The Zymoseptoria tritici Avirulence Factor AvrStb6 Accumulates in Hyphae Close to Stomata and Triggers a Wheat Defense Response Hindering Fungal Penetration.

Zymoseptoria tritici, the causal agent of Septoria tritici blotch, is one of Europe's most damaging wheat pathogens, causing significant economic losses. Genetic resistance is a common strategy to control the disease, Stb6 being a resistance gene used for more than 100 years in Europe. This study investigates the molecular mechanisms underlying Stb6-mediated resistance. Utilizing confocal microscopy imaging, we determined that Z. tritici epiphytic hyphae mainly accumulate the corresponding avirulence factor AvrStb6 in close proximity to stomata. Consequently, the progression of AvrStb6-expressing avirulent strains is hampered during penetration. The fungal growth inhibition co-occurs with a transcriptional reprogramming in wheat characterized by an induction of immune responses, genes involved in stomatal regulation, and cell wall-related genes. Overall, we shed light on the gene-for-gene resistance mechanisms in the wheat-Z. tritici pathosystem at the cytological and transcriptomic level, and our results highlight that stomatal penetration is a critical process for pathogenicity and resistance. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Virulent strains of Zymoseptoria tritici suppress the host immune response and facilitate the success of avirulent strains in mixed infections.

Plants interact with a plethora of pathogenic microorganisms in nature. Pathogen-plant interaction experiments focus mainly on single-strain infections, typically ignoring the complexity of multi-strain infections even though mixed infections are common and critical for the infection outcome. The wheat pathogen Zymoseptoria tritici forms highly diverse fungal populations in which several pathogen strains often colonize the same leaf. Despite the importance of mixed infections, the mechanisms governing interactions between a mixture of pathogen strains within a plant host remain largely unexplored. Here we demonstrate that avirulent pathogen strains benefit from being in mixed infections with virulent strains. We show that virulent strains suppress the wheat immune response, allowing avirulent strains to colonize the apoplast and to reproduce. Our experiments indicate that virulent strains in mixed infections can suppress the plant immune system, probably facilitating the persistence of avirulent pathogen strains in fields planted with resistant host plants.

Major proliferation of transposable elements shaped the genome of the soybean rust pathogen Phakopsora pachyrhizi.

With >7000 species the order of rust fungi has a disproportionately large impact on agriculture, horticulture, forestry and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. A key example is Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease, one of the world's most economically damaging agricultural diseases. Despite P. pachyrhizi's impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. Here, we sequence three independent P. pachyrhizi genomes and uncover a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. We study the incursion and dominant impact of these TEs on the genome and show how they have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity.

A thousand-genome panel retraces the global spread and adaptation of a major fungal crop pathogen.

Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici, a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen.

Epigenetic modifications affect the rate of spontaneous mutations in a pathogenic fungus.

Mutations are the source of genetic variation and the substrate for evolution. Genome-wide mutation rates appear to be affected by selection and are probably adaptive. Mutation rates are also known to vary along genomes, possibly in response to epigenetic modifications, but causality is only assumed. In this study we determine the direct impact of epigenetic modifications and temperature stress on mitotic mutation rates in a fungal pathogen using a mutation accumulation approach. Deletion mutants lacking epigenetic modifications confirm that histone mark H3K27me3 increases whereas H3K9me3 decreases the mutation rate. Furthermore, cytosine methylation in transposable elements (TE) increases the mutation rate 15-fold resulting in significantly less TE mobilization. Also accessory chromosomes have significantly higher mutation rates. Finally, we find that temperature stress substantially elevates the mutation rate. Taken together, we find that epigenetic modifications and environmental conditions modify the rate and the location of spontaneous mutations in the genome and alter its evolutionary trajectory.

Host Adaptation and Virulence in Heteroecious Rust Fungi.

Rust fungi (Pucciniales, Basidiomycota) are obligate biotrophic pathogens that cause rust diseases in plants, inflicting severe damage to agricultural crops. Pucciniales possess the most complex life cycles known in fungi. These include an alternation of generations, the development of up to five different sporulating stages, and, for many species, the requirement of infecting two unrelated host plants during different parts of their life cycle, termed heteroecism. These fungi have been extensively studied in the past century through microscopy and inoculation studies, providing precise descriptions of their infection processes, although the molecular mechanisms underlying their unique biology are poorly understood. In this review, we cover recent genomic and life cycle transcriptomic studies in several heteroecious rust species, which provide insights into the genetic tool kits associated with host adaptation and virulence, opening new avenues for unraveling their unique evolution.

Recent loss of the Dim2 DNA methyltransferase decreases mutation rate in repeats and changes evolutionary trajectory in a fungal pathogen.

