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. 2023 Oct 2;24(12):1522–1534. doi: 10.1111/mpp.13390

The interaction of two Puccinia striiformis f. sp. tritici effectors modulates high‐temperature seedling‐plant resistance in wheat

Xiyue Bao 1, Yangshan Hu 1,2, Yuxiang Li 1, Xianming Chen 3, Hongsheng Shang 1, Xiaoping Hu 1,✉
PMCID: PMC10632793  PMID: 37786323

Abstract

Wheat cultivar Xiaoyan 6 (XY6) has high‐temperature seedling‐plant (HTSP) resistance to Puccinia striiformis f. sp. tritici (Pst). However, the molecular mechanism of Pst effectors involved in HTSP resistance remains unclear. In this study, we determined the interaction between two Pst effectors, PstCEP1 and PSTG_11208, through yeast two‐hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and pull‐down assays. Transient overexpression of PSTG_11208 enhanced HTSP resistance in different temperature treatments. The interaction between PstCEP1 and PSTG_11208 inhibited the resistance enhancement by PSTG_11208. Furthermore, the wheat apoplastic thaumatin‐like protein 1 (TaTLP1) appeared to recognize Pst invasion by interacting with PSTG_11208 and initiate the downstream defence response by the pathogenesis‐related protein TaPR1. Silencing of TaTLP1 and TaPR1 separately or simultaneously reduced HTSP resistance to Pst in XY6. Moreover, we found that PstCEP1 targeted wheat ferredoxin 1 (TaFd1), a homologous protein of rice OsFd1. Silencing of TaFd1 affected the stability of photosynthesis in wheat plants, resulting in chlorosis on the leaves and reducing HTSP resistance. Our findings revealed the synergistic mechanism of effector proteins in the process of pathogen infection.

Keywords: effector protein, high‐temperature seedling‐plant resistance, pathogenesis‐related protein, Puccinia striiformis f. sp. tritici , wheat stripe rust

The Puccinia striiformis f. sp. tritici effector PstCEP1 interacts with PSTG_11208 to disturb recognition by TaTLP1; PstCEP1 could target TaFd1 to inhibit the high‐temperature seedling‐plant resistance of wheat.

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1. INTRODUCTION

Wheat stripe rust, caused by the obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst), can severely reduce grain yield (Wellings, 2011). Control of wheat stripe rust mainly relies on resistant cultivars, fungicide application, and cultivation management measures (Chen et al., 2013; Li & Zeng, 2002). Many resistant cultivars possess only race‐specific resistance and often become susceptible due to the appearance and increase of specific virulence genes in the pathogen population (Chen, 2007). Therefore, it is critical to understand the interactions between wheat and Pst in order to improve the control of stripe rust.

Wheat resistance to Pst is classified as all‐stage resistance (ASR) and adult‐plant resistance (APR) (Chen, 2007). ASR refers to resistance throughout all growth stages of host plants and is usually race‐specific and controlled by qualitatively inherited genes. APR refers to resistance at adult stages of plants that are susceptible to the same pathogen races at the seedling stage. High‐temperature adult‐plant (HTAP) resistance is a major type of APR that is more effective when the temperature is relatively high (Chen, 2013). HTAP resistance is nonrace‐specific and its level is affected by plant growth stage, temperature, and disease pressure. In a previous study, a slightly different type of wheat resistance to Pst was identified, referred to as high‐temperature seedling‐plant (HTSP) resistance (Shang, 1998). Wheat plants with only HTSP resistance are susceptible at relatively low temperatures (e.g., a 15/10°C diurnal cycle), but become resistant when kept at a relatively high temperature (20°C) for 24 h during the latent period of Pst infection (Hu et al., 2012, 2023). So far, numerous HTAP resistance genes and quantitative trait loci have been reported and are widely used in breeding for resistance to stripe rust in the United States (Chen, 2013; Feng et al., 2018; Li et al., 2023; Liu et al., 2018, 2019; Zhou et al., 2023). Previous studies identified 1395 differentially expressed genes (DEGs) in wheat that are involved in HTSP resistance by transcriptome profiling, including genes encoding resistance proteins, transcription factors, and protein kinases (Tao et al., 2018). Several of the DEGs have been determined to influence the HTSP resistance of wheat through different signal pathways; these genes include TaRPM1, TaRPS2, TaWRKY70, TaWRKY49, TaWRKY62, TaCRK10, and TaRIPK (Hu et al., 2021, 2022; Wang et al., 2019, 2021; Wang, Tao, An, et al., 2017; Wang, Tao, Tian, et al., 2017).

In the process of pathogen infection, effector proteins secreted by the pathogen inhibit the host immune response, protect the pathogen, and facilitate infection (Uhse & Djamei, 2018). Pst forms haustoria and releases effector proteins during infection, which target different proteins of the host and perform different functions. PstGSRE1, a glycine‐serine‐rich effector, disrupts the nuclear localization of the reactive oxygen species (ROS)‐associated transcription factor TaLOL2 and suppresses ROS‐induced cell death mediated by TaLOL2, compromising host immunity (Qi et al., 2019). PstGSRE4 inhibits the enzyme activity of the wheat copper zinc superoxide dismutase TaCZSOD2 to facilitate Pst infection (Liu et al., 2022). PsSpg1 enhances the activity of the wheat receptor‐like cytoplasmic kinase TaPsIPK1 to disrupt the immune priming (Wang et al., 2022). Effector proteins perform their functions in the host cytoplasm or apoplast to interfere with plant immunity and assist the pathogen to obtain nutrients (Rocafort et al., 2020). Pst effectors are vital for infection, but it is not clear which effectors affect HTSP resistance and how they interact with wheat genes.

