Wheat near-isogenic line TcLr19 (Tc*6/RL6040), susceptible wheat line Chinese Spring, TaTLP1-overexpressing transgenic line (TaTLP1-OE), and wild type Jinan Wheat No. 1 (JW1) are preserved in the Laboratory of Wheat Leaf Rust, Hebei Agricultural University. Pt race PHNT (isolate 07-10-426-1) was used in all tests according to methods outlined by Roelfs and Martell (1984). The second seedling leaves inoculated with PHNT or distilled water (control) for RNA extraction were harvested at 0, 24, 48, 72, 96, and 120 h post-inoculation (hpi). All samples were immediately frozen in liquid nitrogen and stored at −80°C. Each treatment included three independent biological replicates.

RNA-seq was used to analyze 7 TaPR1 genes involved in the Lr19-mediated resistance to Pt. The molecular size and isoelectric points (pI) of seven TaPR1 genes were predicted using ProtParam.¹ Subcellular localization and chromosome locations were determined using EnsemblPlants.² The signal peptides were identified using SignalP 5.0.³

Total RNA was extracted using an M5 Plant RNeasy Complex Mini Kit (Mei5bio, Beijing) according to the manufacturer’s instructions. cDNA was synthesized using the M5 Super Plus qPCR RT kit with gDNA remover (Mei5bio, Beijing). qPCR was conducted using 2 × M5 HiPer Realtime PCR Super Mix (Mei5bio, Beijing) with an ABI QuantStudio 5 instrument (ABI, Waltham, MA, United States). Expressions of TaPR1-1, TaPR1-4, TaPR1-7, TaPR1-9, TaPR1-16, TaPR1-19, and TaPR1-20 were investigated, and the wheat glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Gene ID: AF251217) gene was used to calibrate the expression levels of queried genes, as previously described (Gao et al., 2015; Supplementary Table 1). Data were analyzed using the 2^–ΔΔCT method (Livak and Schmittgen, 2002). The statistical significance of differences was calculated using one-way analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) with p < 0.05 in SPSS 26.0 (IBM SPSS Statistics, IBM). For each treatment, three technical repeats and three independent biological replicates were used for analysis.

Green fluorescent protein (GFP) fusion constructs were produced by cloning the coding sequence of TaPR1-4 into the vector pEarlyGate103 (with GFP-trap). Resuspended Agrobacterium tumefaciens GV3101 carrying pEarlyGate103-TaPR1-4 and pEarlyGate103 at a final OD₆₀₀ = 0.8. Nicotiana benthamiana infiltrations were performed on plants that were 4–5 weeks old. pEarlyGate103 was used as a negative control. The fluorescence in leaves of N. benthamiana was monitored 48 h after agroinfiltration and then imaged directly using a confocal laser scanning microscope (Olympus FluoView FV1000).

The Matchmaker Gold Yeast Two-Hybrid System (Clontech, Japan) was used to verify the interaction between TaTLP1 and different truncated TaPR1-4 mutants, which included C-terminal deletions of residues 113–164 (C_{Δ 113–164}-TaPR1-4), 119–164 (C_{Δ 119–164}-TaPR1-4), 128–164 (C_{Δ 128–164}-TaPR1-4), and 143–164 (C_{Δ 143–164}-TaPR1-4). These mutants were generated by PCR amplification and subcloned into pGBKT7 as bait (Supplementary Table 1). TaTLP1 was transformed into pGADT7 as prey. The bait and prey plasmids were co-transformed into yeast strain Y2HGold according to the manufacturer’s instructions. A series of site-specific mutant variants (A114S, A115R, G116A, K117A, and V118S) of TaPR1-4 were synthesized, ligated to the pGBKT7 vector, transformed into yeast cells, and assayed for growth on synthetic dropout SD/-Trp-Leu and SD/-Trp-Leu-His-Ade plates containing X-α-galactosidase (X-α-Gal) and aureobasidin A (AbA).

According to the secondary structures of TaPR1-4 protein, truncated TaPR1-4 variants in the N- or C-terminal region and wild type TaPR1-4 were generated by PCR amplification and cloned into pGEX-6P-3 (with GST-tag) (Supplementary Table 1). The recombinant proteins were expressed in E. coli. Crude proteins were induced by isopropyl β-d-1-thiogalactopyranoside (IPTG) with a final concentration of 0.5 mmol L^–1 (Salcedo et al., 2017). A GST-Agarose Label kit (TRAN, Beijing) was used to bind GST-tagged protein following the manufacturer’s instructions. The purified protein products were separated by 15% SDS-PAGE and visualized using Coomassie Blue staining.

