Rust effector PNPi interacting with wheat TaPR1a attenuates plant defense response

NPR1 is a key regulator of systemic acquired resistance (SAR) in plant species. In our previous study, we identified a conserved fungal effector PNPi from Puccinia striiformis f. sp. tritici (Pst) that can suppress acquired resistance in local leaf by directly targeting the wheat NPR1 protein. In this investigation, we identified and validated a novel protein interaction between PNPi and wheat pathogenesis-related TaPR1a in the apoplastic space. TaPR1a-overexpressing wheat lines exhibited enhanced resistance to both Pst and Puccinia triticina (Pt). We further determined that exogenous expression of PNPi RNA in transgenic wheat lines reduced the degree of acquired resistance to Magnaporthe oryzae isolate P131 in the region adjacent to Pseudomonas syringae pv. tomato DC3000 infection area. Additionally, when PNPi was overexpressed, the expression levels of two plant defense responsive genes were suppressed upon P. syringae DC3000 infection in the local infiltration region. These findings established the mechanism of a single rust effector that can suppress multiple defense responses in wheat plants by targeting different components.


Background
Plants are constantly subjected to biotic and abiotic stresses, triggering various plant resistance mechanisms that are mediated by different hormones, such as salicylic acid (SA), jasmonate (JA), and abscisic acid (ABA) (Smith et al. 2017). SA is a crucial component of plant disease resistance, mediating plant immune response to pathogen infection and generating systemic acquired resistance (SAR) beyond the infection area . NPR1 (Nonexpresser of PR genes 1, also known as NIM1 and SAI1) is a key transcriptional regulator of SAR in Arabidopsis. Upon pathogen infection or SA treatment, NPR1 is translocated from the cytoplasm to the nucleus where it interacts with the transcription factor TGA2 to promote the expression of pathogenesis-related (PR) genes (Cao et al. 1994;Delaney et al. 1995;Ryals et al. 1997;Shah et al. 1997;Mou et al. 2003). In Arabidopsis, NPR3 and NPR4 are paralogs of NPR1 that participate in CUL3 E3 ligase-mediated degradation of NPR1 in a SA concentration-dependent manner (Fu et al. 2012). Studies of the post-translational modification of NPR1 revealed complex regulation of this protein (Saleh et al. 2015;Withers and Dong 2016). NPR1 is a key regulator of defense across diverse plant species, making it a strong target gene for genetic engineering to improve disease and stress tolerance in crops (Silva et al. 2018;Backer et al. 2019). NPR1 may also be a target hub of effectors from various pathogens, including Pseudomonas syringae, Puccinia striiformis f. sp. tritici, and Phytophthora capsici (Wang et al. 2016;Chen et al. 2017;Li et al. 2019;Zhao et al. 2020).
The genes downstream of the plant defense response and SAR belong to 18 gene families, and are considered pathogenesis-related (PR) genes that respond to various pathogens (van Loon et al. 2006;Ferreira et al. 2007;Sels et al. 2008;Wang et al. 2018). Among the PR genes characterized in wheat and barley, PR1 is a hallmark of the activation of plant defense pathways (Molina et al. 1999) and is downstream of NPR1 in the acquired resistance triggered by Pseudomonas syringae pv. tomato DC3000 (Wang et al. 2016). The CAPE1 peptide derived from the C-terminal end of the PR1 protein is a damage-associated molecular pattern (DAMP) that triggers the accumulation of reactive oxygen species (ROS) and transcriptional activation of several PR genes (Chen et al. 2014). The wheat TaPR1a protein can physically interact with ToxA to potentially mediate ToxA-induced necrosis in sensitive wheat lines (Lu et al. 2014). A recent genetic and biochemical study showed that binding of PR1 to sterols can inhibit pathogen growth (Gamir et al. 2017). Interestingly, Arabidopsis AtPR1 can be targeted by a cerato-platanin protein, SsCP1 from Sclerotinia sclerotiorum, which suppresses the plant defense response (Yang et al. 2017).
Wheat rusts, including stripe rust, leaf rust, and stem rust, are severe fungal diseases that affect wheat crops (Hovmøller et al. 2010). The successful biotrophic lifestyle of rust fungi depends upon the ability of these fungi to deliver specialized effectors into host cells to suppress or evade plant defense. Recent genome sequencing approaches allow the prediction of hypothetical effectors from different wheat rust species (Duplessis et al. 2011;Cantu et al. 2013a;Zheng et al. 2013;Kiran et al. 2016;Zhao et al. 2020). The few well-characterized rust effector PST02549 from Puccinia striiformis f. sp. tritici (Pst) physically interacts with wheat EDC4 protein and is associated with processing bodies (Petre et al. 2016). Another wheat stripe rust effector, PEC6, inhibits pattern-triggered immunity (PTI) in a host species-independent manner and interacts with wheat adenosine kinases (Liu et al. 2016).