DNA methylation is found throughout all domains of life, yet the extent and function of DNA methylation differ among eukaryotes. Strains of the plant pathogenic fungus Zymoseptoria tritici appeared to lack cytosine DNA methylation (5mC) because gene amplification followed by Repeat-Induced Point mutation (RIP) resulted in the inactivation of the dim2 DNA methyltransferase gene. 5mC is, however, present in closely related sister species. We demonstrate that inactivation of dim2 occurred recently as some Z. tritici isolates carry a functional dim2 gene. Moreover, we show that dim2 inactivation occurred by a different path than previously hypothesized. We mapped the genome-wide distribution of 5mC in strains with or without functional dim2 alleles. Presence of functional dim2 correlates with high levels of 5mC in transposable elements (TEs), suggesting a role in genome defense. We identified low levels of 5mC in strains carrying non-functional dim2 alleles, suggesting that 5mC is maintained over time, presumably by an active Dnmt5 DNA methyltransferase. Integration of a functional dim2 allele in strains with mutated dim2 restored normal 5mC levels, demonstrating de novo cytosine methylation activity of Dim2. To assess the importance of 5mC for genome evolution, we performed an evolution experiment, comparing genomes of strains with high levels of 5mC to genomes of strains lacking functional dim2. We found that presence of a functional dim2 allele alters nucleotide composition by promoting C to T transitions (C→T) specifically at CpA (CA) sites during mitosis, likely contributing to TE inactivation. Our results show that 5mC density at TEs is a polymorphic trait in Z. tritici populations that can impact genome evolution.

Dynamics of transposable elements in recently diverged fungal pathogens: lineage-specific transposable element content and efficiency of genome defenses.

Transposable elements (TEs) impact genome plasticity, architecture, and evolution in fungal plant pathogens. The wide range of TE content observed in fungal genomes reflects diverse efficacy of host-genome defense mechanisms that can counter-balance TE expansion and spread. Closely related species can harbor drastically different TE repertoires. The evolution of fungal effectors, which are crucial determinants of pathogenicity, has been linked to the activity of TEs in pathogen genomes. Here, we describe how TEs have shaped genome evolution of the fungal wheat pathogen Zymoseptoria tritici and four closely related species. We compared de novo TE annotations and repeat-induced point mutation signatures in 26 genomes from the Zymoseptoria species-complex. Then, we assessed the relative insertion ages of TEs using a comparative genomics approach. Finally, we explored the impact of TE insertions on genome architecture and plasticity. The 26 genomes of Zymoseptoria species reflect different TE dynamics with a majority of recent insertions. TEs associate with accessory genome compartments, with chromosomal rearrangements, with gene presence/absence variation, and with effectors in all Zymoseptoria species. We find that the extent of RIP-like signatures varies among Z. tritici genomes compared to genomes of the sister species. The detection of a reduction of RIP-like signatures and TE recent insertions in Z. tritici reflects ongoing but still moderate TE mobility.

Genome compartmentalization predates species divergence in the plant pathogen genus Zymoseptoria.

BACKGROUND: Antagonistic co-evolution can drive rapid adaptation in pathogens and shape genome architecture. Comparative genome analyses of several fungal pathogens revealed highly variable genomes, for many species characterized by specific repeat-rich genome compartments with exceptionally high sequence variability. Dynamic genome structure may enable fast adaptation to host genetics. The wheat pathogen Zymoseptoria tritici with its highly variable genome, has emerged as a model organism to study genome evolution of plant pathogens. Here, we compared genomes of Z. tritici isolates and of sister species infecting wild grasses to address the evolution of genome composition and structure. RESULTS: Using long-read technology, we sequenced and assembled genomes of Z. ardabiliae, Z. brevis, Z. pseudotritici and Z. passerinii, together with two isolates of Z. tritici. We report a high extent of genome collinearity among Zymoseptoria species and high conservation of genomic, transcriptomic and epigenomic signatures of compartmentalization. We identify high gene content variability both within and between species. In addition, such variability is mainly limited to the accessory chromosomes and accessory compartments. Despite strong host specificity and non-overlapping host-range between species, predicted effectors are mainly shared among Zymoseptoria species, yet exhibiting a high level of presence-absence polymorphism within Z. tritici. Using in planta transcriptomic data from Z. tritici, we suggest different roles for the shared orthologs and for the accessory genes during infection of their hosts. CONCLUSION: Despite previous reports of high genomic plasticity in Z. tritici, we describe here a high level of conservation in genomic, epigenomic and transcriptomic composition and structure across the genus Zymoseptoria. The compartmentalized genome allows the maintenance of a functional core genome co-occurring with a highly variable accessory genome.

Host-specialized transcriptome of plant-associated organisms.

