Tritici Strains Research Articles

A fungal endophyte induces local cell wall-mediated resistance in wheat roots against take-all disease.

Take-all disease, caused by the Ascomycete fungus Gaeumannomyces tritici, is one of the most important root diseases of wheat worldwide. The fungus invades the roots and destroys the vascular tissue, hindering the uptake of water and nutrients. Closely related non-pathogenic species in the Magnaporthaceae family, such as Gaeumannomyces hyphopodioides, occur naturally in arable and grassland soils and have previously been reported to reduce take-all disease in field studies. However, the mechanism of take-all protection has remained unknown. Here, we demonstrate that take-all control is achieved via local but not systemic host changes in response to prior G. hyphopodioides root colonisation. A time-course wheat RNA sequencing analysis revealed extensive transcriptional reprogramming in G. hyphopodioides-colonised tissues, characterised by a striking downregulation of key cell wall-related genes, including genes encoding cellulose synthases (CESA), and xyloglucan endotransglucosylase/hydrolases (XTH). In addition, we characterise the root infection biologies of G. tritici and G. hyphopodioides in wheat. We investigate the ultrastructure of previously described "subepidermal vesicles" (SEVs), dark swollen fungal cells produced in wheat roots by non-pathogenic G. hyphopodioides, but not by pathogenic G. tritici. We show that G. hyphopodioides SEVs share key characteristics of fungal resting structures, containing a greater number of putative lipid bodies and a significantly thickened cell wall compared to infection hyphae. We hypothesise that SEVs are fungal resting structures formed due to halted hyphal growth in the root cortex, perhaps as a stress response to locally induced wheat defence responses. In the absence of take-all resistant wheat cultivars or non-virulent G. tritici strains, studying closely related non-pathogenic G. hyphopodioides provides a much needed avenue to elucidate take-all resistance mechanisms in wheat.

Characteristics of Zymoseptoria tritici isolates from the Russian Federation and the Republic of Kazakhstan by azoxystrobin sensitivity

Septoria tritici blotch is one of the harmful diseases of wheat cultivars cultivated in the Russian Federation and the Republic of Kazakhstan. The purpose of the research was to study a sample of Zymoseptoria tritici isolates obtained from the Saratov and Tambov (Russian Federation) and Almaty (Republic of Kazakhstan) regions according to the degree of the azoxystrobin sensitivity, as well as to evaluate these isolates for the presence of the G143A mutation using PCR-RFLP analysis. The azoxystrobin sensitivity of Z. tritici isolates was evaluated by studying the growth restriction of cultures on a nutrient medium (concentrations of active drug in the medium were 100, 10, 1, 0.1, 0.01 mg/l.). In the control, on the 21st day of cultivation, the diameter of the colonies of Z. tritici strains varied from 14.24±1.43 mm to 19.53±0.66 mm. Azoxystrobin had a significant inhibitory effect on the growth of fungal isolates from the Tambov region: EC50 is 1.72 μg/l, from the Almaty region EC50 is 2.36 μg/l, from the Saratov region EC50 is 2.63 μg/l. The results of biotesting made it possible to identify isolates of Z. tritici resistant to azoxystrobin: 277-22-5, 277-22-13 (from Kazakhstan); 104-23-9, 104-23-10 (from the Saratov region); 19-23-5, 19-23-8 (from the Tambov region). PCR-RFLP method made it possible to identify mitochondrial mutations G143A in selected fungal isolates, which are associated with the development of resistance to fungicides in the causative agent of septoria. This work is the beginning of a more in-depth study of the sensitivity of the economically significant phytopathogen Z. tritici to fungicides in the territories of Russia and the Republic of Kazakhstan.

Effector-mediated partial and nonhost disease resistance in wheat.

Effector-mediated partial and nonhost disease resistance in wheat.

