Corn Smut Disease Research Articles

Effects of pollination and plant genotype on Ustilago maydis disease development on the ears of maize inbreds and maize‐teosinte near‐isogenic lines

AbstractUstilago maydis, the fungus that causes corn smut disease, leads to significant economic losses in maize cultivars. A key feature of successful plant pathogens is their ability to utilize the plant–pathogen relationship to influence disease progression. Greenhouse experiments examined how pollination and plant genotype affect disease incidence and severity of U. maydis infection. Four U. maydis susceptible maize inbreds (B73, H95, Mo17, and Golden Bantam), and two U. maydis resistant maize‐teosinte near‐isogenic lines (NIL1 and NIL2) were utilized for this work. Three‐hundred and sixty plants (pollinated and unpollinated) from the six plant genotypes were inoculated with U. maydis and assessed based on five phenotypic traits [(1) disease incidence, (2) gall number, (3) gall weight, (4) disease severity, and (5) area under disease progress curve]. All pollinated plants demonstrated significantly (p < .001) lower disease incidence, gall number, gall weight, area under the disease progress curve, and severity in comparison to the unpollinated plants. Both pollinated resistant NILs demonstrated significantly (p < .001) less disease development than the pollinated susceptible maize plants and two unpollinated NILs. Therefore, disease resistance to U. maydis was dependent upon pollination and plant genotype. This provides novel evidence that pollination can significantly improve resistance to U. maydis in different plant genotypes. Enhanced disease resistance observed in the resistant NILs after pollination indicates pollination‐mediated resistance is one of the resistance mechanisms functioning in the resistant NILs. Integration of pollination‐mediated resistance and resistance introgressed from a maize progenitor will be useful for improving resistance to U. maydis and management of the disease.

Klebsiella michiganensis: a nitrogen-fixing endohyphal bacterium from Ustilago maydis

Ustilago maydis is a pathogenic fungus in Basidiomycota causing corn smut disease. A strain of U. maydis YZZF202006 was isolated from the tumor of corn smut collected from Jingzhou city in China. The intracellular bacteria were confirmed inner hyphal of the strain YZZF202006 by PCR amplification and fluorescence in situ hybridization (FISH) and SYTO-9. An endohyphal bacterium YZUMF202001 was isolated from the protoplasts of the strain YZZF202006. It was gram-negative, short rod-shaped with smooth light yellow colony. The endohyphal bacterium was genomic evidenced as Klebsiella michiganensis on the basis of average nucleotide identity (ANI) analysis and the phylogram. Then K. michiganensis was GFP-Labeled and reintroduced into U. maydis, which confirmed the bacterium can live in hyphae of U.maydis. The bacterium can grow on N-free culture media. Its nitrogenase activity was reached av. 646.25 ± 38.61 nmol·mL− 1·h− 1 C2H4 by acetylene reduction assay. A cluster of nitrogen fixation genes (nifJHDKTXENXUSVWZMFLABQ) was found from its genome. The endohyphal K. michiganensis may play an important role to help nitrogen fixation for fungi in the future.

The Ustilago maydis AA10 LPMO is active on fungal cell wall chitin.

Lytic polysaccharide monooxygenases (LPMOs) can perform oxidative cleavage of glycosidic bonds in carbohydrate polymers (e.g., cellulose, chitin), making them more accessible to hydrolytic enzymes. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. The AA10 LPMOs are active on chitin and/or cellulose and mostly found in bacteria and in some viruses and archaea. Interestingly, AA10-encoding genes are also encountered in some pathogenic fungi of the Ustilaginomycetes class, such as Ustilago maydis, responsible for corn smut disease. Transcriptomic studies have shown the overexpression of the AA10 gene during the infectious cycle of U. maydis. In fact, U. maydis has a unique AA10 gene that codes for a catalytic domain appended with a C-terminal disordered region. To date, there is no public report on fungal AA10 LPMOs. In this study, we successfully produced the catalytic domain of this LPMO (UmAA10_cd) in Pichia pastoris and carried out its biochemical characterization. Our results show that UmAA10_cd oxidatively cleaves α- and β-chitin with C1 regioselectivity and boosts chitin hydrolysis by a GH18 chitinase from U. maydis (UmGH18A). Using a biologically relevant substrate, we show that UmAA10_cd exhibits enzymatic activity on U. maydis fungal cell wall chitin and promotes its hydrolysis by UmGH18A. These results represent an important step toward the understanding of the role of LPMOs in the fungal cell wall remodeling process during the fungal life cycle. IMPORTANCE Lytic polysaccharide monooxygenases (LPMOs) have been mainly studied in a biotechnological context for the efficient degradation of recalcitrant polysaccharides. Only recently, alternative roles and paradigms begin to emerge. In this study, we provide evidence that the AA10 LPMO from the phytopathogen Ustilago maydis is active against fungal cell wall chitin. Given that chitin-active LPMOs are commonly found in microbes, it is important to consider fungal cell wall as a potential target for this enigmatic class of enzymes.