In previous studies, transcriptome sequencing analysis identified 25 DEGs of Pst related to HTSP resistance, of which the gene encoding the Pst candidate effector protein 1 (PstCEP1) was most highly up‐regulated. Moreover, silencing of PstCEP1 reduced the pathogenicity of Pst (Tao et al., 2020). In the present study, we determined the molecular functions of two Pst effectors, PstCEP1 and PSTG_11208. We found that PSTG_11208 interacted with PstCEP1 and induced HTSP resistance in wheat cultivar Xiaoyan 6 (XY6) through recognition by the wheat apoplastic thaumatin‐like protein 1 (TaTLP1). PstCEP1 and PSTG_11208 have different functions in HTSP resistance. We found that the interaction between PstCEP1 and PSTG_11208 affected the recognition of PSTG_11208 by plants. In addition, PstCEP1 targeted wheat ferredoxin 1 (TaFd1) to affect HTSP resistance to Pst.

2. RESULTS

2.1. PstCEP1 interacts with effector PSTG_11208 in vitro and in vivo

In our previous study, we showed that PstCEP1 was up‐regulated in response to HTSP resistance in XY6 and also showed an important effect on the basic pathogenicity of Pst (Tao et al., 2020). In the present study, PstCEP1 ΔSP was cloned into vector pGBKT7 (BD) as bait for the yeast two‐hybrid (Y2H) assay to determine which proteins PstCEP1 interacts with during HTSP resistance. Firstly, we determined that the Y2HGold strain carrying the recombinant plasmid expressing BD‐PstCEP1ΔSP could not grow normally on synthetic defined (SD) medium without tryptophan but supplemented with 5‐bromo‐4‐chloro‐3‐indoxyl‐α‐d‐galactopyranoside (X‐α‐Gal) and aureobasidin A (AbA) (single dropout, SDO/X/A) (Figure S1), indicating that BD‐PstCEP1ΔSP could not activate the reporter gene AbAr and hence that BD‐PstCEP1ΔSP has no self‐activation activity. The size of Y2HGold colonies expressing BD‐PstCEP1ΔSP grown on SDO/X medium was consistent with that of colonies containing empty vector BD (Figure S1). These results indicated that BD‐PstCEP1ΔSP has no toxic effects on yeast growth and does not have self‐activation activity. Therefore, it could be used for further Y2H screening.

We screened the potential interactions between PstCEP1 and different proteins using a Y2H library via yeast mating. Then, we cloned the open reading frames (ORFs) of candidate genes into the pGADT7 (AD) vector for accurate verification. The results showed that the yeast strains co‐transformed with BD‐PstCEP1ΔSP and AD‐PSTG_11208ΔSP or BD‐PstCEP1ΔSP and AD‐TaFd1 grew normally and turned blue on SD medium lacking tryptophan, leucine, histidine, and adenine with X‐α‐gal and AbA (quadruple dropout, QDO/X/A), similar to the positive control (Figure 1a), indicating that PstCEP1 interacted separately with PSTG_11208 and TaFd1 in yeast cells.

FIGURE 1.

FIGURE 1

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Identification of the interaction between PstCEP1 and PSTG_11208. (a) Yeast two‐hybrid analysis indicating that PstCEP1 interacts with PSTG_11208 and TaFd1. Y2HGold cells transformed with the labelled constructs were assayed for growth on synthetic defined (SD) medium lacking tryptophan and leucine (double dropout, DDO) and SD medium lacking tryptophan, leucine, histidine, and adenine with 5‐bromo‐4‐chloro‐3‐indoxyl‐α‐d‐galactopyranoside (X‐α‐gal) and aureobasidin A (AbA) (quadruple dropout, QDO/X/A). Y2HGold cells carrying BD‐murine P53 (53) and AD‐SV40 large T‐antigen (T) or BD‐human lamin C (Lam) and AD‐T were used as the positive and negative controls, respectively. Photographs were taken after incubation at 30°C for 72 h. (b) Bimolecular fluorescence complementation assay indicating that PstCEP1 interacts with PSTG_11208. PSTG_11208ΔSP fused with the N‐terminus of yellow fluorescent protein (YFPN) and PstCEP1ΔSP fused with the C‐terminus of YFP (YFPC) were transiently overexpressed in Nicotiana benthamiana. Coexpression of YFPN‐PSTG_11208ΔSP and the empty YFPC vector or PstCEP1ΔSP‐YFPC and the empty YFPN vector were used as negative controls. The YFP images were taken 48 h later. Bars = 50 μm. (c) Pull‐down assay indicating that PstCEP1 interacts with PSTG_11208. PSTG_11208ΔSP‐GST‐ or GST‐bound resin was incubated with Escherichia coli crude extract containing PstCEP1ΔSP‐MBP. Proteins were detected by western blot using anti‐GST or anti‐MBP. The asterisks represent protein PSTG_11208ΔSP‐GST. GST, glutathione S‐transferase; MBP, maltose‐binding protein; SP, signal peptide.

The interaction between PstCEP1 and PSTG_11208 was confirmed by the bimolecular fluorescence complementation (BiFC) assay. Yellow fluorescence was detected in Nicotiana benthamiana leaves infiltrated with YFPN‐PSTG_11208ΔSP and PstCEP1ΔSP‐YFPC, while no fluorescence was detected in the negative controls (Figure 1b), indicating that PstCEP1 and PSTG_11208 interacted in vivo. To investigate the direct interaction between PstCEP1 and PSTG_11208 in vitro, glutathione S‐transferase (GST), PSTG_11208ΔSP‐GST, and PstCEP1ΔSP‐MBP (maltose‐binding protein) were expressed in bacterial cells in a pull‐down assay. We found that PstCEP1 interacted with PSTG_11208 in vivo (Figure 1c).