Specific virulence patterns in Tunisian Zymoseptoria tritici strains isolated from bread and durum wheat

AbstractSeptoria tritici blotch caused by Zymoseptoria tritici is a serious threat to wheat in Tunisia, causing yield losses of up to 50%. Epidemics occur regularly, mainly on durum wheat (DW) but also to a lesser extent on bread wheat (BW), that are mainly cropped in the north‐western and north‐eastern regions, respectively. Isolates of Z. tritici exhibit both cultivar specificity and host species specificity. In this study, we investigated the extent of host specialization of Z. tritici in the Tunisian context. Cross‐inoculations of different strains derived from either BW or DW were performed at the seedling stage on a panel of the most cultivated BW and DW varieties in Tunisia using the percentage of pycnidia coverage and necrotic leaf area as disease parameters. Results showed that the effect of strains, the strain‐derived species, the wheat species and of their interactions were highly significant based on pycnidia coverage. This indicates the presence of significant virulence variability among Z. tritici strains on the tested wheat genotypes, with some strains being more virulent than others. It also suggests cultivar and host species specificity, as not only genotypes of the strains interacted differently with the genotypes of the host, but also the origin of the strain, whether BW or DW, influenced the disease outcome. Hence, future research based on comparative genomics coupled with transcriptomics of these strains and their progenies could determine genes whose expression control this host specificity or is influenced by host or strain genotype, or alternatively by life cycle stage.

Inactivation of a candidate effector gene of Zymoseptoria tritici affects its sporulation.

Wheat is one of the most important staple crops produced worldwide. Its susceptibility to plant diseases reduces its production significantly. One of the most important diseases of wheat is septoria tritici blotch, a devastating disease observed in fields with wet and temperate conditions. Z. tritici secretes effector proteins to influence the host's defense mechanisms, as is typical of plant pathogens. In this investigation, we evaluated the pathogenicity of some Zymoseptoria tritici effector candidate genes having a signal peptide for secretion with no known function. Three genes named Mycgr3G104383, Mycgr3G104444 and Mycgr3G105826 were knocked out separately through homologous recombination, generating Z. tritici IPO323 mutants lacking the functional copy of the corresponding genes. While KO1 and KO3 mutants did not show any significant differences during phenotypic and virulence investigations, the KO2 mutant generated exclusively macropycnidiospores in artificial media, different from wild-type IPO323 which produce only micropycidiospores. The mycelial growth capability of KO2 was also severely attenuated in all of the investigated growth conditions. These changes were observed independent of growth media and growth temperatures, implying that changes were genetic and inherited through generations. Virulence of knockout mutants in wheat leaves was observed to be similar to the wild-type IPO323. Understanding the biology of Z. tritici and its interactions with wheat will reveal new strategies to fight septoria tritici blotch, enabling breeding wheat cultivars resistant to a broader spectrum of Z. tritici strains. Furthermore, gene knockout via homologous recombination proved to be a powerful tool for discovering novel gene functions.

Measuring Splash Dispersal of a Major Wheat Pathogen in the Field

Capacity for dispersal is a fundamental fitness component of plant pathogens. Characterization of plant pathogen dispersal is important for understanding how pathogen populations change in time and space. We devised a systematic approach to measure and analyze rain splash-driven dispersal of plant pathogens in field conditions, using the major fungal wheat pathogen Zymoseptoria tritici as a case study. We inoculated field plots of wheat ( Triticum aestivum) with two distinct Z. tritici strains. Next, we measured disease intensity as counts of fruiting bodies (pycnidia) using automated image analysis. These measurements characterized primary disease gradients, which we used to estimate effective dispersal of the pathogen population. Genotyping of reisolated pathogen strains with strain-specific PCR confirmed the conclusions drawn from phenotypic data. Consistently with controlled environment studies, we found that the characteristic scale of dispersal is tens of centimeters. We analyzed the data using a spatially explicit mathematical model that incorporates the spatial extent of the source, rather than assuming a point source, which allows for a more accurate estimation of dispersal kernels. We employed bootstrapping methods for statistical testing and adopted a two-dimensional hypotheses test based on kernel density estimation, enabling more robust inference compared with standard methods. We report the first estimates of dispersal kernels of the pathogen in field conditions. However, repeating the experiment in other environments would lead to more robust conclusions. We put forward advanced methodology that paves the way for further measurements of plant pathogen dispersal in field conditions, which can inform spatially targeted plant disease management. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Differential tolerance of Zymoseptoria tritici to altered optimal moisture conditions during the early stages of wheat infection

