The generic position of Ustilago maydis, Ustilago scitaminea, and Ustilago esculenta (Ustilaginales)

Three species of smut fungi (Ustilaginales, Basidiomycota) of economic importance, Ustilago maydis on corn, U. scitaminea on sugar cane, and U. esculenta on Zizania latifolia, were investigated in order to define their systematic position using morphological characteristics of the sori, ultrastructure of teliospore walls, and molecular data of the LSU rDNA. LSU rDNA suggests that U. maydis and U. scitaminea belong to the genus Sporisorium. This has already been proposed for U. scitaminea, which develops sori with whip-shaped axes corresponding to columellae. U. maydis and U. scitaminea, like typical species of Sporisorium, present peridia and columellae in their sori. Therefore, U. scitaminea is called Sporisorium scitamineum. U. maydis, however, is not placed in the genus Sporisorium here, because ongoing investigation of molecular data from the ITS rDNA region yields contradictory results and because the name Sporisorium maydis is occupied by an imperfect fungus. U. esculenta is recognized as Yenia esculenta. This placement in a separate genus is based on molecular data and on unique teliospore ultrastructure, i.e. apically enlarged, partly confluent warts developing on a strongly folded plasmalemma, and the exosporium and endosporium forming part of the ornamentation. Part 193 in the series „Studies in Heterobasidiomycetes“ from the Botanical Institute, University of Tübingen.     

Ustilago maydis Produces Cytokinins and Abscisic Acid for Potential Regulation of Tumor Formation in Maize

The infection of maize (Zea mays) by the basidiomycete fungus Ustilago maydis leads to common smut of corn characterized by the production of tumors in susceptible aboveground plant tissues. LC-(ES)MS/MS profiles of abscisic acid (ABA) and 12 different cytokinins (CKs) were determined for infected and uninfected maize tissues over a time course following fungal exposure. Samples were taken at points corresponding to the appearance of disease symptoms. Axenic cultures of haploid and dikaryon forms of U. maydis were also profiled. This study confirmed the capability of Ustilago maydis to synthesize CKs, ABA, and auxin (IAA). It also provided evidence for the involvement of CK and ABA in the U. maydis-maize infection process. Significant quantities of CKs and ABA were detected from axenic cultures of U. maydis as was IAA. CKs and ABA levels were elevated in leaves and stems of maize after infection; notable was the high level of cis-zeatin 9-riboside. Variation among hormone profiles of maize tissues was observed at different time points during infection and between infections with nonpathogenic haploid and pathogenic dikaryon strains. This suggested that CKs and ABA accumulate and are likely metabolized in maize tissue infected with U. maydis. Because U. maydis produced these phytohormones at significant levels, it is possible that the fungal pathogen is a source of these compounds in infected tissue. This is the first study to confirm the production of CKs and document the production of ABA by U. maydis. This study also established an involvement of these phytohormones and a possible functional role for ABA in U. maydis infection of maize.     

The autophagy genes atg8 and atg1 affect morphogenesis and pathogenicity in Ustilago maydis

Autophagy is a complex degradative process in which cytosolic material, including organelles, is randomly sequestered within double‐membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, the autophagy genes ATG1 and ATG8 are crucial for autophagy induction and autophagosome assembly, respectively, and their deletion has an impact on the autophagic potential of the corresponding mutant strains. We were interested in the role of autophagy in the development and virulence of U. maydis. Using a reverse genetic approach, we showed that the U. maydis ATG8 orthologue, atg8, is associated with autophagy‐dependent processes. Deletion of atg8 abolished autophagosome accumulation in the vacuoles of carbon‐starved cells and drastically reduced the survival of U. maydisΔatg8 mutant strains during these conditions. In addition, atg8 deletion had an impact on the budding process during saprobic haploid growth. The infection of maize with compatible Δatg8 strains resulted in fewer galled plants, and fungal gall colonization was strongly reduced, as reflected by the very low hyphal density in these tissues. Δatg8 infections resulted in the formation of very few teliospores. To corroborate the role of autophagy in U. maydis development, we also deleted the ATG1 orthologue, atg1. Deletion of atg1 yielded phenotypes similar to the Δatg8 strains during saprobic growth, but of lower magnitude. The Δatg1 strains were only slightly less pathogenic than the wild‐type and teliospore production was not affected. Surprisingly, atg1 deletion in the Δatg8 background exacerbated those phenotypes already observed in the Δatg8 and Δatg1 single‐mutant strains, strongly suggesting an additive phenotype. In particular, the double mutant was completely suppressed for plant gall induction.     

