Scott E. Gold

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.

The Ustilago maydis regulatory subunit of a cAMP-dependent protein kinase is required for gall formation in maize.

In the plant, filamentous growth is required for pathogenicity of the corn smut pathogen Ustilago maydis. Earlier, we identified a role for the cAMP signal transduction pathway in the switch between budding and filamentous growth for this fungus. A gene designated ubc1 (for Ustilago bypass of cyclase) was found to be required for filamentous growth and to encode the regulatory subunit of a cAMP-dependent protein kinase (PKA). Here, we show that ubc1 is important for the virulence of the pathogen. Specifically, ubc1 mutants are able to colonize maize plants and, like the wild-type pathogen, cause localized symptoms in association with the presence of hyphae. However, in contrast to plants infected with wild-type cells that often developed galls from initially chlorotic tissue, plants infected with the ubc1 mutant did not produce galls. These data suggest that PKA regulation is critical for the transition from saprophytic to pathogenic growth and from vegetative to reproductive development. Plate mating assays in which exogenous cAMP was applied suggested that the cAMP and b mating-type morphogenetic pathways may be coordinated.

A MAP kinase encoded by the ubc3 gene of Ustilago maydis is required for filamentous growth and full virulence

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this, we have taken a forward genetic approach. Previously, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilagobypass of cyclase, as they no longer require the production of cAMP to grow in the budding morphology. Complementation of one of these suppressor mutants led to the identification of ubc3, which is required for filamentous growth and encodes a MAP kinase most similar to those of the yeast pheromone response pathway. In addition to filamentous growth, the ubc3 gene is required for pheromone response and for full virulence. Mutations in the earlier identified fuz7 MAP kinase kinase also suppress the filamentous phenotype of the uac1 disruption mutant, adding evidence that both ubc3 and fuz7 are members of this same MAP kinase cascade. These results support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.

The Ustilago maydis ubc4 and ubc5 genes encode members of a MAP kinase cascade required for filamentous growth.

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this we have taken a forward genetic approach. Earlier, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cyclic AMP (cAMP) to grow in the budding morphology. Complementation of a subset of these suppressor mutants led to the identification of the ubc4 and ubc5 genes, which are required for filamentous growth and encode a MAP (mitogen-activated protein) kinase kinase kinase and a MAP kinase kinase, respectively. Evidence suggests that they are important in the pheromone response pathway and in pathogenicity. These results further support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.

Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism

Nutrient sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence, for both animal and plant pathogens. Dimorphic pathogens such as the phytopathogenic smut fungi, Ustilago maydis and Microbotryum violaceum, must switch from a yeast‐like to a filamentous form in order to cause disease. Two genes encoding methylammonium permeases (MEPs) were identified from each of these latter fungi and all the encoded proteins were most similar to Mep2p, the high‐affinity permease from Saccharomyces cerevisiae that plays a direct role in pseudohyphal or filamentous growth for that organism. This is the first report of MEPs from pathogenic fungi. The two genes from U. maydis and one of the genes from M. violaceum were expressed in diploid S. cerevisiae mutants deleted for all three mep genes (mep1mep2mep3). Each of the heterologous genes could complement the severe growth defect of the S. cerevisiae mutant on low ammonium. Moreover, the U. maydis ump2 gene, initially detected as an upregulated gene in budding cells, was also able to complement the pseudohyphal defect characteristic of the mutant yeast. This gene is thus one of few heterologous MEP genes capable of efficiently restoring pseudohyphal growth in yeast. For U. maydis, disruption of ump2 eliminated the filamentous phenotype of haploid cells on low ammonium, while ump1 disruption only slightly reduced methylamine uptake. The most significant drop in methylamine uptake was seen for the ump2 and the ump1ump2 double mutants. Moreover, when grown in liquid medium, the ump1ump2 double mutant aggregated and sedimented. Also, the importance of a putative site for phosphorylation by protein kinase A was investigated in both Mep2p and Ump2p via site‐directed mutagenesis of the respective genes. A mutation predicted to prevent phosphorylation of either protein, still allowed each to provide growth on low ammonium, but eliminated their abilities to provide pseudohyphal growth for the S. cerevisiae triple mutant. These findings allow us to present a model of how ammonium transporters play a role in regulating dimorphic growth in fungi.

The ubc2 gene of Ustilago maydis encodes a putative novel adaptor protein required for filamentous growth, pheromone response and virulence.

