CRISPR/Cas9 Ribonucleoprotein-Mediated Mutagenesis in Sporisorium reilianum.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has become the state of the art for mutagenesis in filamentous fungi. Here, we describe a ribonucleoprotein complex (RNP)-mediated CRISPR/Cas9 for mutagenesis in Sporisorium reilianum. The efficiency of the method was tested in vitro with a cleavage assay as well as in vivo with a GFP-expressing S. reilianum strain. We applied this method to generate frameshift- and knock-out mutants in S. reilianum without a resistance marker by using an auto-replicating plasmid for selection. The RNP-mediated CRISPR/Cas9 increased the mutagenesis efficiency, can be applied for all kinds of mutations, and enables a marker-free genome editing in S. reilianum. Key features • First CRISPR/Cas9 application in S. reilianum. • Generation of S. reilianum mutants without genomic integration of resistance marker. • Allows the generation of multiple gene knockouts as well as deletion of large genomic regions.

NIa-Pro of sugarcane mosaic virus targets Corn Cysteine Protease 1 (CCP1) to undermine salicylic acid-mediated defense in maize.

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.

The fungal pathogen Ustilago maydis targets the maize corepressor RELK2 to modulate host transcription for tumorigenesis.

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.

A transcriptional activator effector of Ustilago maydis regulates hyperplasia in maize during pathogen-induced tumor formation.

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.

A conserved extracellular Ribo1 with broad-spectrum cytotoxic activity enables smut fungi to compete with host-associated bacteria.

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.

Combination of transcriptomic, proteomic, and degradomic profiling reveals common and distinct patterns of pathogen-induced cell death in maize.

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.

Combination of in vivo proximity labeling and co-immunoprecipitation identifies the host target network of a tumor-inducing effector in the fungal maize pathogen Ustilago maydis.

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.

Maize Phytocytokines Modulate Pro-Survival Host Responses and Pathogen Resistance.

Phytocytokines are signaling peptides that alert plant cells of danger. However, the downstream responses triggered by phytocytokines and their effect on plant survival are still largely unknown. Here, we have identified three biologically active maize orthologues of phytocytokines previously described in other plants. The maize phytocytokines show common features with microbe-associated molecular patterns (MAMPs), including the induction of immune-related genes and activation of papain-like cysteine proteases. In contrast to MAMPs, phytocytokines do not promote cell death in the presence of wounding. In infection assays with two fungal pathogens, we found that phytocytokines affect the development of disease symptoms, likely due to the activation of phytohormonal pathways. Collectively, our results show that phytocytokines and MAMPs trigger unique and antagonistic features of immunity. We propose a model in which phytocytokines activate immune responses partially similar to MAMPs but, in contrast to microbial signals, they act as danger and survival molecules to the surrounding cells. Future studies will focus on the components determining the divergence of signaling outputs upon phytocytokine activation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A conserved enzyme of smut fungi facilitates cell-to-cell extension in the plant bundle sheath.

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-ß-glucanase activity, which is required to attenuate ß-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.

Many ways to TOPLESS - manipulation of plant auxin signalling by a cluster of fungal effectors.

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.

Detection of Apoplastic Protease Inhibitors Using Convolution Activity-Based Protein Profiling.

Activity-based protein profiling (ABPP) is a powerful tool in biological chemistry to monitor protein activity using chemical probes that bind covalently and irreversible to active site of enzymes such as proteases. To date, there are three different ways to experimentally use ABPP: comparative, competitive, and convolution ABPP. Here we use and describe the convolution ABPP approach, a method used to detect changes in protease inhibitor abundance in different proteomes. We have applied this method to monitor the activity of Lolium perenne apoplastic cysteine proteases during the interaction with the fungal endophyte Epichloë festucae. We describe the method to isolate apoplastic fluids from infected and uninfected L. perenne ryegrass leaves and the protocol to perform a convolution ABPP experiment. Furthermore, we report how to quantify and analyze fluorescent gels obtained from the ABPP labeling.

Secreted Glycoside Hydrolase Proteins as Effectors and Invasion Patterns of Plant-Associated Fungi and Oomycetes.

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.

Cross-species analysis between the maize smut fungi Ustilago maydis and Sporisorium reilianum highlights the role of transcriptional change of effector orthologs for virulence and disease.

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.

Host apoplastic cysteine protease activity is suppressed during the mutualistic association of Lolium perenne and Epichloë festucae.

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.

