivation of both SidF and SidL abolished the biosynthesis of both FusTS and FchTS and caused a growth defect under iron limitation, similar to the inactivation of SidA. Biosynthesis of both FusTS and FchTS depended on both the unique N-terminal and the GNAT motif-containing C-terminal SidF domains. Taken together, SidF is the hitherto unknown FchTS biosynthetic enzyme: in contrast to SidL, SidF is a bifunctional enzyme accepting acetyl-CoA and anhydromevalonyl-CoA as substrates for biosynthesis of both FusTS and FchTS. Moreover, this study revealed interdependence of FusTS and FchTS production, and that peroxisomal localization of FusTS enzymes is important for optimizing FusTS production at the expense of FchTS.

Trichoderma harzianum is a filamentous ascomycete frequently applied as biocontrol agent in agriculture. While mycoparasitism and antagonism of Trichoderma spp. against fungal pathogens are well known, early fungal responses to the presence of a plant await broader investigation. Analyzing early stages of plant-fungus communication we show that T. harzianum B97 chemotropically responds to a plant extract and that both plant and fungus alter secondary metabolite secretion upon recognition. We developed a strategy for omics-analysis simulating conditions of early plant recognition eliciting a chemotropic response in the fungus and found 102 genes to be differentially regulated, including nitrate and nitrite reductases. Additionally, the previously uncharacterized Plant Communication Associated (PCA) gene cluster was strongly induced upon recognition of the plant, comprises a palindromic DNA motif and was essential for plant colonization. The PCA-cluster is only present in the Harzianum clade of Trichoderma and closely related to a homologous cluster in Metarhizium spp. Horizontal gene transfer (HGT) was detected for PCA-cluster genes by plants, while the cluster in T. harzianum is likely under balancing or positive selection. Hence, the PCA-cluster mediates early fungus-plant chemical communication and may be responsible for the high potential of T. harzianum and closely related species for biocontrol applications. Plain language summaryInteractions of plants with fungi - beneficial or pathogenic - are crucial for the ecological function of both partners. Yet, the chemical "language" they use and how or when they use it is still insufficiently known. We describe discovery of a novel gene cluster, which is strongly induced upon plant recognition and essential for plant-fungal interkingdom interaction in the biocontrol-agent Trichoderma harzianum.