TaRBP1 stabilizes TaGLTP and negatively regulates stripe rust resistance in wheat

Abstract

The dynamic balance and distribution of sphingolipid metabolites modulate the level of programmed cell death and plant defence. However, current knowledge is still limited regarding the molecular mechanism underlying the relationship between sphingolipid metabolism and plant defence. In this study, we identified a wheat RNA‐binding protein 1 (TaRBP1) and TaRBP1 mRNA accumulation significantly decreased in wheat after infection by Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaRBP1 via virus‐induced gene silencing conferred strong resistance to Pst by enhancing host plant reactive oxygen species (ROS) accumulation and cell death, indicating that TaRBP1 may act as a negative regulator in response to Pst. TaRBP1 formed a homopolymer and interacted with TaRBP1 C‐terminus in plants. Additionally, TaRBP1 physically interacted with TaGLTP, a sphingosine transfer protein. Knockdown of TaGLTP enhanced wheat resistance to the virulent Pst CYR31. Sphingolipid metabolites showed a significant accumulation in TaGLTP‐silenced wheat and TaRBP1‐silenced wheat, respectively. In the presence of the TaRBP1 protein, TaGLTP failed to be degraded in a 26S proteasome‐dependent manner in plants. Our results reveal a novel susceptible mechanism by which a plant fine‐tunes its defence responses by stabilizing TaGLTP accumulation to suppress ROS and sphingolipid accumulation during Pst infection.

The TaRBP1–TaGLTP complex stabilized the negative regulator TaGLTP and suppressed TaGLTP‐mediated cell death and reactive oxygen species accumulation to promote Puccinia striiformis f. sp. tritici colonization in wheat.

1. INTRODUCTION

Plants activate multilayered immune functions to diminish destructive attacks from many forms of stress, such as extreme climate and microbial pathogens. Intrinsic plant immunity is composed of the physical barrier on the plant surface, such as the cuticle and waxy layer, which prevents pathogens and predators from invading the plant or feeding. Additionally, plants recognize inherent pathogen traits, such as bacterial flagellin, elongation factor Tu (EF‐Tu), and cell wall components (pathogen‐associated molecular patterns [PAMPs]) through pattern recognition receptors. Molecular signalling is transmitted and amplified from the extracellular space to the cytoplasm and nucleus, activating a series of plant immune responses called PAMP‐triggered immunity (PTI) (Boller & He, 2009; Iwasaki & Medzhitov, 2010; Jones & Dangl, 2006). Another plant immune mechanism involves diverse effectors delivered from pathogens that are perceived by plant nucleotide‐binding leucine‐rich repeat (NB‐LRR) proteins, leading to effector‐triggered immunity (ETI), which is accompanied by a hypersensitive response and reactive oxygen species (ROS) accumulation. This prevents pathogen development and colonization at infected sites (Dodds & Rathjen, 2010; Jones & Dangl, 2006).

Previous studies reported that ROS, including superoxide (O²⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH·), are rapidly accumulated in infected cells during early stages of pathogen–plant interactions (Heller & Tudzynski, 2011). Numerous ROS are derived from chloroplasts and mitochondria, causing plant cell damage, nucleic acid breakage, protein oxidation, lipid peroxidation, and cell death (Sachdev et al., 2021). Normally, ROS production is tightly regulated and balanced by the activation of enzymatic antioxidants (superoxide dismutase, catalase, peroxidase) and nonenzymatic antioxidants, such as flavonoids and carotenoids (Sachdev et al., 2021). However, this balance can be quickly altered by ROS regulator activation (including redox‐responsive transcription factor 1, RRTF1) in response to environmental stimuli. RRTF1 is a node of the redox signalling pathway and promotes ROS accumulation and cell death in Arabidopsis thaliana when the plant is subjected to biotic and abiotic stress (Matsuo et al., 2015). Overexpression of the RNA‐binding protein 1 (CaRBP1) of Capsicum annuum rapidly and strongly induces cell death and defence responses in plant cytoplasm (Lee et al., 2012). Recently, the wheat leaf rust resistance gene Lr13 was identified as a necrosis 2 gene (Ne2), producing a range of necrotic symptoms and contributing to wheat resistance to leaf rust fungi (Hewitt et al., 2021; Yan et al., 2021). However, knowledge is limited regarding the network of ROS regulation and how the cell death response is triggered by plant pathogens.

When exposed to pests and pathogens, plants use resources to activate the plant defence response to resist invasion, at a cost to plant growth and development (Deng et al., 2017; Li et al., 2019). Lesion mimic mutants (LMMs), also known as hypersensitive reaction‐like traits, cause defence lesions to arise spontaneously in leaf tissues without being attacked by plant pathogens. Thus, LMMs are valuable genetic resources for studying programmed cell death (PCD) signalling pathways and disease resistance in plants. Plants undergo large‐scale necrosis and tissue disintegration to locally kill pathogenic microorganisms (Brodersen et al., 2002). Mutation of GmLMM1, encoding a malectin‐like receptor kinase, inhibited the receptor kinase FLS2 (flagellin‐sensitive 2)–BAK1 (brassinosteroid insensitive 1 associated kinase receptor 1) interaction and immune activation, thus ultimately regulating plant resistance to bacterial and oomycete pathogens (Wang et al., 2020). The acd11 LMM has robust autoimmunity and a dwarf phenotype, and leads to imbalance between plant development and immunity (Brodersen et al., 2002; Bruggeman et al., 2015; Petersen et al., 2008). AtACD11 was identified as a sphingosine transfer protein, which selectively shifted sphingosine and sphingomyelin between the cytoplasm and membranes (Simanshu et al., 2014). Sphingolipids are divided into the following types: ceramides (Cers), glucosylceramides (GlcCers), hydroxyceramides (hCers), glycosyl inositol phosphoryl ceramides (GIPCs), and free long‐chain bases (Markham et al., 2013). The balance between ceramide and its phosphorylated derivative was proposed to regulate PCD levels in plants (Simanshu et al., 2014). Up‐regulation of ceramide and the long‐chain base sphingosine altered by ACD11 deficiency have been connected to plant PCD in Arabidopsis (Liang et al., 2003). Plant defence responses could be completely suppressed in the acd11 mutant without the NahG gene, indicating that salicylic acid (SA) plays a role in PCD initiation in the ACD11 mutant (Brodersen et al., 2005). Therefore, LMM plants are a good choice for directly exploring the molecular mechanism of PCD, plant hormone metabolism, and disease resistance.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a serious disease that severely reduces grain production worldwide. However, due to the difficulties in developing a stable transformation system in hexaploid plants such as wheat, functional analyses of wheat genes have been severely hindered. Recently, several approaches, including protein–protein interaction assays, virus‐mediated gene silencing (VIGS), and a transient expression system in Nicotiana benthamiana, have been developed to analyse the mechanism of plant resistance and identify defence‐associated proteins and vital nodes of the metabolic pathway in plant–pathogen interactions. Barley stripe mosaic virus (BSMV)‐induced gene silencing (VIGS) has emerged as a useful tool to study loss‐of‐function phenotypes of candidate genes. A 88%–100% similarity in nucleotide sequence can cause the silencing of an endogenous gene target in monocot and dicot plants (Holzberg et al., 2002). For example, use of VIGS to co‐silence TaCBL4 and TaCIPK5 resulted in enhanced wheat susceptibility to Pst (Liu et al., 2018). Wheat kinase START1, encoded by Yr36, phosphorylates PsbO, a photosystem II oxygen‐evolving enhancer protein 1, resulting in the destruction of photosystem II and increasing the susceptibility of wheat to Pst (Wang et al., 2019). However, due to the complexity of the hexaploid wheat genome and high levels of variation of Pst virulence, the molecular mechanism of plant susceptibility is unclear and effectively controlling the disease is difficult.

