A Myosin Light Chain Is Critical for Fungal Growth Robustness in Candida albicans

Hyphal tip growth is critical in a range of fungal pathogens, in particular for invasion into animal and plant tissues. In Candida albicans , as in many filamentous fungi, a cluster of vesicles, called a Spitzenkörper, is observed at the tip of growing hyphae that is thought to function as a vesicle supply center.

Pseudomonas Strains Induce Transcriptional and Morphological Changes and Reduce Root Colonization of Verticillium spp.

Phytopathogenic Verticillia cause Verticillium wilt on numerous economically important crops. Plant infection begins at the roots, where the fungus is confronted with rhizosphere inhabiting bacteria. The effects of different fluorescent pseudomonads, including some known biocontrol agents of other plant pathogens, on fungal growth of the haploid Verticillium dahliae and/or the amphidiploid Verticillium longisporum were compared on pectin-rich medium, in microfluidic interaction channels, allowing visualization of single hyphae, or on Arabidopsis thaliana roots. We found that the potential for formation of bacterial lipopeptide syringomycin resulted in stronger growth reduction effects on saprophytic Aspergillus nidulans compared to Verticillium spp. A more detailed analyses on bacterial-fungal co-cultivation in narrow interaction channels of microfluidic devices revealed that the strongest inhibitory potential was found for Pseudomonas protegens CHA0, with its inhibitory potential depending on the presence of the GacS/GacA system controlling several bacterial metabolites. Hyphal tip polarity was altered when V. longisporum was confronted with pseudomonads in narrow interaction channels, resulting in a curly morphology instead of straight hyphal tip growth. These results support the hypothesis that the fungus attempts to evade the bacterial confrontation. Alterations due to co-cultivation with bacteria could not only be observed in fungal morphology but also in fungal transcriptome. P. protegens CHA0 alters transcriptional profiles of V. longisporum during 2 h liquid media co-cultivation in pectin-rich medium. Genes required for degradation of and growth on the carbon source pectin were down-regulated, whereas transcripts involved in redox processes were up-regulated. Thus, the secondary metabolite mediated effect of Pseudomonas isolates on Verticillium species results in a complex transcriptional response, leading to decreased growth with precautions for self-protection combined with the initiation of a change in fungal growth direction. This interplay of bacterial effects on the pathogen can be beneficial to protect plants from infection, as shown with A. thaliana root experiments. Treatment of the roots with bacteria prior to infection with V. dahliae resulted in a significant reduction of fungal root colonization. Taken together we demonstrate how pseudomonads interfere with the growth of Verticillium spp. and show that these bacteria could serve in plant protection.

Modulation of fungal virulence through CRZ1 regulated F-BAR-dependent actin remodeling and endocytosis in chickpea infecting phytopathogen Ascochyta rabiei

Polarized hyphal growth of filamentous pathogenic fungi is an essential event for host penetration and colonization. The long-range early endosomal trafficking during hyphal growth is crucial for nutrient uptake, sensing of host-specific cues, and regulation of effector production. Bin1/Amphiphysin/Rvs167 (BAR) domain-containing proteins mediate fundamental cellular processes, including membrane remodeling and endocytosis. Here, we identified a F-BAR domain protein (ArF-BAR) in the necrotrophic fungus Ascochyta rabiei and demonstrate its involvement in endosome-dependent fungal virulence on the host plant Cicer arietinum. We show that ArF-BAR regulates endocytosis at the hyphal tip, localizes to the early endosomes, and is involved in actin dynamics. Functional studies involving gene knockout and complementation experiments reveal that ArF-BAR is necessary for virulence. The loss-of-function of ArF-BAR gene results in delayed formation of apical septum in fungal cells near growing hyphal tip that is crucial for host penetration, and impaired secretion of a candidate effector having secretory signal peptide for translocation across the endoplasmic reticulum membrane. The mRNA transcripts of ArF-BAR were induced in response to oxidative stress and infection. We also show that ArF-BAR is able to tubulate synthetic liposomes, suggesting the functional role of F-BAR domain in membrane tubule formation in vivo. Further, our studies identified a stress-induced transcription factor, ArCRZ1 (Calcineurin-responsive zinc finger 1), as key transcriptional regulator of ArF-BAR expression. We propose a model in which ArCRZ1 functions upstream of ArF-BAR to regulate A. rabiei virulence through a mechanism that involves endocytosis, effector secretion, and actin cytoskeleton regulation.