Tetracycline-controlled (TetON) gene expression system for the smut fungus Ustilago maydis.

Ustilago maydis is a biotrophic phytopathogenic fungus that causes corn smut disease. As a well-established model system, U. maydis is genetically fully accessible with large omics datasets available and subject to various biological questions ranging from DNA-repair, RNA-transport, and protein secretion to disease biology. For many genetic approaches, tight control of transgene regulation is important. Here we established an optimised version of the Tetracycline-ON (TetON) system for U. maydis. We demonstrate the Tetracycline concentration-dependent expression of fluorescent protein transgenes and the system's suitability for the induced expression of the toxic protein BCL2 Associated X-1 (Bax1). The Golden Gate compatible vector system contains a native minimal promoter from the mating factor a-1 encoding gene, mfa with ten copies of the tet-regulated operator (tetO) and a codon optimised Tet-repressor (tetR*) which is translationally fused to the native transcriptional corepressor Mql1 (UMAG_05501). The metabolism-independent transcriptional regulator system is functional both, in liquid culture as well as on solid media in the presence of the inducer and can become a useful tool for toxin-antitoxin studies, identification of antifungal proteins, and to study functions of toxic gene products in Ustilago maydis.

Ustilago maydis Metabolic Characterization and Growth Quantification with a Genome-Scale Metabolic Model.

Ustilago maydis is an important plant pathogen that causes corn smut disease and serves as an effective biotechnological production host. The lack of a comprehensive metabolic overview hinders a full understanding of the organism’s environmental adaptation and a full use of its metabolic potential. Here, we report the first genome-scale metabolic model (GSMM) of Ustilago maydis (iUma22) for the simulation of metabolic activities. iUma22 was reconstructed from sequencing and annotation using PathwayTools, and the biomass equation was derived from literature values and from the codon composition. The final model contains over 25% annotated genes (6909) in the sequenced genome. Substrate utilization was corrected by BIOLOG phenotype arrays, and exponential batch cultivations were used to test growth predictions. The growth data revealed a decrease in glucose uptake rate with rising glucose concentration. A pangenome of four different U. maydis strains highlighted missing metabolic pathways in iUma22. The new model allows for studies of metabolic adaptations to different environmental niches as well as for biotechnological applications.

Seventeen Ustilaginaceae High-Quality Genome Sequences Allow Phylogenomic Analysis and Provide Insights into Secondary Metabolite Synthesis.

The family of Ustilaginaceae belongs to the order of Basidiomycetes. Despite their plant pathogenicity causing, e.g., corn smut disease, they are also known as natural producers of value-added chemicals such as extracellular glycolipids, organic acids, and polyols. Here, we present 17 high-quality draft genome sequences (N50 > 1 Mb) combining third-generation nanopore and second-generation Illumina sequencing. The data were analyzed with taxonomical genome-based bioinformatics methods such as Percentage of Conserved Proteins (POCP), Average Nucleotide Identity (ANI), and Average Amino Acid Identity (AAI) analyses indicating that a reclassification of the Ustilaginaceae family might be required. Further, conserved core genes were determined to calculate a phylogenomic core genome tree of the Ustilaginaceae that also supported the results of the other phylogenomic analysis. In addition, to genomic comparisons, secondary metabolite clusters (e.g., itaconic acid, mannosylerythritol lipids, and ustilagic acid) of biotechnological interest were analyzed, whereas the sheer number of clusters did not differ much between species.

