Journal of Fungi

Editorial

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4 pages, 202 KiB

Open AccessEditorial

Smuts to the Power of Three: Biotechnology, Biotrophy, and Basic Biology

by Jan Schirawski, Michael H. Perlin and Barry J. Saville

J. Fungi 2021, 7(8), 660; https://doi.org/10.3390/jof7080660 - 14 Aug 2021

Viewed by 1942

Abstract

Smut fungi are a large group of mainly biotrophic plant pathogens, many of which cause disease on cereal crops [...] Full article

(This article belongs to the Special Issue Smut Fungi)

Research

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21 pages, 26542 KiB

Open AccessArticle

Controlling Unconventional Secretion for Production of Heterologous Proteins in Ustilago maydis through Transcriptional Regulation and Chemical Inhibition of the Kinase Don3

by Kai P. Hussnaetter, Magnus Philipp, Kira Müntjes, Michael Feldbrügge and Kerstin Schipper

J. Fungi 2021, 7(3), 179; https://doi.org/10.3390/jof7030179 - 3 Mar 2021

Cited by 7 | Viewed by 3264

Abstract

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins [...] Read more.

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. The kinase Don3 is essential for functional assembly of the fragmentation zone and hence, for release of Cts1-fusion proteins. Here, we are first to develop regulatory systems for unconventional protein secretion using Don3 as a gatekeeper to control when export occurs. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional don3 regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform. Full article

(This article belongs to the Special Issue Smut Fungi)

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11 pages, 1149 KiB

Open AccessArticle

Transcriptome Profiles of Sporisorium reilianum during the Early Infection of Resistant and Susceptible Maize Isogenic Lines

by Boqi Zhang, Nan Zhang, Qianqian Zhang, Qianya Xu, Tao Zhong, Kaiyue Zhang and Mingliang Xu

J. Fungi 2021, 7(2), 150; https://doi.org/10.3390/jof7020150 - 19 Feb 2021

Cited by 7 | Viewed by 2622

Abstract

The biotrophic fungus Sporisorium reilianum causes destructive head smut disease in maize (Zea mays L.). To explore the pathogenicity arsenal of this fungus, we tracked its transcriptome changes during infection of the maize seedling mesocotyls of two near-isogenic lines, HZ4 and HZ4R, [...] Read more.

The biotrophic fungus Sporisorium reilianum causes destructive head smut disease in maize (Zea mays L.). To explore the pathogenicity arsenal of this fungus, we tracked its transcriptome changes during infection of the maize seedling mesocotyls of two near-isogenic lines, HZ4 and HZ4R, differing solely in the disease resistance gene ZmWAK. Parasitic growth of S. reilianum resulted in thousands of differentially expressed genes (DEGs) compared with growth in axenic culture. The protein synthesis and energy metabolism of S. reilianum were predominantly enriched with down-regulated DEGs, consistent with the arrested hyphal growth observed following colonization. Nutrition-related metabolic processes were enriched with both up- and down-regulated DEGs, which, together with activated transmembrane transport, reflected a potential transition in nutrition uptake of S. reilianum once it invaded maize. Notably, genes encoding secreted proteins of S. reilianum were mostly up-regulated during biotrophy. ZmWAK-mediated resistance to head smut disease reduced the number of DEGs of S. reilianum, particularly those related to the secretome. These observations deepen our understanding of the mechanisms underlying S. reilianum pathogenicity and ZmWAK-induced innate immunity. Full article

(This article belongs to the Special Issue Smut Fungi)

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12 pages, 2190 KiB

Open AccessArticle

Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

by Sarah-Maria Wege, Katharina Gejer, Fabienne Becker, Michael Bölker, Johannes Freitag and Björn Sandrock

J. Fungi 2021, 7(2), 149; https://doi.org/10.3390/jof7020149 - 18 Feb 2021

Cited by 14 | Viewed by 3427

Abstract

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via [...] Read more.

