Stress and sexual reproduction affect the dynamics of the wheat pathogen effector AvrStb6 and strobilurin resistance

Host resistance and fungicide treatments are cornerstones of plant-disease control. Here, we show that these treatments allow sex and modulate parenthood in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that the Z. tritici–wheat interaction complies with the gene-for-gene model by identifying the effector AvrStb6, which is recognized by the wheat resistance protein Stb6. Recognition triggers host resistance, thus implying removal of avirulent strains from pathogen populations. However, Z. tritici crosses on wheat show that sex occurs even with an avirulent parent, and avirulence alleles are thereby retained in subsequent populations. Crossing fungicide-sensitive and fungicide-resistant isolates under fungicide pressure results in a rapid increase in resistance-allele frequency. Isolates under selection always act as male donors, and thus disease control modulates parenthood. Modeling these observations for agricultural and natural environments reveals extended durability of host resistance and rapid emergence of fungicide resistance. Therefore, fungal sex has major implications for disease control

Apoplastic recognition of multiple candidate effectors from the wheat pathogen Zymoseptoria tritici in the nonhost plant Nicotiana benthamiana

The fungus Zymoseptoria tritici is a strictly apoplastic, host-specific pathogen of wheat leaves and causal agent of septoria tritici blotch (STB) disease. All other plants are considered nonhosts, but the mechanism of nonhost resistance (NHR) to Z. tritici has not been addressed previously. We sought to develop Nicotiana benthamiana as a system to study NHR against Z. tritici. Fluorescence microscopy and quantitative reverse transcription polymerase chain reactions were used to establish the interaction between Z. tritici and N. benthamiana. Agrobacteriummediated transient expression was used to screen putative Z. tritici effector genes for recognition in N. benthamiana, and virus-induced gene silencing (VIGS) was employed to determine the role of two receptor-like kinases (RLKs), NbBAK1 and NbSOBIR1, in Z. tritici effector recognition. Numerous Z. tritici putative effectors (14 of 63 tested) induced cell death or chlorosis in N. benthamiana. For most, phenotypes were light-dependent and required effector secretion to the leaf apoplastic space. Moreover, effector-induced host cell death was dependent on NbBAK1 and NbSOBIR1. Our results indicate widespread recognition of apoplastic effectors from a wheat-infecting fungal pathogen in a taxonomically distant nonhost plant species presumably by cell surface immune receptors. This suggests that apoplastic recognition of multiple nonadapted pathogen effectors may contribute to NHR

Regulation of proteinaceous effector expression in phytopathogenic fungi

Effectors are molecules used by microbial pathogens to facilitate infection via effector-triggered susceptibility or tissue necrosis in their host. Much research has been focussed on the identification and elucidating the function of fungal effectors during plant pathogenesis. By comparison, knowledge of how phytopathogenic fungi regulate the expression of effector genes has been lagging. Several recent studies have illustrated the role of various transcription factors, chromosome-based control, effector epistasis, and mobilisation of endosomes within the fungal hyphae in regulating effector expression and virulence on the host plant. Improved knowledge of effector regulation is likely to assist in improving novel crop protection strategies

Identification and functional characterization of putative (a)virulence factors in the fungal wheat pathogen Zymoseptoria tritici