2.2. PSTG_11208 can be transported to the nucleus, cytoplasm, and membranes of plant cells

The sequence of the first 17 amino acids at the N‐terminus of PSTG_11208 was predicted to be a signal peptide (SP) by SignalP v. 5.0. In order to confirm the secretory function of the SP, we observed the growth of the mutant yeast strain YTK12 and performed a chromogenic reaction. We found that the SP could make YTK12 grow normally on YPRAA medium (Figure S2a) and could transform 2,3,5‐triphenyltetrazolium chloride to red insoluble 1,3,5‐triphenylformazan (Figure S2b), consistent with the positive control Avr1b. These results indicated that the SP of PSTG_11208 has a secretory function.

Next, the subcellular localization of PSTG_11208ΔSP in N. benthamiana leaves was analysed. The data showed that the PSTG_11208ΔSP‐eGFP (enhanced green fluorescent protein) fusion protein was distributed in the nucleus, cytoplasm, and membrane (Figure S2c). The western blot assay showed that PSTG_11208ΔSP‐eGFP was successfully expressed in N. benthamiana (Figure S2d). To further confirm the results, the subcellular localization of PSTG_11208ΔSP was analysed in wheat protoplasts (Figure S2e). PSTG_11208ΔSP signals were found in the nucleus, cytoplasm, and membrane of wheat plants.

2.3. PSTG_11208 contributes to HTSP resistance in wheat

To study the effect of PSTG_11208 on the infection of Pst in XY6, a 124‐bp specific sequence of PSTG_11208 was inserted into the barley stripe mosaic virus (BSMV):γ vector to silence PSTG_11208. Ten days after BSMV inoculation, the fourth leaves of XY6 inoculated with BSMV:TaPDS showed photobleaching symptoms, and the leaves inoculated with BSMV:00 and BSMV:PSGT_11208 showed stripe mosaic symptoms, while the leaves treated with FES buffer were normal green (Figure 2a), indicating that the silencing system was successfully constructed. Then, the leaves were inoculated with urediniospores of Pst race CYR32 and put in a growth chamber with either a 16/8 h diurnal cycle at 15/10°C in light/dark (treatment N) or a 16/8 h diurnal cycle at 15/10°C for the first 192 h, followed by 24 h at 20°C and back to 15/10°C with the same light/dark setting (treatment NHN). The data indicated that the silencing efficiency of PSTG_11208 was 36.6%–57.1% (Figure 2d). The hyphal length of Pst was increased in BSMV:PSGT_11208 (Figure 2a; Figure S3a–d). No significant differences were detected in the number of Pst uredinia in BSMV:00 and BSMV:PSGT_11208 after treatment N, whereas a significant increase in the number of uredinia was observed in BSMV:PSGT_11208 after treatment NHN (Figure 2f). In addition, no significant differences in the growth of uredinia were found between BSMV:00 and BSMV:PSGT_11208 after treatment N, but BSMV:PSGT_11208 uredinia were longer than BSMV:00 uredinia after treatment NHN (Figure S3e–h), suggesting that silencing of PSTG_11208 decreased HTSP resistance to Pst.

FIGURE 2.

FIGURE 2

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Barley stripe mosaic virus (BSMV)‐mediated gene silencing of PSTG_11208. (a) Phenotypes of the wheat leaves at 10 days post‐inoculation with BSMV. (b, c) Phenotypes of wheat leaves at 312 hours post‐inoculation (hpi) with Puccinia striiformis f. sp. tritici (Pst) race CYR32 on the fourth leaves of wheat in the normal temperature (N, 15/10°C) treatment and the normal‐high‐normal temperature (NHN, 15/10°C for 192 h followed by 20°C for 24 h and back to 15/10°C) treatment. (d) Silencing efficiency assessment of PSTG_11208. The asterisks indicate significant differences in the relative transcriptional expression levels of PSTG_11208 between BSMV:PSTG_11208‐ and BSMV:00‐inoculated plants (p = 0.05, Student's t test). (e) Hyphal length of Pst at 48 and 120 hpi. Student's t test was conducted to test for statistical significance (p = 0.05). (f) Uredinial number per unit leaf area of Pst‐inoculated nonsilenced and PSTG_11208‐silenced wheat plants at 312 hpi in the different temperature treatments. Duncan's multiple range test was conducted to test for statistical significance (p = 0.05).

2.4. Overexpression of PSTG_11208 and PstCEP1 affects resistance to Pst

It has been proven that overexpression of PstCEP1 could enhance the pathogenicity of Pst to affect HTSP resistance (Tao et al., 2020). To determine the roles of the PSTG_11208–PstCEP1 interaction in HTSP resistance to Pst, we overexpressed PSTG_11208 in wheat. Overexpression of PSTG_11208 significantly increased the accumulation of callose in wheat, while the accumulation of callose after co‐overexpression of PstCEP1 and PSTG_11208 in wheat was not significantly different from that of wildtype EtHAn or after overexpression of the red fluorescent protein dsRed (Figure 3a). After inoculation with Pst, the resistance of wheat plants overexpressing PSTG_11208 was significantly enhanced in both treatments N and NHN, while co‐overexpression with PstCEP1 inhibited the resistance enhancement (Figure 3b–d).

FIGURE 3.

FIGURE 3

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Bacterial type III secretion system‐mediated overexpression of PSTG_11208 and PstCEP1. (a) Callose deposition of wheat leaves infiltrated with MgCl2, EtHAn, pEDV6‐dsRed, pEDV6‐PstCEP1, pEDV6‐PSTG_11208, and pEDV6‐PstCEP1 and pEDV6‐PSTG_11208. Blue fluorescent spots represent callose deposition. Bars = 500 μm. (b) Stripe rust phenotypes of wheat plants infiltrated with wildtype EtHAn and EtHAn carrying dsRed or PSTG_11208 or PSTG_11208 and PstCEP1 at 15 days after Puccinia striiformis f. sp. tritici (Pst) inoculation in the different temperature treatments. (c) Average number of callose deposition spots per mm2 in wheat leaves. Duncan's multiple range test was conducted to test for statistical significance (p = 0.05). (d) Uredinial number per unit leaf area of Pst‐inoculated wheat plants. Duncan's multiple range test was conducted to test for statistical significance (p = 0.05).