In our previous study, we used yeast two-hybrid screening to identify an effector protein PNPi from Pst that directly targets the wheat homolog of NPR1 protein (wNPR1) (Wang et al. 2016). PNPi interacts with the NPR1/NIM1-like domain of wNPR1 via its C-terminal DPBB_1 domain, and bimolecular fluorescence complementation (BiFC) assay revealed interaction between PNPi and wNPR1 in the nuclei of Nicotiana benthamiana protoplasts. In the same study, a transgenic barley line overexpressing the coding region of the signal peptide-truncated PNPi was generated to functionally characterize this effector. The induction of NPR1mediated PR genes during SAR triggered by P. syringae DC3000 was suppressed by overexpression of PNPi, which might reflect a competitive effect of PNPi on the interaction between wNPR1 and wTGA2. The signal peptide of PNPi was sufficient to induce protein secretion from fungal yeast, but not from plant cells. Additionally, an RxLR-dEER motif-like region (RSLL-DEEP) of 24 amino acids was identified after the end of the predicted signal peptide.
In this investigation, we found that the wheat pathogenesis-related protein TaPR1a is targeted by PNPi in the apoplastic space. This interaction and TaPR1a function were further analyzed, and the results established the mechanism of a single rust fungal effector able to suppress multiple defense responses in wheat plants by targeting different components during infections. The generated transgenic wheat line TaPR1a-OE provides a valuable resource for the genetic improvement of wheat resistance to rust fungi.

Results
The wheat pathogenesis-related protein TaPR1a was targeted by the rust effector PNPi A yeast two-hybrid (Y2H) library screening approach was used to identify proteins that interact with the rust effector PNPi (GenBank accession KT764125). The signal peptidetruncated PNPi was used as the bait, and the screen yielded 12 protein interactors (Additional file 1: Table S1). The gene expression patterns of these PNPi interactors during barley SAR-like response triggered by P. syringae DC3000 were examined using data from one of our previously established transcriptome databases (Additional file 2: Figure S1) . Since the expression level of the gene encoding barley pathogenesis-related protein HvPR1a_MLOC_52081 exhibited significant NPR1dependent up-regulation upon P. syringae DC3000 infection, we selected wheat TaPR1a (GenBank accession FJ815169) for further characterization as a PNPi interactor. Analysis of the TaPR1a sequence using SignalP 4.1 revealed a 24-amino acid signal peptide located at the N-terminus of TaPR1a. To prevent the secretion of the tested proteins, we applied Y2H validation using versions of these proteins lacking their respective signal peptides, TaPR1a  and PNPi  . A strong interaction was observed between TaPR1a  and PNPi (23-333) on synthetic dropout (SD) selective medium lacking leucine, tryptophan, histidine, and adenine (SD-Leu-Trp-His-Ade) (Fig. 1a).
To determine the regions of PNPi required for its interaction with TaPR1a, two truncated PNPi proteins, PNPi  and PNPi  , were prepared to examine their potential to interact with TaPR1a. PNPi (23-235) contains the N-terminal region of PNPi and PNPi  contains the C-terminal region of PNPi, including the DPBB_1 domain (pfam03330) of PNPi. The results showed interactions of TaPR1a with both truncated PNPi proteins (Fig. 1a). We next cloned a homologous gene of PNPi from the wheat leaf rust pathogen, PtPNPi (Puccinia triticina PNPi homolog, GenBank accession OAV94594). Our initial Y2H result indicated that the interactions were conserved between PtPNPi (23-309) and wNPR1 (Genbank accession JX424315) or TaPR1a (25-164) (Additional file 2: Figure S2).
The C-terminal end of TaPR1a protein (CNYE_PAGN-VIGQKPY) aligned well with the CAPE1 peptide found in most PR1 proteins (Fig. 1b). This peptide is responsible for the induction of CAPE1-mediated plant defense responses (Chen et al. 2014). To investigate whether the PNPi protein can interact with the CAPE1 region of TaPR1a, the coding regions of two CAPE1-truncated TaPR1a proteins were constructed into the Y2H vector as TaPR1a (25-153) -AD and TaPR1a (25-151) -AD. The Y2H results showed that either truncation of the CAPE1 region in TaPR1a fully abolished interaction with PNPi (Fig. 1a).