Zymoseptoria tritici (Desm.) Quaedvlieg & Crous (previously known as Mycosphaerella graminicola) is the causal agent of septoria tritici blotch (STB), which is a devastating foliar wheat disease worldwide. It is responsible for significant yield losses occurring annually in all major wheat-growing areas and threatens global food security. Z. tritici is a hemi-biotrophic fungal pathogen that, after stomatal penetration, establishes a stealthy biotrophic and symptomless relation with its host plant that is followed by a sudden switch to a necrotrophic growth phase coinciding with chlorosis that eventually develops in large necrotic blotches containing many pycnidia producing asexual splash-borne conidia. Under natural conditions - once competent mating partners are present and conditions are conducive- pseudothecia are formed producing airborne ascospores. Disease management of STB is primarily achieved through fungicide applications and growing commercial cultivars carrying Stb resistance genes. However, the efficacy of both strategies is limited as strains resistant to fungicides frequently develop and progressively dominate natural populations, which hampers disease management; also the deployed Stb genes are often overcome by existing or newly developed isolates of the fungus. Hence, there is a need for discovery research to better understand the molecular basis of the host-pathogen interaction that enables breeders to identify and deploy new Stb genes, which will eventually contribute to more sustainable disease control. Chapter 1 introduces the subject of the thesis and describes various aspects of the lifestyle of Z. tritici with emphasis on dissecting the various stages and physiological processes during pathogenesis on wheat. In addition, it includes a short summary and discussion of the current understanding of the role of (a)virulence factors in the Z. tritici–wheat pathosystem. Chapter 2 describes new gateway technology-driven molecular tools comprising 22 entry constructs facilitating rapid construction of binary vectors for functional analyses of fungal genes. The entry vectors for single, double or triple gene deletion mutants were developed using hygromycin, geneticin and nourseothricin resistance genes as selection markers. Furthermore, these entry vectors contain the genes encoding green fluorescent (GFP) or red fluorescent (RFP) protein in combination with the three selection markers, which enables simultaneous tagging of gene deletion mutants for microscopic analyses. The functionality of these entry vectors was validated in Z. tritici and described in Chapters 3, 4 and 5. Chapter 3 describes the functional characterization of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans. ZtWor1 is up-regulated during initiation of colonization and fructification, and regulates expression of candidate effector genes, including one that was discovered after comparative proteome analysis of Z. tritici wild-type and ΔZtWor1 strains. Cell fusion and anastomosis occurred frequently in ΔZtWor1 strains, which is reminiscent of mutants of MgGpb1, the β-subunit of the heterotrimeric G protein. Comparative expression profiling of ΔZtWor1, ΔMgGpb1 and ΔMgTpk2 (the catalytic subunit of protein kinase A) strains, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenicity of many fungal plant pathogens. Chapter 4 describes combined bioinformatics and expression profiling studies during pathogenesis in order to discover candidate effectors of Z. tritici important for virulence. In addition, a genetic approach was followed to map quantitative trait loci (QTLs) in Z. tritici carrying putative effectors. Functional analysis of two top effector candidates, small-secreted proteins SSP15 and SSP18, which were selected based on their expression profile in planta, showed that they are dispensable for virulence of Z. tritici. These analyses suggest that generally adopted criteria for effector discovery, such as protein size, number of cysteine residues and up-regulated expression during pathogenesis, should be taken with caution and cannot be applied to every pathosystem, as they likely represent only a subset of effector genes. Chapter 5 describes the functional characterization of ZtCpx1 and ZtCpx2 encoding a secreted and a cytoplasmic catalase-peroxidase (CP) in Z. tritici, respectively. Gene replacement of ZtCpx1 resulted in mutant strains that were sensitive to exogenously added H2O2 and in planta phenotyping showed they are significantly less virulent compared to wild-type. All mutant phenotypes could be restored to wild-type by complementation with the wild-type allele of ZtCpx1 driven by its native promoter. Additionally, functional analysis of ZtCpx2 confirmed that this gene encodes a secreted CP and is, however, dispensable for virulence of Z. tritici on wheat. However, we showed that both genes act synergistically, as the generated double knock-out strain showed a significantly stronger reduction in virulence than the individual single knock-out strains. Hence, both genes are required by Z. tritici for successful infection and colonization of wheat. In Chapter 6 I discuss and summarize the genetic approaches used in this study, reflect on the major findings and bottlenecks encountered, and propose new strategies to identify effectors of Z. tritici in the future