The Basidiomycete fungus Ustilago maydis causes corn smut disease and alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. Previous work demonstrated that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Suppressor mutants of a uac1 disruption strain, named ubc for Ustilagobypass of cyclase, no longer require cAMP for the budding morphology. The ubc2 gene was isolated by complementation and is required for filamentous growth. The deduced amino acid sequence encoded by ubc2 shows localized homology to Sterile Alpha Motif (SAM), Ras Association (RA) and Src homology 3 (SH3) protein–protein interaction domains. A K78E missense mutation within the SAM domain, revealed a genetic interaction between ubc2 and ubc4, a pheromone‐responsive MAP kinase kinase kinase. This indicates involvement of ubc2 in the pheromone‐responsive MAP kinase cascade and ubc2 is required for pheromone‐responsive morphogenesis. The ubc2 gene is a critical virulence factor. Thus, ubc2 encodes a putative novel adaptor protein that may act directly upstream of the pheromone‐responsive MAP kinase cascade in U. maydis.

Genetics of morphogenesis and pathogenic development of Ustilago maydis.

Ustilago maydis has emerged as an important model system for the study of fungi. Like many fungi, U. maydis undergoes remarkable morphological transitions throughout its life cycle. Fusion of compatible, budding, haploid cells leads to the production of a filamentous dikaryon that penetrates and colonizes the plant, culminating in the production of diploid teliospores within fungal-induced plant galls or tumors. These dramatic morphological transitions are controlled by components of various signaling pathways, including the pheromone-responsive MAP kinase and cAMP/PKA (cyclic AMP/protein kinase A) pathways, which coregulate the dimorphic switch and sexual development of U. maydis. These signaling pathways must somehow cooperate with the regulation of the cytoskeletal and cell cycle machinery. In this chapter, we provide an overview of these processes from pheromone perception and mating to gall production and sporulation in planta. Emphasis is placed on the genetic determinants of morphogenesis and pathogenic development of U. maydis and on the fungus-host interaction. Additionally, we review advances in the development of tools to study U. maydis, including the recently available genome sequence. We conclude with a brief assessment of current challenges and future directions for the genetic study of U. maydis.

MAP kinase and cAMP signaling pathways modulate the pH-induced yeast-to-mycelium dimorphic transition in the corn smut fungus Ustilago maydis.

Acid pH induces the yeast-to-mycelium transition in haploid cells of Ustilago maydis. We tested two signal transduction pathways known to be involved in dimorphism for roles in acid-induced filamentation. In wild-type cells intracellular cAMP levels were reduced under acid growth. A mutant defective in the regulatory subunit of PKA, ubc1, failed to respond to acid induction on solid medium, but in liquid medium showed a mycelial phenotype at acid pH. Mutants in the pheromone-responsive MAP kinase pathway lost the capacity to grow as mycelium at acid pH, while a mutant in the pheromone response-transcriptional regulator, prf1, behaved as wild-type. Filamentation by both ubc1 and prf1 mutants was inhibited by addition of cAMP. A putative MAP kinase cascade adaptor protein gene, ubc2, complemented a previously identified myc mutant strain defective in pH-induced myceliation. These results indicate that pH-dependent dimorphism is regulated by two known signaling pathways but that an effector for cAMP signaling alternative to Ubc1 is present in U. maydis and that Prf1 is not the sole downstream target of MAP kinase signaling.

Three selectable markers for transformation of Ustilago maydis.

Although Ustilago maydis is readily amenable to molecular genetic experimentation, few antibiotic-resistance markers are available for DNA-mediated transformation. This poses constraints on experiments involving targeted gene disruption and complementation. To address this problem, we constructed vectors using one of three additional genes as dominant selectable markers for transformation. Two genes, sat-1 (encoding streptothricin acetyltransferase) and Sh-ble (encoding a phleomycin-resistance polypeptide), are of bacterial origin and have been engineered for expression in Ustilago sp. The third gene encodes an allele of U. maydis beta-tubulin that confers resistance to the fungicide benomyl.

Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis.

The phytopathogenic fungus Ustilago maydis exhibits a dimorphic transition in which non‐pathogenic, yeast‐like cells mate to form a pathogenic, filamentous dikaryon. Northern analysis indicated that two chitin synthase genes, chs1 and chs2, from U. maydis are expressed at similar levels in yeast‐like cells and in cells undergoing the mating reaction leading to the filamentous cell type. A mutation was constructed in each of the chitin synthase genes by targeted gene disruption. Each mutant showed a reduction in the level of trypsin‐activated enzyme activity, compared with a wild‐type strain, but retained the wild‐type morphology, the ability to mate and the ability to form the filamentous pathogenic cell type.