Two wheat (Triticum aestivum L.) cultivars, tobacco (Nicotiana benthamiana), and Pst pathotype CYR32 were used in this study. Wheat AvocetS (AvS) and its near-isogenic line AvSYr10NIL (AvS+Yr10) wheat seedlings carrying the Yr10 resistance gene were grown in a growth chamber at 14°C under a 16h light/8h dark photoperiod. N. benthamiana was maintained in an artificial climate room with a temperature range of 20 to 22°C, a light intensity of 20,000Lux, and a 16-h photoperiod.

The Pst race CYR32 that is compatible to wheat AvS and incompatible to AvS+Yr10 was provided by the State Key Laboratory of Crop Stress Biology for Arid Areas, NWAFU, China. The uredospores of the CYR32 pathotype were suspended in isohexadecane (IHD) and sprayed evenly on the first leaves of AvS and AvS+Yr10 wheat seedlings using a small air pump. After inoculation, these seedlings were maintained for 24h in a dark chamber with 100% relative humidity. These were subsequently returned to the original photoperiod condition. The seedlings in the control group were treated similarly with IHD containing no uredospores.

The wheat leaves were sampled at 0, 12, 24, 48, 72, and 120h post-inoculation (hpi) and immediately frozen in liquid nitrogen and stored at −80°C before extraction of total RNA. The total RNA of wheat leaves was extracted using the Qiagen RNeasy Plant Mini Kit (Qiagen, Valencia, CA, United States) following the manufacturer’s instructions. The quality and concentration of RNA were assessed using a 1.5% agarose gel electrophoresis and 260/280_abs measurement using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States), respectively. Next, cDNAs were synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, United States) following the manufacturer’s protocol.

To clone the TaMYB29 gene, a pair of primers (M29-F/R) were designed using the Primer 5.0 software. The primers were designed based on the TaMYB29 sequence released by the Chinese Academy of Agricultural Sciences in 2012 (NCBI GenBank Accession No. JF951912.1); these were largely used to amplify the open reading frame (ORF) of the TaMYB29 gene. The PCR program was as follows: 3min at 95°C, followed by 35cycles of 30s at 95°C, 30s at 55°C, and 60s at 72°C, and finally 10min at 72°C. The PCR mixture contained the following: 0.125μl of Takara Ex Taq, 2.5μl of 10× Ex Tag buffer, 2μl of dNTP mixture, 2μl of template, and 1μl of each primer. Next, the total volume was made up to 20μl with ddH₂O. The target fragments were inserted into the pGEM-T Easy vector (Promega, Madison, WI, United States) and sequenced. The TaMYB29 sequence was used to BLAST the related MYB genes at the National Center for Biotechnology Information¹ and the Ensemble plant database at http://plants.ensembl.org/index.html. The DNAman program (Lynnon Biosoft, Quebec, Canada) was used to align all nucleotide sequences of these genes and corresponding deduced protein sequences.

To determine the expression profiles of TaMYB29 after different treatments, a pair of primers (M29-qRT-F/R) were designed. In addition, four pairs of primers were designed to detect the expression patterns of TaPR1, TaPR2, TaPR5, and TaCAT genes by quantitative real-time PCR (qRT-PCR). Wheat housekeeping gene actin was used as an internal control. All primers used are listed in Supplementary Table S1. The qRT-PCRs were performed on a CFX96 Real-Time System (Bio-Rad, Munich, Germany). The reactions were performed in a 20μl volume containing 10μl of 2×UltraSYBR mixture, 2μl of diluted cDNA, 1μl of each primer, and 6μl of ddH₂O. The amplification conditions were as follows: a pre-denaturation for 10min at 95°C, followed by 40cycles of 15s at 95°C and 60s at 60°C. A dissociation curve was generated for every reaction to ensure specific amplification. Each reaction was performed in triplicate, and reactions without templates were used as negative controls. Three independent biological replicates were used for each time point and treatment. The 2^−ΔΔCt method was applied to analyze the experimental results (Livak and Schmittgen, 2001).