Karyotype Variability in Plant-Pathogenic Fungi

Recent advances in genetic and molecular technologies gradually paved the way for the transition from traditional fungal karyotyping to more comprehensive chromosome biology studies. Extensive chromosomal polymorphisms largely resulting from chromosomal rearrangements (CRs) are widely documented in fungal genomes. These extraordinary CRs in fungi generate substantial genome plasticity compared to other eukaryotic organisms. Here, we review the most recent findings on fungal CRs and their underlying mechanisms and discuss the functional consequences of CRs for adaptation, fungal evolution, host range, and pathogenicity of fungal plant pathogens in the context of chromosome biology. In addition to a complement of permanent chromosomes called core chromosomes, the genomes of many fungal pathogens comprise distinct unstable chromosomes called dispensable chromosomes (DCs) that also contribute to chromosome polymorphisms. Compared to the core chromosomes, the structural features of DCs usually differ for gene density, GC content, housekeeping genes, and recombination frequency. Despite their dispensability for normal growth and development, DCs have important biological roles with respect to pathogenicity in some fungi but not in others. Therefore, their evolutionary origin is also reviewed in relation to overall fungal physiology and pathogenicity

In silico maturation of affinity and selectivity of DNA aptamers against aflatoxin B1 for biosensor development

A high affinity and selectivity DNA aptamer for aflatoxin B1 (AFB1) was designed through Genetic Algorithm (GA) based in silico maturation (ISM) strategy. The sequence of a known AFB1 aptamer (Patent: PCT/CA2010/001292, Apt1) applied as a probe in many aptasensors was modified using seven GA rounds to generate an initial library and three different generations of ss DNA oligonucleotides as new candidate aptamers. Molecular docking methodology was used to screen and analyze the best aptamer�AFB1 complexes. Also, a new pipeline was proposed to faithfully predict the tertiary structure of all single stranded DNA sequences. By the second generation, aptamer Apt1 sequence was optimized in the local search space and five aptamers including F20, g12, C52, C32 and H1 were identified as the best aptamers for AFB1. The selected aptamers were applied as probes in an unmodified gold nanoparticles-based aptasensor to evaluate their binding affinity to AFB1 and their selectivity against other mycotoxins (aflatoxins B2, G1, G2, M1, ochratoxin A and zearalenone). In addition, a novel direct fluorescent anisotropy aptamer assay was developed to confirm the binding interaction of the selected aptamers over AFB1. The ISM allowed the identification of an aptamer, F20, with up to 9.4 and 2 fold improvement in affinity and selectivity compared to the parent aptamer, respectively. © 2020 Elsevier B.V

The ZtVf1 transcription factor regulates development and virulence in the foliar wheat pathogen Zymoseptoria tritici

The dimorphic fungal pathogen, Zymoseptoria tritici undergoes discrete developmental changes to complete its life cycle on wheat. Molecular mechanisms underlying morphogenesis during infection process of Z. tritici are poorly understood. In this study, we have investigated the role of ZtVf1 gene encoding a transcription factor belonging to C2-H2 subfamily. In planta assays revealed that ZtVf1 is required for virulence. Reduced necrotic lesions and low pycnidia density within the lesions resulted in significantly reduced virulence of ZtVf1 mutants. Cytological analysis showed that the impaired virulence of ZtVf1 mutants attributed to reduced penetration and colonization along with hampered pycnidia differentiation. In vitro phenotyping showed that ZtVf1 deletion affects hyphal branching and biomass production suggesting that the reduced tissue colonization by the ZtVf1 mutant might be due to lower hyphal branching and less fungal biomass production. In addition, the majority of infected substomatal cavities by the ZtVf1 mutant filled with compacted mycelia mat that did not differentiate to mature pycnidia indicating that the impaired melanization negatively affected pycnidia formation and maturation. The ZtVf1 might target multiple genes belonging to different cellular processes whose identification is of eminent interest to increase our understanding of this pathosystem. Overall, the data provided in this study indicates that attenuated pathogenicity of ZtVf1 mutant is due to involvement of this gene in the regulation of both early and late stages of infection