https://orcid.org/0000-0001-6147-4312
Figures
Abstract
Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), one of the most economically destructive wheat foliar diseases. In this study, we explore the physiological and molecular changes elicited in two wheat cultivars with divergent responses (Taichung 29 = susceptible, and Shafir = resistant) upon infection by Z. tritici. Our aim is to uncover novel insights into the intricate mechanisms that govern wheat defense against Z. tritici infection. Our quantitative histopathological study showed that H2O2 accumulated in the resistant cultivar to a higher degree compared to the susceptible cultivar at the biotrophic and switching phase. Additionally, we combined qPCR with a targeted quantitative HPLC technique to evaluate the expression profiles of 13 defense-related genes and profile the polyphenolic compounds induced differentially in the STB susceptible and resistant cultivar. Our finding indicated that five out of 13 genes were strongly up-regulated in the resistant cultivar compared with that of the susceptible one at eight days post-inoculation (dpi), corresponding to the transition phase present in the infection process of Z. tritici. Finally, our targeted HPLC analysis demonstrated that the traced phenolic compounds were highly elevated in the susceptible cultivar infected by Z. tritici compared with that of the resistant cultivar. In conclusion, our comprehensive analysis unveils a robust defense response in the resistant wheat cultivar Shafir, characterized by heightened H2O2 accumulation, significant up-regulation of key defense-related genes during the transition phase, and a distinct profile of polyphenolic compounds, shedding light on the intricate mechanisms contributing to its resistance against Z. tritici, thereby providing valuable insights for the development of more resilient wheat varieties.
Citation: Mirzadi Gohari A, Ghiasi Noei F, Ebrahimi A, Ghanbari MA, Didaran F, Farzaneh M, et al. (2024) Physiological and molecular responses of a resistant and susceptible wheat cultivar to the fungal wheat pathogen Zymoseptoria tritici. PLoS ONE 19(10): e0308116. https://doi.org/10.1371/journal.pone.0308116
Editor: Hao-Xun Chang, National Taiwan University, TAIWAN
Received: December 1, 2023; Accepted: July 10, 2024; Published: October 4, 2024
Copyright: © 2024 Mirzadi Gohari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: We acknowledge financial support from the Iran National Science Foundation Grant NO 97011958. We additionally thank Prof. Gert Kema for providing the biological materials (IPO323 isolate and wheat cultivars) used in this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Zymoseptoria tritici (Desm.) Quaedvlieg & Crous [1] causes Septoria tritici blotch (STB), which is a notorious disease of wheat, threatening global food security. Under favourable environmental conditions for disease development, STB leads to huge economic yield losses and results in decreasing grain quality [2]. Control of STB is based on a combination of chemical compounds and using resistant cultivars bearing Stb resistance genes [3]. Nevertheless, both employed measures pose some limitations in providing an efficient control. These limitations include emerging fungicide resistant isolates in natural populations [4], hindering disease management, and breaking the conferred resistance through Stb genes [5]. It is worth noting that Z. tritici has an active sexual cycle allowing this damaging pathogen to overcome unfavorable environmental (fungicide resistance) and biological (host resistance) conditions [6]. Consequently, it is of great importance to understand the molecular basis of the host-pathogen relationship and manipulate host resistance towards this harmful pathogen.
Z. tritici has a hemibiotrophic lifestyle with two distinct invasion phases comprising a stealth biotrophic phase followed by a necrotic phase. The fungus seems to remain endophytic during the biotrophic stage lasting for 7–10 days before a sudden switch to the advanced necrotrophic phase, coinciding with the occurrence of disease symptoms [7]. Nevertheless, recent studies indicated that asynchronous development of Zymoseptoria tritici infection in wheat exists, and an epiphytic growth of this fungus before the invasion of the host plant is demonstrated [8, 9]. The necrotrophy is suppressed in an incompatible interaction where fungal biomass remains unchanged, while it increases in a compatible interaction [7]. Therefore, it seems that the transition phase is a crucial determinant of compatibility or incompatibility in the Z. tritici-wheat interaction. This switch has features reminiscent of programmed cell death (PCD) corresponding with a loss of cell-membrane integrity, dramatic increases in apoplastic metabolites, and a sharp increase in fungal biomass in infected leaves [10]. However, Rudd et al. (2008) demonstrated that resistance in the Z. tritici-wheat pathosystem is not associated with PCD, an effective resistance mechanism toward biotrophic fungal pathogens such as powdery mildew (Blumeria graminis) or rust (Puccinia graminis f.sp. tritici [11–13]. Interestingly, no sign of programmed cell death, a hallmark of an incompatible interaction between biotrophic pathogens and cereal hosts, was observed during the biotrophic infection process of Z. tritici. It is worth mentioning that recent studies indicated that the prevention of penetration through stomatal closures is a crucial step in suppressing STB development, similar to switching from biotrophy to necrotrophy [14, 15].
The Z. tritici–wheat relationship follows the gene-for-gene model via the recognition of the effector AvrStb6, which is recognized by the corresponding resistance protein Stb6 [6, 16]. It is presumed that this recognition leads to a wide array of defensive responses in cv. Shafir, which harbors the resistance gene Stb6. These responses suppress further colonization of Z. tritici IPO323 beyond the transition phase [14].
Induced expression of defense-related genes is a common response to fungal infection and molecular markers can indicate compatibility/incompatibility [17]. Some studies have compared transcriptional reactions of cereal plants such as wheat and rice during compatible and incompatible interactions with hemibiotrophic plant pathogens [18, 19]. These investigations revealed that there is a differential expression of defense-related genes between susceptible and resistant plants [20]. Additionally, it is reported that differential changes in the timing of the activated defense-related genes depend on compatibility/incompatibility [21] and even types of the applied race/cultivar [22, 23].
Rapid accumulation of phenolic compounds at the attempted infection sites by the fungal pathogen is another tactic employed by plants to combat pathogen attack [24]. Plant phenolics are secondary metabolites constituting structurally diverse products generated through the shikimate—phenylpropanoid pathways. They are pre-existing barriers implicating in plant defense strategies. These compounds have potent antioxidant properties and roles in neutralizing the free radical molecules generated during the oxidative burst [25]. Several lines of evidence suggest that phenolic compounds are quantitatively and temporally different between incompatible and compatible interactions. It claimed that they could be applied as promising markers to determine susceptibility and resistance of pathosystems such as the Z. tritici-wheat interactions [14, 26–29].
Revealing the molecular basis of Z. tritici-wheat interactions may aid in defining the differences between compatibility and incompatibility. More importantly, discovering the molecular mechanisms underlying defense responses would facilitate the development of effective management strategies against STB. Therefore, the purpose of this research was to extend previous efforts to determine molecular markers associated with compatibility and incompatibility in Z. tritici-wheat pathosystem enabling us to delineate the differences between compatible and incompatible responses that occurred in wheat attacked by the Z. tritici. [22, 23, 30]. To accomplish this, we performed a quantitative histochemical analysis coupled with staining with DAB and tryptophan blue to monitor the occurred events in the resistant and susceptible plants. Additionally, we combined qPCR with a targeted quantitative HPLC technique to either evaluate the expression profiles of 13 defense-related genes in the investigated wheat cultivars upon infection by Z. tritici and to profile the polyphenolic compounds induced differentially. Genes for expression profiling were elected to cover the key defense-related genes, playing an essential role in host-microbe interactions, including pathogenesis-related protein PR-1 (PR-1), peroxidase (Per), phenylalanine ammonia-lyase (PAL), and serine carboxypeptidase (SCP) [22]; β-1,3-endoglucanase (PR-2), thaumatin-like protein (PR-5) and lipoxygenase (LOX) [30]; chitinase (Chit) [31]. Three antioxidant enzymes comprising catalase (CAT), mitochondrial manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPX) were selected [32]. Mitogen-activated protein kinases (TaMPK3), known to transcriptionally activate at the symptomless biotrophic stage in both compatible and incompatible interactions in Z. tritici-wheat pathosystem, were also included [12]. A protein disulphide isomerase (PDI), a molecular chaperone, was chosen since previous report showed that it was strongly induced in resistant cultivar challenged with the Z. tritici [30]. Time points were selected to represent consecutive stages of Z. tritici pathogenesis, comprising 2, 4 days post-inoculation (dpi) as well as 12, 16 and 21 dpi corresponded with biotrophy and necrotrophy, respectively. Eight dpi was considered as the transition phase expected to play a central role in determining the outcome of the interaction in this pathosystem. Additionally, the amount of three phenolic chemicals traced in this study, investigated recently in the AvrStb6-Stb6 interaction, was quantified in the applied resistant and susceptible plants [14].