2.5. PSTG_11208 can be recognized by wheat TaTLP1

In order to confirm the function of PSGT_11208 in the HTSP resistance of XY6, the target of PSGT_11208 was identified by a Y2H assay. First, the self‐activation of BD‐PSTG_11208ΔSP was examined. Y2HGold cells co‐transformed with BD‐PSTG_11208ΔSP and AD plasmid grew normally and turned blue on SDO/X/A medium (Figure S4a), demonstrating that BD‐PSTG_11208ΔSP has obvious self‐activation properties. Different concentrations of 3‐amino‐1,2,4‐triazole (3‐AT) were screened to find the best concentration to inhibit the self‐activation of BD‐PSTG_11208ΔSP. Based on the results, a 3‐AT concentration of 80 mM was selected for further screening of the yeast cDNA library (Figure S4b).

The candidate protein TaTLP1 was found to interact with PSGT_11208 by Y2H screening. Y2HGold cells co‐transformed with BD‐PSTG_11208ΔSP and AD‐TaTLP1ΔSP or BD‐TaTLP1ΔSP and AD‐PSTG_11208ΔSP grew normally and turned blue on QDO/X/A medium supplemented with 3‐AT (80 mM) (Figure 4a), whereas cells co‐transformed with BD‐TaTLP1ΔSP and empty vector AD did not grow (Figure 4a). These results indicated that PSTG_11208 interacted with TaTLP1. The interaction between PSTG_11208 and TaTLP1 was further confirmed through the BiFC assay. Yellow fluorescence was detected in N. benthamiana leaves coexpressing YFPN‐PSTG_11208ΔSP and TaTLP1ΔSP‐YFPC, but no fluorescence was detected in the negative controls (Figure 4b), confirming that TaTLP1 and PSTG_11208 interacted in plants. The pull‐down assay showed that TaTLP1 interacted with PSTG_11208 in vivo (Figure 4c).

FIGURE 4.

FIGURE 4

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Identification of the interaction between PSTG_11208 and TaTLP1. (a) Yeast two‐hybrid analysis indicating that PSTG_11208 interacts with TaTLP1. Y2HGold cells transformed with the labelled constructs were analysed for growth on synthetic defined (SD) medium lacking tryptophan, leucine, histidine, and adenine with 5‐bromo‐4‐chloro‐3‐indoxyl‐α‐d‐galactopyranoside (X‐α‐gal), aureobasidin A (AbA), and 3‐AT (quadruple dropout, QDO/X/A/3AT). Y2HGold cells carrying BD‐murine P53 (53) and AD‐SV40 large T‐antigen (T) or BD‐human lamin C (Lam) and AD‐T were used as the positive and negative controls, respectively. Photographs were taken after incubation at 30°C for 72 h. (b) Bimolecular fluorescence complementation assay indicating that PSTG_11208 interacts with TaTLP1. PSTG_11208ΔSP fused with the N‐terminus of yellow fluorescent protein (YFPN) and TaTLP1ΔSP fused with the C‐terminus of YFP (YFPC) were transiently overexpressed in Nicotiana benthamiana leaves. Coexpression of YFPN‐PSTG_11208ΔSP and the empty YFPC vector or TaTLP1ΔSP‐YFPC and the empty YFPN vector were used as negative controls. The YFP images were taken 48 h later. Bars = 50 μm. (c) Pull‐down assay indicating that PSTG_11208 interacts with TaTLP1. PSTG_11208ΔSP‐GST‐ or GST‐bound resin was incubated with Escherichia coli crude extract containing TaTLP1ΔSP‐His. Proteins were detected by western blot using anti‐GST or anti‐His. The asterisks represent protein PSTG_11208ΔSP‐GST. (d) PstCEP1 competes with TaTLP1 to interact with PSTG_11208 in vivo. PSTG_11208ΔSP‐GST‐bound resin was incubated with E. coli crude extract containing TaTLP1ΔSP‐His, PstCEP1‐MBP, and MBP. Proteins were detected by western blot using anti‐GST, anti‐His, or anti‐MBP. GST, glutathione S‐transferase; MBP, maltose‐binding protein; SP, signal peptide.

Co‐overexpression of PSTG_11208 and PstCEP1 inhibited the resistance enhancement by PSTG_11208. We speculated that the interaction between PstCEP1 and PSTG_11208 inhibited the recognition of PSTG_11208 by TaTLP1. To test this hypothesis, PSTG_11208ΔSP‐GST, TaTLP1ΔSP‐His, PstCEP1ΔSP‐MBP, and MBP were expressed to perform a pull‐down assay. When the abundance of PstCEP1ΔSP‐MBP increased, the abundance of TaTLP1ΔSP‐His interacting with PSTG_11208ΔSP‐GST was reduced, indicating that PstCEP1ΔSP could interfere with the interaction between TaTLP1 and PSTG_11208 (Figure 4d).

