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Papain-like cysteine proteases (PLCPs) play pivotal roles in plant defense against pathogen invasions. While pathogens can secrete effectors to target and inhibit PLCP activities, the roles of PLCPs in plant-virus interactions and the mechanisms through which viruses neutralize PLCP activities remain largely uncharted. Here, we demonstrate that the expression and activity of a maize PLCP CCP1 (Corn Cysteine Protease), is upregulated following sugarcane mosaic virus (SCMV) infection. Transient silencing of CCP1 led to a reduction in PLCP activities, thereby promoting SCMV infection in maize. Furthermore, the knockdown of CCP1 resulted in diminished salicylic acid (SA) levels and suppressed expression of SA-responsive pathogenesis-related genes. This suggests that CCP1 plays a role in modulating the SA signaling pathway. Interestingly, NIa-Pro, the primary protease of SCMV, was found to interact with CCP1, subsequently inhibiting its protease activity. A specific motif within NIa-Pro termed the inhibitor motif was identified as essential for its interaction with CCP1 and the suppression of its activity. We have also discovered that the key amino acids responsible for the interaction between NIa-Pro and CCP1 are crucial for the virulence of SCMV. In conclusion, our findings offer compelling evidence that SCMV undermines maize defense mechanisms through the interaction of NIa-Pro with CCP1. Together, these findings shed a new light on the mechanism(s) controlling the arms races between virus and plant. Author summary: PLCPs perform crucial roles in plant defense against invasions by pathogens. While pathogens can secrete effectors that target and inhibit PLCP activities, the functions of PLCPs in plant-virus interactions and the mechanisms by which viruses counteract PLCP activities remain largely unexplored. In this study, we demonstrate that a maize PLCP termed CCP1 confers resistance to SCMV infection via SA signaling pathway. Conversely, SCMV NIa-Pro interacts with CCP1 and inhibits its protease activity to allow efficient viral infection. Furthermore, we have discovered that the critical amino acids responsible for the interaction between NIa-Pro and CCP1 are crucial for the virulence of SCMV. [ABSTRACT FROM AUTHOR]

Summary: Ustilago maydis is a biotrophic fungus that causes tumor formation on all aerial parts of maize. U. maydis secretes effector proteins during penetration and colonization to successfully overcome the plant immune response and reprogram host physiology to promote infection.In this study, we functionally characterized the U. maydis effector protein Topless (TPL) interacting protein 6 (Tip6). We found that Tip6 interacts with the N‐terminus of RELK2 through its two Ethylene‐responsive element binding factor‐associated amphiphilic repression (EAR) motifs. We show that the EAR motifs are essential for the virulence function of Tip6 and critical for altering the nuclear distribution pattern of RELK2.We propose that Tip6 mimics the recruitment of RELK2 by plant repressor proteins, thus disrupting host transcriptional regulation. We show that a large group of AP2/ERF B1 subfamily transcription factors are misregulated in the presence of Tip6.Our study suggests a regulatory mechanism where the U. maydis effector Tip6 utilizes repressive domains to recruit the corepressor RELK2 to disrupt the transcriptional networks of the host plant. [ABSTRACT FROM AUTHOR]

Summary: Ribotoxins are secreted ribonucleases that specifically target and cleave the universally conserved sarcin–ricin loop sequence of rRNA, which leads to inhibition of protein biosynthesis and subsequently to cell death. We have identified and characterized a secreted Ribo1 protein of plant pathogenic smut fungi.Heterologous expression in different model systems showed that smut Ribo1 has cytotoxic activity against bacteria, yeast, host and nonhost plants.Recombinant expression of Ribo1 in Nicotiana benthamiana induced plant cell death; however, an active site mutant induced cell death only when expressed as a secreted protein. In the maize smut Ustilago maydis, transcription of Ribo1 is specifically induced in early infection stages. While a knockout mutant revealed that Ribo1 is dispensable for U. maydis virulence, the overexpression of Ribo1 in planta had a strong dominant negative effect on virulence and induced host defense responses including cell death.Our findings suggest a function of Ribo1 during the epiphytic development rather than for invasive colonization of the host. Accordingly, in the presence of the biocontrol bacteria Pantoea sp., which were isolated from maize leaves, the ribo1 knockout mutant was significantly impaired in virulence. Together, we conclude that Ribo1 enables smut fungi to compete with host‐associated bacteria during epiphytic development. [ABSTRACT FROM AUTHOR]

Ustilago maydis causes common smut in maize, which is characterized by tumor formation in aerial parts of maize. Tumors result from the de novo cell division of highly developed bundle sheath and subsequent cell enlargement. However, the molecular mechanisms underlying tumorigenesis are still largely unknown. Here, we characterize the U. maydis effector Sts2 (Small tumor on seedlings 2), which promotes the division of hyperplasia tumor cells. Upon infection, Sts2 is translocated into the maize cell nucleus, where it acts as a transcriptional activator, and the transactivation activity is crucial for its virulence function. Sts2 interacts with ZmNECAP1, a yet undescribed plant transcriptional activator, and it activates the expression of several leaf developmental regulators to potentiate tumor formation. On the contrary, fusion of a suppressive SRDX-motif to Sts2 causes dominant negative inhibition of tumor formation, underpinning the central role of Sts2 for tumorigenesis. Our results not only disclose the virulence mechanism of a tumorigenic effector, but also reveal the essential role of leaf developmental regulators in pathogen-induced tumor formation. Ustilago maydis causes tumor on maize seedlings. Here we show that U. maydis secrets the transcriptional activator effector Sts2 to induce leaf developmental regulators which activate the bundle sheath cell division for tumor formation. [ABSTRACT FROM AUTHOR]