Living organisms respond to their immediate environment by modulating their genetic programme to perform adapted functions. Eukaryotic organisms that associate with plants (fungi, oomycetes, insects, …) alter their transcriptome in a host-specific manner. Recent comparative transcriptomic studies revealed that host-specialized transcriptomes consist of a limited set of genes. Such a set typically encodes proteins that modulate host structures and functions (predicted effectors and other secreted proteins), control nutrient assimilation (proteases, transporters), and maintain cellular homeostasis (oxidoreductases, detoxification enzymes). We conclude by discussing open mechanistic and evolutionary questions and integrated approaches to move beyond descriptive studies.

Structural genomics applied to the rust fungus Melampsora larici-populina reveals two candidate effector proteins adopting cystine knot and NTF2-like protein folds.

Rust fungi are plant pathogens that secrete an arsenal of effector proteins interfering with plant functions and promoting parasitic infection. Effectors are often species-specific, evolve rapidly, and display low sequence similarities with known proteins. How rust fungal effectors function in host cells remains elusive, and biochemical and structural approaches have been scarcely used to tackle this question. In this study, we produced recombinant proteins of eleven candidate effectors of the leaf rust fungus Melampsora larici-populina in Escherichia coli. We successfully purified and solved the three-dimensional structure of two proteins, MLP124266 and MLP124017, using NMR spectroscopy. Although both MLP124266 and MLP124017 show no sequence similarity with known proteins, they exhibit structural similarities to knottins, which are disulfide-rich small proteins characterized by intricate disulfide bridges, and to nuclear transport factor 2-like proteins, which are molecular containers involved in a wide range of functions, respectively. Interestingly, such structural folds have not been reported so far in pathogen effectors, indicating that MLP124266 and MLP124017 may bear novel functions related to pathogenicity. Our findings show that sequence-unrelated effectors can adopt folds similar to known proteins, and encourage the use of biochemical and structural approaches to functionally characterize effector candidates.

Advances in understanding obligate biotrophy in rust fungi.

Contents Summary 1190 I. Introduction 1190 II. Rust fungi: a diverse and serious threat to agriculture 1191 III. The different facets of rust life cycles and unresolved questions about their evolution 1191 IV. The biology of rust infection 1192 V. Rusts in the genomics era: the ever-expanding list of candidate effector genes 1195 VI. Functional characterization of rust effectors 1197 VII. Putting rusts to sleep: Pucciniales research outlooks 1201 Acknowledgements 1202 References 1202 SUMMARY: Rust fungi (Pucciniales) are the largest group of plant pathogens and represent one of the most devastating threats to agricultural crops worldwide. Despite the economic importance of these highly specialized pathogens, many aspects of their biology remain obscure, largely because rust fungi are obligate biotrophs. The rise of genomics and advances in high-throughput sequencing technology have presented new options for identifying candidate effector genes involved in pathogenicity mechanisms of rust fungi. Transcriptome analysis and integrated bioinformatics tools have led to the identification of key genetic determinants of host susceptibility to infection by rusts. Thousands of genes encoding secreted proteins highly expressed during host infection have been reported for different rust species, which represents significant potential towards understanding rust effector function. Recent high-throughput in planta expression screen approaches (effectoromics) have pushed the field ahead even further towards predicting high-priority effectors and identifying avirulence genes. These new insights into rust effector biology promise to inform future research and spur the development of effective and sustainable strategies for managing rust diseases.

A rust fungal effector binds plant DNA and modulates transcription.

The basidiomycete Melampsora larici-populina causes poplar rust disease by invading leaf tissues and secreting effector proteins through specialized infection structures known as haustoria. The mechanisms by which rust effectors promote pathogen virulence are poorly understood. The present study characterized Mlp124478, a candidate effector of M. larici-populina. We used the models Arabidopsis thaliana and Nicotiana benthamiana to investigate the function of Mlp124478 in plant cells. We established that Mlp124478 accumulates in the nucleus and nucleolus, however its nucleolar accumulation is not required to promote growth of the oomycete pathogen Hyaloperonospora arabidopsidis. Stable constitutive expression of Mlp124478 in A. thaliana repressed the expression of genes involved in immune responses, and also altered leaf morphology by increasing the waviness of rosette leaves. Chip-PCR experiments showed that Mlp124478 associats'e with the TGA1a-binding DNA sequence. Our results suggest that Mlp124478 exerts a virulence activity and binds the TGA1a promoter to suppress genes induced in response to pathogen infection.

Show me the way: rust effector targets in heterologous plant systems.