2.6. TaTLP1 and TaPR1 synergistically regulate HTSP resistance in XY6

A previous study showed that the TaTLP1–TaPR1 interaction positively contributes to wheat resistance to Puccinia triticina, the leaf rust pathogen (Wang, Yuan, et al., 2020). In order to determine the roles of TaTLP1 and TaPR1 in HTSP resistance to Pst, gene silencing experiments were conducted with specific sequences of TaTLP1 and TaPR1. Ten days after BSMV inoculation, the wheat leaves inoculated with BSMV:TaPDS displayed photobleaching symptoms and the leaves inoculated with BSMV:00, BSMV:TaTLP1, BSMV:TaPR1, and BSMV:TaTLP1 + TaPR1 showed stripe mosaic symptoms, while the leaves treated with FES buffer were normal green (Figure 5a), showing that the silencing system was successfully constructed. After we inoculated XY6 plants with CYR32, we determined the expression levels of TaTLP1 and TaPR1. The silencing efficiencies of TaTLP1 and TaPR1 were 41.6%–79.4% and 47.8%–82.9% in the single‐gene silencing treatments and 29.3%–66.6% and 28.3%–73.4% in the cosilencing treatment, respectively, in both treatments N and NHN (Figure 5e,f). The number of uredinia on wheat leaves was counted 14 days after Pst inoculation. The results showed that silencing of either TaTLP1 or TaPR1 in treatment N had no significant effects on the number of uredinia (Figure 5b,d), whereas silencing of either TaTLP1 or TaPR1 in treatment NHN significantly increased the number of uredinia (Figure 5c,d). Cosilencing of TaTLP1 and TaPR1 had no additive effect.

FIGURE 5.

FIGURE 5

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Barley stripe mosaic virus (BSMV)‐mediated gene cosilencing of TaTLP1 and TaPR1. (a) Phenotypes of wheat leaves at 10 days after inoculation with BSMV. (b, c) Phenotypes at 312 h post‐inoculation (hpi) with Puccinia striiformis f. sp. tritici (Pst) race CYR32 on the fourth leaves of wheat in the normal temperature (N, 15/10°C) treatment and the normal‐high‐normal temperature (NHN, 15/10°C followed by 20°C for 24 h and back to 15/10°C) treatment. (d) Uredinial number per unit leaf area of Pst‐inoculated wheat plants at 312 hpi in the different temperature treatments. (e) Silencing efficiency assessment of TaTLP1. (f) Silencing efficiency assessment of TaPR1. Duncan's multiple range test was conducted to test for statistical significance (p = 0.05).

2.7. TaFd1 accumulates in the chloroplasts and participates in HTSP resistance

OsFd1 has been reported to affect photosynthetic electron transport and carbon assimilation in rice (He et al., 2020). We aligned the amino acid sequences of TaFd1 and OsFd1 and found that their similarity exceeded 70% (Figure S5). Firstly, we fused TaFd1 with an eGFP fluorescent tag to determine the localization of TaFd1 and found that TaFd1 was localized in the chloroplasts of N. benthamiana leaves (Figure S6a). The TaFd1‐eGFP fusion protein was successfully detected in N. benthamiana by western blot analysis (Figure S6b).

To study the function of TaFd1 in the HTSP resistance of XY6, we selected two specific fragments of TaFd1 to perform gene silencing. Ten days after BSMV inoculation, BSMV:TaPDS showed photobleaching symptoms, while BSMV:00, BSMV:TaFd1‐1, and BSMV:TaFd1‐2 showed stripe mosaic symptoms (Figure S6c). The silencing efficiencies of TaFd1‐1 and TaFd1‐2 were 24.1%–42.2% and 32.2%–60.8%, respectively (Figure S6f). At 312 h after inoculation with Pst, stripe rust was observed on wheat leaves. The number of uredinia on BSMV:TaFd1‐1 and BSMV:TaFd1‐2 leaves was similar to that on BSMV:00 leaves in treatment N (Figure S6d,g), whereas the number of uredinia on BSMV:TaFd1‐1 and BSMV:TaFd1‐2 leaves was significantly higher than that on BSMV:00 leaves in treatment NHN (Figure S6e,g). In addition, TaFd1‐silenced wheat leaves showed photobleaching and chlorosis (Figure S6d,e).

TaFd1 was localized in chloroplasts of N. benthamiana leaves. We further verified the localization of PstCEP1. A 22–62‐amino‐acid sequence was predicted to be a chloroplast‐targeting sequence (Figure S7a). We fused truncated proteins to the N‐terminus of eGFP to perform the localization of PstCEP1. In N. benthamiana cells, the fusion proteins PstCEP1‐eGFP and PstCEP163‐243‐eGFP were observed in chloroplasts, and removing the chloroplast‐targeting sequence disturbed the subcellular localization of PstCEP1 (Figure S7b). The subcellular localization of PstCEP1 was analysed in wheat protoplasts, and the results showed that the PstCEP1 signal overlapped with the partial autofluorescence of chloroplasts (Figure S7c). These data showed that PstCEP1 could be a chloroplast‐targeted protein.

3. DISCUSSION

Previous studies have shown that effector genes are up‐regulated in the process of wheat infection by Pst, such as Pst_8713, Pst30, PSTha5a23, Pst18363, and PstGSRE4 (Cheng et al., 2017; Liu et al., 2022; Wang et al., 2022; Wang, Fan, et al., 2020; Yang et al., 2020; Zhao et al., 2018). Tao et al. (2020) found that the expression level of PstCEP1 was rapidly up‐regulated in treatment NHN, and silencing of PstCEP1 reduced the pathogenicity of Pst in both treatments N and NHN, indicating that PstCEP1 affects not only HTSP resistance of wheat, but also the pathogenicity of Pst. In the present study, we determined that PstCEP1 interacts with effector protein PSTG_11208, but they have different functions in HTSP resistance to Pst. Transient overexpression of PstCEP1 enhanced the pathogenicity of Pst, while transient overexpression of PSTG_11208 induced resistance to stripe rust in XY6.