Materials and methods
Fungal isolate
The fully sequenced Z. tritici reference strain IPO323 was used [33].This strain was stored at -80 °C and was re-cultured on potato dextrose agar (PDA) (Sigma-Aldrich Chemie, Steinheim, Germany) at 18 °C once desired for experimentation. Yeast-like spores were produced in yeast glucose broth (YGB) medium (yeast extract 10 g/L, glucose 30 g/L) after placement in a shaker incubator set at 125 rpm (Innova 4430; New Brunswick Scientific, Nijmegen, The Netherlands) at 18 °C and their concentrations were calculated by a disposable hemocytometer (VWR, The Netherlands).
Plant material and infection assay
Wheat cultivar (cv.) Shafir expressing resistance against avirulent IPO323 due to the presence of resistance Stb6 protein interacting with AvrStb6 in IPO323 was utilized as a resistant plant while wheat cv. Taichung 29 was susceptible plant [34]. Plants used in this study were cultivated in a greenhouse under controlled conditions, maintained at a temperature of 18/16 °C (day/night rhythm) and a relative humidity (RH) of 70%. They were grown until reaching the stage where the first leaves were fully unfolded. For each pot, 3–5 seeds were initially sown, and after 7 days, three seedlings were selected in each pot, each with their first fully unfolded leaves. It’s important to note that inoculation was performed at this stage, prior to the emergence of the second set of leaves. The inoculum was produced in YGB (yeast extract 10 g/L, Glucose 30 g/L) at 18 °C for 7 days in a shaker incubator adjusted at 125 rpm (Innova 4430; New Brunswick Scientific, Nijmegen, The Netherlands), and yeast-like spores were obtained after centrifugation at 330 ×g and two washing steps to remove the residual medium. Subsequently, the spore concentrations were adjusted to 1 × 107 spores mL-1 and the resulting suspension was supplemented with 0.15% Tween 20 as a surfactant (MERCK®, Nottingham, UK). In each biological replicate, seeds of cvs. Shafir and Taichung 29 were sown in a 5 cm pot until the first leaves were fully unfolded. Infection assays were performed by a hand sprayer till run off and inoculated plants were incubated in transparent plastic bags for 48 hours under 100% RH and then transferred to a greenhouse compartment (22 °C, relative humidity > 90%- and 16-hours light).
Infection biology
The infection biology of Z. tritici was examined by collecting inoculated leaves at 2, 4, 8, 12, 16, and 21 dpi for compatible (IPO323-cv. Taichung 29) and incompatible interactions (IPO323-cv. Shafir). These leaves were then cleared using 100% ethanol, acetic acid, and glycerol (3:1:1) for 30 minutes at 80 °C to remove the leaves’ natural color (Shetty et al., 2003). In the next step, microscopic slides made from the infected area of the leaves were created using a lactophenol solution (Shetty et al., 2003). A light microscope from Olympus® was used to examine the prepared slides (Olympus, Tokyo, Japan). Based on the previously described technique, a study of the quantitative developmental phases of the strains used in planta was performed [35]. These assays were carried out in three biological samples and was repeated three times independently.
In planta detection of H2O2 and cell death
The 3,3-diaminobenzidine (DAB, D-8001, Sigma) staining technique was used to detect H2O2 in planta [36]. At 2, 4, 8, 12, 16, and 21 days post-inoculation, the infected leaves were collected and placed in a container with a dilute acid (pH 3.8) containing 1 mg/ml DAB while remaining in the dark. The container was kept in a desiccator equipped with a vacuum pump to generate a suction force of 0.2 bar for 30 min, and this was maintained overnight. The following day, a clearing technique was carried out to remove chlorophyll using 100% ethanol, acetic acid, and glycerol (3:1:1) for 30 min at 80 °C [35]. According to Koch and Slusarenko (1990), dead cells and fungal structures were stained by trypan blue [37]. In this assay, 3 infected leaves per each biological sample were collected. These tests were carried out in three biological samples and was repeated three times independently.
Image processing
Using PlantCV, the integrated optical density (IOD) of DAB- or trypan-blue-stained leaf regions was determined [38]. For this, a multiclass naive Bayes procedure based on RGB pixel values was applied. Three staining strengths (low, medium, and high), background, and unstained areas were all covered in the classes. These categorizations are based on how strongly the color is present in the stained tissues; these strengths were first visually categorized, and then Photoshop was used to detect the color spectrum. The three intensities level were defined by machine learning using a table of red, green, and blue color values made up of 50 pixels in each class, with each column representing a different class [39]. Finally, using machine learning data, hundreds of pixels in all leaves were classified into these classifications. For each of the classes, probability density functions (PDFs) were created using the table. Adobe Photoshop was applied to extract the RGB values from the leaf photos (Version 23.1.0).
Quantification of gene transcription
Transcript accumulation of 13 genes of interest (S1 Table) was investigated using quantitative reverse transcription PCR (qRT-PCR). Each replicate contained three separate seedlings that carried one fully unfolded leaf. Three infected leaves per each replicate were harvested at 2, 4, 8, 12, 16, and 21 dpi, flash frozen and ground in liquid nitrogen using a mortar and pestle. This assay was conducted in three biological samples (three different plants). Total RNA was extracted from ground leaves using the RNeasy plant mini kit (Qiagen, location, USA), and subsequently, DNA contamination was removed using the DNA-free kit (Ambion, Cambridgeshire, U.K.). First-strand cDNA was synthesized from two μg of total RNA primed with oligo (dT) using the Invitrogen SuperScript III Reverse Transcriptase according to the manufacturer’s instructions. One μl of the resulting cDNA (15 ng) was used in a 25 μl PCR reaction using a QuantiTect SYBR Green PCR Kit and run and analyzed using an ABI 7500 Real-Time PCR System. The relative expression of each gene was initially normalized with the constitutively expressed wheat beta-tubulin gene and then calculated based on the comparative C(t) method described previously [40]. The R package pheatmap was applied to draw the heatmaps, with the special parameters cluster rows = TRUE to classify the genes based on the hierarchical clustering (https://cran.r-project.org/web/packages/pheatmap/index.html).