Previous studies reported that a rust fungus effector Pst_A23 acts as a splicing factor to regulate host pre‐mRNA splicing by direct engagement of the splicing sites, thereby interfering with host immunity (Tang et al., 2022). One of the candidate target host proteins, TaRBP1, was confirmed using Pst_A23 as the bait in a screen of a yeast two‐hybrid (Y2H) library (Tang et al., 2022). To better understand the role of TaRBP1 during the Pst–wheat interaction, in this study the transcript levels of TaRBP1 were first measured and found to be markedly reduced at early infection in response to Pst. As a negative regulator, TaRBP1 weakened wheat resistance to Pst, which targeted and manipulated the amount of TaGLTP in a 26S proteasome‐dependent manner. The susceptible mechanism of the TaRBP1–TaGLTP complex stabilized the negative regulator TaGLTP and suppressed TaGLTP‐mediated cell death and sphingolipid accumulation to promote fungal colonization in the Pst–wheat interaction.

2. RESULTS

2.1. Analysis of the TaRBP1 protein

A cDNA fragment was amplified from a library constructed from wheat leaves infected with stripe rust. The cDNA fragment was 858 bp long, with a predicted open reading frame of 735 bp. The SMART database (http://smart.embl‐heidelberg.de/) predicted the presence of an RNA recognition motif (RRM) at the N‐terminus (Figure S1a). RRM‐containing proteins, known as RNA‐binding proteins, are ubiquitous and involved in the development and growth of living plants, as well as the plant response to biotic and abiotic stresses (Guerra et al., 2015; Skelly et al., 2016). Compared to the RRM domain with other orthologous proteins in various species, the sequence alignment showed that the RRM domain possesses conserved RNP1 and RNP2 motifs in the four β‐sheet and two α‐helix structures (Figure S1a). Phylogenetic analysis indicated that it was highly similar to other RNA‐binding proteins, so it was designated as TaRBP1. TaRBP1 and its Poaceae homologue shared the greatest similarity, indicating that TaRBP1‐like homologues are conserved in the Poaceae (Figure S1b). We used a fragment of TaRBP1 to search for similar sequences in the wheat genome using BLAST and found three copies of TaRBP1 with 97% nucleic acid sequence similarity located on chromosomes 2A, 2B, and 2D (Figure S2). TaRBP1 on chromosome 2A was amplified and obtained from wheat. To identify its localization in plants, TaRBP1 fused with green fluorescent protein (TaRBP1:GFP) was transiently expressed in N. benthamiana. The fluorescence signal of the TaRBP1:GFP protein was observed in the cytoplasm and nucleus of N. benthamiana (Figure S3). These results indicate that TaRBP1 may be a wheat RNA‐binding protein in the plant's cytoplasm and nucleus.

2.2. Knockdown of TaRBP1 enhances wheat tolerance to Pst

To examine the role of TaRBP1 in the Pst–wheat interaction, the expression of TaRBP1 was previously analysed using wheat RNA‐Seq data (Borrill et al., 2016). TaRBP1‐2A and TaRBP1‐2B were significantly down‐regulated on infection with an avirulent Pst CYR23 at 24 h postinoculation (hpi), while no significant difference existed on infection with virulent Pst CYR31 (Figure S4a). The expression profile of TaRBP1‐2A and TaRBP1‐2B was further confirmed by reverse transcription‐quantitative PCR (RT‐qPCR) during the Pst infection of wheat. The transcript level of TaRBP1 was decreased by 0.6‐ to 0.7‐fold after the plants were inoculated with the avirulent strain CYR23 at 24 and 48 hpi. The transcript level of TaRBP1 was insignificant compared with the control infected by virulent Pst CYR31 (Figure S4b).

To further confirm the function of TaRBP1 in the Pst–wheat interaction, two specific fragments were chosen to transiently silence the three copies of TaRBP1 using BSMV‐mediated VIGS, because of the high similarity between the three gene copies. Strong photobleaching on the fourth leaves 10 days after inoculation with BSMV:TaPDS indicated that TaPDS gene was successfully silenced, and thus the silencing system functioned in the wheat plants (Figure 1a). The disease phenotype was observed 14 days after Pst CYR31 infection of the wheat plant carrying VIGS constructs. The number of uredinia decreased, with abundant necrotic cell death, in wheat plants expressing the VIGS construct of TaRBP1 compared to the empty vector control (BSMV:00) (Figure 1b). In comparison with the control plants, the expression level of TaRBP1 decreased by 65%–70% in TaRBP1‐silenced plants (Figure 1c). Pst biomass was measured in infected plants to test whether this disease phenotype correlated with fungal growth. Pst biomass decreased by 60%–70% in TaRBP1‐silenced plants compared to that in control plants (Figure 1d), consistent with the reduced disease phenotype.

FIGURE 1.

Silencing TaRBP1 enhanced plant resistance to Puccinia striiformis f. sp. tritici (Pst). (a) The second leaves of two‐leaf‐stage wheat cultivar Suwon 11 were inoculated with barley stripe mosaic virus BSMV:00 and recombinant BSMV:TaPDS, BSMV:TaRBP1‐1, and BSMV:TaRBP1‐2. Mild chlorotic mosaic symptoms were photographed 10 days after BSMV inoculation. (b) The disease phenotype was photographed 14 days after Pst CYR31 inoculation on the fourth leaf of BSMV‐inoculated wheat cultivar Suwon 11. (c) The relative expression level of TaRBP1 in leaves inoculated with Pst CYR31 was assayed by reverse transcription‐quantitative PCR at 24, 48, and 120 h postinoculation (hpi). (d) The Pst:wheat biomass ratio was assayed at 120 hpi by quantitative PCR. The corresponding RNA was isolated from the fourth leaves of the same set of wheat plants at 24, 48, and 120 hpi. Mean and standard deviations were calculated with data from three independent replicates. The asterisk marks a significant difference based on the two‐tailed Student's t test (p < 0.05).

Cell death, H₂O₂ accumulation, and plant defence‐related genes were observed for changes in plant resistance to Pst on infected wheat leaves. H₂O₂ accumulation and cell death were rarely observed in silenced plants at 24 hpi in BSMV:00‐inoculated wheat leaves. However, H₂O₂ accumulation increased in plants infected with BSMV:TaRBP1‐1 and BSMV:TaRBP1‐2 at 48 hpi (Figure 2a,b) compared to the control. The area of cell death at infected sites significantly increased in plants in which TaRBP1 expression was knocked down (Figure 2a,c). Histological observation showed that the hyphal length and infection areas of Pst at 24 and 48 hpi also decreased when TaRBP1 was silenced (Figure S5). Furthermore, the wheat defence‐related genes TaPR1 and TaPR2 were increased two‐ to three‐fold in the TaRBP1 knockdown plants (Figure 2d), indicating their possible role as a negative regulator in response to Pst.