Population Genomics of the Maize Pathogen Ustilago maydis: Demographic History and Role of Virulence Clusters in Adaptation.

The tight interaction between pathogens and their hosts results in reciprocal selective forces that impact the genetic diversity of the interacting species. The footprints of this selection differ between pathosystems because of distinct life-history traits, demographic histories, or genome architectures. Here, we studied the genome-wide patterns of genetic diversity of 22 isolates of the causative agent of the corn smut disease, Ustilago maydis, originating from five locations in Mexico, the presumed center of origin of this species. In this species, many genes encoding secreted effector proteins reside in so-called virulence clusters in the genome, an arrangement that is so far not found in other filamentous plant pathogens. Using a combination of population genomic statistical analyses, we assessed the geographical, historical, and genome-wide variation of genetic diversity in this fungal pathogen.We report evidence of two partially admixed subpopulations that are only loosely associated with geographic origin. Using the multiple sequentially Markov coalescent model, we inferred the demographic history of the two pathogen subpopulations over the last 0.5 Myr. We show that both populations experienced a recent strong bottleneck starting around 10,000 years ago, coinciding with the assumed time of maize domestication. Although the genome average genetic diversity is low compared with other fungal pathogens, we estimated that the rate of nonsynonymous adaptive substitutions is three times higher in genes located within virulence clusters compared with nonclustered genes, including nonclustered effector genes. These results highlight the role that these singular genomic regions play in the evolution of this pathogen.

An Unconventional Melanin Biosynthesis Pathway in Ustilago maydis.

Ustilago maydis is a phytopathogenic fungus responsible for corn smut disease. Although it is a very well-established model organism for the study of plant-microbe interactions, its potential to produce specialized metabolites, which might contribute to this interaction, has not been studied in detail. By analyzing the U. maydis genome, we identified a biosynthetic gene cluster whose activation led to the production of a black melanin pigment. Single deletion mutants of the cluster genes revealed that five encoded enzymes are required for the accumulation of the black pigment, including three polyketide synthases (pks3, pks4, and pks5), a cytochrome P450 monooxygenase (cyp4), and a protein with similarity to versicolorin B synthase (vbs1). Metabolic profiles of deletion mutants in this gene cluster suggested that Pks3 and Pks4 act in concert as heterodimers to generate orsellinic acid (OA), which is reduced to the corresponding aldehyde by Pks5. The OA-aldehyde can then react with triacetic acid lactone (TAL), also derived from Pks3/Pks4 heterodimers to form larger molecules, including novel coumarin derivatives. Our findings suggest that U. maydis synthesizes a novel type of melanin based on coumarin and pyran-2-one intermediates, while most fungal melanins are derived from 1,8-dihydroxynaphthalene (DHN) or l-3,4-dihydroxyphenylalanine (l-DOPA). Along with these observations, this work also provides insight into the mechanisms of polyketide synthases in this filamentous fungus.IMPORTANCE The fungus Ustilago maydis represents one of the major threats to maize plants since it is responsible for corn smut disease, which generates considerable economical losses around the world. Therefore, contributing to a better understanding of the biochemistry of defense mechanisms used by U. maydis to protect itself against harsh environments, such as the synthesis of melanin, could provide improved biological tools for tackling the problem and protect the crops. In addition, the fact that this fungus synthesizes melanin in an unconventional way, requiring more than one polyketide synthase for producing melanin precursors, gives a different perspective on the complexity of these multidomain enzymes and their evolution in the fungal kingdom.