To measure the endogenous SA concentration under both compatible and incompatible interactions, the wheat leaves were sampled at 0, 12, 24, 48, 72, and 120hpi with CYR32 pathotype. These were frozen in liquid nitrogen immediately. The SA concentration was analyzed using liquid chromatography-mass spectrometry (LC-MS) on an LC-30A+TripleTOF5600+ machine (AB Sciex, Singapore) according to Wang et al.’s (2017) method. In brief, 200mg of the sample was ground into a powder in liquid nitrogen and subsequently extracted with 750μl of MeOH–H₂O–HOAc (90:9:1, v/v/v). The solution was centrifuged at a high speed of 10,000rpm. Next, the supernatant was collected and dried under nitrogen. The extract was dissolved in 1,000μl of HPLC-grade MeOH and filtered using a Millex-HV 0.22μm filter (Millipore, Bedford, United States). Three independent biological replicates were used for each time point.

To analyze the expression profiles of TaMYB29 under different hormone treatments, the one-leaf stage wheat seedlings of AvS+Yr10 were sprayed with 2μm methyl jasmonate (MeJA), 2μm ethylene (ET), 2μm abscisic acid (ABA), and 20μm salicylic acid (SA), following Wang et al.’s (2013) method. The mock control seedlings were similarly treated with an equal volume of distilled water. Subsequently, the seedlings were cut with a sterilized scissor for sampling at 0h, 2h, 6h, 12h, 24h, and 48h post-treatment (hpt). The samples were immediately frozen in liquid nitrogen and stored at −80°C. Three independent biological replicates were used for each time point and control.

To study the subcellular localization of TaMYB29 protein, a pair of primers (163-M29-F/R) with restriction enzyme HindIII and BamHI sites were designed. The PCR product of TaMYB29 was subcloned into the 16318GFP vector. Next, the recombinant plasmid was amplified in Escherichia coli strain DH5α and extracted using the OMEGA Plasmid Maxi Kit. The wheat leaves were processed with cellulase R10 (Yakult Honsha) and macerozyme R10 (Yakult Honsha) to isolate protoplasts as previously described (Yoo et al., 2007). The TaMYB29-eGFP fusion vector and only eGFP vector (control) were transformed into the wheat protoplasts separately using PEG4000. The treated wheat protoplasts were maintained in the dark at 23°C for 24h and subsequently observed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

In addition, the gateway cloning technology was used to link the TaMYB29 gene to the pK7WGF2.0 vector carrying an enhanced green fluorescent protein (eGFP) tag. The pK7-TaMYB29-eGFP and pK7-eGFP vectors served as controls were transformed into the Agrobacterium strain GV3101 (pSoup-p19) by electroporation. Strains carrying different recombinant vectors were cultured to an optical density of 0.8 at 600nm (OD600) and injected into 4-week-old N. benthamiana leaves using a 1.0ml syringe without a needle. The transformed N. benthamiana were maintained in a greenhouse at 20 to 22°C, a light intensity of 20,000Lux, and a 16-h photoperiod. Finally, the fluorescence of eGFP was observed under a laser confocal fluorescence microscope at 48h post-infiltration.

The gene of full-length TaMYB29_1–261 protein, its N-terminal region TaMYB29_1–116, and C-terminal region TaMYB29_117–261 were amplified using three pairs of primers containing NdeI and EcoRI restriction sites, namely, M29-BD-F/R, M29-BD-F/R₁₁₆, and M29-BD-F₁₁₇/R, respectively. The PCR products were purified and linked to the pGBKT7 vectors linearized by restriction endonucleases. The three pGBKT7-TaMYB29 recombinant plasmids were directly transformed into DH5α E. coli strain and amplified. Subsequently, the pGBKT7-TaMYB29 recombinant plasmids and the pGBKT7 plasmid (negative control) were introduced into the AH109 yeast strain containing the HIS3, ADE2, MEL1, and lacZ reporter genes. The transformed yeast strains were plated on a medium lacking tryptophan (SD/-Trp) and incubated at 30°C for 2 to 3days. The positive clones on the Trp-deficient media were selected and transferred into the yeast peptone dextrose adenine (YPDA) liquid media for culturing. Next, the yeast solutions were diluted to OD₆₀₀ of 1, 10⁻¹ 10⁻², 10⁻³, and 10⁻⁴. Lastly, these yeast solutions were inoculated into a medium lacking adenine, histidine, and tryptophan (SD-His/-Ade/-Trp) as well as a medium lacking the above three amino acids (SD-His/-Ade/-Trp) and supplemented with X-α-D-galactoside (X-α-Gal). These cultures were incubated at 30°C for 3 to 4days.

