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.

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.

Natural variation in Avr3D1 from Zymoseptoria sp. contributes to quantitative gene-for-gene resistance and to host specificity.

Successful host colonization by plant pathogens requires the circumvention of host defense responses, frequently through sequence modifications in secreted pathogen proteins known as avirulence factors (Avrs). Although Avr sequences are often polymorphic, the contribution of these polymorphisms to virulence diversity in natural pathogen populations remains largely unexplored. We used molecular genetic tools to determine how natural sequence polymorphisms of the avirulence factor Avr3D1 in the wheat pathogen Zymoseptoria tritici contributed to adaptive changes in virulence. We showed that there is a continuous distribution in the magnitude of resistance triggered by different Avr3D1 isoforms and demonstrated that natural variation in an Avr gene can lead to a quantitative resistance phenotype. We further showed that homologues of Avr3D1 in two nonpathogenic sister species of Z. tritici are recognized by some wheat cultivars, suggesting that Avr-R gene-for-gene interactions can contribute to nonhost resistance. We suggest that the mechanisms underlying host range, qualitative resistance, and quantitative resistance are not exclusive.

Asexual reproductive potential trumps virulence as a predictor of competitive ability in mixed infections.

Natural infections frequently involve several co-infecting pathogen strains. These mixed infections can affect the extent of the infection, the transmission success of the pathogen and the eventual epidemic outcome. To date, few studies have investigated how mixed infections affect transmission between hosts. Zymoseptoria tritici is a highly diverse wheat pathogen in which multiple strains often coexist in the same lesion. Here we demonstrate that the most competitive strains often exclude their competitors during serial passages of mixed infections. The outcome of the competition depended on both the host genotype and the genotypes of the competing pathogen strains. Differences in virulence among the strains were not associated with competitive advantages during transmission, while differences in reproductive potential had a strong effect on strain competitive ability. Overall, our findings suggest that host specialization is determined mainly by the ability to successfully transmit offspring to new hosts during mixed infections.

The Arabidopsis stomatal polarity protein BASL mediates distinct processes before and after cell division to coordinate cell size and fate asymmetries.

In many land plants, asymmetric cell divisions (ACDs) create and pattern differentiated cell types on the leaf surface. In the Arabidopsis stomatal lineage, BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) regulates division plane placement and cell fate enforcement. Polarized subcellular localization of BASL is initiated before ACD and persists for many hours after the division in one of the two daughters. Untangling the respective contributions of polarized BASL before and after division is essential to gain a better understanding of its roles in regulating stomatal lineage ACDs. Here, we combine quantitative imaging and lineage tracking with genetic tools that provide temporally restricted BASL expression. We find that pre-division BASL is required for division orientation, whereas BASL polarity post-division ensures proper cell fate commitment. These genetic manipulations allowed us to uncouple daughter-cell size asymmetry from polarity crescent inheritance, revealing independent effects of these two asymmetries on subsequent cell behavior. Finally, we show that there is coordination between the division frequencies of sister cells produced by ACDs, and this coupling requires BASL as an effector of peptide signaling.

Tuning self-renewal in the Arabidopsis stomatal lineage by hormone and nutrient regulation of asymmetric cell division.

Asymmetric and self-renewing divisions build and pattern tissues. In the Arabidopsis stomatal lineage, asymmetric cell divisions, guided by polarly localized cortical proteins, generate most cells on the leaf surface. Systemic and environmental signals modify tissue development, but the mechanisms by which plants incorporate such cues to regulate asymmetric divisions are elusive. In a screen for modulators of cell polarity, we identified CONSTITUTIVE TRIPLE RESPONSE1, a negative regulator of ethylene signaling. We subsequently revealed antagonistic impacts of ethylene and glucose signaling on the self-renewing capacity of stomatal lineage stem cells. Quantitative analysis of cell polarity and fate dynamics showed that developmental information may be encoded in both the spatial and temporal asymmetries of polarity proteins. These results provide a framework for a mechanistic understanding of how nutritional status and environmental factors tune stem-cell behavior in the stomatal lineage, ultimately enabling flexibility in leaf size and cell-type composition.

Mixed infections alter transmission potential in a fungal plant pathogen.