FIGURE 2.

The plant resistance response to Puccinia striiformis f. sp. tritici (Pst) in TaRBP1 knockdown plants. (a) Histological observation of H₂O₂ accumulation and hypersensitive response (HR) in TaRBP1 wheat leaves of cultivar Suwon 11 silenced by BSMV‐induced gene silencing and inoculated with Pst CYR31 at 48 h postinoculation (hpi). Bar = 10 μm. IH, infectous hypha; SV, substomatal vesicle. (b) H₂O₂ accumulation was measured by calculating the 3,3′‐diaminobenzidine‐stained area at each infection site. (c) CellSens Entry software estimated the area of cell death. Means were assayed from 50 infection sites of three biological replicates. The asterisks indicate significant differences using the two‐tailed Student's t test (p < 0.05). (d) Transcripts of defence‐related genes TaPR1 and TaPR2 were detected 48 h after Pst CYR31 inoculation on the TaRBP1‐silenced plants. Averages and standard deviations were calculated from three replicates. The asterisks mark significant differences by the two‐tailed Student's t test (p < 0.05).

2.3. TaRBP1 forms a homopolymer and interacts with its C‐terminus

In a Y2H assay, yeast cells expressing pBD:TaRBP1 and pAD:TaRBP1 grew on synthetic dropout medium (SD) (−Leu/−Trp/−His/−Ade) plates (Figure 3a). This result fortuitously indicated that TaRBP1 could interact with itself in yeast. To confirm this relationship, TaRBP1 was inserted into pSPYCE(M) and pSPYNE(R)173 for a bimolecular fluorescence complementation (BiFC) assay, in which the fluorescence mainly remained in the cytoplasm and nucleus (Figure 3b). A co‐immunoprecipitation (Co‐IP) assay was used to test whether this interaction could occur in plants. TaRBP1:GFP (green fluorescent protein) and TaRBP1:FLAG proteins were expressed on N. benthamiana leaves, and total protein was extracted and pulled down using anti‐FLAG beads. Protein analysis showed that TaRBP1:GFP could be pulled down from total proteins (Figure 3c), further confirming its interaction. We artificially truncated the RRM region from the TaRBP1 protein to better understand its interaction (Figure S6a). Yeast cells expressing pBD‐TaRBP1 (full‐length protein) and pAD‐mRRM (lacking the N terminus and RRM region) grew on SD (−Leu/−Trp/−His/−Ade) plates, while transformed strains containing pBD‐TaRBP1 and pAD‐RRM (N‐terminus and RRM present but lacking the C‐terminus) did not (Figure S6b). Additionally, the BiFC assay confirmed that TaRBP1 could interact with its C‐terminus instead of its N‐terminus (Figure S6c). A homologous protein of TaRBP1 from N. benthamiana was found (NbRBP) with 54% amino acid sequence similarity that could also interact with TaRBP1 (Figure S6b,c), therefore TaRBP1 forms a homopolymer and the interaction site is at the C‐terminus of the protein.

FIGURE 3.

TaRBP1 forms homopolymer in vivo. (a) The yeast two‐hybrid analysis indicates that TaRBP1 interacts with itself. The transformant with the labelled constructs grew on the SD medium lacking LW (−Leu/−Trp), LWH (−Leu/−Trp/−His), and LWHA (−Leu/−Trp/−His/−Ade). (b) The bimolecular fluorescence complementation assay confirmed the interaction in plants. pNE and pCE‐TaRBP1 were the negative controls. pNE‐TaSGT1 and pCE‐TaRar1 were the positive controls. Bar = 20 μm. (c) The co‐immunoprecipitation assay confirmed the interactions in Nicotiana benthamiana. Western blots and immunoprecipitation of proteins were identified with the anti‐GFP or anti‐FLAG antibody. The protein marker is labelled on the left.

2.4. TaRBP1 interacts with a glycolipid transfer protein, TaGLTP

To further explore the mechanism by which TaRBP1 influences the plant immune response, we used TaRBP1 to screen the Y2H library constructed with RNA isolated from Pst CYR31‐infected wheat leaves of the cultivar Suwon 11. A candidate protein was identified to be a wheat glycolipid transfer protein, the full length of the TaGLTP gene was extracted from wheat cDNA, and protein–protein interaction was confirmed using the Y2H system. Except for the negative control, all yeast cells grew on SD (−Leu/−Trp/−His) and SD (−Leu/−Trp/−His/−Ade) plates (Figure 4a). The gene encoding AtACD11 (At2g34690) from Arabidopsis thaliana, which is 64% identical to TaGLTP, was amplified (Figure S7a), and the interaction between TaRBP1 and AtACD11 was also confirmed by Y2H assays (Figure S7b). The BiFC assay was used to further test the interaction between TaRBP1 and TaGLTP in plants. The full‐length TaRBP1 and TaGLTP genes were inserted into pSPYCE and pSPYNE, respectively. Strong yellow fluorescent protein (YFP) signals of the interaction between TaRBP1 and TaGLTP were observed in the cytoplasm and nucleus in N. benthamiana leaves (Figure 4b), whereas negative controls had no fluorescence signal in plant cells (Figure 4b), indicating that TaRBP1 proteins interacted with TaGLTP. Additionally, the TaRBP1–TaGLTP interaction was confirmed by Co‐IP assays in plants. Total TaGLTP:GFP and TaRBP1:HA proteins in N. benthamiana were amplified and exposed to GFP‐Trap gel beads. Finally, both TaGLTP and TaRBP1 were detected in western blots of total proteins and proteins eluted from GFP beads using anti‐HA and anti‐GFP polyclonal antibodies, respectively (Figure 4c). These data therefore confirmed that TaRBP1 could physically interact with TaGLTP.

FIGURE 4.

TaRBP1 interacts with TaGLTP in vivo. (a) The interaction between TaRBP1 and TaGLTP was detected via yeast two‐hybrid assays. Yeast cells transformed with the labelled constructs were assayed for growth on the SD medium lacking LW (−Leu/−Trp), LWH (−Leu/−Trp/−His), and LWHA (−Leu/−Trp/−His/−Ade). (b) The TaRBP1–TaGLTP interaction was confirmed via bimolecular fluorescence complementation assays. pNE with pCE‐TaRBP1 and pNE‐TaGLTP with pCE were the negative controls. pNE‐TaSGT1 and pCE‐TaRar1 were the positive controls. Bar = 20 μm. (c) Interaction between TaRBP1 and TaGLTP was confirmed by co‐immunoprecipitation assays. Western blots of the total proteins from Nicotiana benthamiana leaves and proteins eluted from GFP beads were identified with the anti‐GFP or anti‐HA antibody. The sizes of TaGLTP:GFP and TaRBP1:HA bands were 47 and 27 kDa, respectively. The protein marker is labelled on the left.