Puccinia triticina race PHNT was tested for disease response in the presence of TaPR1-4, N_{Δ 25–64}-TaPR1-4, C_{Δ 119–164}-TaPR1-4 (-αIV), C_{Δ 128–164}-TaPR1-4 (+αIV), and CAPE1 peptide, respectively. According to the experiment used to detect germination of urediniospores, petri dishes were filled with 20 ml of agar medium containing purified TaPR1-4, N_{Δ 25–64}-TaPR1-4, C_{Δ 119–164}-TaPR1-4 (-αIV), C_{Δ 128–164}-TaPR1-4 (+αIV), and CAPE1 peptide (1 mg/ml). The plates were incubated at 25°C in the dark, and the germination of spores and hyphal length were observed using the Nikon Ti2-LAPP Ti2 Laser Application System (Nikon Corporation, Minato-ku, Tokyo, Japan). Negative controls were also carried out using sterile water, elution buffer, and GST tag protein.

To identify the antifungal activity of CAPE1 in TaPR1-4 in vivo, the susceptible wheat line Chinese Spring was used for infiltration assays. TaPR1-4, CAPE1, and C_{Δ 128–164}-TaPR1-4 (+αIV) proteins were diluted to 0.1 mg/ml in 1 × PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 2 mM KH₂PO₄) and infiltrated into the abaxial side of the first leaves of 14-day-old seedlings of Chinese Spring using a 0.5-ml syringe and GST proteins as a negative control. Inoculations with Pt race PHNT were carried out 24 h after protein infiltration, and disease responses were observed after 14 days. Leaves at 0, 24, 48, and 120 hpi were sampled and RNA extracted, then transcription levels of several resistance-related genes, including TaPR1-4, TaTLP1, and TaSOD (superoxide dismutase, SOD), were analyzed following protein infiltration and inoculation with Pt. A similar experiment was conducted on 21-day-old TaTLP1-OE and JW1 wheat lines to further explore the role of CAPE1 in the TaTLP1-mediated resistance response, then the transcription levels of TaPR1-4, TaCAT (catalase, CAT), TaSOD, and TaNOX (NADPH oxidase, NOX) at 0, 24, 48, and 120 hpi were investigated using qPCR. In all these experiments, each treatment included 3–5 plants and was repeated at least two times. The numbers of urediniospores were quantified using Image J software.

Harvested samples were decolorized as described previously (Wang et al., 2007). Hyphae were stained using Fluorescent Brightener 28. Autofluorescence of attacked mesophyll cells was observed in necrotic areas using an Olympus IX-53 microscope (Olympus Corporation, Tokyo, Japan) (excitation filter, 488 nm; dichromic mirror, 510 nm; and barrier filter, 520 nm). Necrotic areas and infection areas were quantified using Image J software. For each treatment, at least 50 different infection sites were examined on each of five randomly selected leaf segments.

To detect H₂O₂ accumulation, 3, 3-diaminobenzidine (DAB; Solarbio, Beijing) staining was conducted following the protocols described previously (Thordal Christensen et al., 1997) and was then viewed by differential interference contrast optics. Areas of H₂O₂ were quantified using Image J software. A minimum of 50 infection sites were examined on each of five randomly selected leaf segments for every treatment.

In our previous study, transcriptome sequencing was conducted on the resistant line TcLr19 and the sensitive variety Chinese Spring inoculated with Pt race PHNT (Raw sequence reads have been deposited in the NCBI Sequence Read Archive under the BioProject PRJNA694214). In an RNA-seq library, seven TaPR1 genes, namely TaPR1-1, TaPR1-4, TaPR1-7, TaPR1-9, TaPR1-16, TaPR1-19, and TaPR1-20, were identified and divided into three groups following Lu et al. (2011) (Supplementary Table 2). Amino acid sequence analyses indicated that the open reading frames (ORFs) of these TaPR1 genes ranged from 495 to 522 base pairs (bp), and molecular weights ranged from 17 to 18 kDa. Predicted isoelectric points (pI) of the proteins by Expasy ranged from 4.2 to 8.7 (Supplementary Table 2). Predicted subcellular localization by EnsemblPlants showed that all seven TaPR1 proteins were located in the apoplastic space. Predicted chromosome locations in EnsemblPlants indicated that TaPR1-1 and TaPR1-7 were located in chromosome 5B, TaPR1-9 and TaPR1-19 were located in chromosome 5A, TaPR1-16 was located in chromosome 5D, and TaPR1-4 and TaPR1-20 were located in chromosome 7D. Signal peptide (SP) structure analysis using SignalP 5.0 indicated that all TaPR1 proteins contained an SP motif at the N-terminus (Supplementary Table 2).