Infections by more than one strain of a pathogen predominate under natural conditions. Mixed infections can have significant, though often unpredictable, consequences for overall virulence, pathogen transmission and evolution. However, effects of mixed infection on disease development in plants often remain unclear and the critical factors that determine the outcome of mixed infections remain unknown. The fungus Zymoseptoria tritici forms genetically diverse infections in wheat fields. Here, for a range of pathogen traits, we experimentally decompose the infection process to determine how the outcomes and consequences of mixed infections are mechanistically realized. Different strains of Z. tritici grow in close proximity and compete in the wheat apoplast, resulting in reductions in growth of individual strains and in pathogen reproduction. We observed different outcomes of competition at different stages of the infection. Overall, more virulent strains had higher competitive ability during host colonization, and less virulent strains had higher transmission potential. We showed that within-host competition can have a major effect on infection dynamics and pathogen population structure in a pathogen and host genotype-specific manner. Consequently, mixed infections likely have a major effect on the development of septoria tritici blotch epidemics and the evolution of virulence in Z. tritici.

A wheat cysteine-rich receptor-like kinase confers broad-spectrum resistance against Septoria tritici blotch.

The poverty of disease resistance gene reservoirs limits the breeding of crops for durable resistance against evolutionary dynamic pathogens. Zymoseptoria tritici which causes Septoria tritici blotch (STB), represents one of the most genetically diverse and devastating wheat pathogens worldwide. No fully virulent Z. tritici isolates against synthetic wheats carrying the major resistant gene Stb16q have been identified. Here, we use comparative genomics, mutagenesis and complementation to identify Stb16q, which confers broad-spectrum resistance against Z. tritici. The Stb16q gene encodes a plasma membrane cysteine-rich receptor-like kinase that was recently introduced into cultivated wheat and which considerably slows penetration and intercellular growth of the pathogen.

Chromatin Dynamics Contribute to the Spatiotemporal Expression Pattern of Virulence Genes in a Fungal Plant Pathogen.

Dynamic changes in transcription profiles are key for the success of pathogens in colonizing their hosts. In many pathogens, genes associated with virulence, such as effector genes, are located in regions of the genome that are rich in transposable elements and heterochromatin. The contribution of chromatin modifications to gene expression in pathogens remains largely unknown. Using a combination of a reporter gene-based approach and chromatin immunoprecipitation, we show that the heterochromatic environment of effector genes in the fungal plant pathogen Zymoseptoria tritici is a key regulator of their specific spatiotemporal expression patterns. Enrichment in trimethylated lysine 27 of histone H3 dictates the repression of effector genes in the absence of the host. Chromatin decondensation during host colonization, featuring a reduction in this repressive modification, indicates a major role for epigenetics in effector gene induction. Our results illustrate that chromatin modifications triggered during host colonization determine the specific expression profile of effector genes at the cellular level and, hence, provide new insights into the regulation of virulence in fungal plant pathogens.IMPORTANCE Fungal plant pathogens possess a large repertoire of genes encoding putative effectors, which are crucial for infection. Many of these genes are expressed at low levels in the absence of the host but are strongly induced at specific stages of the infection. The mechanisms underlying this transcriptional reprogramming remain largely unknown. We investigated the role of the genomic environment and associated chromatin modifications of effector genes in controlling their expression pattern in the fungal wheat pathogen Zymoseptoria tritici Depending on their genomic location, effector genes are epigenetically repressed in the absence of the host and during the initial stages of infection. Derepression of effector genes occurs mainly during and after penetration of plant leaves and is associated with changes in histone modifications. Our work demonstrates the role of chromatin in shaping the expression of virulence components and, thereby, the interaction between fungal pathogens and their plant hosts.

Root diffusion barrier control by a vasculature-derived peptide binding to the SGN3 receptor.

The root endodermis forms its extracellular diffusion barrier by developing ringlike impregnations called Casparian strips. A factor responsible for their establishment is the SCHENGEN3/GASSHO1 (SGN3/GSO1) receptor-like kinase. Its loss of function causes discontinuous Casparian strips. SGN3 also mediates endodermal overlignification of other Casparian strip mutants. Yet, without ligand, SGN3 function remained elusive. Here we report that schengen2 (sgn2) is defective in an enzyme sulfating peptide ligands. On the basis of this observation, we identified two stele-expressed peptides (CASPARIAN STRIP INTEGRITY FACTORS, CIF1/2) that complement sgn2 at nanomolar concentrations and induce Casparian strip mislocalization as well as overlignification-all of which depend on SGN3. Direct peptide binding to recombinant SGN3 identifies these peptides as SGN3 ligands. We speculate that CIF1/2-SGN3 is part of a barrier surveillance system, evolved to guarantee effective sealing of the supracellular Casparian strip network.