The TaMYB29 gene was amplified using a pair of primers (PVX-M29-F/R) containing SmaI and NotI restriction enzyme sites. The PCR product was subcloned into the pMD18-T simple vector. Afterward, the recombinant plasmid was amplified in DH5α E. coli strain and digested by SmaI and NotI restriction enzymes. Subsequently, the gene digestion product was inserted into the PVX vector which carries a 3×HA tag and was linearized by the same restriction enzymes. The PVX-TaMYB29 construct was transformed into the Agrobacterium strain GV3101 (pSoup-p19). Strains were grown in the Luria–Bertani (LB) medium containing 50mg/ml rifampicin and 25mg/ml kanamycin at 28°C for 24h. The bacteria were harvested by centrifugation and resuspended in the acetosyringone (AS) buffer containing 10mm MES (pH 5.6), 10mm MgCl₂, and 150μm acetosyringone after being washed with 10mm MgCl₂. Strains carrying different recombinant vectors were adjusted to a density of 0.6 at 600nm (OD₆₀₀) with AS buffer and incubated for 2h in the dark at room temperature before leaf infiltration. These leaves were sampled at 1, 2, and 3days post-infiltration (dpi) and stained with 3,3′-diaminobenzidine (DAB) or trypan blue (Bai et al., 2012). The PVX-GFP construct was used as a control and treated in the same manner. These experiments were repeated at least three times and got the same result.

A conserved region of about 200bp was selected from the cloned TaMYB29 sequence to design the primers (M29-vigs-F/R) for silencing the fragments. The PCR product was linked to the BSMV:γ modified vector to construct the BSMV:γ-TaMYB29 recombinant plasmid as previously described (Wang et al., 2020a). Next, the positive plasmid was amplified in DH5α E. coli strain. A construct carrying only the BSMV genome was used as a negative control and named BSMV:00. A construct carrying a 183-bp phytoene desaturase (PDS) gene was used as a positive control and named BSMV:PDS. The recombinant γ virus vector linearized by BssHII and RNAα, RNAγ linearized with MluI, and RNAβ with SpeI were transcribed in vitro using the mMessage mMachine T7 in vitro transcription kit (Ambion, Austin, TX, United States) following the manufacturer’s protocol. The transcription products of BSMV:α, β, γ (or γ-target) were diluted thrice with diethylpyrocarbonate (DEPC) water and mixed in a ratio of 1:1:1. Next, 70μl of FES buffer which is a kind of inoculation buffer containing a wounding agent was added to every 30μl of the mixture. The second leaves of AvS and AvS+Yr10 wheat seedlings were infected with BSMV using the method described by Holzberg et al. (2002). The BSMV-infected wheat seedlings were maintained in a 16h/8h photoperiod at 25°C. In addition, the seedlings were mock inoculated with FES buffer devoid of BSMV transcripts. At 10 to 14days after the virus inoculation, photobleaching was observed on BSMV:PDS seedlings. The third and fourth leaves were further inoculated with uredospores of Pst pathotype CYR32. Next, these plants were directly maintained in a growth chamber for 24h in the dark with 100% relative humidity at 15°C. The leaves were sampled at 0, 24, 48, and 120hpi for histological changes and analyzed by qRT-PCR.