Initiation of Meiotic Recombination in Ustilago maydis

A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar⁺ recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.     

Differential gene expression during teliospore germination in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Ustilago maydis is a model fungal pathogen that induces the formation of tumors in maize. The tumor provides an environment for hyphal differentiation, leading to the formation of thick-walled, diploid teliospores. Such spores serve as a dispersal agent for smut and rust fungi, and their germination leads to new rounds of infection. The morphological changes that occur during teliospore germination in U. maydis have been described in detail. However, the specific molecular events that facilitate this process have not been identified. Through the construction and hybridization of microarrays containing a set of 3918 non-redundant cDNAs, we have identified genes that are differentially regulated during teliospore germination. Teliospores induced to germinate for 4 and 11 h were selected for comparison with dormant teliospores. Genes identified as differentially expressed included many that are presumably involved in as yet undescribed molecular events during teliospore germination, as well as characterized genes previously shown to be required for the process. This study represents the first large-scale investigation of changes in gene expression during teliospore germination.Electronic Supplementary Material Supplementary material is available for this article at     

Conditional gene expression reveals stage-specific functions of the unfolded protein response in the Ustilago maydis–maize pathosystem

Ustilago maydis is a model organism for the study of biotrophic plant–pathogen interactions. The sexual and pathogenic development of the fungus are tightly connected since fusion of compatible haploid sporidia is prerequisite for infection of the host plant, maize (Zea mays). After plant penetration, the unfolded protein response (UPR) is activated and required for biotrophic growth. The UPR is continuously active throughout all stages of pathogenic development in planta. However, since development of UPR deletion mutants stops directly after plant penetration, the role of an active UPR at later stages of development remained to be determined. Here, we established a gene expression system for U. maydis that uses endogenous, conditionally active promoters to either induce or repress expression of a gene of interest during different stages of plant infection. Integration of the expression constructs into the native genomic locus and removal of resistance cassettes were required to obtain a wild-type-like expression pattern. This indicates that genomic localization and chromatin structure are important for correct promoter activity and gene expression. By conditional expression of the central UPR regulator, Cib1, in U. maydis, we show that a functional UPR is required for continuous plant defence suppression after host infection and that U. maydis relies on a robust control system to prevent deleterious UPR hyperactivation.     

Identification of a biosynthesis gene cluster for flocculosin a cellobiose lipid produced by the biocontrol agent Pseudozyma flocculosa

Flocculosin is an antifungal glycolipid produced by the biocontrol fungus Pseudozyma flocculosa. It consists of cellobiose, O‐glycosidically linked to 3,15,16‐trihydroxypalmitic acid. The sugar moiety is acylated with 2‐hydroxy‐octanoic acid and acetylated at two positions. Here we describe a gene cluster comprising 11 genes that are necessary for the biosynthesis of flocculosin. We compared the cluster with the biosynthesis gene cluster for the highly similar glycolipid ustilagic acid (UA) produced by the phytopathogenic fungus Ustilago maydis. In contrast to the cluster of U. maydis, the flocculosin biosynthesis cluster contains an additional gene encoding an acetyl‐transferase and is lacking a gene homologous to the α‐hydroxylase Ahd1 necessary for UA hydroxylation. The functions of three acyl/acetyl‐transferase genes (Fat1, Fat2 and Fat3) including the additional acetyl‐transferase were studied by complementing the corresponding U. maydis mutants. While P. flocculosa Fat1 and Fat3 are homologous to Uat1 in U. maydis, Fat2 shares 64% identity to Uat2, a protein involved in UA biosynthesis but with so far unknown function. By genetic and mass spectrometric analysis, we show that Uat2 and Fat2 are necessary for acetylation of the corresponding glycolipid. These results bring unique insights into the biocontrol properties of P. flocculosa and opportunities for enhancing its activity.     