Targeted Quantification of Phosphorylation Sites Identifies STRIPAK-Dependent Phosphorylation of the Hippo Pathway-Related Kinase SmKIN3

ABSTRACT We showed recently that the germinal center kinase III (GCKIII) SmKIN3 from the fungus Sordaria macrospora is involved in sexual development and hyphal septation. Our recent extensive global proteome and phosphoproteome analysis revealed that SmKIN3 is a target of the striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex. Here, using protein samples from the wild type and three STRIPAK mutants, we applied absolute quantification by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in SmKIN3 and other septation initiation network (SIN) components, such as CDC7 and DBF2, as well as BUD4, acting downstream of SIN. For SmKIN3, we show that phosphorylation of S668 and S686 is decreased in mutants lacking distinct subunits of STRIPAK, while a third phosphorylation site, S589, was not affected. We constructed SmKIN3 mutants carrying phospho-mimetic and phospho-deficient codons for phosphorylation sites S589, S668, and S686. Investigation of hyphae in a ΔSmkin3 strain complemented by the S668 and S686 mutants showed a hyper-septation phenotype, which was absent in the wild type, the ΔSmkin3 strain complemented with the wild-type gene, and the S589 mutant. Furthermore, localization studies with SmKIN3 phosphorylation variants and STRIPAK mutants showed that SmKIN3 preferentially localizes at the terminal septa, which is distinctly different from the localization of the wild-type strains. We conclude that STRIPAK-dependent phosphorylation of SmKIN3 has an impact on controlled septum formation and on the time-dependent localization of SmKIN3 on septa at the hyphal tip. Thus, STRIPAK seems to regulate SmKIN3, as well as DBF2 and BUD4 phosphorylation, affecting septum formation. IMPORTANCE Phosphorylation and dephosphorylation of proteins are fundamental posttranslational modifications that determine the fine-tuning of their biological activity. Involved in this modification process is the recently identified striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex, which is evolutionarily conserved from fungi to humans. STRIPAK functions as a macromolecular assembly communicating through physical interactions with other conserved signaling protein complexes to constitute larger dynamic protein networks. Its function is implied in many cellular processes, such as signal transduction pathways, growth, and cellular differentiation. We applied absolute quantification of protein phosphorylation by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in signaling components that are linked to the STRIPAK complex. Using the filamentous fungus Sordaria macrospora, we provide evidence for the phosphorylation-dependent role of the Hippo-like germinal center kinase SmKIN3, which controls septum formation, and localize it in a time-dependent manner on septa at the hyphal tip.

Ramularia leaf spot: PCR-based methods reveal widespread distribution of Ramulariopsis pseudogycines and limited presence of R. gossypii in Brazil

Whilst Brazil is the fourth largest cotton producer globally, incidence of ramularia leaf spot (RLS) has decreased yield across all growing regions. In 2017-18 and 2018-19 growing seasons, ca. 300 fungal samples were collected from seven Brazilian states. Hyphal tip cultures were obtained for DNA extraction and amplification of partial sequences of the RNA polymerase II gene (RPB2), 28S rRNA, the ribosomal DNA internal transcribed spacers (ITS), actin (ACT), elongation factor (EF1-α) and histone H3 (HIS3) gene regions. Genetic diversity of the isolates was evaluated using fourteen molecular markers. Clade assignments based on the concatenated-sequence tree (RPB2, LSU, EF1-α, ITS, ACT, and HIS3) were identical to those in tree clades generated by RPB2-sequences, as well as in an RPB2 haplotype network, in an ISSR (TGTC)4 dendrogram, and based on morphological comparisons. In total, 252 out of 267 isolates were identified as Ramulariopsis pseudoglycines, indicating this species as the most widespread causal agent of cotton RLS in the Brazilian growing regions today. Validation of the ISSR (TGTC)4 primer as a tool to study the diversity and distribution of Ramulariopsis species will make it possible to carry out extensive RLS sampling studies worldwide.

Trade-off between Plasticity and Velocity in Mycelial Growth

ABSTRACT Tip-growing fungal cells maintain cell polarity at the apical regions and elongate by de novo synthesis of the cell wall. Cell polarity and tip growth rate affect mycelial morphology. However, it remains unclear how both features act cooperatively to determine cell shape. Here, we investigated this relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 μm-width channels of microfluidic devices. Since the channels are much narrower than the diameter of hyphae, any hypha growing through the channel must adapt its morphology. Live-cell imaging analyses revealed that hyphae of some species continued growing through the channels, whereas hyphae of other species often ceased growing when passing through the channels, or had lost apical polarity after emerging from the other end of the channel. Fluorescence live-cell imaging analyses of the Spitzenkörper, a collection of secretory vesicles and polarity-related proteins at the hyphal tip, in Neurospora crassa indicates that hyphal tip growth requires a very delicate balance of ordered exocytosis to maintain polarity in spatially confined environments. We analyzed the mycelial growth of seven fungal species from different lineages, including phytopathogenic fungi. This comparative approach revealed that the growth defects induced by the channels were not correlated with their taxonomic classification or with the width of hyphae, but, rather, correlated with the hyphal elongation rate. This report indicates a trade-off between morphological plasticity and velocity in mycelial growth and serves to help understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology, and pathogenicity. IMPORTANCE Cell morphology, which is controlled by polarity and growth, is fundamental for all cellular functions. However how polarity and growth act cooperatively to control cell shape remains unclear. Here we investigated their relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 μm-width channels of microfluidic devices. We found that most fast growing hyphae often lost the cell polarity after emerging from the channels, whereas slow growing hyphae retained polarity and continued growing, indicating a trade-off between plasticity and velocity in mycelial growth. These results serve to understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology and pathogenicity.

