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.

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.

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.

The Interspecific Fungal Hybrid Verticillium longisporum Displays Subgenome-Specific Gene Expression.

Hybridization is an important evolutionary mechanism that can enable organisms to adapt to environmental challenges. It has previously been shown that the fungal allodiploid species Verticillium longisporum, the causal agent of verticillium stem striping in rapeseed, originated from at least three independent hybridization events between two haploid Verticillium species. To reveal the impact of genome duplication as a consequence of hybridization, we studied the genome and transcriptome dynamics upon two independent V. longisporum hybridization events, represented by the hybrid lineages "A1/D1" and "A1/D3." We show that V. longisporum genomes are characterized by extensive chromosomal rearrangements, including between parental chromosomal sets. V. longisporum hybrids display signs of evolutionary dynamics that are typically associated with the aftermath of allodiploidization, such as haploidization and more relaxed gene evolution. The expression patterns of the two subgenomes within the two hybrid lineages are more similar than those of the shared A1 parent between the two lineages, showing that the expression patterns of the parental genomes homogenized within a lineage. However, as genes that display differential parental expression in planta do not typically display the same pattern in vitro, we conclude that subgenome-specific responses occur in both lineages. Overall, our study uncovers genomic and transcriptomic plasticity during the evolution of the filamentous fungal hybrid V. longisporum and illustrates its adaptive potential. IMPORTANCEVerticillium is a genus of plant-associated fungi that includes a few plant pathogens that collectively affect a wide range of hosts. On several occasions, haploid Verticillium species hybridized into the stable allodiploid species Verticillium longisporum, which is, in contrast to haploid Verticillium species, a Brassicaceae specialist. Here, we studied the evolutionary genome and transcriptome dynamics of V. longisporum and the impact of the hybridization. V. longisporum genomes display a mosaic structure due to genomic rearrangements between the parental chromosome sets. Similar to other allopolyploid hybrids, V. longisporum displays an ongoing loss of heterozygosity and more relaxed gene evolution. Also, differential parental gene expression is observed, with enrichment for genes that encode secreted proteins. Intriguingly, the majority of these genes display subgenome-specific responses under differential growth conditions. In conclusion, hybridization has incited the genomic and transcriptomic plasticity that enables adaptation to environmental changes in a parental allele-specific fashion.

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.

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.

Target the core: durable plant resistance against filamentous plant pathogens through effector recognition.

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.

Molecular Interactions Between Smut Fungi and Their Host Plants.

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.

Dynamic virulence-related regions of the plant pathogenic fungus Verticillium dahliae display enhanced sequence conservation.

Plant pathogens continuously evolve to evade host immune responses. During host colonization, many fungal pathogens secrete effectors to perturb such responses, but these in turn may become recognized by host immune receptors. To facilitate the evolution of effector repertoires, such as the elimination of recognized effectors, effector genes often reside in genomic regions that display increased plasticity, a phenomenon that is captured in the two-speed genome hypothesis. The genome of the vascular wilt fungus Verticillium dahliae displays regions with extensive presence/absence polymorphisms, so-called lineage-specific regions, that are enriched in in planta-induced putative effector genes. As expected, comparative genomics reveals differential degrees of sequence divergence between lineage-specific regions and the core genome. Unanticipated, lineage-specific regions display markedly higher sequence conservation in coding as well as noncoding regions than the core genome. We provide evidence that disqualifies horizontal transfer to explain the observed sequence conservation and conclude that sequence divergence occurs at a slower pace in lineage-specific regions of the V. dahliae genome. We hypothesize that differences in chromatin organisation may explain lower nucleotide substitution rates in the plastic, lineage-specific regions of V. dahliae.

In silico prediction and characterisation of secondary metabolite clusters in the plant pathogenic fungus Verticillium dahliae.

Fungi are renowned producers of natural compounds, also known as secondary metabolites (SMs) that display a wide array of biological activities. Typically, the genes that are involved in the biosynthesis of SMs are located in close proximity to each other in so-called secondary metabolite clusters. Many plant-pathogenic fungi secrete SMs during infection in order to promote disease establishment, for instance as cytocoxic compounds. Verticillium dahliae is a notorious plant pathogen that can infect over 200 host plants worldwide. However, the SM repertoire of this vascular pathogen remains mostly uncharted. To unravel the potential of V. dahliae to produce SMs, we performed in silico predictions and in-depth analyses of its secondary metabolite clusters. Using distinctive traits of gene clusters and the conserved signatures of core genes 25 potential SM gene clusters were identified. Subsequently, phylogenetic and comparative genomics analyses were performed, revealing that two putative siderophores, ferricrocin and TAFC, DHN-melanin and fujikurin may belong to the SM repertoire of V. dahliae.

The Genome of the Fungal Pathogen Verticillium dahliae Reveals Extensive Bacterial to Fungal Gene Transfer.

