Skip to main content
NCBI home page
Molecular Plant Pathology logoLink to Molecular Plant Pathology
. 2016 Jan 6;17(6):818–831. doi: 10.1111/mpp.12332

L eptosphaeria maculans effector AvrLm4‐7 affects salicylic acid (SA) and ethylene (ET) signalling and hydrogen peroxide (H2O2) accumulation in B rassica napus

Miroslava Nováková 1,2, Vladimír Šašek 1, Lucie Trdá 1, Hana Krutinová 1,3, Thomas Mongin 4, Olga Valentová 2, Marie‐HelEne Balesdent 4, Thierry Rouxel 4, Lenka Burketová 1,✉
PMCID: PMC6638468  PMID: 26575525

Summary

To achieve host colonization, successful pathogens need to overcome plant basal defences. For this, (hemi)biotrophic pathogens secrete effectors that interfere with a range of physiological processes of the host plant. AvrLm4‐7 is one of the cloned effectors from the hemibiotrophic fungus Leptosphaeria maculans ‘brassicaceae’ infecting mainly oilseed rape (Brassica napus). Although its mode of action is still unknown, AvrLm4‐7 is strongly involved in L. maculans virulence. Here, we investigated the effect of AvrLm4‐7 on plant defence responses in a susceptible cultivar of B. napus. Using two isogenic L. maculans isolates differing in the presence of a functional AvrLm4‐7 allele [absence (‘a4a7’) and presence (‘A4A7’) of the allele], the plant hormone concentrations, defence‐related gene transcription and reactive oxygen species (ROS) accumulation were analysed in infected B. napus cotyledons. Various components of the plant immune system were affected. Infection with the ‘A4A7’ isolate caused suppression of salicylic acid‐ and ethylene‐dependent signalling, the pathways regulating an effective defence against L. maculans infection. Furthermore, ROS accumulation was decreased in cotyledons infected with the ‘A4A7’ isolate. Treatment with an antioxidant agent, ascorbic acid, increased the aggressiveness of the ‘a4a7’ L. maculans isolate, but not that of the ‘A4A7’ isolate. Together, our results suggest that the increased aggressiveness of the ‘A4A7’ L. maculans isolate could be caused by defects in ROS‐dependent defence and/or linked to suppressed SA and ET signalling. This is the first study to provide insights into the manipulation of B. napus defence responses by an effector of L. maculans.

Keywords: AvrLm4‐7, Brassica napus, effector, ethylene, Leptosphaeria, ROS, salicylic acid

Introduction

Plant immunity consists of multi‐layered defence responses (Chisholm et al., 2006) as represented by the ‘zig‐zag’ model designed by Jones and Dangl (2006). The first layer is based on the recognition of pathogen‐ (or microbe‐) associated molecular patterns (PAMPs or MAMPs), components common to whole classes of microbes, by surface transmembrane pattern recognition receptors (PRRs). The perception activates PAMP‐triggered immunity (PTI), also referred to as basal innate immunity. However, adapted pathogens have acquired effector proteins that overcome PTI. This phase in the plant–pathogen interaction is called effector‐triggered susceptibility (ETS) and leads to a compatible interaction (i.e. susceptibility of the plant). In the co‐evolutionary arms race, plants have struck back by the recognition of these effectors through additional intracellular receptors. This second layer of plant immunity is called effector‐triggered immunity (ETI) (Dodds and Rathjen, 2010) and usually provides an incompatible interaction (i.e. resistance of the plant and avirulence of the pathogen). As the effectors are recognized by plant immunity on ETI, they are termed avirulence (Avr) effectors encoded by Avr genes. A plethora of cellular events follows the activation of both PTI and ETI: a rapid influx of calcium ions, an oxidative burst characterized by the production of reactive oxygen species (ROS), hormonal changes and transcriptional reprogramming. Often, ETI is followed by localized cell death in the form of a hypersensitive response (HR) (Dodds and Rathjen, 2010; Tsuda and Katagiri, 2010).

The key components of the plant immune system are the plant hormones that operate signal transduction after the perception of a pathogen (Pieterse et al., 2009). Although salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are the principal defence hormones in plants, other phytohormones, such as abscisic acid (ABA), auxin (indole‐3‐acetic acid, IAA), cytokinins (CKs), brassinosteroids (BRs) and gibberellins (GAs), also intervene in plant defences. Hormonal homeostasis and crosstalk between signalling pathways are crucial for the fine regulation of plant immunity (Pieterse et al., 2012; Robert‐Seilaniantz et al., 2011). The changes in phytohormone levels (or sensing) lead to transcriptional reprogramming to favour defence over other cellular processes, such as growth and development (Buscaill and Rivas, 2014; Denancé et al., 2013; Robert‐Seilaniantz et al., 2011). SA, JA and ET signalling can all be activated during some cases of PTI and ETI (Tsuda and Katagiri, 2010). Although there are exceptions, SA signalling is generally implicated in defence against biotrophs, the types of pathogen that grow and retrieve nutrients from living plant tissue, whereas JA/ET signalling mediates defence against necrotrophic pathogens that kill plant cells and feed on dead tissue (Glazebrook, 2005). Naturally, plant pathogens have evolved a variety of strategies to overcome plant hormone‐mediated immunity or to induce host susceptibility by interfering with various hormonal processes (Dou and Zhou, 2012).

Typically, effectors are defined as small secreted proteins (c. 300 amino acids) that are highly expressed during host infection, do not include known conserved domains or motifs and are often enriched in cysteine residues engaged in disulphide bridges for protein stability after secretion (Doehlemann and Hemetsberger, 2013). A more general definition describes effectors as ‘small secreted proteins or molecules that alter host‐cell structure or function’ (Hogenhout et al., 2009). The recent advances in fungal genomics have allowed the prediction of hundreds of effector genes in the genomes of different fungal pathogens (Schmidt and Panstruga, 2011).

Although more is known about the interference of bacterial effectors with the plant immune system (Block and Alfano, 2011; Deslandes and Rivas, 2012), notably in the Pseudomonas–Arabidopsis pathosystem, recent progress has also been made in deciphering the roles of fungal effectors (Giraldo and Valent, 2013; Rafiqi et al., 2012). Despite the diversity of fungal effectors and the differences between fungal and bacterial effectors, it seems that they impair the host plant immune system at similar key steps, such as the perception of PAMPs and downstream signalling, hormonal homeostasis and crosstalk, plant cell death or the production of antimicrobial compounds (Doehlemann and Hemetsberger, 2013; Dou and Zhou, 2012).

Leptosphaeria maculans is a fungal plant extracellular pathogen belonging to the Dothideomycetes. The fungus infects mainly Brassica crops. In oilseed rape (Brassica napus), L. maculans causes blackleg (or phoma stem canker), the most damaging disease of this crop in Australia, Canada and Europe (Howlett, 2004; West et al., 2001). The infection cycle begins with the germination of ascospores on the leaf surface, which penetrate cotyledons and younger leaves via stomata or wounds. The fungus grows in the extracellular space (apoplast) and initially colonizes the tissue as a biotroph, but, behind the hyphal front, the fungus becomes necrotrophic and kills plant cells. Concomitantly, hyphae spread down the petiole in an endophytic manner, eventually reaching the stem cortex and causing black/brown blackleg necrotic lesions (Hammond and Lewis, 1987; West et al., 2001). In L. maculans, the genomic location of 11 Avr genes, designated AvrLm1, 2, 3, 4, 5, 6, 7, 8, 9, 11, AvrLepR1 and AvrLmJ1, has been identified to date (Balesdent et al., 2002, 2005; Ghanbarnia et al., 2012; Van de Wouw et al., 2014). Among these, six have been cloned: AvrLm1 (Gout et al., 2006), AvrLm2 (Ghanbarnia et al., 2015), AvrLm4‐7 (Parlange et al., 2009), AvrLm6 (Fudal et al., 2007), AvrLm11 (Balesdent et al., 2013) and AvrLmJ1 (Van de Wouw et al., 2014). These are involved in PTI with a series of Brassica hosts, such as B. napus, B. rapa and/or B. juncea. Although some of the L. maculans effectors have been shown to be involved in fungal aggressiveness (Huang et al., 2010, 2006), we currently have no mechanistic understanding of their function and how they may interfere with plant defences or other metabolic pathways.

One of the most interesting candidates for such an interaction is the Avr protein AvrLm4‐7, recognized by two resistance proteins coded by Rlm4 and Rlm7. During adaptation to resistance, a single non‐synonymous base mutation in AvrLm4‐7 led to the escape of effector recognition by Rlm4, whereas the recognition by Rlm7 was conserved (Parlange et al., 2009). Moreover, comparison of two near‐isogenic L. maculans isolates differing in the presence of an AvrLm4‐7 allele showed that the virulent avrLm4 allele was associated with a decrease in fungal aggressiveness (Huang et al., 2010, 2006). The importance of the effector is underlined by the fact that, in nature and (before the use of Rlm7) in agronomic practice, all the examined strains virulent on susceptible genotypes of B. napus (rlm4 rlm7) possessed AvrLm4‐7 (Balesdent et al., 2006). The AvrLm4‐7 effector was thus postulated to be implicated as a virulence factor of L. maculans (Rouxel and de Wit, 2012). Although AvrLm4‐7 has been studied mainly as an Avr protein, we focus here on its activity as an effector and investigate how the AvrLm4‐7 effector alters the B. napus immune system during ETS. For this objective, we investigated the effect of two isogenic isolates of L. maculans, harbouring or not the AvrLm4‐7 allele, following inoculation on a susceptible B. napus cultivar (rlm4 rlm7). Different defence responses were monitored, including the production of plant hormones, expression of defence‐related genes and ROS accumulation. Our additional scope was to characterize the defence responses triggered after the recognition of AvrLm4‐7 by Rlm4 in B. napus in the incompatible interaction and compare them with previously reported AvrLm1‐triggered defence responses (Šašek et al., 2012).