Wheat leaves sampled at 0, 24, 48, and 120hpi in control and silenced plants were observed for ROS accumulation by DAB staining and HR response areas because of the auto-fluorescence of necrotic cells using an Olympus BX-51 fluorescence microscope (Olympus Corp. Tokyo, Japan). Then, the leaf segments were stained with wheat germ agglutinin (WGA) and observed for the infection areas and hyphae development of Pst as previously described (Wang et al., 2017). At least 30 infection sites were examined on each of five randomly selected leaf segments per treatment. Infection sites with substomatal vesicles were considered successfully penetrated. Standard deviations were calculated and Tukey’s test was performed for statistical analysis using the SPSS software (SPSS, Inc. Chicago, United States).

A full-length cDNA fragment was successfully isolated due to its high expression under incompatible interaction of wheat cv. AvS+Yr10 infected with avirulent Pst CYR32. The cDNA sequence displayed 100% identity with the referred TaMYB29 sequence in the NCBI GenBank (accession no. JF951912.1). In addition, the ORF was cloned using a pair of primers to create a 786-bp fragment. The International Wheat Genomic Sequence Consortium (IWGSC) revealed homologs of TaMYB29 located on chromosomes 5A, 5B, and 5D in wheat cv. Chinese Spring. The alignment results of these nucleotide sequences also showed a high similarity ranging from 96 to 99%, referred to as TaMYB29-5A, TaMYB29-5B, and TaMYB29-5D (Supplementary Figure S1). The TaMYB29 sequence of AvS+Yr10 displayed the highest similarity to the nucleotide sequence of the Chinese Spring TaMYB29-5B.

The deduced protein of TaMYB29 had 261 amino acids, with a predicted molecular weight of 29.4 kD and an isoelectric point (pI value) of 9.05. Alignments of all wheat alleles of TaMYB29 with wheat TaMYB4 (AEG64799.1), Arabidopsis AtMYB30 (AEE77505.1), and Arabidopsis AtMYB4 (AEE86955.1) revealed two highly conserved regions R2 and R3, which contained three uniformly spaced tryptophan residues responsible for the interaction between the MYB protein and specific DNA sequences (Figure 1). The second and third tryptophan residues were conserved in all R2R3-MYB protein members, except in R3 repeats, where the first tryptophan was replaced by phenylalanine (Zhang et al., 2012a). These results revealed highly conserved amino acid sequences in these MYB proteins, indicating their similar functions in mediating stress response.

To study the subcellular localization of TaMYB29 in plants, the target gene fused with the eGFP vector and only eGFP vector (control) was separately transformed into wheat protoplasts. To avoid the interference of chlorophyll, its distribution was studied individually. After analyzing the fluorescence microscopy observations, the exact localization of TaMYB29-eGFP fusion protein was found to be the nucleus of wheat protoplast, whereas green signals of only eGFP were observed both in the cytoplasm and the nucleus (Figure 2A). We subsequently used the leaves of N. benthamiana as the transformed material and repeated the subcellular localization experiment and obtained the same results (Figure 2B). Therefore, we conclude that TaMYB29 is a nuclear protein.

To identify whether the TaMYB29 protein is a transcriptional activator or repressor, pGBKT7-TaMYB29_1–261 and its two truncated protein plasmids pGBKT7-TaMYB29_1–116 and pGBKT7-TaMYB29_117–261 were transformed into the AH109 yeast strain. In addition, the pGBKT7 plasmid (negative control) was transformed simultaneously. All transformants grew well on a selective synthetic defined (SD) medium lacking tryptophan (SD/-Trp); however, only pGBKT7-TaMYB29_1–261 and pGBKT7-TaMYB29_117–261 yeast strains showed growth on the selective medium without tryptophan, histidine, and adenine (SD-Trp/-His/-Ade). Furthermore, the colonies turned blue on the same medium supplemented with X-α-Gal (SD-Trp/-His/-Ade+X-α-Gal; Figure 3). Therefore, we conclude that TaMYB29 functions as a transcriptional activator. What is more, the transactivation domain of TaMYB29 is located in the C-terminal region.