Extraction of phenolic compounds and targeted HPLC-UV analysis
The first infected leaves of cvs. Shafir and Taichung 29 were harvested at 2, 4, 8, 12, 16, and 21 dpi (days post-inoculation), flash-frozen, and ground in liquid nitrogen. For each biological sample, three infected leaves were collected. Subsequently, the powdered material from three infected first leaves of each pot was combined to create one biological replicate. This approach ensured that each replicate represented a homogenized mixture of leaves from the same pot, minimizing potential variability between replicates originating from different pots. Subsequently, 10 mL of methanol was added to each powdered leaf, incubated for 30 min in an ultrasonic bath, and centrifuged at 13000 ×g for 5 min. The extraction solvent was evaporated using a rotary evaporator at 45 °C, and then the dry participate was dissolved in 1 mL of methanol. This assay was conducted in three biological samples (three different plants). The HPLC profile analysis of methanolic extracts was done by Waters 2695 Alliance HPLC system equipped with 996 PDA detector to monitor phenolic compounds as previously reported [27]. The mobile phase included of (A), methanol+ 0.02%TFA; and (B), HPLC grade water+ 0.02% trifluoroacetic acid (TFA); with the flow-rate of 0.5 mL min-1, on the C18 column (Novapack C18, 4.6 × 15 mm, 4 μm) were used in the gradient program for 60 min. The Peaks were monitored at 200–400 nm wavelength and identification of each phenolic compound was carried out by comparison of its spectra and retention time with the 14 targeted commercial standards indicated in the S2 Table.
Statistical analysis
This study employed a factorial design conducted in a completely randomized design with three replications to ensure robustness and reliability of the results. Statistical analyses were performed using SPSS 26 software (IBM SPSS, Armonk, NY, USA), and the effects of the independent variables were assessed through a two-way ANOVA. Mean differences were further examined using the LSD test at a significance level of 1% to compare the levels of the factors. Furthermore, meticulous melting and amplification curve analyses were conducted on the gene expression data to ensure data accuracy. Heatmap clustering analysis was performed using the heatmap.2 function in the gplots (Version: 3.1.3.1) R-package, employing the coefficient of Euclidean distance with the Ward method for clustering.
Results
Disease symptoms development
The selected time points correspond with consecutive stages of pathogenesis in the Z. tritici IPO323-wheat interaction covering from inoculation to the onset of pycnidial formation. No symptoms were observed at earlier time points (2 and 4 dpi) corresponding to the biotrophic stage in both interactions. Chlorosis lesions initiating from leaf tips formed on the leaves of cv. Taichung 29 (susceptible cultivar) at 8 dpi which is at the switch to necrotrophy while inoculated leaves of cv. Shafir (resistant cultivar) remained green at 8 dpi. Formed lesions on cv. Taichung 29 expanded over time and eventually developed into large necrotic lesions covered by asexual fruiting bodies containing abundant pycnidiospores (14–21 dpi) whereas small chlorotic flecks appeared on the leaves of cv. Shafir at 21 dpi. S1 Fig displays symptoms once the final samples were harvested on day 21 after inoculation.
Infection biology
Microscopical observation showed that the landed spores in both interactions germinated by producing thin germ tubes and penetrated via stomata by 2 dpi (Fig 1A and 1F). At investigated time points, the proportion of germinated spores was low (15–29%; S3 Table), but it did not differ substantially between the incompatible (cv.Shafir-IPO323) and compatible interactions (cv. Taichung 29-IPO323). Our observations led to the identification of three categories that depict the directions in which germ tubes produced by spores are developing. These include the germ tubes growing towards a stoma, germ tubes ending on a stoma, and germ tubes growing past a stoma. H2O2 accumulated in stained cells 2–8 dpi to a greater extent in the incompatible interaction compared with susceptible plants infected with Z. tritici IPO323 (Figs 1B and 1C; 2A). This event happened particularly around the substomatal cavity where the penetration occurred (Fig 1B). H2O2 gathered around the fungal hyphae, attempting to enter the apoplastic space, and no further hyphal growth was seen (Fig 1C). In the incompatible context at 8 dpi, cell collapse combined with cytoplasmic-like shrinking was also observed (Fig 1D). In the compatible interaction, intercellular hyphal growth was noticed at the 4–8 dpi, possibly as the generated H2O2 was insufficient to halt the hyphal progression (Fig 1G and 1H). The initial formation of the a-sexual fruiting body structures (Fig 1I) was seen 12 days post-inoculation, coinciding with tissue collapse and mature pycnidia releasing the yeast-like cells were observed at 21 dpi (Fig 1J).
(A and F) The fungal strain germinated by 2 dpi and pierced directly through stomata (S stands for the stomata and arrow reflects the germinated spores). (B and G) Accumulation of H2O2 as observed by the formation of red-brown staining in the incompatible context (upper panel) and intercellular growth of the penetrated infective hyphae in the compatible interaction (lower panel), respectively at 4 dpi. (C and H) Extensive accumulation of H2O2 in the cv. Shafir infected by Zymoseptoria tritici IPO323, blocks further hyphal growth while intercellular hyphal growth of WT strain was seen in the cv. Taichung 29 at 8 dpi. (D) Cytoplasmic-like shrinkage happened in the incompatible context at 8 dpi. (I) Initiating the formation of the immature asexual flask-shaped fruiting bodies in the compatible interaction at 12 dpi. (E and J) Healthy cells were observed in the incompatible context while the release of asexual spores (pycnidiospore) was found in the compatible interaction at 21 dpi. The scale bars are 20 μm.
Accumulation of hydrogen peroxide (H2O2) following staining by DAB signified by the red-brown areas (A) and the occurrence of cell death indicated as dark-blue areas stained by the trypan blue (B). The wheat leaves of cvs. Shafir and Taichung 29 were inoculated by Z. tritici IPO323 strain by a hand sprayer. Pictures were taken at 2, 4, 8, 12, 16, and 21 dpi. The images were analyzed by the PlantCV software to obtain the integrated optical density (IOD), displaying the percentage of the stained cells by DAB and trypan blue. Data are indicated as mean ± SD (Standard Deviation) of three biological samples from three independent experiments.
H2O2 accumulation and cell death occurrence
To investigate the functional significance of H2O2 and cell death in the resistant and susceptible plants, we evaluated the accumulation of H2O2 and the onset of cell death at several time intervals, including 2, 4, 8, 12, 16, and 21 dpi. In the incompatible interaction (cv. Shafir-IPO323), the temporal accumulation of H2O2 and cell death was discovered at low levels between 2–4 dpi, peaking at 8 dpi (Fig 2). Following the switch phase (8 dpi), low accumulation of H2O2 and low cell death was observed at the necrotrophic stages (12, 16, and 21 dpi). In contrast, susceptible plants (cv. Taichung 29) inoculated by the IPO323 (compatible interaction) displayed increasing H2O2 accumulation and cell death from 2 dpi onwards. The hallmarks of these plants were high levels of H2O2 accretions and the incidence of cell death at the necrotrophic development stages (12, 16, and 21 dpi). Therefore, high levels of H2O2 accumulations occur, which result in the infected leaves dying completely at 21 dpi (Fig 2A). The PlantCV tool was also employed to quantify the stained area of infected leaves by DAB. Our findings revealed that the incompatible interaction generates substantially more H2O2 than the susceptible plants during the transition stage (8 dpi) (Fig 2A; S4 Table). Similar results were obtained for trypan blue staining by applying the PlantCV program. At 8 dpi, cell death levels peaked in the incompatible interaction compared with the compatible interaction. As we expected, cv. Taichung 29 experienced high levels of cell death at the necrotrophic stages (onwards 8 dpi) (Fig 2B; S4 Table).