The interaction between PNPi and TaPR1a was next validated by co-immunoprecipitation (Co-IP) assay. To do this, green florescent protein (GFP) or GFP-TaPR1a (25- Fig. 1 Validation of the protein interaction between PNPi and TaPR1a by Y2H and Co-IP assays. a Yeast transformants co-expressing different bait and prey constructs were assayed on SD-Leu-Trp-His-Ade selective medium. EV, empty vector. b A multiple sequence alignment was generated by MEGA software using CAPE1 regions of PR1 proteins from Solanum lycopersicum (Sl), Triticum aestivum (Ta), Hordeum vulgare (Hv), Nicotiana tabacum (Nt), Zea mays (Zm), and Oryza sativa (Os). c Schematic diagram of wheat transgenic construct. d Validation of the interaction between PNPi and TaPR1a by Co-IP assay. Total proteins from tobacco leaves transiently-expressing GFP-TaPR1a  and transgenic wheat line overexpressing SP (TaPR1a) -PNPi-cMYC were extracted and immunoprecipitated with an α-GFP antibody (α-GFP IP). Input and bound proteins were detected via immunoblot using α-GFP and α-cMYC antibodies, respectively 164) was transiently expressed in tobacco leaves. Next, to promote the secretion of PNPi in wheat plant, we recombined the sequence encoding the signal peptide of TaPR1a and the signal peptide-truncated PNPi into our transgenic construct (Fig. 1c). Transgenic wheat lines overexpressing the recombinant transcript of SP (TaPR1a) -PNPi-cMYC under the control of the maize Ubiquitin promoter (SP-PNPi-OE) were generated using Agrobacterium-mediated transformation in the spring common wheat cultivar "JW1". For the Co-IP assay, total proteins were extracted from Agrobacterium-infiltrated tobacco leaves and a transgenic wheat line overexpressing SP (TaPR1a) -PNPi-cMYC. A specific signal for SP (TaPR1a) -PNPi-cMYC was clearly detected in the GFP-TaPR1a (25-164) immunoprecipitate, but not in the GFP control (Fig. 1d), confirming the physical association of PNPi with TaPR1a.

Protein interaction between PNPi and TaPR1a in the apoplastic space was validated in N. benthamiana
To validate the function of the signal peptide of TaPR1a, a red florescent protein (RFP) tag was fused to the Cterminus of TaPR1a. The RFP-fused protein was transiently expressed in N. benthamiana by Agrobacterium-mediated transformation. Next, plasmolysis was induced by addition of 800 mM mannitol, and then clear apoplastic signals were observed for TaPR1a (1-164) -RFP, but not for the empty vector (EV-RFP) control (Fig. 2a). To validate the secretion of the recombinant SP (TaPR1a) -PNPi  , another construct of SP (TaPR1a) -PNPi (23-333) -GFP was prepared and transiently expressed in tobacco leaves. Under plasmolysis treatment, dot-like apoplastic signals were observed for SP (TaPR1a) -PNPi (23-333) -GFP (Fig. 2b). Interestingly, we also observed GFP signals for the recombinant SP (TaPR1a) -PNPi (23-333) -GFP in both the cytoplasm and nucleus.
To further validate the interaction between PNPi and TaPR1a, we performed a bimolecular fluorescence complementation (BiFC) assay in N. benthamiana. Co-expression of SP (TaPR1a) -PNPi (23-333) -YFP N and TaPR1a-YFP C generated yellow fluorescent protein (YFP) fluorescent signals in the apoplastic space (Fig. 3). Clear YFP fluorescent signals were also observed for the positive control, using AvrLm1-YFP N and BnMPK9-YFP C (Ma et al. 2018). Co-expression of each recombinant vector with the corresponding TaPR1a signal peptide-fused YFP N and YFP C vectors (SP (TaPR1a) -YFP N and SP (TaPR1a) -YFP C ) served as negative controls, which showed no fluorescent signals.
Transgenic wheat lines overexpressing TaPR1a showed enhanced resistance to both Pst and Pt To further explore the function of the TaPR1a gene, we generated transgenic wheat lines overexpressing TaPR1a in the spring common wheat cultivar "JW1". The expression levels of the overall endogenous and transgenic TaPR1a in the transgenic wheat line TaPR1a-OE ranged from 6.5-to 13.0-fold of that in wild-type plants (Fig. 4a). The third leaves of the transgenic wheat line TaPR1a-OE and wild-type plants were inoculated with urediniospores of the Pst pathotype CYR32, and CYR32 exhibited high virulence on wild-type plants (Fig. 4b). The susceptible phenotype (sporulation) in response to stripe rust pathogen infection was observed for all tested transgenic lines and wild-type plants. The percentage of Pst sporulation area for each inoculated leaf was calculated using ASSESS software (Additional file 2: Figure S3a). Compared with wild-type plants, significantly improved resistance to Pst was observed in TaPR1a-OE as evidenced by altered sporulation area (*P < 0.05, **P < 0.01, 6-10 biological replicates) ( Fig. 4b and Additional file 1: Table S2).
