Necrotrophic Effectors SnTox from the Stagonospora nodorum (Berk.) Manipulate the Redox Metabolism of the Host Plant to Hijack Its Early Defense Response ^†

Abstract

Reactive oxygen species (ROS) play a central role in plant immune responses. The most important virulence factors of Stagonospora nodorum are multiple fungal necrotrophic effectors (NEs) (SnTox) that affect the redox status and cause necrosis and/or chlorosis in wheat lines possessing dominant susceptibility genes (Snn). However, the effect of NEs on ROS generation in the early stages of infection has not been studied. In this study, our aim was to research the effect of S. nodorum effectors SnToxA, SnTox1, and SnTox3 on development of disease symptoms, the generation of hydrogen peroxide, and the enzyme activity of the redox metabolism in the early stage of infection in various wheat genotypes infected with isolates of S. nodorum (Sn4VD, SnB, and Sn9MN) carrying a set of various NE genes. Our results indicate that all three NEs of SnToxA, SnTox1, and SnTox3 play an important role in the inhibition of ROS in the initial stage of infection. Tsn1–SnToxA and Snn3–SnTox3 inhibited ROS production in wheat by affecting NADPH oxidases, peroxidases, superoxide dismutase, and catalase. Snn1–SnTox1 inhibited the production of ROS in wheat by mainly affecting peroxidase. NEs suppress ROS production only in the presence of the susceptibility genes Tsn1, Snn1, Snn3.

1. Introduction

Plants have developed several levels of defense against microbial pathogens, which have been described in the «zig-zag» model of the plant immune system [1]. The first line of defense in plants is through the perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), which leads to the development of basal immunity, known as PAMP-triggered immunity (PTI) [1]. However, pathogens can suppress PTI using effectors, which leads to the development of effector-triggered susceptibility (ETS). The second line of defense in plants is called effector-triggered immunity (ETI) and develops when an effector is recognized by products of effector-specific resistance genes [1]. The development of PTI and ETI induces similar responses in plants: Both lines of defense can be separated in time and space, but both are closely related to the production of reactive oxygen species (ROS) [2]. PAMP-triggered ROS perform two functions. Apoplastic ROS are cytotoxic to kill pathogens, acting as signaling molecules to activate plant defenses [2]. The development of ETI leads to an oxidative burst, as well as the formation of necrosis and the restriction of the biotrophic pathogens growth [1].

Until recently, necrotrophic pathogens were considered universal and non-host-specific. However, recent studies have revealed that approximately 20 necrotrophic fungal species in the Dothideomycete class produce effector proteins also known as host-selective toxins (HSTs) or necrotrophic effectors (NEs) that interact either directly or indirectly with dominant sensitivity/susceptibility gene products to induce disease [3]. When a specific NE is recognized by the corresponding host gene, a host response follows that allows necrotrophs to penetrate, grow, and sporulate. Additional studies of the host response’s recognition of NE have discovered hallmarks of an ETI response [4]. Thus, NEs use the host’s ETI pathway to develop sensitivity, resulting in NE-triggered susceptibility (NETS) [3,4]. The classical pathogens that produce HST are pathogens of the genera Cochliobolus, Alternaria, Pyrenophora, and Stagonospora [3].

The pathogenic fungus Stagonospora nodorum Berk. (syn. Septoria, Parastagonospora; teleo, Phaeosphaeria) is the causative agent of Septoria nodorum blotch (SNB) of wheat. The most important factors of Stagonospora nodorum virulence include numerous fungal necrotrophic effectors (NEs) encoded by SnTox genes [5,6]. To date, three effector genes have been identified in the genome of S. nodorum (SnToxA, SnTox1, and SnTox3) [5]. SnToxA causes necrosis of wheat varieties that contain the Tsn1 gene [7]. Sensitivity to SnTox1 is defined by the Snn1 gene located on wheat chromosome 1BS [5,8]. Sensitivity to SnTox3 is conferred by Snn3-B1 and Snn3-D1 located on wheat chromosomes 5BS and 5DS, respectively [5].