Gene transcription profiling
The expression levels of genes involved in the defense response were assessed using the qRT-PCR technique in two wheat cultivars exhibiting contrasting responses to Z. tritici infection. Our analysis revealed significant differences in the relative expression of the studied genes between the two interactions, with the exception of PDI, which did not show statistical significance (Table 1; S5 Table).
The analysis derived from qRT-PCR data of gene expression of 13 wheat genes on two cultivar/Zymoseptoria tritici IPO323 combinations over six time points including 2, 4, 8, 12, 16, and 21 days post-inoculation covered the entire infection process of this damaging fungal pathogen.
The observed distinctions were highly significant, with a P-value < 0.001, except for CAT, which reached statistical significance at P < 0.05. These findings suggest a substantial modulation of the majority of the genes in response to Z. tritici attack. Furthermore, significant alterations in the expression levels of the tested genes were observed at the selected time points, with PAL showing significance at P < 0.05 and other genes at P < 0.001. This indicates that the transcript accumulation of the studied genes was statistically different in both interactions. Additionally, the interaction of genotype and time course for all examined genotypes, except for PAL, exhibited differential expression at a significance level of P < 0.01 (Table 1).
Genes expressed differentially during biotrophic and transition phases.
The expression analysis demonstrated that seven defense-related genes, including PR-1, PR-2, and PR-5, Per, Chit, PDI, and PAL were significantly up-regulated in an incompatible interaction compared with that of the compatible interaction. PR-5, Per, and Chit follow the same expression patterns: gradually up-regulated at early time points (2 and 4 dpi), peaking at eight dpi, whereas PR-1 and PR-2 show the opposite trend: gradually down-regulated during biotrophy (2 and 4 dpi), followed by the highest peak at transition stage (8 dpi). There were no remarkable differences in gene expression of PDI during biotrophy in an incompatible interaction but was significantly higher than that of a compatible context. The highest transcript level recorded at 8 dpi was around 178, belonging to PR-5 and PR-2, while the lowest transcript accumulation belonged to PDI (30). Lastly, PAL was expressed at a low level in both interactions ranging from 0.02–0.3. Nevertheless, this gene was significantly up-regulated at the incompatible interaction compared to the compatible one during the biotrophic stage (2 dpi) (Fig 3).
Inoculated leaves of resistant cv. Shafir, as well as susceptible cv. Taichung 29 by Zymoseptoria tritici IPO323 was harvested at 2, 4, 8-, 12-, 16-, and 21-days post-inoculation. Subsequently, the expression level of targeted genes was initially normalized with the constitutively expressed wheat beta-tubulin gene and then calculated based on the comparative C(t) method.
Genes have bimodal expression profiles.
In contrast to the previous genes, CAT and GPX showed a bimodal expression profile, indicating they were up-regulated steadily during the early stage of infection (2 and 4 dpi) in cv. Shafir, then dropped to 16 dpi. Notably, pycnidial formation, which typically occurs at 21 dpi in other cultivars such as Taichung, was not observed in the Shafir cultivar. Despite this, CAT and GPX were significantly up-regulated again at 21 dpi in Shafir, suggesting potential roles beyond pycnidial formation. TaMPK3 and MnSOD exhibited the same trend, but the transcript accumulation of these genes at 21 dpi was significantly higher than that of the transition phase (8 dpi). The transcript accumulation of these genes varied from 6–16 in cv. Shafir challenged with IPO323 compared with that of cv. Taichung 29 (Fig 4).
Inoculated leaves of resistant cv. Shafir, as well as susceptible cv. Taichung 29 by Zymoseptoria tritici IPO323 was harvested at 2, 4, 8-, 12-, 16-, and 21-days post-inoculation. Subsequently, the expression level of targeted genes was initially normalized with the constitutively expressed wheat beta-tubulin gene and then calculated based on the comparative C(t) method.
Genes up-regulated in the compatible interaction.
Two genes, including SCP, and LOX were expressed at low levels in planta, but the highest peaks were found in the compatible interaction compared to the incompatible context. SCP was induced significantly at 2 dpi in cv. Shafir inoculated by IPO323 compared with that of cv. Taichung 29, but the highest transcript induction of this gene was noticed in the compatible interaction at the late stage of infection (21 dpi). There was no significant accumulation of LOX in both interactions during early time points by 12 dpi, where this gene was expressed greatly in cv. Taichung 29 challenged by IPO323 and continued to be expressed by 16 dpi, followed by a sharp reduction to 21 dpi (Fig 5).
Inoculated leaves of resistant cv. Shafir, as well as susceptible cv. Taichung 29 by Zymoseptoria tritici IPO323 was harvested at 2, 4, 8-, 12-, 16-, and 21-days post-inoculation. Subsequently, the expression level of targeted genes was initially normalized with the constitutively expressed wheat beta-tubulin gene and then calculated based on the comparative C(t) method.
Heat map analysis.
We applied a heat map analysis for better visualization of genes differentially expressed between incompatible and compatible interactions. Our analysis indicated that the expression level of three genes, including LOX, PAL, and, SCP remained without major changes in both interactions while that of five genes (MnSOD, TaMPK3, GPX, PDI and CAT) had moderate differences in the incompatible and compatible interactions. Interestingly, expression profiles of five genes (Per, PR-1, PR-.2, and PR-5 and Chit) displayed strong differential alternations in the investigated interactions. The highest discriminating expression patterns between the two investigated interactions were observed for PR-2 and PR-5 at 8 dpi, corresponding to the switch from the biotrophic phase (Fig 6).
The R package pheatmap was applied to draw the heatmaps. Number 1 (blue colour) and 7 (red colour) indicate the lowest and highest expression level of tested genes, respectively. Sh represents the cv. Shafir (the resistant cultivar) and Ta indicates the cv. Taichung 29 (the susceptible cultivar). 2, 4, 8, 12, 16 and 21 dpi are selected time courses indicating days post inoculation.
Targeted quantitative HPLC analysis
Out of 14 targeted polyphenolic compounds, three ones (Table 2; S6 Table) were detected and quantified in both investigated interactions through a targeted quantitative-HPLC technique to obtain novel insights into how these naturally occurring compounds play roles in mediating resistance in the applied wheat cultivars following infection by Z. tritici IPO323. Our analysis traced chlorogenic acid, apigenin, and rutin as dominant compounds differentially triggered in the incompatible and compatible interactions. Results are expressed in microgram/gram fresh weight (μg/g fw). Chlorogenic acid was upregulated strongly at 2 and 16 dpi in the susceptible cv. Taichung 29 (compatible interaction) compared with that of resistant cv. Shafir (incompatible context), indicating this compound has a bimodal induction profile. The highest amount of this compound (619 μg/g fw) was observed in the cv. Taichung 29 inoculated by IPO323 at 16 dpi, whereases this compound was low in the incompatible interaction, ranging from 15–121 μg/g fw. Apigenin profiles showed a trimodal pattern, reaching the highest peak at 2, 8 and 21 dpi in the compatible interaction compared with incompatible interaction. The highest amount of apigenin was found to be 185 μg/g fw that was quantified in the Taichung 29 cv. at 8 dpi. Furthermore, this compound was remained almost constant over the tested time courses in the cv. Shafir. Contrastingly, rutin was significantly expressed at 4 and 12 dpi, 66 and 30 μg/g fw, respectively, in the incompatible interaction compared with that of the compatible one, 36 and 19 μg/g fw, respectively. However, rutin was remarkably expressed in the compatible context compared with the incompatible one at other tested time courses and the highest amount of rutin (91 μg/g fw) was traced in the cv. Taichung 29 at 2 dpi (Table 2). This experiment was repeated independently twice to ensure robustness and reliability of our observations.