Transgenic wheat line TaPR1a-OE and wild-type plants were next inoculated with urediniospores of the Pt pathotype THTT. A moderately susceptible phenotype (sporulation with necrosis) in response to leaf rust pathogen infection was observed in both TaPR1a-OE and wild-type plants (Fig. 4c). The percentage of Pt sporulation area in TaPR1a-OE was significantly lower (**P < 0.01, 16-39 biological replicates) than that in wild-type plants (Fig. 4c, Additional file 1: Table S2, and Additional file 2: Figure S3b).

Exogenous expression of SP (TaPR1a) -PNPi-cMYC-OE in transgenic wheat lines reduced the degree of acquired resistance
The successful establishment of the transgenic wheat line SP (TaPR1a) -PNPi-cMYC-OE was confirmed by PCR of genomic DNA and RT-qPCR. The expression levels of the SP (TaPR1a) -PNPi-cMYC transgene ranged from 24.2-to 80.5-fold of the levels of the endogenous control gene, TaActin (GenBank accession AB181991) (Fig. 5a). We did not detect definite signals corresponding to the SP (TaPR1a) -PNPi-cMYC transcript in wild-type plants. P. syringae DC3000 was injected into wheat leaves to induce a SAR-like response (acquired resistance). To evaluate the degree of the acquired resistance, M. oryzae isolate P131 was inoculated adjacent to the P. syringae DC3000 infiltration area as the secondary pathogen. This inoculation was performed at 48 hours post-infiltration (hpi), when a clear cell death phenotype caused by P. syringae DC3000 was observed in the infiltration area (Fig. 5b). In wild-type plants, we observed significantly (**P = 0.009) improved resistance to M. oryzae isolate P131 in the region adjacent to the P. syringae DC3000 infiltration area, whereas the acquired resistance response was consistently suppressed in two independent transgenic wheat lines of SP-PNPi-OE ( Fig. 5c and Additional file 1: Table S3). We further evaluated the acquired resistance to M. oryzae isolate P131 in transgenic wheat line TaPR1a-OE, but did not observe any differences between TaPR1a-OE and wild-type plants.
We also examined the possible function of the TaPR1a gene during the acquired resistance triggered by P. syringae DC3000. To do this, RT-qPCR assay was employed to evaluate the expression levels of TaPR2 and TaPR4b in the adjacent region of P. syringae DC3000 infection in the transgenic wheat line TaPR1a-OE and wild-type plants. However, we did not detect any significant differences in the expression levels of these two genes between the transgenic line and wild-type plants (Additional file 1: Table S5 and Additional file 2: Figure S4).
We used P. syringae DC3000 to trigger the plant defense response and performed RT-qPCR assay to monitor the expression levels of TaAdi3 (GenBank accession XM_ 020291623) and TaPR7 (XP_020149865) in transgenic wheat lines TaPR1a-OE and SP-PNPi-OE in the local infiltration region at 6 hpi. Expression levels of both TaAdi3 (**P = 0.0005, 6-8 biological replicates) and TaPR7 (**P = 0.0002) were significantly induced by P. syringae DC3000 infection in wild-type plants and increased to even higher levels (**P < 0.01) in the transgenic wheat line TaPR1a-OE ( Fig. 6 and Additional file 1: Table S5). Compared with wild-type plants, we detected significant decreased induction of TaAdi3 (**P = 0.0002) and TaPR7 (**P = 0.0012) in the transgenic wheat line SP-PNPi-OE (Fig. 6).

Discussion
In our previous study, we discovered a virulent rust effector PNPi that can suppress systemic acquired resistance by directly targeting the wheat NPR1 protein (Wang et al. 2016). In this work, using a classic Y2H library screening approach, we identified 12 potential PNPi interactors (Additional file 1: Table S1). Based on the expression patterns of these interactor-encoding genes during SAR-like response triggered by P. syringae DC3000 in barley (Additional file 2: Figure S1), we selected TaPR1a, a wheat pathogenesisrelated protein as a putative interactor of PNPi for further characterization. The physical interaction between PNPi and TaPR1a was further validated by Co-IP assay (Fig. 1d). In our previous investigation, we determined the DPBB_1 domain of PNPi was required for interaction with wNPR1 (Wang et al. 2016). However, we did not identify specific domains in PNPi responsible for interaction with TaPR1a (Fig. 1a). The observed interactions of PtPNPi (homolog of PNPi in Pt) with both wNPR1 and TaPR1a by Y2H assay (Additional file 2: Figure S2) suggest potential conserved features among PNPi homologs from different rust species.