Foliar plant pathogens require liquid or vapour water for at least part of their development, but their response and their adaptive tolerance to moisture conditions have been much less studied than other meteorological factors to date. We examined the impact of altering optimal moisture conditions conducive to infection on the wheat-Zymoseptoria tritici interaction. We assessed the responses in planta of 48 Z. tritici strains collected in two climatologically distinct locations (Ireland and Israel) to four high moisture regimes differing in the timing and the duration of uninterrupted exposure to saturated relative humidity (100% RH) during the first three days of infection. Individual- and population-level moisture reaction norms expressing how the sporulating area of a lesion change with the RH conditions were established based on visual assessments of lesion development at 14, 17 and 20 days post-inoculation (dpi). Our findings highlighted: (i) a critical time-dependent effect on lesion development of uninterrupted periods of exposure to 100% RH during these earliest infection stages; (ii) a marked interindividual variation in the sensitivity to RH conditions both in terms of strain average moisture response and plasticity; (iii) a higher tolerance – expressed at 14 dpi, not later – of the Israeli population to early interruption of optimal moisture conditions. By indicating that sensitivity to sub-optimal moisture conditions may vary between individuals and populations, this study highlights the evidence of moisture adaptation signature in a plant pathogen. Understanding such variation is helpful to predict the response of pathogen populations to changing climatic conditions.

Resistance of the Wheat Cultivar 'Renan' to Septoria Leaf Blotch Explained by a Combination of Strain Specific and Strain Non-Specific QTL Mapped on an Ultra-Dense Genetic Map.

Quantitative resistance is considered more durable than qualitative resistance as it does not involve major resistance genes that can be easily overcome by pathogen populations, but rather a combination of genes with a lower individual effect. This durability means that quantitative resistance could be an interesting tool for breeding crops that would not systematically require phytosanitary products. Quantitative resistance has yet to reveal all of its intricacies. Here, we delve into the case of the wheat/Septoria tritici blotch (STB) pathosystem. Using a population resulting from a cross between French cultivar Renan, generally resistant to STB, and Chinese Spring, a cultivar susceptible to the disease, we built an ultra-dense genetic map that carries 148,820 single nucleotide polymorphism (SNP) markers. Phenotyping the interaction was done with two different Zymoseptoria tritici strains with contrasted pathogenicities on Renan. A linkage analysis led to the detection of three quantitative trait loci (QTL) related to resistance in Renan. These QTL, on chromosomes 7B, 1D, and 5D, present with an interesting diversity as that on 7B was detected with both fungal strains, while those on 1D and 5D were strain-specific. The resistance on 7B was located in the region of Stb8 and the resistance on 1D colocalized with Stb19. However, the resistance on 5D was new, so further designated Stb20q. Several wall-associated kinases (WAK), nucleotide-binding and leucine-rich repeats (NB-LRR) type, and kinase domain carrying genes were present in the QTL regions, and some of them were expressed during the infection. These results advocate for a role of Stb genes in quantitative resistance and for resistance in the wheat/STB pathosystem being as a whole quantitative and polygenic.

Genotypic and Phenotypic Characterization of Two Triticum aestivum L.—Dasypyrum villosum Translocations Lines in the Same Wheat Genetic Background

A wheat 660K chip was used to genotype two wheat-Dasypyrum villosum 6V#4S.6DL and 6V#2S.6AL translocation lines (A303 and B303) and their common wheat recurrent parent Wan7107. The results showed that these three lines have similar characteristics of base composition except for the translocation chromosomes. The alien translocation chromosomes have fewer homozygous and more heterozygous genotypes with more invalid probes. Distributions of SNPs between the translocation lines and Wan7107 were mainly dense on the regions of 6AS or 6DS as expected, but unexpectedly also on near the telomere of 2BS, and some regions of other wheat chromosomes. Meanwhile, the translocation lines A303 and B303 have 99.44% and 98.81% identical genotypes to Wan7107, respectively. Under the same genetic background, A303 and B303 showed different reactions to Blumeria graminis f. sp. tritici (Bgt) strains of powdery mildew. Both translocation lines have higher grain weight and plant height, and B303 has fewer spikelets compared to Wan7107. These results provide us a new insight into the genomic variation between the backcross generation plant and the recurrent parent, which is valuable information for understanding the relationship between wheat and the 6VS chromosome of D. villosum as well as the application potential of the alien chromosome arms.