2.5. Silencing of TaGLTP increases the wheat defence response to Pst

In A. thaliana, AtACD11 genes encode a sphingosine transfer protein, and its mutant is dwarfed with a cell death phenotype, negatively regulating plant immunity (Brodersen et al., 2002, 2005). TaGLTP and AtACD11 from A. thaliana were 64% similar in amino acids (Figure S7a). Additionally, the structure models of the TaGLTP and AtACD11 proteins were highly consistent (Figure S8), indicating the possibility for similar functions. To seek conclusive evidence, we transiently silenced TaGLTP in wheat. TaGLTP transcript accumulation was reduced by 70%–80% in the TaGLTP knockdown plants (Figure S9). The seedlings of TaGLTP‐silenced wheat showed the dwarf phenotype compared to the control after 15 days of BSMV inoculation, but macroscopic lesion spots and a change in TaGLTP‐silenced wheat leaf width were not observed compared with the control plants (Figure 5a). The height of TaGLTP knockdown wheat was 7–8 cm shorter than the control plant (Figure 5b,c). We also assessed the defence priming of TaGLTP‐silenced wheat. A strong induction of ROS and cell death production was detected by 3,3′‐diaminobenzidine (DAB) and trypan blue staining compared with that in the control plant (Figure 5d). Additionally, the transcript level of the defence‐related genes TaPR1 and TaPR2 in the TaGLTP knockdown plants was significantly up‐regulated four‐fold compared to that in the control (Figure 5e). Therefore silencing TaGLTP elevated wheat defence priming and triggered spontaneous autoimmunity.

FIGURE 5.

Knockdown of TaGLTP enhances plant immunity. (a) The second leaves of two‐leaf stage wheat cultivar Suwon 11 were inoculated with BSMV or recombinant BSMV. The phenotype was photographed 10 days after BSMV inoculation. (b) Plant heights were photographed after 15 days of BSMV or recombinant BSMV inoculation. (c) Data mean and standard deviations were calculated from three independent replicates. The asterisks mark significant differences based on the two‐tailed Student's t test (p < 0.05). (d) Reactive oxygen species production in TaGLTP‐silenced wheat leaves was detected via 3,3′‐diaminobenzidine (DAB) and trypan blue staining. (e) The TaPR1 and TaPR2 expressions were detected via reverse transcription‐quantitative PCR after silencing TaGLTP. Mean and standard deviations were calculated from three independent replicates. The asterisks mark significant differences based on the two‐tailed Student's t test (p < 0.05).

To determine whether TaGLTP has a role in wheat defence against Pst, we first measured the transcript levels of TaGLTP exposed to biotic stress. A two‐fold significant increase in TaGLTP expression was observed on infection with virulent Pst CYR31 at 12 hpi, while its expression was rapidly reduced on infection with Pst CYR23 (Figure S10). When the Pst CYR31 was inoculated on BSMV‐infected leaves, fewer uredinia were produced on TaGLTP‐knockdown plants than in the control at 14 days postinoculation (dpi) (Figure 6a). The expression of the plant defence‐related genes TaPR1 and TaPR2 was assessed to detect the host resistance response. Compared with the control leaves expressing BSMV, TaPR1 and TaPR2 expression increased three‐ and four‐fold in TaGLTP‐knockdown plants, respectively (Figure 6b,c). The Pst biomass decreased by 50%–60% in TaGLTP‐silenced plants compared to that in the control (Figure 6d), indicating that TaGLTP‐silenced genes enhanced the wheat defence response.

FIGURE 6.

Knockdown of TaGLTP enhances wheat resistance to Puccinia striiformis f. sp. tritici (Pst). (a) The disease phenotype was photographed 14 days after Pst CYR31 inoculation on the fourth BSMV‐inoculated wheat leaf. (b) The transcript levels of TaPR1 and TaPR2 measured 48 h after Pst CYR31 inoculation on the TaGLTP‐silenced plant. (c) Mean and standard deviations were calculated from three independent replicates. The asterisks mark significant differences based on the two‐tailed Student's t test (p < 0.05). (d) The Pst:wheat biomass ratio was measured at 120 h postinoculation via quantitative PCR. The asterisks mark significant differences based on the two‐tailed Student's t test (p < 0.05).

2.6. TaRBP1 protein stabilizes TaGLTP

To explore the effect of TaRBP1 on TaGLTP in vivo, TaRBP1‐GFP was transiently expressed with TaGLTP‐FLAG in N. benthamiana. The western blot showed that more TaGLTP‐HA protein accumulated in vivo when co‐expressed with TaRBP1 than when co‐expressed with the negative control GFP (Figure 7a). Based on this result, we speculated that it may be difficult to maintain the stability of TaGLTP protein in plants, whereas the presence of TaRBP1 protein was beneficial to the stability of TaGLTP. Because most protein degradation is accomplished via the ubiquitin/26S proteasome pathway, we used proteasome inhibitor MG132 to examine this pathway. As shown in Figure 7a, compared with that in the control without MG132, the protein abundance of TaGLTP was greater when co‐infiltrating MG132, suggesting that TaGLTP was degraded through the ubiquitin/26S proteasome pathway in plants. To rule out the effect of gene expression levels on protein abundance, we measured the gene expression by RT‐qPCR and found that TaRBP1 did not affect the transcript level of TaGLTP (Figure 7b). To explore the effect of TaRBP1 on TaGLTP during Pst–wheat interaction, we transiently expressed TaRBP1 and TaGLTP on the second leaves of wheat cultivars Suwon 11 and infected them with Pst CYR23, which is avirulent on this cultivar. Co‐expression of TaRBP1 and TaGLTP increased plant susceptibility compared with the wheat leaves expressing TaGLTP and TaRBP1, respectively (Figure S11a). The Pst biomass was significantly increased in plants overexpressing TaRBP1 and TaGLTP (Figure S11b). In addition, we also measured TaGLTP protein abundance in western blots of total proteins. In wheat cells expressing TaGLTP‐HA alone, little TaGLTP‐HA protein was detected. However, we detected a strong accumulation of TaGLTP‐HA in leaves co‐expressing TaGLTP and TaRBP1 (Figure S11c). These results were consistent with the degradation of TaGLTP in N. benthamiana. Taken together, our data confirmed that TaRBP1 stabilized TaGLTP in plants by protecting TaGLTP from proteolytic degradation mediated by the ubiquitin/26S proteasome pathway.

FIGURE 7.

TaRBP1 stabilizes TaGLTP protein in plants. Western blotting was used to analyse the protein stability of TaGLTP. (a) TaGLTP‐HA was co‐expressed with TaRBP1‐GFP or GFP in Nicotiana benthamiana and infiltrated with 1% DMSO or the proteasome inhibitor MG132 at 24 h postinfiltration (hpi). Western blots of total proteins from N. benthamiana leaves were extracted at 48 hpi and probed with the anti‐GFP or anti‐HA antibody. Ponceau S represents equal protein loading. (b) The transcript level of TaGLTP was evaluated via reverse transcription‐quantitative PCR. The relative expression of TaGLTP was calculated by the comparative threshold method (2^−ΔΔCt). Means and standard deviations were calculated from three independent replicates.