Results

The presence of a functional allele of AvrLm4‐7 promotes the aggressiveness of L. maculans during a compatible interaction

It has been reported previously that the AvrLm4‐7 effector contributes to L. maculans fitness and increases its virulence, and that even differences in alleles (i.e. AvrLm4‐AvrLm7 versus avrLm4‐AvrLm7 alleles) could lead to fitness differences, with the virulent allele being responsible for a fitness deficit (Huang et al., 2010, 2006). Here, we expanded the experiment by comparing 26 isogenic L. maculans isolates differing at the AvrLm4‐7 locus for aggressiveness on two susceptible B. napus cultivars, Eurol and ES Astrid, both lacking the corresponding resistance genes (rlm4 rlm7) (Fig. 1A). The wild‐type (WT) isolate Nzt‐4 (a4a7) contains a highly degenerated non‐functional allele of AvrLm4‐7. A series of Nzt‐4 isolates complemented with either the AvrLm4‐AvrLm7 (A4A7, 11 isolates) or avrLm4‐AvrLm7 (a4A7, eight isolates) allele of the gene (Parlange et al., 2009) was compared with control WT Nzt‐4 isolates that underwent the transformation process but did not integrate the construct (a4a7, seven isolates). In both B. napus cultivars, the group of L. maculans isolates complemented with the A4A7 allele caused significantly larger cotyledon lesions than the control group of isolates lacking AvrLm4‐7 (Fig. 1A). Cotyledons of cv. ES Astrid infected with isolates complemented with the a4A7 allele exhibited more severe symptoms in comparison with the control group, whereas, on cv. Eurol, the L. maculans infection with isolates carrying the a4A7 allele increased the lesion size only slightly, but without statistical significance (Fig. 1A).

Figure 1.

Figure 1

Open in a new tab

Introduction of a functional allele of AvrLm4‐7 increases Leptosphaeria maculans aggressiveness on susceptible Brassica napus cultivars lacking corresponding resistance genes (rlm4 and rlm7). (A) Isogenic isolates of Nzt‐4, with the a4a7, a4A7 or A4A7 allele of AvrLm4‐7, were puncture inoculated on cv. ‘Eurol’ and cv. ‘ES Astrid’. At 12 days after inoculation (dai), the cotyledons were scanned. The box‐plots represent the median lesion area obtained for each isolate/plant genotype combination. For a given plant genotype, groups with the same letter do not induce significantly different median leaf lesions (red trait) (Kruskall–Wallis test, 5% threshold). (B) Disease symptoms at 13 dai caused by two isogenic β‐glucuronidase (GUS)‐tagged Nzt‐4 isolates lacking (a4a7) or complemented (A4A7) with the AvrLm4‐7 gene. (C) Quantification of disease symptoms caused by the two isogenic GUS‐tagged Nzt‐4 isolates. Lesion areas were measured using image analysis. Values represent means ± standard error (SE) (n > 22). Asterisks indicate statistically significant differences between ‘a4a7’ and ‘A4A7’ isolates (**P < 0.01, Student's t‐test).The experiment is a representative from three independent biological experiments.

In further experiments, a pair of isogenic L. maculans Nzt‐4 isolates differing only in the presence of the AvrLm4‐7 gene (Parlange et al., 2009) were chosen, transformed by the β‐glucuronidase (GUS) gene from Escherichia coli using Agrobacterium tumefaciens (Gardiner and Howlett, 2004), and referred to as ‘a4a7’ and ‘A4A7’ hereafter. Although the GUS transformation increased slightly the aggressiveness of the ‘a4a7’ isolate compared with the WT (Fig. S2, see Supporting Information), the difference between the transformed isolates ‘a4a7’ and ‘A4A7’ was conserved. On susceptible B. napus cv. Columbus, the ‘A4A7’ isolate exhibited about 40% larger lesions than the ‘a4a7’ isolate at 13 days after inoculation (dai) (Fig. 1B,C).

In vitro and in planta growth of isogenic L. maculans Nzt‐4 isolates and expression of AvrLm4‐7

We first monitored the in vitro growth of the ‘a4a7’ and ‘A4A7’ isolates to check whether any growth difference could be observed that might have an impact on symptom severity. Both L. maculans isolates differing in AvrLm4‐7 grew similarly on V8 juice agar in vitro (Fig. S1 and Table S1, see Supporting Information). The spread of infection in planta was then monitored in susceptible B. napus Columbus (rlm4 rlm7) cotyledons infected by puncture with either an L. maculans ‘a4a7’ or ‘A4A7’ isolate starting from 4 dai. None of the isolates developed macroscopically visible symptoms until 8 dai. To precisely monitor the growth of L. maculans isolates in infected plant tissue, we adopted a fluorescence measurement method of GUS activity using 4‐methylumbilliferone glucuronide (MUG). As prerequisite, the GUS activity in in vitro‐grown mycelia was shown to be similar (Fig. S3, see Supporting Information). Both isolates colonized the plant tissue similarly until 8 dai (Fig. 2A,B). Later, at 10 dai, the colonization by the ‘A4A7’ isolate was more intense and its mycelium quantity reached three times that of the ‘a4a7’ isolate (Fig. 2B).

Figure 2.

Figure 2

Open in a new tab

In planta growth of Leptosphaeria maculans isolates differing in the presence of the AvrLm4‐7 gene on susceptible Brassica napus ‘Columbus’ (rlm4 and rlm7), visualized and quantified using β‐glucuronidase (GUS) activity. Plants (14 days old) were puncture inoculated by either water (mock) or a spore suspension of GUS‐tagged L. maculans Nzt‐4 isolates lacking (a4a7) or complemented (A4A7) with the AvrLm4‐7 effector. (A) Disease symptoms and histochemical visualization of fungal mycelia by GUS staining at 6 or 7, 8 and 10 days after inoculation (dai). (B) GUS activity in inoculated cotyledons measured by a 4‐methylumbilliferone glucuronide (MUG) assay at 4, 6, 8 and 10 dai. Relative fluorescence is reported to fungal mass at the infection site. Values represent means ± standard error (SE) from three biological replicates. Asterisks indicate statistically significant differences between ‘a4a7’ and ‘A4A7’ isolates (*P < 0.05 and **P < 0.01; Student's t‐test). The experiment was repeated three times with similar results.

Typically, the expression of L. maculans effectors is linked to the biotrophic stage of infection. To confirm this, we analysed the expression of AvrLm4‐7 in L. maculans isolate Nzt‐4 complemented with an AvrLm4‐7 allele originating from JN3 (‘A4A7’ isolate) during infection of susceptible B. napus cv. Columbus and after in vitro growth. The expression of AvrLm4‐7 in L. maculans isolate JN3 was also determined in vitro. Consistent with previous observations (Parlange et al. 2009; Soyer et al., 2014), the expression of AvrLm4‐7 was strongly repressed in vitro (Fig. 3). During infection, almost 10 000 times more AvrLm4‐7 transcripts were detected in the complemented ‘A4A7’ isolate compared with in vitro growth (Fig. 3). The peak occurred during the asymptomatic stage of infection until 8 dai.

Figure 3.

Figure 3

Open in a new tab

Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) relative expression of AvrLm4‐7 during the growth of Leptosphaeria maculans isolates JN3 and Nzt‐4 ‘A4A7’ in vitro and during infection on susceptible Brassica napus ‘Columbus’ (rlm4 and rlm7) infected with isolate Nzt‐4 ‘A4A7’. In vitro spore suspensions of JN3 and Nzt‐4 were grown in Gamborg B5 medium for 10 days. Plants (14 days old) were inoculated by puncture inoculation with a spore suspension of L. maculans Nzt‐4 isolate complemented with (A4A7) the AvrLm4‐7 gene. Expression of AvrLm4‐7 is normalized to LmITS1 and relative to AvrLm4‐7 expression in JN3 in vitro. Values represent means ± standard error from three biological replicates.

AvrLm4‐7 decreases SA and ABA levels during compatible interactions

We hypothesized that the presence of AvrLm4‐7 could subvert plant immunity. First, in infected B. napus cv. Columbus cotyledons, we checked the levels of the principal plant hormones by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis.

The SA concentration increased strongly with infection. Differences between isolates were observed at 6 dai, but reached a maximum at 8 dai. Being surprisingly low, the level of SA in ‘A4A7’‐infected cotyledons then increased strongly, reaching a comparable level to that of cotyledons infected with the ‘a4a7’ isolate at 10 dai (Fig. 4).