Deuterium-labeled Raman tracking of glucose accumulation and protein metabolic dynamics in Aspergillus nidulans hyphal tips

Filamentous fungi grow exclusively at their tips, where many growth-related fungal processes, such as enzyme secretion and invasion into host cells, take place. Hyphal tips are also a site of active metabolism. Understanding metabolic dynamics within the tip region is therefore important for biotechnology and medicine as well as for microbiology and ecology. However, methods that can track metabolic dynamics with sufficient spatial resolution and in a nondestructive manner are highly limited. Here we present time-lapse Raman imaging using a deuterium (D) tracer to study spatiotemporally varying metabolic activity within the hyphal tip of Aspergillus nidulans. By analyzing the carbon–deuterium (C–D) stretching Raman band with spectral deconvolution, we visualize glucose accumulation along the inner edge of the hyphal tip and synthesis of new proteins from the taken-up D-labeled glucose specifically at the central part of the apical region. Our results show that deuterium-labeled Raman imaging offers a broadly applicable platform for the study of metabolic dynamics in filamentous fungi and other relevant microorganisms in vivo.

The CHY-type zinc finger protein FgChy1 regulates polarized growth, pathogenicity, and microtubule assembly in Fusarium graminearum

Microtubules, as transport tracks, play important roles in hyphal tip growth in filamentous fungi, but microtubule-associated proteins involved in polarized growth remain unknown. Here, we found that one novel zinc finger protein, FgChy1, is required for microtubule morphology and polarized growth in Fusarium graminearum. The Fgchy1 mutant presented curved and directionless growth of hyphae. Importantly, the conidia and germ tubes of the Fgchy1 mutant exhibited badly damaged and less organized beta-tubulin cytoskeletons. Compared with the wild type, the Fgchy1 mutant lost the ability to maintain polarity and was also more sensitive to the anti-microtubule drugs carbendazim and nocodazole, likely due to the impaired microtubule cytoskeleton. Indeed, the hyphae of the wild type treated with nocodazole exhibited a morphology consistent with that of the Fgchy1 mutant. Interestingly, the disruption of FgChy1 resulted in the off-center localization of actin patches and the polarity-related polarisome protein FgSpa2 from the hyphal tip axis. A similar defect in FgSpa2 localization was also observed in the nocodazole-treated wild-type strain. In addition, FgChy1 is also required for conidiogenesis, septation, sexual reproduction, pathogenicity and deoxynivalenol production. Overall, this study provides the first demonstrations of the functions of the novel zinc finger protein FgChy1 in polarized growth, development and virulence in filamentous fungi.

FpgMBV1 Alleviates The Pathogenicity of Fusarium Pseudograminearum to Wheat Plants: A Megabirnavirus Case Study

Background: Fusarium pseudograminearum is the predomenant causal agent of devastating crown rot diseases in cereal crops around the world. Mycoviruses have attracted increasing attention as potential biological control agents on plant diseases. The unique mycoviruse isolated from F. pseudograminearum is Fusarium pseudograminearum megabirnavirus 1 (FpgMBV1), which is a new member of the family Megabirnaviridae. To determine the hypovirulence effects of FpgMBV1 on F. pseudograminearum to wheat plants is critical for the potential application of FpgMBV1 in the control of cereal crown rot disease. Methods: Hyphal tip cultures were conducted to obtain a FpgMBV1-free strain, named as FC136-2A-V－. A hyg gene was transformed into a highly virulent virus-negative stain WZ-8A of F. pseudograminearum to obtain the deduced strain WZ-8A-HygR-V－. WZ-8A-HygR-V－ was used in pairing culture with the FpgMBV1-carrying F. pseudograminearum strain FC136-2A to obtain a FpgMBV1-positive strain WZ-8A-HygR-V+. Then the two pairs of strains, FC136-2A-V－/FC136-2A and WZ-8A-HygR-V－/WZ-8A-HygR-V+, were used to determine the potential effect on F. pseudograminearum by the infection of FpgMBV1 through tests on the growth, sensitivity to stress and cellophane penetrating ability in vitro and the pathogenicity to wheat plants.Results: FpgMBV1 could be cured by hyphal tip culture and horizontally transferred between F. pseudograminearum strains through pairing culture. Reduction of FpgMBV1-carrying strains on hyphal growth were found under the treatment of 0.5% SDS. No differences in the growth rates of tested strains in the treatments with 1 M NaCl, 1 M KCl, or 15 mM H2O2. Comparing to virus-negative strains, the In vitro cellophane penetrating ability was lost in FpgMBV1-carrying strains. The disease index of wheat plants inoculated with FC136-2A-V- was significantly higher than that inoculated with FC136-2A, while the pathogenicity of WZ-8A-HygR-V+ reduced significantly comparing to that of WZ-8A-HygR-V－.Conclusions: FpgMBV1 is the causal agent of the low pathogenicity to wheat plants of its original host F. pseudograminearum strain FC136-2A. And FpgMBV1 could be horizontally transferred to another F. pseudograminearum strain and reduce its pathogenicity to wheat plants.