2.7. Silencing of wheat TaGLTP perturbs sphingolipid levels

To elucidate whether TaGLTP is involved in plant defence by altering sphingolipid metabolism, we examined sphingolipid levels in TaGLTP knockdown plant leaves using high‐performance liquid chromatography (HPLC) electrospray tandem mass spectrometry. The total sphingolipid amount including ceramides (Cers), hydroxyceramides (hCers), and glucosylceramides (GlcCer) in TaGLTP‐knockdown plants were four‐, three‐, and three‐fold higher than that in wild‐type (WT) plants, respectively (Figure 8a). In repeated assays, the transcript level of a sphingolipid metabolic pathway‐related gene LCB2b was two‐ and three‐fold higher in TaGLTP‐knockdown plants than in WT plants at 24 and 48 hpi, respectively (Figure 8b). As expected, the total amounts of Cers, hCers, and GlcCer in TaRBP1‐knockdown plants were three‐fold higher than in control plants (Figure 8c). Moreover, the transcript level of a gene related to the sphingolipid metabolic pathway, long‐chain base subunit 2b (LCB2b), was also significantly increased by two‐fold at 24 and 48 hpi (Figure 8d). These results indicated that TaRBP1 and TaGLTP may be involved in sphingolipid metabolism during Pst–wheat interaction.

FIGURE 8.

The role of TaRBP1 and TaGLTP in sphingolipid accumulation during wheat–Puccinia striiformis f. sp. tritici (Pst) interaction. (a, c) Sphingolipid accumulation in TaGLTP‐silenced wheat leaves. Sphingolipids were extracted by high‐performance liquid chromatography (HPLC) electrospray tandem mass spectrometry. Data are shown as three sphingolipid classes including ceramides (Cer), hydroxyceramides (hCer), and glucosylceramides (GlcCer). (b, d) The expression of LCB2b in the TaGLTP and TaRBP1 knockdown plants, respectively. The gene expression values presented are relative to the control (set as 1). Data represent the mean ± SD and asterisks mark significant differences based on two‐tailed Student's t test (p < 0.05). (e) The possible model of TaRBP1 and TaGLTP in the wheat–Pst interaction. During Pst infection of wheat, the expression of TaRBP1 might be down‐regulated by fungal stimulation in the resistant reaction. TaGLTP is unstable and degraded via the 26S proteasome under the reduction of the TaRBP1 protein, increasing cell death and reactive oxygen species accumulation. In the susceptible reaction, increasing the amount of TaRBP1 protein could cause its interaction with TaGLTP and stabilization to suppress plant immunity.

3. DISCUSSION

Obligate biotrophic fungi, including stripe rust, must establish a close association with the living host's tissues to interrupt the flow of nutrients, such as carbohydrates, amino acids, and nucleic acids (Roman‐Reyna & Rathjen, 2017; Voegele & Mendgen, 2003). A common and efficient resistance strategy in host plants is ROS accumulation and cell death at infection sites to restrict absorption of nutrients of obligate biotrophic fungi (Singh et al., 2021). In this study, knocking down TaRBP1 increased ROS accumulation and cell death at Pst infection sites, leading to the restriction of Pst hyphal development. Additionally, the expression of TaRBP1 significantly suppressed the resistance reaction, while the expression rehabilitated in the susceptible reaction. VIGS technology has been successfully applied in monocotyledonous and dicotyledonous plants and their fungal pathogens, including rust fungi and powdery mildew. Silencing of Pst_8713 and Pst_12806, two Pst effectors, compromises the pathogenicity of Pst due to accumulation of host H₂O₂ (Xu et al., 2019; Zhao et al., 2018). Silencing of the WsWRKY1 gene of Withania somnifera by VIGS has a negative effect on the expression of defence genes, resulting in reduced tolerance to biotic stress (Singh et al., 2017). BSMV‐VIGS of target genes results in systemic spread of silencing in wheat plants and is homogenous and consistent, probably due to viral replication and movement. When virus is inoculated at the second leaf, a few viral symptoms are observed at the base of the third leaf and then obvious symptoms in the fourth and fifth leaves. In addition, mosaic symptoms become more obvious at 10–14 dpi, and with the increase of inoculation time, leaves that emerge after 21 dpi (leaf 7 upwards for the main shoot) are more likely to show wild‐type BSMV symptoms (Baulcombe, 2015; Holzberg et al., 2002). In this study, seedlings were inoculated with the BSMV‐VIGS constructs on the second leaf for 10 days and then inoculated with urediniospores of Pst on the fourth leaf. The gene silencing efficiency was above 65% in the fourth leaf when we knocked down TaRBP1 by VIGS. Silencing TaRBP1 enhanced the host's immune response, including up‐regulation of defence‐related genes and ROS accumulation in wheat treated with virulent Pst. This resulted in a reduction of rust uredinia, indicating that TaRBP1 might act as a susceptibility factor to negatively regulate ROS and cell death in infection sites.

A recent study reported that the ubiquitin ligase XBAT35.2 from A. thaliana controls the proteasome‐dependent degradation of ACD11 to regulate salt and drought stress and plant defence against Pseudomonas syringae (Li et al., 2019; Liu et al., 2017), indicating that the turnover of ACD11 modulates the plants' response to biotic and abiotic stress. Fortunately, TaGLTP was screened as a candidate interacting protein, which has not been functionally reported in the wheat–Pst interaction. This study showed that TaGLTP had a similar amino acid sequence and protein three‐dimensional structure to AtACD11, which indicated that they may have similar function. More importantly, TaRBP1 also interacted with AtACD11 using a Y2H assay. In the Arabidopsis acd11 mutant, the amount of ceramide‐1‐phosphate increased, disturbing the sphingolipid metabolite balance and promoting PCD (Simanshu et al., 2014). We also detected that the sphingolipid metabolites significantly accumulated in the leaves in TaGLTP‐knockdown wheat plants. As expected, TaRBP1‐knockdown wheat had similarly increasing sphingolipid metabolites and ROS accumulation. Earlier studies on ceramide function showed that the absence of ceramide kinase related to sphingolipid metabolism exhibited mitochondrial ROS and PCD, which reduced the response to Botrytis cinerea at early infection (Bi et al., 2014). This study showed that silencing three TaGLTP copies in wheat increased ROS accumulation and cell death and reduced Pst colonization. However, due to the transient silencing system, no significant differences in growth and development in TaGLTP‐silenced plants existed compared to the control. Thus, further research will be conducted to create stable transgenic wheat for its lesion mimic phenotype, to explore how the balance between crop yield and immunity might be achieved in plant breeding.