An RXLR-DEER-like (RSLL-DEEP) region was identified in the N-terminal region of the PNPi protein after the signal peptide, but the translocation mechanism of this rust effector from the apoplastic space to the cytoplasm and nucleus was not previously characterized. Under plasmolysis condition, apoplastic signals were observed for both TaPR1a-RFP and SP (TaPR1a) -PNPi (23-333) -GFP in N. benthamiana (Fig. 2). In a recent investigation, a similar apoplastic localization of TaPR1a-GFP was observed in tobacco leaves under plasmolysis treatment (Wang et al. 2020). We also observed cytoplasmic and nuclear signals for the recombinant SP (TaPR1a) -PNPi (23-333) -GFP protein, which might be caused by the cell re-entry process of the PNPi protein via its N-terminal RSLL-DEEP region, or could be generated by traffic congestion of the secretion pathway in the Agrobacterium-mediated transient expression assay (Wawra et al. 2017). In a previous study, transiently expressed TaPR1-GFP (approximately 40 kDa in size) in tobacco leaves was very stable, with no cleavage of the C-terminal CAPE1 region (Breen et al. 2016). Thus, we speculated that cleavage of CAPE1 did not occur on the time scale of our transient expression of  and TaPR1a-YFP C in the Co-IP and BiFC assays. Based on the detection of recombined YFP signals The phenotypes of TaPR1a-OE and wild-type plants in response to the highly virulent Pst pathotype CYR32. c The susceptibility phenotypes of TaPR1a-OE and wild-type plants to the Pt pathotype THTT. The numbers below the images of the leaves represent the average percentages of the rust sporulation area in each inoculated leaf. The asterisks (*P < 0.05, **P < 0.01) indicate the significance of the differences between the transgenic line and wild-type plants as assayed by Dunnett's test Fig. 5 The NPR1-mediated acquired resistance to Magnaporthe oryzae isolate P131 was suppressed in the transgenic wheat line SP-PNPi-OE. a Expression levels of the SP (TaPR1a) -PNPi-cMYC transgene in the transgenic wheat line SP-PNPi-OE were determined by RT-qPCR. b P. syringae DC3000 was employed to induce the acquired resistance in the region adjacent to the infiltration area. c The M. oryzae isolate P131 was inoculated in the adjacent region as a secondary pathogen to evaluate the degree of the acquired resistance. The numbers below the images of the leaves represent the average lesion size caused by M. oryzae. d The expression levels of TaPR1a, TaPR2, and TaPR4b in transgenic wheat line SP-PNPi-OE and wild-type plants during acquired resistance were determined by RT-qPCR. The whole experiment was repeated twice using two independent transgenic lines of SP-PNPi-OE. Statistical analyses are presented in Additional file 1: Table S5 in N. benthamiana co-expressing SP (TaPR1a) -PNPi (23-333) -YFP N and TaPR1a-YFP C (Fig. 3), we predicted interation of the rust effector PNPi with wheat TaPR1a in the apoplastic space.
PR1 family proteins have been reported to be involved in various biological processes, including sterol binding, defense signaling, and as targeting hubs of pathogen effectors (Breen et al. 2017). Resistance to both Pst and Pt was significantly improved in the transgenic wheat line TaPR1a-OE ( Fig. 4 and Additional file 1: Table S2). Although we detected no high-resistance phenotypes in TaPR1a-OE, the significantly diminished or delayed infection processes of both the virulent Pst pathotype CYR32 and the Pt pathotype THTT were observed. Therefore, the generated transgenic wheat line TaPR1a-OE provides a valuable resource for future efforts to improve wheat resistance to rust diseases.
In our previous investigation, we validated the role of PNPi in the suppression of SAR using transgenic barley line overexpressing the transcript that encodes the signal peptide-truncated PNPi   (Wang et al. 2016). Previous work showed that the signal peptide of PNPi can guide the secretion of a protein from an yeast fungus, but not from a plant cell (Wang et al. 2016). To confirm the function of PNPi in the natural wheat host and imitate the apoplastic state of this rust effector, we developed a transgenic wheat line overexpressing the recombinant transcript of SP (Ta-PR1a) -PNPi-cMYC. The model bacterial pathogen P. syringae DC3000 and the fungal pathogen M. oryzae isolate P131 were applied as an inducer and a secondary invader, respectively, to evaluate the degree of NPR1-mediated acquired resistance in wheat plants (Colebrook et al. 2012;Wang et al. 2016;Gao et al. 2018). Reduced acquired resistance to M. oryzae isolate P131 was observed in the transgenic wheat line SP-PNPi-OE, with significant suppression of TaPR1a, TaPR2, and TaPR4b genes ( Fig. 5 and Additional file 1: Table S3). These findings were similar to those we observed previously in the HvNPR1-silenced transgenic barley line .