Incompatibility between proliferation and plant invasion is mediated by a regulator of appressorium formation in the corn smut fungus Ustilago maydis

Plant pathogenic fungi often developed specialized infection structures to breach the outer surface of a host plant. These structures, called appressoria, lead the invasion of the plant by the fungal hyphae. Studies in different phytopathogenic fungi showed that appressorium formation seems to be subordinated to the cell cycle. This subordination ensures the loading in the invading hypha of the correct genetic information to proceed with plant infection. However, how the cell cycle transmits its condition to the genetic program controlling appressorium formation and promoting the plant's invasion is unknown. Our results have uncovered how this process occurs for the appressorium of Ustilago maydis, the agent responsible for corn smut disease. Here, we described that the complex Clb2-cyclin-dependent kinase (Cdk)1, one of the master regulators of G2/M cell cycle progression in U. maydis, interacts and controls the subcellular localization of Biz1, a transcriptional factor required for the activation of the appressorium formation. Besides, Biz1 can arrest the cell cycle by down-regulation of the gene encoding a second b-cyclin Clb1 also required for the G2/M transition. These results revealed a negative feedback loop between appressorium formation and cell cycle progression in U. maydis, which serves as a "toggle switch" to control the fungal decision between infecting the plant or proliferating out of the plant.

A fungal substrate mimicking molecule suppresses plant immunity via an inter-kingdom conserved motif

Ustilago maydis is a biotrophic fungus causing corn smut disease in maize. The secreted effector protein Pit2 is an inhibitor of papain-like cysteine proteases (PLCPs) essential for virulence. Pit2 inhibitory function relies on a conserved 14 amino acids motif (PID14). Here we show that synthetic PID14 peptides act more efficiently as PLCP inhibitors than the full-length Pit2 effector. Mass spectrometry shows processing of Pit2 by maize PLCPs, which releases an inhibitory core motif from the PID14 sequence. Mutational analysis demonstrates that two conserved residues are essential for Pit2 function. We propose that the Pit2 effector functions as a substrate mimicking molecule: Pit2 is a suitable substrate for apoplastic PLCPs and its processing releases the embedded inhibitor peptide, which in turn blocks PLCPs to modulate host immunity. Remarkably, the PID14 core motif is present in several plant associated fungi and bacteria, indicating the existence of a conserved microbial inhibitor of proteases (cMIP).

New Insights of Ustilago maydis as Yeast Model for Genetic and Biotechnological Research: A Review.

The basidiomycete Ustilago maydis is a biotrophic organism responsible for corn smut disease. In recent years, it has become one of the most promising models for biochemical and biotechnological research due to advantages, such as rapid growth, and easy genetic manipulation. In some aspects, this yeast is more similar to complex eukaryotes, such as humans, compared to standard laboratory yeast models. U. maydis can be employed as a tool to explore physiological processes with more versatility than other fungi. Previously, U. maydis was only considered as a phytopathogenic fungus, but different studies have shown its potential as a research model. Therefore, numerous promising studies have focused on deepening our understanding of the natural interactions, enzyme production, and biotechnological capacity. In this review, we explore general characteristics of U. maydis, both as pathogenic and "innocuous" basidiomycete. Additionally, a comparison with other yeast models focusing on genetic, biochemical, and biotechnological research are analyzed, to emphasize the versatility, dynamism, and novelty that U. maydis has as a research model. In this review, we highlight the applications of the yeast form of the fungus; however, since the filamentous form is also of relevance, it is addressed in the present work, as well.

The core effector Cce1 is required for early infection of maize by Ustilago maydis.

The biotrophic pathogen Ustilago maydis, the causative agent of corn smut disease, infects one of the most important crops worldwide – Zea mays. To successfully colonize its host, U. maydis secretes proteins, known as effectors, that suppress plant defense responses and facilitate the establishment of biotrophy. In this work, we describe the U. maydis effector protein Cce1. Cce1 is essential for virulence and is upregulated during infection. Through microscopic analysis and in vitro assays, we show that Cce1 is secreted from hyphae during filamentous growth of the fungus. Strikingly, Δcce1 mutants are blocked at early stages of infection and induce callose deposition as a plant defense response. Cce1 is highly conserved among smut fungi and the Ustilago bromivora ortholog complemented the virulence defect of the SG200Δcce1 deletion strain. These data indicate that Cce1 is a core effector with apoplastic localization that is essential for U. maydis to infect its host.

The fungal UmSrt1 and maize ZmSUT1 sucrose transporters battle for plant sugar resources.