Transcription levels of the seven TaPR1 genes in TcLr19 following infection with Pt were determined by qPCR. TaPR1-1, TaPR1-9, and TaPR1-16 expression peaked at 48 hpi and then decreased in the incompatible combination. However, transcription of these genes in the Thatcher control, which is susceptible to all tested Pt races, was almost unchanged over the same time periods (Figures 1A,D,E). TaPR1-7 and TaPR1-20 were highly expressed in both TcLr19 and Thatcher (Figures 1C,G). TaPR1-4 and TaPR1-19 expression peaked at 96 hpi in TcLr19 when the expression level was 253.93-fold that at 0 hpi, and the expression of TaPR1-4 at 96 hpi was 358.08-fold higher than at 0 hpi. However, the expression levels of TaPR1-4 and TaPR1-19 in Thatcher were lower than that in TcLr19 (Figures 1B,F). Our findings demonstrated that TaPR1-4 is most induced by Pt. In accordance with our previous studies (Gao et al., 2015; Wang F. et al., 2020), TaPR1-4 was used for further study.

To further assess the secretion and localization of TaPR1-4 protein in vivo, we generated the fusion construct pEarlyGate103-TaPR1-4 in which TaPR1-4 was fused to GFP at its C-terminus for the agrobacterium-mediated transformation of N. benthamiana. The empty vector pEarlyGate103 was used as a negative control. Confocal microscopic observation showed that the control GFP protein was localized in both the cytoplasm and nucleus (Supplementary Figure 1). In contrast, pEarlyGate103-TaPR1-4 signals were clearly visualized in the apoplastic space (AP), as evidenced by the plasmolysis (Supplementary Figure 1), and no GFP signals were observed in the nucleus. Based on these observations, we concluded that TaPR1-4 was secreted outside of plant cell, which was consistent with the predicted subcellular localization.

In a previous study, we showed that 15 amino acids from 113 to 127 located in the C-terminal region of TaPR1-4 are required for interaction with TaTLP1 (Wang F. et al., 2020). The detailed structure of TaPR1-4 was analyzed to further identify critical residues of TaPR1-4 interacting with TaTLP1. We found that amino acids A114–V118 were exposed on the surface (Supplementary Figure 2). Site-specific mutagenesis with a focus on residues A114–V118, respectively, was performed as these amino acids were less likely to be structurally disruptive (Protein ID: Q94F73). Yeast two-hybrid (Y2H) results showed that TaTLP1-pGADT7 and TaPR1-4-pGBKT7 as positive controls produced blue coloring (Figure 2, rows 1 and Supplementary Figure 3, rows 1), whereas interactions between all five mutants and TaTLP1 were weakened, but not negative (Supplementary Figure 3, rows 5–9). The negative controls pGADT7 and pGBKT7 caused a loss of blue coloration (Figure 2, row 2 and Supplementary Figure 3, row 2). These results indicate that a single residue from A114 to V118 cannot abolish the interaction between TaPR1-4 and TaTLP1.

Kirby et al. (2002) reported that the αIII and αIV helices in the PR1 fold likely interact with other proteins. Truncated variants C_{Δ 119–164}-TaPR1-4 without the αIV helix structure (-αIV) and C_{Δ 128–164}-TaPR1-4 including the αIV helix structure (+αIV) were generated to identify the effect of αIV helix on the interaction between TaPR1-4 and TaTLP1. Truncated variants C_{Δ 113–164}-TaPR1-4 and C_{Δ 143–164}-TaPR1-4 from our previous study (Wang F. et al., 2020) were used as controls (Supplementary Figure 4 and Figure 2). Y2H results showed that the truncated variants C_{Δ 128–164}-TaPR1-4 (+αIV) and C_{Δ 143–164}-TaPR1-4 interacted with TaTLP1 (Figure 2, rows 5–6), whereas C_{Δ 113–164}-TaPR1-4 failed to interact with TaTLP1, and C_{Δ 119–164}-TaPR1-4 (-αIV) with TaTLP1 still remained a trace of weak interaction (Figure 2, rows 3–4). These results showed that interaction between C_{Δ 119–164}-TaPR1-4 (-αIV) and TaTLP1 was abolished or extremely weakened, indicating that the αIV helix in TaPR1-4 contains critical residues for binding with TaTLP1, which means the critical residue of TaPR1-4 binding with TaTLP1 was further narrowed based on our previous study.