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi. Full article

(This article belongs to the Special Issue Smut Fungi)

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16 pages, 1292 KiB

Open AccessArticle

Hungry for Sex: Differential Roles for Ustilago maydisb Locus Components in Haploid Cells vis à vis Nutritional Availability

by R. Margaret Wallen, Kirsten Richardson, Madison Furnish, Hector Mendoza, Allison Dentinger, Sunita Khanal and Michael H. Perlin

J. Fungi 2021, 7(2), 135; https://doi.org/10.3390/jof7020135 - 12 Feb 2021

Cited by 7 | Viewed by 3800

Abstract

Mating-types allow single-celled eukaryotic organisms to distinguish self from non-self in preparation for sexual reproduction. The components of mating-type loci provide initial self/non-self-recognition through pheromone and receptor interactions that control early cell fusion events. However, they may also provide a second level of [...] Read more.

Mating-types allow single-celled eukaryotic organisms to distinguish self from non-self in preparation for sexual reproduction. The components of mating-type loci provide initial self/non-self-recognition through pheromone and receptor interactions that control early cell fusion events. However, they may also provide a second level of scrutiny that requires differences in alleles leading to production of a transcription factor required for successful downstream developmental pathways after initial cell fusion. Interestingly, the protein subunits of these transcription factors have not been thoroughly examined for their roles, if any, in the haploid cells themselves. In Ustilago maydis, the causative agent of galls in maize plants, the b locus, encoding bEast (bE) and bWest (bW), components of the eventual requisite transcription factor, has been extensively studied for its role in formation of the stable dikaryon after mating and subsequent pathogenic program. Little is known, however, about any roles for bE or bW in haploid cells. Since mating in fungi is often induced under conditions of nitrogen starvation, we have explored connections between the b locus and the nitrogen-sensing and response pathways in U. maydis. We previously identified a connection in haploid cells between the b locus and Ump2, the high-affinity transceptor, a protein that both transports ammonium and triggers filamentous growth as a response to nitrogen starvation. Deletion of the entire b locus abrogates the filamentous response to low ammonium, a phenotype that is rescued by overexpression of Ump2. Here we further investigated the individual roles of bE and bW in haploid cells. We show that bE and bW are expressed differentially in haploid cells starved for ammonium. Their respective deletion elicits different effects on transcription of mating and pathogenic-related genes and, importantly, on the degree of pathogenic development in host plants. This is the first demonstration of a role for these mating locus components on haploid development and the first to demonstrate a connection to the ammonium transceptors. Full article

(This article belongs to the Special Issue Smut Fungi)

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10 pages, 1184 KiB

Open AccessArticle

Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in Tilletia indica Isolates Inciting Karnal Bunt of Wheat

by Malkhan Singh Gurjar, Rashmi Aggarwal, Shekhar Jain, Sapna Sharma, Jagmohan Singh, Sangeeta Gupta, Shweta Agarwal and Mahender Singh Saharan

J. Fungi 2021, 7(2), 103; https://doi.org/10.3390/jof7020103 - 2 Feb 2021

Cited by 11 | Viewed by 2739

Abstract

Karnal bunt of wheat is an internationally quarantined disease affecting trade, quality, and production of wheat. During 2015–2016, a severe outbreak of Karnal bunt disease occurred in north-western plain zone of India. The present study was undertaken to decipher genetic variations in Indian [...] Read more.

Karnal bunt of wheat is an internationally quarantined disease affecting trade, quality, and production of wheat. During 2015–2016, a severe outbreak of Karnal bunt disease occurred in north-western plain zone of India. The present study was undertaken to decipher genetic variations in Indian isolates of Tilletia indica collected from different locations. Seven multilocus sequence fragments were selected to differentiate and characterize these T. indica isolates. A phylogenetic tree constructed based on pooled sequences of actin-related protein 2 (ARP2), β-tubulin (TUB), eukaryotic translation initiation factor 3 subunit A (EIF3A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 2B (H2B), phosphoglycerate kinase (PGK), and serine/threonine-protein kinase (STPK) showed that isolate KB-11 (Kaithal, Haryana) was highly conserved as it was located in cluster 1 and has the maximum sequence similarity with the reference strain. Other isolates in cluster 1 included KB-16 and KB-17, both from Uttar Pradesh, and KB-19 from Haryana. Isolates KB-07 (Jind, Haryana) and KB-18 (Mujaffar Nagar, Uttar Pradesh) were the most diverse and grouped in a subgroup of cluster 2. Maximum numbers of single nucleotide polymorphisms (SNPs) (675) were in the PGK gene across the T. indica isolates. The minimum numbers of SNPs (67) were in KB-11 (Kaithal, Haryana), while the maximum number of SNPs (165) was identified in KB-18, followed by 164 SNPs in KB-14. KB-18 isolate was found to be the most diverse amongst all T. indica isolates. This first study on multilocus sequence typing (MLST) revealed that the population of T. indica was highly diverse. Full article

