Gero Steinberg

Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant–microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no ‘true’ virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

Hyphal Growth: a Tale of Motors, Lipids, and the Spitzenkörper

Filamentous fungi are a large and evolutionarily successful group of organisms of enormous ecological importance ([27][1], [114][2]). Fungi also have a considerable impact on our economy because they serve as bio-factories for the industrial production of proteins ([90][3], [130][4]) and because

A dynein loading zone for retrograde endosome motility at microtubule plus‐ends

In the fungus Ustilago maydis, early endosomes move bidirectionally along microtubules (MTs) and facilitate growth by local membrane recycling at the tip of the infectious hypha. Here, we set out to elucidate the molecular mechanism of this process. We show that endosomes travel by Kinesin‐3 activity into the hyphal apex, where they reverse direction and move backwards in a dynein‐dependent manner. Our data demonstrate that dynein, dynactin and Lis1 accumulate at MT plus‐ends within the hyphal tip, where they provide a reservoir of inactive motors for retrograde endosome transport. Consistently, endosome traffic is abolished after depletion of the dynein activator Lis1 and in Kinesin‐1 null mutants, which was due to a defect in targeting of dynein and dynactin to the apical MT plus‐ends. Furthermore, biologically active GFP‐dynein travels on endosomes in retrograde and not in anterograde direction. Surprisingly, a CLIP170 homologue was neither needed for dynein localization nor for endosome transport. These results suggest an apical dynein loading zone in the hyphal tip, which ensure that endosomes reach the expanding growth region before they reverse direction.

A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis.

We identified a temperature‐sensitive mutant of the plant pathogenic fungus Ustilago maydis that is defective in the polar distribution of cell wall components and shows abnormal morphology. The affected gene, yup1, was cloned by complementation. It encodes a putative target soluble N‐ethylmaleimide‐sensitive fusion protein attachment protein receptor (t‐SNARE), suggesting a function in membrane fusion. A Yup1–GFP fusion protein localized to vesicles that showed rapid saltatory motion along microtubules. These vesicles are part of the endocytic pathway and accumulate at sites of active growth, thereby supporting the expansion of the hyphal tip. In yup1ts cells, endocytosis is impaired and accumulation of Yup1‐carrying endosomes at cell poles is abolished, resulting in apolar distribution of wall components and morphological alterations. This suggests that a membrane recycling process via early endosomes supports polar growth of U.maydis.

Kinesin is essential for cell morphogenesis and polarized secretion in Neurospora crassa

Kinesin is a force‐generating molecule that is thought to translocate organelles along microtubules, but its precise cellular function is still unclear. To determine the role of kinesin in vivo, we have generated a kinesin‐deficient strain in the simple cell system Neurospora crassa. Null cells exhibit severe alterations in cell morphogenesis, notably hyphal extension, morphology and branching. Surprisingly, the movement of organelles visualized by video microscopy is hardly affected, but apical hyphae fail to establish a Spitzenkörper, an assemblage of secretory vesicles intimately linked to cell elongation and morphogenesis in Neurospora and other filamentous fungi. As cell morphogenesis depends on polarized secretion, our findings demonstrate that a step in the secretory pathway leading to cell shape determination and cell elongation cannot tolerate a loss of kinesin function. The defect is suggested to affect the transport of small, secretory vesicles to the site involved in protrusive activity, resulting in the uncoordinated insertion of new cell wall material over much of the cell surface. These observations have implications for the presumptive function of kinesin in more complex cell systems.

A balance of KIF1A-like kinesin and dynein organizes early endosomes in the fungus Ustilago maydis

In Ustilago maydis, bidirectional transport of early endosomes is microtubule dependent and supports growth and cell separation. During early budding, endosomes accumulate at putative microtubule organizers within the bud, whereas in medium‐budded cells, endosome clusters appear at the growing ends of microtubules at the distal cell pole. This suggests that motors of opposing transport direction organize endosomes in budding cells. Here we set out to identify these motors and elucidate the molecular mechanism of endosome reorganization. By PCR we isolated kin3, which encodes an UNC‐104/KIF1‐like kinesin from U.maydis. Recombinant Kin3 binds microtubules and has ATPase activity. Kin3–green fluorescent protein moves along microtubules in vivo, accumulates at sites of growth and localizes to endosomes. Deletion of kin3 reduces endosome motility to ∼33%, and abolishes endosome clustering at the distal cell pole and at septa. This results in a transition from bipolar to monopolar budding and cell separation defects. Double mutant analysis indicates that the remaining motility in Δkin3‐mutants depends on dynein, and that dynein and Kin3 counteract on the endosomes to arrange them at opposing cell poles.