Data are shown as mean ± SD (Standard Deviation) of three biological replicates.
Discussion
In this study, our histopathological study corroborated the previous studies proposing that H2O2 plays an essential player in Z. tritici-wheat interaction to restrict and halt the fungal growth in the incompatible interaction and resistant cultivars [35, 41]. It should be noticed that Z. tritici is a hemi-biotrophic fungus, exploiting two distinct pathogenicity phases to complete its infection cycle [7]. We found that H2O2 accumulated in the resistant plants to a higher amount at the biotrophic and switching phase in comparison with the susceptible plants (Fig 2A). This observation verified its promising role in halting the fungal growth, similar to that documented previously for the biotrophic fungal pathogens such as Blumeria graminis f.sp. hordei [36] and for Z. tritici [35, 41]. Following the transition phase, susceptible plants experienced significant hydrogen peroxide (H2O2) accumulation in mesophyll cells, coinciding with Septoria tritici blotch (STB) symptom onset and tissue collapse. While H2O2 typically serves as a signal for resistance, its presence may indicate increased oxidative stress induced by the pathogen. This stress likely exacerbates cellular damage, contributing to STB progression and tissue collapse. Therefore, Z. tritici may hijack the defense system at the necrotrophic growth stage, as with Botrytis cinerea and Colletotrichum lindemuthianum, to facilitate the infection process [42, 43].
Here, the genes Per, Chit, PR-1, PR-2, and PR-5 were differentially expressed at 8 dpi, coinciding with the onset of the disease symptom expression and corresponding to the biotrophic switch. Per encodes a peroxidase enzyme, playing a central role not only in the diverse physiological processes of plant development but also in plant defense responses through the establishment of a physical barrier or the generation of a highly toxic environment in host tissues to restrict fungal invasion [44] whereas Chit encodes a plant chitinase that hydrolyze the N-acetylglucosamine polymer chitin, a key structural component in the cell wall of Z. tritici. PR-1 is induced abundantly in host tissues encountered by invading phytopathogenic agents to limit their colonization through its antifungal activity [45] while PR-2 is a well-known protein to play a pivotal role in plant defense response against biotic stress through degrading of the β-glucan, which is a major polysaccharide in the fungal cell wall and functions as a pathogen associated molecular pattern (PAMP) molecule after release from the cell wall to trigger further defense response [46]. PR-5 encodes a thaumatin-like protein accumulating to high levels in response to biotic stress and shows antifungal activity in several plant species [47]. PDI, encodes a protein disulfide isomerase and catalyzes the generation and breakage of disulfide bonds between cysteine residues within proteins. We anticipated that this gene might play a role in degrading the cysteine-rich proteins secreted by the Z. tritici, particularly at the transition phase [29].
Our results showed that up-regulation of these five-defense-related genes are strongly induced in the incompatible interaction (Fig 3) suggesting that it might be due to recognition of AvrStb6 secreted by IPO323 through the resistance protein Stb6 present in the cv. Shafir. The transition phase is a turning-point in the battlefield between Z. tritici and wheat, determining the outcome of interactions in this pathosystem, and these genes could be applied as molecular signatures to define the compatibility/incompatibility in Z. tritici-wheat relationship. This finding indicates that wheat defense response towards Z. tritici attack is not solely restricted to the early stage of infection (e.g., the first 24 hours), but rather it is often extended into the transition point when fungal biomass increases dramatically in the compatible interaction [7]. Our results are in disagreement with the study conducted previously by Ray et al., (2003), where PDI, PR-1, PR-2 and PR-5 were differentially upregulated in resistant cv. Tadinia (containing Stb4 resistance gene) compared with that of the susceptible cv. Yecora Rojo at 12 hours after inoculation (hai) [30]. However, the later time points corresponded with the transition phase and the onset of lesion development was not included in their study. In another study, PR-1 upregulated strongly in incompatible interaction at early time points, coinciding with the biotrophic stage while it was lowly expressed in the susceptible cultivar [22]. Shetty et al., (2003) indicated that PR-2 expression was elevated at early time points (1 and 3 dai) in the resistant cv. Stakado while that of was induced in the susceptible cv. Sevin from 9 dai, coinciding with switching phase and entering pathogen to the necrotrophic stage [35]. Interestingly, the highest transcript levels of Chit were observed at 9 dpi (transition phase) in the cv. Sevin challenged by IPO323 while it was elevated in the resistant cvs. Tadinia and W7984 inoculated with T48 isolate at early time points. These differences among various studies suggest that the transcript accumulations of the defense-related genes in wheat in response to Z. tritici attack may depend on combinations of the used host cultivars and fungal genotypes [48].
CAT displayed a bimodal expression pattern, reaching a peak at the switching phase (8 dpi) and the late infection phase (21 dpi) in the incompatible interaction (Fig 4). Catalase is one of the most important antioxidant enzymes, playing a vital role in scavenging the ROS molecules such as hydrogen peroxide (H2O2) accumulated in the infected cells in response to fungal attack. Increasing evidence indicates that oxidative burst, a sudden increase of free radical concentrations in the invaded cells, provide a strong resistance response accompanied with HR reaction against biotrophic plant pathogens to halt further fungal colonization in the attempted sites while an increased ROS level is beneficial for the necrotrophic fungus by triggering HR reaction in such a way to kill more cells [42]. Our result suggested that CAT probably plays an essential role in preventing damage to plant cells because the extensive accumulation of H2O2 generated at 8 and 21 dpi in the incompatible interaction is harmful to plant cells. In addition, results provided in this study revealed that the fine-tune inactivation of H2O2 is a crucial event in the Z. tritici-wheat interaction. This event avoids the induction of HR in the biotrophic stage as these free radical functions as a signalling molecule to trigger HR in other pathosystems. Our data is consistent with the previous report demonstrating CAT’s transcript remarkably induced in the resistant cv. Stakado infected by IPO323 compared with that of the cv. Sevin [35]. Additionally, gene expression profiling of catalase (CAT) in the Macrophomina phaseolina-sesame pathosystem revealed dynamic changes in expression levels. Specifically, during the early stage of infection in the incompatible interaction, CAT expression was found to be downregulated compared to the compatible interaction. However, at the necrotrophic stage of the incompatible interaction, CAT expression showed an upregulation. This differential expression pattern suggested that CAT may not be prominently involved in the initial defense response during incompatible interactions but may play a role in managing oxidative stress associated with the necrotrophic phase of the pathogen [49]. It is worth mentioning that the failure of high H2O2 levels to trigger HR in compatible interactions is likely due to a combination of pathogen-mediated suppression of defense mechanisms, modulation of ROS homeostasis, and co-evolutionary dynamics between plants and pathogens [42, 50].