Comparative transcriptome profiling identifies maize line specificity of fungal effectors in the maize-Ustilago maydis interaction.

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.

The Ustilago hordei-Barley Interaction Is a Versatile System for Characterization of Fungal Effectors.

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.

A fungal member of the Arabidopsis thaliana phyllosphere antagonizes Albugo laibachii via a GH25 lysozyme.

Plants are not only challenged by pathogenic organisms but also colonized by commensal microbes. The network of interactions these microbes establish with their host and among each other is suggested to contribute to the immune responses of plants against pathogens. In wild Arabidopsis thaliana populations, the oomycete pathogen Albugo laibachii plays an influential role in structuring the leaf phyllosphere. We show that the epiphytic yeast Moesziomyces bullatus ex Albugo on Arabidopsis, a close relative of pathogenic smut fungi, is an antagonistic member of the A. thaliana phyllosphere, which reduces infection of A. thaliana by A. laibachii. Combination of transcriptomics, reverse genetics, and protein characterization identified a GH25 hydrolase with lysozyme activity as a major effector of this microbial antagonism. Our findings broaden the understanding of microbial interactions within the phyllosphere, provide insights into the evolution of epiphytic basidiomycete yeasts, and pave the way for novel biocontrol strategies.

Much like the 'good bacteria' that live in our guts, many microscopic organisms can co-exist with and even benefit the plants they live on. For instance, the yeast Moesziomyces bullatus ex Albugo (MbA for short) can shield the leaves of its plant host against white rust, a disease caused by the organism Albugo laibachii. Studies have started to unveil how the various microbes at the surface of leaves interact and regulate their own community, yet the genetic mechanisms at play are less well-known. To investigate these processes, Eitzen et al. examined the genes that were switched on when MbA cells were in contact with A. laibachii on a leaf. This experiment revealed a few gene candidates that were then deleted, one by one, in MbA cells. As a result, a gene emerged as being key to protect the plant from white rust. It produces an enzyme known as the GH25 hydrolase, which, when purified, could reduce A. laibachii infections on plant leaves. Bacteria, fungi and other related microorganisms cause many diseases which, like white rust, can severely affect crops. Chemical methods exist to prevent these infections but they can have many biological and ecological side effects. A solution inspired by natural interactions may be safer and more effective at managing plant diseases that affect valuable crops. Harnessing the interactions between microbes living on plants, and the GH25 enzyme, may offer better disease control.

Shaping the leaf microbiota: plant-microbe-microbe interactions.

The aerial portion of a plant, namely the leaf, is inhabited by pathogenic and non-pathogenic microbes. The leaf's physical and chemical properties, combined with fluctuating and often challenging environmental factors, create surfaces that require a high degree of adaptation for microbial colonization. As a consequence, specific interactive processes have evolved to establish a plant leaf niche. Little is known about the impact of the host immune system on phyllosphere colonization by non-pathogenic microbes. These organisms can trigger plant basal defenses and benefit the host by priming for enhanced resistance to pathogens. In most disease resistance responses, microbial signals are recognized by extra- or intracellular receptors. The interactions tend to be species specific and it is unclear how they shape leaf microbial communities. In natural habitats, microbe-microbe interactions are also important for shaping leaf communities. To protect resources, plant colonizers have developed direct antagonistic or host manipulation strategies to fight competitors. Phyllosphere-colonizing microbes respond to abiotic and biotic fluctuations and are therefore an important resource for adaptive and protective traits. Understanding the complex regulatory host-microbe-microbe networks is needed to transfer current knowledge to biotechnological applications such as plant-protective probiotics.

Cas9HF1 enhanced specificity in Ustilago maydis.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely used as a tool to precisely manipulate genomic sequence targeted by sgRNA (single guide RNA) and is adapted in different species for genome editing. One of the major concerns of CRISPR-Cas9 is the possibility of off-target effects, which can be remedied by the deployment of high fidelity Cas9 variants. Ustilago maydis is a maize fungal pathogen, which has served as a model organism for biotrophic pathogens for decades. The successful adaption of CRISPR-Cas9 in U. maydis greatly facilitated effector biology studies. Here, we constructed an U. maydis reporter strain that allows in vivo quantification of efficiency and target specificity of three high fidelity Cas9 variants, Cas9HF1, Cas9esp1.1 and Cas9hypa. This approach identified Cas9HF1 as most specific Cas9 variant in U. maydis. Furthermore, whole genome sequencing showed absence of off-target effects in U. maydis by CRISPR-Cas9 editing.

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

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.