As the αIV helix is indispensable for TaPR1-4 interaction with TaTLP1, we speculated that the αIV helix was the functional region of TaPR1-4 that contributed to resistance to Pt. Truncated variants of C_{Δ 128–164}-TaPR1-4 (+αIV) and C_{Δ 119–164}-TaPR1-4 (-αIV) were ligated to the pGEX-6P-3 vector to express in E. coli, and all the pure proteins were observed by SDS-PAGE analysis [Figure 3A-(3–4), lane 5–6 and Supplementary Figure 5]. However, growth-inhibition assays showed that there was no significant difference in the germination of urediniospores and hyphal growth between the C_{Δ 128–164}-TaPR1-4 (+αIV) and C_{Δ 119–164}-TaPR1-4 (-αIV) (Figures 3E,F,J,K), indicating that the C_{Δ 128–164}-TaPR1-4 (+αIV) and C_{Δ 119–164}-TaPR1-4 (-αIV) peptides had the same antifungal activity, and the αIV helix did not affect the function of TaPR1-4.

Sung et al. (2021) reported that the CAPE1 motif in TaPR1-1 can induce an immune response and repress infection by Parastagonospora nodorum. To assess the requirement of the CAPE1 region within the TaPR1-4 protein for disease repression, the CAPE1 peptide (amino acids 154–164) of TaPR1-4 was synthesized and ligated to the pGEX-6P-3 vector to express in E. coli [Figure 3A-(5), lane 7 and Supplementary Figure 5]. The wild-type TaPR1-4 [Figure 3A-(1), lane 3 and Supplementary Figure 5] and N-terminus truncations N_{Δ 25–64}-TaPR1-4 pure protein [from our previous study, Wang F. et al. (2020)] [Figure 3A-(2), lane 4 and Supplementary Figure 5] that contain CAPE1 used as positive controls were observed by SDS-PAGE analysis. Antifungal activity results showed that the hyphae lengths treated with N_{Δ 25–64}-TaPR1-4, TaPR1-4, and CAPE1 peptide were significantly shorter than the negative control groups (elution buffer, sterile water, and GST) (Figure 3), indicating that the germination of urediniospores and hyphal growth were significantly restricted with N_{Δ 25–64}-TaPR1-4, TaPR1-4, and CAPE1 peptide. In addition, we found that residues A114 and V118 in TaPR1-4 and TaPR1-1 differed from other TaPR1s according to multiple sequences alignment of seven TaPR1 proteins (Supplementary Figure 4). To identify whether amino acids A114 and V118 are related to the disease resistance function of TaPR1-4, A114 and V118 in TaPR1-4 were replaced with S, respectively, and growth-inhibition assays showed no effects on disease response (Supplementary Figure 6). Taken together, all these results emphasized that the CAPE1 peptide of TaPR1-4 plays a major role in the antifungal activity, αIV helix just has a minor role in antifungal activity.

To further demonstrate whether CAPE1 is indispensable for the antifungal activity of TaPR1-4 protein, the TaPR1-4 and CAPE1 proteins were infiltrated into leaves of susceptible wheat Chinese Spring, respectively. First, elution buffer, sterile water, and pGEX-6P-3 vector (GST) as negative control were infiltrated into wheat leaves to test whether contaminants from E. coli may elicit host immunity and prime resistance against leaf rust. After 14 days post inoculated (dpi) with Pt race PHNT, phenotype observation, and DAB staining results showed that GST had no effect on phenotype and H₂O₂ accumulation, indicating that the expression system can be used for wheat infiltration assays (Supplementary Figure 7). There were lots of urediniospores on leaves infiltrated with GST as a negative control at 14 dpi (Figure 4A). However, similar to TaPR1-4, the number of urediniospores on leaves infiltrated with the CAPE1 protein was significantly less than that infiltrated with C_{Δ 128–164}-TaPR1-4 (+αIV) protein (Figure 4A). These results indicated that CAPE1 protein reduced the number of urediniospores and enhanced wheat resistance to Pt.