PH effect on strain-specific transcriptomes of the take-all fungus

The soilborne fungus Gaeumannomyces tritici (G. tritici) causes the take-all disease on wheat roots. Ambient pH has been shown to be critical in different steps of G. tritici life cycle such as survival in bulk soil, saprophytic growth, and pathogenicity on plants. There are however intra-specific variations and we previously found two types of G. tritici strains that grow preferentially either at acidic pH or at neutral/alkaline pH; gene expression involved in pH-signal transduction pathway and pathogenesis was differentially regulated in two strains representative of these types. To go deeper in the description of the genetic pathways and the understanding of this adaptative mechanism, transcriptome sequencing was achieved on two strains (PG6 and PG38) which displayed opposite growth profiles in two pH conditions (acidic and neutral). PG6, growing better at acidic pH, overexpressed in this condition genes related to cell proliferation. In contrast, PG38, which grew better at neutral pH, overexpressed in this condition genes involved in fatty acids and amino acid metabolisms, and genes potentially related to pathogenesis. This strain also expressed stress resistance mechanisms at both pH, to assert a convenient growth under various ambient pH conditions. These differences in metabolic pathway expression between strains at different pH might buffer the effect of field or soil variation in wheat fields, and explain the success of the pathogen.

Response to vanadate exposure in Ochrobactrum tritici strains.

Vanadium is a transition metal that has been added recently to the EU list of Raw Critical Metals. The growing needs of vanadium primarily in the steel industry justify its increasing economic value. However, because mining of vanadium sources (i. e. ores, concentrates and vanadiferous slags) is expanding, so is vanadium environmental contamination. Bioleaching comes forth as smart strategy to deal with supply demand and environmental contamination. It requires organisms that are able to mobilize the metal and at the same time are resistant to the leachate generated. Here, we investigated the molecular mechanisms underlying vanadium resistance in Ochrobactrum tritici strains. The highly resistant strain 5bvl1 was able to grow at concentrations > 30 mM vanadate, while the O. tritici type strain only tolerated < 3 mM vanadate concentrations. Screening of O. tritici single mutants (chrA, chrC, chrF and recA) growth during vanadate exposure revealed that vanadate resistance was associated with chromate resistance mechanisms (in particular ChrA, an efflux pump and ChrC, a superoxide dismutase). We also showed that sensitivity to vanadate was correlated with increased accumulation of vanadate intracellularly, while in resistant cells this was not found. Other up-regulated proteins found during vanadate exposure were ABC transporters for methionine and iron, suggesting that cellular responses to vanadate toxicity may also induce changes in unspecific transport and chelation of vanadate.

Rapid loss of virulence during submergence of Z. tritici asexual spores

Zymoseptoria tritici, the causal agent of Septoria tritici blotch, is a notable pathogen of temperate-grown wheat. To better understand the mechanisms underpinning pathogenicity, leaf infection assays are commonly used to compare either the virulence of Z. tritici wildtype or mutant strains, or the susceptibility of wheat cultivars. These assays, which control for many biotic, abiotic and experimental variables, involve the application of known spore numbers to leaves. To achieve this, spore numbers are quantified during a period of aqueous suspension. Published methods rarely state the period in which spores are held in suspension, suggesting that this variable may be uncontrolled. Using simple, agar-based plating experiments, this work firstly demonstrates that blastospore culturability (the ability to form a colony when plated on appropriate agar) decreases rapidly over time during maintenance in aqueous suspension. It is subsequently shown that this reduction in culturability correlates to a reduction in the virulence of the blastospore population. This is shown in three wild type Z. tritici strains. From this, it is concluded that suspension time is a variable of major importance in experimental design and one which, if not controlled, may lead to erroneous conclusions from inter-strain comparisons. The conidia of the unrelated fungus Magnaporthe oryzae also rapidly lose culturability when stored in aqueous suspension, whereas the microspores of Fusarium oxysporum f. sp. cubense do not, suggesting that this phenomenon occurs in some but not all other fungi. Finally, a droplet method of inoculations is proposed to decrease the variability in the numbers of spores applied, within and between experiments.