Figure 4.

Figure 4

Open in a new tab

Liquid chromatography–mass spectrometry analysis of plant hormone levels in susceptible Brassica napus ‘Columbus’ (rlm4 and rlm7) infected with Leptosphaeria maculans isolates differing in the presence of the AvrLm4‐7 gene. Salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA) and its catabolic product phaseic acid (PA), indole‐3‐acetic acid (IAA) and its oxidized product 2‐oxo‐IAA (OxIAA), gibberellin 1 (GA1) and gibberellin 4 (GA4) were determined in extracts from cotyledons. Plants (14 days old) were inoculated by puncture inoculation with either water (mock) or a spore suspension of L. maculans Nzt‐4 isolates lacking (a4a7) or complemented with (A4A7) the AvrLm4‐7 gene. Values represent means ± standard error (SE) from three independent experiments. Asterisks indicate statistically significant differences between cotyledons infected with ‘a4a7’ and ‘A4A7’ isolates (*P < 0.05 and **P < 0.01; one‐tailed Student's t‐test).

Interestingly, the ABA content was significantly reduced in cotyledons infected with the ‘A4A7’ isolate compared with ‘a4a7’‐infected cotyledons at 8 dai (Fig. 4). The level of the ABA degradation product, phaseic acid (PA), increased more slowly in ‘A4A7’‐infected plants and, at 10 dai, the content of PA was significantly reduced in comparison with plants infected with the ‘a4a7’ isolate.

The contents of gibberellins, auxin and its degradation product 2‐oxo‐IAA (OxIAA) exhibited no change in comparison with either the mock‐treated cotyledon or between the two isolates (Fig. 4).

The JA concentration increased similarly in the mock‐treated and infected cotyledons (Fig. 4). We explain the slight JA increase in mock‐treated and infected cotyledons between 6 and 10 dai as being the effect of puncture inoculation, as JA responds to wounding (Bell et al., 1995).

SA‐ and ET‐dependent signalling is lowered in susceptible B. napus cotyledons infected with the ‘A4A7’ isolate

To confirm our findings, we examined whether decreased concentrations of plant hormones are also reflected at the transcript level. For this, we used a set of B. napus defence signalling marker genes that were identified in our previous study (Šašek et al., 2012).

The expression induction of a gene coding for the key enzyme in the SA biosynthetic pathway, ISOCHORISMATE SYNTHASE 1 (ICS1), was lower in cotyledons infected with the ‘A4A7’ isolate compared with the virulent recipient at 8 dai (Fig. 5). Moreover, we observed a delayed expression induction of the widely used SA marker PATHOGENESIS‐RELATED GENE 1 (PR1) in cotyledons infected with the ‘A4A7’ isolate. In cotyledons infected with the ‘a4a7’ isolate, the transcription of marker genes increased almost linearly from 6 to 10 dai, whereas, in cotyledons infected with the ‘A4A7’ isolate, the increase in expression was only seen from 8 dai (Fig. 5).

Figure 5.

Figure 5

Open in a new tab

Relative expression of salicylic acid (SA), ethylene (ET), jasmonic acid (JA) and abscisic acid (ABA) marker genes in susceptible Brassica napus ‘Columbus’ (rlm4 and rlm7) infected with Leptosphaeria maculans isolates differing in the presence of the AvrLm4‐7 gene. Plants (14 days old) were inoculated by puncture inoculation with either water (mock) or a spore suspension of L. maculans Nzt‐4 isolates lacking (a4a7) or complemented with (A4A7) the AvrLm4‐7 gene. The expression of B. napus signalling marker genes was determined by reverse transcription‐quantitative polymerase chain reaction and normalized to B. napus Actin. Values represent means ± standard error (SE) from three independent experiments. Asterisks indicate statistically significant differences between cotyledons infected with ‘a4a7’ and ‘A4A7’ isolates (*P < 0.05; one‐tailed Student's t‐test).

In addition, we also analysed the transcription of genes related to ET signalling. The transcription rate of a gene involved in the biosynthesis of 1‐aminocyclopropane (ET precursor), 1‐AMINO‐CYCLOPROPANE‐1‐CARBOXYLATE SYNTHASE 2 (ACS2), was always lower in cotyledons infected with the ‘A4A7’ isolate compared with those infected with ‘a4a7’. Similarly, hevein‐like protein (HEL; also classified as PR4), responding to ET and JA signalling pathways concomitantly, was induced to a lesser degree in cotyledons infected with the ‘A4A7’ isolate (Fig. 5). The transcription of the ABA‐related marker genes NCED3, a gene coding for the key enzyme of ABA biosynthesis, and RD26, another ABA‐inducible gene, was not significantly affected (Fig. 5).

Hydrogen peroxide (H2O2) accumulation is decreased in cotyledons infected with the ‘A4A7’ isolate at late stages of infection

Reactive oxygen species (ROS) are components of importance to the plant immune system, acting either as signalling molecules or exerting a direct toxic effect on invading microbes (Apel and Hirt, 2004; Foyer and Noctor, 2013). As several plant pathogens subvert ROS production (Doehlemann and Hemetsberger, 2013), we examined the possibility that AvrLm4‐7 also affects ROS levels. The production of H2O2 was monitored from 6 to 10 dai by diaminobenzidine (DAB) staining. At 6 dai, no cotyledon showed brown staining (Fig. 6A). At this stage of the infection, only a little L. maculans mycelium is detected at the site of infection (Fig. 2B). In infected cotyledons, H2O2 stained by DAB appeared from 8 dai, showing more intense accumulation at 10 dai. We detected significantly less DAB‐stained area in cotyledons infected with the ‘A4A7’ isolate than in those infected with the ‘a4a7’ isolate at both time points (Fig. 6). Furthermore, we examined the expression of NADPH oxidase genes during the response of cv. Columbus to ‘a4a7’ and ‘A4A7’ L. maculans isolates. NADPH oxidases are involved in the production of superoxide, an important precursor of several ROS. Amongst these, the transcription of rbohF was slightly induced in cotyledons when infected with the ‘a4a7’ isolate only (Fig. S4, see Supporting Information).

Figure 6.

Figure 6

Open in a new tab

Detection of hydrogen peroxide in susceptible Brassica napus ‘Columbus’ cotyledons infected with Leptosphaeria maculans isolates differing in the presence of the AvrLm4‐7 effector using diaminobenzidine (DAB) staining. Plants (14 days old) were inoculated by puncture inoculation with either water (mock) or a spore suspension of L. maculans Nzt‐4 isolates lacking (a4a7) or complemented with (A4A7) the AvrLm4‐7 gene. (A) DAB‐stained cotyledons at 6, 8 and 10 days after inoculation (dai). (B) DAB‐stained area was measured using image analysis at 8 and 10 dai. Values represent means ± standard error from 22 cotyledons. Asterisks indicate statistically significant differences between L. maculans isolates (*P < 0.05; Student's t‐test). The experiment was performed twice with similar results.

Treatment with an antioxidant agent increases ‘a4a7’ aggressiveness

Ascorbic acid belongs to the natural antioxidants in plants and, together with other antioxidants, determines the lifetime of ROS (Foyer and Noctor, 2013). Infected cotyledons were treated with water or 5 mm ascorbic acid at 3 and 6 dai, and disease symptoms were quantified at 13 dai. Although the necrotic lesion area caused by the ‘A4A7’ isolate was unaffected by treatment, the lesions produced by the ‘a4a7’ isolate increased after ascorbic acid treatment to reach the level obtained following ‘A4A7’ infection (Fig. 7). This result indicates that H2O2 is involved in the defence response of B. napus to L. maculans at late stages of the primary infection, and that its accumulation is affected by the action of AvrLm4‐7.

Figure 7.

Figure 7

Open in a new tab

Treatment with ascorbic acid increases the aggressiveness of Leptosphaeria maculans isolate Nzt‐4 ‘a4a7’ on susceptible Brassica napus ‘Columbus’. Plants (14 days old) were inoculated by puncture inoculation by water (mock) or a spore suspension of L. maculans Nzt‐4 isolates lacking (a4a7) or complemented with (A4A7) the AvrLm4‐7 gene. At 3 and 6 days after inoculation (dai), inoculation sites were treated with either water (mock) or 5 mm ascorbic acid (Asc). (A) Quantification of disease symptoms at 13 dai. The necrotic lesion area was measured using image analysis. Values represent means ± standard error (SE) (n ≥ 40). Different letters above the columns indicate statistically significant differences according to Student's t‐test (P < 0.05). (B) Representative cotyledons at 13 dai.

Recognition of AvrLm4‐7 by Rlm4 induces SA and ET signalling

In addition to the effect of AvrLm4‐7 during a compatible interaction, we also aimed to characterize the defence pathways triggered by the recognition of AvrLm4‐7 in B. napus. For this purpose, the same two isogenic isolates were also inoculated onto an Rlm4 B. napus genotype (cv. Pixel). In the incompatible interaction, the recognition of AvrLm4‐7 by Rlm4 led to the inhibition of infection spread. Indeed, only small greyish lesions with sharp margins developed on cotyledons and were restricted to a small area surrounding the site of inoculation. In contrast, the ‘a4a7’ isolate lacking the AvrLm4‐7 effector (compatible interaction) caused large greyish necrotic lesions that spread up to the cotyledon margin at 10 dai (Fig. 8A).