Effectors SnToxA, SnTox1, and SnTox3 are considered the main ones in the pathogen S. nodorum and are quite widespread among strains and isolates [8]. Effectors SnToxA, SnTox1, and SnTox3 cause necrosis and chlorosis in susceptible wheat genotypes; it follows from this that they have an impact on the redox metabolism of the host plant. Unfortunately, the whole signal transduction pathway from recognition of the effector by the receptor to necrosis development is unknown [4,6]. However, the role of NEs SnToxA, SnTox1, and SnTox3 in the suppression of PTI and the development of ETS is expected [6]. It is believed that suppression of a primary ROS burst during PTI by effector proteins as virulence factors is a common adaptation of many virulent pathogens [2]. A lot of data have been accumulated on the effectors of various pathogens that suppress an ROS burst during PTI [2]. However, there are no such data on NEs SnToxA, SnTox1, and SnTox3.

In this study, our aim was to research the effect of S. nodorum effectors SnToxA, SnTox1, and SnTox3 on the development of disease symptoms, the generation of hydrogen peroxide, the expression of oxidoreductase genes, and the activity of their protein products at an early stage of infection in various cultivars of soft spring wheat infected with isolates of S nodorum—Sn4VD, SnB, and Sn9MN, carrying a various set of NE genes. Herein, we evaluated the roles of each compatible interaction (Tsn1–SnToxA, Snn3–SnTox3, and Snn1–SnTox1) in suppressing ROS production in the initial stage of infection.

2. Material and Methods

2.1. Research Objects and Seedling Resistance

The objects of this study were three cultivars of soft spring wheat (Triticum aestivum L.) contrasting in resistance to S. nodorum Berk.: Omskaya 35 (Om35), Kazakhstanskaya 10 (Kaz10), and Zhnitsa. Plants were hydroponically grown on a 10% solution of Hoagland–Arnon nutrient medium in a KS-200 SPU growth chamber (Russia) at 20/24 °C (night/day) at an irradiance of 146 W/m² FAR (Osram lamps L 36W/77) and a 16 h photoperiod for seven days. The pathogen objects were three isolates of the fungus S. nodorum: SnB, Sn4VD, and Sn9MN.

The evaluation of seedling resistance of the cultivars was carried out by the lawns method, as previously described [9]. The development of SNB symptoms on wheat leaves was registered on the sixth day after infection with S. nodorum isolates using an SP-800UZ Image Stabilization camera (Olympus, Bekasi, Indonesia); the damage area was measured using the ImageJ 1,44 computer program (rsbweb.nih.gov/ij/download.html) and expressed as a percentage of the total leaf area. In addition, the degree of lesion was also evaluated according to the International scale based on the percentage of the damage area of plant organs: RR (0%–5%)—varieties with very high and high resistance; R (up to 10%–15%)—resistant varieties; M (up to 25%)—slightly susceptible varieties; S (up to 40%–65%)—susceptible varieties; SS (over 65%)—varieties with very high and high susceptibility. Detriment was also scored on a qualitative scale rating the type of lesion from 0 to 5, as described by Liu et al. (2004) [10].

2.2. Isolation of DNA and RNA and Performing the Polymerase Chain Reaction (PCR) and Biochemical Parameters

DNA was isolated from wheat seedlings and 7-day fungus cultures by the phenol-detergent method [11]. Identification of the NE genes SnToxA, SnTox1, and SnTox3 in S. nodorum isolates Sn4VD, SnB, and Sn9MN was performed by PCR with gene-specific primers SnToxA (JX997419), SnTox1 (JX997402) [4], and SnTox3 (FJ823644) [12]. Primers for the housekeeping gene tubulin (S56922) [11] were used as an internal control for the presence of fungal DNA. PCR with the cDNA template was performed in a TP4-PCR-01-Tertsik-type PCR machine (DNK-Tekhnologia, Moscow, Russia).

The dominant allele of the Tsn1 gene was identified in the wheat cultivars by PCR with primers for the Xfcp623 microsatellite marker on the internal region of the Tsn1 gene [7]. The dominant allele of the Snn1 gene was identified with primers for the intragenic marker Snn1 (KP085710) [4]. The allelic state of the Snn3-B1 locus was determined by PCR with primers for the Xcfd20 and Xgwm234 microsatellite markers [11]. In all cases, the PCR products were resolved in 7% PAAG stained with ethidium bromide using the Gene Ruler DNA Ladder (Fermentas). The gels were photographed using a documenting system of GelDoc XR (BioRad Laboratories, Hercules, CA, USA).

Total RNA was isolated from the control and infected with S. nodorum isolates (Sn4VD, SnB, and Sn9MN) of wheat leaves of three varieties Om35, Kaz10, and Zhnitsa, fixed in liquid nitrogen with Trizol reagent according to manufacturer’s (Merck KGaA, Sigma-Aldrich, Darmstadt, Germany) recommendations. Analysis of the gene expression for the NEs (SnToxA, SnTox1, and SnTox3) in different isolates of S. nodorum during inoculation of the wheat plants and the expression of the genes for the wheat oxidoreductases (TaRbohF, TaSod, and TaPrx) was performed by quantitative real-time polymerase chain reaction (RT-PCR) with an iCycler iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and SYBR Green I intercalating dye (Sintol, Russia). Real-time PCR was performed using primers for genes encoding NADPH oxidase (TaRbohF, AY561153), superoxide dismutase (SOD) (TaSod, JX398977.1), anionic peroxidase (TaPrx, TC151917) [11], SnToxA (JX997419), SnTox1 (JX997402) [4], and SnTox3 (FJ823644) [12]. To normalize the expression results of the studied genes, primers were used for the tubulin gene of the fungus S. nodorum (S56922) [11] and for the gene of constitutively expressed protein, a wheat ribonuclease inhibitor (RNase L inhibitor-like) RLI (AY059462) [11]. Changes in the expression of the gene of interest were estimated by the level of normalized gene expression calculated with iCycler iQ5 Real-Time Detection System Software (Bio-Rad, USA).

The content of hydrogen peroxide (H₂O₂) and the enzyme activities of peroxidase (POX) and catalase in the wheat leaves of the three varieties Om35, Kaz10, and Zhnitsa were measured 24 h after inoculation with S. nodorum isolates, as previously described [9].

2.3. Statistical Analysis

All experiments were carried out three times with three biological and three analytical replications (n = 9 in total), except for the measurements of the infected area, which were performed in no less than 30 biological replications (n = 90 altogether). The figures and table report the mean values and their confidence intervals calculated from their standard errors. Statistical analysis consisted of Student’s t-test at a significance level of p ≤ 0.05.

3. Results

3.1. The Role of Compatible Interactions in Causing Disease

The Tsn1–SnToxA, Snn3–SnTox3, and Snn1–SnTox1 interactions were investigated in this study. Alleles of the susceptibility genes Snn1 and Tsn1 and the locus Snn3-B1 were identified by PCR in three varieties of soft spring wheat (Figure 1). Thus, Om 35 (tsn1/Snn1/snn3) was sensitive to NE of SnTox1. Variety Kaz10 (tsn1/Snn1/Snn3) was sensitive to NE of SnTox1 and SnTox3. Zhnitsa (Tsn1/Snn1/Snn3) was sensitive to all three effectors (Figure 1).

The three S. nodorum isolates Sn4VD, SnB, and Sn9MN were tested for the presence of the three NE genes SnToxA, SnTox3, and SnTox1 by PCR (Figure 2a). The isolates Sn4VD and Sn9MN contained the three NE genes SnToxA, SnTox3, and SnTox1 in their genome (Figure 2a). The isolate SnB contained only the two NE genes SnToxA and SnTox3 in its genome (Figure 2a).

Investigation of the transcriptional activity of NE genes in the three S. nodorum isolates SnB, Sn4VD, and Sn9MN during inoculation of the susceptible genotype of Zhnitsa (Tsn1/Snn1/Snn3) revealed differences in expression (Figure 2b). In isolate Sn4VD, expression of all three NE genes in planta was not detected (Figure 2b). The SnB isolate expressed only two NE genes, SnToxA and SnTox3 (Figure 2b). In isolate Sn9MN, expression of the three NE genes SnTox1, SnToxA, and SnTox3 was found (Figure 2b).

Thus, incompatible interactions or resistance were observed when cultivars were inoculated with the Sn4VD isolate, because the isolate did not produce NEs (

Table 1. Reaction to the damage of wheat varieties infected with S. nodorum SnB, Sn9MN, and Sn4VD isolates.

Variety	Reaction to Damage	Isolate S. nodorum
		Sn4VD (toxa/tox3/tox1)	SnB (ToxA/Tox3/tox1)	Sn9MN (ToxA/Tox3/Tox1)
Omskaya 35 (tsn1/snn3/Snn1)	Necrosis, %	0.05 ± 0.002	5 ± 0.7	23 ± 2
	Chlorosis, %	0	3 ± 0.5	0
	Damage zone, %	0.05 ± 0.001	8 ± 1	23 ± 2
	Damage score	1	2	3
	Resistance group *	RR	R	M
Kazahstanskaya 10 (tsn1/Snn3/Snn1)	Necrosis, %	0.05 ± 0.002	16 ± 2	31 ± 3
	Chlorosis, %	0	35 ± 3	25 ± 2
	Damage zone, %	0.05 ± 0.001	51 ± 5	56 ± 4
	Damage score	1	4	4
	Resistance group *	RR	S	S
Zhnitsa (Tsn1/Snn3/Snn1)	Necrosis, %	1 ± 0.1	18 ± 2	27 ± 2
	Chlorosis, %	2 ± 0.2	55 ± 4	57 ± 5
	Damage zone, %	3 ± 0.3	73 ± 6	84 ± 6
	Damage score	1	5	5
	Resistance group *	RR	SS	SS

Note: * RR (0–5%)—varieties with very high and high resistance; R (up to 10%–15%)—resistant varieties; M (up to 25%)—slightly susceptible varieties; S (up to 40%–65%)—susceptible varieties; SS (up to 90–100%)—varieties with very high and high susceptibility.

). This incompatible interaction was observed in the combination cultivar/isolate Om35/SnB (tsn1/snn3/Snn1—ToxA/Tox3/tox1). Minimal damage areas were observed in all incompatible interactions in variants of Om35/Sn4VD, Kaz10/Sn4VD, Zh/Sn4VD, and Om35/SnB (Table 1). This can be explained by hypersensitivity reactions or by the influence of other genes or effectors with minor effects [5]. The Snn1–SnTox1 interaction and the effect of SnTox1 on the development of the disease were observed in the combination of Om35/Sn9MN (the combination of genotypes is presented in Table 1). The Snn1–SnTox1 interaction led to the formation of necrosis, occupying almost 25% of the leaf area, but did not cause the formation of chlorosis (Table 1). The Snn3–SnTox3 interaction and the effect of SnTox3 on disease progression were observed in the combination Kaz10/SnB (Table 1). The Snn3–SnTox3 interaction led to the formation of necrosis and significant chlorosis, together occupying more than 50% of the leaf area (Table 1). The Snn3–SnTox3 and Snn1–SnTox1 interactions were observed in the combination Kaz10/Sn9MN (Table 1). However, we did not find the expected significant increase in the total lesion, but there was an increase in necrotic spots compared to the Kaz10/SnB variant (Snn3–SnTox3) (Table 1). This reaction could be explained by the fact that the Snn1–SnTox1 interaction is epistatic to Snn3–SnTox3 [5]. Tsn1–SnToxA and Snn3–SnTox3 interactions were observed in the combination Zh/SnB (Table 1). These interactions led to a significant increase in damage areas compared to the Kaz10/SnB variant (Snn3–SnTox3), and chlorosis and necrosis occupied more than 70% of the leaf area (Table 1). Tsn1–SnToxA, Snn3–SnTox3, and Snn1–SnTox1 interactions were observed in the combination Zh/Sn9MN (Table 1). In this variant, an increase in affected areas was found in comparison to the Zh/SnB variant (Snn3–SnTox3 and Tsn1–SnToxA), mainly due to an increase in the formation of necrosis (Table 1). Thus, according to our results, NE SnTox1 caused the development of necrosis, NE SnTox3 stimulated the development of both necrosis and chlorosis, and NE SnToxA induced the development of extensive chlorosis in susceptible wheat plants, which is consistent with the literature [5,8,12].

3.2. The Role of Compatible Interactions in the Suppression of ROS Production

It is known that one of the earliest responses to the invasion of pathogens is the local generation of ROS—an oxidative “burst,” which plays an important role in the development of systemic resistance and is controlled by enzymes of the pro-/antioxidant system [13]. NADPH oxidases, also known as respiratory burst oxidase homologs (RBOHs), are responsible for the production of ROS in plants during pathogen infection [14]. In addition, apoplastic peroxidases play an important role in the production of ROS in PTI [14].

Earlier, we found that the resistance of T. aestivum to the pathogen S. nodorum was determined by the intensive generation of ROS, mainly H₂O₂, due to an increase in peroxidase activity and a decrease or absence of an increase in catalase activity in the initial stage of infection [9]. It is known that peroxidases are involved in both the generation processes and the processes of ROS utilization in the apoplast, and catalase activates the decomposition reaction of H₂O₂ molecules [13]. In this study, a two- and threefold increase in the H₂O₂ content was observed in variants with incompatible interactions (Om35/Sn4VD, Kaz10/Sn4VD, Zh/Sn4VD, and Om35/SnB) (Figure 3a). The increase in H₂O₂ production in wheat upon inoculation with the avirulent isolate Sn4VD occurred mainly due to high activity of peroxidase (Figure 3c) and the high transcript level of the gene encoding anionic peroxidase (TaPrx) (Figure 3f), as well as due to low activity of catalase (Figure 3e) in the early stage of infection (24 h). Regarding the genes TaRbohF and TaSod, a small increase in the transcript level was observed in the early stage of infection upon inoculation with the avirulent isolate Sn4VD (Figure 3b,d). When the resistant variety Om35 was inoculated with the SnB virulent isolate and an incompatible interaction developed, a large increase in the transcript level of the genes TaRbohF and TaSod was observed (Figure 3b,d). Thus, a typical reaction of resistance to S. nodorum was observed in all incompatible interactions, which led to the development of an oxidative burst and restriction of pathogen growth.

All compatible interactions inhibited H₂O₂ production in susceptible wheat varieties in the early stage of infection compared to incompatible interactions (Figure 3a). The Snn1–SnTox1 (Om35/Sn9MN) and Snn3–SnTox3 (Kaz10/SnB) interactions reduced H₂O₂ production by two times compared to the incompatible interaction in variant Om35/SnB (Figure 3a). The presence of two or three compatible interactions in wheat plants led to an even greater decrease in H₂O₂ production (Figure 3a). All three interactions (Tsn1–SnToxA, Snn3–SnTox3, and Snn1–SnTox1), individually or in combination, led to a decrease in peroxidase activity and a decline of the transcript level of the genes TaPrx and TaSod in wheat in the early stage of infection (Figure 3c,d,f).

It should be noted that the Snn1–SnTox1 interaction did not lead to a decrease in the transcript level of the gene TaRbohF or an increase in the activity of catalase compared to the incompatible interaction in the variant Om35/SnB, unlike the other interactions (Tsn1–SnToxA and Snn3–SnTox3) (Figure 3b,e). The transcript level of the gene TaRbohF and the activity of catalase in the variants Om35/SnB and Om35/Sn9MN were found to be similar. Our results show that in the Kaz10/Sn9MN and Zh/Sn9MN variants, the Snn1–SnTox1 interaction did not affect the transcript level of the gene TaRbohF (Figure 3b). We found similar results in the changes in catalase activity (Figure 3e). The Snn3–SnTox3 or Tsn1–SnToxA interactions increased the catalase activity by 1.5 times in the Kaz10/SnB and Zh/SnB variants, respectively (Figure 3e). In the Kaz10/Sn9MN and Zh/Sn9MN variants, the Snn1–SnTox1 interaction did not affect the catalase activity (Figure 3e).

4. Discussion

The suppression of the host defense response by the effectors of S. nodorum—SnToxA, SnTox1, and SnTox3—was carried out due to a decrease in the generation of H₂O₂ in the leaves of susceptible varieties in the initial stage of infection. The Tsn1–SnToxA and Snn3–SnTox3 interactions inhibited H₂O₂ production in wheat in the early stage of infection by affecting four enzymes of the redox metabolism: NADPH oxidases, peroxidases, superoxide dismutase, and catalase. The Snn1–SnTox1 interaction inhibited the production of H₂O₂ in wheat by mainly affecting the peroxidase activity and the transcript level of the gene encoding anionic peroxidase (TaPrx).

5. Conclusions

The results herein indicate that all three NEs of S. nodorum—SnToxA, SnTox1, and SnTox3—played an important role in the inhibition of ROS during PTI in the initial stage of infection, despite the fact that in the late stage of infection, all three NEs caused the formation of necrosis and chlorosis on the leaves of wheat of the susceptible genotypes. Our results also showed that inhibition of ROS in PTI by NEs occurred only in the presence of the susceptibility genes Tsn1, Snn1, and Snn3, which is also supported by other studies [6]. Therefore, our results suggest that effector–host sensitivity gene interactions have the ability not only to hijack the host’s own ETI pathway, but also to suppress the host’s own PTI pathway, resulting in NE-triggered susceptibility (NETS).