Thaumatin‐like proteins (TLPs) belong to the pathogenesis‐related (PR) protein 5 family, which plays an important role in antifungal processes in plants (Petre et al., 2011; van Loon et al., 2006). Studies have shown that the resistance of transgenic plants to Fusarium head blight could be improved by transferring rice TLPs into wheat (Chen et al., 1999), and the resistance of transgenic plants to powdery mildew was improved after transforming TaTlp into wheat cultivar Yangmai 58 (Xing et al., 2008). TLPs mostly participate in plant disease resistance by interacting with resistance proteins, but may also be attacked by pathogenic effector proteins (González et al., 2017; Zhang et al., 2019). PR proteins TaTLP1 and TaPR1 have N‐terminal signal peptides and can be secreted into the apoplast. Moreover, TaTLP1 triggers the immune response by interacting with TaPR1, playing an important role in wheat resistance to leaf rust (Wang, Yuan, et al., 2020). The apoplast is the first interface between a pathogen and a plant after the pathogen enters the plant cell. We found that TaTLP1 interacts with PSTG_11208 through Y2H and BiFC assays, and silencing of TaTLP1 and TaPR1 also reduced HTSP resistance to Pst. According to studies of the regulatory effects of TaTLP1–TaPR1 on the resistance response of wheat (Cui et al., 2021; Wang, Yuan, et al., 2020), we speculate that TaTLP1 recognizes the effector protein PSTG_11208 of Pst in the apoplast and triggers the downstream defence response through TaPR1. Overexpression of PstCEP1 could inhibit the defence response caused by overexpression of PSTG_11208, indicating that the interaction between PstCEP1 and PSTG_11208 may prevent TaTLP1 from recognizing PSTG_11208. Furthermore, PstCEP1 is an intracellular effector protein (Tao et al., 2020), which may escape the recognition of plants by transferring PSTG_11208 from the apoplast to the cytoplasm. In addition, PSTG_11208 exhibits strong self‐activation and is partially distributed in the host nucleus. Therefore, PSTG_11208 may directly bind with gene promoters to act as a transcription factor‐like protein.

Ferredoxins (Fds) are small proteins containing iron–sulphur clusters that receive electrons from photosynthetic system I and transfer them to downstream Fd‐dependent enzymes to participate in photosynthesis (Hanke & Mulo, 2013). In addition, Fds are involved in the regulation of the ROS burst and the hypersensitivity response triggered by pathogen‐associated molecular patterns upon biotic stress (Dayakar et al., 2003; Huang et al., 2004, 2007). OsFd1 can receive electrons from photosystem I and transfer them to NADP+ reductase; OsFd1 deletion rice mutants show chlorosis and seedling death in the three‐leaf stage (He et al., 2020). In the present study, we found that TaFd1, a homologous protein of OsFd1, is a target protein of PstCEP1. The amino acid sequence similarity between the two proteins is over 70%, and their C‐terminal functional domains are highly conserved (Figure S5). Silencing of TaFd1 produced chlorosis symptoms in wheat leaves and reduced the HTSP resistance of wheat to Pst. Therefore, we speculate that the effector protein PstCEP1 targets TaFd1 to interfere with the stability of the wheat photosynthetic system and then responds to HTSP resistance. In addition, chloroplasts play an important role in the production of ROS and salicylic acid‐mediated immune signalling pathways (de Torres Zabala et al., 2015; Serrano et al., 2016). Overexpression of PstCEP1 in wheat could inhibit the accumulation of ROS induced by HTSP resistance to Pst (Tao et al., 2020), which may be achieved by targeting the chloroplastic protein TaFd1.

In conclusion, our results reveal a new molecular regulatory mechanism of Pst effector proteins in response to the HTSP resistance of XY6. The Pst effector protein PSTG_11208 can be recognized by TaTLP1 and then induces HTSP resistance in wheat through TaPR1 during Pst infection. The interaction between PstCEP1 and PSTG_11208 avoids the recognition of plants. Additionally, PstCEP1 targets TaFd1 and inhibits HTSP resistance in wheat by inhibiting ROS production and interfering with the stability of the photosynthetic system (Figure 6).

FIGURE 6.

FIGURE 6

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A working model of interaction of Puccinia striiformis f. sp. tritici (Pst) effectors regulating high‐temperature seedling‐plant (HTSP) resistance. Wheat TaTLP1 recognizes the Pst secreted protein PSTG_11208 and triggers downstream defence through TaPR1. Another Pst effector, PstCEP1, interacts with PSTG_11208 to avoid recognition by plants. PstCEP1 targets TaFd1 for interfering with the stability of the photosystems and reactive oxygen species (ROS) production. PSTG_11208 may localize in the nucleus and perform its transcription activation function. Solid lines represent well‐studied results, while the dotted lines represent our hypothesis.

4. EXPERIMENTAL PROCEDURES

4.1. Plant and fungal materials, inoculations, and temperature treatments

In this study, wheat cultivar XY6, N. benthamiana, and Pst race CYR32 were used. N. benthamiana plants were grown in a greenhouse under a diurnal cycle with 16/8 h of light/dark at 25°C. For Pst infection, urediniospores of CYR32 were mixed with engineered fluid (3M Novec 7100) at a concentration of 10 mg/mL. Ten microlitres of the suspension was inoculated evenly on 3–5 cm of leaf with a pipette and the entire leaf was inoculated with 10–20 μL of the suspension. Wheat plants inoculated with Pst were moisturized in a growth chamber at 10°C for 24 h and then grown in two temperature treatments: (1) N, wheat plants were grown under 16/8 h light/dark conditions at 15/10°C; (2) NHN, wheat plants were grown under 16/8 h light/dark conditions at 15/10°C until 192 hours post‐inoculation (hpi), transferred to and kept at 20°C for 24 h, and then returned to 15/10°C (Tao et al., 2018). Plant samples were treated in liquid nitrogen and stored at −80°C until use.

4.2. Y2H assay

The Y2H assay was conducted with the yeast strain Y2HGold using the GAL4 two‐hybrid system. The regions of PstCEP1ΔSP and PSTG_11208ΔSP were cloned separately into the BD vector, and the interacting proteins were cloned into the AD vector. The recombinant constructs were cotransformed into yeast strain Y2HGold (Weidibio). The growth of Y2HGold strains carrying recombinant constructs was observed on SD medium lacking tryptophan and leucine (double dropout, DDO) and QDO/X/A medium. The Y2HGold strains carrying BD‐murine P53 (53) and AD‐SV40 large T‐antigen (T) or BD‐human lamin C (Lam) and AD‐T were used as a positive control and negative control, respectively. Photographs were taken after incubation at 30°C for 72 h.