Figure 8.

Figure 8

Open in a new tab

Compatible and incompatible interaction of Leptosphaeria maculans isolates differing in the presence of the AvrLm4‐7 effector with Brassica napus ‘Pixel’ carrying the resistance gene (Rlm4). Plants (14 days old) were inoculated by puncture inoculation with either water (mock) or a spore suspension of L. maculans Nzt‐4 isolates lacking (a4a7) or complemented with (A4A7) the AvrLm4‐7 gene. (A) Disease symptoms developed at 10 days after inoculation (dai). (B) Expression of salicylic acid (SA), ethylene (ET), jasmonic acid (JA) and abscisic acid (ABA) marker genes relative to mock at 6 dai determined by reverse transcription‐quantitative polymerase chain reaction. Values represent means ± standard error (SE) from three biological replicates. Asterisks indicate statistically significant differences between L. maculans isolates (*P < 0.01; Student's t‐test).

Compared with the isolate lacking AvrLm4‐7, the ‘A4A7’ isolate induced higher transcription of the SA marker genes ICS1 and PR1. The transcription of WRKY 70, an SA‐responsive transcription factor, was also induced on AvrLm4‐7 recognition, but without statistical significance (Fig. 8B). Similar to PR1 and ICS1, the ET‐related marker gene ACS2 was also strongly induced in cotyledons infected with the ‘A4A7’ isolate. In addition, HEL and chitinase (CHI), which respond concomitantly to ET and JA, were induced. Here, we attribute the increase to the effect of ET, as the JA‐dependent marker genes AOS and LOX3 revealed no difference in transcription (Fig. 8B). Interestingly, the recognition of AvrLm4‐7 induced a slight but statistically significant increase in the transcription of the ABA‐dependent genes NCED3 and RD26 (Fig. 8B).

In summary, the recognition of L. maculans AvrLm4‐7 by Rlm4 triggered strong activation of SA‐ and ET‐dependent signalling pathways, whereas ABA signalling was only slightly induced and JA‐dependent signalling exhibited no activation. The successful elimination of L. maculans growth by the defence triggered by the SA and ET signalling pathways also illustrated the expression decrease of activated marker genes at 10 dai (Fig. S5, see Supporting Information). In ‘A4A7’‐infected cotyledons, SA and ET signalling marker genes exhibited a lower transcription rate at 10 dai in comparison with 6 or 8 dai, whereas the expression increased steadily from 6 to 10 dai in cotyledons infected with the virulent isolate ‘a4a7’ (Fig. S5).

Discussion

This study aimed to confirm and find a cause for the increased aggressiveness of L. maculans isolates harbouring the AvrLm4‐7 effector by investigating its effect on B. napus defence responses. This work is the first attempt so far to investigate the role of AvrLm4‐7 as an effector during compatible interactions with B. napus, with the working hypothesis that it may interfere with plant defence signalling.

First, using a set of L. maculans isolates differing at the AvrLm4‐7 locus, we showed that the absence of a functional allele of AvrLm4‐7 was responsible for the reduced aggressiveness on cotyledons, as measured by the size of the lesions in control conditions. In contrast with AvrLm1, which has also been shown to contribute to pathogen aggressiveness to some extent (Huang et al., 2010, 2006), the increase in aggressiveness caused by the presence of the AvrLm4‐7 gene was much higher and cultivar independent, as three different susceptible cultivars of B. napus, namely Eurol, Columbus and ES Astrid, revealed greater symptom development during primary infection when infected with isolates complemented with the AvrLm4‐7 gene. Huang et al. (2006) have reported previously decreased aggressiveness associated with the loss of AvrLm4 specificity as a result of a single base mutation in the AvrLm4‐7 gene, but did not analyse the effect of the loss of the gene. In our study, we found decreased aggressiveness following the loss of the gene, but no significant effect of the single amino acid change. The reasons for these different findings are unclear, but we stress the very different starting material and inoculation protocols between the two studies. In particular, Huang et al. (2006) departed from two field isolates that showed, or not, AvrLm4 specificity, and used the progeny of isolates obtained following a series of backcrosses to generate batches of progeny harbouring, or not, the AvrLm4 allele in a related, but not isogenic, genetic background. In this study, genetic complementation allowed us to compare the different alleles of AvrLm4‐7 (including the virulent degenerated allele) in the same genetic background. Moreover, Huang et al. (2006) used inoculations with ascospores without wounding the cotyledons; this is a tedious procedure, more closely resembling what happens in the field than the usual cotyledon inoculation test involving the inoculation of conidia on wounded cotyledons. It is possible that the inoculation with ascospores is more sensitive and may reveal fitness differences as a result of a single amino acid change that cannot be seen using the cotyledon inoculation test; for example, the ascospore inoculation procedure may reveal a fitness deficit linked to the penetration step that is not accessible when wounding the tissues prior to inoculation. All in all, the importance of the a4A7 allele in fungal aggressiveness strongly suggests that the single mutation in the AvrLm4‐7 gene, allowing it to escape Rlm4 resistance only slightly (if at all), compromises the effector function of the gene.

Further, we demonstrated that the two selected isogenic isolates ‘a4a7’ and ‘A4A7’, differing by the presence of AvrLm4‐7, grew similarly in vitro, in accordance with the findings of Huang et al. (2006), substantiating the fact that AvrLm4‐7, as a regular effector, does not have an intrinsic function for the fungus, but rather exerts its effect on the plant during the interaction. However, it must be remembered that the expression of AvrLm4‐7, similar to that of other effector genes located in AT isochores of L. maculans, is strongly repressed during vegetative growth (Soyer et al., 2014). Once in the plant, the repression of effector gene expression was relieved, and a sharp increase in expression was observed between 3 and 6–8 dai, followed by reduced levels of expression at later stages (Parlange et al., 2009; this study). Both isolates ‘a4a7’ and ‘A4A7’ colonized the plant tissue comparably and grew asymptomatically on susceptible B. napus cotyledons until 8 dai. We assume that the effect of AvrLm4‐7 is linked to the biotrophic stage of the primary infection, as the wave of effector expression to which that of AvrLm4‐7 belongs peaked during the asymptomatic stage of leaf colonization prior to the development of leaf lesions. This is a commonly found feature for many biotrophic and hemibiotropic filamentous phytopathogens for which waves of expression of effectors associated with the biotrophic stage of infection are common (Gan et al., 2013; Lee and Rose, 2010). In other cases, however, such as that of Zymoseptoria tritici, waves of expression of putative effectors seem to be linked rather to the necrotic stage of hemibiotrophic behaviour (Rudd et al., 2015). In later stages of infection, AvrLm4‐7 had a significant impact on the development of necrotic lesions. Similarly, the presence of the AvrLm4‐AvrLm7 allele of AvrLm4‐7 increased the hyphal growth towards the petiole to reach the stem (Huang et al., 2006).

Analogy in signalling after perception of AvrLm1 and AvrLm4‐7

The recognition of AvrLm4‐7 by Rlm4 examined in this study revealed a strong induction of SA‐ and ET‐dependent signalling pathways early in the incompatible interaction. The same signalling pathways mediated defence in our previous study (Šašek et al., 2012), in which the incompatible interaction based on the recognition of AvrLm1 by Rlm1 was examined. AvrLm1 is recognized by the LepR3 resistance gene, which is an RLP (Larkan et al., 2013), and by Rlm1 (not cloned to date), but there is still debate on whether or not the two R genes may be the same (Rouxel and Balesdent, 2013). Nevertheless, one difference between the defence signalling mediated by AvrLm1 and AvrLm4‐7 recognition still exists. The perception of the AvrLm4‐7 effector by Rlm4 in B. napus cv. Pixel also slightly induced marker genes related to ABA signalling. Moreover, all compatible interactions in the two studies revealed elevated ABA levels, but we were unable to detect the up‐regulation of the ABA‐dependent marker genes NCED3 and RD26 (Šašek et al., 2012). Considering our previous finding that the induction of ABA signalling prior to infection decreased symptoms caused by a virulent isolate (Šašek et al., 2012), the role of ABA in the L. maculans–B. napus interaction remains obscure and requires further investigation.

Increased aggressiveness is linked to the suppression of signalling pathways by AvrLm4‐7

In B. napus, SA and ET signalling pathways mount an effective defence response to L. maculans (Šašek et al., 2012), as was also shown here during the course of the AvrLm4–Rlm4 interaction. Our study suggests that, in a compatible interaction, these signalling pathways may be the primary targets of the AvrLm4‐7 effector. Our results indicate that the presence of AvrLm4‐7 in infected cotyledons causes suppression of SA signalling as a whole, affecting biosynthesis, the SA level and the response of the SA‐related marker gene PR1. Moreover, ET signalling seems to be lowered in infected cotyledons when AvrLm4‐7 is present: the transcription of the ET‐responsive genes ACS2 and HEL was repressed from 6 to 10 dai.