Polarly localized kinase SGN1 is required for Casparian strip integrity and positioning.

Casparian strips are precisely localized and aligned ring-like cell wall modifications in the root of all higher plants. They set up an extracellular diffusion barrier analogous to animal tight junctions, and are crucial for maintaining the homeostatic capacity of plant roots. Casparian strips become localized because of the formation of a highly stable plasma membrane domain, consisting of a family of small transmembrane proteins called Casparian strip membrane domain proteins (CASPs). Here we report a large-scale forward genetic screen directly visualizing endodermal barrier function, which allowed us to identify factors required for the formation and integrity of Casparian strips. We present the identification and characterization of one of the mutants, schengen1 (sgn1), a receptor-like cytoplasmic kinase that we show localizes in a strictly polar fashion to the outer plasma membrane of endodermal cells and is required for the positioning and correct formation of the centrally located CASP domain.

Transcriptome dynamics of the stomatal lineage: birth, amplification, and termination of a self-renewing population.

Developmental transitions can be described in terms of morphology and the roles of individual genes, but also in terms of global transcriptional and epigenetic changes. Temporal dissections of transcriptome changes, however, are rare for intact, developing tissues. We used RNA sequencing and microarray platforms to quantify gene expression from labeled cells isolated by fluorescence-activated cell sorting to generate cell-type-specific transcriptomes during development of an adult stem-cell lineage in the Arabidopsis leaf. We show that regulatory modules in this early lineage link cell types that had previously been considered to be under separate control and provide evidence for recruitment of individual members of gene families for different developmental decisions. Because stomata are physiologically important and because stomatal lineage cells exhibit exemplary division, cell fate, and cell signaling behaviors, this dataset serves as a valuable resource for further investigations of fundamental developmental processes.

A receptor-like kinase mutant with absent endodermal diffusion barrier displays selective nutrient homeostasis defects.

The endodermis represents the main barrier to extracellular diffusion in plant roots, and it is central to current models of plant nutrient uptake. Despite this, little is known about the genes setting up this endodermal barrier. In this study, we report the identification and characterization of a strong barrier mutant, schengen3 (sgn3). We observe a surprising ability of the mutant to maintain nutrient homeostasis, but demonstrate a major defect in maintaining sufficient levels of the macronutrient potassium. We show that SGN3/GASSHO1 is a receptor-like kinase that is necessary for localizing CASPARIAN STRIP DOMAIN PROTEINS (CASPs)--major players of endodermal differentiation--into an uninterrupted, ring-like domain. SGN3 appears to localize into a broader band, embedding growing CASP microdomains. The discovery of SGN3 strongly advances our ability to interrogate mechanisms of plant nutrient homeostasis and provides a novel actor for localized microdomain formation at the endodermal plasma membrane.

Plant Defensin type 1 (PDF1): protein promiscuity and expression variation within the Arabidopsis genus shed light on zinc tolerance acquisition in Arabidopsis halleri.

Plant defensins are recognized for their antifungal properties. However, a few type 1 defensins (PDF1s) were identified for their cellular zinc (Zn) tolerance properties after a study of the metal extremophile Arabidopsis halleri. In order to investigate whether different paralogues would display specialized functions, the A. halleri PDF1 family was characterized at the functional and genomic levels. Eleven PDF1s were isolated from A. halleri. Their ability to provide Zn tolerance in yeast cells, their activity against Fusarium oxysporum f. sp. melonii, and their level of expression in planta were compared with those of the seven A. thaliana PDF1s. The genomic organization of the PDF1 family was comparatively analysed within the Arabidopsis genus. AhPDF1s and AtPDF1s were able to confer Zn tolerance and AhPDF1s also displayed antifungal activity. PDF1 transcripts were constitutively more abundant in A. halleri than in A. thaliana. Within the Arabidopsis genus, the PDF1 family is evolutionarily dynamic, in terms of gain and loss of gene copy. Arabidopsis halleri PDF1s display no superior abilities to provide Zn tolerance. A constitutive increase in AhPDF1 transcript accumulation is proposed to be an evolutionary innovation co-opting the promiscuous PDF1 protein for its contribution to Zn tolerance in A. halleri.

A mechanism for localized lignin deposition in the endodermis.