4.3. Pull‐down assay

The PSTG_11208ΔSP, PstCEP1ΔSP, and TaTLP1ΔSP coding sequences were cloned separately into pGEX‐4T‐1, pMAL‐C4X, and pET28a vectors to obtain recombinant plasmids PSTG_11208ΔSP‐GST, PstCEP1ΔSP‐MBP, and TaTLP1ΔSP‐His. The recombinant plasmids were expressed in Escherichia coli BL21 (Weidibio). The pull‐down assay was performed using the Pierce GST Protein Interaction Pull‐Down kit (Thermo Scientific) according to the instructions. The recombinant proteins were separated by 10% SDS‐PAGE. Anti‐GST, anti‐MBP, and anti‐His antibodies (Beyotime) were used for western blot analysis.

4.4. RNA extraction and transcriptional level analysis

The total RNA of plant samples was extracted using the SV Total RNA Isolation kit (Promega). The first‐strand cDNA was synthesized using the PrimeScript RT Reagent kit (TaKaRa). To investigate the transcriptional expression of PSTG_11208 after silencing, leaf samples were collected at 24, 48, 72, and 96 hpi. Reverse transcription‐quantitative PCR (RT‐qPCR) was performed using Ultra SYBR Mixture (Kangwei) in a LightCycler 480 (Roche). The wheat gene Ta26S (ATP‐dependent 26S proteasome regulatory subunit) and the housekeeping gene elongation factor 1 (EF1) of Pst were used as the reference genes (Tao et al., 2018, 2020), and specific primer pairs were designed for RT‐qPCR (Table S1). The relative expression level was analysed using the comparative 2−ΔΔCt method (Livak & Schmittgen, 2001). Three independent biological replicates and three technical replicates were included for each treatment in the RT‐qPCR assays.

4.5. BiFC assay

The BiFC assay (Miller et al., 2015) was conducted to confirm the interaction between PSTG_11208 and PstCEP1. The coding sequences of PSTG_11208 ΔSP and PstCEP1 ΔSP were introduced into the vectors pER8‐YFPN and pER8‐YFPC to generate recombinant plasmids YFPN‐PSTG_11208ΔSP and PstCEP1ΔSP‐YFPC, respectively. The recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101 (Weidibio). Positive clones containing YFPN‐PSTG_11208ΔSP or PstCEP1ΔSP‐YFPC plasmids were co‐injected into 5‐week‐old N. benthamiana leaves. N. benthamiana leaves with YFPN‐PSTG_11208ΔSP and YFPC or YFPN and PstCEP1ΔSP‐YFPC were used as negative controls. Leaf samples were collected 3–4 days after injection and examined for changes under an FV3000 microscope (Olympus).

4.6. Functional verification of the signal peptide

The signal peptide of PSTG_11208 was predicted by SignalP v. 5.0 (Almagro Armenteros et al., 2019), and the secretory function was verified by yeast signal sequence trap (Jacobs et al., 1997). The sequence of the signal peptide of PSTG_11208 was introduced into vector pSUC2. The recombinant plasmid and empty plasmid pSUC2 were transformed into the yeast strain YTK12 and we screened for positive clones on CMD−W medium. The positive yeast clones were transferred to YPRAA medium to observe growth. Besides, we detected the invertase enzymatic activity. If the signal peptide was secretory, SUC2 could be secreted to the extracellular space and convert the colourless soluble 2,3,5‐triphenyltetrazolium chloride into red insoluble 1,3,5‐triphenylformazan (Jiang et al., 2020). The functional signal peptide of the reported effector Avr1b was used as a positive control (Dou et al., 2008).

4.7. Subcellular localization

The transit peptide was determined by LOCALIZER (http://localizer.csiro.au/index.html). The subcellular localization assay was carried out in N. benthamiana and wheat protoplasts. For subcellular localization in N. benthamiana leaves, we cloned truncated proteins separately into the pBINGFP (eGFP) or p1302GFP (eGFP) vectors. The recombinant plasmid was transformed into A. tumefaciens GV3101 to infiltrate N. benthamiana leaves. Leaf samples were examined for changes 48–72 h after infiltration under an FV3000 microscope. For western blot analysis, the total protein was extracted from N. benthamiana leaves carrying the recombinant protein and separated by 10% SDS‐PAGE. GFP was detected using anti‐GFP (Beyotime).

The sequence of PSTG_11208 ΔSP was cloned into the p16318‐GFP vector, and the subcellular localization in wheat protoplasts was analysed using the Wheat Protoplast Preparation and Transformation Kit (Coolaber). Leaf samples were examined under an FV3000 microscope.

4.8. BSMV‐mediated PSTG_11208 gene silencing

A BLASTn search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was conducted to select the specific sequence as the silent fragment for constructing a recombinant vector using γ‐PSGT_11208. Vectors α, β, γ, γ‐TaPDS (TaPDS is a wheat phytoene desaturase gene), and γ‐PSGT_11208 were linearized and transcribed in vitro using the RiboMAX Large Scale RNA Production System—T7 (Promega) according to the manufacturer's instructions. The inoculation of wheat plants with BSMV and gene silencing was carried out using a previously described method (Holzberg et al., 2002; Zhu et al., 2017). The mixture of α, β, and γ transcripts is referred to as BSMV:00 without silencing any gene; the mixture of α, β, and γ‐TaPDS is referred to as BSMV:TaPDS as a positive control; and the mixture of α, β, and γ‐PSGT_11208 transcripts is referred to as BSMV:PSGT_11208. BSMV:00, BSMV:TaPDS, and BSMV:PSGT_11208 were mixed separately with FES buffer and used to inoculate the second leaves of XY6 wheat seedlings at the three‐leaf stage. The BSMV‐inoculated wheat plants were placed in a dew chamber at 25°C for 24 h in the dark and then grown at 25°C with a 16/8 h light/dark cycle. The wheat seedlings treated with FES buffer were used as a mock control.