The CAPE1 peptide of PR1 protein was reported as a DAMP during plant immune response that could act to transcriptionally activate several PR genes (Chen et al. 2014). RT-qPCR results confirmed that the observed increases of TaAdi3 and TaPR7 genes were directly associated with the increased expression level of TaPR1a transgene in the local region inoculated with P. syringae DC3000 (Fig. 6). These results were consistent with those of a previous study in which the chemically synthesized CAPE1 peptide was applied as an inducer of PR genes in tomato plants (Chen et al. 2014). Regulation of the TaAdi3 and TaPR7 genes may independent of NPR1 based on our previous transcriptome data (Additional file 2: Figure S5) , making these genes ideal candidates for further investigation of the wNPR1independent effect of PNPi on TaPR1a. The detected significant suppression of TaAdi3 and TaPR7 in the transgenic wheat line SP-PNPi-OE (Fig. 6), together with the evidence of the requirement of the CAPE1 region for Fig. 6 The transcriptional levels of TaAdi3 and TaPR7 were determined by RT-qPCR in transgenic wheat lines TaPR1a-OE and SP-PNPi-OE upon P. syringae DC3000 infection. Two independent transgenic lines of TaPR1a-OE and SP-PNPi-OE were tested. Statistical analyses are presented in Additional file 1: Table S5 interaction between PNPi and TaPR1a (Fig. 1a) provide valuable clues to help elucidate the possible working model of the rust effector PNPi.

Conclusions
During the early stage of infection, rust fungus induces the cleavage of the conserved CAPE1 peptide from the Cterminal end of the TaPR1a protein, allowing recognition by a pattern recognition receptor (PRR). The signal will be transduced to eventually drive transcriptional activation of TaAdi3 and TaPR7 genes. In the apoplastic space or extrahaustorial matrix, rust fungus secretes the effector PNPi to target the CAPE1 region of the TaPR1a protein, and this interaction may suppress the expression of TaAdi3 and TaPR7. PNPi can be translocated into the plant cell and nucleus to suppress the expression burst of PR genes by directly targeting wheat NPR1.

Screening of Y2H library using PNPi as a bait
The Y2H cDNA library tested here was previously constructed from Puccinia striiformis f. sp. tritici-infected leaves of the Triticum turgidum subsp. durum cultivar "Langdon" (line RSL65) (Yang et al. 2013). The wheat line RSL65 carries the Yr36 resistant gene, which confers partial resistance to Pst at temperature higher than 25°C (Fu et al. 2009). Seedlings of RSL65 were grown in a growth chamber with a nocturnal temperature of 10°C, a diurnal temperature of 15°C, and 16 h of light. Under these conditions, Yr36 resistance is not active, so plants are partially susceptible to Pst. RSL65 seedlings were inoculated with urediniospores of Pst pathotype PST-133 as previously described (Fu et al. 2009). Pst-infected leaves were sampled at 0, 3, 9, and 15 days post-inoculation (dpi). Total RNA were extracted from samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Extracted RNAs were pooled and reverse-transcribed into cDNA, using the Make Your Own "Mate & Plate" Library System following the manufacturer's protocol (Clontech, Mountain View, CA, USA). The resulting cDNAs were then recombined into the prey vector (pGADT7Rec) using Clontech's SMART technology, and the final library was transformed into the yeast strain Y187. Next, the transcript encoding PNPi  was cloned into the bait vector pLAW10 (BD) and introduced into the yeast strain "Y2H Gold" using the lithium acetate method (Cantu et al. 2013b). The pLAW10-PNPi (23-333) construct was tested against an empty vector and was not auto-activated on SD-Leu-Trp-His-Ade. The bait colonies of pLAW10-PNPi  were grown to approximately 10 8 cfu/mL in 50 mL SD-Trp liquid medium. Yeast cells were pelleted, washed once with sterile H 2 O, and re-suspended in 50 mL of liquid media consisting of 2× YPDA. One aliquot of the Y187 target yeast (> 2 × 10 7 cells) was mixed with the bait. After incubation at 30°C for 20-24 h with slight shaking, the yeast cells were isolated, washed twice with sterile water, and then plated on SD-Leu-Trp-His-Ade medium. Yeast putative positive diploids from primary screenings were isolated and plasmids were extracted using Zymoprep I™ Yeast Plasmid Minipreparation Kit (Zymo Research, CA, USA). Matchmaker AD primers were used to amplify the inserted gene fragments. Sequence annotations were performed with BLASTx homology searches against the NCBI GenBank nr database (https://www.ncbi.nlm.nih.gov/).