Quantitative PCR was conducted to test the expression levels of several resistance-related genes, including TaPR1-4, TaTLP1, and TaSOD, after protein infiltration. The results showed that all of these genes were upregulated at 24 hpi in different treatments, and the expression levels of TaPR1-4, TaTLP1, and TaSOD were higher after infiltrated with CAPE1 than C_{Δ 128–164}-TaPR1-4 (+αIV) (Figures 4B–D), which suggests that TaTLP1 and TaSOD genes were induced by the TaPR1-4, CAPE1, and C_{Δ 128–164}-TaPR1-4 (+αIV) proteins. In the meanwhile, these results provide another proof that the CAPE1 plays a key role in TaPR1-4 protein-induced plant defense.

To further assess whether CAPE1 could be related to TaTLP1-induced defense responses, the GST, TaPR1-4, CAPE1, and C_{Δ 128–164}-TaPR1-4 (+αIV) proteins were infiltrated into TaTLP1-OE lines, with the susceptible wheat JW1 (WT) as a negative control. Phenotype after 14 days inoculated with fresh Pt race PHNT showed that more urediniospores appeared on WT lines infiltrated with GST protein than that infiltrated with TaPR1-4, CAPE1, and C_{Δ 128–164}-TaPR1-4 (+αIV) (Figure 5A). In addition, we found that the number of urediniospores on WT lines infiltrated with TaPR1-4 and CAPE1 was significantly less than that infiltrated with C_{Δ 128–164}-TaPR1-4 (+αIV) proteins (Figure 5A). In accordance with our previous reports (Cui et al., 2021), lots of necrotic spots were observed on TaTLP1-OE lines infiltrated with different proteins. Moreover, a few numbers of Pt urediniospores were produced around the necrotic spots on TaTLP1-OE lines infiltrated with GST and C_{Δ 128–164}-TaPR1-4 (+αIV), but not on wheat leaves infiltrated with TaPR1-4 and CAPE1 (Figure 5A). All these results suggest that wheat leaves infiltrated with TaPR1-4 and CAPE1 showed significantly more resistance to PHNT compared to C_{Δ 128–164}-TaPR1-4 (+αIV).

To further understand how CAPE1 participates in wheat resistance, the transcription levels of TaPR1-4, TaSOD, TaCAT, and TaNOX in the TaTLP1-OE and WT lines following infection with Pt were determined. qPCR results showed that TaCAT was significantly downregulated, while those of TaPR1-4, TaSOD, and TaNOX were significantly upregulated. Moreover, the expression levels of TaPR1-4, TaSOD, and TaNOX were higher after being infiltrated with CAPE1 and TaPR1-4 than that infiltrated with C_{Δ 128–164}-TaPR1-4 (+αIV), but TaCAT expression was lower after infiltrated with CAPE1 and TaPR1-4 than C_{Δ 128–164}-TaPR1-4 (+αIV) (Figures 5B–E). TaSOD, TaCAT, and TaNOX are related to ROS accumulation, which suggests that the TaPR1-4 or CAPE1 positively modulates wheat resistance to Pt in a ROS-dependent manner.

Histological observation of TaTLP1-OE and WT lines infected with Pt following CAPE1 and TaPR1-4 infiltration were recorded. It showed that the infected areas after CAPE1 and TaPR1-4 infiltration were lower than GST and C_{Δ 128–164}-TaPR1-4 (+αIV) (Figures 6A,B). TaTLP1-OE lines infiltrated with CAPE1 and TaPR1-4 displayed larger necrotic areas compared to GST and C_{Δ 128–164}-TaPR1-4 (+αIV) (Figure 6C), indicating that TaPR1-4 and CAPE1 in TaTLP1-OE can enhance the resistance response to Pt infection. In addition, we further analyzed TaPR1-4 and CAPE1-triggered ROS accumulation upon inoculation of wheat with Pt race PHNT. The results showed that the H₂O₂ accumulation was higher after infiltration with CAPE1 and TaPR1-4 than C_{Δ 128–164}-TaPR1-4 (+αIV) in TaTLP1-OE and WT lines (Figure 7). Taken together, all of these results prove that CAPE1 or TaPR1-4 and TaTLP1 function additively in wheat resistance to Pt infection.