(This article belongs to the Special Issue Smut Fungi)

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19 pages, 3378 KiB

Open AccessArticle

Ustilaginaceae Biocatalyst for Co-Metabolism of CO₂-Derived Substrates toward Carbon-Neutral Itaconate Production

by Lena Ullmann, An N. T. Phan, Daniel K. P. Kaplan and Lars M. Blank

J. Fungi 2021, 7(2), 98; https://doi.org/10.3390/jof7020098 - 29 Jan 2021

Cited by 14 | Viewed by 2897

Abstract

The family Ustilaginaceae (belonging to the smut fungi) are known for their plant pathogenicity. Despite the fact that these plant diseases cause agricultural yield reduction, smut fungi attracted special attention in the field of industrial biotechnology. Ustilaginaceae show a versatile product spectrum such [...] Read more.

The family Ustilaginaceae (belonging to the smut fungi) are known for their plant pathogenicity. Despite the fact that these plant diseases cause agricultural yield reduction, smut fungi attracted special attention in the field of industrial biotechnology. Ustilaginaceae show a versatile product spectrum such as organic acids (e.g., itaconate, malate, succinate), polyols (e.g., erythritol, mannitol), and extracellular glycolipids, which are considered value-added chemicals with potential applications in the pharmaceutical, food, and chemical industries. This study focused on itaconate as a platform chemical for the production of resins, plastics, adhesives, and biofuels. During this work, 72 different Ustilaginaceae strains from 36 species were investigated for their ability to (co-) consume the CO₂-derived substrates acetate and formate, potentially contributing toward a carbon-neutral itaconate production. The fungal growth and product spectrum with special interest in itaconate was characterized. Ustilago maydis MB215 and Ustilago rabenhorstiana NBRC 8995 were identified as promising candidates for acetate metabolization whereas Ustilago cynodontis NBRC 7530 was identified as a potential production host using formate as a co-substrate enhancing the itaconate production. Selected strains with the best itaconate production were characterized in more detail in controlled-batch bioreactor experiments confirming the co-substrate utilization. Thus, a proof-of-principle study was performed resulting in the identification and characterization of three promising Ustilaginaceae biocatalyst candidates for carbon-neutral itaconate production contributing to the biotechnological relevance of Ustilaginaceae. Full article

(This article belongs to the Special Issue Smut Fungi)

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14 pages, 1756 KiB

Open AccessArticle

Self-Generated Hypoxia Leads to Oxidative Stress and Massive Death in Ustilago maydis Populations under Extreme Starvation and Oxygen-Limited Conditions

by Jelena Petkovic, Milorad Kojic and Mira Milisavljevic

J. Fungi 2021, 7(2), 92; https://doi.org/10.3390/jof7020092 - 28 Jan 2021

Cited by 1 | Viewed by 2206

Abstract

Ustilago maydis and Saccharomyces cerevisiae differ considerably in their response to water-transfer treatments. When stationary phase cells were transferred to pure water and incubated under limited supply of oxygen, the U. maydis cells suffered a catastrophic loss of viability while the S. cerevisiae [...] Read more.