SUMMARY: Regulated cell death (RCD) is crucial for plant development, as well as in decision‐making in plant‐microbe interactions. Previous studies revealed components of the molecular network controlling RCD, including different proteases. However, the identity, the proteolytic network as well as molecular components involved in the initiation and execution of distinct plant RCD processes, still remain largely elusive. In this study, we analyzed the transcriptome, proteome, and N‐terminome of Zea mays leaves treated with the Xanthomonas effector avrRxo1, the mycotoxin Fumonisin B1 (FB1), or the phytohormone salicylic acid (SA) to dissect plant cellular processes related to cell death and plant immunity. We found highly distinct and time‐dependent biological processes being activated on transcriptional and proteome levels in response to avrRxo1, FB1, and SA. Correlation analysis of the transcriptome and proteome identified general, as well as trigger‐specific markers for cell death in Zea mays. We found that proteases, particularly papain‐like cysteine proteases, are specifically regulated during RCD. Collectively, this study characterizes distinct RCD responses in Z. mays and provides a framework for the mechanistic exploration of components involved in the initiation and execution of cell death. Significance Statement: This work combines a large‐scale transcriptomic, proteomic, and N‐terminomic analysis of maize leaves to obtain insight into the molecular processes taking place during effector‐ and toxin‐triggered cell death in maize. We provide a framework of components involved in the initiation and execution of cell death in maize, which will enable the identification of common elements with conserved functions in pathogen‐induced cell death across plant species. [ABSTRACT FROM AUTHOR]

Plant pathogens secrete effectors, which target host proteins to facilitate infection. The Ustilago maydis effector UmSee1 is required for tumor formation in the leaf during infection of maize. UmSee1 interacts with maize SGT1 (suppressor of G2 allele of skp1) and blocks its phosphorylation in vivo. In the absence of UmSee1, U. maydis cannot trigger tumor formation in the bundle sheath. However, it remains unclear which host processes are manipulated by UmSee1 and the UmSee1–SGT1 interaction to cause the observed phenotype. Proximity-dependent protein labeling involving the turbo biotin ligase tag (TurboID) for proximal labeling of proteins is a powerful tool for identifying the protein interactome. We have generated transgenic U. maydis that secretes biotin ligase-fused See1 effector (UmSee1–TurboID-3HA) directly into maize cells. This approach, in combination with conventional co-immunoprecipitation, allowed the identification of additional UmSee1 interactors in maize cells. Collectively, our data identified three ubiquitin–proteasome pathway-related proteins (ZmSIP1, ZmSIP2, and ZmSIP3) that either interact with or are close to UmSee1 during host infection of maize with U. maydis. ZmSIP3 represents a cell cycle regulator whose degradation appears to be promoted in the presence of UmSee1. Our data provide a possible explanation of the requirement for UmSee1 in tumor formation during U. maydis – Zea mays interaction. [ABSTRACT FROM AUTHOR]

Summary: Plant biotrophic pathogens employ secreted molecules, called effectors, to suppress the host immune system and redirect the host's metabolism and development in their favour. Putative effectors of the gall‐inducing maize pathogenic fungus Ustilago maydis were analysed for their ability to induce auxin signalling in plants.Using genetic, biochemical, cell‐biological, and bioinformatic approaches we functionally elucidate a set of five, genetically linked effectors, called Topless (TPL) interacting protein (Tips) effectors that induce auxin signalling.We show that Tips induce auxin signalling by interfering with central corepressors of the TPL family. CRISPR‐Cas9 mutants and deletion strain analysis indicate that the auxin signalling inducing subcluster effectors plays a redundant role in virulence.Although none of the Tips seem to have a conserved interaction motif, four of them bind solely to the N‐terminal TPL domain and, for Tip1 and Tip4, we demonstrate direct competition with auxin/indole‐3‐acetic acid transcriptional repressors for their binding to TPL class of corepressors. Our findings reveal that TPL proteins, key regulators of growth–defence antagonism, are a major target of the U. maydis effectome. [ABSTRACT FROM AUTHOR]

Smut fungi comprise one of the largest groups of fungal plant pathogens causing disease in all cereal crops. They directly penetrate host tissues and establish a biotrophic interaction. To do so, smut fungi secrete a wide range of effector proteins, which suppress plant immunity and modulate cellular functions as well as development of the host, thereby determining the pathogen's lifestyle and virulence potential. The conserved effector Erc1 (enzyme required for cell-to-cell extension) contributes to virulence of the corn smut Ustilago maydis in maize leaves but not on the tassel. Erc1 binds to host cell wall components and displays 1,3-β-glucanase activity, which is required to attenuate β-glucan-induced defense responses. Here we show that Erc1 has a cell type-specific virulence function, being necessary for fungal cell-to-cell extension in the plant bundle sheath and this function is fully conserved in the Erc1 orthologue of the barley pathogen Ustilago hordei. Smut fungi secrete effector proteins that suppress host plant immunity. Here the authors show that the corn smut effector Erc1 has 1,3-β-glucanase activity and promotes virulence in maize leaves by attenuating β-glucan-induced defense responses. [ABSTRACT FROM AUTHOR]