Y2H validation
Primers were designed to amplify the coding region of TaPR1a (Additional file 1: Table S4). The complete coding region of TaPR1a was amplified from cDNA synthesized from RNA isolated from the seedlings of the common wheat line "Chinese Spring". SignalP 4.1 server (http:// www.cbs.dtu.dk/services/SignalP/) was employed to identify the signal peptide. The coding regions of the signal peptide-truncated PNPi and TaPR1a were cloned into Gateway Y2H vectors pLAW10 (BD) and pLAW11 (AD), respectively. Recombinant Y2H vectors carrying different regions of the PNPi gene were constructed and described in our previous study (Wang et al. 2016). The coding sequence of PNPi (23-235) started immediately after the end of the 22-amino-acid-long predicted signal peptide and included 213 amino acids from the N-terminal region of the PNPi protein.  included the DPBB_1 domain located in the C-terminal region of the protein.
TaPR1a  encoded the signal peptide-truncated TaPR1a. TaPR1a  and TaPR1a  are derivatives of TaPR1a with both the signal peptide and the C-terminal CAPE1 peptide region truncated. Co-transformed yeast strains were assayed on plates of SD-Leu-Trp-His-Ade selective medium.
We cloned the coding region of PtPNPi using cDNA synthesized from RNA isolated from wheat seedlings of the common wheat line "Chinese Spring" that were inoculated with Pt pathotype PHTT(P) . The coding region of the signal peptide-truncated PtPNPi was cloned into Gateway Y2H vector pLAW11 (AD). The wNPR1-BD construct was constructed in our previous study (Cantu et al. 2013b). The co-transformed yeast strains were assayed on selective medium (SD-Leu-Trp-His-Ade).

Subcellular localization and BiFC
To confirm the signal peptide of TaPR1a predicted by the SignalP 4.1, a RFP tag was fused to the C-terminus of TaPR1a and a GFP tag was fused to the C-terminal end of the recombinant SP (TaPR1a) -PNPi  protein.
The resulting constructs were transformed into the Agrobacterium strain GV3101, and then used to infiltrate four-week-old N. benthamiana plants. An empty pGWB5 vector expressing only GFP and an empty pGWB454 vector expressing only RFP served as controls. Green or red fluorescence was detected 48 hpi using a Nikon Ti-2 fluorescence microscope with a filter specific to GFP or RFP, respectively. Epidermal peels from N. benthamiana leaves were plasmolyzed in 800 mM mannitol for 6 min.
BiFC assay was conducted using a split YFP system in N. benthamiana leaves, as previously described (Ma et al. 2018). The complete coding regions of TaPR1a, as well as the recombinant transcript of SP (TaPR1a) -PNPi  , were fused to the N-and C-terminal regions of YFP in Gateway destination vectors pDEST-GW VYNE and pDEST-GW-VYCE, respectively. The fusion proteins were then coexpressed in N. benthamiana leaves using Agrobacteriummediated transformation. Fluorescence was monitored approximately 48 hpi, using a Nikon fluorescence microscope with a YFP filter. Co-transformation of AvrLm1-YFP N and BnMPK9-YFP C was utilized as a positive control (Ma et al. 2018). Signal peptide-fused empty vectors of SP (TaPR1a) -YFP N and SP (TaPR1a) -YFP C were used as negative controls.

Co-IP assay
The coding sequence of the signal peptide-truncated TaPR1a was cloned into the Gateway compatible GFPtagged expression vector pMDC43 (Curtis and Grossniklaus 2003) as 35S::GFP-TaPR1a  . Tobacco leaves were infiltrated with Agrobacterium carrying 35S::GFP-TaPR1a  , and infiltration with 35S::GFP was performed as a control. Protein samples were extracted from Agrobacteriuminfiltrated tobacco leaves at 3 dpi and the transgenic wheat line overexpressing SP (TaPR1a) -PNPi-cMYC. Next, α-GFP IP was performed using GFP-trap beads (LABLEAD, Beijing, China) as previously described (Du et al. 2015). Total proteins and immunoprecipitates were separated by SDS-PAGE, and then detected by immunoblots using α-GFP antibody (Solarbio, Beijing, China) or α-cMYC antibody (Solarbio, Beijing, China). Original, uncropped blots are provided as Additional file 2: Figure S6.