Previous studies have demonstrated that posttranslational modification of proteins, including acetylation, phosphorylation, and ubiquitination, manipulate protein stability via multiple mechanisms. In Oryza sativa, APIP10, a negative factor RING‐type E3 ligase, interacts with two rice transcription factors, OsVOZ1 and OsVOZ2, to promote their degradation, thus regulating plant PTI and Piz‐t‐mediated immunity (Wang et al., 2021). The positive regulator PBS3 suppressed the degradation of EDS1 by protecting its protein from the cullin3‐based E3 ligases, NPR3 and NPR4, to fine‐tune defence responses (Chang et al., 2019). In this study, TaRBP1 formed a homopolymer and interacted with its C‐terminus, possibly contributing to TaRBP1 protein stability in plants. Ectopic co‐expression of the TaRBP1 and TaGLTP proteins resulted in accumulation of TaGLTP by suppressing the 26S proteasome‐mediated degradation system. Because of a lack of ubiquitination‐related protein, TaRBP1 might indirectly operate the proteasome to stabilize TaGLTP. The low expression of TaRBP1 increased TaGLTP protein instability in the resistance reaction, finally exacerbating resistance to Pst but failing in the susceptible reaction. A large variety of effectors of pathogens also use this idiomatic strategy to effectively reshape and control host target proteins involved in plant immunity. The rust effector Pst18363 stabilizes protein accumulation levels of TaNUDX23, a negative regulator, by suppressing the 26S proteasome‐mediated degradation, suppressing ROS accumulation during the Pst–wheat interaction (Yang et al., 2020). Another virulence factor, Pi02860 from Phytophthora infestans, interacts with a susceptibility factor, NRL1, and promotes proteasome‐mediated degradation of the immune regulator SWAP70 to suppress plant immunity (He et al., 2018). It is unknown whether rust virulence factors operate the differential expression of TaRBP1 or directly act as a 26S proteasome regulator to stabilize the TaGLTP protein. However, in A. thaliana, the Phytophthora capsici effector RxLR207 interacts with BPA1, promoting its degradation and disrupting ACD11 stabilization, resulting in the transition from biotroph to necrotroph (Li et al., 2019). In wheat cells expressing TaGLTP and TaRBP1, a strong accumulation of TaGLTP protein was observed in leaves infected by Pst, indicating that TaRBP1 might stabilize TaGLTP in plants. Therefore, the regulation of TaGLTP is a key node of plant immunity. Further exploration of the molecular mechanism by which this occurs is necessary to determine whether TaGLTP can act as a candidate target for broad‐spectrum resistance and whether TaRBP1 acts as a molecular switch at the protein level of TaGLTP to alleviate its autoimmunity in plant molecular breeding.

We speculate that TaGLTP is unstable, degrading in a 26S proteasome‐dependent manner under the reduction of TaRBP1, although the transcript level of TaGLTP is slightly elevated in the resistant reaction (Figure 8e). Conversely, increasing the TaRBP1 protein could cause an interaction with TaGLTP and stabilize it, suppressing sphingolipid and ROS accumulation and cell death at the infection site. The TaRBP1–TaGLTP complex in sphingolipid accumulation and cell death and plant immunity will provide valuable insight for understanding and managing wheat stripe rust.

4. EXPERIMENTAL PROCEDURES

4.1. Biological material, fungal inoculation, and culture conditions

Wheat cultivars MX169 and Suwon 11 in this study were obtained from the culture collection of the Triticeae Research Institute. Wheat seedlings were grown in a soil mixture in 10‐cm diameter pots in an incubator with 16 h of light and 8 h of darkness at 16°C. N. benthamiana, used for transiently expression, was cultured in a greenhouse at 20–23°C (16 h of light and 8 h of dark). For propagating Pst races, fresh urediniospores of Pst CYR23 and CYR31 were produced on the second leaves of wheat cultivars MX169 and Suwon 11, respectively (Kang et al., 2002). Briefly, a total of 1 mg of urediniospores was suspended in 2 mL of water and then used to inoculate leaves of wheat seedlings at the two‐leaf stage using a fine paintbrush. After inoculation, seedlings were kept in a humid chamber for 18 h at 16°C and then were returned to the growth chamber. The susceptible reaction was that the virulent Pst CYR31 was inoculated on the wheat Suwon 11 cultivar and the resistant reaction was avirulent Pst CYR23 inoculated on wheat Suwon 11 cultivar. For bacterial material, Escherichia coli DH5α was cultured on Luria‐Bertani (LB) plates at 37°C and Agrobacterium tumefaciens GV3101 were cultured on medium with 30 μg/mL rifampicin at 28°C.

4.2. Total RNA extraction and RT‐qPCR analysis

Wheat samples for the transcript level detection of TaRBP1 were collected at 0, 12, 24, 48, 72, and 120 hpi in susceptible reaction and resistant reaction. The total RNA of samples was extracted using a MiniBEST Plant RNA Extraction Kit (TaKaRa) following the manufacturer's instructions. First‐strand cDNA was synthesized using the GoScript reverse transcription system (Promega Corp.). qPCR was performed in a 20‐μL reaction mixture containing ultraSYBR mixture (CWBIO), 10 pmol each of the forward and reverse gene‐specific primers, and 2 μL of diluted cDNA (1:20). qPCR was carried out using the Bio‐Rad CFX Manager with the following conditions: preheating at 95°C for 8 min, 39 cycles at 95°C for 15 s, 56°C for 30 s to assess the cycle threshold, and melt curves were obtained at 95°C for 20 s, 60°C for 1 min, and 95°C for 15 s. Primers were designed using the qPCR primer database (https://biodb.swu.edu.cn/qprimerdb/) (Table S1). Biomass was determined by absolute quantification using double‐standard curves (Panwar et al., 2013). Double‐standard curves of absolute quantitation were derived from wheat and Pst cDNA. The wheat cDNA and Pst cDNA were diluted in a gradient series at 10×, 20×, 50×, 100×, 200×, 500×, and 1000×. The correlation coefficient of the standard curve was above 0.99, and its slope was approximately 3.3. The biomass of wheat and Pst was indicated by the internal control genes Ta_EF and Pst_EF, respectively.

4.3. Sequence analysis

TaRBP1 and TaGLTP were amplified from the cDNA library of wheat leaves infected with virulent Pst CYR31 via PCR using the specific primers (Table S1). For the cDNA library, the total RNA was extracted from Pst‐infected wheat Suwon 11 leaves at 24 and 48 hpi with the MiniBEST Plant RNA Extraction Kit (TaKaRa). The amino acid sequence and protein domain of TaRBP1 and TaGLTP were retrieved from the SMART website (http://smart.embl‐heidelberg.de/smart/). Multiple sequence alignments were carried out by MULTALIN (http://multalin.toulouse.inra.fr/multalin/multalin). The Compute pI/Mw tool predicted the protein molecular structure (http://web.expasy.org/compute_pi/). Structure models of TaGLTP were built and superposed using the SWISS‐MODEL website (https://swissmodel.expasy.org/). Polygenetic relationships were constructed using MEGA 5.0 software using the neighbour‐joining method.

4.4. Plasmid constructs

Plasmids used for VIGS were based on previously described constructs (Xu et al., 2019). Selected gene fragments were amplified by PCR from cDNA using the primers. Two small fragments of TaRBP1 and TaGLTP were inserted into restriction sites (NotI/PacI) of the virus vector γ for plasmid constructs for the silencing system. For the Y2H system, TaRBP1 was inserted into pGADT7 and pGBKT7 at the EcoRI and BamHI sites, respectively. For transient expression in N. benthamiana, TaRBP1 and TaGLTP were amplified using the combination of primers and were inserted into restriction sites (NcoI/BgIII) of the pCAMBIA1302 vector for sublocalization and linked to pSPYNE and pSPYCE for BiFC (Waadt et al., 2008). TaRBP1 was inserted into the pICH86988 vector with a FLAG and HA tag. All plasmids in this study were validated by Beijing AuGCT DNA‐SYN Biotechnology (Beijing, China). Table S1 shows all primers used in this study.