During host colonization, plant-associated microbes, including fungi and oomycetes, deliver a collection of glycoside hydrolases (GHs) to their cell surfaces and surrounding extracellular environments. The number and type of GHs secreted by each organism is typically associated with their lifestyle or mode of nutrient acquisition. Secreted GHs of plant-associated fungi and oomycetes serve a number of different functions, with many of them acting as virulence factors (effectors) to promote microbial host colonization. Specific functions involve, for example, nutrient acquisition, the detoxification of antimicrobial compounds, the manipulation of plant microbiota, and the suppression or prevention of plant immune responses. In contrast, secreted GHs of plant-associated fungi and oomycetes can also activate the plant immune system, either by acting as microbe-associated molecular patterns (MAMPs), or through the release of damage-associated molecular patterns (DAMPs) as a consequence of their enzymatic activity. In this review, we highlight the critical roles that secreted GHs from plant-associated fungi and oomycetes play in plant–microbe interactions, provide an overview of existing knowledge gaps and summarize future directions. [ABSTRACT FROM AUTHOR]

Summary: The constitution and regulation of effector repertoires shape host–microbe interactions. Ustilago maydis and Sporisorium reilianum are two closely related smut fungi, which both infect maize but cause distinct disease symptoms. Understanding how effector orthologs are regulated in these two pathogens can therefore provide insights into the evolution of different infection strategies.We tracked the infection progress of U. maydis and S. reilianum in maize leaves and used two distinct infection stages for cross‐species RNA‐sequencing analyses. We identified 207 of 335 one‐to‐one effector orthologs as differentially regulated during host colonization, which might reflect the distinct disease development strategies.Using CRISPR‐Cas9‐mediated gene conversion, we identified two differentially expressed effector orthologs with conserved function between two pathogens. Thus, differential expression of functionally conserved genes might contribute to species‐specific adaptation and symptom development. Interestingly, another differentially expressed orthogroup (UMAG_05318/Sr10075) showed divergent protein function, providing a possible case for neofunctionalization.Collectively, we demonstrated that the diversification of effector genes in related pathogens can be caused both by alteration on the transcriptional level and through functional diversification of the encoded effector proteins. [ABSTRACT FROM AUTHOR]

SUMMARY: The biotrophic pathogen Ustilago maydis causes smut disease on maize (Zea mays) and induces the formation of tumours on all aerial parts of the plant. Unlike in other biotrophic interactions, no gene‐for‐gene interactions have been identified in the maize–U. maydis pathosystem. Thus, maize resistance to U. maydis is considered a polygenic, quantitative trait. Here, we study the molecular mechanisms of quantitative disease resistance (QDR) in maize, and how U. maydis interferes with its components. Based on quantitative scoring of disease symptoms in 26 maize lines, we performed an RNA sequencing (RNA‐Seq) analysis of six U. maydis‐infected maize lines of highly distinct resistance levels. The different maize lines showed specific responses of diverse cellular processes to U. maydis infection. For U. maydis, our analysis identified 406 genes being differentially expressed between maize lines, of which 102 encode predicted effector proteins. Based on this analysis, we generated U. maydis CRISPR/Cas9 knock‐out mutants for selected candidate effector sets. After infections of different maize lines with the fungal mutants, RNA‐Seq analysis identified effectors with quantitative, maize line‐specific virulence functions, and revealed auxin‐related processes as a possible target for one of them. Thus, we show that both transcriptional activity and virulence function of fungal effector genes are modified according to the infected maize line, providing insights into the molecular mechanisms underlying QDR in the maize–U. maydis interaction. Significance Statement: Resistance of maize to the fungal pathogen Ustilago maydis is a multiallelic, quantitative trait. Transcriptomics of U. maydis and maize lines with different levels of resistance, followed by CRISPR‐Cas9 mediated mutagenesis of the pathogen revealed that both transcriptional activity and virulence function of fungal effector genes can be specific to maize lines, providing insight in the complexity of co‐adaptation between an adapted fungal pathogen and its host. [ABSTRACT FROM AUTHOR]