Plant materials and induction of acquired resistance
All transgenic wheat lines used in this study were generated in our lab with technical help from Jinan Bangdi Ltd. Company, Shandong, China. The transgenic background material "JW1" was selected by this company from a segregating population of the cross of spring common wheat cultivars "Fielder" and "NB1". Briefly, the full-length ORF of TaPR1a and the recombinant transcript of SP (TaPR1a) -PNPi-cMYC (PNPi with the signal peptide of TaPR1a and a C-terminal cMYC tag), were cloned into a wheat transgenic vector, pLGY-02, that contained the maize (Zea mays) ubiquitin 1 promoter and T-DNA insertion site. Transgenic wheat lines were generated using Agrobacterium-mediated transformation. Acquired resistance was induced by inoculation with P. syringae DC3000, as previously described (Colebrook et al. 2012). Briefly, P. syringae DC3000 was grown on King's B medium supplemented with 50 μg/mL rifampicin antibiotics and then diluted to OD600 = 0.6 in sterile water. Third leaves of transgenic wheat lines SP-PNPi-OE and TaPR1a-OE as well as wild-type plants were inoculated using a 1-mL needless syringe by pressure infiltration of bacterial suspensions through the leaf abaxial surface. The borders of each infiltrated region were marked using a marker pen and control seedlings were similarly infiltrated with sterile water. After bacterial inoculation, the seedlings were transferred under a constant temperature of 23°C to facilitate bacterial growth. Samples for RT-qPCR assay were collected from regions adjacent to the infiltration area (approximately 1 cm from the border of the infiltrated area) at 48 hpi.
M. oryzae inoculation was performed on adjacent regions of leaves to evaluate the degree of acquired resistance in the transgenic wheat line SP-PNPi-OE and wildtype plants. The M. oryzae isolate P131 was grown on tomato-oat medium for 11 d at 25°C under a 16/8 h light-dark cycle. Ten mL of the P131 conidia suspension (5.0× 10 5 spores/mL) containing 0.05% Tween-20 was dropped onto press-injured spots in adjacent region. The plants were then incubated in a mist chamber at 25°C in the dark for the first 24 hpi before transfer to a growth chamber at 23°C and 80% humidity under a photoperiod of 16 h light and 8 h dark. The inoculated leaves were photographed 5 days later and the size of the lesion caused by P131 on each leaf was measured. Six to ten biological replicates were performed for each treatment and phenotype combination, and the whole experiment was repeated twice using two independent transgenic lines. Dunnett's test was performed using SAS software v9.4 (SAS Institute, Cary, NC, USA).

Wheat rust inoculation
Seedlings of the transgenic wheat line TaPR1a-OE and wild-type plants were maintained and inoculated with Pst pathotype CYR32 and Pt pathotype THTT according to the previously described procedures and conditions (Huang et al. 2019;Wu et al. 2019). The fully expanded third leaves of wheat seedlings were spray-inoculated with uredospores of CYR32 and THTT in water solution. The disease symptoms were scored at 14 dpi. The percentages of the Pst or Pt sporulation area for each inoculated leaf were determined using ASSESS image analysis software v2.0 for plant disease quantification (Lamari 2008). This software was previously applied for evaluation of wheat resistance to stem rust (Zhang et al. 2017), stripe rust, and powdery mildew (Li et al. 2020). The ASSESS software recognizes rust uredinium in an inoculated leaf based on individual pixel color (Additional file 2: Figure S3). For Pst inoculation, two independent transgenic lines were employed with 6-10 biological replicates. Data were transformed for normality restoration, and Dunnett's test was performed using SAS software v9.4 (SAS Institute, Cary, NC, USA). For Pt inoculation, the whole experiment was repeated twice using two independent transgenic lines. Each experiment of 16-39 biological replicates was considered a block. The data were log-transformed to restore normality, and two-sample t-test was conducted using SAS software v9.4.

RNA extraction and RT-qPCR
Total RNA samples were prepared by extraction using a RNA Extraction kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions, and then first-strand cDNA samples were synthesized using a Reverse Transcription kit (Takara, Dalian, China). Equal amounts of RNA were used for each reverse transcription reaction. The primers utilized for RT-qPCR assay are listed in Additional file 1: Table S4. The wheat TaActin gene (GenBank accession AB181991) was employed as an internal reference for the analyses (Paolacci et al. 2009). The amplification efficiency of each pair of primers was calculated by a preliminary RT-qPCR assay using six 2-fold diluted cDNA samples (1:1, 1:2, 1:4, 1:8, 1:16, and 1:32). Next, dissociation curves were generated at temperatures ranging from 60 to 94°C to ensure that a single and specific product was amplified. Gene expression was quantified using the threshold values (Ct) generated by the Roche LightCycler 96 (Roche, Indianapolis, IN, USA) based on the 2 -ΔCt method (Wang et al. 2016), and all transcript levels were expressed relative to those of the endogenous control TaActin. The whole experiment was repeated twice with independent transgenic lines. Each experiment of 4-12 biological replicates was considered as a block. Calculations of the mean and standard error were performed using Microsoft Excel software. Data were log-transformed to restore normality, and the general linearized model (GLM) ANOVA, Dunnett's test, and two-sample t-test were performed using SAS software version v9.4.