4.5. Virus‐induced gene silencing

Two 200–300 bp specific fragments of TaRBP1 or TaGLTP served as the RNAi targets to transiently silence TaRBP1 using the BSMV‐VIGS system (Baulcombe, 2015; Holzberg et al., 2002). The corresponding recombinant vectors (TaPDS‐γ, TaRBP1‐1‐γ, TaRBP1‐2‐γ, TaGLTP‐1‐γ, and TaGLTP‐2‐γ), α, β, and γ, were linearized and transcribed into RNA in vitro using the RiboMAX large‐scale RNA production system‐T7 (Promega). Then, 15 μL of the BSMV RNA (α, β) were mixed with TaPDS‐γ, TaRBP1‐1‐γ, TaRBP1‐2‐γ, and γ in 300 μL of the FES buffer (0.06 M K₂HPO₄, 0.1 M glycine pH 8.9, 1% wt/vol sodium pyrophosphate, 1% wt/vol celite, and 1% wt/vol bentonite), respectively. Seedlings of wheat Suwon 11 cultivar received this inoculation on their second leaves and were maintained in the incubator at 27–30°C for 10–12 days. Following this, the fourth leaves of the Suwon 11 cultivar were inoculated with urediniospores of Pst CYR31, and samples were harvested at 24, 48, and 120 hpi for histological observation and detection of silencing efficiency. The disease phenotype was examined and captured after 14 days of Pst inoculation.

4.6. Sphingolipid analyses

Sphingolipid analysis was performed via HPLC–tandem mass spectrometry (Bielawski et al., 2009). After 10–12 days of inoculation with BSMV, 300 mg of leaf materials of wheat cultivar Suwon 11 infected by Pst CYR31 were harvested from three biological replicates for each treatment and ground to a powder in liquid nitrogen. This was homogenized in an extraction buffer (methyl tert‐butyl ether/methanol [vol/vol], 3:1) at 4°C for 1 h, and then methanol/water (1:1 vol/vol) was added to the mixture. The mixture was centrifuged at 15,000 g for 20 min. The upper liquid was transferred to a 2‐mL tube and the organic extract was evaporated under nitrogen until dry. Finally, the dry residue was reconstituted by methylene chloride/methanol (1:1 vol/vol) for the HPLC‐MS system.

4.7. Transient expression of proteins

For sublocalization in N. benthamiana, Agrobacterium tumefaciens GV3101 carrying GFP or TaRBP1‐GFP was resuspended in the AS buffer (10 mM MgCl₂, 10 mM MES‐KOH, pH 5.6 (2‐[N‐morpholino] ethanesulfonic acid), 10 μM acetosyringone) with an OD₆₀₀ = 0.6 and then infiltrated into 4‐week‐old N. benthamiana leaves. The GFP signal was observed at 48 hpi with an Olympus BX‐51 fluorescence microscope. For transient expression in wheat, the recombinant plasmids containing TaRBP1‐GFP or TaGLTP‐HA driven by the CaMV 35S promoter were prepared. The second leaves from 14‐day‐old wheat cultivar Suwon 11 were fixed in an agar plates with 75 μg/mL 6‐benzylaminopurine and were bombarded using He/1100 particles (Bio‐Rad) at a bombardment distance of 6 cm as previously described (Wang et al., 2022). After bombardment, the leaves were cultured at 16°C for 12 h and inoculated with Pst CYR23. For the BiFC assay, the sequences of TaRBP1 and TaGLTP were inserted into the vector pSPYCE and pSPYNE, respectively, and transformed into A. tumefaciens GV3101 (Waadt et al., 2008). The Agrobacterium strains carrying corresponding vectors had mixed and infiltrated at an OD₆₀₀ of 0.5. Three days after infiltration (dpi), YFP fluorescence was captured via confocal microscopy. Agrobacteria carrying TaGLTP‐GFP and TaRBP1‐HA were co‐infiltrated into N. benthamiana leaves for the Co‐IP assay. The infiltrated leaves were harvested at 72 hpi, their veins were removed, and the remainder was ground to a powder in liquid nitrogen and homogenized in an extraction buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP‐40, 1 mM EDTA, 10 mM dithiothreitol, 1× protease inhibitor, and 1 mM PMSF). The extract solution was centrifuged at 15,000 g for 20 min, and the supernatant was transferred into a 2‐mL tube for the Co‐IP assay. The GFP‐Trap agarose beads (gta‐20, ChromoTek) were washed three times with 600 μL of the extraction buffer, and the total protein solution was incubated with agarose beads at 4°C for 3–4 h. The bound beads were precipitated after washing three times with 500 μL of the 50 mM Tris–HCl buffer pH 7.4 with 0.5% Tween 20. Protein bound to agarose beads was boiled for 10 min and detected via western blotting. For immunodetection, the proteins on the PVDF membrane were detected by the corresponding anti‐GFP mouse monoclonal antibody (Beyotime Biotechnology), anti‐HA‐ ag mouse monoclonal antibody (Beyotime Biotechnology), and anti‐FLAG tag mouse monoclonal antibody (Sangon Biotech) with a secondary horseradish peroxidase‐conjugated goat anti‐mouse IgG (Sangon Biotech).

4.8. Yeast two‐hybrid assay

The Matchmaker GAL4 system (Clontech Laboratories) identified candidate targets from wheat following the Y2H system protocol. The binding domain fusion of TaRBP1 (pBD‐TaRBP1) and activating domain fusion of TaGLTP (pAD‐TaGLTP) and other target candidates were co‐transformed into Saccharomyces cerevisiae AH109 and selected on the selective media (SD/−Leu/−Trp, SD/−Leu/−Trp/−His, and SD/−Leu/−Trp/−His/−Ade).

4.9. Histochemical assays

To observe fungi development, leaf tissues of wheat cultivar Suwon 11 infected by Pst CYR31 were cleared with ethanol and autoclaved in 2 mL of 1 M KOH at 121°C for 5 min. The wheat fragments were carefully washed three times with 50 mM Tris–HCl pH 7.4 and stained by wheat germ agglutinin (Alexa‐488; Thermo Fisher Scientific). Fifty infection sites were counted with CellSens Entry software (v. 1.7) by an Olympus BX‐51 microscope (Cheng et al., 2015). To assess the H₂O₂ assay, wheat leaves infected by Pst CYR31 were incubated in DAB solution (HCl solution pH 3.8) under 8 h of light. The samples were treated with ethanol as previously described to remove chlorophyll and H₂O₂ was visualized as a reddish‐brown colour under bright field microscopy (Thordal‐Christensen et al., 1997). The reddish‐brown coloured area was measured via the CellSens Entry program with the BX‐53 microscope (Olympus) connected, which measured the closed polygon's area when the distribution of ROS was circled. Fifty infection sites were observed on five randomly selected leaf segments per treatment.

CONFLICT OF INTEREST STATEMENT

The authors declare no competing interests.