Ustilago maydis and Saccharomyces cerevisiae differ considerably in their response to water-transfer treatments. When stationary phase cells were transferred to pure water and incubated under limited supply of oxygen, the U. maydis cells suffered a catastrophic loss of viability while the S. cerevisiae population was virtually unaffected by the treatment. The major factor underlying the death of the U. maydis cells under those conditions was an oxygen-consuming cellular activity that generated a hypoxic environment, thereby inducing oxidative stress and accumulation of reactive oxygen species, which resulted in lethality. Importantly, a small residue of U. maydis cells that did survive was able to resume growth and repopulate up to the initial culture density when sufficient aeration was restored. The regrowth was dependent on the cellular factors (Adr1, Did4, Kel1, and Tbp1), previously identified as required for repopulation, after killing with hydrogen peroxide. Surprisingly, the survivors were also able to resume growth under apparently hypoxic conditions, indicating that these remnant cells likely switched to a fermentative mode of growth. We discuss the findings in terms of their possible relevance to the eco-evolutionary adaptation of U. maydis to risky environments. Full article

(This article belongs to the Special Issue Smut Fungi)

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17 pages, 5188 KiB

Open AccessArticle

The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

by Bilal Ökmen, Daniela Schwammbach, Guus Bakkeren, Ulla Neumann and Gunther Doehlemann

J. Fungi 2021, 7(2), 86; https://doi.org/10.3390/jof7020086 - 27 Jan 2021

Cited by 15 | Viewed by 4575

Abstract

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies [...] Read more.

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley. Full article

(This article belongs to the Special Issue Smut Fungi)

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14 pages, 1718 KiB

Open AccessArticle

Carbon and Nitrogen Sources Have No Impact on the Organization and Composition of Ustilago maydis Respiratory Supercomplexes

by Deyamira Matuz-Mares, Oscar Flores-Herrera, Guadalupe Guerra-Sánchez, Lucero Romero-Aguilar, Héctor Vázquez-Meza, Genaro Matus-Ortega, Federico Martínez and Juan Pablo Pardo

J. Fungi 2021, 7(1), 42; https://doi.org/10.3390/jof7010042 - 11 Jan 2021

Cited by 7 | Viewed by 2996

Abstract

Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in principle should increase the respiratory chain efficiency and ATP synthesis. In addition to the four classic respiratory complexes and the [...] Read more.

Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in principle should increase the respiratory chain efficiency and ATP synthesis. In addition to the four classic respiratory complexes and the ATP synthase, U. maydis mitochondria contain three type II NADH dehydrogenases (NADH for reduced nicotinamide adenine dinucleotide) and the alternative oxidase. Changes in the composition of the respiratory supercomplexes due to energy requirements have been reported in certain organisms. In this study, we addressed the organization of the mitochondrial respiratory complexes in U. maydis under diverse energy conditions. Supercomplexes were obtained by solubilization of U. maydis mitochondria with digitonin and separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The molecular mass of supercomplexes and their probable stoichiometries were 1200 kDa (I₁:IV₁), 1400 kDa (I₁:III₂), 1600 kDa (I₁:III₂:IV₁), and 1800 kDa (I₁:III₂:IV₂). Concerning the ATP synthase, approximately half of the protein is present as a dimer and half as a monomer. The distribution of respiratory supercomplexes was the same in all growth conditions. We did not find evidence for the association of complex II and the alternative NADH dehydrogenases with other respiratory complexes. Full article

(This article belongs to the Special Issue Smut Fungi)

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14 pages, 5791 KiB

Open AccessArticle

Genetic Manipulation of the Brassicaceae Smut Fungus Thecaphora thlaspeos

by Lesley Plücker, Kristin Bösch, Lea Geißl, Philipp Hoffmann and Vera Göhre

J. Fungi 2021, 7(1), 38; https://doi.org/10.3390/jof7010038 - 9 Jan 2021

Cited by 6 | Viewed by 3328

Abstract

Investigation of plant–microbe interactions greatly benefit from genetically tractable partners to address, molecularly, the virulence and defense mechanisms. The smut fungus Ustilago maydis is a model pathogen in that sense: efficient homologous recombination and a small genome allow targeted modification. On the host [...] Read more.

Investigation of plant–microbe interactions greatly benefit from genetically tractable partners to address, molecularly, the virulence and defense mechanisms. The smut fungus Ustilago maydis is a model pathogen in that sense: efficient homologous recombination and a small genome allow targeted modification. On the host side, maize is limiting with regard to rapid genetic alterations. By contrast, the model plant Arabidopsis thaliana is an excellent model with a vast amount of information and techniques as well as genetic resources. Here, we present a transformation protocol for the Brassicaceae smut fungus Thecaphora thlaspeos. Using the well-established methodology of protoplast transformation, we generated the first reporter strains expressing fluorescent proteins to follow mating. As a proof-of-principle for homologous recombination, we deleted the pheromone receptor pra1. As expected, this mutant cannot mate. Further analysis will contribute to our understanding of the role of mating for infection biology in this novel model fungus. From now on, the genetic manipulation of T. thlaspeos, which is able to colonize the model plant A. thaliana, provides us with a pathosystem in which both partners are genetically amenable to study smut infection biology. Full article

(This article belongs to the Special Issue Smut Fungi)

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14 pages, 2606 KiB

Open AccessArticle

An Optimized Ustilago maydis for Itaconic Acid Production at Maximal Theoretical Yield

by Johanna Becker, Hamed Hosseinpour Tehrani, Philipp Ernst, Lars Mathias Blank and Nick Wierckx

J. Fungi 2021, 7(1), 20; https://doi.org/10.3390/jof7010020 - 31 Dec 2020

Cited by 42 | Viewed by 5085

Abstract

Ustilago maydis, a member of the Ustilaginaceae family, is a promising host for the production of several metabolites including itaconic acid. This dicarboxylate has great potential as a bio-based building block in the polymer industry, and is of special interest for pharmaceutical [...] Read more.

Ustilago maydis, a member of the Ustilaginaceae family, is a promising host for the production of several metabolites including itaconic acid. This dicarboxylate has great potential as a bio-based building block in the polymer industry, and is of special interest for pharmaceutical applications. Several itaconate overproducing Ustilago strains have been generated by metabolic and morphology engineering. This yielded stabilized unicellular morphology through fuz7 deletion, reduction of by-product formation through deletion of genes responsible for itaconate oxidation and (glyco)lipid production, and the overexpression of the regulator of the itaconate cluster ria1 and the mitochondrial tricarboxylate transporter encoded by mttA from Aspergillusterreus. In this study, itaconate production was further optimized by consolidating these different optimizations into one strain. The combined modifications resulted in itaconic acid production at theoretical maximal yield, which was achieved under biotechnologically relevant fed-batch fermentations with continuous feed. Full article

(This article belongs to the Special Issue Smut Fungi)

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14 pages, 2371 KiB

Open AccessArticle

Effectors with Different Gears: Divergence of Ustilago maydis Effector Genes Is Associated with Their Temporal Expression Pattern during Plant Infection

by Jasper R. L. Depotter, Weiliang Zuo, Maike Hansen, Boqi Zhang, Mingliang Xu and Gunther Doehlemann

J. Fungi 2021, 7(1), 16; https://doi.org/10.3390/jof7010016 - 29 Dec 2020

Cited by 9 | Viewed by 3293

Abstract

Plant pathogens secrete a variety of effector proteins that enable host colonization but are also typical pathogen detection targets for the host immune system. Consequently, effector genes encounter high selection pressures, which typically makes them fast evolving. The corn smut pathogen Ustilago maydis [...] Read more.

Plant pathogens secrete a variety of effector proteins that enable host colonization but are also typical pathogen detection targets for the host immune system. Consequently, effector genes encounter high selection pressures, which typically makes them fast evolving. The corn smut pathogen Ustilago maydis has an effector gene repertoire with a dynamic expression across the different disease stages. We determined the amino acid divergence of U. maydis effector candidates with Sporisorium reilianum orthologs, a close relative of U. maydis. Intriguingly, there are two distinct groups of effector candidates, ones with a respective conserved and diverged protein evolution. Conservatively evolving effector genes especially have their peak expression during the (pre-)penetration stages of the disease cycle. In contrast, expression of divergently evolving effector genes generally peaks during fungal proliferation within the host. To test if this interspecific effector diversity corresponds to intraspecific diversity, we sampled and sequenced a diverse collection of U. maydis strains from the most important maize breeding and production regions in China. Effector candidates with a diverged interspecific evolution had more intraspecific amino acid variation than candidates with a conserved evolution. In conclusion, we highlight diversity in evolution within the U. maydis effector repertoire with dynamically and conservatively evolving members. Full article

(This article belongs to the Special Issue Smut Fungi)

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26 pages, 4579 KiB

Open AccessArticle

Leaping into the Unknown World of Sporisorium scitamineum Candidate Effectors

by Natália Sousa Teixeira-Silva, Patrícia Dayane Carvalho Schaker, Hugo Vianna Silva Rody, Thiago Maia, Christopher M. Garner, Walter Gassmann and Claudia Barros Monteiro-Vitorello

J. Fungi 2020, 6(4), 339; https://doi.org/10.3390/jof6040339 - 4 Dec 2020

Cited by 9 | Viewed by 4972

Abstract

Sporisorium scitamineum is a biotrophic fungus causing sugarcane smut disease. In this study, we set up a pipeline and used genomic and dual transcriptomic data previously obtained by our group to identify candidate effectors of S. scitamineum and their expression profiles in infected [...] Read more.

Sporisorium scitamineum is a biotrophic fungus causing sugarcane smut disease. In this study, we set up a pipeline and used genomic and dual transcriptomic data previously obtained by our group to identify candidate effectors of S. scitamineum and their expression profiles in infected smut-resistant and susceptible sugarcane plants. The expression profile of different genes after infection in contrasting sugarcane genotypes assessed by RT-qPCR depended on the plant genotypes and disease progression. Three candidate effector genes expressed earlier only in resistant plants, four expressed in both genotypes, and three later in susceptible plants. Ten genes were cloned and transiently expressed in N. benthamiana leaves to determine their subcellular location, while four localized in more than one compartment. Two candidates, g3890 having a nucleoplasmic and mitochondrial location and g5159 targeting the plant cell wall, were selected to obtain their possible corresponding host targets using co-immunoprecipitation (CoIP) experiments and mass spectrometry. Various potential interactors were identified, including subunits of the protein phosphatase 2A and an endochitinase. We investigated the presence of orthologs in sugarcane and using transcriptome data present their expression profiles. Orthologs of sugarcane shared around 70% similarity. Identifying a set of putative fungal effectors and their plant targets provides a valuable resource for functional characterization of the molecular events leading to smut resistance in sugarcane plants and uncovers further opportunities for investigation. Full article

(This article belongs to the Special Issue Smut Fungi)

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33 pages, 9409 KiB

Open AccessArticle

UhAVR1, an HR-Triggering Avirulence Effector of Ustilago hordei, Is Secreted via the ER–Golgi Pathway, Localizes to the Cytosol of Barley Cells during in Planta-Expression, and Contributes to Virulence Early in Infection

by Ana Priscilla Montenegro Alonso, Shawkat Ali, Xiao Song, Rob Linning and Guus Bakkeren

J. Fungi 2020, 6(3), 178; https://doi.org/10.3390/jof6030178 - 18 Sep 2020

Cited by 9 | Viewed by 4191

Abstract

The basidiomycete Ustilago hordei causes covered smut disease of barley and oats. Virulence effectors promoting infection and supporting pathogen lifestyle have been described for this fungus. Genetically, six avirulence genes are known and one codes for UhAVR1, the only proven avirulence effector identified [...] Read more.

The basidiomycete Ustilago hordei causes covered smut disease of barley and oats. Virulence effectors promoting infection and supporting pathogen lifestyle have been described for this fungus. Genetically, six avirulence genes are known and one codes for UhAVR1, the only proven avirulence effector identified in smuts to date that triggers complete immunity in barley cultivars carrying resistance gene Ruh1. A prerequisite for resistance breeding is understanding the host targets and molecular function of UhAVR1. Analysis of this effector upon natural infection of barley coleoptiles using teliospores showed that UhAVR1 is expressed during the early stages of fungal infection where it leads to HR triggering in resistant cultivars or performs its virulence function in susceptible cultivars. Fungal secretion of UhAVR1 is directed by its signal peptide and occurs via the BrefeldinA-sensitive ER–Golgi pathway in cell culture away from its host. Transient in planta expression of UhAVR1 in barley and a nonhost, Nicotiana benthamiana, supports a cytosolic localization. Delivery of UhAVR1 via foxtail mosaic virus or Pseudomonas species in both barley and N. benthamiana reveals a role in suppressing components common to both plant systems of Effector- and Pattern-Triggered Immunity, including necrosis triggered by Agrobacterium-delivered cell death inducers. Full article