Life cycle and nuclear behavior in three rust fungi (Uredinales)

Kuehneola japonica has a microcyclic life cycle with a regular alternation of generations. Single basidiospore inoculations onto Rosa wichuraiana resulted in teliospore production, indicating its homothallic nature. Dikaryotization in a vegetative mycelium in the host seemed to occur through nuclear division that was not followed by septum formation. Karyogamy and meiosis took place through teliospore and metabasidium development; this fungus was considered to reproduce genetically homogeneous progenies. Puccinia lantanae and P. patriniae were also microcyclic in their life cycle; however, these fungi differed from K. japonica in the mode of nuclear behavior. In the former two fungi, both vegetative and reproductive cells were uninucleate. No karyogamy was observed, and nuclear division in the metabasidium development was thought to be mitotic. In P. lantanae, a basidiospore was formed on a sterigma, whereas a whiplike hypha emerged from each metabasidium cell in P. patriniae. Inoculations of Justicia procumbens with a single basidiospore of P. lantanae resulted in teliospore production. The fungus seemed to remain uninucleate, either haploid or diploid, throughout the life cycle. Thus, reproduction was considered to be apomictic. Received: August 16, 2001 / Accepted: October 1, 2001     

Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut

The corn smut fungus, Ustilago maydis, is a global pathogen responsible for extensive agricultural losses. Control of corn smut using traditional breeding has met with limited success because natural resistance to U. maydis is organ specific and involves numerous maize genes. Here, we present a transgenic approach by constitutively expressing the Totivirus antifungal protein KP4, in maize. Transgenic maize plants expressed high levels of KP4 with no apparent negative impact on plant development and displayed robust resistance to U. maydis challenges to both the stem and ear tissues in the greenhouse. More broadly, these results demonstrate that a high level of organ independent fungal resistance can be afforded by transgenic expression of this family of antifungal proteins.     

Induction of apoptosis‐like cell death and clearance of stress‐induced intracellular protein aggregates: dual roles for Ustilago maydis metacaspase Mca1

Metacaspases primarily associate with induction and execution of programmed cell death in protozoa, fungi and plants. In the recent past, several studies have also demonstrated cellular functions of metacaspases other than cell death in different organisms including yeast and protozoa. This study shows similar dual function for the only metacaspase of a biotrophic phytopathogen, Ustilago maydis. In addition to a conventional role in the induction of cell death, Mca1 has been demonstrated to play a key role in maintaining the quality of the cellular proteome. On one hand, Mca1 could be shown to bring about apoptosis‐like phenotypic changes in U. maydis on exposure to oxidative stress, on the other hand, the protein was found to regulate cellular protein quality control. U. maydis metacaspase has been found to remain closely associated with the insoluble intracellular protein aggregates, generated during an event of stress exposure to the fungus. The study, therefore, provides direct evidence for a role of U. maydis metacaspase in the clearance of the stress‐induced intracellular insoluble protein aggregates. Furthermore, host infection assays with mca1 deletion strain also revealed a role of the protein in the virulence of the fungus.     

Sporisorium scitamineum colonisation of sugarcane genotypes susceptible and resistant to smut revealed by GFP‐tagged strains

Sporisorium scitamineum is the causal agent of sugarcane smut disease. The fungus establishes a biotrophic interaction with sugarcane tissues, and unlike smut fungi of other monocot hosts, the primary meristem of sugarcane plants develops a whip‐like structure instead of a tumour‐like galls emerging from floral structures (tassels and ears). We examined (GFP)‐tagged S. scitamineum infecting tissues of three sugarcane genotypes with distinct responses to smut (susceptible, intermediate resistant and resistant). Mating compatible haploid cells gfp‐expressing were obtained by Agrobacterium tumefaciens‐mediated transformation (ATMT) using the integrative vector pFAT‐gfp. Regardless of the inoculation method (drop inoculation and hypodermal syringe inoculation), all genotypes were colonised by the fungus. GFP‐tagged strains of opposite mating reaction were able to: (a) grow in vitro as fluorescent yeast‐like cells; (b) generate infectious dikaryon; (c) penetrate sugarcane tissues; (d) colonise tissues by growing a filamentous network; and (e) form the characteristic highly branched hyphae within host cells. Fungal colonisation 160 DAI revealed an association of the fungus with vascular vessels disrupting their organisation in all three genotypes analysed. However, the resistant plants did not develop whips spanning the experiment time. The first whips emerged 76 DAI from plants of the susceptible genotype whereas for intermediate resistant plants whips were detected at 137 DAI. These whips were dissected and fluorescent sporogenesis and teliospore maturation were analysed. In vitro germination of recovered teliospores revealed after meiosis the formation of a three‐celled hyphal filament, where the fourth cell was likely maintained in the teliospore coat. These cells showed independent segregation of the gfp marker, as a result of gfp insertions in different chromosomes of each compatible haploid strain. This work presents the complete fungal life cycle of GFP‐marked S. scitamineum to study developmental stages in planta.