For years, the study of rust fungal effectors has been impeded by the lack of molecular genetic tools in rust pathosystems. The recent use of heterologous plants to perform effector screens (effectoromics)-including effector localisation (cellular targets) and protein interactors (molecular targets) in plant cells-has changed the game. These screens revealed that many candidate effectors from various rust fungi target specific plant cell compartments, including chloroplasts, and associate with specific plant protein complexes. Such information represents unparalleled opportunities to understand how effectors sustain extreme parasitic interactions and obligate biotrophy. Despite their limitations, we here portray how the use of heterologous expression systems has been essential for gaining new insight into rust effectors.

The Rust Fungus Melampsora larici-populina Expresses a Conserved Genetic Program and Distinct Sets of Secreted Protein Genes During Infection of Its Two Host Plants, Larch and Poplar.

Mechanisms required for broad-spectrum or specific host colonization of plant parasites are poorly understood. As a perfect illustration, heteroecious rust fungi require two alternate host plants to complete their life cycles. Melampsora larici-populina infects two taxonomically unrelated plants, larch, on which sexual reproduction is achieved, and poplar, on which clonal multiplication occurs, leading to severe epidemics in plantations. We applied deep RNA sequencing to three key developmental stages of M. larici-populina infection on larch: basidia, pycnia, and aecia, and we performed comparative transcriptomics of infection on poplar and larch hosts, using available expression data. Secreted protein was the only significantly overrepresented category among differentially expressed M. larici-populina genes between the basidial, the pycnial, and the aecial stages, highlighting their probable involvement in the infection process. Comparison of fungal transcriptomes in larch and poplar revealed a majority of rust genes were commonly expressed on the two hosts and a fraction exhibited host-specific expression. More particularly, gene families encoding small secreted proteins presented striking expression profiles that highlight probable candidate effectors specialized on each host. Our results bring valuable new information about the biological cycle of rust fungi and identify genes that may contribute to host specificity.

Heterologous Expression Screens in Nicotiana benthamiana Identify a Candidate Effector of the Wheat Yellow Rust Pathogen that Associates with Processing Bodies.

Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal candidate effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. We propose that P-bodies are a novel plant cell compartment targeted by pathogen effectors.

Rust fungal effectors mimic host transit peptides to translocate into chloroplasts.

Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane-rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified chloroplast-targeted protein 1 (CTP1), a candidate effector from the poplar leaf rust fungus Melampsora larici-populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora-specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in an N-terminal signal-dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant-specific sorting signals to traffic within plant cells.

Effector-Mining in the Poplar Rust Fungus Melampsora larici-populina Secretome.

The poplar leaf rust fungus, Melampsora larici-populina has been established as a tree-microbe interaction model. Understanding the molecular mechanisms controlling infection by pathogens appears essential for durable management of tree plantations. In biotrophic plant-parasites, effectors are known to condition host cell colonization. Thus, investigation of candidate secreted effector proteins (CSEPs) is a major goal in the poplar-poplar rust interaction. Unlike oomycetes, fungal effectors do not share conserved motifs and candidate prediction relies on a set of a priori criteria established from reported bona fide effectors. Secretome prediction, genome-wide analysis of gene families and transcriptomics of M. larici-populina have led to catalogs of more than a thousand secreted proteins. Automatized effector-mining pipelines hold great promise for rapid and systematic identification and prioritization of CSEPs for functional characterization. In this review, we report on and discuss the current status of the poplar rust fungus secretome and prediction of candidate effectors from this species.

Candidate Effector Proteins of the Rust Pathogen Melampsora larici-populina Target Diverse Plant Cell Compartments.

Rust fungi are devastating crop pathogens that deliver effector proteins into infected tissues to modulate plant functions and promote parasitic growth. The genome of the poplar leaf rust fungus Melampsora larici-populina revealed a large catalog of secreted proteins, some of which have been considered candidate effectors. Unraveling how these proteins function in host cells is a key to understanding pathogenicity mechanisms and developing resistant plants. In this study, we used an effectoromics pipeline to select, clone, and express 20 candidate effectors in Nicotiana benthamiana leaf cells to determine their subcellular localization and identify the plant proteins they interact with. Confocal microscopy revealed that six candidate effectors target the nucleus, nucleoli, chloroplasts, mitochondria, and discrete cellular bodies. We also used coimmunoprecipitation (coIP) and mass spectrometry to identify 606 N. benthamiana proteins that associate with the candidate effectors. Five candidate effectors specifically associated with a small set of plant proteins that may represent biologically relevant interactors. We confirmed the interaction between the candidate effector MLP124017 and TOPLESS-related protein 4 from poplar by in planta coIP. Altogether, our data enable us to validate effector proteins from M. larici-populina and reveal that these proteins may target multiple compartments and processes in plant cells. It also shows that N. benthamiana can be a powerful heterologous system to study effectors of obligate biotrophic pathogens.