Draft Genome Sequence of the Phenazine-Producing \u3ci\u3ePseudomonas fluorescens\u3c/i\u3e Strain 2-79

Pseudomonas fluorescens strain 2-79, a natural isolate of the rhizosphere of wheat (Triticum aestivum L.), possesses antagonistic potential toward several fungal pathogens. We report the draft genome sequnce of strain 2-79, which comprises 5,674 protein-coding sequences

Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem

VelB/VeA/LaeA Complex Coordinates Light Signal with Fungal Development and Secondary Metabolism

Differentiation and secondary metabolism are correlated processes in fungi that respond to light. In Aspergillus nidulans , light inhibits sexual reproduction as well as secondary metabolism. We identified the heterotrimeric velvet complex VelB/VeA/LaeA connecting light-responding developmental regulation and control of secondary metabolism. VeA, which is primarily expressed in the dark, physically interacts with VelB, which is expressed during sexual development. VeA bridges VelB to the nuclear master regulator of secondary metabolism, LaeA. Deletion of either velB or veA results in defects in both sexual fruiting-body formation and the production of secondary metabolites

The velvet protein Vel1 controls initial plant root colonization and conidia formation for xylem distribution in Verticillium wilt.

The conserved fungal velvet family regulatory proteins link development and secondary metabolite production. The velvet domain for DNA binding and dimerization is similar to the structure of the Rel homology domain of the mammalian NF-κB transcription factor. A comprehensive study addressed the functions of all four homologs of velvet domain encoding genes in the fungal life cycle of the soil-borne plant pathogenic fungus Verticillium dahliae. Genetic, cell biological, proteomic and metabolomic analyses of Vel1, Vel2, Vel3 and Vos1 were combined with plant pathogenicity experiments. Different phases of fungal growth, development and pathogenicity require V. dahliae velvet proteins, including Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 heterodimers, which are already present during vegetative hyphal growth. The major novel finding of this study is that Vel1 is necessary for initial plant root colonization and together with Vel3 for propagation in planta by conidiation. Vel1 is needed for disease symptom induction in tomato. Vel1, Vel2, and Vel3 control the formation of microsclerotia in senescent plants. Vel1 is the most important among all four V. dahliae velvet proteins with a wide variety of functions during all phases of the fungal life cycle in as well as ex planta

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.ISSN:1664-302

Transcription Factor SomA Is Required for Adhesion, Development and Virulence of the Human Pathogen <i>Aspergillus fumigatus</i>

<div><p>The transcription factor Flo8/Som1 controls filamentous growth in <i>Saccharomyces cerevisiae</i> and virulence in the plant pathogen <i>Magnaporthe oryzae</i>. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS) domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from <i>Aspergillus fumigatus</i> rescued in yeast <i>flo8</i> mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. <i>A</i>. <i>fumigatus</i> SomA acts similarly to yeast Flo8 on the promoter of <i>FLO11</i> fused with reporter gene (<i>LacZ</i>) in <i>S</i>. <i>cerevisiae</i>. <i>FLO11</i> expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the <i>somA</i> gene in <i>A</i>. <i>fumigatus</i> resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of <i>somA</i> using the inducible Tet-on system. A adherence assay with the conditional <i>somA</i> expression strain indicated that SomA is required for biofilm formation. A <i>ptaB</i> deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls <i>A</i>. <i>fumigatus</i> biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of <i>A</i>. <i>fumigatus</i>. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen <i>A</i>. <i>fumigatus</i>.</p></div

SomA interacts with PtaB in <i>A</i>. <i>fumigatus</i>.

<p>(A) The abundance of SomA and PtaB was measured by LC/MS and estimated based on MaxQuant’s logarithmized label free quantification (log2 LFQ) intensities. High and intermediate LFQ intensities are shown in red and blue. Absence of peptides and low LFQ intensities are presented in black. (B) Western hybridization of GFP-Trap and RFP-Trap enrichments with α-GFP antibody. The single band in the RFP-Trap indicated by an arrow was identified as SomA-GFP by LC/MS with the given peptides. (C) Reciprocal western to (B) but an α-RFP antibody instead of the α-GFP antibody as a different probe. The double band (arrow) correspond to PtaB and SomA by LC/MS. For all experiments, the strains were grown in MM medium for 24 h at 37°C. Protein extracts were performed with either GFP-Trap or RFP-Trap beads and the eluted proteins were separated by 12% SDS-PAGE.</p

Model of the SomA and SomA-PtaB genetic network in <i>A</i>. <i>fumigatus</i>.

<p>The solid arrows indicate the presented data and the results of previous studies [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005205#ppat.1005205.ref025" target="_blank">25</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005205#ppat.1005205.ref028" target="_blank">28</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005205#ppat.1005205.ref030" target="_blank">30</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005205#ppat.1005205.ref056" target="_blank">56</a>]. We showed that SomA/PtaB complex is a transcriptional activator for downstream targets (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005205#ppat.1005205.g006" target="_blank">Fig 6</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005205#ppat.1005205.s003" target="_blank">S3 Fig</a>) and these proteins had different cellular functions in <i>A</i>. <i>fumigatus</i> (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005205#sec010" target="_blank">Discussion</a>).</p