Plants secrete various defence-related proteins into the apoplast, including proteases. Papain-like cysteine proteases (PLCPs) are central components of the plant immune system. To overcome plant immunity and successfully colonize their hosts, several plant pathogens secrete effector proteins inhibiting plant PLCPs. We hypothesized that not only pathogens, but also mutualistic microorganisms interfere with PLCP-meditated plant defences to maintain endophytic colonization with their hosts. Epichloë festucae forms mutualistic associations with cool season grasses and produces a range of secondary metabolites that protect the host against herbivores. In this study, we performed a genome-wide identification of Lolium perenne PLCPs, analysed their evolutionary relationship, and classified them into nine PLCP subfamilies. Using activity-based protein profiling, we identified four active PLCPs in the apoplast of L. perenne leaves that are inhibited during endophyte interactions. We characterized the L. perenne cystatin LpCys1 for its inhibitory capacity against ryegrass PLCPs. LpCys1 abundance is not altered during the mutualistic interaction and it mainly inhibits LpCP2. However, since the activity of other L. perenne PLCPs is not sensitive to LpCys1, we propose that additional inhibitors, likely of fungal origin, are involved in the suppression of apoplastic PLCPs during E. festucae infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plant pathogens colonize their host through the secretion of effector proteins that modulate plant metabolism and immune responses to their benefit. Plants evolve towards effector recognition, leading to host immunity. Typically, pathogen effectors are targets for recognition through plant receptors that are encoded by resistance genes. Resistance gene mediated crop immunity puts a tremendous pressure on pathogens to adapt and alter their effector repertoire to overcome recognition. We argue that the type of effector that is recognized by the host may have considerable implications on the durability of resistance against filamentous plant pathogens. Effector genes that are conserved among pathogens and reside in core genome regions are most likely to hold indispensable virulence functions. Consequently, the cost for the pathogen to overcome recognition by the host is higher than for diversified, host‐specific effectors with a quantitative impact on virulence. Consequently, resistance genes that directly target conserved effector proteins without the interception of other effector proteins are potentially excellent resistance resources. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Smut fungi are a large group of biotrophic plant pathogens that infect mostly monocot species, including economically relevant cereal crops. For years, Ustilago maydis has stood out as the model system to study the genetics and cell biology of smut fungi as well as the pathogenic development of biotrophic plant pathogens. The identification and functional characterization of secreted effectors and their role in virulence have particularly been driven forward using the U. maydis–maize pathosystem. Today, advancing tools for additional smut fungi such as Ustilago hordei and Sporisorium reilianum, as well as an increasing number of available genome sequences, provide excellent opportunities to investigate in parallel the effector function and evolution associated with different lifestyles and host specificities. In addition, genome analyses revealed similarities in the genomic signature between pathogenic smuts and epiphytic Pseudozyma species. This review elaborates on how knowledge about fungal lifestyles, genome biology, and functional effector biology has helped in understanding the biology of this important group of fungal pathogens. We highlight the contribution of the U. maydis model system but also discuss the differences from other smut fungi, which raises the importance of comparative genomic and genetic analyses in future research. [ABSTRACT FROM AUTHOR]

The basidiomycete smut fungi are predominantly plant parasitic, causing severe losses in some crops. Most species feature a saprotrophic haploid yeast stage, and several smut fungi are only known from this stage, with some isolated from habitats without suitable hosts, e.g. from Antarctica. Thus, these species are generally believed to be apathogenic, but recent findings that some of these might have a plant pathogenic teleomorph counterpart cast doubts on the validity of this hypothesis. Here, four genomes of species previously assigned to the polyphyletic genus Pseudozyma were re-annotated and compared with published smut pathogens. It was found that 113 genes coding for putative secreted effector proteins were conserved among smut-causing and Pseudozyma genomes. Among these were several validated effector genes, including Pep1. Orthologs of this well-characterised effector from Pseudozyma yeasts were further analysed and checked for their ability to complement a Pep1-deficient mutants of Ustilago maydis. By genetic complementation, we show that Pep1 homologs from the supposedly apathogenic yeasts restore virulence in Pep1-deficient mutants Ustilago maydis. Thus, it is concluded that Pseudozyma species have likely retained a suite of effectors, which hints at the possibility that Pseudozyma species have kept an unknown plant pathogenic stage for sexual recombination. However, it cannot be excluded that these effectors might also have positive effects also when colonising plant surfaces. [ABSTRACT FROM AUTHOR]

Plant proteases are key regulators of plant cell processes such as seed development, immune responses, senescence and programmed cell death (PCD). Apoplastic papain-like cysteine proteases (PL) are hubs in plant-microbe interactions and play an important role during abiotic stresses. The apoplast is a crucial interface for the interaction between plant and microbes. So far, apoplastic maize PL and their function have been mostly described for aerial parts. In this study, we focused on apoplastic PLCPs in the roots of maize plants. We have analyzed the phylogeny of maize PLCPs and investigated their protein abundance after salicylic acid (SA) treatment. Using activity-based protein profiling (ABPP) we have identified a novel root-specific PLCP belonging to the RD21-like subfamily, as well as three SA activated PLCPs. The root specific PLCP CP1C shares sequence and structural similarities to known CP1-like proteases. Biochemical analysis of recombinant CP1C revealed different substrate specificities and inhibitor affinities compared to the related proteases. This study characterized a root-specific PLCP and identifies differences between the SA-dependent activation of PLCPs in roots and leaves. [ABSTRACT FROM AUTHOR]

Summary: The success of plant‐pathogenic fungi mostly relies on their arsenal of virulence factors which are expressed and delivered into the host tissue during colonization. The biotrophic fungal pathogen Ustilago hordei causes covered smut disease on both barley and oat. In this study, we combined cytological, genomics and molecular biological methods to achieve a better understanding of the molecular interactions in the U. hordei–barley pathosystem. Microscopic analysis revealed that U. hordei densely colonizes barley leaves on penetration, in particular the vascular system. Transcriptome analysis of U. hordei at different stages of host infection revealed differential expression of the transcript levels of 273 effector gene candidates. Furthermore, U. hordei transcriptionally activates core effector genes which may suppress even non‐host early defence responses. Based on expression profiles and novelty of sequences, knockout studies of 14 effector candidates were performed in U. hordei, which resulted in the identification of four virulence factors required for host colonization. Yeast two‐hybrid screening identified potential barley targets for two of the effectors. Overall, this study provides a first systematic analysis of the effector repertoire of U. hordei and identifies four effectors (Uvi1–Uvi4) as virulence factors for the infection of barley. [ABSTRACT FROM AUTHOR]