Supporting information

FIGURE S1 Phylogenetic analysis of TaRBP1 in main crops. (a) The RNA‐binding domain was conserved in the main crop species. RNP1 and RNP2 are RNP. α, α‐helices; β, β‐sheet. (b) MEGA 5 software’s phylogenetic analysis of TaRBP1 from main crops is shown. GenBank accession numbers and the organisms are labelled in branches. Triticum aestivum (Ta), Glycine max (Gm), Hordeum vulgare (Hv), Arabidopsis thaliana (At), Gossypium hirsutum (Gh), Solanum tuberosum (St), Sorghum bicolor (Sb), Solanum lycopersicum (Sl), Nicotiana tabacum (Nt), Oryza sativa (Os), Zea mays (Zm)

FIGURE S2 Three homologues of TaRBP1 are significantly similar on the wheat genome. The sequences of three homologues of TaRBP1 genes were obtained from the Ensembl Plant database (http://plants.ensembl.org/index.html). (a) The nucleic acid sequence alignment of TaRBP1 is shown. (b) The amino acid sequence alignment of TaRBP1 is shown. The sequence alignment was generated using http://multalin.toulouse.inra.fr/multalin/multalin.html

FIGURE S3 TaRBP1 protein mainly accumulates in plant cytoplasm and nucleus. Protein TaRBP1:GFP and green fluorescent protein (GFP) alone were expressed in Nicotiana benthamiana, and the epidermis was observed after 48 h of Agrobacterium inoculation on an Olympus BX‐51 microscope. A marker protein PAT4 (protein S‐acyl transferase 4), fused with the red fluorescent protein (RFP), was used as a plasma membrane marker. Bar = 20 μm

FIGURE S4 TaRBP1 expression was reduced early during infection with Puccinia striiformis f. sp. tritici (Pst). (a) Three gene homeologs (TaRBP1‐2A, TaRBP1‐2B, and TaRBP1‐2D) down‐regulated during early Pst infection in the resistant reaction. Expression levels were assessed with wheat RNA‐Seq data (http://www.wheat‐expression.com/). tpm, transcripts per million; d, day. (b) The transcript level of TaRBP1 was detected via reverse transcription‐quantitative PCR. The TaRBP1 expression was assayed with RNA isolated from wheat cultivars Suwon 11 leaves inoculated with urediniospores of Pst CYR31 or CYR23 at 0, 12, 24, 48, 72, and 120 h postinoculation (hpi). The transcript level of TaRBP1 in the leaves at time 0 was standardized as 1. All values and standard deviations from the three biological replicates are shown. An asterisk marks a significant difference based on the two‐tailed Student’s t test (p < 0.05)

FIGURE S5 Detection of Puccinia striiformis f. sp. tritici (Pst) development in BSMV:00, BSMV:TaRBP1‐1, and BSMV:TaRBP1‐2 plants after Pst inoculation. (a) Fungal growth observation in wheat cultivars Suwon 11 plants expressing BSMV:00, BSMV:TaRBP1‐1, and BSMV:TaRBP1‐2 after Pst CYR31 inoculation is shown. SV, substomatal vesicle; H, haustorium. Bar = 20 μm. (b) Statistical analysis of the hyphae length and infection areas in the silencing plant at 24 and 48 h postinoculation (hpi) with Pst. Means and standard deviations were assayed from three different replicates. An asterisk marks the significant difference based on Student’s t test (p < 0.05).

FIGURE S6 TaRBP1 interacts with its C‐terminus. (a) The artificial truncation of the TaRBP1 protein is shown. RRM; mRRM. The C‐terminus of TaRBP1 without RRM structure is shown. (b) TaRBP1 interacts with its C‐terminus and homologues from Nicotiana benthamiana via yeast two‐hybrid analysis. The transformant with the labelled constructs grew on the SD medium lacking LW, LWHA, and LWHA with X‐α‐gal. (c) The bimolecular fluorescence complementation assay confirmed the interaction in plants. pNE and pCE‐TaRBP1 are the negative controls. Bar = 20 μm

FIGURE S7 TaRBP1 also interacts with AtACD11, the homologue of TaGLTP, in yeast. (a) Amino acid sequence alignment of TaGLTP and AtACD11. The sequence alignment was generated at http://multalin.toulouse.inra.fr/multalin/multalin.html. (b) TaRBP1 interacts with AtACD11 in yeast

FIGURE S8 A comparison of the structure and sequence of TaGLTP and AtACD11 proteins. (a) The structural model of TaGLTP and AtACD11 overlapped with the SWISS‐MODEL. (b) Consistency was calculated with the selected chains of these models. Structure models were built and superposed at https://swissmodel.expasy.org/

FIGURE S9 Analysis of the silencing efficiency in the TaGLTP‐knockdown plant. The silencing efficiency of the TaGLTP gene was detected at 0, 24, and 48 h postinoculation (hpi) in wheat cultivars Suwon 11 plants after Puccinia striiformis f. sp. tritici CYR31 inoculation. Values and standard deviations from three biological replicates are shown. The asterisk marks a significant difference based on the two‐tailed Student’s t test (p < 0.05)

FIGURE S10 TaGLTP expression was up‐regulated during early Puccinia striiformis f. sp. tritici (Pst) infection. The transcript level of TaGLTP was detected via reverse transcription‐quantitative PCR. The TaGLTP expression was assayed with RNA isolated from wheat cultivar Suwon 11 plants infected by Pst CYR31 and CYR23, respectively. The transcript level of TaGLTP in the leaves at time 0 was standardized as 1. All values and standard deviations from the three biological replicates are shown. An asterisk marks a significant difference based on the two‐tailed Student’s t test (p < 0.05)

FIGURE S11 Transiently expressing TaRBP1 and TaGLTP regulated TaGLTP protein abundance and increased wheat susceptibility. (a) Seedlings of cultivar Suwon 11 transiently expressing TaRBP1 and TaGLTP constructs on the second leaf were inoculated with urediniospores of Puccinia striiformis f. sp. tritici (Pst) CYR23. Leaves infected with Pst were examined at 14 days postinoculation. (b) The Pst:wheat biomass ratio was assayed by quantitative PCR with RNA isolated from leaves of the same set of wheat plants as Figure S11a at 120 h postinoculation. All values and standard deviations from the three biological replicates are shown. An asterisk marks a significant difference based on the two‐tailed Student’s t test (*p < 0.05, **p < 0.01). (c) TaGLTP protein abundance in the same leaf as in Figure S11a was detected by western blot with an anti‐HA antibody. Coomassie brilliant blue (CBB) staining shows equal loading

TABLE S1 Primers used in this study

Li, Y. , Zhang, R. , Wu, Y. , Wu, Q. , Jiang, Q. , Ma, J. et al. (2023) TaRBP1 stabilizes TaGLTP and negatively regulates stripe rust resistance in wheat. Molecular Plant Pathology, 24, 1205–1219. Available from: 10.1111/mpp.13364

DATA AVAILABILITY STATEMENT

All sequence data in this article can be found in GenBank at www.ncbi.nlm.nih.gov with the accession numbers: TaRBP1 (KAF7011826), TaGLTP (KAF7068041), TaPR1 (AF384143), TaPR2 (DQ090946), and TaEF‐1a (Q03033).