Horizontal gene transfer (HGT) involves the transmission of genetic material between distinct evolutionary lineages and can be an important source of biological innovation. Reports of interkingdom HGT to eukaryotic microbial pathogens have accumulated over recent years. Verticillium dahliae is a notorious plant pathogen that causes vascular wilt disease on hundreds of plant species, resulting in high economic losses every year. Previously, the effector gene Ave1 and a glucosyltransferase-encoding gene were identified as virulence factor-encoding genes that were proposed to be horizontally acquired from a plant and a bacterial donor, respectively. However, to what extent HGT contributed to the overall genome composition of V. dahliae remained elusive. Here, we systematically searched for evidence of interkingdom HGT events in the genome of V. dahliae and provide evidence for extensive horizontal gene acquisition from bacterial origin.

The Emerging British Verticillium longisporum Population Consists of Aggressive Brassica Pathogens.

Verticillium longisporum is an economically important fungal pathogen of brassicaceous crops that originated from at least three hybridization events between different Verticillium spp., leading to the hybrid lineages A1/D1, A1/D2, and A1/D3. Isolates of lineage A1/D1 generally cause stem striping on oilseed rape (Brassica napus), which has recently been reported for the first time to occur in the United Kingdom. Intriguingly, the emerging U.K. population is distinct from the north-central European stem striping population. Little is known about the pathogenicity of the newly emerged U.K. population; hence, pathogenicity tests were executed to compare British isolates to previously characterized reference strains. In addition to the model plant Arabidopsis thaliana, the pathogenicity of four British isolates was assessed on four cultivars of three Brassica crop species: oilseed rape (Quartz and Incentive), cauliflower (Clapton), and Chinese cabbage (Hilton). To this end, vascular discoloration of the roots, plant biomass accumulations, and fungal stem colonization upon isolate infection were evaluated. The British isolates appeared to be remarkably aggressive, because plant biomass was significantly affected and severe vascular discoloration was observed. The British isolates were successful stem colonizers and the extent of fungal colonization negatively correlated with plant biomass of cauliflower and Quartz oilseed rape. However, in Quartz, the fungal colonization of A1/D1 isolates was significantly lower than that of the virulent reference isolate from lineage A1/D3, PD589. Moreover, despite levels of stem colonization similar to those of A1/D1 strains, PD589 did not cause significant disease on Incentive. Thus, A1/D1 isolates, including British isolates, are aggressive oilseed rape pathogens despite limited colonization levels in comparison with a virulent A1/D3 isolate.

A distinct and genetically diverse lineage of the hybrid fungal pathogen Verticillium longisporum population causes stem striping in British oilseed rape.

Population genetic structures illustrate evolutionary trajectories of organisms adapting to differential environmental conditions. Verticillium stem striping disease on oilseed rape was mainly observed in continental Europe, but has recently emerged in the United Kingdom. The disease is caused by the hybrid fungal species Verticillium longisporum that originates from at least three separate hybridization events, yet hybrids between Verticillium progenitor species A1 and D1 are mainly responsible for Verticillium stem striping. We reveal a hitherto un-described dichotomy within V. longisporum lineage A1/D1 that correlates with the geographic distribution of the isolates with an 'A1/D1 West' and an 'A1/D1 East' cluster. Genome comparison between representatives of the A1/D1 West and East clusters excluded population distinctiveness through separate hybridization events. Remarkably, the A1/D1 West population that is genetically more diverse than the entire A1/D1 East cluster caused the sudden emergence of Verticillium stem striping in the UK, whereas in continental Europe Verticillium stem striping is predominantly caused by the more genetically uniform A1/D1 East population. The observed genetic diversity of the A1/D1 West population argues against a recent introduction of the pathogen into the UK, but rather suggests that the pathogen previously established in the UK and remained latent or unnoticed as oilseed rape pathogen until recently.

Verticillium longisporum, the invisible threat to oilseed rape and other brassicaceous plant hosts.

INTRODUCTION: The causal agents of Verticillium wilts are globally distributed pathogens that cause significant crop losses every year. Most Verticillium wilts are caused by V. dahliae, which is pathogenic on a broad range of plant hosts, whereas other pathogenic Verticillium species have more restricted host ranges. In contrast, V. longisporum appears to prefer brassicaceous plants and poses an increasing problem to oilseed rape production. TAXONOMY: Kingdom Fungi; Phylum Ascomycota; Class Sordariomycetes; Subclass Hypocreomycetida; Family Plectosphaerellaceae; genus Verticillium. DISEASE SYMPTOMS: Dark unilateral stripes appear on the stems of apparently healthy looking oilseed rape plants at the end of the growing season. Microsclerotia are subsequently formed in the stem cortex beneath the epidermis. GENOME: Verticillium longisporum is the only non-haploid species in the Verticillium genus, as it is an amphidiploid hybrid that carries almost twice as much genetic material as the other Verticillium species as a result of interspecific hybridization. DISEASE MANAGEMENT: There is no effective fungicide treatment to control Verticillium diseases, and resistance breeding is the preferred strategy for disease management. However, only a few Verticillium wilt resistance genes have been identified, and monogenic resistance against V. longisporum has not yet been found. Quantitative resistance exists mainly in the Brassica C-genome of parental cabbage lines and may be introgressed in oilseed rape breeding lines. COMMON NAME: Oilseed rape colonized by V. longisporum does not develop wilting symptoms, and therefore the common name of Verticillium wilt is unsuitable for this crop. Therefore, we propose 'Verticillium stem striping' as the common name for Verticillium infections of oilseed rape.