Summary: Fungalysins from several phytopathogenic fungi have been shown to be involved in cleavage of plant chitinases. While fungal chitinases are responsible for cell wall remodeling during growth and morphogenesis, plant chitinases are important components of immunity. This study describes a dual function of the Ustilago maydis fungalysin UmFly1 in modulation of both plant and fungal chitinases. Genetic, biochemical and microscopic experiments were performed to elucidate the in vitro and in planta functions of U. maydis UmFly1. U. maydis ∆umfly1 mutants show significantly reduced virulence, which coincides with reduced cleavage of the maize chitinase ZmChiA within its chitin‐binding domain. Moreover, deletion of umfly1 affected the cell separation of haploid U. maydis sporidia. This phenotype is associated with posttranslational activation of the endogenous chitinase UmCts1. Genetic complementation of the ∆umfly1 mutant with a homologous gene from closely related, but nonpathogenic, yeast fully rescued the cell separation defect in vitro, but it could not recover the ∆umfly1 defect in virulence and cleavage of the maize chitinase. We report on the dual function of the secreted fungalysin UmFly1. We hypothesize that co‐evolution of U. maydis with its host plant extended the endogenous function of UmFly1 towards the modulation of plant chitinase activity to promote infection. [ABSTRACT FROM AUTHOR]

Summary: The biotrophic fungus Ustilago maydis causes smut disease on maize (Zea mays), which is characterized by immense plant tumours. To establish disease and reprogram organ primordia to tumours, U. maydis deploys effector proteins in an organ‐specific manner. However, the cellular contribution to leaf tumours remains unknown. We investigated leaf tumour formation at the tissue‐ and cell type‐specific levels. Cytology and metabolite analysis were deployed to understand the cellular basis for tumourigenesis. Laser‐capture microdissection was performed to gain a cell type‐specific transcriptome of U. maydis during tumour formation.In vivo visualization of plant DNA synthesis identified bundle sheath cells as the origin of hyperplasic tumour cells, while mesophyll cells become hypertrophic tumour cells. Cell type‐specific transcriptome profiling of U. maydis revealed tailored expression of fungal effector genes. Moreover, U. maydis See1 was identified as the first cell type‐specific fungal effector, being required for induction of cell cycle reactivation in bundle sheath cells. Identification of distinct cellular mechanisms in two different leaf cell types and of See1 as an effector for induction of proliferation of bundle sheath cells are major steps in understanding U. maydis‐induced tumour formation. Moreover, the cell type‐specific U. maydis transcriptome data are a valuable resource to the scientific community. [ABSTRACT FROM AUTHOR]

Yeasts of the now unused asexually typified genus Pseudozyma belong to the smut fungi (Ustilaginales) and are mostly believed to be apathogenic asexual yeasts derived from smut fungi that have lost pathogenicity on plants. However, phylogenetic studies have shown that most Pseudozyma species are phylogenetically close to smut fungi parasitic to plants, suggesting that some of the species might represent adventitious isolations of the yeast morph of otherwise plant pathogenic smut fungi. However, there are some species, such as Moesziomyces aphidis (syn. Pseudozyma aphidis) that are isolated throughout the world and sometimes are also found in clinical samples and do not have a known plant pathogenic sexual morph. In this study, it is revealed by phylogenetic investigations that isolates of the biocontrol agent Moesziomyces aphidis are interspersed with M. bullatus sexual lineages, suggesting conspecificity. This raises doubts regarding the apathogenic nature of asexual morphs previously placed in Pseudozyma, but suggests that there might also be pathogenic sexual morph counterparts for those species known only from asexual morphs. The finding that several additional species currently only known from their yeast morphs are embedded within the genus Moesziomyces, suggests that the yeast morph might play a more dominant role in this genus as compared to other genera of Ustilaginaceae. In addition, phylogenetic reconstructions demonstrated that Moesziomyces bullatus has a narrow host range and that some previously described but not widely used species names should be applied for Moesziomyces on other host genera than Echinochloa. [ABSTRACT FROM AUTHOR]

Many filamentous fungal pathogens induce drastic modulation of host cells causing abnormal infectious structures such as galls, or tumors that arise as a result of re-programming in the original developmental cell fate of a colonized host cell. Developmental consequences occur predominantly with biotrophic phytopathogens. This suggests that these host structures result as an outcome of efficient defense suppression and intimate fungal-host interaction to suit the pathogen's needs for completion of its infection cycle. This mini-review mainly summarizes host cell re-programming that occurs in the Ustilago maydis - maize interaction, in which the pathogen deploys cell-type specific effector proteins with varying activities. The fungus senses the physiological status and identity of colonized host cells and re-directs the endogenous developmental program of its host. The disturbance of host cell physiology and cell fate leads to novel cell shapes, increased cell size, and/or the number of host cells. We particularly highlight the strategies of U. maydis to induce physiologically varied host organs to form the characteristic tumors in both vegetative and floral parts of maize. [ABSTRACT FROM AUTHOR]