GPX, a key ROS-scavenging enzyme that catalyzes the reduction of H2O2 to protect the plant cells from oxidative damage generated through oxidative burst events followed the same expression trend similar to that of CAT. It was previously demonstrated that the GPX expression was significantly upregulated in the moderately resistant cv. Cellule in response to IPO323 at 3 dpi [51]. SOD is a critical antioxidant enzyme contributing to scavenge free radicals accumulated in a plant under biotic and abiotic stress conditions, expressed gradually in the resistant cv. Shafir to reach the highest peak at 21 dpi (Fig 4), indicating its potential role in protecting plant cells against oxidative burst events. Keon et al., (2007) monitored the transcript accumulation of SOD in the susceptible cv. Riband challenged with the IPO323 and they found that this gene upregulated particularly at the late stage of infection (14 and 21 dpi) [10]. TaMPK3 was expressed significantly in the incompatible interaction at all investigated time points compared with the compatible interaction (Fig 4). The highest peaks of TaMPK3 were observed at the transition (8 dpi) and 21 dpi (Fig 4). This result suggested TaMPK3 probably plays a role in rendering resistance response triggered by Avr-R interaction as stimulation of this gene was reported in the Avr-R-mediated disease resistance reactions [52]. However, these results were inconsistent with the previous report indicating this gene was upregulated remarkedly at the compatible context preceding the onsets of symptom development (8 dpi) [12]. PAL was significantly induced in the cv. Shafir at 2 dpi corresponded to the biotrophic stage (Fig 3). This gene encodes the first enzyme implicated in the phenylpropanoid pathway leading to the salicylic acid (SA) biosynthesis [53]. It is worth noticing the plant hormone SA plays an essential role in mediating disease resistance response against the biotrophic phytopathogens. This finding is similar to the previous report demonstrating that PAL differentially and significantly triggered in the resistant cv. Tadinia compared with the susceptible cv. Yecora Rojo at the early stage of infection, coinciding with the biotrophic growth [22].
Arrays of molecular players like secondary metabolites are involved in the infected plants to provide an efficient reaction in response to the infection by pathogens. It is well documented that the level of phenolic contents is usually elevated in plants faced with diverse biotic or abiotic stresses [24, 54, 55]. It is well documented phenolic compounds or secondary metabolites are not certainly the only key components of resistance mechanisms in the wheat toward STB. In the current study, we compared the amount of three polyphenolic compounds, including chlorogenic acid, apigenin, and rutin in the resistant and susceptible wheat genotypes in response to Z. tritici infection (Table 2). Our HPLC analysis revealed that the traced phenolic compounds were elevated markedly in the Z tritici infected susceptible background than in the resistant cultivar. In Shafir, the level of phenolic compounds was unchanged without extreme fluctuations during infection except for the apigenin that was significantly increased in the incompatible interaction at 12 dpi compared with that of the compatible one. This result disagrees with the recent study investigating the role of the phenolic compounds in the system complying with the AvrStb6-Stb6 interaction [14]. It seems that the type of plant genotype determines the expression of the phenolic compounds in the Z. tritici-wheat interaction, as three traced phenolic chemicals here expressed at a high level in the susceptible cv. Taichung 29 compared with the resistant one. The gained data indicates additional players besides other phenolic compounds such as waxes, cuticle thickness, and primary metabolites must contribute to wheat resistance towards STB. Somai-Jemmali et al (2017) measured the total phenolic content of bread wheat (BW) and durum wheat (DW) inoculated by Z. tritici isolates, and they found no significant differences in phenolic compound content [56]. Furthermore, polyphenol deposition at the attempted infection sites were found on both wheat species. Results provided in this study is consistent with the report on phenolic profiles of raspberry canes in response to infection by Didymella applanata and Leptosphaeria coniothyrium, where it was shown that the level of phenolic compounds in the canes could be attributed to the differences in disease susceptibility [57]. Additionally, our results are in agreement with reports on the Venturia inaequalis-apple interactions, where it was shown that the amounts of phenolic compounds such as apigenin and rutin increased significantly in the susceptible background compared to the control plants [27]. However, there is accumulating evidence displaying the key roles of phenolic compounds in providing resistance towards fungal colonization. It was reported that chlorogenic acid plays an essential role in conferring tomato resistance against Alternaria alternata. This response is mediated through inhibiting the alternariol biosynthesis, a central factor to colonize the tomato plants [58]. Furthermore, several studies indicated that rutin and apigenin have an antimicrobial impact on the growth of diverse phytopathogens such as Aspergillus ochraceus and Verticillium dahlia in vitro [59].
Conclusion
Ttransition point (8 dpi) is a turning point in mounting compatibility/incompatibility in Z. tritici-wheat relationship since expression analysis demonstrated that five defense-related genes were remarkably and highly up-regulated in the cv. Shafir at 8 dpi. However, recent studies researching how Sb6/Stb16q mediated incompatible interaction confirmed that temporal closure of stomata is a key step in hindering the Z. tritici colonization [14, 15]. We proposed that both events occur in these two crucial stages playing central roles in mediating the incompatible interaction in Z. tritici-wheat pathosystem. Defense-related genes attributed to the primary metabolites play an important role in providing resistance response against incompatible interaction, while secondary metabolites such as phenolic compounds play an important role in a susceptible genotype to defend themselves, but they are an unsuccessful biological weapon to restrict Z. tritici colonization in the investigated compatible interaction. However, it seems that the type of applied cultivars defines the type/amount of generated secondary metabolites in this pathosystem based on the results obtained by Seybold et al. 2020, which it was shown that genes implicated in the phenylpropanoids pathway are induced differentially in resistant cultivar (Chinese Spring) at 4 and 8 dpi compared with the susceptible cultivar (Obelisk). To elaborate the presented conclusions in further detail, employing a global transcriptomics/metabolomics approach coupled with a TaqMan real-time PCR to accurately determine the fungal biomass in the inoculated leaves is needed. These would eventually enable us to precisely compare transcripts/metabolites that are differentially accumulated in the incompatible and compatible interactions and to discover instrumental genes mediating wheat resistance towards STB.
Supporting information
S1 Fig. Disease symptoms development on wheat cultivars infected by Zymoseptoria tritici IPO323.
(a) Plant infected by distilled water (Mock plant). (b) Cultivar Shafir is resistant to IPO323 (incompatible interaction) and no STB symptoms were observed on the inoculated leaves at 21 days post-inoculations. (c) Cultivar Taichung 29 is susceptible to IPO323 (compatible interaction), and necrotic lesions covered by abundant asexual fruiting bodies (pycnidia) were found on the infected leaves at 21 days post-inoculations. The first leaves of the applied cultivars were inoculated with IPO323, and photographs were taken at 21 days post-inoculation.
https://doi.org/10.1371/journal.pone.0308116.s001
(TIF)
S2 Table. The targeted phenolic compounds standard used in this study.
https://doi.org/10.1371/journal.pone.0308116.s003
(PDF)
S3 Table. Observed incidence of various developmental phases in the infection course of Zymoseptoria tritici IPO323 on cv. Shafir that is resistant to IPO323 (incompatible interaction and cv. Taichung 29 that is susceptible to IPO323 (compatible interaction).
Values given are percentages.
https://doi.org/10.1371/journal.pone.0308116.s004
(PDF)
S4 Table. The raw data generated by PlantCV tool to quantify the DAB and trypan blue staining.
https://doi.org/10.1371/journal.pone.0308116.s005
(XLSX)
S5 Table. The raw data (Ct value) generated during qRT-PCR to profile the expression pattern of the investigated defense-related genes.
https://doi.org/10.1371/journal.pone.0308116.s006
(XLS)
S6 Table. The raw data generated during targeted HPLC to quantify the phenolic compounds identified in the examined interactions.
https://doi.org/10.1371/journal.pone.0308116.s007
(XLSX)
Acknowledgments
We thank Prof. Gert Kema for providing the biological materials (IPO323 isolate and wheat cultivars) used in this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We greatly appreciate Dr. Angela Feechan’s invaluable support in critically reviewing and revising our paper.
References
- 1. Quaedvlieg W, Kema G, Groenewald J, Verkley G, Seifbarghi S, Razavi M, et al. Zymoseptoria gen. nov.: a new genus to accommodate Septoria-like species occurring on graminicolous hosts. Persoonia-Molecular Phylogeny and Evolution of Fungi. 2011;26(1):57–69.