A combination of target-site alterations, overexpression and enhanced efflux activity contribute to reduced azole sensitivity present in the Irish Zymoseptoria tritici population

Control of septoria tritici blotch in Irish winter wheat crops has been reliant on the application of azoles fungicides for over a decade. The resulting intensive applications of azole fungicides have placed the Irish Zymoseptoria tritici populations under immense pressures to adapt. Evidence of this adaptation is observed in the decreasing sensitivity of the Irish population to the azole fungicide epoxiconazole, where the mean sensitivity has decreased almost 16x over the period 2006–2015. This decrease in sensitivity has occurred in a stepwise manner, with the sensitive proportion of the population being gradually replaced by those more resistant. The decrease in sensitivity was also accompanied by an increase in frequency of Z. tritici strains with an insert (120, 862 or 866 bp) in their CYP51 promoter region. Neither the 862 bp nor 866 bp inserts impacted the expression of CYP51 in either absence or presence of epoxiconcazole. Sequencing of the CYP51 gene in a sub-sample of the 2015 collection identified 25 different CYP51 haplotypes, the majority of which combined previously identified mutations, including V136A, I381V and S524 T. The presence of the 862 or 866 bp insert in the different strains, whilst not exclusive was strongly associated with specific CYP51 haplotypes. This was also reflected in their sensitivity to a range of azoles, most notably metconazole and tebuconazole. Strains with inserts in their MFS1 gene promoter region and identical in size to those known confer multiple drug resistance (MDR) phenotypes were also identified in the 2015 sub-collection. Although the frequencies of strains with those inserts known to confer moderate-high levels of MDR were extremely low, their presence further highlights the adaptability and continued erosion of azole sensitivity in the Irish Z. tritici population.

Distribution of Zymoseptoria Tritici Mating Types in Western Poland

Zymoseptoria tritici is one of the most common and economically important pathogens of winter wheat in Poland. Sexual reproduction of the species requires two opposite strains, MAT1-1 and MAT1-2 and it plays a significant role in the genetic structure of the pathogen population. The aim of the study was to determine the distribution of mating types of Z. tritici in western Poland between 2013 and 2016. In total 296 Z. tritici strains were isolated and analysed by means of multiplex PCR assay to identify the mating type. The ratio between MAT1-1 and MAT1-2 was individually assessed each year and showed bidirectional, but statistically insignificant fluctuations in frequency. For the four years the total ratio between MAT1-1 and MAT1-2 isolates was almost equal (1.18). Balanced distribution of mating types in the Z. tritici population may suggest that sexual reproduction has considerable impact on the pathogen’s population structure.

Transposable element insertions shape gene regulation and melanin production in a fungal pathogen of wheat

BackgroundFungal plant pathogens pose major threats to crop yield and sustainable food production if they are highly adapted to their host and the local environment. Variation in gene expression contributes to phenotypic diversity within fungal species and affects adaptation. However, very few cases of adaptive regulatory changes have been reported in fungi and the underlying mechanisms remain largely unexplored. Fungal pathogen genomes are highly plastic and harbor numerous insertions of transposable elements, which can potentially contribute to gene expression regulation. In this work, we elucidated how transposable elements contribute to variation in melanin accumulation, a quantitative trait in fungi that affects survival under stressful conditions.ResultsWe demonstrated that differential transcriptional regulation of the gene encoding the transcription factor Zmr1, which controls expression of the genes in the melanin biosynthetic gene cluster, is responsible for variation in melanin accumulation in the fungal plant pathogen Zymoseptoria tritici. We show that differences in melanin levels between two strains of Z. tritici are due to two levels of transcriptional regulation: (1) variation in the promoter sequence of Zmr1 and (2) an insertion of transposable elements upstream of the Zmr1 promoter. Remarkably, independent insertions of transposable elements upstream of Zmr1 occurred in 9% of Z. tritici strains from around the world and negatively regulated Zmr1 expression, contributing to variation in melanin accumulation.ConclusionsOur studies identified two levels of transcriptional control that regulate the synthesis of melanin. We propose that these regulatory mechanisms evolved to balance the fitness costs associated with melanin production against its positive contribution to survival in stressful environments.