The biotrophic fungus Ustilago maydis causes corn smut disease, inducing tumor formation in its host Zea mays. Upon infection, the fungal hyphae invaginate the plasma membrane of infected maize cells, establishing an interface where pathogen and host are separated only by their plasma membranes. At this interface the fungal and maize sucrose transporters, UmSrt1 and ZmSUT1, compete for extracellular sucrose in the corn smut/maize pathosystem. Here we biophysically characterized ZmSUT1 and UmSrt1 in Xenopus oocytes with respect to their voltage-, pH- and substrate-dependence and determined affinities toward protons and sucrose. In contrast to ZmSUT1, UmSrt1 has a high affinity for sucrose and is relatively pH- and voltage-independent. Using these quantitative parameters, we developed a mathematical model to simulate the competition for extracellular sucrose at the contact zone between the fungus and the host plant. This approach revealed that UmSrt1 exploits the apoplastic sucrose resource, which forces the plant transporter into a sucrose export mode providing the fungus with sugar from the phloem. Importantly, the high sucrose concentration in the phloem appeared disadvantageous for the ZmSUT1, preventing sucrose recovery from the apoplastic space in the fungus/plant interface.

Characterization of ApB73, a virulence factor important for colonization of Zea mays by the smut Ustilago maydis.

SummaryThe biotrophic fungus Ustilago maydis, the causal agent of corn smut disease, uses numerous small secreted effector proteins to suppress plant defence responses and reshape the host metabolism. However, the role of specific effectors remains poorly understood. Here, we describe the identification of ApB73 (Apathogenic in B73), an as yet uncharacterized protein essential for the successful colonization of maize by U. maydis. We show that apB73 is transcriptionally induced during the biotrophic stages of the fungal life cycle. The deletion of the apB73 gene results in cultivar‐specific loss of gall formation in the host. The ApB73 protein is conserved among closely related smut fungi. However, using virulence assays, we show that only the orthologue of the maize‐infecting head smut Sporisorium reilianum can complement the mutant phenotype of U. maydis. Although microscopy shows that ApB73 is secreted into the biotrophic interface, it seems to remain associated with fungal cell wall components or the fungal plasma membrane. Taken together, the results show that ApB73 is a conserved and important virulence factor of U. maydis that localizes to the interface between the pathogen and its host Zea mays.

Phytohormone sensing in the biotrophic fungus Ustilago maydis - the dual role of the transcription factor Rss1.

SummaryThe phenolic compound salicylic acid (SA) is a key signalling molecule regulating local and systemic plant defense responses, mainly against biotrophs. Many microbial organisms, including pathogens, share the ability to degrade SA. However, the mechanism by which they perceive SA is unknown. Here we show that Ustilago maydis, the causal agent of corn smut disease, employs a so far uncharacterized SA sensing mechanism. We identified and characterized the novel SA sensing regulator, Rss1, a binuclear zinc cluster protein with dual functions as putative SA receptor and transcriptional activator regulating genes important for SA and tryptophan degradation. Rss1 represents a major component in the identified SA sensing pathway during the fungus’ saprophytic stage. However, Rss1 does not have a detectable impact on virulence. The data presented in this work indicate that alternative or redundant sensing cascades exist that regulate the expression of SA‐responsive genes in U. maydis during its pathogenic development.

The putative phospholipase Lip2 counteracts oxidative damage and influences the virulence of Ustilago maydis.

Ustilago maydis is an obligate biotrophic fungal pathogen which causes common smut disease of corn. To proliferate in host tissue, U. maydis must gain access to nutrients and overcome plant defence responses, such as the production of reactive oxygen species. The elucidation of the mechanisms by which U. maydis meets these challenges is critical for the development of strategies to combat smut disease. In this study, we focused on the contributions of phospholipases (PLs) to the pathogenesis of corn smut disease. We identified 11 genes encoding putative PLs and characterized the transcript levels for these genes in the fungus grown in culture and during infection of corn tissue. To assess the contributions of specific PLs, we focused on two genes, lip1 and lip2, which encode putative phospholipase A2 (PLA2 ) enzymes with similarity to platelet-activating factor acetylhydrolases. PLA2 enzymes are known to counteract oxidative damage to lipids in other organisms. Consistent with a role in the mitigation of oxidative damage, lip2 mutants were sensitive to oxidative stress provoked by hydrogen peroxide and by increased production of reactive oxygen species caused by inhibitors of mitochondrial functions. Importantly, mutants defective in lip2, but not lip1, were attenuated for virulence in corn seedlings. Finally, a comparative analysis of fatty acid and cardiolipin profiles in the wild-type strain and a lip2 mutant revealed differences consistent with a protective role for Lip2 in maintaining lipid homeostasis and mitochondrial health during proliferation in the hostile host environment.