- 2. Fones H, Gurr S. The impact of Septoria tritici Blotch disease on wheat: An EU perspective. Fungal Genetics and Biology. 2015;79:3–7. pmid:26092782
- 3. Torriani SF, Melichar JP, Mills C, Pain N, Sierotzki H, Courbot M. Zymoseptoria tritici: a major threat to wheat production, integrated approaches to control. Fungal Genetics and Biology. 2015;79:8–12. pmid:26092783
- 4. Garnault M, Duplaix C, Leroux P, Couleaud G, Carpentier F, David O, et al. Spatiotemporal dynamics of fungicide resistance in the wheat pathogen Zymoseptoria tritici in France. Pest management science. 2019;75(7):1794–807. pmid:30680908
- 5. Brown JK, Chartrain L, Lasserre-Zuber P, Saintenac C. Genetics of resistance to Zymoseptoria tritici and applications to wheat breeding. Fungal Genetics and Biology. 2015;79:33–41. pmid:26092788
- 6. Kema GH, Mirzadi Gohari A, Aouini L, Gibriel HA, Ware SB, van Den Bosch F, et al. Stress and sexual reproduction affect the dynamics of the wheat pathogen effector AvrStb6 and strobilurin resistance. Nature genetics. 2018;50(3):375–80. pmid:29434356
- 7. Kema GH, Yu D, Rijkenberg FH, Shaw MW, Baayen RP. Histology of the pathogenesis of Mycosphaerella graminicola in wheat. Phytopathology. 1996;86(7):777–86.
- 8. Fones HN, Eyles CJ, Kay W, Cowper J, Gurr SJ. A role for random, humidity-dependent epiphytic growth prior to invasion of wheat by Zymoseptoria tritici. Fungal Genetics and Biology. 2017;106:51–60. pmid:28694096
- 9. Fantozzi E, Kilaru S, Gurr SJ, Steinberg G. Asynchronous development of Zymoseptoria tritici infection in wheat. Fungal Genetics and Biology. 2021;146:103504. pmid:33326850
- 10. Keon J, Antoniw J, Carzaniga R, Deller S, Ward JL, Baker JM, et al. Transcriptional adaptation of Mycosphaerella graminicola to programmed cell death (PCD) of its susceptible wheat host. Molecular Plant-Microbe Interactions. 2007;20(2):178–93. pmid:17313169
- 11. Tiburzy R, Reisener H. Resistance of wheat to Puccinia graminis f. sp. tritici: association of the hypersensitive reaction with the cellular accumulation of lignin-like material and callose. Physiological and Molecular Plant Pathology. 1990;36(2):109–20.
- 12. Rudd JJ, Keon J, Hammond-Kosack KE. The wheat mitogen-activated protein kinases TaMPK3 and TaMPK6 are differentially regulated at multiple levels during compatible disease interactions with Mycosphaerella graminicola. Plant Physiology. 2008;147(2):802–15. pmid:18441220
- 13. Prats E, Gay AP, Roberts PC, Thomas BJ, Sanderson R, Paveley N, et al. Blumeria graminis interactions with barley conditioned by different single R genes demonstrate a temporal and spatial relationship between stomatal dysfunction and cell death. Phytopathology. 2010;100(1):21–32. pmid:19968546
- 14. Noei FG, Imami M, Didaran F, Ghanbari MA, Zamani E, Ebrahimi A, et al. Stb6 mediates stomatal immunity, photosynthetic functionality, and the antioxidant system during the Zymoseptoria tritici-wheat interaction. Frontiers in plant science. 2022;13:1004691. pmid:36388590
- 15. Battache M, Lebrun M-H, Sakai K, Soudière O, Cambon F, Langin T, et al. Blocked at the stomatal gate, a key step of wheat Stb16q-mediated resistance to Zymoseptoria tritici. Frontiers in Plant Science. 2022;13.
- 16. Saintenac C, Lee W-S, Cambon F, Rudd JJ, King RC, Marande W, et al. Wheat receptor-kinase-like protein Stb6 controls gene-for-gene resistance to fungal pathogen Zymoseptoria tritici. Nature genetics. 2018;50(3):368–74. pmid:29434355
- 17. van Loon LC, Rep M, Pieterse CM. Significance of inducible defense-related proteins in infected plants. Annual review of phytopathology. 2006;44:135–62. pmid:16602946
- 18. McDonald MC, McDonald BA, Solomon PS. Recent advances in the Zymoseptoria tritici—wheat interaction: insights from pathogenomics. Frontiers in plant science. 2015;6:102. pmid:25759705
- 19. Wang Y, Kwon SJ, Wu J, Choi J, Lee Y-H, Agrawal GK, et al. Transcriptome analysis of early responsive genes in rice during Magnaporthe oryzae infection. The plant pathology journal. 2014;30(4):343. pmid:25506299
- 20. Bagnaresi P, Biselli C, Orrù L, Urso S, Crispino L, Abbruscato P, et al. Comparative transcriptome profiling of the early response to Magnaporthe oryzae in durable resistant vs susceptible rice (Oryza sativa L.) genotypes. PloS one. 2012;7(12):e51609. pmid:23251593
- 21. Bell J, Ryder T, Wingate V, Bailey J, Lamb C. Differential accumulation of plant defense gene transcripts in a compatible and an incompatible plant-pathogen interaction. Molecular and Cellular Biology. 1986;6(5):1615–23. pmid:3785174
- 22. Adhikari TB, Balaji B, Breeden J, Goodwin SB. Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression. Physiological and Molecular Plant Pathology. 2007;71(1–3):55–68.
- 23. Orton E, Rudd J, Brown J. Early molecular signatures of responses of wheat to Zymoseptoria tritici in compatible and incompatible interactions. Plant pathology. 2017;66(3):450–9. pmid:28356604
- 24.
Tak Y, Kumar M. Phenolics: a key defence secondary metabolite to counter biotic stress. Plant phenolics in sustainable agriculture: Springer; 2020. p. 309–29.
- 25. Dai J, Mumper RJ. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules. 2010;15(10):7313–52. pmid:20966876
- 26. Arici SE, Kafkas E, Kaymak S, Koc NK. Phenolic compounds of apple cultivars resistant or susceptible to Venturia inaequalis. Pharmaceutical Biology. 2014;52(7):904–8. pmid:24446864
- 27. Mansoor S, Sharma V, Mir MA, Mir JI, un Nabi S, Ahmed N, et al. Quantification of polyphenolic compounds and relative gene expression studies of phenylpropanoid pathway in apple (Malus domestica Borkh) in response to Venturia inaequalis infection. Saudi journal of biological sciences. 2020;27(12):3397–404. pmid:33304148
- 28. Seybold H, Demetrowitsch TJ, Hassani MA, Szymczak S, Reim E, Haueisen J, et al. A fungal pathogen induces systemic susceptibility and systemic shifts in wheat metabolome and microbiome composition. Nature Communications. 2020;11(1):1910. pmid:32313046
- 29. Rudd JJ, Kanyuka K, Hassani-Pak K, Derbyshire M, Andongabo A, Devonshire J, et al. Transcriptome and metabolite profiling of the infection cycle of Zymoseptoria tritici on wheat reveals a biphasic interaction with plant immunity involving differential pathogen chromosomal contributions and a variation on the hemibiotrophic lifestyle definition. Plant physiology. 2015;167(3):1158–85. pmid:25596183
- 30. Ray S, Anderson JM, Urmeev FI, Goodwin SB. Rapid induction of a protein disulfide isomerase and defense-related genes in wheat in response to the hemibiotrophic fungal pathogen Mycosphaerella graminicola. Plant molecular biology. 2003;53(5):741–54.
