Rhamnose synthase activity is required for pathogenicity of the vascular wilt fungus Verticillium dahliae.

The initial interaction of a pathogenic fungus with its host is complex and involves numerous metabolic pathways and regulatory proteins. Considerable attention has been devoted to proteins that play a crucial role in these interactions, with an emphasis on so-called effector molecules that are secreted by the invading microbe to establish the symbiosis. However, the contribution of other types of molecules, such as glycans, is less well appreciated. Here, we present a random genetic screen that enabled us to identify 58 novel candidate genes that are involved in the pathogenic potential of the fungal pathogen Verticillium dahliae, which causes vascular wilt diseases in over 200 dicotyledonous plant species, including economically important crops. One of the candidate genes that was identified concerns a putative biosynthetic gene involved in nucleotide sugar precursor formation, as it encodes a putative nucleotide-rhamnose synthase/epimerase-reductase (NRS/ER). This enzyme has homology to bacterial enzymes involved in the biosynthesis of the nucleotide sugar deoxy-thymidine diphosphate (dTDP)-rhamnose, a precursor of L-rhamnose, which has been shown to be required for virulence in several human pathogenic bacteria. Rhamnose is known to be a minor cell wall glycan in fungi and has therefore not been suspected as a crucial molecule in fungal-host interactions. Nevertheless, our study shows that deletion of the VdNRS/ER gene from the V. dahliae genome results in complete loss of pathogenicity on tomato and Nicotiana benthamiana plants, whereas vegetative growth and sporulation are not affected. We demonstrate that VdNRS/ER is a functional enzyme in the biosynthesis of uridine diphosphate (UDP)-rhamnose, and further analysis has revealed that VdNRS/ER deletion strains are impaired in the colonization of tomato roots. Collectively, our results demonstrate that rhamnose, although only a minor cell wall component, is essential for the pathogenicity of V. dahliae.

Colonization, competition, and dispersal of pathogens in fluid flow networks.

The colonization of bacteria in complex fluid flow networks, such as those found in host vasculature, remains poorly understood. Recently, it was reported that many bacteria, including Bacillus subtilis [1], Escherichia coli [2], and Pseudomonas aeruginosa [3, 4], can move in the opposite direction of fluid flow. Upstream movement results from the interplay between fluid shear stress and bacterial motility structures, and such rheotactic-like behavior is predicted to occur for a wide range of conditions [1]. Given the potential ubiquity of upstream movement, its impact on population-level behaviors within hosts could be significant. Here, we find that P. aeruginosa communities use a diverse set of motility strategies, including a novel surface-motility mechanism characterized by counter-advection and transverse diffusion, to rapidly disperse throughout vasculature-like flow networks. These motility modalities give P. aeruginosa a selective growth advantage, enabling it to self-segregate from other human pathogens such as Proteus mirabilis and Staphylococcus aureus that outcompete P. aeruginosa in well-mixed non-flow environments. We develop a quantitative model of bacterial colonization in flow networks, confirm our model in vivo in plant vasculature, and validate a key prediction that colonization and dispersal can be inhibited by modifying surface chemistry. Our results show that the interaction between flow mechanics and motility structures shapes the formation of mixed-species communities and suggest a general mechanism by which bacteria could colonize hosts. Furthermore, our results suggest novel strategies for tuning the composition of multi-species bacterial communities in hosts, preventing inappropriate colonization in medical devices, and combatting bacterial infections.

Spatial gene expression analysis in tomato hypocotyls suggests cysteine as key precursor of vascular sulfur accumulation implicated in Verticillium dahliae defense.

Verticillium dahliae is a prominent generator of plant vascular wilting disease and sulfur (S)-enhanced defense (SED) mechanisms contribute to its in-planta elimination. The accumulation of S-containing defense compounds (SDCs) including elemental S (S(0) ) has been described based on the comparison of two near-isogenic tomato (Solanum lycopersicum) lines differing in fungal susceptibility. To better understand the effect of S nutrition on V. dahliae resistance both lines were supplied with low, optimal or supraoptimal sulfate-S. An absolute quantification demonstrated a most effective fungal elimination due to luxury plant S nutrition. High-pressure liquid chromatography (HPLC) showed a strong regulation of Cys levels and an S-responsive GSH pool rise in the bulk hypocotyl. High-frequency S peak accumulations were detected in vascular bundles of resistant tomato plants after fungal colonization by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Global transcriptomic analysis suggested that early steps of the primary S metabolism did not promote the SDCs synthesis in the whole hypocotyl as gene expression was downregulated after infection. Enhanced S fertilization mostly alleviated the repressive fungal effect but did not reverse it. Upregulation of glutathione (GSH)-associated genes in bulk hypocotyls but not in vascular bundles indicated a global antioxidative role of GSH. To finally assign the contribution of S metabolism-associated genes to high S(0) accumulations exclusively found in the resistant tomato line, a spatial gene expression approach was applied. Laser microdissection of infected vascular bundles revealed a switch toward transcription of genes connected with cysteine (Cys) synthesis. The upregulation of LeOASTLp1 suggests a role for Cys as key precursor for local S accumulations (possibly S(0) ) in the vascular bundles of the V. dahliae-resistant tomato line.