(This article belongs to the Special Issue Smut Fungi)

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Review

Jump to: Editorial, Research

19 pages, 3727 KiB

Open AccessReview

Structural, Evolutionary, and Functional Analysis of the Protein O-Mannosyltransferase Family in Pathogenic Fungi

by María Dolores Pejenaute-Ochoa, Carlos Santana-Molina, Damien P. Devos, José Ignacio Ibeas and Alfonso Fernández-Álvarez

J. Fungi 2021, 7(5), 328; https://doi.org/10.3390/jof7050328 - 23 Apr 2021

Cited by 6 | Viewed by 3264

Abstract

Protein O-mannosyltransferases (Pmts) comprise a group of proteins that add mannoses to substrate proteins at the endoplasmic reticulum. This post-translational modification is important for the faithful transfer of nascent glycoproteins throughout the secretory pathway. Most fungi genomes encode three O-mannosyltransferases, usually named Pmt1, [...] Read more.

Protein O-mannosyltransferases (Pmts) comprise a group of proteins that add mannoses to substrate proteins at the endoplasmic reticulum. This post-translational modification is important for the faithful transfer of nascent glycoproteins throughout the secretory pathway. Most fungi genomes encode three O-mannosyltransferases, usually named Pmt1, Pmt2, and Pmt4. In pathogenic fungi, Pmts, especially Pmt4, are key factors for virulence. Although the importance of Pmts for fungal pathogenesis is well established in a wide range of pathogens, questions remain regarding certain features of Pmts. For example, why does the single deletion of each pmt gene have an asymmetrical impact on host colonization? Here, we analyse the origin of Pmts in fungi and review the most important phenotypes associated with Pmt mutants in pathogenic fungi. Hence, we highlight the enormous relevance of these glycotransferases for fungal pathogenic development. Full article

(This article belongs to the Special Issue Smut Fungi)

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13 pages, 2583 KiB

Open AccessReview

How Do Smut Fungi Use Plant Signals to Spatiotemporally Orientate on and In Planta?

by Karina van der Linde and Vera Göhre

J. Fungi 2021, 7(2), 107; https://doi.org/10.3390/jof7020107 - 2 Feb 2021

Cited by 17 | Viewed by 4174

Abstract

Smut fungi represent a large group of biotrophic plant pathogens that cause extensive yield loss and are also model organisms for studying plant–pathogen interactions. In recent years, they have become biotechnological tools. After initial penetration of the plant epidermis, smut fungi grow intra—and [...] Read more.

Smut fungi represent a large group of biotrophic plant pathogens that cause extensive yield loss and are also model organisms for studying plant–pathogen interactions. In recent years, they have become biotechnological tools. After initial penetration of the plant epidermis, smut fungi grow intra—and intercellularly without disrupting the plant-plasma membrane. Following the colonialization step, teliospores are formed and later released. While some smuts only invade the tissues around the initial penetration site, others colonize in multiple plant organs resulting in spore formation distal from the original infection site. The intimate contact zone between fungal hyphae and the host is termed the biotrophic interaction zone and enables exchange of signals and nutrient uptake. Obviously, all steps of on and in planta growth require fine sensing of host conditions as well as reprogramming of the host by the smut fungus. In this review, we highlight selected examples of smut fungal colonization styles, directional growth in planta, induction of spore formation, and the signals required, pointing to excellent reviews for details, to draw attention to some of the open questions in this important research field. Full article

(This article belongs to the Special Issue Smut Fungi)

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18 pages, 7411 KiB

Open AccessReview

Physiological Basis of Smut Infectivity in the Early Stages of Sugar Cane Colonization

by Carlos Vicente, María-Estrella Legaz and Elena Sánchez-Elordi

J. Fungi 2021, 7(1), 44; https://doi.org/10.3390/jof7010044 - 12 Jan 2021

Cited by 8 | Viewed by 8916

Abstract

Sugar cane smut (Sporisorium scitamineum) interactions have been traditionally considered from the plant’s point of view: How can resistant sugar cane plants defend themselves against smut disease? Resistant plants induce several defensive mechanisms that oppose fungal attacks. Herein, an overall view [...] Read more.