To understand the regulation of TaMYB29 by plant hormones, the AvS+Yr10 wheat seedling leaves were treated with four different exogenous hormones (SA, ABA, JA, and ET) at the two-leaf stage. The TaMYB29 transcript levels were detected by qRT-PCR at six different time points (Figure 4). Regarding SA treatment, the relative expression of TaMYB29 showed a 2.59-fold increase at 12h and a 10-fold increase at 24h. Eventually, the increase at 48h was 11 times more than that in the control at 0h. The TaMYB29 expression of leaves treated with ABA showed a similar increasing trend. However, the increase was considerably high at 48h, 50 times as high as that in the control. With respect to JA treatment, the highest level was measured at 12h, with upregulation recorded from 12h to 48h. The upregulation was also observed in ET-treated leaves, especially at 2h, 12h, and 24h. However, the expression peak of TaMYB29 induced by JA or ET treatment was not as high as that detected with the ABA treatment. These results demonstrate that TaMYB29 exert an effect in the plant through complex hormone signal pathways.

To study the expression profile of TaMYB29 during the interaction between wheat and stripe rust pathogen, the relative expression of TaMYB29 in the wheat leaves was measured by qRT-PCR at 0, 12, 24, 48, 72, and 120h in two different wheat cultivars post-inoculation with CYR32 (Figure 5A). Wheat AvocetS (AvS), which is susceptible to Pst pathotype CYR32, forms a compatible interaction, and its near-isogenic line AvSYr10NIL (AvS+Yr10), carrying the Yr10 resistance gene and highly resistant to CYR32, forms an incompatible interaction. The expression of TaMYB29 in the leaves of AvS+Yr10 plants was significantly upregulated at four different time points, except 12hpi in contrast to the 0hpi control and peaked at 48hpi, nearly 3.5 times more than that in the control. However, the expression of TaMYB29 in the leaves of AvS plants showed an opposite trend, declining slightly until 24hpi at first. Although the transcript abundance increased slightly at 48hpi compared with 0hpi, it decreased to nearly half the level at 0hpi in the end at 120 hpi. The expression of TaMYB29 in the incompatible interaction was always higher than that in the compatible interaction at five different time points. These results indicate that TaMYB29 functions during the resistance of wheat against Pst infection, especially in the incompatible interaction.

We measured the endogenous levels of SA in both compatible and incompatible interactions at six time points (Figure 5B). In the incompatible interaction, the first significant increase in the SA levels appeared at 12 hpi, earlier than the TaMYB29 expression, and subsequently reduced at 24 and 48hpi, contrary to the observation in the TaMYB29 expression compared with the 0hpi control. The highest SA levels were obtained at 72hpi. Next, the SA levels rapidly returned to the basal levels at 120hpi. The SA levels in the compatible interaction presented a similar trend as in the incompatible system although the levels were lower than those in the incompatible system at every time point.

Furthermore, the expression of the wheat PR gene TaPR1 was measured by qRT-PCR (Figure 5C). TaPR1 was upregulated in both interactions during pathogenesis. In the incompatible interaction, the expression of TaPR1 significantly increased at 24hpi and reached the 20-fold increase at 48hpi, and maintained significantly high levels at 72hpi, which is considerably similar to the expression of TaMYB29. Next, it decreased at 120hpi, different from the expression of TaMYB29. In addition, the expression of TaPR1 in the compatible interaction was slightly upregulated; however, the level was lower as compared with that in the incompatible interaction.

The recognition of certain molecules released by pathogens triggers plant cell death and a subsequent defense response known as the HR response (Coll et al., 2011). To confirm whether TaMYB29 can induce ROS and cell death, the HR symptoms were detected in N. benthamiana leaves transformed by PVX:TaMYB29 and PVX:GFP constructs. After 1 to 2days post-infiltration (dpi), the accumulation of ROS was detected successfully with DAB staining (Figure 6A). The ROS accumulation in leaf areas infiltrated with PVX:TaMYB29 was apparent in contrast to that in control PVX:GFP. Subsequently, apoptosis was observed using trypan blue staining to further investigate the effects of TaMYB29 expression during ROS accumulation and pathogen-independent cell death (Figure 6B). The TaMYB29-induced cell death on N. benthamiana leaves was apparent. However, PVX:GFP control did not trigger cell death. The results showed that the expression of TaMYB29 successfully led to ROS accumulation and cell death in contrast to the GFP control, indicating that TaMYB29 played a crucial role in ROS accumulation and pathogen-independent cell death.