Susceptibility of maize hybrids to corn smut diseas

Corn smut disease is one of the most common maize diseases. In the previous years the disease lost from its importance, however nowadays it is becoming more and more important and widespread. The protection against it encounters great difficulties, therefore it is important to emphasize prevention and resistance based protection. The aim of the research was to investigate under laboratory circumstances the resistance of Hungary’s most commonly cultivated maize hybrids to corn smut disease. Twelve species were investigated. We distinguished two susceptibility groups within the hybrids based on the investigated parameters. The results are intended to assist the protection against corn smut disease and to make a suggestion for the farmers so that they could apply more environmental friendly and economical production.

White collar 1-induced photolyase expression contributes to UV-tolerance of Ustilago maydis.

Ustilago maydis is a phytopathogenic fungus causing corn smut disease. It also is known for its extreme tolerance to UV‐ and ionizing radiation. It has not been elucidated whether light‐sensing proteins, and in particular photolyases play a role in its UV‐tolerance. Based on homology analysis, U. maydis has 10 genes encoding putative light‐responsive proteins. Four amongst these belong to the cryptochrome/photolyase family (CPF) and one represents a white collar 1 ortholog (wco1). Deletion mutants in the predicted cyclobutane pyrimidine dimer CPD‐ and (6–4)‐photolyase were impaired in photoreactivation. In line with this, in vitro studies with recombinant CPF proteins demonstrated binding of the catalytic FAD cofactor, its photoreduction to fully reduced FADH − and repair activity for cyclobutane pyrimidine dimers (CPDs) or (6–4)‐photoproducts, respectively. We also investigated the role of Wco1. Strikingly, transcriptional profiling showed 61 genes differentially expressed upon blue light exposure of wild‐type, but only eight genes in the Δwco1 mutant. These results demonstrate that Wco1 is a functional blue light photoreceptor in U. maydis regulating expression of several genes including both photolyases. Finally, we show that the Δwco1 mutant is less tolerant against UV‐B due to its incapability to induce photolyase expression.

Influence of chitosan and its derivatives on cell development and physiology of Ustilago maydis

Ustilago maydis, a dimorphic fungus causing corn smut disease, serves as an excellent model to study different aspects of cell development. This study shows the influence of chitosan, oligochitosan and glycol chitosan on cell growth and physiology of U. maydis. These biological macromolecules affected the cell growth of U. maydis. In particular, it was found that chitosan completely inhibited U. maydis growth at 1mg/mL concentration. Microscopic studies revealed swellings on the surface of the cells treated with the polymers, and chitosan caused complete destruction of the membrane and formation of vesicles on the periphery of the cell. Oligochitosan and chitosan caused changes in oxygen consumption, K+ efflux and H+-ATPase activity. Oligochitosan induced a faster consumption of oxygen in the cells, while glycol chitosan provoked slower oxygen consumption. It is noteworthy that chitosan completely inhibited the fungal respiratory activity. The strongest effects were exhibited by chitosan in all evaluated aspects. These findings showed high sensitivity of U. maydis to chitosan and provided evidence for antifungal effects of chitosan derivatives. To our knowledge, this is a first report showing that chitosan and its derivatives affect the cell morphology and physiological processes in U. maydis.

Old-new disease is maize smut (Ustilago maydis)

Corn smut disease (Ustilago maydis) is one of the most common maize diseases. In the previous years the disease lost some from its importance, however nowadays it turns into more and more important and widespread one. The control encounters great difficulties, therefore it should be emphasized the preventive and resistance-based protection.