The precise localization of extracellular matrix and cell wall components is of critical importance for multicellular organisms. Lignin is a major cell wall modification that often forms intricate subcellular patterns that are central to cellular function. Yet the mechanisms of lignin polymerization and the subcellular precision of its formation remain enigmatic. Here, we show that the Casparian strip, a lignin-based, paracellular diffusion barrier in plants, forms as a precise, median ring by the concerted action of a specific, localized NADPH oxidase, brought into proximity of localized peroxidases through the action of Casparian strip domain proteins (CASPs). Our findings in Arabidopsis provide a simple mechanistic model of how plant cells regulate lignin formation with subcellular precision. We speculate that scaffolding of NADPH oxidases to the downstream targets of the reactive oxygen species (ROS) that they produce might be a widespread mechanism to ensure specificity and subcellular precision of ROS action within the extracellular matrix.

The endodermis--development and differentiation of the plant's inner skin.

Controlling external compound entrance is essential for plant survival. To set up an efficient and selective sorting of nutrients, free diffusion via the apoplast in vascular plants is blocked at the level of the endodermis. Although we have learned a lot about endodermal specification in the last years, information regarding its differentiation is still very limited. A differentiated endodermal cell can be defined by the presence of the "Casparian strip" (CS), a cell wall modification described first by Robert Caspary in 1865. While the anatomical description of CS in many vascular plants has been very detailed, we still lack molecular information about the establishment of the Casparian strips and their actual function in roots. The recent isolation of a novel protein family, the CASPs, that localizes precisely to a domain of the plasma membrane underneath the CS represents an excellent point of entry to explore CS function and formation. In addition, it has been shown that the endodermis contains transporters that are localized to either the central (stele-facing) or peripheral (soil-facing) plasma membranes. These features suggest that the endodermis functions as a polar plant epithelium.

A novel protein family mediates Casparian strip formation in the endodermis.

Polarized epithelia are fundamental to multicellular life. In animal epithelia, conserved junctional complexes establish membrane diffusion barriers, cellular adherence and sealing of the extracellular space. Plant cellular barriers are of independent evolutionary origin. The root endodermis strongly resembles a polarized epithelium and functions in nutrient uptake and stress resistance. Its defining features are the Casparian strips, belts of specialized cell wall material that generate an extracellular diffusion barrier. The mechanisms localizing Casparian strips are unknown. Here we identify and characterize a family of transmembrane proteins of previously unknown function. These 'CASPs' (Casparian strip membrane domain proteins) specifically mark a membrane domain that predicts the formation of Casparian strips. CASP1 displays numerous features required for a constituent of a plant junctional complex: it forms complexes with other CASPs; it becomes immobile upon localization; and it sediments like a large polymer. CASP double mutants display disorganized Casparian strips, demonstrating a role for CASPs in structuring and localizing this cell wall modification. To our knowledge, CASPs are the first molecular factors that are shown to establish a plasma membrane and extracellular diffusion barrier in plants, and represent a novel way of epithelial barrier formation in eukaryotes.

A developmental framework for endodermal differentiation and polarity.

The endodermis is a root cell layer common to higher plants and of fundamental importance for root function and nutrient uptake. The endodermis separates outer (peripheral) from inner (central) cell layers by virtue of its Casparian strips, precisely aligned bands of specialized wall material. Here we reveal that the membrane at the Casparian strip is a diffusional barrier between the central and peripheral regions of the plasma membrane and that it mediates attachment to the extracellular matrix. This membrane region thus functions like a tight junction in animal epithelia, although plants lack the molecular modules that establish tight junction in animals. We have also identified a pair of influx and efflux transporters that mark both central and peripheral domains of the plasma membrane. These transporters show opposite polar distributions already in meristems, but their localization becomes refined and restricted upon differentiation. This "central-peripheral" polarity coexists with the apical-basal polarity defined by PIN proteins within the same cells, but utilizes different polarity determinants. Central-peripheral polarity can be already observed in early embryogenesis, where it reveals a cellular polarity within the quiescent center precursor cell. A strict diffusion block between polar domains is common in animals, but had never been described in plants. Yet, its relevance to endodermal function is evident, as central and peripheral membranes of the endodermis face fundamentally different root compartments. Further analysis of endodermal transporter polarity and manipulation of its barrier function will greatly promote our understanding of plant nutrition and stress tolerance in roots.