- 31. Motteram J, Küfner I, Deller S, Brunner F, Hammond-Kosack KE, Nürnberger T, et al. Molecular characterization and functional analysis of MgNLP, the sole NPP1 domain—containing protein, from the fungal wheat leaf pathogen Mycosphaerella graminicola. Molecular Plant-Microbe Interactions. 2009;22(7):790–9. pmid:19522561
- 32. Baek K-H, Skinner DZ. Alteration of antioxidant enzyme gene expression during cold acclimation of near-isogenic wheat lines. Plant Science. 2003;165(6):1221–7.
- 33. Goodwin SB, M’Barek SB, Dhillon B, Wittenberg AH, Crane CF, Hane JK, et al. Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis. PLoS Genetics. 2011;7(6):e1002070. pmid:21695235
- 34. Kema GH, Verstappen EC, Waalwijk C. Avirulence in the wheat Septoria tritici leaf blotch fungus Mycosphaerella graminicola is controlled by a single locus. Molecular Plant-Microbe Interactions. 2000;13(12):1375–9. pmid:11106030
- 35. Shetty N, Kristensen B, Newman M-A, Møller K, Gregersen PL, Jørgensen HL. Association of hydrogen peroxide with restriction of Septoria tritici in resistant wheat. Physiological and Molecular Plant Pathology. 2003;62(6):333–46.
- 36. Thordal‐Christensen H, Zhang Z, Wei Y, Collinge DB. Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley—powdery mildew interaction. The Plant Journal. 1997;11(6):1187–94.
- 37. Koch E, Slusarenko A. Arabidopsis is susceptible to infection by a downy mildew fungus. The Plant Cell. 1990;2(5):437–45. pmid:2152169
- 38. Gehan MA, Fahlgren N, Abbasi A, Berry JC, Callen ST, Chavez L, et al. PlantCV v2: Image analysis software for high-throughput plant phenotyping. PeerJ. 2017;5:e4088. pmid:29209576
- 39. Díaz‐Tielas C, Grana E, Sotelo T, Reigosa MJ, SÁNCHEZ‐MOREIRAS AM. The natural compound trans‐chalcone induces programmed cell death in Arabidopsis thaliana roots. Plant, Cell & Environment. 2012;35(8):1500–17.
- 40. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nature protocols. 2008;3(6):1101–8. pmid:18546601
- 41. Shetty NP, Mehrabi R, Lütken H, Haldrup A, Kema GH, Collinge DB, et al. Role of hydrogen peroxide during the interaction between the hemibiotrophic fungal pathogen Septoria tritici and wheat. New Phytologist. 2007;174(3):637–47. pmid:17447918
- 42. Govrin EM, Levine A. The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Current biology. 2000;10(13):751–7. pmid:10898976
- 43. Shams E, Javan-Nikkhah M, Mirzadi Gohari A. Dissecting molecular events and gene expression signatures involved in Colletotrichum lindemuthianum-Phaseolus vulgaris pathosystem in compatible and incompatible interactions. European journal of plant pathology. 2020;156:925–37.
- 44. Passardi F, Cosio C, Penel C, Dunand C. Peroxidases have more functions than a Swiss army knife. Plant cell reports. 2005;24(5):255–65. pmid:15856234
- 45. Breen S, Williams SJ, Outram M, Kobe B, Solomon PS. Emerging insights into the functions of pathogenesis-related protein 1. Trends in plant science. 2017;22(10):871–9. pmid:28743380
- 46. Balasubramanian V, Vashisht D, Cletus J, Sakthivel N. Plant β-1, 3-glucanases: their biological functions and transgenic expression against phytopathogenic fungi. Biotechnology letters. 2012;34(11):1983–90.
- 47. Petre B, Major I, Rouhier N, Duplessis S. Genome-wide analysis of eukaryote thaumatin-like proteins (TLPs) with an emphasis on poplar. BMC plant biology. 2011;11(1):1–16. pmid:21324123
- 48. Reilly A, Karki SJ, Twamley A, Tiley AM, Kildea S, Feechan A. Isolate-Specific Responses of the Nonhost Grass Brachypodium distachyon to the Fungal Pathogen Zymoseptoria tritici Compared with Wheat. Phytopathology®. 2021;111(2):356–68. pmid:32720875
- 49. Chowdhury S, Basu A, Kundu S. Biotrophy-necrotrophy switch in pathogen evoke differential response in resistant and susceptible sesame involving multiple signaling pathways at different phases. Scientific reports. 2017;7(1):17251. pmid:29222513
- 50. Dou D, Zhou J-M. Phytopathogen effectors subverting host immunity: different foes, similar battleground. Cell host & microbe. 2012;12(4):484–95. pmid:23084917
- 51. Samain E, Aussenac T, Selim S. The Effect of Plant Genotype, Growth Stage, and Mycosphaerella graminicola Strains on the Efficiency and Durability of Wheat-Induced Resistance by Paenibacillus sp. Strain B2. Frontiers in plant science. 2019;10:587. pmid:31143198
- 52. Stulemeijer IJ, Stratmann JW, Joosten MH. Tomato mitogen-activated protein kinases LeMPK1, LeMPK2, and LeMPK3 are activated during the Cf-4/Avr4-induced hypersensitive response and have distinct phosphorylation specificities. Plant Physiology. 2007;144(3):1481–94. pmid:17478632
- 53. Dixon RA, Achnine L, Kota P, Liu CJ, Reddy MS, Wang L. The phenylpropanoid pathway and plant defence—a genomics perspective. Molecular plant pathology. 2002;3(5):371–90. pmid:20569344
- 54. Naikoo MI, Dar MI, Raghib F, Jaleel H, Ahmad B, Raina A, et al. Role and regulation of plants phenolics in abiotic stress tolerance: An overview. Plant signaling molecules. 2019:157–68.
- 55. Sharma A, Shahzad B, Rehman A, Bhardwaj R, Landi M, Zheng B. Response of phenylpropanoid pathway and the role of polyphenols in plants under abiotic stress. Molecules. 2019;24(13):2452. pmid:31277395
- 56. Somai-Jemmali L, Siah A, Harbaoui K, Fergaoui S, Randoux B, Magnin-Robert M, et al. Correlation of fungal penetration, CWDE activities and defense-related genes with resistance of durum wheat cultivars to Zymoseptoria tritici. Physiological and Molecular Plant Pathology. 2017;100:117–25.
- 57. Mikulic‐Petkovsek M, Schmitzer V, Stampar F, Veberic R, Koron D. Changes in phenolic content induced by infection with Didymella applanata and Leptosphaeria coniothyrium, the causal agents of raspberry spur and cane blight. Plant Pathology. 2014;63(1):185–92.
- 58. Wojciechowska E, Weinert CH, Egert B, Trierweiler B, Schmidt-Heydt M, Horneburg B, et al. Chlorogenic acid, a metabolite identified by untargeted metabolome analysis in resistant tomatoes, inhibits the colonization by Alternaria alternata by inhibiting alternariol biosynthesis. European Journal of Plant Pathology. 2014;139(4):735–47.
- 59. Báidez AG, Gómez P, Del Río JA, Ortuño A. Dysfunctionality of the xylem in Olea europaea L. plants associated with the infection process by Verticillium dahliae Kleb. Role of phenolic compounds in plant defense mechanism. Journal of agricultural and food chemistry. 2007;55(9):3373–7. pmid:17394331