Identification of a Motor Protein Required for Filamentous Growth in Ustilago maydis

The phytopathogenic fungus Ustilago maydis exists in two stages, the yeast‐like haploid form and the filamentous dikaryon. Both pathogenicity and dimorphism are genetically controlled by two mating‐type loci, with only the filamentous stage being pathogenic on corn. We have identified two genes (kin1 and kin2) encoding motor proteins of the kinesin family. Kin1 is most similar to the human CENP‐E gene product, while Kin2 is most closely related to the conventional kinesin Nkin of Neurospora crassa. Deletion mutants of kin1 had no discernible phenotype; Δkin2 mutants, however, were severely affected in hyphal extension and pathogenicity. The wild‐type dikaryon showed rapid tip growth, with all the cytoplasm being moved to the tip compartment. Left behind are septate cell wall tubes devoid of cytoplasm. In Δkin2 mutants, dikaryotic cells were formed after cell fusion, but these hyphal structures remained short and filled with cytoplasm. A functional green fluorescent protein (GFP)–Kin2 fusion was generated and used to determine the localization of the motor protein by fluorescence microscopy. Inspection of the hyphal tips by electron microscopy revealed a characteristic accumulation of darkly stained vesicles which was absent in mutant cells. We suggest that the motor protein Kin2 is involved in organizing this specialized growth zone at the hyphal tip, probably by affecting the vectorial transport of vesicles.

Heterotrophic flagellates and other protists associated with oceanic detritus throughout the water column in the mid North Atlantic

Heterotrophic protists, mostly flagellates, encountered in association with marine detritus from various collections in the mid North Atlantic are described. About 40 species have been identified and are reported. Taxa reported here for the first time are: Caecitellus gen. nov. (Protista incertae sedis) and Ministeria marisola gen. nov., sp. nov. (Protista incertae sedis). The flagellates form a subset of the community of heterotrophic marine flagellates encountered in more productive marine sites. Most species are bacterivorous and small. The community extends to the ocean floor but the diversity is reduced in samples taken from greater depths. The decline in species diversity is linked also to a decline in numbers of individuals. We discuss these changes in relation to food supply and pressure effects.

Transient binding of dynein controls bidirectional long-range motility of early endosomes

In many cell types, bidirectional long-range endosome transport is mediated by the opposing motor proteins dynein and kinesin-3. Here we use a fungal model system to investigate how both motors cooperate in early endosome (EE) motility. It was previously reported that Kin3, a member of the kinesin-3 family, and cytoplasmic dynein mediate bidirectional motility of EEs in the fungus Ustilago maydis. We fused the green fluorescent protein to the endogenous dynein heavy chain and the kin3 gene and visualized both motors and their cargo in the living cells. Whereas kinesin-3 was found on anterograde and retrograde EEs, dynein motors localize only to retrograde organelles. Live cell imaging shows that binding of retrograde moving dynein to anterograde moving endosomes changes the transport direction of the organelles. When dynein is leaving the EEs, the organelles switch back to anterograde kinesin-3–based motility. Quantitative photobleaching and comparison with nuclear pores as an internal calibration standard show that single dynein motors and four to five kinesin-3 motors bind to the organelles. These data suggest that dynein controls kinesin-3 activity on the EEs and thereby determines the long-range motility behavior of the organelles.

Polar Localizing Class V Myosin Chitin Synthases Are Essential during Early Plant Infection in the Plant Pathogenic Fungus Ustilago maydis

Fungal chitin synthases (CHSs) form fibers of the cell wall and are crucial for substrate invasion and pathogenicity. Filamentous fungi contain up to 10 CHSs, which might reflect redundant functions or the complex biology of these fungi. Here, we investigate the complete repertoire of eight CHSs in the dimorphic plant pathogen Ustilago maydis. We demonstrate that all CHSs are expressed in yeast cells and hyphae. Green fluorescent protein (GFP) fusions to all CHSs localize to septa, whereas Chs5-GFP, Chs6-GFP, Chs7-yellow fluorescent protein (YFP), and Myosin chitin synthase1 (Mcs1)-YFP were found at growth regions of yeast-like cells and hyphae, indicating that they participate in tip growth. However, only the class IV CHS genes chs7 and chs5 are crucial for shaping yeast cells and hyphae ex planta. Although most CHS mutants were attenuated in plant pathogenicity, Δchs6, Δchs7, and Δmcs1 mutants were drastically reduced in virulence. Δmcs1 showed no morphological defects in hyphae, but Mcs1 became essential during invasion of the plant epidermis. Δmcs1 hyphae entered the plant but immediately lost growth polarity and formed large aggregates of spherical cells. Our data show that the polar class IV CHSs are essential for morphogenesis ex planta, whereas the class V myosin-CHS is essential during plant infection.