To study the effects of the TaMYB29 gene during the interaction between wheat and Pst, the target gene was silenced using the barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) assay. At the beginning of the knockdown experiment, the PDS gene was used to ensure that the VIGS system worked normally and correctly. The photobleaching occurred on BSMV:PDS leaves, proving the effectiveness of the VIGS system (Figure 7A).

AvS and AvS+Yr10 wheat seedlings infected with FES buffer (mock), BSMV:00, and BSMV:TaMYB29 were further inoculated with uredospores of CYR32. Infection types were assessed based on McNeal measurement (Mcneal et al., 1971) to evaluate the differences between the phenotypes of mock, BSMV:00, and BSMV:TaMYB29. AvS+Yr10 displayed a resistant response after inoculation with CYR32 on the mock and BSMV:00 controls, characterized by a large necrosis area at the infection site. While a small number of fungal spores appeared on TaMYB29-silenced leaves. These results indicated that the resistance of wheat AvS+Yr10 with silenced TaMYB29 gene resulted in a significant decline from highly resistant to moderately susceptible. There was no susceptible phenotype change in TaMYB29-silenced AvS wheat compared with the control mock and BSMV:00 (Figure 7A). The relative expression of TaMYB29 was detected by qRT-PCR after TaMYB29 gene silencing before Pst inoculations labeled as 0hpi, and 24hpi, and 48hpi during Pst infection. The results showed that the TaMYB29 transcription level decreased in both AvS+Yr10 and AvS, which was knocked down up to 86% in incompatible interaction and 55% under compatible interaction compared with BSMV:00 labeled as CK (control check) in Figure 7B.

Pathogenesis-related genes are vital for mounting disease resistance responses in plants. TaPR1, TaPR2, and TaPR5 have been reported to be involved in systemic acquired resistance (SAR)—a type of plant immunization (Wang et al., 2020b). Therefore, we selected three PR genes as defense-related genes to investigate the effects of the knockdown of TaMYB29 before and during Pst inoculation. The results showed that the decline of TaPR1, TaPR2, and TaPR5 gene expression in BSMV:TaMYB29 compared with BSMV:00 was observed at four different time points in the incompatible interaction (Figure 7C), proving that the silencing of TaMYB29 reduced the wheat resistance to disease.

The infection area of Pst in TaMYB29-silenced AvS+Yr10 wheat leaves was examined microscopically after inoculation with CYR32. The results showed that the total infection area in TaMYB29-silenced leaves was similar to that in BSMV:00 control until 48hpi. However, significant differences between the two groups were observed at 120hpi (Figures 7D,E). Furthermore, the hyphae development of Pst in BSMV:TaMYB29 was stronger than that in BSMV:00, especially at 120hpi (Figures 7D,E).

In conclusion, TaMYB29 knockdown effectively reduced wheat resistance to stripe rust by downregulating the expression of PR genes and promoting the development of hyphae.

We observed the accumulation of ROS and cell necrosis of AvS+Yr10 wheat leaves upon pathogen challenge. The production of H₂O₂ and the necrotic area in BSMV:TaMYB29 leaves were significantly less than those in BSMV:00 at 48hpi and 120hpi, creating a gap of about 2,000μm² at 120hpi (Figures 8A,B,D). In addition, the expression patterns of TaCAT (X94352) that can eliminate H₂O₂ as a catalase gene in wheat were detected by qRT-PCR (Coleto et al., 2021). The results showed that the expression of TaCAT was significantly upregulated in TaMYB29-silenced leaves at 24hpi, peaked at 48hpi as compared with control non-TaMYB29-silenced leaves (Figure 8C), implying that a high expression of TaCAT reduced the ROS accumulation in TaMYB29-silenced wheat leaves. We hypothesized that the silencing of the TaMYB29 gene positively regulated the expression of the TaCAT gene, eventually decreasing the accumulation of ROS and partially eliminating the HR response in wheat mesophyll cells, thereby weakening the resistance of wheat to stripe rust.