Sugar cane smut (Sporisorium scitamineum) interactions have been traditionally considered from the plant’s point of view: How can resistant sugar cane plants defend themselves against smut disease? Resistant plants induce several defensive mechanisms that oppose fungal attacks. Herein, an overall view of Sporisorium scitamineum’s mechanisms of infection and the defense mechanisms of plants are presented. Quorum sensing effects and a continuous reorganization of cytoskeletal components, where actin, myosin, and microtubules are required to work together, seem to be some of the keys to a successful attack. Full article

(This article belongs to the Special Issue Smut Fungi)

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9 pages, 689 KiB

Open AccessReview

Understanding Ustilago maydis Infection of Multiple Maize Organs

by Alex C Ferris and Virginia Walbot

J. Fungi 2021, 7(1), 8; https://doi.org/10.3390/jof7010008 - 27 Dec 2020

Cited by 14 | Viewed by 5887

Abstract

Ustilago maydis is a smut fungus that infects all aerial maize organs, namely, seedling leaves, tassels, and ears. In all organs, tumors are formed by inducing hypertrophy and hyperplasia in actively dividing cells; however, the vast differences in cell types and developmental stages [...] Read more.

Ustilago maydis is a smut fungus that infects all aerial maize organs, namely, seedling leaves, tassels, and ears. In all organs, tumors are formed by inducing hypertrophy and hyperplasia in actively dividing cells; however, the vast differences in cell types and developmental stages for different parts of the plant requires that U. maydis have both general and organ-specific strategies for infecting maize. In this review, we summarize how the maize–U. maydis interaction can be studied using mutant U. maydis strains to better understand how individual effectors contribute to this interaction, either through general or specific expression in a cell type, tissue, or organ. We also examine how male sterile maize mutants that do not support tumor formation can be used to explore key features of the maize anthers that are required for successful infection. Finally, we discuss key unanswered questions about the maize–U. maydis interaction and how new technologies can potentially be used to answer them. Full article

(This article belongs to the Special Issue Smut Fungi)

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26 pages, 2397 KiB

Open AccessReview

Investigating the Smuts: Common Cues, Signaling Pathways, and the Role of MAT in Dimorphic Switching and Pathogenesis

by Teeratas Kijpornyongpan and M. Catherine Aime

J. Fungi 2020, 6(4), 368; https://doi.org/10.3390/jof6040368 - 16 Dec 2020

Cited by 18 | Viewed by 4782

Abstract

The corn smut fungus Ustilago maydis serves as a model species for studying fungal dimorphism and its role in phytopathogenic development. The pathogen has two growth phases: a saprobic yeast phase and a pathogenic filamentous phase. Dimorphic transition of U. maydis involves complex [...] Read more.

The corn smut fungus Ustilago maydis serves as a model species for studying fungal dimorphism and its role in phytopathogenic development. The pathogen has two growth phases: a saprobic yeast phase and a pathogenic filamentous phase. Dimorphic transition of U. maydis involves complex processes of signal perception, mating, and cellular reprogramming. Recent advances in improvement of reference genomes, high-throughput sequencing and molecular genetics studies have been expanding research in this field. However, the biology of other non-model species is frequently overlooked. This leads to uncertainty regarding how much of what is known in U. maydis is applicable to other dimorphic fungi. In this review, we will discuss dimorphic fungi in the aspects of physiology, reproductive biology, genomics, and molecular genetics. We also perform comparative analyses between U. maydis and other fungi in Ustilaginomycotina, the subphylum to which U. maydis belongs. We find that lipid/hydrophobicity is a potential common cue for dimorphic transition in plant-associated dimorphic fungi. However, genomic profiles alone are not adequate to explain dimorphism across different fungi. Full article

(This article belongs to the Special Issue Smut Fungi)

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