To our knowledge, this is the first study to indicate the manipulation of SA pathways by an effector from hemibiotrophic fungi. A strategy of other (hemi)biotrophic fungi involves effectors targeting JA signalling to promote susceptibility in a host (Kazan and Lyons, 2014). For example, Fusarium oxysporum secretes the Fo‐SIX4 effector that induces JA signalling in Arabidopsis thaliana and contributes to disease development (Thatcher et al., 2009). The SA pathway is also manipulated by bacterial effectors (Kazan and Lyons, 2014). The type III effector XopD from Xanthomonas campestris pv vesicatori represses SA‐dependent gene expression and SA production in tomato plants (Kim et al., 2008). Recently, Kim et al. (2013) have reported that XopD interacts directly with the tomato ET response factor ERF4 in subnuclear foci and also suppresses ET‐stimulated immunity late in infection. In this aspect, the effect of AvrLm4‐7 on B. napus defence signalling resembles that exerted by XopD in tomato.

H2O2 as the key player in the playground?

Previously, Jindřichová et al. (2011) have shown that H2O2 accumulates during L. maculans infection. In this study, we demonstrated that the removal of H2O2 by ascorbic acid during the biotrophic stage of the infection increased the lesion development of the less aggressive isolate ‘a4a7’ lacking the AvrLm4‐7 effector. This illustrates the importance of H2O2 in the defence response to L. maculans infection. Interestingly, the presence of AvrLm4‐7 in the interaction led to a decreased accumulation of H2O2 during the later stages of the primary infection. Moreover, the transcription of rbohF, a gene coding for one of the NADPH oxidases, which are a source of ROS in the apoplast, was reduced when cotyledons were infected with the ‘A4A7’ isolate harbouring the AvrLm4‐7 effector. These results suggest a role for AvrLm4‐7 in affecting the accumulation of H2O2. Among fungal effectors known to interfere with ROS production in plants, the pep1 effector of the biotrophic pathogen Ustilago maydis has been shown to inhibit the maize peroxidase POX12 in the apoplast (Hemetsberger et al., 2012). Restricted colonization of the plant tissue by U. maydis is associated with the accumulation of ROS when pep1 is absent in the interaction. Until the effect of purified AvrLm4‐7 protein on ROS‐producing enzymes is examined, the possibility that AvrLm4‐7 also acts in the apoplast by direct inhibition of ROS production in a pep1‐like manner cannot be excluded. However, alternative explanations of the H2O2 decrease are possible. ROS have been proposed to act synergistically in a signal amplification loop with SA to drive the HR and the establishment of systemic defences (Draper, 1997; Herrera‐Vásquez et al., 2015). Possibly, the modest ROS accumulation at the infection site of cotyledons infected by isolates harbouring AvrLm4‐7 could reflect the decreased SA level and SA signalling. Nevertheless, the difference in SA level between the isolates disappeared at 10 dai, whereas the difference in ROS accumulation increased. A noticeable role in the regulation of H2O2 accumulation could also be played by ET signalling. ET can induce programmed cell death and senescence, processes that are also associated with the action of ROS (de Jong et al., 2002). Therefore, in the absence of AvrLm4‐7, the accumulation of ROS at 10 dai could be stimulated by increased ET signalling.

Taken together, we speculate that the increased aggressiveness of L. maculans isolates harbouring the functional AvrLm4‐7 allele could be caused by defects in ROS accumulation or by the complex effects (involving ROS accumulation) exerted by AvrLm4‐7 on the B. napus defence system through the suppression of SA and ET signalling. Recent findings of Blondeau et al. (2015), showing that AvrLm4‐7 is translocated into the plant cell, rather suggest that SA and/or ET signalling is the primary target of AvrLm4‐7. The identification of the interacting partner of AvrLm4‐7 upstream of these processes represents the next step in our understanding of the molecular mechanisms of this unique L. maculans effector.

Experimental Procedures

Plant and pathogen cultivation

Brassica napus plants were grown in soil mixture Atami BioGrowmix (GROWMAN PLAINS, Prague, Czech Republic) in a cycle of 14 h of daylight (150 μE/m/s, 24°C) and 10 h of night (19°C) at 70% relative humidity in a cultivation chamber (Snijders Labs, Tilburg, the Netherlands). Leptosphaeria maculans isolates Nzt‐4 AvrLm4_6 and Nzt‐4 AvrLm4_3 (Parlange et al., 2009), here referred to as ‘a4a7’ and ‘A4A7’, respectively, were cultivated on V8 juice agar medium at 26°C in the dark. Sporulation was obtained as described by Ansan‐Melayah et al. (1995). Spores were washed once with distilled water after harvesting, diluted to 108 spores/mL and stored at −20°C. For aggressiveness studies following inoculation on compatible B. napus varieties, additional isogenic isolates were used: (i) a total of seven single‐conidia isolates of Nzt‐4, lacking AvrLm4‐7; (ii) a total of eight Nzt‐4 isogenic isolates having integrated the avrLm4‐AvrLm7 allele of AvrLm4‐7 (a4A7); and (iii) a total of 11 isogenic isolates having integrated the AvrLm4‐AvrLm7 allele of AvrLm4‐7 (A4A7) (Parlange et al., 2009).

Plant inoculation and treatments

Plants were inoculated by puncture inoculation, i.e. by placing 10 μL of spore suspension (106 spores/mL) on a cotyledon that was punctured by a sterile needle.

For experiments with antioxidant agent, 10 µL of water (mock) or 5 mm ascorbic acid (Farmakon, Olomouc, Czech Republic) were placed on the puncture at 3 and 6 dai.

For aggressiveness studies, isolates were inoculated with the puncture method on the two compatible varieties, i.e. devoid of Rlm4 and Rlm7, Eurol and ES‐Astrid, using 10–12 inoculation points per isolate per plant variety. At 12 dai, cotyledons were scanned to measure lesion areas. The experiment was repeated twice. Median lesion areas were calculated and the data were subjected to non‐parametric tests (Kruskall–Wallis) to test the significance of the differences observed between the three groups of isolates.

Transformation of L. maculans

Leptosphaeria maculans isolates Nzt‐4 AvrLm4_6 and Nzt‐4 AvrLm4_3 were transformed with a pSO1 construct (Persson et al., 2009) carrying the gene for GUS from E. coli. The transformation was performed using A. tumefaciens LBA4404 according to Gardiner and Howlett (2004). GUS‐transformed isolates were assessed for GUS activity in vitro and compared with WT (recipient) isolates for phenotype changes and virulence to exclude possible alterations caused by transformation. Unless stated otherwise, all inoculations were performed using GUS‐tagged isolates.

GUS staining

Discs (diameter, 12 mm) surrounding the inoculation sites were cut from B. napus cotyledons and stained as described previously (Šašek et al., 2012). In addition, cotyledon discs were submerged in 10% (w/w) NaOH (Lachema, Brno, Czech Republic) and incubated for 1 h at 37°C with shaking at 130 rpm. Rehydrated cotyledon discs were scanned in a thin layer of water under transmission light.

MUG assay

Fluorescence measurements of GUS activity were performed according to a method described by Jefferson et al. (1987) with several modifications.

For the determination of GUS activity in mycelia of GUS‐tagged L. maculans isolates, 15 mg of mycelia filtered from the cultivation medium Gamborg B5‐MES were homogenized in a 2‐mL screw cap filled with 0.5 g of 1.3‐mm‐diameter silica beads with 500 μL of GUS extraction buffer (Jefferson et al., 1987) using a FastPrep®‐24 Instrument (MP Biomedicals, Santa Ana, CA, USA), centrifuged and 50 μL of supernatants were pipetted to a 96‐well plate maintained at 37°C. To each well, 200 μL of 1 mm MUG (Duchefa Biochemie, Haarlem, the Netherlands), preheated to 37°C, was added. Immediately, 50 μL were removed to 300 μL of STOP buffer (0.2 m Na2CO3) in a black Nunc™ F96 Microwell Polystyrene plate (Thermo Fisher Scientific, Waltham, MA, USA) for blank measurement. The mixture was incubated for 15 min at 37°C and stopped as described above. The fluorescence of 4‐methylumbelliferone (MU) formed by GUS activity was measured in a Tecan Infinite F200 plate reader (Tecan, Männedorf, Switzerland) equipped with a 360/20‐nm excitation filter and 465/25‐nm emission filter.

For the determination of GUS activity in B. napus cotyledons infected with GUS‐tagged L. maculans isolates, four discs (approximately 50 mg fresh weight) surrounding the inoculation sites were cut by Harris Uni‐Core® (diameter, 6 mm) from four plants. The extraction of GUS, the reaction with MUG and the detection of fluorescence proceeded similarly to that with L. maculans mycelium, except that the reaction time was 30 min.

Analysis of plant hormones

Plant hormones were extracted from approximately 200 mg of fresh tissue and determined as described previously (Dobrev and Kamínek, 2002; Dobrev and Vankova, 2012). The addition of appropriate internal standards to the samples preceded the analyses. Quantification was performed on an Ultimate 3000 high‐performance liquid chromatograph (Dionex, Bannockburn, IL, USA) coupled to a 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA, USA).