902I.902II.903III.903IV.903V.905VI.905VII.905906References906 Summary: Plants deploy a sophisticated immune system to cope with different microbial pathogens and other invaders. Recent research provides an increasing body of evidence for papain‐like cysteine proteases (PLCPs) being central hubs in plant immunity. PLCPs are required for full resistance of plants to various pathogens. At the same time, PLCPs are targeted by secreted pathogen effectors to suppress immune responses. Consequently, they are subject to a co‐evolutionary host–pathogen arms race. When activated, PLCPs induce a broad spectrum of defense responses including plant cell death. While the important role of PLCPs in plant immunity has become more evident, it remains largely elusive how these enzymes are activated and which signaling pathways are triggered to orchestrate different downstream responses. See also the Commentary on this article by Ökmen & Doehlemann, 212: 799–801. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR), a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER), coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker’s yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs) in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP) analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors. [ABSTRACT FROM AUTHOR]

The secreted fungal effector Pep1 is essential for penetration of the host epidermis and establishment of biotrophy in the Ustilago maydis-maize pathosystem. Previously, Pep1 was found to be an inhibitor of apoplastic plant peroxidases, which suppresses the oxidative burst, a primary immune response of the host plant and enables fungal colonization., To investigate the conservation of Pep1 in other pathogens, genomes of related smut species were screened for pep1 orthologues. Pep1 proteins were produced in Escherichia coli for functional assays. The biological function of Pep1 was tested by heterologous expression in U. maydis and Hordeum vulgare., Pep1 orthologues revealed a remarkable degree of sequence conservation, indicating that this effector might play a fundamental role in virulence of biotrophic smut fungi. Pep1 function and its role in virulence are conserved in different pathogenic fungi, even across the monocot-dicot border of host plants., The findings described in this study classify Pep1 as a phylogenetically conserved fungal core effector. Furthermore, we documented the influence of Pep1 on the disease caused by Blumeria graminis f. sp. hordei which is a non-smut-related pathosystem. [ABSTRACT FROM AUTHOR]

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize. [ABSTRACT FROM AUTHOR]

Although phylogenetically unrelated, filamentous oomycetes and fungi establish similar structures to colonize plants and they represent economically the most important microbial threat to crop production. In mutualistic interactions established by root-colonizing fungi, clear differences to pathogens can be seen, but there is mounting evidence that their infection strategies and molecular interactions have certain common features. To infect the host, fungi and oomycetes employ similar strategies to circumvent plant innate immunity. This process involves the suppression of basal defence responses which are triggered by the perception of conserved molecular patterns. To establish biotrophy, effector proteins are secreted from mutualistic and pathogenic microbes to the host tissue, where they play central roles in the modulation of host immunity and metabolic reprogramming of colonized host tissues. This review article discusses key effector mechanisms of filamentous pathogens and mutualists, how they modulate their host targets and the fundamental differences or parallels between these different interactions. The orchestration of effector actions during plant infection and the importance of their localization within host tissues are also discussed. [ABSTRACT FROM AUTHOR]

This article is a Commentary on De Wit, 212: 805–813; Derevnina et al., 212: 888–895; Rovenich et al., 212: 896–901 and Misas‐Villamil et al., 212: 902–907. [ABSTRACT FROM AUTHOR]

With the exception of Ustilago maydis, smut fungi infecting monocotyledonous hosts systemically colonize infected plants and cause symptoms exclusively in the inflorescences. Ustilago maydis infects primordia of all aerial organs of maize ( Zea mays L.) and results in the formation of large plant tumours. Previously, we have found that U. maydis infection of seedling leaves, adult leaves and tassels causes organ-specific transcriptional changes in both the pathogen and the host. Of particular interest, U. maydis genes encoding secreted proteins are differentially expressed depending on the colonized maize organ. Therefore, we hypothesized that the fungus secretes virulence-related proteins (effectors) that act in an organ-specific manner. Here, we present the identification and functional characterization of 20 presumptive organ-specific U. maydis effector genes. Ustilago maydis deletion strains for these genes were generated and tested for infectivity of maize seedling leaves and tassels. This approach identified 11 effector genes required for the full virulence of U. maydis. In nine cases, virulence was only affected in one of the tested plant organs. These results demonstrate that individual fungal effector proteins contribute to fungal virulence in an organ-specific manner. [ABSTRACT FROM AUTHOR]

In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function. [ABSTRACT FROM AUTHOR]

Salicylic acid ( SA) is a key plant defence hormone which plays an important role in local and systemic defence responses against biotrophic pathogens like the smut fungus Ustilago maydis. Here we identified Shy1, a cytoplasmic U. maydis salicylate hydroxylase which has orthologues in the closely related smuts Ustilago hordei and Sporisorium reilianum. shy1 is transcriptionally induced during the biotrophic stages of development but not required for virulence during seedling infection. Shy1 activity is needed for growth on plates with SA as a sole carbon source. The trigger for shy1 transcriptional induction is SA, suggesting the possibility of a SA sensing mechanism in this fungus. [ABSTRACT FROM AUTHOR]

