Effect of the host-specific toxin SnTOX3 from Stagonospora nodorum on ethylene signaling pathway regulation and redox-state in common wheat

The fungus Stagonospora nodorum Berk. 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. They interact with the matching products of host susceptibility genes ( Snn ). SnTox- Snn interactions are mirror images of classical gene-for-gene interactions and lead to the development of disease. We have studied the SnTox3- Snn3 interaction, resulting in the development of infection on leaves and formation of extensive lesions. The mechanism of SnTox3 action is likely to be linked to the regulation of redox metabolism and the influence on ethylene synthesis in the wheat plants, although the molecular mechanisms are not fully unveiled. To characterize the SnTox3- Snn3 interaction, we used S. nodorum isolates differing in the expression of the NEs genes SnTox3 (SnB (Tox3 + ), Sn4VD (Tox3 – )) and two soft spring wheat ( Triticum aestivum L.) cultivars, contrasting in resistance to the SNB agent and differing in the allelic composition of the susceptibility locus Snn3-B1 : Kazakhstanskaya 10 (susceptible) and Omskaya 35 (resistant). We carried out a comparative assessment of the transcriptional activity patterns of genes responsible for ethylene biosynthesis ( TaACS1 , TaACО ) and signaling pathway ( TaEIL1 , TaERF1 ) by real-time PCR and estimated the redox state of wheat plants infected with different isolates of S. nodorum by spectrometry. The induction of ethylene biosynthesis and signaling has been shown to result from gene-for-gene interaction between Snn3-B1 and SnTox3. The results of plant redox status estimation showed that ethylene inhibited accumulation of hydrogen peroxide in SnTox3-sensitive genotypes by regulating the operation of various pro-/antioxidant enzymes at the transcriptional and posttranslational levels. Our results suggest that NE SnTox3 influences ethylene biosynthesis and signaling, thereby regulating redox metabolism in infected wheat plants as necessary for successful host colonization at the initial phases of infection, which ultimately leads to extensive lesions due to fast pathogen reproduction. host-specific toxin


Introduction
Wheat, a staple crop, has been attacked by various kinds of leaf spot diseases in recent decades, and Septoria nodorum blotch (SNB) ranks among the most injurious ones. It is caused by the fungal pathogen Stagonospora nodorum Berk. Yield losses inflicted by this pest reach up to 30 % in susceptible wheat cultivars under permissive environmental conditions (Bertucci et al., 2014). Studies of SNB have been intensively conducted over the past three decades, but there is still no clear understanding of the mechanisms that underlie wheat resistance/susceptibility to infection, on the one hand, and pathogen virulence, on the other hand (Fraaije et al., 2002;Bertucci et al., 2014;Winterberg et al., 2014;Phan et al., 2016;Shi et al., 2016).
It has been shown that among the most important factors of virulence of S. nodorum are the numerous necrotrophic effectors (NEs), formerly referred to as host-specific (selective) toxins (Phan et al., 2016;McDonald, Solomon, 2018). The interaction in the wheat-S. nodorum pathosystem is of the gene-for-gene type (McDonald, Solomon, 2018). These relationships are confirmed by the fact that products of the pathogen virulence genes (=host-specific toxins) (SnTox) cause compatibility, i. e. disease expansion, when interacting with products of the host plant susceptibility genes (Snn) (Phan et al., 2016). The effect of each SnTox-Snn interaction is incomplete and is complemented by other interactions. To date, eight SnTox-Snn interactions are known, while only three genes encoding NEs (SnToxA, SnTox1, and SnTox3) have been cloned from the pathogen, and only two susceptibility genes (Tsn1 and Snn1) have been cloned from wheat (Phan et al., 2016;Shi et al., 2016).
The genetics of the relationship between wheat and S. nodorum is very complex, race-specific resistance explaining only about 40 % of phenotypic manifestations (Shi et al., 2016). In addition, it was shown in the last four years that some of the characterized SnTox-Snn interactions causing susceptibility are aimed at manipulating nonspecific plant defense pathways associated with redox metabolism, secondary metabolism and pathogenicity-related proteins (Winterberg et al., 2014;Phan et al., 2016;Shi et al., 2016).
The Snn3-B1-SnTox3 interaction plays a significant role in SNB development (Shi et al., 2016;McDonald, Solomon, 2018). It is assumed that the result of this interaction is the development of infection on leaves with the formation of extensive lesions, which is associated with the influence of SnTox3 on the generation of reactive oxygen species (ROS); unfortunately, the mechanism underlying this effect remains obscure (Winterberg et al., 2014). Nevertheless, a recent study shows that SnTox3 induces methionine accumulation and ethylene synthesis in wheat plants within 24 h after infection (Winterberg et al., 2014).
One of the earliest plant responses to the penetration of a pathogen is known to be local ROS generation, which plays an important role in the development of systemic resistance (Barna et al., 2012). Currently, the mechanisms regulating apoplastic ROS synthesis during immune response are intensively studied but still insufficiently understood. Recent studies have shown that the pro-/antioxidant state of plants is under the strict control of plant hormones involved in the formation of defense reactions during stress (Barna et al., 2012). They include ethylene, whose role in biotic stress is complex and depends on the type of pathogen and plant species (Vleesschauver et al., 2010;Barna et al., 2012). Earlier, we showed the negative role of ethylene in the development of wheat plant resistance to S. nodorum (Veselova et al., 2016). Unfortunately, the mechanisms of action of plant hormones, including ethylene, on ROS generation under biotic stress are poorly known (Barna et al., 2012).
In this regard, the aim of this work was a comparative assessment of the transcriptional pattern of genes involved in ethylene biosynthesis and signaling pathway genes and of the redox state of wheat plants infected with Stagonospora nodorum isolates differing in NE SnTox3 expression.

Materials and methods
The objects of the study were two cultivars of soft spring wheat (Triticum aestivum L.) contrasting in resistance to S. nodorum Berk.: susceptible cv. Kazakhstanskaya 10 (Kaz10) and resistant cv. Omskaya 35 (Om35). The pathogen objects were two isolates of the fungus S. nodorum: Sn4VD (Republic of Belarus) and SnB (Republic of Bashkortostan). Fungi were grown on potato-glucose agar (PGA). Plants were hydroponically grown on 10 % solution of Hoagland-Arnon nutrient medium in a KS-200 SPU growth chamber (Russia) at Effect of the SnTOX3 on the ethylene signaling pathway and redox-status in common wheat 20/24 °С (night/day) at the irradiance 146 W/m 2 FAR (Osram lamps L 36W/77) and the 16-h photoperiod for seven days. The assessment of seedling resistance of cultivars was carried out by the lawns method, as described in (Veselova et al., 2016). The resistance/susceptibility of cultivars was assessed from the lesion area seven days after inoculation with S. nodorum isolates. The development of SNB symptoms on wheat leaves was photographed with an SP-800UZ Image Stabilization camera (Olympus, Indonesia). The lesion area was measured with ImageJ program (rsbweb. nih.gov/ij/download.html) and expressed as percent leaf area infected. The degree of damage was also assessed according to the International scale based on the percentage of the affected area of plant organs: RR (0-5 %) -caltivars with very high and high resistance; R (up to 10-15 %) -resistant caltivars; M (up to 25 %) -slightly susceptible caltivars; S (up to 40-65 %) -susceptible caltivars; SS (over 65 %) -caltivars with very high and high susceptibility.
DNA was isolated from wheat seedlings and 7-day fungus culture by the phenol-detergent method (Maniatis et al., 1984). SnTox3 gene (FJ823644) identification in S. nodorum isolates was performed by PCR with gene-specific primers (5´ 3´): F-CGAGCTGATATCCCGTTTGA; R-GGGACAGT-GACAATAGGTAAGG (Winterberg et al., 2014); primers for the housekeeping gene tubulin (S56922) (Fraaije et al., 2002) being used as an internal control for the presence of fungal DNA. Analysis of SnTox3 gene expression in different isolates of S. nodorum during inoculation of wheat plants was performed with the same primers using semi-quantitative PCR. Total RNA was isolated with Trizol reagent (Sigma, Germany) according to manufacturer's recommendations from leaves of susceptible wheat cv. Kaz10 and resistant cv. Om35 fixed in liquid nitrogen after their inoculation with a pathogen. To obtain cDNA based on RNA from the studied samples, reverse transcription reaction was performed using reverse transcriptase in accordance with manufacturer's protocol (Synthol, Russia). PCR with the cDNA template was performed in a TP4-PCR-01-Tertsik type PCR machine (DNK-Tekhnologia, Russia).
To reveal the effect of SnTox3 on the biosynthesis and signaling pathway of ethylene, part of the wheat seedlings were treated with 1.5 mM solution of ethephone (ET) (2-chloroethylphosphonic acid), an ethylene-releasing compound (Sigma, Germany) (Veselova et al., 2016), 24 h before inoculation with various S. nodorum isolates, while the remainder of the wheat seedlings were treated with 50 μM aminoethoxyvinylglycine (AVG), ethylene biosynthesis inhibitor (Sigma, Germany). After the treatment, the vessels were closed and kept in the dark. The content of hydrogen peroxide (H 2 O 2 ) and the ac-tivities of peroxidase enzymes (PO), oxalate oxidase (ОXО), and catalase were measured 24 and 72 h after inoculation with S. nodorum isolates as previously described (Veselova et al., 2018).
All experiments were carried out three times with three biological and three analytical replications (n = 9 in total), except for the measurements of infected area, which were performed in not less than 30 biological replications (n = 90 altogether). The Figures 1-3 and Tables 1-3 report mean values and their confidence intervals calculated from their standard errors. Significance of differences between experimental variants was estimated by Student's t-test at the confidence level p ≤ 0.05.

Results
SnTox3 gene and Snn3-B1 susceptibility locus. Two S. nodorum isolates, SnB and Sn4VD, were tested for the presence/ absence of the SnTox3 gene by PCR. The gene was found in both ( Fig. 1, a).
However, analysis of the transcriptional activity of this NE gene showed no expression in the avirulent Sn4VD isolate and accumulation of SnTox3 transcripts after inoculation of the susceptible cv. Kaz10 and the resistant cv. Om35 with the virulent isolate SnB (see Fig. 1, b). PCR diagnostics of the allelic state of the Snn3-B1 locus was performed in two soft spring wheat cultivars, Kaz10 and Om35. For this purpose, specific primers for two Xcfd20 and Xgwm234 microsatellite markers flanking the Snn3-B1 locus were used (Bertucci et al., 2014;Shi et al., 2016). The null allele was not found in these varieties. However, the cultivars differed in the allelic устойчивость растений к биотическим факторам / Resistance of plants to biotic factoRs composition of the Snn3-B1 locus (see Fig. 1, c). In particular, the Xcfd20 marker was represented by two alleles in both cultivars, and Xgwm234 was represented by one allele in Om35 and by two in Kaz10 (see Fig. 1, c).
A complete compatibility reaction was detected in the S/Tox3 + cultivar/isolate combination (Kaz10/SnB), where inoculation with the pathogen led to the formation of large lesions covering up to 80 % of the total leaf area (Table 1). Resistance reactions were observed in the remaining cultivar/ isolate combinations (Om35/SnB, Kaz10/Sn4VD, Om35/ Sn4VD) (see Table 1). Pretreatment of both Tox3-sensitive (Kaz10) and Tox3-insensitive (Om35) plants with ET increased their susceptibility only to the SnB isolate (Tox3 + ) but did not affect the susceptibility to the Sn4VD isolate (Tox3 -) (see Table 1). Pretreatment of Tox3-insensitive plants (Om35) with ethylene biosynthesis inhibitor AVG did not affect their defense response, regardless of the isolate that was used for plant inoculation: SnB (Tox3 + ) or Sn4VD (Tox3 -) (see Table 1). Treatment of Tox3-sensitive plants (Kaz10) with AVG increased their resistance to the SnB isolate but did not affect the resistance to the Sn4VD isolate (see Table 1).
The biosynthesis and signaling pathway of ethylene in infected plants. Analysis of the transcriptional activity of ethylene biosynthesis genes (TaACS for ACC synthase and TaACO for ACC oxidase) and signaling pathway genes (TaEIL1 and TaERF1) showed an increase in the mRNA contents of these genes during the compatibility reaction developed in susceptible plants (Kaz10/SnB) and in plants treated with ET (Kaz10/SnB + ET, Om35/SnB + ET) ( Table 2). In Tox3-sensitive plants (Kaz10), the transcripts contents of the ethylene biosynthesis genes and the gene coding for TaERF1 (transcription factor involved in the primary response to ethylene) increased about three to four fold, and the mRNA content of the TaEIL1 gene (coding for the main regulatory factor of the ethylene signaling pathway) increased 14-18 times on the 24th hours after inoculation (see Table 2). However, in Tox3-insensitive plants (Om35/SnB + ET cultivar/isolate combination), the accumulation of mRNAs of the genes responsible for biosynthesis and signaling pathway of ethylene was lower than in other cultivar/isolate combinations, which led to a compatibility reaction (see Table 2).
When the incompatibility reaction developed in Tox3insensitive plants in the Om35/SnB, Om35/Sn4VD cultivar/ isolate combinations, suppression or absence of the accumulation of transcripts of genes involved in ethylene biosynthesis and signaling pathway was found regardless of ET or AVG treatments (see Table 2). When the incompatibility reaction developed in Tox3-sensitive plants, either inoculated with Sn4VD isolate (Tox3 -) (Kaz10/Sn4VD, Kaz10/Sn4VD + AVG, Kaz10/Sn4VD + ET) or with SnB isolate (Tox3 + ) (Kaz10/SnB + AVG), there was no accumulation of mRNAs of the TaACS, TaACO, or TaERF1 genes and the accumula-   Effect of the SnTOX3 on the ethylene signaling pathway and redox-status in common wheat tion of TaEIL1 mRNA was low as compared to control plants (see Table 2).

Redox state of infected plants.
We studied the components of the pro-/antioxidant system in order to elucidate the role of SnTox3 in the regulation of redox metabolism in the host plant via the biosynthesis and signaling pathway of ethylene in suppressing defense reactions in infected plants. The compatibility reactions in susceptible plants (Kaz10/SnB) and in plants treated with ET (Kaz10/SnB + ET, Om35/SnB + ET) were characterized by a decrease in H 2 O 2 content (Fig. 2, a, b) due to elevated activity of catalase (CAT) (Fig. 3, e, f), reduced peroxidase (PO) (see Fig. 3, a, b) and oxalate oxidase (OXO) (see Fig. 3, c, d) activities, and lack of transcript accumulation of the genes encoding oxidoreductases NADPH oxidase (TaRbohF), SOD (TaSod), and PO (TaPrx) ( Table 3). However, in Tox3-insensitive plants (Om35/SnB + ЕТ cultivar/isolate combination), the suppression reaction of oxidative burst was less pronounced than in other combinations leading to a compatibility reaction (see Fig. 2, 3, Table 3).
The incompatibility reactions in the Om35/SnB, Om35/ Sn4VD, Kaz10/Sn4VD cultivar/isolate combinations and in AVG-treated plants from all combinations were characterized by an increase in H 2 O 2 generation (see Fig. 2, a, b) due to a decrease or absence of increase in CAT activity, a drastic increase in the activity of PO and OXO (see Fig. 3), and the accumulation of TaRbohF, TaSod, and TaPrx transcripts at the early stage of infection (24 h) (see Table 3), which led to the development of a hypersensitive-type response and arrest     Effect of the SnTOX3 on the ethylene signaling pathway and redox-status in common wheat of pathogen growth (see Table 1). Treatment with ET did not affect the nature of the response to inoculation with the Sn4VD isolate in either Tox3-sensitive or insensitive plants (see Fig. 2, 3, Table 3).

Discussion
At present, it is known that S. nodorum produces eight NEs associated with the virulence of pathogen isolates (Phan et al., 2016;Shi et al., 2016). The main NEs are SnToxA, SnTox1, SnTox3 toxins, which are considered to be the key factors of the virulence of pathogen strains and isolates, as shown in experiments with mutant S. nodorum strains and different wheat lines sensitive and insensitive to these effectors (Phan et al., 2016;Shi et al., 2016).
In our study, high transcriptional activity of the SnTox3 gene was found in the virulent isolate SnB (Fig. 1, b). The avirulent isolate Sn4VD did not express this gene (see Fig.  1, b), which may be indicative of SnTox3 inactivation in this isolate (Tan, Oliver, 2017). Here we studied two cultivars of soft spring wheat contrasting in resistance to S. nodorum and differing in the allelic composition of the Snn3-B1 locus (see Fig. 1, c). This difference is presumed to be associated with their sensitivity and insensitivity to NE SnTox3 (Shi et al., 2016). In the referred study, two BG220 and Sumai3 wheat lines carrying different alleles of the Snn3-B1 locus showed different degrees of sensitivity to NE SnTox3. Despite the fact that the Snn3-B1 null allele was not detected in cv. Om35, this cultivar was insensitive or weakly sensitive to SnTox3. This may indicate a large deletion in the locus between the Xcfd20 and Xgwm234 markers, which is in agreement with the literature data (Shi et al., 2016). Thus, out of 17 Sumai3 mutants insensitive to SnTox3, the Snn3-B1 null allele was detected only in 5 of them: two lines harbored null alleles for three microsatellite markers, Xgwm234, Xmag705 and Xcfb306, and three lines had a null allele for one microsatellite marker Xcfb306 (Shi et al., 2016). Thus, four different cultivar/isolate combinations were selected with two isolates of S. nodorum SnB (Tox3 + ) and Sn4VD (Tox3 -) and two cultivars of spring common wheat with different genotypes to study the role of SnTox3 in the development of infection.
On the one hand, the main function of NE SnTox3 is the formation of lesion zones on the wheat leaves of sensitive genotypes by hijacking host's nonspecific signaling defense pathways and manipulating them for pathogen growth and propagation (Winterberg et al., 2014). Furthermore, SnTox3 has been shown to increase ethylene synthesis in infected plants (Winterberg et al., 2014).
To elucidate the role of SnTox3 in ethylene biosynthesis and signaling pathways, we treated part of wheat plants of two cultivars, Kaz10 and Om35, with ethylene chemical precursor ET and ethylene biosynthesis inhibitor AVG. The results showed that both the elevated susceptibility of the ET-treated plants to S. nodorum and elevated resistance of the AVG-treated plants depended on the pathogen genotype: Tox3 + in SnB or Tox3in Sn4VD (see Table 1). This observation suggests that NE SnTox3 acts as a virulence factor and affects the plant defensive system by regulating ethylene biosynthesis and signaling pathways. It has been shown that ethylene production by some pathogens is closely associated with their virulence (Ма K.-W., Ma W., 2016). For instance, the XopD effector of the pathogenic bacterium Xanthomonas euvesicatoria manipulates the ethylene signaling pathway, affecting transcription factor ERF4 (Ма K.-W., Ma W., 2016), and the necrotrophic fungus Cochliobolus miyabeanus induces the ethylene signaling pathway in rice to produce and secrete ethylene as an effector to accelerate infection .
In our work, the analysis of the transcriptional activity of the genes controlling the biosynthesis and signaling pathway of ethylene also showed that the activation of genes involved in this pathway in infected plants depended on the pathogen isolate genotype and the sensitivity of the wheat genotype to NE SnTox3 (see Table 2). These results suggest that the biosynthesis and signaling pathway of ethylene are induced in the gene-for-gene interaction between Snn3-B1 and SnTox3.
Interestingly, in SnTox3 insensitive plants (Om35/SnB + ET cultivar/isolate combination), ET treatment increased plant sensitivity to this NE (see Table 3), suggesting that such a reaction could result from a mutation in genes regulated by the Snn3-B1-SnTox3 interaction (Shi et al., 2016). However, the activation of genes for ethylene biosynthesis and the signaling pathway in the Om35/SnB + ET combination was weaker than in case of the compatible interaction in plants sensitive to SnTox3 (Kaz10/SnB, Kaz10/SnB + ET) (see Table 2). This suggests that resistant plants possess a mechanism for efficient suppression of ethylene biosynthesis and signaling pathway to induce defense responses that are inhibited by ethylene. For example, ethylene inhibited salicylic acid (SA) biosynthesis and suppressed the expression of the SA-mediated signaling pathway marker genes PR-1 and PR-2 in Arabidopsis plants infected with Pseudomonas syringae (Chen et al., 2009), as well as in wheat plants infected with S. nodorum (Veselova et al., 2016). Silicon blocked ethylene production by the pathogen C. miyabeanus, which improved the resistance of rice plants . Thus, the obtained results prove the influence of SnTox3 on the biosynthesis and signaling pathway of ethylene in the course of Snn3-B1-SnTox3 interaction according to the gene-for-gene type with ultimate suppression of the defense reactions of wheat plants to facilitate colonization.
Our previous studies showed that ethylene provided comfortable conditions for the penetration and development of S. nodorum in wheat plant tissues at the initial stage of infection due to the regulation of redox metabolism and reduction of Н 2 О 2 generation (Veselova et al., 2016(Veselova et al., , 2018. On the contrary, the accumulation of ROS in wheat plants at the initial stage of infection with the pathogen S. nodorum determined the resistance of the cultivar, inducing the expression of the genes encoding pathogenicity-related proteins (Veselova et al., 2016(Veselova et al., , 2018. The change in the redox state of infected wheat plants in our experiments completely depended on the activation or inhibition of the biosynthesis and signaling pathway of ethylene, and this effect was due to the Snn3-B1-SnTox3 interaction of the gene-for-gene type (see Fig. 2, 3). Our results demonstrate that ethylene suppresses Н 2 О 2 accumulation in plants sensitive to SnTox3 via increasing CAT activity, reducing PO and OXO activities, and lowering the transcript contents of genes encoding NADPH oxidase and SOD, in consistency with literature data (Golemiec et  Formerly, it was shown that NE SnTox3 regulated genes involved in redox metabolism and the formation of necrosis (Winterberg et al., 2014), but the mechanisms effecting the influence of SnTox3 on ROS generation remain obscure. It is known from the literature that specific effectors of pathogens can induce hypersensitive response in plant cells and suppress the oxidative burst in plants during the infection process in various ways (Jwa, Hwang, 2017). For example, two cytoplasmic effectors of Phytophthora sojae interact with catalases to regulate Н 2 О 2 concentration. The Pep1 effector of U. maydis interacts with POX12 maize peroxidase in vivo and suppresses early immune responses in maize (Hemetsberger et al., 2012).

Conclusion
The results of our work suggest that the pathogen effector SnTox3 influences biosynthesis and signaling pathway of ethylene in order to regulate the redox metabolism of infected wheat plants in the way promoting successful colonization of the host at initial stages of infection, which subsequently gives rise to extensive damage lesions due to fast pathogen reproduction.