Gene expression analysis

Discs surrounding the inoculation sites were cut by Harris Uni‐Core® (diameter, 6 mm) from at least six plants. Homogenization, total RNA extraction, treatment with DNA‐Free kit (Ambion, Austin, TX, USA) and reverse transcription of RNA to cDNA were performed as described previously (Šašek et al., 2014). The reverse transcription‐quantitative polymerase chain reactions (RT‐qPCRs) were performed as described previously (Janda et al., 2015). For ACS2a, the annealing conditions were modified to 55°C for 20 s in all reactions. The relative expression of B. napus and L. maculans genes was calculated with efficiency correction and normalization to B. napus Actin and L. maculans ITS1 (Persson et al., 2009), respectively. Primer sets for B. napus signalling marker genes were characterized, designed and verified in our previous study, where a complete list is given (Šašek et al., 2012). Primers for LmITS1 (FJ172239) and AvrLm4‐7 were designed by Persson et al. (2009) and Parlange et al. (2009), respectively.

H2O2 detection using DAB

Detection of H2O2 in B. napus cotyledons using staining with DAB (Sigma‐Aldrich) was performed as described previously (Šašek et al., 2012).

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's website:

Figure S1 Phenotypes of aerial colonies of Leptosphaeria maculans wild‐type Nzt‐4 and β‐glucuronidase (GUS)‐tagged Nzt‐4 isolates harbouring (A4A7) or lacking (a4a7) the AvrLm4‐7 effector. The transformed and wild‐type (WT) isolates were grown on V8 juice agar medium and the photographs were taken 7 days after subculture.

Click here for additional data file. (2.4MB, jpg)

Table S1 In vitro growth rate (mm/day) of wild‐type Leptosphaeria maculans and β‐glucuronidase (GUS)‐tagged isolates

Click here for additional data file. (14.3KB, docx)

Figure S2 Aggressiveness of Leptosphaeria maculans wild‐type (recipient) Nzt‐4 and β‐glucuronidase (GUS)‐tagged Nzt‐4 isolates harbouring (A4A7) or lacking (a4a7) the AvrLm4‐7 effector following inoculation on the susceptible Brassica napus ‘Columbus’ (rlm4 and rlm7). Plants (14 days old) were inoculated by infiltration of a spore suspension of wild‐type L. maculans (WT) or GUS‐tagged (GUS) Nzt‐4 isolates (a4a7 and A4A7). The area of the necrotic lesion relative to the total cotyledon area was measured using image analysis at 10 days after inoculation. Values represent means ± standard error (SE) (n > 21). Different letters above the columns indicate statistically significant differences according to Student's t‐test (P < 0.05).

Click here for additional data file. (101.4KB, jpg)

Figure S3 β‐Glucuronidase (GUS) activity in mycelia of GUS‐tagged Leptosphaeria maculans isolates Nzt‐4 differing in the presence of the AvrLm4‐7 effector. Leptosphaeria maculans mycelium was harvested from axenic culture in Gamborg B5 by filtration at 10 days after inoculation of the medium. GUS activity was measured by a 4‐methylumbilliferone glucuronide (MUG) assay. Relative fluorescence indicates the fluorescence of 4‐methylumbelliferone formed by GUS activity. Values represent means ± standard errors from three biological replicates. No statistically significant difference was observed between ‘a4a7’ and ‘A4A7’ isolates (P > 0.05, Student's t‐test).

Click here for additional data file. (162.8KB, jpg)

Figure S4 Relative expression of rboh genes in susceptible Brassica napus ‘Columbus’ (rlm4 and rlm7) infected with Leptosphaeria maculans isolates differing in the AvrLm4‐7 effector, determined by reverse transcription‐quantitative polymerase chain reaction. Plants (14 days old) were inoculated by puncture inoculation with either water (mock) or a spore suspension of L. maculans Nzt‐4 isolates lacking (a4a7) or harbouring (A4A7) the AvrLm4‐7 effector. Expression was normalized to B. napus Actin. Values represent means ± standard error (SE) from three independent experiments. Asterisks indicate statistically significant differences between cotyledons infected with ‘a4a7’ and ‘A4A7’ isolates (*P < 0.05; one‐tailed Student's t‐test).

Click here for additional data file. (494.6KB, jpg)

Figure S5 Relative expression of salicylic acid (SA), ethylene (ET), jasmonic acid (JA) and abscisic acid (ABA) marker genes in resistant Brassica napus ‘Pixel’ (Rlm4) infected with Leptosphaeria maculans virulent (a4a7) and avirulent (A4A7) Nzt‐4 isolates, determined by reverse transcription‐quantitative polymerase chain reaction. Plants (14 days old) were inoculated by puncture inoculation with either water (mock) or a spore suspension of L. maculans Nzt‐4 isolates lacking (a4a7) or harbouring (A4A7) the AvrLm4‐7 effector. Values represent means ± standard error from three biological replicates. Asterisks indicate statistically significant differences between L. maculans isolates (*P < 0.05 and **P < 0.01; Student's t‐test).

Click here for additional data file. (3.9MB, jpg)

Acknowledgements

We thank M. Pařízková and E. L. Maseda for their excellent technical support, P. Dobrev for LC‐MS/MS analysis and M. Janda for advice and discussions. This research was supported by grant from the Czech Science Foundation (GA13‐26798S). All authors declared no conflicts of interest.