1001I.1001II.1003III.1004IV.1005V.1006VI.1007VII.10111011References1011 Summary: Microbial plant pathogens have evolved a variety of strategies to enter plant hosts and cause disease. In particular, biotrophic pathogens, which parasitize living plant tissue, establish sophisticated interactions in which they modulate the plant's metabolism to their own good. The prime decision, whether or not a pathogen can accommodate itself in its host tissue, is made during the initial phase of infection. At this stage, the plant immune system recognizes conserved molecular patterns of the invading microbe, which initiate a set of basal immune responses. Induced plant defense proteins, toxic compounds and antimicrobial proteins encounter a broad arsenal of pathogen‐derived virulence factors that aim to disarm host immunity. Crucial regulatory processes and protein–protein interactions take place in the apoplast, that is, intercellular spaces, plant cell walls and defined host–pathogen interfaces which are formed between the plant cytoplasm and the specialized infection structures of many biotrophic pathogens. This article aims to provide an insight into the most important principles and components of apoplastic plant immunity and its modulation by filamentous microbial pathogens. [ABSTRACT FROM AUTHOR]

The basidiomycete Ustilago maydis causes smut disease in maize, with large plant tumors being formed as the most prominent disease symptoms. During all steps of infection, U. maydis depends on a biotrophic interaction, which requires an efficient suppression of plant immunity. In a previous study, we identified the secreted effector protein Pit2, which is essential for maintenance of biotrophy and induction of tumors. Deletion mutants for pit2 successfully penetrate host cells but elicit various defense responses, which stops further fungal proliferation. We now show that Pit2 functions as an inhibitor of a set of apoplastic maize cysteine proteases, whose activity is directly linked with salicylic-acid-associated plant defenses. Consequently, protease inhibition by Pit2 is required for U. maydis virulence. Sequence comparisons with Pit2 orthologs from related smut fungi identified a conserved sequence motif. Mutation of this sequence caused loss of Pit2 function. Consequently, expression of the mutated protein in U. maydis could not restore virulence of the pit2 deletion mutant, indicating that the protease inhibition by Pit2 is essential for fungal virulence. Moreover, synthetic peptides of the conserved sequence motif showed full activity as protease inhibitor, which identifies this domain as a new, minimal protease inhibitor domain in plant-pathogenic fungi. [ABSTRACT FROM AUTHOR]

Mutations in the l(3)mbt tumour suppressor result in overproliferation of Drosophila larval brains. Recently, the derepression of different gene classes in l(3)mbt mutants was shown to be causal for transformation. However, the molecular mechanisms of dL(3)mbt-mediated gene repression are not understood. Here, we identify LINT, the major dL(3)mbt complex of Drosophila. LINT has three core subunits--dL(3)mbt, dCoREST, and dLint-1--and is expressed in cell lines, embryos, and larval brain. Using genome-wide ChIP--Seq analysis, we show that dLint-1 binds close to the TSS of tumour-relevant target genes. Depletion of the LINT core subunits results in derepression of these genes. By contrast, histone deacetylase, histone methylase, and histone demethylase activities are not required to maintain repression. Our results support a direct role of LINT in the repression of brain tumour-relevant target genes by restricting promoter access. [ABSTRACT FROM AUTHOR]

The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1) as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H2O2 strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction. [ABSTRACT FROM AUTHOR]

Ustilago maydis is a biotrophic pathogen causing maize (Zea mays) smut disease. Transcriptome profiling of infected maize plants indicated that a gene encoding a putative cystatin (CC9) is induced upon penetration by U. maydis wild type. By contrast, cc9 is not induced after infection with the U. maydis effector mutant Δ pep1 , which elicits massive plant defenses. Silencing of cc9 resulted in a strongly induced maize defense gene expression and a hypersensitive response to U. maydis wild-type infection. Consequently, fungal colonization was strongly reduced in cc9 -silenced plants, while recombinant CC9 prevented salicylic acid (SA)–induced defenses. Protease activity profiling revealed a strong induction of maize Cys proteases in SA-treated leaves, which could be inhibited by addition of CC9. Transgenic maize plants overexpressing cc9-mCherry showed an apoplastic localization of CC9. The transgenic plants showed a block in Cys protease activity and SA-dependent gene expression. Moreover, activated apoplastic Cys proteases induced SA-associated defense gene expression in naïve plants, which could be suppressed by CC9. We show that apoplastic Cys proteases play a pivotal role in maize defense signaling. Moreover, we identified cystatin CC9 as a novel compatibility factor that suppresses Cys protease activity to allow biotrophic interaction of maize with the fungal pathogen U. maydis. [ABSTRACT FROM AUTHOR]