When photobleaching was observed in the fourth leaves of wheat with BSMV:TaPDS and chlorosis was observed in the fourth leaves with BSMV:00 and BSMV:PSGT_11208, the fourth leaves were inoculated with Pst race CYR32 and the plants were grown at 15/10°C. The silencing efficiency of PSGT_11208 was determined at 24, 48, 72, and 96 hpi. At 48 and 120 hpi, the leaves were collected to measure the hyphal length of Pst. The fourth leaves were used to score responses to Pst infection and photographed at 312 hpi, and the uredinia per unit leaf area of Pst‐inoculated wheat plants were counted. The gene silencing experiments for TaTLP1 and TaPR1 were conducted following the same procedure.

To measure the hyphal length of Pst, the wheat leaves were decolourized and stained using WGA‐Alexa 488 (20 μg/mL) according to previously described methods (Cheng et al., 2015). The hyphal length was measured under a BX‐51 microscope (Olympus). Three biological replicates were included for each treatment, with at least 10 random wheat leaves for each replicate.

4.9. Overexpression mediated by bacterial type III secretion system

The ORFs of PSTG_11208 and PstCEP1 were cloned into the pEDV6 vector to construct recombinant vectors pEDV6‐PSTG_11208 and pEDV6‐PstCEP1, respectively. The recombinant vectors were transformed into Pseudomonas fluorescens EtHAn. The second leaves of 14‐day‐old wheat plants were infiltrated with EtHAn containing pEDV6‐PstCEP1 or pEDV6‐PSTG_11208 plasmids. Leaves infiltrated with MgCl2 were used as a blank control, and leaves infiltrated with EtHAn containing pEDV6‐dsRed were used as a negative control. Leaf samples were collected at 96 h after injection of MgCl2, pEDV6‐dsRed, pEDV6‐PstCEP1, or pEDV6‐PSTG_11208 or coexpression of pEDV6‐PstCEP1 and pEDV6‐PSTG_11208 to observe callose deposition after aniline blue staining (Hood & Shew, 1996). Pst race CYR32 was inoculated 96 h after infiltrating with EtHAn on the second wheat leaves. The second leaves were photographed at 312 hpi with Pst, and the uredinia per unit leaf area of Pst‐inoculated wheat plants were counted. Three biological replicates were included for each treatment, with at least 10 random wheat leaves for each replicate.

4.10. Primers and applications

Primers and their use in experiments are listed in Table S1.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

Supporting information

Figure S1. Evaluation of self‐activation and toxicity of pGBKT7 (BD)‐PstCEP1ΔSP.

Click here for additional data file. (569.2KB, docx)

Figure S2. Verification of the secretory function of the signal peptide of PSTG_11208 and the subcellular localization of PSTG_11208ΔSP.

Click here for additional data file. (1.3MB, docx)

Figure S3. Histological observation of the effects of no silencing and PSTG_11208 silencing on wheat seedlings after inoculation.

Click here for additional data file. (1MB, docx)

Figure S4. Evaluation of self‐activation and toxicity of pGBKT7 (BD)‐PSTG_11208ΔSP.

Click here for additional data file. (406.5KB, docx)

Figure S5. Amino acid sequence alignment of OsFd1 and TaFd1.

Click here for additional data file. (255KB, docx)

Figure S6. Subcellular localization of TaFd1 and barley stripe mosaic virus (BSMV)‐mediated gene silencing of TaFd1.

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

Figure S7. Subcellular localization of PstCEP1.

Click here for additional data file. (1.4MB, docx)

Table S1. Primers used in this study.

Click here for additional data file. (24.7KB, docx)

ACKNOWLEDGEMENTS

This work was supported by the National Key Research and Development Program of China (2021YFD1401000), the China Agriculture Research System for Wheat (CARS‐03‐37), and the National Natural Science Foundation of China (31972219 and 31271985). We thank Professor Xiaojie Wang and Dr Siwei Zhang of Northwest A&F University for providing the plasmids. We also thank Jingchen Zhao of Northwest A&F University for technical assistance during manuscript preparation.

Bao, X. , Hu, Y. , Li, Y. , Chen, X. , Shang, H. & Hu, X. (2023) The interaction of two Puccinia striiformis f. sp. tritici effectors modulates high‐temperature seedling‐plant resistance in wheat. Molecular Plant Pathology, 24, 1522–1534. Available from: 10.1111/mpp.13390

Xiyue Bao and Yangshan Hu contributed equally to this work.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

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

Supplementary Materials

Figure S1. Evaluation of self‐activation and toxicity of pGBKT7 (BD)‐PstCEP1ΔSP.

Click here for additional data file. (569.2KB, docx)

Figure S2. Verification of the secretory function of the signal peptide of PSTG_11208 and the subcellular localization of PSTG_11208ΔSP.

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Figure S3. Histological observation of the effects of no silencing and PSTG_11208 silencing on wheat seedlings after inoculation.

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Figure S4. Evaluation of self‐activation and toxicity of pGBKT7 (BD)‐PSTG_11208ΔSP.

Click here for additional data file. (406.5KB, docx)

Figure S5. Amino acid sequence alignment of OsFd1 and TaFd1.

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Figure S6. Subcellular localization of TaFd1 and barley stripe mosaic virus (BSMV)‐mediated gene silencing of TaFd1.

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Figure S7. Subcellular localization of PstCEP1.

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Table S1. Primers used in this study.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Molecular Plant Pathology are provided here courtesy of Wiley

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