References

  1. Ansan‐Melayah, D. , Balesdent, M. , Buée, M. and Rouxel, T. (1995) Genetic characterization of AvrLm1, the first avirulence gene of Leptosphaeria maculans . Phytopathology, 85, 1525–1529. [Google Scholar]
  2. Apel, K. and Hirt, H. (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–399. [DOI] [PubMed] [Google Scholar]
  3. Balesdent, M. , Attard, A. , Kühn, M. and Rouxel, T. (2002) New avirulence genes in the phytopathogenic fungus Leptosphaeria maculans . Phytopathology, 92, 1122–1133. [DOI] [PubMed] [Google Scholar]
  4. Balesdent, M. , Barbetti, M. , Li, H. , Sivasithamparam, K. , Gout, L. and Rouxel, T. (2005) Analysis of Leptosphaeria maculans race structure in a worldwide collection of isolates. Phytopathology, 95, 1061–1071. [DOI] [PubMed] [Google Scholar]
  5. Balesdent, M.H. , Fudal, I. , Ollivier, B. , Bally, P. , Grandaubert, J. , Eber, F. , Chèvre, A.M. , Leflon, M. and Rouxel, T. (2013) The dispensable chromosome of Leptosphaeria maculans shelters an effector gene conferring avirulence towards Brassica rapa . New Phytol. 198, 887–898. [DOI] [PubMed] [Google Scholar]
  6. Balesdent, M.‐H. , Louvard, K. , Pinochet, X. and Rouxel, T. (2006) A large‐scale survey of races of Leptosphaeria maculans occurring on oilseed rape in France. Eur. J. Plant Pathol. 114, 53–65. [Google Scholar]
  7. Bell, E. , Creelman, R.A. and Mullet, J.E. (1995) A chloroplast lipoxygenase is required for wound‐induced jasmonic acid accumulation in Arabidopsis . Proc. Natl. Acad. Sci. USA, 92, 8675–8679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Block, A. and Alfano, J.R. (2011) Plant targets for Pseudomonas syringae type III effectors: virulence targets or guarded decoys? Curr. Opin. Microbiol. 14, 39–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Blondeau, K. , Blaise, F. , Graille, M. , Kale, S.D. , Linglin, J. , Ollivier, B. , Labarde, A. , Lazar, N. , Daverdin, G. , Balesdent, M.H. Choi, D.H.Y. , Tyler, B.M. , Rouxel, T. , van Tilbeurgh, H. and Fudal, I. (2015) Crystal structure of the effector AvrLm4–7 of Leptosphaeria maculans reveals insights into its translocation into plant cells and recognition by resistance proteins. Plant J. 83, 610–624. doi: 10.1111/tpj.12913. [DOI] [PubMed] [Google Scholar]
  10. Buscaill, P. and Rivas, S. (2014) Transcriptional control of plant defence responses. Curr. Opin. Plant Biol. 20, 35–46. [DOI] [PubMed] [Google Scholar]
  11. Chisholm, S.T. , Coaker, G. , Day, B. and Staskawicz, B.J. (2006) Host–microbe interactions: shaping the evolution of the plant immune response. Cell, 124, 803–814. [DOI] [PubMed] [Google Scholar]
  12. Denancé, N. , Sánchez‐Vallet, A. , Goffner, D. and Molina, A. (2013) Disease resistance or growth: the role of plant hormones in balancing immune responses and fitness costs. Front. Plant Sci. 4, 155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Deslandes, L. and Rivas, S. (2012) Catch me if you can: bacterial effectors and plant targets. Trends Plant Sci. 17, 644–655. [DOI] [PubMed] [Google Scholar]
  14. Dobrev, P.I. and Kamínek, M. (2002) Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed‐mode solid‐phase extraction. J. Chromatogr. A, 950, 21–29. [DOI] [PubMed] [Google Scholar]
  15. Dobrev, P.I. and Vankova, R. (2012) Quantification of abscisic acid, cytokinin, and auxin content in salt‐stressed plant tissues In: Plant Salt Tolerance, pp. 251–261. Springer. [DOI] [PubMed] [Google Scholar]
  16. Dodds, P.N. and Rathjen, J.P. (2010) Plant immunity: towards an integrated view of plant–pathogen interactions. Nat. Rev. Genet. 11, 539–548. [DOI] [PubMed] [Google Scholar]
  17. Doehlemann, G. and Hemetsberger, C. (2013) Apoplastic immunity and its suppression by filamentous plant pathogens. New Phytol. 198, 1001–1016. [DOI] [PubMed] [Google Scholar]
  18. Dou, D. and Zhou, J.‐M. (2012) Phytopathogen effectors subverting host immunity: different foes, similar battleground. Cell Host Microbe, 12, 484–495. [DOI] [PubMed] [Google Scholar]
  19. Draper, J. (1997) Salicylate, superoxide synthesis and cell suicide in plant defence. Trends Plant Sci. 2, 162–165. [Google Scholar]
  20. Foyer, C.H. and Noctor, G. (2013) Redox signaling in plants. Antioxid. Redox. Signal. 18, 2087–2090. [DOI] [PubMed] [Google Scholar]
  21. Fudal, I. , Ross, S. , Gout, L. , Blaise, F. , Kuhn, M. , Eckert, M. , Cattolico, L. , Bernard‐Samain, S. , Balesdent, M.H. and Rouxel, T. (2007) Heterochromatin‐like regions as ecological niches for avirulence genes in the Leptosphaeria maculans genome: map‐based cloning of AvrLm6 . Mol. Plant–Microbe Interact. 20, 459–470. [DOI] [PubMed] [Google Scholar]
  22. Gan, P. , Ikeda, K. , Irieda, H. , Narusaka, M. , O'Connell, R.J. , Narusaka, Y. , Takano, Y. , Kubo, Y. and Shirasu, K. (2013) Comparative genomic and transcriptomic analyses reveal the hemibiotrophic stage shift of Colletotrichum fungi. New Phytol. 197, 1236–1249. [DOI] [PubMed] [Google Scholar]
  23. Gardiner, D.M. and Howlett, B.J. (2004) Negative selection using thymidine kinase increases the efficiency of recovery of transformants with targeted genes in the filamentous fungus Leptosphaeria maculans . Curr. Genet. 45, 249–255. [DOI] [PubMed] [Google Scholar]
  24. Ghanbarnia, K. , Fudal, I. , Larkan, N.J. , Links, M.G. , Balesdent, M.H. , Profotova, B. , Fernando, W. , Rouxel, T. and Borhan, M.H. (2015) Rapid identification of the Leptosphaeria maculans avirulence gene AvrLm2, using an intraspecific comparative genomics approach. Mol. Plant Pathol. 16, 699–709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ghanbarnia, K. , Lydiate, D.J. , Rimmer, S.R. , Li, G. , Kutcher, H.R. , Larkan, N.J. , McVetty, P.B. and Fernando, W.D. (2012) Genetic mapping of the Leptosphaeria maculans avirulence gene corresponding to the LepR1 resistance gene of Brassica napus . Theor. Appl. Genet. 124, 505–513. [DOI] [PubMed] [Google Scholar]
  26. Giraldo, M.C. and Valent, B. (2013) Filamentous plant pathogen effectors in action. Nat. Rev. Microbiol. 11, 800–814. [DOI] [PubMed] [Google Scholar]
  27. Glazebrook, J. (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43, 205–227. [DOI] [PubMed] [Google Scholar]
  28. Gout, L. , Fudal, I. , Kuhn, M.L. , Blaise, F. , Eckert, M. , Cattolico, L. , Balesdent, M.H. and Rouxel, T. (2006) Lost in the middle of nowhere: the AvrLm1 avirulence gene of the Dothideomycete Leptosphaeria maculans . Mol. Microbiol. 60, 67–80. [DOI] [PubMed] [Google Scholar]
  29. Hammond, K.E. and Lewis, B. (1987) The establishment of systemic infection in leaves of oilseed rape by Leptosphaeria maculans . Plant Pathol. 36, 135–147. [Google Scholar]
  30. Hemetsberger, C. , Herrberger, C. , Zechmann, B. , Hillmer, M. and Doehlemann, G. (2012) The Ustilago maydis effector Pep1 suppresses plant immunity by inhibition of host peroxidase activity. PLoS Pathog. 8, e1002684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Herrera‐Vásquez, A. , Salinas, P. and Holuigue, L. (2015) Salicylic acid and reactive oxygen species interplay in the transcriptional control of defense genes expression. Front. Plant Sci. 6, 1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hogenhout, S.A. , Van der Hoorn, R.A. , Terauchi, R. and Kamoun, S. (2009) Emerging concepts in effector biology of plant‐associated organisms. Mol. Plant–Microbe Interact. 22, 115–122. [DOI] [PubMed] [Google Scholar]
  33. Howlett, B.J. (2004) Current knowledge of the interaction between Brassica napus and Leptosphaeria maculans . Can. J. Plant Pathol. 26, 245–252. [Google Scholar]
  34. Huang, Y.‐J. , Balesdent, M.‐H. , Li, Z.‐Q. , Evans, N. , Rouxel, T. and Fitt, B.D. (2010) Fitness cost of virulence differs between the AvrLm1 and AvrLm4 loci in Leptosphaeria maculans (phoma stem canker of oilseed rape). Eur. J. Plant Pathol. 126, 279–291. [Google Scholar]
  35. Huang, Y.‐J. , Li, Z.‐Q. , Evans, N. , Rouxel, T. , Fitt, B.D. and Balesdent, M.‐H. (2006) Fitness cost associated with loss of the AvrLm4 avirulence function in Leptosphaeria maculans (phoma stem canker of oilseed rape). Eur. J. Plant Pathol. 114, 77–89. [Google Scholar]
  36. Janda, M. , Šašek, V. , Chmelařová, H. , Andrejch, J. , Nováková, M. , Hajšlová, J. , Burketová, L. and Valentová, O. (2015) Phospholipase D affects translocation of NPR1 to the nucleus in Arabidopsis thaliana . Front. Plant Sci. 6, 59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Jefferson, R.A. , Kavanagh, T.A. and Bevan, M.W. (1987) GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Jindřichová, B. , Fodor, J. , Šindelářová, M. , Burketová, L. and Valentová, O. (2011) Role of hydrogen peroxide and antioxidant enzymes in the interaction between a hemibiotrophic fungal pathogen, Leptosphaeria maculans, and oilseed rape. Environ. Exp. Bot. 72, 149–156. [Google Scholar]
  39. Jones, J.D. and Dangl, J.L. (2006) The plant immune system. Nature, 444, 323–329. [DOI] [PubMed] [Google Scholar]
  40. de Jong, A.J. , Yakimova, E.T. , Kapchina, V.M. and Woltering, E.J. (2002) A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells. Planta, 214, 537–545. [DOI] [PubMed] [Google Scholar]
  41. Kazan, K. and Lyons, R. (2014) Intervention of phytohormone pathways by pathogen effectors. Plant Cell, 26, 2285–2309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Kim, J.‐G. , Stork, W. and Mudgett, M.B. (2013) Xanthomonas type III effector XopD desumoylates tomato transcription factor SlERF4 to suppress ethylene responses and promote pathogen growth. Cell Host Microbe, 13, 143–154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Kim, J.‐G. , Taylor, K.W. , Hotson, A. , Keegan, M. , Schmelz, E.A. and Mudgett, M.B. (2008) XopD SUMO protease affects host transcription, promotes pathogen growth, and delays symptom development in Xanthomonas‐infected tomato leaves. Plant Cell, 20, 1915–1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Larkan, N. , Lydiate, D. , Parkin, I. , Nelson, M. , Epp, D. , Cowling, W. , Rimmer, S. and Borhan, M. (2013) The Brassica napus blackleg resistance gene LepR3 encodes a receptor‐like protein triggered by the Leptosphaeria maculans effector AVRLM1 . New Phytol. 197, 595–605. [DOI] [PubMed] [Google Scholar]
  45. Lee, S.‐J. and Rose, J.K. (2010) Mediation of the transition from biotrophy to necrotrophy in hemibiotrophic plant pathogens by secreted effector proteins. Plant Signal. Behav. 5, 769–772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Parlange, F. , Daverdin, G. , Fudal, I. , Kuhn, M.L. , Balesdent, M.H. , Blaise, F. , Grezes‐Besset, B. and Rouxel, T. (2009) Leptosphaeria maculans avirulence gene AvrLm4‐7 confers a dual recognition specificity by the Rlm4 and Rlm7 resistance genes of oilseed rape, and circumvents Rlm4‐mediated recognition through a single amino acid change. Mol. Microbiol. 71, 851–863. [DOI] [PubMed] [Google Scholar]
  47. Persson, M. , Staal, J. , Oide, S. and Dixelius, C. (2009) Layers of defense responses to Leptosphaeria maculans below the RLM1‐ and camalexin‐dependent resistances. New Phytol. 182, 470–482. [DOI] [PubMed] [Google Scholar]
  48. Pieterse, C.M. , Leon‐Reyes, A. , Van der Ent, S. and Van Wees, S.C. (2009) Networking by small‐molecule hormones in plant immunity. Nat. Chem. Biol. 5, 308–316. [DOI] [PubMed] [Google Scholar]
  49. Pieterse, C.M. , Van der Does, D. , Zamioudis, C. , Leon‐Reyes, A. and Van Wees, S.C. (2012) Hormonal modulation of plant immunity. Annu. Rev. Cell Dev. Biol. 28, 489–521. [DOI] [PubMed] [Google Scholar]
  50. Rafiqi, M. , Ellis, J.G. , Ludowici, V.A. , Hardham, A.R. and Dodds, P.N. (2012) Challenges and progress towards understanding the role of effectors in plant–fungal interactions. Curr. Opin. Plant Biol. 15, 477–482. [DOI] [PubMed] [Google Scholar]
  51. Robert‐Seilaniantz, A. , Grant, M. and Jones, J.D. (2011) Hormone crosstalk in plant disease and defense: more than just jasmonate–salicylate antagonism. Annu. Rev. Phytopathol. 49, 317–343. [DOI] [PubMed] [Google Scholar]
  52. Rouxel, T. and Balesdent, M.H. (2013) From model to crop plant–pathogen interactions: cloning of the first resistance gene to Leptosphaeria maculans in Brassica napus . New Phytol. 197, 356–358. [DOI] [PubMed] [Google Scholar]
  53. Rouxel, T. and de Wit, P.J. (2012) Dothideomycete effectors facilitating biotrophic and necrotrophic lifestyles In: Effectors in Plant–Microbe Interactions (Francis M. and Sophien K., eds). John Wiley & Sons. [Google Scholar]
  54. Rudd, J. , Kanyuka, K. , Hassani‐Pak, K. , Derbyshire, M. , Devonshire, J. , Saqi, M. , Desai, N. , Powers, S. , Hooper, J. , Ambroso, L. , Bharti, A. , Farmer, A. , Hammond‐Kosack, K. , Dietrich, R. and Courbot, M. (2015) Transcriptome and metabolite profiling the infection cycle of Zymoseptoria tritici on wheat (Triticum aestivum) reveals a biphasic interaction with plant immunity involving differential pathogen chromosomal contributions, and a variation on the hemibiotrophic lifestyle definition. Plant Physiol. 167, 1158–1185. DOI: 10.1104/pp.114.255927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Šašek, V. , Janda, M. , Delage, E. , Puyaubert, J. , Guivarc'h, A. , López Maseda, E. , Dobrev, P.I. , Caius, J. , Bóka, K. and Valentová, O. (2014) Constitutive salicylic acid accumulation in pi4kIIIβ1β2 Arabidopsis plants stunts rosette but not root growth. New Phytol. 203, 805–816. [DOI] [PubMed] [Google Scholar]
  56. Šašek, V. , Nováková, M. , Jindrichová, B. , Bóka, K. , Valentová, O. and Burketová, L. (2012) Recognition of avirulence gene AvrLm1 from hemibiotrophic ascomycete Leptosphaeria maculans triggers salicylic acid and ethylene signaling in Brassica napus . Mol. Plant–Microbe Interact. 25, 1238–1250. [DOI] [PubMed] [Google Scholar]
  57. Schmidt, S.M. and Panstruga, R. (2011) Pathogenomics of fungal plant parasites: what have we learnt about pathogenesis? Curr. Opin. Plant Biol. 14, 392–399. [DOI] [PubMed] [Google Scholar]
  58. Soyer, J.L. , El Ghalid, M. , Glaser, N. , Ollivier, B. , Linglin, J. , Grandaubert, J. , Balesdent, M.‐H. , Connolly, L.R. , Freitag, M. , Rouxel, T. and Fudal, I. (2014) Epigenetic control of effector gene expression in the plant pathogenic fungus Leptosphaeria maculans . PLoS Genet. 10, e1004227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Thatcher, L.F. , Manners, J.M. and Kazan, K. (2009) Fusarium oxysporum hijacks COI1‐mediated jasmonate signaling to promote disease development in Arabidopsis . Plant J. 58, 927–939. [DOI] [PubMed] [Google Scholar]
  60. Tsuda, K. and Katagiri, F. (2010) Comparing signaling mechanisms engaged in pattern‐triggered and effector‐triggered immunity. Curr. Opin. Plant Biol. 13, 459–465. [DOI] [PubMed] [Google Scholar]
  61. Van de Wouw, A.P. , Lowe, R.G. , Elliott, C.E. , Dubois, D.J. and Howlett, B.J. (2014) An avirulence gene, AvrLmJ1, from the blackleg fungus, Leptosphaeria maculans, confers avirulence to Brassica juncea cultivars. Mol. Plant Pathol. 15, 523–530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. West, J. , Kharbanda, P. , Barbetti, M. and Fitt, B.D. (2001) Epidemiology and management of Leptosphaeria maculans (phoma stem canker) on oilseed rape in Australia, Canada and Europe. Plant Pathol. 50, 10–27. [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional Supporting Information may be found in the online version of this article at the publisher's website:

Figure S1 Phenotypes of aerial colonies of Leptosphaeria maculans wild‐type Nzt‐4 and β‐glucuronidase (GUS)‐tagged Nzt‐4 isolates harbouring (A4A7) or lacking (a4a7) the AvrLm4‐7 effector. The transformed and wild‐type (WT) isolates were grown on V8 juice agar medium and the photographs were taken 7 days after subculture.

Click here for additional data file. (2.4MB, jpg)

Table S1 In vitro growth rate (mm/day) of wild‐type Leptosphaeria maculans and β‐glucuronidase (GUS)‐tagged isolates

Click here for additional data file. (14.3KB, docx)

Figure S2 Aggressiveness of Leptosphaeria maculans wild‐type (recipient) Nzt‐4 and β‐glucuronidase (GUS)‐tagged Nzt‐4 isolates harbouring (A4A7) or lacking (a4a7) the AvrLm4‐7 effector following inoculation on the susceptible Brassica napus ‘Columbus’ (rlm4 and rlm7). Plants (14 days old) were inoculated by infiltration of a spore suspension of wild‐type L. maculans (WT) or GUS‐tagged (GUS) Nzt‐4 isolates (a4a7 and A4A7). The area of the necrotic lesion relative to the total cotyledon area was measured using image analysis at 10 days after inoculation. Values represent means ± standard error (SE) (n > 21). Different letters above the columns indicate statistically significant differences according to Student's t‐test (P < 0.05).

Click here for additional data file. (101.4KB, jpg)

Figure S3 β‐Glucuronidase (GUS) activity in mycelia of GUS‐tagged Leptosphaeria maculans isolates Nzt‐4 differing in the presence of the AvrLm4‐7 effector. Leptosphaeria maculans mycelium was harvested from axenic culture in Gamborg B5 by filtration at 10 days after inoculation of the medium. GUS activity was measured by a 4‐methylumbilliferone glucuronide (MUG) assay. Relative fluorescence indicates the fluorescence of 4‐methylumbelliferone formed by GUS activity. Values represent means ± standard errors from three biological replicates. No statistically significant difference was observed between ‘a4a7’ and ‘A4A7’ isolates (P > 0.05, Student's t‐test).

Click here for additional data file. (162.8KB, jpg)

Figure S4 Relative expression of rboh genes in susceptible Brassica napus ‘Columbus’ (rlm4 and rlm7) infected with Leptosphaeria maculans isolates differing in the AvrLm4‐7 effector, determined by reverse transcription‐quantitative polymerase chain reaction. Plants (14 days old) were inoculated by puncture inoculation with either water (mock) or a spore suspension of L. maculans Nzt‐4 isolates lacking (a4a7) or harbouring (A4A7) the AvrLm4‐7 effector. Expression was normalized to B. napus Actin. Values represent means ± standard error (SE) from three independent experiments. Asterisks indicate statistically significant differences between cotyledons infected with ‘a4a7’ and ‘A4A7’ isolates (*P < 0.05; one‐tailed Student's t‐test).

Click here for additional data file. (494.6KB, jpg)

Figure S5 Relative expression of salicylic acid (SA), ethylene (ET), jasmonic acid (JA) and abscisic acid (ABA) marker genes in resistant Brassica napus ‘Pixel’ (Rlm4) infected with Leptosphaeria maculans virulent (a4a7) and avirulent (A4A7) Nzt‐4 isolates, determined by reverse transcription‐quantitative polymerase chain reaction. Plants (14 days old) were inoculated by puncture inoculation with either water (mock) or a spore suspension of L. maculans Nzt‐4 isolates lacking (a4a7) or harbouring (A4A7) the AvrLm4‐7 effector. Values represent means ± standard error from three biological replicates. Asterisks indicate statistically significant differences between L. maculans isolates (*P < 0.05 and **P < 0.01; Student's t‐test).

Click here for additional data file. (3.9MB, jpg)

Articles from Molecular Plant Pathology are provided here courtesy of Wiley

RESOURCES