Summary Ustilago maydis is a biotrophic fungal pathogen that colonizes living tissue of its host plant maize. Based on transcriptional upregulation during biotrophic development we identified the pit ( proteins important for tumours) cluster, a novel gene cluster comprising four genes of which two are predicted to encode secreted effectors. Disruption of the gene cluster abolishes U. maydis-induced tumour formation and this phenotype can be caused by deleting either pit1 encoding a transmembrane protein or pit2 encoding a secreted protein. Pit1 localizes to the fungal plasma membrane at hyphal tips, endosomes and vacuoles while Pit2 is secreted to the biotrophic interface. Both Δ pit1 and Δ pit2 mutants are able to penetrate maize epidermis and grow intracellularly at sites of infection but fail to spread in the infected leaf. Microarray analysis shows an indistinguishable response of the plant to infection by Δ pit1 and Δ pit2 mutant strains. Transcriptional activation of maize defence genes in infections with Δ pit1/2 mutant strains indicates that the mutants have a defect in suppressing plant immune responses. Our results suggest that the activity of Pit1 and Pit2 during tumour formation might be functionally linked and we discuss possibilities for a putative functional connection of the two proteins. [ABSTRACT FROM AUTHOR]

Fungi inhabit every natural and anthropogenic environment on Earth. They have highly varied life-styles including saprobes (using only dead biomass as a nutrient source), pathogens (feeding on living biomass), and symbionts (co-existing with other organisms). These distinctions are not absolute as many species employ several life styles (e.g. saprobe and opportunistic pathogen, saprobe and mycorrhiza). To efficiently survive in these different and often changing environments, fungi need to be able to modify their physiology and in some cases will even modify their local environment. Understanding the interaction between fungi and their environments has been a topic of study for many decades. However, recently these studies have reached a new dimension. The availability of fungal genomes and development of postgenomic technologies for fungi, such as transcriptomics, proteomics and metabolomics, have enabled more detailed studies into this topic resulting in new insights. Based on a Special Interest Group session held during IMC9, this paper provides examples of the recent advances in using (post-)genomic approaches to better understand fungal interactions with their environments. [ABSTRACT FROM AUTHOR]

Infection of maize (Zea mays) plants with the corn smut fungus Ustilago maydis leads to the formation of large tumors on the stem, leaves and inflorescences. In this biotrophic interaction, plant defense responses are actively suppressed by the pathogen, and previous transcriptome analyses of infected maize plants showed massive and stage-specific changes in host gene expression during disease progression. To identify maize genes that are functionally involved in the interaction with U. maydis, we adapted a virus-induced gene silencing (VIGS) system based on the brome mosaic virus (BMV) for maize. Conditions were established that allowed successful U. maydis infection of BMV-preinfected maize plants. This set-up enabled quantification of VIGS and its impact on U. maydis infection using a quantitative real-time PCR (qRT-PCR)-based readout. In proof-of-principle experiments, an U. maydis-induced terpene synthase was shown to negatively regulate disease development while a protein involved in cell death inhibition was required for full virulence of U. maydis. The results suggest that this system is a versatile tool for the rapid identification of maize genes that determine compatibility with U. maydis. [ABSTRACT FROM AUTHOR]

Class V chitin synthases are fungal virulence factors required for plant infection. They consist of a myosin motor domain fused to a membrane-spanning chitin synthase region that participates in fungal cell wall formation. The function of the motor domain is unknown, but it might deliver the myosin chitin synthase-attached vesicles to the growth region. Here, we analyze the importance of both domains in Mcs1, the chitin synthase V of the maize smut fungus Ustilago maydis. By quantitative analysis of disease symptoms, tissue colonization, and single-cell morphogenic parameters, we demonstrate that both domains are required for fungal virulence. Fungi carrying mutations in the chitin synthase domain are rapidly recognized and killed by the plant, whereas fungi carrying a deletion of the motor domain show alterations in cell wall composition but can invade host tissue and cause a moderate plant response. We also show that Mcs1-bound vesicles exhibit long-range movement for up to 20 μm at a velocity of ~1.75 μm/s. Apical Mcs1 localization depends on F-actin and the motor domain, whereas Mcs1 motility requires microtubules and persists when the Mcs1 motor domain is deleted. Our results suggest that the myosin motor domain of ChsV supports exocytosis but not long-range delivery of transport vesicles. [ABSTRACT FROM AUTHOR]

The basidiomycete Ustilago maydis is the causal agent of corn smut disease and induces tumor formation during biotrophic growth in its host maize (Zea mays). We have conducted a combined metabolome and transcriptome survey of infected leaves between 1 d post infection (dpi) and 8 dpi, representing infected leaf primordia and fully developed tumors, respectively. At 4 and 8 dpi, we observed a substantial increase in contents of the nitrogen-rich amino acids glutamine and asparagine, while the activities of enzymes involved in primary nitrogen assimilation and the content of ammonia and nitrate were reduced by 50% in tumors compared with mock controls. Employing stable isotope labeling, we could demonstrate that U. maydis-induced tumors show a reduced assimilation of soil-derived 15NO3 and represent strong sinks for nitrogen. Specific labeling of the free amino acid pool of systemic source leaves with [15N]urea revealed an increased import of organic nitrogen from systemic leaves to tumor tissue, indicating that organic nitrogen provision supports the formation of U. maydis-induced tumors. In turn, amino acid export from systemic source leaves was doubled in infected plants. The analysis of the phloem amino acid pool revealed that glutamine and asparagine are not transported to the tumor tissue, although these two amino acids were found to accumulate within the tumor. Photosynthesis was increased and senescence was delayed in systemic source leaves upon tumor development on infected plants, indicating that the elevated sink demand for nitrogen could determine photosynthetic rates in source leaves. [ABSTRACT FROM AUTHOR]