Thermal generalist behaviour of invasive Puccinia striiformis f. sp. tritici strains under current and future climate conditions

Yellow rust is a devastating wheat disease. Since 2000, Puccinia striiformis f. sp. tritici strains PstS1 and PstS2 have become adapted to high temperatures and have spread worldwide. By 2011, Warrior strains had invaded both warm and cold areas of Europe. This study questioned whether thermal aptitude promoted the spread of Warrior strains, similar to PstS1/PstS2, by comparing infection efficiency (IE) at five temperatures and latent period (LP) under warm and cold regimes for Warrior isolates and pre‐2011 reference strains on two susceptible wheat varieties. The Warrior isolates showed a range of IE and LP responses to temperature that was intermediate between the northern reference isolates adapted to cold conditions and both the southern and invasive PstS2 isolates adapted to warm conditions. Warrior isolates had the highest IE under optimal temperatures of 10 and 15 °C, and displayed reduced infectivity under the warmest (20 °C) and coldest (5 °C) temperatures. Warrior strains acted as thermal generalists and the reference isolates acted as specialists. An IE thermal response was used to simulate the development of each isolate under future climate scenarios in a temperate and Mediterranean region. Isolates had the same ranking for yearly IE over the three 30‐year periods (1971–2000, 2021–2050, 2071–2100) and both locations, with a slight infection increase in the future. However, in the future IEs increased in earlier months. The thermal generalist profile of Warrior isolates for IE was confirmed, with an intermediate capacity to tolerate warming climate, whereas the southern isolates are better adapted to warm conditions, but do not have the virulences necessary to develop on current varieties.

Temporal and spatial field evaluations highlight the importance of the presymptomatic phase in supporting strong partial resistance in Triticum aestivum against Zymoseptoria tritici

Zymoseptoria tritici, the causal agent of septoria tritici blotch (STB), remains a significant threat to European wheat production with the continuous emergence of fungicide resistance in Z. tritici strains eroding the economic sustainability of wheat production systems. The life cycle of Z. tritici is characterized by a presymptomatic phase (latent period, LP) after which the pathogen switches to an aggressive necrotrophic stage, when lesions bearing pycnidia quickly manifest on the leaf. As minimal knowledge of the possible role of the LP in supporting STB resistance/susceptibility exists, the goal of this study was to investigate the spatial and temporal association between the LP and disease progression across three locations (Ireland – Waterford, Carlow; UK – Norwich) that represent commercially high, medium and low STB pressure environments. Completed over two seasons (2013–2015) with commercially grown cultivars, the potential of the LP in stalling STB epidemics was significant as identified with cv. Stigg, whose high level of partial resistance was characterized by a lengthened LP (c. 36 days) under the high disease pressure environment of Waterford. However, once the LP concluded it was followed by a rate of disease progression in cv. Stigg that was comparable to that observed in the more susceptible commercial varieties. Complementary analysis, via logistic modelling of intensive disease assessments made at Carlow and Waterford in 2015, further highlighted the value of a lengthened LP in supporting strong partial resistance against STB disease of wheat.

A gene locus for targeted ectopic gene integration in Zymoseptoria tritici

Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. In Zymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single “soft-landing” site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1R allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z. tritici, and thus the sdi1 locus is a useful tool for virulence analysis in genetically modified Z. tritici strains. Furthermore, we have developed a vector which facilitates yeast recombination cloning and thus allows assembly of multiple overlapping DNA fragments in a single cloning step for high throughput vector and strain generation.

Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici

The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici.

Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq)

The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein–DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen.