Glycine-serine-rich effector PstGSRE4 in Puccinia striiformis f. sp. tritici inhibits the activity of copper zinc superoxide dismutase to modulate immunity in wheat.

Puccinia striiformis f. sp. tritici (Pst) secretes an array of specific effector proteins to manipulate host immunity and promote pathogen colonization. In a previous study, we functionally characterized a glycine-serine-rich effector PstGSRE1 with a glycine-serine-rich motif (m9). However, the mechanisms of glycine-serine-rich effectors (GSREs) remain obscure. Here we report a new glycine-serine-rich effector, PstGSRE4, which has no m9-like motif but inhibits the enzyme activity of wheat copper zinc superoxide dismutase TaCZSOD2, which acts as a positive regulator of wheat resistance to Pst. By inhibiting the enzyme activity of TaCZSOD2, PstGSRE4 reduces H2O2 accumulation and HR areas to facilitate Pst infection. These findings provide new insights into the molecular mechanisms of GSREs of rust fungi in regulating plant immunity.

Introduction

In nature, plants are exposed to a variety of biotic and abiotic stresses, including the invasion of numerous pathogenic microorganisms. In their interactions, plants and pathogens confront processes of defense and pathogenicity and co-evolve. Upon pathogen infection, pattern recognition receptors (PRRs) in plants recognize the pathogen-associated molecular pattern (PAMP) and activate PAMP-triggered immunity (PTI) to form the first level of defense [1]. Pathogens have formed a large number of virulence factors during the long-term evolution with the host, and successfully infect and colonize the host by acting on the host plant cells [1]. Effectors, as a type of very important virulence factors, are secreted from the pathogen into the host primarily to inhibit the host’s defense response, and thus cause host plant susceptibility. In addition, when certain avirulence effectors from the pathogen are directly or indirectly recognized by plant disease-resistant proteins, the plant immune system is strongly activated to induce the host cell hypersensitive response (HR), which has been termed effector-triggered immunity (ETI) [2]. Therefore, the effectors of a pathogen have a dual function of virulence and avirulence, which is not only an important weapon of pathogenicity, but also an important target of the plant immune system. Both PTI and ETI include the induction of reactive oxygen species (ROS), a key component of the defense system [3,4].

The sharp increase of ROS is a common manifestation when plants are confronted with various pathogens, indicating that ROS play a vital role in the process of plant resistance to pathogens. ROS burst is generally defined as a rapid production of high levels of ROS in response to external stimuli [5]. Superoxide radicals (O2-) and hydrogen peroxide (H2O2) are considered important ROS in response to biotic stress [6]. During plant-pathogen interaction, penetration of pathogen into host plasma membrane triggers the early O2− burst by an NADPH oxidase, then they are rapidly converted to H2O2 by dismutation [5]. Most of the data seem to indicate that the major ROS building the oxidative burst is H2O2, with possible participation of O2−. On the one hand, O2- and H2O2 are directly toxic to pathogens. For instance, the accumulation of O2- or H2O2 caused by Pseudomonas syringae pv. tabaci significantly decreased the number of bacteria in Nicotiana benthamiana, and then the number of bacteria significantly increased following the addition of SOD or other reactive oxygen scavengers [7]. On the other hand, H2O2 can also act as signaling molecules to directly or indirectly activate the expression of resistance genes and defense genes. H2O2 can induce the increase of antioxidant enzyme activity in plants to resist the invasion of pathogens. Exogenous H2O2 can induce a significant increase in glutathione S-transferase (GST) transcription in soybean suspension cells, and H2O2 scavengers can prevent this effect [8]. In addition, H2O2 also participate in the lignification of cell walls and the cross-linking of proteins to cell walls to strengthen plant cell walls against pathogen invasion. After infection with diseased substances, synthesis of H2O2 was observed in lignification sites of plant tissues [7]. H2O2 can also induce the occurrence of plant HR response. A large number of experiments proved that exogenous H2O2 can induce HR in cells of Arabidopsis thaliana [9]. Interestingly, many studies have shown that effector proteins can control the host immune response by interfering with the host ROS signaling pathway [10-13]. Understanding the mechanism of effectors regulating ROS-related targets will increase our knowledge of molecular mechanisms underlying the interaction between plants and phytopathogens, and provide a theoretical foundation to achieve durable disease resistance.

Superoxide dismutase (SOD) is an important component of the antioxidant enzyme system and is widely distributed in microorganisms, plants and animals. It catalyzes superoxide anion (O2−) radical disproportionation to produce O2 and H2O2, and plays an important role in the balance between oxidation and oxidation resistance [14]. Based on their metal cofactors, protein folds, and subcellular distribution, SODs are mainly categorized as CuZnSODs, FeSODs, and MnSODs [15]. A previous study indicated that infection of grape with grapevine fanleaf virus caused the accumulation of ROS and activated its enzyme defense system, including SOD [16]. Among the isoenzymes of SOD in sunflower, the expression of CuZnSOD under biological stress is the most affected, indicating that CuZnSOD is the main antioxidant defense enzyme [17]. When the CuZnSOD gene in tomato chloroplasts was transferred into two N. benthamiana strains, it enhanced the resistance to anthrax by changing the expression of the antioxidant enzyme [18]. In the Phaseolus vulgaris-Uromyces appendiculatus interaction, the expression of CuZnSOD was increased greatly during the incompatible interaction [19]. Recently, in the study of barley-powdery mildew interaction, loss-of-function mutations in Mla and Rar1 both resulted in the reduced accumulation of copper-zinc superoxide dismutase 1 (HvSOD1), whereas loss of function in Rom1 re-established HvSOD1 levels [20]. In the study of rice-Magnaporthe oryzae interaction, different SODs in miR398b regulated resistance to rice blast disease, and miR398b increased total SOD activity to upregulate the H2O2 concentration and thereby improve disease resistance [21]. However, there have been no reports on phytopathogenic effectors targeting and regulating CuZnSODs from plants to suppress host immune response.

Among the diseases caused by rust fungi, the diseases on Gramineae and Leguminosae seriously threaten the safety of food production in China and throughout the world [22]. Stripe rust is one of the most serious diseases of wheat in the world [23]. Wheat has evolved resistance genes to protect against disease. However, Pst constantly mutates to overcome these resistance genes, and the effectors contributed significantly to the virulence diversity of Pst [24]. Due to the importance of effector proteins in the interaction between pathogens and plants, more and more attention has been paid to the study of effector proteins. Recently, stripe rust effector Pst18363 has been reported to stabilize a negative regulator of wheat defense, TaNUDX23, which suppresses ROS accumulation and facilitates Pst infection [25]. Another stripe rust effector, Pst_12806, is translocated into chloroplasts and perturbs photosynthesis, avoiding triggering cell death and supporting pathogen survival on living plants [26]. In several organisms, glycine- or serine-rich proteins have been shown to participate in RNA splicing, metabolism and signal transduction [27,28]. Pathogen effectors with a high content of glycine or serine could potentially modify the host’s metabolism or signal transduction [29]. In Pst, a glycine-serine-rich effector protein PstGSRE1 containing a glycine-serine-rich motif (m9) has been shown to disrupt the nuclear localization of TaLOL2 and suppress ROS-mediated cell death induced by TaLOL2, thus compromising host immunity [29]. However, the mechanisms of glycine-serine-rich effectors, remain obscure, and further investigation is required. In this study, we characterized a new glycine-serine-rich effector protein PSTCY32_07414 (alias PstGSRE4), which lacks the m9-like motif, targets a wheat copper zinc superoxide dismutase TaCZSOD2. PstGSRE4 is required for full virulence of Pst in wheat. Further analyses showed that TaCZSOD2 is a positive regulator of wheat resistance to Pst, and PstGSRE4 reduces H2O2 accumulation by inhibiting the activity of TaCZSOD2 to facilitate Pst infection. Our results provide new insights into the molecular mechanisms of glycine-serine-rich effectors of rust fungi regulating host immunity.

Results

PstGSRE4 is a glycine-serine-rich effector protein lacking m9-like motif in Pst

In our previous study, four glycine-serine-rich effectors were identified from Pst [29]. Sequence analysis showed that PSTCYR32_07414 encodes a 232-amino-acid secreted protein which was enriched in glycine (12.93%) and serine (19.40%) and did not contain any known functional domains except for a 22 amino acid (aa) signal peptide at its N-terminus (). BLASTp analyses revealed that homologs of PstGSRE4 can be found only in rust fungi, including 13 in Pst, seven in Puccinia triticina (Pt) and four in Puccinia graminis f. sp. tritici (Pgt) (), indicating that GSREs constitute a large family within the rust fungi. Using MEME, we found that PSTCYR32_07414 did not have the motif as the m9 motif of PstGSRE1 (). Moreover, yeast two-hybrid (Y2H) assay indicated that PstGSRE4 does not interact with TaLOL2 (), suggesting that it is functionally diverged. Thus, PSTCYR32_07414 was designated Puccinia striiformis Glycine-Serine-Rich Effector 4 (PstGSRE4) and selected for further study.

Relative transcript levels of PstGSRE4 at different Pst infection stages

In order to characterize the expression pattern of PstGSRE4 during Pst infection stages, we analyzed its relative transcript levels by qRT-PCR at different time points during the infection process. We used fresh ungerminated urediniospores of CYR32 and infected wheat tissues collected from 6 to 264 hpi to detect the transcripts of PstGSRE4. qRT-PCR assays showed that PstGSRE4 was upregulated during Pst infection (6–72 hpi), and reached higher expression levels at 24–48 hpi (approximately 158-fold, 145-fold, and 140-fold, respectively), corresponding to the formation of the haustorium (). The transcript levels were downregulated at the late ‘parasitic/biotroph’ stage (168 hpi) and ‘sporulation’ stage (216–264 hpi) ().

PstGSRE4 suppresses Pst322- and Bax-induced cell death by decreasing H2O2 accumulation

SignalP 5.0 analysis showed that PstGSRE4 has a signal peptide encoded by the first 22 amino acids. Secretion of PstGSRE4 was verified through a signal sequence trap system [29]. pSUC2T7M13ORI-PstGSRE4 was transferred into the yeast SUC2-minus strain, YTK12. The fusion of the signal peptide of PstGSRE4 to the mature sequence of SUC2 promoted the successful secretion of invertase, which enables the yeast cells to hydrolyze raffinose and grow on YPRAA media (). In addition, we found that the TTC-treated PstGSRE4 culture filtrates turned red, confirming invertase activity (). The oomycete effector Avr1b was used as positive control, and the YTK12 strains with or without pSUC2 vector were used as negative controls. These results indicate that the signal peptide of PstGSRE4 is functional.

To examine the function of PstGSRE4, we used agro-infiltration to transiently express it in N. benthamiana. We observed that PstGSRE4 inhibited an elicitor-like protein [28] Pst322-induced cell death (). In addition, PstGSRE4 suppressed the pro-apoptotic protein Bax-induced cell death (). Accumulation of PstGSRE4, Pst18363, eGFP, Pst322 and Bax proteins in infiltrated tissue were confirmed by Western blots ().

Because H2O2 is a crucial trigger of cell death and PstGSRE4 inhibits Bax- or Pst322-triggered cell death, we also test if PstGSRE4 suppresses Bax- or Pst322-triggered cell death by preventing H2O2 accumulation. DAB staining was used to examine the H2O2 levels in the infiltrated leaves. The DAB staining in the leaf regions infiltrated with Bax or Pst322 accumulated a large number of H2O2 compared with those infiltrated with MgCl2 buffer (). And the DAB staining in the leaf regions infiltrated with PstGSRE4/Bax or PstGSRE4/Pst322 was much weaker compared with those infiltrated with buffer/Bax or buffer/Pst322 (). These results suggested that PstGSRE4 can suppress Bax- or Pst322-triggered cell death by preventing H2O2 accumulation.

PstGSRE4 suppresses callose deposition and Pst-induced H2O2 accumulation

The important function of fungal effectors is to suppress PTI and/or ETI in order to create a suitable environment for infection. To analyze the ability of PstGSRE4 to inhibit PAMP-triggered responses, we used the bacterial type III secretion assay [29] to deliver PstGSRE4 into wheat leaves (). Callose deposition triggered by Pseudomonas fluorescence strain EtHAn carrying pEDV6-PstGSRE4 was significantly reduced at 24 and 48 hpi compared with the control EtHAn with or without pEDV6-RFP (). We further analyzed H2O2 accumulation triggered by Pst race CYR23 which is incompatible with Suwon11. The results showed that after transient transformation, H2O2 accumulation was significantly suppressed with EtHAn-PstGSRE4 at 24 and 48 hpi compared with the control EtHAn with or without pEDV6-RFP (). These results indicated that delivering PstGSRE4 into wheat inhibits PTI-associated callose deposition as well as H2O2 accumulation in wheat.

Silencing of PstGSRE4 reduces virulence of Pst

To test whether PstGSRE4 is involved in pathogenicity of Pst, host-induced gene silencing (HIGS) mediated by barley stripe mosaic virus (BSMV) was used to silence PstGSRE4 expression in Pst during the infection process. Two silencing fragments of PstGSRE4 were designed to generate two different BSMV constructs () for specifically silencing PstGSRE4. All of the wheat leaves infected with BSMV:γ, BSMV:PstGSRE4-1/2as expressed similar phenotypes of mild chlorotic mosaic symptoms at 12 d post-inoculation (dpi), whereas mock-inoculated leaves remained green and healthy (). Obvious photo-bleaching was observed in the BSMV:TaPDS-inoculated wheat, indicating that the BSMV-HIGS system functioned effectively (). Subsequently, the fourth leaves of BSMV-inoculated wheat were inoculated with freshly collected ungerminated urediniospores of CYR32. The number of rust pustules was significantly reduced in the wheat leaves inoculated with BSMV:PstGSRE4 compared with the leaves of BSMV:γ-inoculated wheat (). qRT-PCR analysis of total RNA extracted from silenced leaves, which were sampled at 24, 48 and 120 hpi, revealed that the expression of PstGSRE4 was significantly reduced in the wheat leaves inoculated with BSMV:PstGSRE4 (). The biomass of Pst/wheat also decreased in the leaves inoculated with BSMV:PstGSRE4 compared with the leaves of mock- and BSMV:γ-inoculated wheat (). In addition, histological analysis by fluorescence microscopy revealed that initial haustorium formation and growth of secondary hyphae both were reduced in BMSV:PstGSRE4-1/2as infected plants ().

To further confirm the virulence function of PstGSRE4 in Pst infection using stable transgenic plants, we prepared the RNA interference (RNAi) construct pAHC25-PstGSRE4-RNAi () and delivered it into wheat cv. XN1376 by particle bombardment. Two T4 transgenic wheat lines (L19 and L76) containing pAHC25-PstGSRE4-RNAi displayed significantly enhanced resistance against the virulent Pst race CYR31 (). Transcript levels of PstGSRE4 during Pst infection of transgenic lines L19 and L76 were significantly reduced (). The results showed that both lines L19 and L76 contained at least one copy of the transgene (). Compared with control plants, fungal biomass in infected leaves of transgenic lines L19 and L76 was significantly reduced (). We also detected the contents of H2O2 and O2- in infected leaves of transgenic lines L19 and L76 at 6, 12, 24 and 48 hpi (Figs ). The result showed that the accumulation of H2O2 was significantly increased and the accumulation of O2− was significantly reduced in transgenic lines L19 and L76, suggesting that PstGSRE4 regulates the accumulation of H2O2 in wheat. The formation of mature haustoria and the development of secondary hyphae were assessed by microscopy. The results showed that hyphal areas during infection of L19 and L76 were significantly reduced at 120 hpi (Figs ). H2O2 accumulation was also measured during Pst infection of transgenic plants at 72 and 120 hpi. We found that H2O2 accumulation was significantly increased in the transgenic plants relative to the control (). These results indicated that PstGSRE4 contributs to virulence of Pst on wheat leaves.

RNAi-mediated stable silencing of PstGSRE4 significantly reduces Virulence of Pst and increases H2O2 accumulation in Wheat.

(A) Phenotypes of the second leaves of the fourth generation of wheat plants at 14 dpi with Pst. (B) Relative transcript levels of PstGSRE4 in different transgenic lines at 48 hpi with Pst. Values represent the means ± SE (n = 3). (C) Ratio of fungal to wheat nuclear content using fungal PstEF-1 and wheat TaEF-1α genes, respectively. Genomic DNA was extracted from the second leaf from three different plants at 14 dpi. Values represent the means ± SE (n = 3). (D) Content of H2O2 in different transgenic lines at 6, 12, 24 and 48 hpi. Values represent the means ± SE of three independent samples. (E) Quantification of hyphal areas in different transgenic lines at 48 and 120 hpi. Values represent the means ± SE (n = 30). Differences were assessed using Student’s t-test. Asterisk indicates P < 0.05.

Overexpression of PstGSRE4 reduces wheat resistance to Pst

To further confirm the virulence function of PstGSRE4 in Pst infection in stable transgenic plants, we prepared the overexpression construct pCAMBIA3301-PstGSRE4-overexpression () and delivered it into wheat cv. Fielder by A. tumefaciens-mediated stable transformation. Two T3 transgenic lines (L2 and L3) containing pCAMBIA3301-PstGSRE4-overexpression displayed significantly reduced wheat resistance against Pst race CYR23 (). The results showed that both L2 and L3 contained at least one copy of the transgene (). RT-PCR () and qRT-PCR () showed that the expression of PstGSRE4 was different in each line, and it influenced the resistance against Pst race CYR23 to a different extent. Compared with control plants, fungal biomass in infected leaves of transgenic lines L2 and L3 was significantly increased (). We also detected the contents of H2O2 and O2- in infected leaves of transgenic lines L2 and L3 at 6, 12, 24 and 48 hpi (Figs ). The result showed that the accumulation of H2O2 was significantly reduced and the accumulation of O2− was significantly increased in transgenic lines L2 and L3, suggesting that PstGSRE4 regulates the accumulation of H2O2 in wheat. As shown in Figs and , H2O2 accumulation and HR triggered by Pst race CYR23 were significantly decreased in PstGSRE4-overexpression plants at 48 hpi. These findings indicate that PstGSRE4 is an important pathogenicity factor that regulates the accumulation of H2O2 during Pst infection.

Overexpression of PstGSRE4 significantly increases virulence of Pst and reduces H2O2 accumulation in wheat.

(A) Phenotypes of the second leaves of the third generation of wheat plants at 14 dpi with Pst. (B) Relative transcript levels of PstGSRE4 in different transgenic lines at 48 hpi with Pst. Values represent the means ± SE (n = 3). (C) Ratio of fungal to wheat nuclear content determined using the contents of fungal PstEF1 and wheat TaEF-1α genes, respectively. Genomic DNA was extracted from the second leaf from three different plants at 14 dpi. Values represent the means ± SE (n = 3). (D) Content of H2O2 in different transgenic lines at 6, 12, 24 and 48 hpi. Values represent the means ± SE of three independent samples. (E) Quantification of necrotic cell death area in different transgenic lines at 48 hpi. Values represent the means ± SE (n = 20). Differences were assessed using Student’s t-test. Asterisk indicates P < 0.05, and double asterisk indicates P < 0.01.

PstGSRE4 specifically targets wheat copper zinc superoxide dismutase TaCZSOD2

To understand the potential virulence function of PstGSRE4 in wheat, a yeast two-hybrid (Y2H) library was constructed to screen constructs and identify potential host targets of PstGSRE4. With PstGSRE4 (ΔSP) as the bait, several candidate targets were identified (). We selected the candidate ROS-associated genes for further study. A candidate target sequence was annotated as superoxide dismutase [Cu-Zn] (TraesCS7A02G292100.1) (http://plants.ensembl.org/index.html). According to a previous study, we collected 26 TaSODs, 8 AtSODs and 8 OsSODs from the Arabidopsis Information Resource (TAIR10) database (http://www.arabidopsis.org/index.jsp), the Rice Genome Annotation Project (RGAP) database (http://rice.plantbiology.msu.edu/) and the wheat reference genome IWGSC v1.1 (E-value < 1e–5), respectively. A phylogenetic tree was constructed, which revealed that the CuZuSOD gene obtained in this study lies within the same clade as AtCSD2 (). Based on this evidence, we designated the wheat CuZnSOD as TaCZSOD2. BlastN analyses of the wheat genome showed that there were three copies of TaCZOD2 located on chromosomes 7A, 7B and 7D, respectively. Sequence alignment showed that the three copies, designated as TaCZSOD2-7A, TaCZSOD2-7B and TaCZSOD2-7D, and the TaCZSOD2 obtained in this study share 99.3% nucleotide identity (). We cloned TaCZSOD2 and the other three TaCZSODs (TaCZSOD1, TaCZSOD3, TaCZSOD4) from wheat cultivar Suwon11 to further confirm the preliminary results of the Y2H assay (). The results showed that PstGSRE4 specifically interacts with TaCZSOD2.

To determine whether PstGSRE4 can directly interact with TaCZSOD2, we used recombinant proteins PstGSRE4-GST, TaCZSOD2-His and GST, TaCZSOD1-His (as negative control) expressed from E. coli BL21 to conduct GST pull-down assay (). We detected immunoprecipitated protein complexes by western blotting. TaCZSOD2-His was detected in PstGSRE4-GST pull-down fractions but not the TaCZSOD1-His, indicating that PstGSRE4 specifically interacts with TaCZSOD2 in vitro. To obtain further experimental evidence, we conducted a Co-IP experiment based on A. tumefaciens-mediated transient expression in N. benthamiana. PstGSRE4 mature protein fused to GFP and TaCZSOD2 or TaCZSOD1 fused to HA were co-expressed in N. benthamiana. Western blotting analysis showed that PstGSRE4 specifically interacts with TaCZSOD2 in vivo (). We also co-expressed PstGSRE4 and TaCZSOD2 in N. benthamiana cells and found that PstGSRE4 and TaCZSOD2 were co-localized in the cytoplasm (), suggesting that PstGSRE4 and TaCZSOD2 interact in the cytoplasm. To further confirm that PstGSRE4 and TaCZSOD2 interact in the cytoplasm, coding sequences of ΔTP-TaCZSOD2 (TaCZSOD2 without chloroplast transit peptide) was ligated into pBINGFP2 to construct the recombinant plasmid ΔTP-TaCZSOD2-GFP. And we co-expressed ΔTP-TaCZSOD2-GFP with PstGSRE4-RFP, TaCZSOD2-GFP with PstGSRE4-RFP, ΔTP-TaCZSOD2-GFP with RFP, and TaCZSOD2-GFP with RFP in N. benthamiana, and detected the activity of CuZnSOD. The results showed that TaCZSOD2 had enzyme activity in the cytoplasm, while PstGSRE4 inhibited the activity of TaCZSOD2 in the cytoplasm (). Compared with full length-TaCZSOD2, PstGSRE4 significantly inhibited the activity of ΔTP-TaCZSOD2 ().

PstGSRE4 specifically interacts with wheat TaCZSOD2 in vitro and in vivo.

(A) PstGSRE4 interacts with TaCZSOD2 in yeast. Only the yeast co-expressing PstGSRE4 and TaCZSOD2 grew on the medium SD-Trp-Leu-His-Ade and yielded X-α-gal activity. Co-expressing PstGSRE1 and TaCZSOD2 cannot grow on the medium SD-Trp-Leu-His-Ade. (B) PstGSRE4 specifically interacts with TaCZSOD2 in yeast. Only the yeast co-expressing PstGSRE4 and TaCZSOD2 grew on the medium SD-Trp-Leu-His-Ade and yielded X-α-gal activity. Co-expressing PstGSRE4 and TaCZSOD1, TaCZSOD3 or TaCZSOD4 cannot grow on the medium SD-Trp-Leu-His-Ade. (C) PstGSRE4 interacts with TaCZSOD2 in vitro. A GST pull-down assay was used to detect the interaction between PstGSRE4-GST and TaCZSOD2-His. TaCZSOD2-His and PstGSRE4-GST were detected with anti-His or anti-GST antibodies, respectively. TaCZSOD1-His was used as negative control. (D) PstGSRE4 interacts with TaCZSOD2 in vivo. Co-immunoprecipitation (IP) was performed on extracts of N. benthamiana leaves expressing both PstGSRE4-GFP and TaCZSOD2-HA. GFP was detected by western blot with anti-GFP antibodies. HA was detected by western blot with anti-HA antibodies. The TaCZSOD1-HA was used as negative control.

TaCZSOD2 positively regulates wheat resistance against Pst

To confirm the function of TaCZSOD2 in wheat resistance to Pst, we knocked down expression of TaCZSOD2 in wheat leaves by BSMV-VIGS. Two specific fragments were designed for silencing TaCZSOD2 (). qRT-PCR analysis showed that, during the interaction between wheat and Pst, transcript levels of TaCZSOD2 were up-regulated at 12, 48 and 96 hpi with the avirulent Pst race CYR23, and up-regulated at 96 and 120 hpi with the virulent Pst race CYR31 (). After virus inoculation, plants displayed mild chlorotic mosaic symptoms at 10 dpi. Compared with the control plants, fewer necrotic spots and sporadic uredia appeared on leaves of TaCZSOD2-knockdown plants after inoculation with the avirulent race CYR23, whereas no significant differences were found on leaves of TaCZSOD2-knockdown plants after inoculation with the virulent race CYR31 (). The silencing efficiency monitored by qRT-PCR indicated that TaCZSOD2-1as/2as transcript levels in knockdown plants were significantly reduced (). Moreover, the transcript levels of the other TaCZSODs were not influenced after the expression of TaCZSOD2 was knocked down (). The biomass of Pst/wheat showed an increase in the leaves inoculated with BSMV:TaCZSOD2 after inoculation with the avirulent race CYR23 compared with the leaves of BSMV:γ-inoculated wheat (). The biomass of Pst/wheat showed no significant changes in the leaves inoculated with BSMV:TaCZSOD2 after inoculation with the virulent race CYR31 compared with the leaves of BSMV:γ-inoculated wheat (). We detected the activity of CuZnSOD in TaCZSOD2-knockdown plants after inoculation with the avirulent race CYR23. Compared with the control plants, the enzyme activity of TaCZSOD2-knockdown plants was significantly reduced (). We also detected the contents of H2O2 and O2- in infected leaves of TaCZSOD2-knockdown plants at 6, 12, 24 and 48 hpi (Figs ). The results showed that knockdown of TaCZSOD2 significantly reduced H2O2 accumulation and increased O2− accumulation in wheat. Histological changes in TaCZSOD2-knockdown plants infected with CYR23 were observed by microscopy. As shown in Figs and , H2O2 accumulation and HR triggered by the avirulent Pst race CYR23 were significantly decreased in TaCZSOD2-knockdown plants at 48 hpi.

TaCZSOD2 is a positive regulator of wheat immunity.

(A) Phenotypes of the fourth leaves of knockdown plants inoculated with Pst race CYR23 and CYR31 at 12 dpi. (B) Relative transcript levels of TaCZSOD2 in TaCZSOD2-knockdown plants challenged by CYR23 and CYR31. TaEF-1α was used for normalization. Values represent the means ± SE (n = 3). (C) Relative transcript levels of other TaCZSODs in TaCZSOD2-knockdown plants challenged by CYR23 at 48 hpi. TaEF-1α was used for normalization. Values represent the means ± SE (n = 3). (D) After inoculation with CYR23, ratio of fungal to wheat nuclear content using fungal PstEF-1 and wheat TaEF1α genes, respectively. Values represent the means ± SE (n = 3). (E) The enzyme activity of CuZnSOD in TaCZSOD2-knockdown plants. Values represent the means ± SE of three independent samples. (F) Content of H2O2 in TaCZSOD2-knockdown plants at 6, 12, 24 and 48 hpi. Values represent the means ± SE of three independent samples. (G) Quantification of necrotic cell death area in TaCZSOD2-knockdown plants at 48 hpi. Values represent the means ± SE (n = 20). Differences between time-course points were assessed using Student’s t-tests. Asterisks indicate P < 0.05, double asterisks indicate P < 0.01.

To further test the function of TaCZSOD2 in wheat defense against Pst, we also performed transient delivery of TaCZSOD2 using the bacterial T3SS system in wheat. At 48 h post-infiltration with a plasmid carrying EtHAn strains, wheat plants were inoculated with Pst virulent race CYR31. The number of uredia was significantly reduced at 14 dpi (). In addition, the area of H2O2 accumulation and HR areas were significantly increased ().

To further confirm the function of TaCZSOD2 during Pst infection of stable transgenic plants, we prepared the overexpression construct CUB-TaCZSOD2-overexpression () and delivered it into wheat cv. Fielder by A. tumefaciens-mediated stable transformation. Three T2 transgenic lines (L1, L4 and L9) containing CUB-TaCZSOD2-overexpression displayed significantly increased wheat resistance against Pst race CYR31 (). PCR and Western blot assays indicated that L1, L4 and L9 contained at least one copy of the transgene (). qRT-PCR showed that the expression of TaCZSOD2 increased in each line (). And the fungal biomass in infected leaves of transgenic lines L1, L4 and L9 were significantly reduced (). Also, compared with control plants, the enzyme activities of CuZnSOD in transgenic lines L1, L4 and L9 were significantly increased (). We also detected the contents of H2O2 and O2- in infected leaves of TaCZSOD2-overexpression plants at 6, 12, 24 and 48 hpi (Figs ). The results showed that overexpression of TaCZSOD2 significantly increased H2O2 accumulation and reduced O2− accumulation in wheat. Histological changes in TaCZSOD2-overexpression plants infected with CYR31 were observed by microscopy. As shown in Figs and , H2O2 accumulation and HR area triggered by Pst race CYR31 were significantly increased in TaCZSOD2-overexpression plants at 48 hpi/72 hpi with the increasing enzyme activity of CuZnSOD. These findings indicate that TaCZSOD2 positively regulates wheat resistance against Pst.

Overexpression of TaCZSOD2 significantly increases H2O2 accumulation and confers the resistance of wheat to Pst.

(A) Phenotypes of the third leaves of the second generation of wheat plants at 14 dpi with Pst. (B) Ratio of fungal to wheat nuclear content determined using the contents of fungal PstEF1 and wheat TaEF-1α genes, respectively. Genomic DNA was extracted from the second leaf from three different plants at 14 dpi. Values represent the means ± SE (n = 3). (C) The enzyme activity of CuZnSOD in TaCZSOD2-overexpression plants at 48 hpi with Pst. Values represent the means ± SE of three independent samples. (D) Content of H2O2 in different transgenic lines at 6, 12, 24 and 48 hpi. Values represent the means ± SE of three independent samples. (E) Quantification of necrotic cell death area in different transgenic lines at 72 hpi. Values represent the means ± SE (n = 30). Differences were assessed using Student’s t-test. Asterisk indicates P < 0.05.

TaCZSOD2 increases H2O2 accumulation

In order to further confirm that TaCZSOD2 can increase H2O2 accumulation, we use chitin to induce a rapid oxidative burst to determine the capacity of TaCZSOD2 to maintain redox balance in TaCZSOD2-knockdown plants and TaCZSOD2-overexpression transgenic plants. qRT-PCR analysis showed that the transcript levels of TaCZSOD2 in knockdown plants were significantly reduced () and next we found that the chitin-induced H2O2 accumulation in TaCZSOD2-knockdown plants were markedly reduced compared with the wild-type (). Meanwhile, qRT-PCR analysis showed that the transcript levels of TaCZSOD2 in transgene plants were significantly increased () and the chitin-induced H2O2 accumulation in TaCZSOD2-overexpression transgenic plants were markedly increased compared with the wild-type (). In summary, these results suggested that TaCZSOD2 has the ability to increase H2O2 accumulation.

PstGSRE4 inhibits the activity of TaCZSOD2

In order to confirm the effect of PstGSRE4 on the activity of TaCZSOD2, we used recombinant proteins PstGSRE4-His, TaCZSOD2-His and eGFP-His, TaCZSOD1-His (as negative control) expressed in E. coli BL21 to conduct enzyme activity assays by the NBT photoreduction method (). The results demonstrated that PstGSRE4 reduces the enzyme activity of TaCZSOD2 in vitro, but not TaCZSOD1. Meanwhile, we used agro-infiltration to transiently co-express PstGSRE4 and TaCZSODs in N. benthamiana to determine whether PstGSRE4 inhibits the activity of TaCZSOD2 (). The results showed that PstGSRE4 reduces the activity of TaCZSOD2 in vivo. To further explore the effect of PstGSRE4 on TaCZSOD2 in vivo, we also used PstGSRE4-overexpression transgenic lines L2 and L3 to detect the activity of CuZnSOD in wheat (). The results showed that the activity of CuZnSOD was reduced when the expression of PstGSRE4 increased. These results indicated that PstGSRE4 can reduce the enzyme activity of TaCZSOD2.

PstGSRE4 inhibits the activity of TaCZSOD2 in vitro and in vivo.

(A) SDS-PAGE analysis of recombinant proteins purified from E. coli. The protein bands were visualized by Coomassie staining. M, Marker. P1, recombinant protein eGFP-His with induction by isopropyl-β-D-thiogalactopyranoside (IPTG). P2, purified recombinant protein eGFP-His. P3, recombinant protein PstGSRE4-His with induction by IPTG. P4, purified recombinant protein PstGSRE4-His. P5, recombinant protein TaCZSOD2-His with induction by IPTG. P6, purified recombinant protein TaCZSOD2-His. P7, recombinant protein TaCZSOD1-His with induction by IPTG. P8, purified recombinant protein TaCZSOD1-His. (B) Nitro-blue tetrazolium (NBT) photoreduction method was used to detect the activity of SOD. The TaCZSOD1-His was used as negative control. (C-D) Using the co-expression method to detect the activity of CuZnSOD in tobacco. Western blot was performed to show normal expression of PstGSRE4-GFP with anti-GFP antibody, TaCZSOD1-HA and TaCZSOD2-HA with anti-HA antibody. Co-express PstGSRE4-GFP and TaCZSOD1-HA/TaCZSOD2-HA respectively in tobacco, then detect the activity of CuZnSOD by using the CuZnSOD assay kit after 48 hpt (hours post treatment). (E-F) Using PstGSRE4-overexpression lines to detect the activity of CuZnSOD in wheat. The activity of CuZnSOD in different transgene lines were detected by using the CuZnSOD assay kit at 48 hpi with Pst. RT-PCR was used to detect the expression of PstGSRE4. Expression of TaEF-1α showed equal loading. WT, wild type, cv. Fielder. L2-L3, the third generation of PstGSRE4-overexpression wheat lines. Values represent the means ± SE of three independent samples. Asterisks indicate P < 0.05, double asterisks indicate P < 0.01.

Discussion

Glycine- or serine-rich proteins perform important, even decisive roles during infection in various pathogens [27-29,30-33]. However, few glycine-serine-rich effectors (GSREs) have been characterized in Pst. In our previous study, we identified four glycine-serine-rich effectors in Pst. Among these candidates, we focused on PstGSRE1, which contains the m9 motif, and was found to target the ROS-associated transcription factor TaLOL2 [29]. Further sequence analysis of the GSREs revealed that only PstGSRE4 lacks the m9-like motif (), suggesting the functional divergence with other GSREs. Silencing PstGSRE4 decreased Pst growth and development (Figs ), due to the increased ROS accumulation in wheat. ROS have been proposed to orchestrate the establishment of plant defenses following HR [3,34]. In plant cells, ROS has been identified as playing a key role in the development of HR and systemic immunity [35]. Inhibition of this reaction is an important strategy for the successful infection and colonization by obligate biotrophic pathogens. Overexpression of PstGSRE4 suppressed ROS accumulation induced by the avirulent Pst race CYR23 and the deposition of callose induced by EtHAn (), which promoted the infection by Pst. Like PstGSRE4, Pst effector PstGSRE1 was also reported to be involved in suppression of callose deposition and ROS accumulation [29]. In M. oryzae, some glycine- or serine-rich effectors regulate the activity of a variety of antioxidant enzymes, then inhibit the level of ROS in the host, and finally decrease the host immune response [30,31]. Therefore, we speculated that GSRE proteins play an important role in regulating the host immune response, and they may specifically regulate the ROS signal transduction pathway of higher plants. However, although PstGSRE4 contains 12.93% glycine and 19.40% serine, it does not interact with TaLOL2 (), suggesting that it regulates ROS signal pathway in wheat via a different mechanism.

In this study, we found that PstGSRE4 specially interacts with wheat copper zinc superoxide dismutase TaCZSOD2 (), an important isoform of SOD in plants. The plant CuZnSOD isoenzymes differ in their subcellular location, either plastid or cytosolic. Distinct subcellular localization of the individual CuZnSOD isoforms indicates the necessity of superoxide removal within specialized cellular compartments [36]. In Arabidopsis, the three CuZnSOD isoforms (CSD1, CSD2, and CSD3) are localized in the cytoplasm and nucleus, chloroplast and peroxisome, respectively. However, in this study, we found that TaCZSOD2, the ortholog of CSD2 of Arabidopsis, is not only localized in the chloroplast, but also in the cytoplasm (). Overexpression of ΔTP-TaCZSOD2 in N. benthamiana improved the CuZnSOD enzyme activity, but slightly lower than overexpression of full length of TaCZSOD2 (), suggesting that TaCZSOD2 of the chloroplast can also be activated via the CCS-independent pathway when localized in cytoplasm like CSD2 in Arabidopsis [37]. Meanwhile, PstGSRE4 inhibits the CuZnSOD enzyme activity that is increased by overexpression of TaCZSOD2 or ΔTP-TaCZSOD2 in N. benthamiana (). Thus, our data suggest that TaCZSOD2 can simultaneously carry out the function of the enzyme in the cytoplasm and chloroplast. The mechanism by which PstGSRE4 interacts with TaCZSOD2 in the cytoplasm remains to be further investigated. In addition, PstGSRE4 interacts only with TaCZSOD2, suggesting that there is a specific recognition site between PstGSRE4 and TaCZSOD2, but that site remains to be identified. Moreover, like rice CSD2 [21], silencing of TaCZSOD2 resulted in reduced CuZnSOD enzyme activity (), whereas overexpression of TaCZSOD2 led to higher CuZnSOD enzyme activity (), suggesting that TaCZSOD2 is an important CuZnSOD enzyme and positively contributes to CuZnSOD activity. In the process of plant-pathogen interaction, the activity of superoxide dismutase (SOD) positively or negatively correlates with plant disease resistance [38-41], this may be because each pathogen has its own unique mechanism of pathogenicity. However, no reports define the function of CuZnSOD in the interaction between wheat and rust fungi. In our study, silencing TaCZSOD2 in wheat reduced the H2O2 accumulation triggered by the avirulent race CYR23 (Figs ). Meanwhile, compared with the control plants there were fewer necrotic spots and only sporadic uredia on TaCZSOD2-knockdown plants (). Moreover, bacterial delivery of TaCZSOD2 into wheat tissue () and TaCZSOD2-overexpression lines (Figs ) increased wheat resistance to Pst in a ROS-dependent manner. Thus, our results indicated that TaCZSOD2 is a positive regulator of wheat resistance to Pst by increasing the accumulation of H2O2 in wheat.

H2O2 acts as a signal molecule to trigger resistance to various biotic and abiotic stresses [42-44]. Therefore, the pathogens usually secrete effectors to inhibit H2O2 accumulation [45,46]. Moreover, H2O2 also functions as intracellular and intercellular signal molecules to amplify the cellular ROS signals and trigger the HR [47-50]. HR is very effective against obligate biotrophic pathogens. During the interaction between wheat and Pst, the early burst of O2− may be induced by the contact of surface structures of the haustorial initials with the plasma membrane of mesophyll cells, whereas H2O2 is induced to activate HR and other resistance responses [6]. In our study, PstGSRE4 inhibited the activity of TaCZSOD2 in vitro and in vivo (), a detrimental response for wheat to accumulate more H2O2 and successfully resist Pst. Meanwhile, the conclusion is confirmed again by the results that H2O2 and HR induced by CYR23 were reduced in PstGSRE4-overexpression transgenic lines (Figs ) and in TaCZSOD2-knockdown plants (Figs ), along with the increase of O2− level. Thus, our results proved that PstGSRE4 inhibits the activity of total CuZnSOD by inhibiting the enzyme activity of TaCZSOD2 to control moderate H2O2 accumulation upon Pst infection, and further promote stripe rust disease (). In this study, we also identified other three highly conversed TaCZSODs, TaCZSOD1, TaCZSOD3, and TaCZSOD4. The enzyme activity of TaCZSOD1 has been also detected in vitro (), suggesting that TaCZSOD1 may catalyze the production of H2O2 from O2- during Pst infection. Future work will be performed to investigate the functions of the three TaCZSODs during wheat-Pst interaction and reveal the molecular mechanisms of other Pst effectors targeting the CuZnSODs involved in wheat immunity.

In summary, we identified a new glycine-serine-rich effector protein lacking the m9-like motif, PstGSRE4, which inhibits the enzyme activity of the wheat TaCZSOD2 to modulate ROS-associated defense responses. To our knowledge, this is the first direct evidence demonstrating that an effector in phytopathogens regulates the activity of CuZnSOD isoenzymes to suppress plant immunity. Previous studies indicated that CSD genes (mainly CSD1 and CSD2) from wheat and rice also play a positive role during abiotic stress [51,52]. Future studies will focus on using gene editing or overexpression technology to obtain broad-spectrum disease resistance and abiotic stress tolerance materials to advance novel strategies for protecting the wheat crop.

A working model illustrating how PstGSRE4 suppresses ROS-associated wheat immunity by decreasing the activity of TaCZSOD2.

During infection, Pst effector PstGSRE4 is secreted and translocated into host cells. It targets TaCZSOD2 and inhibits the enzyme activity of TaCZSOD2, preventing TaCZSOD2 from carrying out its normal role of adjusting the ROS-mediated HR and disease resistance during the interaction between wheat and Pst.

Materials and methods

Plant materials and fungal Strains

In this study, we used wheat cultivar Suwon11 (Su11), Fielder and N. benthamiana. Wheat cultivar Su11 is highly susceptible to CYR31 and CYR32 and highly resistant to CYR23 [53]. Wheat seedlings were planted, inoculated with Pst and maintained in accordance with the procedures and conditions described previously [54]. Wheat cultivar Fielder was used for transgenic transformation. Co-immunoprecipitation (CoIP) analysis was performed in four-leaf tobacco seedlings permeated with agrobacterium tumefaciens GV3101.

Freshly collected urediniospores of Pst race CYR23 were obtained from the leaves of wheat cultivar Thatcher (MX169), while CYR31 and CYR32 were obtained from Suwon11. The wheat cultivars Su11 and MX169 were grown at 16°C in an artificial climate chamber.

Plasmid construction

The PstGSRE4 gene was cloned using complementary DNA from Pst CYR32. Full-length TaCZSOD genes were cloned from Su11. The amplicons were prepared using the appropriate restriction enzymes () and ligated into pBINGFP2 (a plasmid containing green fluorescent protein, GFP) for transient expression in tobacco, and pEDV6 for transient expression in wheat as well as pSUC2T7M13ORI (pSUC2), pEGX4T-1-GST, pET32a-His with the ClonExpress II One Step Cloning Kit (Vazyme Biotechnology, Nanjing, China). For VIGS analysis, specific cDNA segments of PstGSRE4 and TaCZSODs were predicted by siRNA finder software Si-Fi and then inserted into BSMV-γ carriers with NotI and PacI restriction sites [55]. For Co-IP assays, coding sequences of PstGSRE4 were ligated into pBINGFP2, and TaCZSOD2 and TaCZSOD1 were ligated into pICH86988 (a plasmid containing HA-tag), respectively. For Y2H assays, the coding sequences of PstGSRE4 and TaCZSODs were separately prepared using appropriate restriction enzymes () and ligated into pGADT7 and pGBKT7 vectors. The PstGSRE4 was ligated into pCAMBIA3301 for overexpression in wheat by Agrobacterium-mediated transformation, and the TaCZSOD2 was ligated into CUB for overexpression in wheat by Agrobacterium-mediated transformation.

qRT-PCR analysis

To assay expression levels of PstGSRE4, urediniospores and leaves of wheat Suwon11 infected with CYR32 at 6, 12, 18, 24, 36, 48, 72, 120, 168, 216 and 264 h post-inoculation (hpi) were harvested. To analyze the transcript levels of TaCZSOD2, leaves of Su11 inoculated with CYR23 and CYR31 at 0, 6, 12, 24, 48, 72, 96, 120 hpi were sampled. RNA of all samples was extracted with the Quick RNA isolation Kit (Huayueyang Biotechnology, China, Beijing, 0416-50GK). Approximately 2 μg of the total RNA were also used for reverse transcription using RevertAid First Strand cDNA Synthesis Kit (MNI, K1622). qRT-PCR on a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) was performed in a 25-μl reaction mixture containing 12.5 μl of LightCycler SYBR Green I Master Mix, 2 μl of diluted cDNA (1:5), 8.9 μl of distilled H2O, 0.8 μl of forward primer (10 mM) and 0.8 μl of reverse primer (10 mM). The primers used are listed in . Real-time PCR data were analyzed by the comparative 2-ΔΔCT method to quantify relative gene expression [25]. The expression levels of PstGSRE4 and TaCZSOD2 were normalized to PstEF1 and TaEF-1α, respectively. Each sample was analyzed in three biological replications, and each PCR analysis included three technical repeats. The statistical significance was evaluated by Student’s t-test.

Yeast signal sequence trap system

To validate the function of the predicted signal peptide of PstGSRE4, the yeast signal sequence trap system was used as described previously [56]. The predicted signal peptide sequence of PstGSRE4 was cloned into vector pSUC2T7M13ORI (pSUC2) using the specific primers () and then transformed into the invertase mutant yeast strain YTK12 [57]. To test the secretion function of the recombinant plasmid, positive clones were selected from the CMD-W medium then transferred to YPRAA medium to determine whether the recombinant plasmid had secretory function. In addition, invertase enzymatic activity was detected by the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to insoluble red colored 1,3,5-triphenylformazan (TPF) according to procedures and conditions described previously [58].

Agrobacterium tumefaciens infiltration assays

The sequence encoding PstGSRE4 without the signal peptide (PstGSRE4(ΔSP)) was ligated into pGR107 carrier to construct the agrobacterium recombinant plasmid PVX-PstGSRE4-HA. The Avr1b gene from Phytophthora sojae and eGFP-HA were used as controls (). A. tumefaciens cultures were prepared as described previously [59]. Resuspended A. tumefaciens cultures carrying each effector gene or eGFP at a final OD600 of 0.2 and 10 mM MgCl2 buffer were infiltrated into the leaves of 4-week-old N. benthamiana using a syringe without a needle. After 24 h, A. tumefaciens cultures for delivery of Bax or Pst322 at a final OD600 of 0.2 were also infiltrated into the same site of N. benthamiana leaves. Expression of genes in all infiltration sites was detected by immunoblot three days after infiltration. Symptoms were monitored and recorded from 3 to 8 d after infiltration. Three independent biological replicates were conducted for each experiment.

Yeast two hybrid (Y2H) assay

TaCZSODs was constructed into pGBKT7 as bait, while PstGSRE4(ΔSP) was constructed into pGADT7 as prey (). Then they were co-transformed into yeast strain AH109, plated on SD-Trp-Leu and SD-Trp-Leu-His medium, and cultured at 30°C for 3 to 5 d. The monoclonals grown on SD-Trp-Leu-His were selected and diluted with water, then the interactions were confirmed by growth on the SD-Trp-Leu-His-Ade medium containing X-α-gal.

Bacterial T3SS-mediated overexpression in wheat plants

pEDV6-PstGSRE4(ΔSP), pEDV6-TaCZSOD2 were transformed into P. fluorescens strain EtHAn by electroporation. pEDV6-RFP was used as a control. Infiltration into wheat leaves was performed according to the method described previously [60]. The involvement in Pst pathogenicity or host defense response was tested by challenging the second leaves in pEDV6-PstGSRE4-inoculated wheat plants with Pst avirulent race CYR23 after 24 h. For determination of H2O2 measurements, according to the previously described method [61], the inoculated leaves were sampled at 24 and 48 hpi and determined by 3–3’diaminobenzidine (DAB) staining. To examine the suppression of callose deposition, pEDV6-, pEDV6-PstGSRE4- and pEDV6-RFP-inoculated wheat plants were sampled at 48 hpi. Leaf samples were stained with 0.05% aniline blue in 67 mM K2HPO4 (pH 9.0) overnight in darkness [29]. Leaves were rinsed in water and mounted in 50% glycerol and examined under an Olympus BX-53 fluorescence microscope (Olympus Corporation, Tokyo, Japan) using a DAPI filter. Images were acquired using a constant setting with 1000-ms exposure time. The number of callose deposits was quantified using ImageJ software [62].

Glutathione S-transferase (GST) pull-down assay

PstGSRE4(ΔSP) and TaCZSOD2/TaCZSOD1 were separately ligated into pGEX-4T-1 and pET22b/pET32a through enzyme digestion and ligation. Vectors were transformed into E. coli BL21 cells for protein expression. The corresponding protein was expressed and purified according to the prokaryotic expression procedure. GST-pull down kit (Thermo, Shanghai, China, UB281159) was used to validate the protein interactions in vitro. Another protein was detected by Western blot analysis. Horseradish peroxidase (HRP)-conjugated anti GST-Tag rabbit polyclonal antibody (Cwbiotech, cat. no. CW0144M) and HRP conjugated anti His-Tag mouse monoclonal antibody (Cwbiotech, cat. no. CW0285M) were used for Western blots.

Co-immunoprecipitation assays

PstGSRE4(ΔSP) and TaCZSOD2/TaCZSOD1 were ligated into pBINGFP2 and pICH86988 carriers, respectively. In addition, agrobacterium-mediated transient gene expression technology was used to co-express the above combinations in N. benthamiana. At 48 h after agroinfiltration, 100 μL of co-injected leaf proteins were extracted as the control (Input). Twenty μL of GFP Trap beads were added to the remaining extracts and incubated for 1 h, and centrifuged at 12000g at 4°C for 1min. After removal of the supernatant, the beads in 60 uL volume of wash buffer were mixed with 20 uL of loading buffer, and heated at 100°C for 5min. Precipitated proteins and crude proteins (Input) were detected by immunoblotting with an anti-GFP antibody (#A02020; Abbkine, Wuhan, China) and an anti-HA antibody (Beyotime, AF5057).

Activity assays of CuZnSOD

PstGSRE4-15bs, TaCZSOD2-15bs/TaCZSOD1-15bs and GFP-15bs were expressed in vitro by a prokaryotic expression system, then diluted to the same concentration after purifying by His-tag Purification Resin (BeyoGold, P2210) according to the protocol of manufacturer. The activity of TaCZSOD2/TaCZSOD1 was determined by nitroblue tetrazolium (NBT) reaction [63] in different combinations. The 3 mL reaction mixture contained 39 mM L-methionine 1.5 mL, 225 μM nitroblue tetrazolium (NBT) 0.3 mL, 8 μM riboflavin (dissolve in 30 μM EDTA-Na2 buffer) 0.3 mL, 10 μL purified enzyme and 50 mM potassium phosphate buffer (pH 7.8) 890 μL. The reaction was initiated by illuminating the reaction mixture for 20 min, and photochemically produced superoxide reacted with NBT. Absorbance of formazan, the product of NBT reduction, was then recorded at 560 nm. One unit of SOD activity was defined as the amount of enzyme that caused 50% of the maximum inhibition of NBT reduction. These experiments were repeated three times. A standard curve of protein concentration was obtained with bovine serum albumin as standard [64].

In vivo, we determined the activity of CuZnSOD by using the CuZnSOD assay kit (colorimetry) (Jian Cheng, Nanjing, China, A001-4-1) according to the protocol of manufacturer. Weigh 0.2 g plant tissue sample accurately, add 4 times volume homogenate medium according to mass(g)-volume(ml) ratio of 1:4, cut tissue to small pieces, make homogenate in ice-water bath. Centrifugate at 3500 rpm for 10 min, take supernatant for assay. Take 0.1 ml 20% homogenate supernatant, add 0.2 ml homogenate medium (equals to 3 times dilution), mix sufficiently, take 3 samples of different volumes (10 μl, 30 μl, 50 μl), do pre-test according to operation table in order to determine optimal sample volume. Curve appears direct proportion while inhibition percentage is between 15–55%. Take the tube which inhibition percentage is between 45% to 50% as optimal sample volume. Use xanthine and xanthine oxidase reaction system to produce superoxide anion radicals (O2−), the latter will oxidate hydroxylamine to form nitrite, appears prunosus color under effect of chromogenic agent, its absorbance can be measured by visible range spectrophotometer. If sample to assay contains SOD, then it has a narrow spectrum depressant effect for superoxide anion radicals, as result, absorbance in sample tube will be lower than absorbance in contrast tube, SOD activity can be calculated by formula. MnSOD and FeSOD loss activity in pretreated samples and CuZnSOD activity keeps stable. These experiments were repeated three times.

Barley stripe mosaic virus (BSMV)-mediated silencing

Based on the cloned PstGSRE4 and TaCZSOD2 genes, non-conserved regions were analyzed, and Premier Primer 5.0 was used to design gene silencing vector primers. According to previously described methods [65], two fragments of PstGSRE4 or TaCZSOD2 were cloned and inserted into BSMV to produce BSMV:PstGSRE4-1/2as, BSMV:TaCZSOD2-1/2as. The wheat phytoene desaturase gene (PDS) was silenced as a positive control. BSMV:α and BSMV:β were mixed with BSMV:γ or recombinant γ-gene, in 1:1:1, and then the appropriate amount of FES buffer (2.613g dipotassium phosphate, 1.877 g glycine, 0.5 g sodium pyrophosphate, 0.5 g diatomite, 0.5 g porphyritic soil, 50 ml constant volume, 20 min sterilization by autoclaving) was added. Each independent experiment set FES buffer as a negative control, BSMV:γ as a blank control and BSMV:γ-TaPDS as positive controls for about 10 d to observe the symptoms of virus infection. After 10 to 14 d following inoculation, Pst races CYR23 and CYR31 (fresh urediniospores were collected from the infected leaves of Su11 that were grown at 16°C in artificial climate chamber) were separately inoculated on the fourth leaf of wheat plants, which were placed in a dark and high humidity environment at 12°C for 24 h, then grown in a normal 16/8 h light-dark cycle. The fourth leaves were sampled at 24, 48 or 120 hpi for assessment of silencing efficiency and histological observation. The phenotypes of the fourth leaves were photographed at 12 d after inoculation with Pst. These experiments were repeated three times.

Cytological observations of fungal growth and host response

The observation of necrotic death area hyphae and H2O2 detection assay were performed as previously described [26]. Leaf segments were fixed and decolorized in a mixture of acetic acid/ethanol (1:1) for 3 d. Autofluorescence of mesophyll cells was observed to determine necrotic death area using epifluorescence microscopy (excitation filter, 485 nm; dichromic mirror, 510 nm; barrier filter, 520 nm). H2O2 accumulation was detected by staining with DAB (Amresco, Solon, OH, USA). Hyphae were stained with WGA conjugated to Alexa-488 (Invitrogen, Carlsbad, CA, USA) and observed under blue-light excitation (excitation wavelength 450–480 nm, emission wavelength 515 nm). Only the site where an appressorium had formed over a stoma was considered to be a successful penetration. The H2O2 accumulation, necrotic areas, hyphal length, and hyphal areas were observed with a BX-53 microscope (Olympus) and calculated using DP-BSW software.

Western blotting

Proteins were separated by SDS-PAGE. Gels were blotted onto a PVDF membrane (Merck Millipore, Burlington, MA, USA) with transfer buffer at 64V for 2h. Membranes were blocked for 1 h at room temperature, followed by washing. The antibodies—anti-GFP (1:2,000; #A02020; Abbkine, Wuhan, China), anti-RFP (1:2,000; #A02120; Abbkine), anti-His (1:3,000; #A02050; Abbkine), or anti-GST (1:2,000; #A02030; Abbkine)—were added and incubated at 4°C overnight, followed by three washes. Membranes were then incubated with goat anti-mouse antibody (ab6789; Abcam), or goat anti-rabbit (ab205718; Abcam) at a ratio of 1:10,000 in the blotting buffer at room temperature for 2 h. After three washes, membranes were incubated with chemiluminescence HRP substrate (#WBKLS0100, Merck Millipore) for 5 min, and then visualized by excitation at 780 or 800 nm.

Determination of the accumulation of O2− and H2O2

In vivo, we determined the content of O2− by using the O2− assay kit (SA-2-G, Comin Biotechnology, Suzhou, China) according to the protocol of manufacturer. Weigh 0.1 g inoculated leaves in different hours accurately, add 10 times 65 mM phosphate buffer (pH 7.8) according to mass (g)- volume(mL) ratio of 1:10, cut tissue to small pieces, make homogenate in ice-water bath. Centrifugate at 10000 g for 20 min, take supernatant for assay. The 900 μL reaction mixture contained 0.5 mL homogenate, 0.4 mL 10 mM hydroxylamine solution, 37°C for 20 min. Then add 0.3 mL 17mM 4-aminobenzenesulfonic acid and 0.3 mL 7mM α-naphthylamine, 37°C for 20 min. Then add 0.5 mL 1 chloroform, centrifugate at 8000 g for 5 min, take 1 mL supernatant for assay. Recorded absorbance at 530 nm against a distilled water blank.

We determined the content of H2O2 by using the H2O2 assay kit (A064-1-1, Comin Biotechnology, Suzhou, China) according to the protocol of manufacturer. Weigh 0.1 g inoculated leaves in different hours accurately, add 10 times propanone according to mass (g)—volume (mL) ratio of 1:10, cut tissue to small pieces, make homogenate in ice-water bath. Centrifugate at 8000 g for 10 min, take supernatant for assay. The 1.3 mL reaction mixture contained 1 mL homogenate, 0.1 mL titanic sulfate solution and 0.2 mL ammonium water, centrifugate at 8000 g for 5 min, take sediment for assay. Then add 1 mL sulphuric acid solution to dissolve the sediment, let stand for 10 min at the room temperature. Recorded absorbance at 415 nm against a distilled water blank. These experiments were repeated three times.

Oxidative burst measurement

Leaves from 6-wk-old WT and TaCZSOD2-knockdown or TaCZSOD2-overexpression transgenic lines were sliced into 10 mm2 discs, and maintained overnight in water in a 96-well plate. Then, the leaf discs were treated with 200 μL of solution containing 8nM chitin (hexa-N-acetyl-chitohexaose), 20 μg/ml peroxidase (Sigma-Aldrich) and 20 nM luminol. Luminescence was recorded for 30 min using a multiscan spectrum. Each data point consisted of six replicates. These experiments were repeated three times.

Phylogenetic relationship analysis

Multiple alignment was performed by Muscle in MEGA6.0 [66]. The phylogenetic relationship was inferred based on the multiple alignment in MEGA6.0 by the Maximum Likelihood (ML) method based on LG model with bootstrap 1000. The unrooted tree was performed by Interactive Tree of Life (IToL) Version 3.2.3 (http://itol.embl.de/).

Statistical analyses

Statistical analyses of each treatment were performed with the statistical software version package of IBM SPSS Statistics 21 (IBM SPSS Statistics, IBM Corporation, Armonk, New York, USA).

PstGSRE4 is a Glycine- and Serine-rich secreted protein.

(A) Sequence analysis indicates that PstGSRE4 is a glycine- and serine-rich secreted protein. Triangles indicate serine and asterisks represent glycine residues. Multi-sequence alignment of PstGSRE4 and other three glycine- and serine-rich secreted proteins was performed using CLC Sequence Viewer. (B) The motif of PstGSRE4 (PSTCY32_07414) and other three glycine- and serine-rich secreted proteins were predicted by MEME suit (http://meme-suite.org/). The black borders represent m9 region of PstGSRE1 (PSTCY32_24327). (C) PstGSRE4 cannot interact with TaLOL2. Only the yeast co-expressing PstGSRE4 and TaCZSOD2 or PstGSRE1 and TaLOL2 grew on the medium SD-Trp-Leu-His-Ade and yielded X-α-gal activity. Yeast strains co-expressing PstGSRE4 and TaLOL2 or PstGSRE1 and TaCZSOD2 cannot grow on the medium SD-Trp-Leu-His-Ade. This experiment was repeated three times.

(TIF)

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Relative transcript levels of PstGSRE4 at different Pst infection stages.

Wheat leaves (Suwon11) inoculated with freshly collected urediniospores (CYR32) were sampled at different time points according to the infection stage of Pst. US (Urediniospores) was used as a control. Relative transcript levels of PstGSRE4 were calculated by the comparative threshold (2-ΔΔCT) method. The quantitative RT-PCR values were normalized to the expression level for PstEF-1. The transcript level of PstGSRE4 at US stage was standardized as 1. Values represent the means ± SE of three independent replicates. Differences between time-course points were assessed using Student’s t-test. Double asterisks indicate P < 0.01.

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PstGSRE4 has functional signal peptide.

Functional validation of the putative N-terminal signal peptide of PstGSRE4 using the yeast invertase secretion assay. Yeast YTK12 strains carrying pSUC2-SP (Avr1b) and pSUC2-SP (PstGSRE4), which express two different signal peptides fused in frame to the mature invertase gene SUC2, were able to grow in YPRAA (Yeast-Peptone-Raffinose-Antimycin A) medium with raffinose as sole carbon source. YTK12 or YTK12 strains carrying empty vector pSUC2T7M13ORI were used as negative control. Invertase activity was detected with 2,3,5-triphenyltetrazolium chloride (TTC). The red color indicates invertase activity.

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PstGSRE4 inhibits Pst322- and Bax-induced cell death.

(A) PstGSRE4 suppressed Pst322-induced cell death. Photos of N. benthamiana leaves were taken under ultraviolet light. (B) PstGSRE4 suppressed Bax-induced cell death. Photos of N. benthamiana leaves were taken under ultraviolet light. (C) Western blot with anti-HA antibody was performed to show normal expression of eGFP-HA (25kDa), Pst18363-HA (20kDa), PstGSRE4-HA (22kDa), Pst322-HA (20kDa) and Bax-HA (25kDa) in tobacco leaves.

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PstGSRE4 suppresses Pst322- and Bax-triggered cell death by decreasing ROS accumulation.

(A) H2O2 production in N. benthamiana leaves was determined by DAB staining. The measurement was performed at 3 d after infiltration with Pst322 or Bax (left). And overexpression of Bax or Pst322 in Nicotiana benthamiana triggered programmed cell death (PCD) at 4 d after infiltration with Pst322 or Bax (right). (B) Content of H2O2 in N. benthamiana leaves was determined at 3 d after infiltration with Pst322 or Bax. Values represent the means ± SE of three independent samples. (C) H2O2 production in N. benthamiana leaves was determined by DAB staining. N. benthamiana leaves were infiltrated with A. tumefaciens cells carrying the PstGSRE4-HA or MgCl2 buffer, followed after 24 h by infiltration with A. tumefaciens cells carrying the Bax or Pst322. The measurement was performed at 3 d after infiltration with Bax or Pst322 (left). Overexpression of PstGSRE4 in Nicotiana benthamiana suppressed programmed cell death (PCD) triggered by Bax or Pst322 at 4 d (right). (D) Content of H2O2 in N. benthamiana leaves was determined at 3 d after infiltration with PstGSRE4, MgCl2, PstGSRE4/Bax, PstGSRE4/Pst322, MgCl2/Bax or MgCl2/Pst322. Values represent the means ± SE of three independent samples.

(TIF)

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Overexpression of PstGSRE4 in wheat suppresses PTI-associated callose deposition and H2O2 accumulation.

(A) Phenotypes of wheat leaves (Suwon11) inoculated with CYR23 after being injected with Pseudomonas fluorescens strain EtHAn alone or carrying plasmids pEDV6-RFP (a red autofluorescent protein, DsRed), or pEDV6-PstGSRE4 at 14 dpi. HR, hypersensitive response. (B-C) Wheat leaves inoculated as above were examined for callose deposition by epifluorescence microscopy after aniline blue staining. Scale bars, 100 μm. The average number of callose deposits per mm2 at 24 and 48 hpi was counted using ImageJ software. Values represent the means ± SE (n = 20). (D-E) H2O2 production in leaves infiltrated with EtHAn, EtHAn pEDV6-RFP, or EtHAn pEDV6-PstGSRE4 at 24 and 48 hpi with Pst. SV, substomatal vesicle. Tissues were stained with DAB. Scale bars, 20 μm. The amount of H2O2 production was measured by calculating the DAB-stained area at each infection site using DP-BSW software. Values represent the means ± SE (n = 30). Asterisks indicate a significant difference (P < 0.05) relative to the control sample according to Student’s t-test, double asterisks indicate P < 0.01.

(TIF)

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BSMV-mediated host-induced gene silencing (HIGS) of PstGSRE4 reduces virulence of Pst.

(A) Two specific sequence regions were selected for BSMV-mediated transient silencing. (B) Wheat leaves infected with BSMV:TaPDS, which showed photobleaching phenotype, was used as control. Mild chlorotic mosaic symptoms were observed on the fourth leaves of the wheat inoculated with BSMV:γ, BSMV:PstGSRE4-1as, and BSMV:PstGSRE4-2as. Phenotypes of the fourth leaves of knockdown plants or control plants inoculated with Pst race CYR32 at 12 dpi. (C) Relative transcript levels of PstGSRE4 in PstGSRE4-knockdown plants challenged by CYR32. PstEF-1 was used for normalization. Values represent the means ± SE (n = 3). (D) Ratio of fungal to wheat nuclear content using fungal PstEF-1 and wheat TaEF-1α genes, respectively. Genomic DNA was extracted from the second leaf from three different plants at 14 dpi. Values represent the means ± SE (n = 3). Differences were assessed using Student’s t-test, and asterisks indicate P < 0.05, double asterisks indicate P < 0.01.

(TIF)

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Histological changes of Pst growth in PstGSRE4-knockdown plants.

(A) Fungal growth at 24, 48 and 120 hpi in wheat leaves inoculated with BSMV:γ, BSMV:PstGSRE4-1as and BSMV:PstGSRE4-2as. (B-C) Hyphal lengths (48 and 120 hpi) and colony sizes (120 hpi) in PstGSRE4-knockdown plants were stained with WGA and quantified with DP-BSW software. SV, substomatal vesicle. HMC, haustorial mother cell. IH, infection hypha. Values represent the means ± SE (n = 30, n = 20). Differences were assessed using Student’s t-test, and asterisks indicate P < 0.05.

(TIF)

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Cytological observation of host response in PstGSRE4-RNAi wheat leaves inoculated with Pst race CYR31.

(A) Diagram showing the RNAi cassette in the wheat transformation construct pAHC25-PstGSRE4-RNAi. Ubi1, maize ubiquitin1 promoter. Adh1, Zea mays alcohol dehydrogenase 1. Noc Term, Nos terminator. Bar, Biolaphos resistance gene. LB, left border. RB, right border. (B) Transgenic plants were analyzed by genomic PCR for the presence of the selectable marker Bar gene and the fragment of the RNAi cassette (PstGSRE4). (C) Leaves inoculated with Pst race CYR31 were sampled at 48 and 120 hpi and examined under epifluorescence after staining with WGA conjugated to Alexa-488. SV, substomatal vesicle. HMC, haustorial mother cell. IH, infection hypha. Scale bars, 20 μm (left) and 50 μm (right). (D) H2O2 accumulation was measured in transgenic plants at 72 and 120 hpi. DAB was used to detect H2O2 viewed under differential interference contrast optics. Scale bars, 20 μm. (E) Content of O2− accumulation in different transgene lines at 6, 12, 24 and 48 hpi. Values represent the means ± SE of three independent samples. (F) Quantification of H2O2 accumulation in different transgenic lines at 72 and 120 hpi. Values represent the means ± SE (n = 30). Differences were assessed using Student’s t-test. Asterisk indicates P < 0.05.

(TIF)

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Molecular analysis of PstGSRE4-overexpression wheat.

(A) Diagram showing the overexpression cassette in the wheat transformation construct pCAMBIA3301-PstGSRE4-overexpression. LB, left border. RB, right border. (B-C) Transgenic plants were analyzed by genomic PCR for the presence of the selectable marker Bar gene and the pCAMBIA3301 primer. (D) Expression of PstGSRE4 in T3 lines (L2 and L3) was analyzed by RT-PCR. Expression of TaEF-1α showed equal loading. (E) Content of O2- in different transgenic lines at 6, 12, 24 and 48 hpi. Values represent the means ± SE of three independent samples. (F) Quantification of H2O2 accumulation in different transgenic lines at 48 hpi. (G) H2O2 accumulation at infection sites were observed by microscopy after DAB staining. SV, substomatal vesicle. Scale bars, 20 μm. Values represent the means ± SE (n = 20). (H) Observation of necrotic cell death by epifluorescence in transgenic plants. NC, necrotic cell death. SV, substomatal vesicle. Scale bars, 20 μm. Differences were assessed using Student’s t-test, and asterisks indicate P < 0.05, double asterisks indicate P < 0.01.

(TIF)

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Phylogenetic analysis of SOD in Arabidopsis thaliana, rice and wheat.

Branches are labeled with protein names and GenBank accession number. Os, Oryza sativa. At, Arabidopsis thaliana. The red line represents TaCZSOD2-7A. The tree was created with bootstrap of 1000 by maximum likelihood method in MEGA6. And the tree was drawn using Interactive Tree of Life (IToL).

(TIF)

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Sequence analysis of TaCZSOD2.

Multi-alignment of the coding sequences of the three copies of TaCZSOD2 in the genome database of wheat cultivar Chinese Spring.

(TIF)

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PstGSRE4 interacts with TaCZSOD2 in the cytoplasm of N. benthamiana.

(A) Confocal microscopy images showing the subcellular localization of PstGSRE4 and TaCZSOD2. Western blotting analysis shows the total protein of PstGSRE4-GFP and TaCZSOD2-GFP in N. benthamiana leaves. Scale bars, 50 μm. (B) Co-localization of PstGSRE4-RFP (48 kDa) and TaCZSOD2-GFP (47 kDa) in N. benthamiana. In all panels, proteins were expressed in N. benthamiana through agroinfiltration. Fluorescence was detected in epidermal cells of the infiltrated leaves by confocal microscopy at 48 h after agroinfiltration (prior to any cell death). Scale bars, 50 μm. Localization of RFP-fusion of PstGSRE4 was uniformly located in the cytoplasm and nucleus, GFP-fusion of TaCZSOD2 was primarily located in the chloroplast, but also in the cytoplasm. When expressed together, co-localization of PstGSRE4 and TaCZSOD2 accumulated in the cytoplasm. Western blotting analysis shows the total protein of PstGSRE4-RFP and TaCZSOD2-GFP in N. benthamiana leaves. (C) Co-localization of GFP fusion of chloroplast transit peptide-deleted TaCZSOD2 (ΔTP-TaCZSOD2) and RFP-fusion of PstGSRE4 in N. benthamiana. They accumulated in the cytoplasm and nucleus. Scale bars, 50 μm. (D) Co-expressed GFP fusion of ΔTP-TaCZSOD2 with RFP-fusion of PstGSRE4, GFP fusion of TaCZSOD2 with RFP-fusion of PstGSRE4, GFP fusion of ΔTP-TaCZSOD2 with RFP, GFP fusion of TaCZSOD2 with RFP in N. benthamiana, and detected the activity of CuZnSOD. RFP was expressed as control. Values represent the means ± SE of three independent samples. These experiments were repeated three times and obtained the similar result.

(TIF)

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TaCZSOD2 positively regulates wheat resistance against Pst in an ROS-dependent manner.

(A) Two specific sequence regions were selected for BSMV-mediated transient silencing. (B) Wheat leaves (Suwon11) inoculated with freshly collected urediniospores (CYR23 and CYR31) were sampled at different time points according to the infection stage of Pst. Relative transcript levels of TaCZSOD2 were calculated by the comparative threshold (2-ΔΔCT) method. The quantitative qRT-PCR values were normalized to the expression level for TaEF-1α. Differences between time-course points were assessed using Student’s t-tests. Asterisks indicate P < 0.05, double asterisks indicate P < 0.01. Values represent the means ± SE (n = 3). (C) After inoculated with CYR31, ratio of fungal to wheat nuclear content using fungal PstEF-1 and wheat TaEF-1α genes, respectively. Values represent the means ± SE (n = 3). (D) Content of O2− accumulation in TaCZSOD2-knockdown plants at 6, 12, 24 and 48 hpi. Values represent the means ± SE of three independent samples. (E) Quantification of H2O2 accumulation in TaCZSOD2-knockdown plants at 48 hpi. Values represent the means ± SE (n = 20). (F) H2O2 accumulation at infection sites was observed by microscopy after DAB staining. SV, substomatal vesicle. Scale bars, 10 μm. (G) Observation of necrotic cell death by epifluorescence in TaCZSOD2-knockdown wheat plants. NC, necrotic cell death. SV, substomatal vesicle. Scale bars, 20 μm.

(TIF)

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Transient overexpression of TaCZSOD2 increases wheat resistance against Pst in an ROS-dependent manner.

(A) Phenotypes of pEDV6-RFP- and pEDV6-TaCZSOD2-treated wheat plants inoculated with the virulent Pst race CYR31 at 12 dpi. (B) Quantification of H2O2 accumulation at 48 hpi in pEDV6-RFP- and pEDV6-TaCZSOD2-treated wheat plants inoculated with virulent Pst race CYR31. Values represent the means ± SE (n = 30). (C) Quantification of necrotic cell death area in TaCZSOD2-overexpression plants at 48 hpi. Values represent the means ± SE (n = 20). Differences between time-course points were assessed using Student’s t-tests. Asterisks indicate P < 0.05, double asterisks indicate P < 0.01. (D) H2O2 accumulation was observed by microscopy after DAB staining and necrotic cell death observed by epifluorescence. SV, substomatal vesicle. NC, necrotic cell death. Scale bars, 20 μm.

(TIF)

Click here for additional data file.

Molecular analysis of TaCZSOD2-overexpression transgenic wheat.

(A) Diagram showing the overexpression cassette in the wheat transformation construct CUB-TaCZSOD2-overexpression. LB, left border. RB, right border. (B-C) Transgenic plants were analyzed by genomic PCR and western blotting. (D) Relative transcript levels of TaCZSOD2 in TaCZSOD2-overexpression plants challenged by CYR31. TaEF-1α was used for normalization. Values represent the means ± SE (n = 3). (E) Content of O2− accumulation in different transgene lines at 6, 12, 24 and 48 hpi. Values represent the means ± SE of three independent samples. (F) H2O2 accumulation at infection sites were observed by microscopy after DAB staining. SV, substomatal vesicle. Scale bars, 20 μm. (G) Observation of necrotic cell death by epifluorescence in transgenic plants. NC, necrotic cell death. SV, substomatal vesicle. Scale bars, 20 μm. (H) Quantification of H2O2 accumulation in different transgenic lines at 48 hpi. Values represent the means ± SE (n = 30). Differences between time-course points were assessed using Student’s t-tests. Asterisks indicate P < 0.05.

(TIF)

Click here for additional data file.

TaCZSOD2 increases ROS accumulation.

(A) Relative transcript levels of TaCZSOD2 in TaCZSOD2-knockdown plants were calculated by the comparative threshold (2-ΔΔCT) method. The quantitative qRT-PCR values were normalized to the expression level for TaEF-1α. Values represent the means ± SE (n = 3). (B) Reactive oxygen species (ROS) burst induced by 8 nM chitin in discs of TaCZSOD2-knockdown and WT leaves. Values represent the means ± SE (n = 6). (C) Relative transcript levels of TaCZSOD2 in TaCZSOD2-overexpression transgene lines were calculated by the comparative threshold (2-ΔΔCT) method. The quantitative qRT-PCR values were normalized to the expression level for TaEF-1α. Values represent the means ± SE (n = 3). (D) Reactive oxygen species (ROS) burst induced by 8 nM chitin in discs of TaCZSOD2-overexpression and WT leaves. Values represent the means ± SE (n = 6).

(TIF)

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BlastP analysis of protein sequence similarities between PstGSRE4 and homologous proteins.

(XLSX)

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PstGSRE4 is a specific effector in rust fungi.

(XLSX)

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Partial candidate targets of PstGSRE4 through Y2H screening.

(XLSX)

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Primers used in this study.

(XLSX)

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Alignment of the proteins for phylogenetic analysis of SOD.

(DOCX)

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Statistics to support this study.

(XLS)

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14 Mar 2022

Dear Prof. Guo,

Thank you very much for submitting your manuscript "Glycine-serine-rich effector PstGSRE4 in Puccinia striiformis f. sp. tritici inhibits the activity of copper zinc superoxide dismutase to modulate immunity in wheat" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. A number of additional experiments are requested to address the concerns raised by reviewers. Detailed information for methodology and number of replicates in many of the experiments is missing. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Jie Zhang

Guest Editor

PLOS Pathogens

Hui-Shan Guo

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The work presented by Liu et al shows that a new glycine-serine-rich effector, PstGSRE4, reduces H2O2 accumulation and HR areas to facilitate Puccinia striiforms f. sp. Tritici (Pst) infection, and is highly induced during the early stages of infection. Moreover, PstGSRE4 inhibits the enzyme activity of wheat copper zinc superoxide dismutase TaCZSOD2, which acts as a positive regulator of wheat resistance to Pst. Such systematic research results provide new insights into the molecular mechanisms of GSREs of rust fungi in regulating plant immunity. Overall, I think this research is a major advancement in the plant-microbe interaction field, especially the pathogenic mechanism of biotrophic fungi.

Reviewer #2: The paper describes the function of a glycine-serine-rich effector protein from Puccinia striiformis f.sp. tritici (Pst). The authors demonstrated that this protein inhibits the activity of one of the wheat copper zinc superoxide dismutases (TaCZSOD2) and this way prevents H2O2 accumulation and promotes virulence. The results are well described and the conclusions taken are correct. The results provide a mechanistic understanding on how fungal pathogens manipulate plant immunity. Although this has been addressed in many works, it still remains to be unveiled. My main concern is related to the information provided in the materials and methods. Sometimes I found this information scarce or not clear enough.

Reviewer #3: Liu et al. present work on the molecular characterization of the effector PstGSRE4 and its host target TaCuZnSOD2. Based on previous work on the effector PstGSRE1, they identified members of the glycine/serine-rich effector family (GSRE). PstGSRE4 was found to lack the m9 domain previously found in PstGSRE1 and yeast two-hybrid experiments found that PstGSRE4 did not interact with TaLOL2, the target of PstGSRE1. Secretion assays in yeast found that the signal peptide of PstGSRE4 is functional. PstGSRE4 is expressed early in the interaction with wheat and most highly expressed between 24-48 hpi. They found that infiltration of Pseudomonas fluorescence carrying PstGSRE4 reduced callose formation, indicating that PstGSRE4 can contribute to the suppression of PTI responses. Similar results were observed for transient expression in wheat using Agrobacterium-mediated transformation. Host induced gene silencing of PstGSRE4 using two distinct fragments were integrated into BSMV and caused a reduction in PstGSRE4 transcript accumulation and led to reduced Pst biomass. Transgenic wheat lines expressing an RNAi construct targeting PstGSRE4. Similar to HIGS experiments with BSMV, the stable RNAi caused a reduction in Pst development and an increase in ROS formation (H2O2). Transgenic wheat lines carrying overexpression constructs of PstGSRE4 led to a decrease in ROS formation and increase in Pst development. A yeast two-hybrid screen identified TaCuZnSOD2 as a putative interactor. The interaction was confirmed with expression in E. coli based on pulldown of recombinant PstGSRE4 and TaCuZnSOD2 and Co-IP in Nicotiana benthamiana. Fluorescently tagged PstGSRE4 and TaCuZnSOD2 were found to colocalise in the cytoplasm. Transient silencing of TaCuZnSOD2 using BSMV found that it positively regulates immunity to Pst to an avirulent isolate (CYR23) but not a virulent isolate (CYR31). Enzymatic activity was also reduced. Expression of recombinant proteins of PstGSRE4 and TaCuZnSOD2 showed that PstGSRE4 inhibits the activity of TaCuZnSOD2 in vitro and in vivo. Collectively, this is a systematic study that used almost every available molecular technique to investigate the molecular function of PstGSRE4 and its target, TaCuZnSOD2.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: - In the introduction, it will be important to include information about the differences in activity between O2- and H2O2 since this is critical to understand the results of the work.

- My main concern is related to the description on how the activity oassays of CuZnSOD were performed. Was the protein purified? If so, the methodology should be described. How was the NBT reaction performed? How was the concentration of the proteins determined?

- In the infection assays, line 639: How was the inoculum obtained?

- Determination of accumulation of O2 and H2O2. How was it performed? what is the kit consisting in?

- For most of the experiments, what is considered a biological replicate? Were the experiments independent?

- I could not find information about how the qPCR was performed.

- Why the estimation of H2O2 content was performed using different methodologies in fig1 and 2? Both experiments should use the same methodology.

- The legend of figure 6 is not complete. Specially 6D and 6F need more information about the experiment.

Reviewer #3: No single major issue was identified, but considerable minor issues were found that collectively become a major issue, particularly in data presentation.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Here I list several points or suggestions that I believe would improve the manuscript.

1. In the manuscript, the author described that PstGSRE4 inhibits the enzyme activity of TaCZSOD2 to facilitate Pst infection, but the part of “TaCZSOD2 positively regulates wheat resistance against Pst”, showed that transcript levels of TaCZSOD2 were up-regulated at 12, 48, and 96 hpi with the avirulent Pst race CYR23, and up-regulated at 96 and 120 hpi with the virulent Pst race CYR31. Here, I have several questions. First, does the transcript levels increasing represent the increasing the protein level? Second, CYR23 is an avirulent strain, while CYR31 is a virulent strain, the authors need to illustrate why these two strains cause different expression profiles of TaCZSOD2. Third, which strain includes the effector PstGSRE4? In the model, I can see PstGSRE4 is from the CYR31 strain, but the part of “Relative transcript levels of PstGSRE4 at different Pst infection stages”, showed that PstGSRE4 is from CYR32.

2. The author should add some experiments to prove that PstGSRE4 depends on which pathway to suppresses Pst322- and Bax-induced cell death.

3. ROS burst as an important signal after pathogen infection, and the manuscript proved that TaCZSOD2 is a positive regulator of wheat immunity. I would suggest to identify the ROS burst level in the knockdown and overexpression lines of TaCZSOD2 treated with flg22 or Chitin.

4. Whether GSRE1 interact with CZSOD2 in the Y2H system?

5. Please correct the spelling mistakes in lines 178, 518, 586, 653, et al.

6. Lines 254 and 255, “The hyphal areas during infection of L19 and L76 were significantly reduced at 48 and…..”, I think “significantly reduced” is not an acceptable description based on the result showed in Fig. 1D.

7. The description is not clear in lines 445-447.

8. “d” or “day”, for example in line 564 and line 571, they should be uniform.

Reviewer #2: - A large section of the abstract contains information about a previosly identified effector protein. I would recommend to reduce this part to a minimum.

- Line 325: Indicate what is the meaning of TP

- Material and methods. There are several references of the products that are missing : Line 609, Line 613

- Lines 490-493. What do the authors mean with "at certain levels"? Could this sentence be more precise?

- In figure 1D, hyphal areas are quantified. What is this exactly and how was it performed?

Reviewer #3: For all bar plots in the manuscript, raw data points should be included within each figure. This is even more important when only three biological replicates were performed. Also include the number of fields that were analysed for all the microscopy experiments.

L 377. Change transient for stable transformation.

Figure 3A. Include a negative control for TaCZSOD2 in the yeast-two-hybrid experiment.

Figure 3B. There doesn’t seem to be enrichment of PstGSRE4-GST in the GST pulldown experiment with TaCZSOD1-His. Do you have any idea of why this might have happened? The pulldown should be enriching the GST-tagged effector.

Figure 3C. PstGSRE4-GFP is not present in the input of the CoIP experiments, yet it is detected in other experiments (e.g. Fig. S4C, Fig. S12A). Can you explain why this might be?

Figure 4C. "BSMV:" should be removed. The expression of the other TaCZSODs was checked, but these were not targeted with BSMV.

S1 Fig. State which of the four proteins in panel A are GSRE1 and GSRE4. State the number of replicate experiments performed for panel C.

S4 Fig and S12 Fig. Within the figure legend, list the predicted protein sizes for all five recombinant proteins shown in panel C.

The legend title for S5 Fig is incorrect, change ‘S’ to 5. Which wheat accession was used in this experiment?

S6 Fig. State what is shown in Panel A. Is this genomic DNA or cDNA?

S7 Fig. How many sites were evaluated for Panel B/C? State in legend.

S8 Fig. B. Marker instead of Maker.

For length of scale bar, state “left” and “right” for 20 and 50 um, respectively.

S10 Fig. Alignment of the proteins needs to publicly deposited (Figshare) or provided as supplemental data. The number of bootstraps performed should be included the legend. The methods for tree construction were not present in the Materials and Methods. State that this is an unrooted tree. State explicitly in the legend that this is based on a protein alignment.

S12 Fig. State how many experiments were performed and the consistency of the observations.

S13 Fig. State what is shown in Panel A (cDNA I assume).

S15 Fig C. Lane for L9 in western blot seems to be cropped from a different photo. Make a clear differentiation of photos of different blots.

S3 Table. The IWGSC identifiers should be included for all putative interactors.

**********

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Reviewer #2: No

Reviewer #3: Yes: Matthew Moscou

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13 May 2022

Submitted filename: Response to comments on PstGSRE4 ms.docx

Click here for additional data file.

11 Jun 2022

Dear Dr. Guo,

Thank you very much for submitting your manuscript "Glycine-serine-rich effector PstGSRE4 in Puccinia striiformis f. sp. tritici inhibits the activity of copper zinc superoxide dismutase to modulate immunity in wheat" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Reviewer 4 raised a minor issue on the actitvty of TaCZSOD1/3/4. Please address or discuss about this issue.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Jie Zhang

Guest Editor

PLOS Pathogens

Hui-Shan Guo

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In the revised manuscript, the authors have fully addressed all of my concerns. I believe the data to be convincing and sufficient to demonstrate the role of Puccinia striiformis f. sp. tritici (Pst) secretes Glycine-serine-rich effector protein PstGSRE4 manipulate plant immunity through inhibition of the enzyme activity of wheat copper zinc superoxide dismutase TaCZSOD2, and reduction of H2O2 accumulation and HR areas to facilitate Pst infection. Clearly, many interesting avenues for follow up will emerge from this work from the authors as well as other groups in the field of plant-microbe interaction. I’m looking forward to seeing this work published.

Reviewer #2: The authors replied to all the comments I made on the previous version of the manuscript and I do not have further comments or queestions

Reviewer #4: This study reported a new glycine-serine-rich effector protein lacking the m9-like motif, PstGSRE4, which inhibits the enzyme activity of the wheat TaCZSOD2 to modulate ROS-associated defense responses. By inhibiting the enzyme activity of TaCZSOD2, PstGSRE4 reduces H2O2 accumulation and HR to facilitate Puccinia striiformis f. sp. tritici infection. These findings provide new insights into the molecular mechanisms of GSREs of phytopathogenic fungi in regulating plant immunity.

In this revision, authors have address all concerns and suggestions raised by reviewers. Thus, I think that this revision could be accepted for publication in PLoS Pathogens.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: NO.

Reviewer #2: (No Response)

Reviewer #4: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: NO.

Reviewer #2: (No Response)

Reviewer #4: I have only one minor point that could be discussed. In addition to CZSOD2, the wheat (cultivar Suwon11) contains other three highly conversed TaCZSODs (TaCZSOD1, TaCZSOD3, TaCZSOD4). Since PstGSRE4 can directly interact with TaCZSOD2, and inhibit its’ activity. Why cannot TaCZSOD1/3/4 catalyze the production of H2O2 from O2- during Pst infection？

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Reviewer #1: No

Reviewer #2: No

Reviewer #4: No

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20 Jun 2022

Submitted filename: Response to comments on PstGSRE4 msR2.docx

Click here for additional data file.

23 Jun 2022

Dear Dr. Guo,

We are pleased to inform you that your manuscript 'Glycine-serine-rich effector PstGSRE4 in Puccinia striiformis f. sp. tritici inhibits the activity of copper zinc superoxide dismutase to modulate immunity in wheat' has been provisionally accepted for publication in PLOS Pathogens.

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Hui-Shan Guo

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Kasturi Haldar

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PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

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Reviewer Comments (if any, and for reference):

12 Jul 2022

Dear Dr. Guo,

We are delighted to inform you that your manuscript, "Glycine-serine-rich effector PstGSRE4 in Puccinia striiformis f. sp. tritici inhibits the activity of copper zinc superoxide dismutase to modulate immunity in wheat," has been formally accepted for publication in PLOS Pathogens.

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Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064