Functional Analysis of Wheat NAC Transcription Factor, TaNAC069, in Regulating Resistance of Wheat to Leaf Rust Fungus

NAC transcription factors are one of the largest transcription factor families having functions in a variety of stress responses. Few NACs have been reported for interactions between wheat and the wheat rust fungus Puccinia triticina (Pt). In this study, based on analysis of RNA-seq data from wheat line TcLr19 inoculated by Pt, the NAC transcription factor TaNAC069 was cloned from wheat, and its transcriptional activity and homologous dimer formation were verified. Quantitative real-time PCR analysis showed that the expression of TaNAC069 was induced by Pt and associated signaling molecules. To further characterize the function of the TaNAC069 gene in wheat resistance to Pt, virus-induced gene silencing (VIGS) was utilized, and it revealed that Pt resistance in TaNAC069-silenced plants was significantly reduced. Potential interaction targets of TaNAC069 from wheat and Pt were screened and identified by yeast two-hybrid technology. Eukaryotic elongation factor eEF1A, CBSX3 protein, and cold acclimation protein WCOR410c were screened by yeast one-hybrid technology. The results indicate that the TaNAC069 gene plays a positive regulatory role in wheat resistance to Pt, laying a good foundation to analyze the molecular mechanisms of TaNAC069 and its functional role in wheat resistance to Pt.


INTRODUCTION
At the transcriptional level, controlling gene expression is key to regulate many plant cell responses, including normal signal transduction and cell morphological changes in response to stress (Hu et al., 2010). Many studies have shown that transcription factors regulate the expression of stressrelated genes to activate or repress the expression of target genes involved in stress responses (Erpen et al., 2018). NAC transcription factors are one of the largest families of plant-specific transcription factors that respond to biotic and abiotic stresses by participating in plant stress signal transduction pathways and regulating the expression of downstream target genes . A common feature of NAC transcription factor family members is that they contain a conserved NAC domain in the N-terminus, which is usually divided into five subdomains-A, B, C, D, and E-as well as a highly variable regulatory domain in the C-terminus (Shao et al., 2015). The C-terminus of NAC is rich in serine, threonine, proline, and some acidic amino acids and can activate or repress the expression of downstream target genes (Olsen et al., 2005). Additionally, the C-terminus may have transcriptional activity, containing different motifs having specific functions.
NAC transcription factors generally function by forming homodimers or heterodimers of NAC proteins. Different NAC proteins have different biological functions (Ooka et al., 2003). Many reports have demonstrated that NAC transcription factors play important roles in enhancing drought and salt resistance (Nguyen et al., 2019), improving tolerance to dehydration and high salt stress (Nakashima et al., 2007), enhancing tolerance to Erysiphe cichoracearum and Phytophthora parasitica var. nicotianae (Zhu et al., 2012), and regulating plant defense responses against fungal and bacterial pathogens (Wang et al., 2007).
Many studies have shown that NAC proteins play significant roles in different signaling pathways. NAC proteins respond to biotic and abiotic stresses through regulation of downstream gene expression or through interaction with other proteins (Wu et al., 2018). GhirNAC2 plays a positive role in improving drought tolerance in cotton by regulating abscisic acid (ABA) biosynthesis (Shang et al., 2020). The NAC transcription factor CrNAC036 cooperates with CrMYB68 transcription factors to repress ABA biosynthesis by negatively regulating the expression of the CrNCED5 (9-cis-epoxycarotenoid dioxygenase 5) gene in citrus fruit . TaNACL-D1 (Triticum aestivum NAClike transcription factor) can interact with TaFROG (T. aestivum fusarium resistance orphan gene) and positively enhances wheat resistance to Fusarium head blight disease (Perochon et al., 2019). The expression levels of the TaNAC4 and TaNAC8 genes were significantly up-regulated during incompatible interactions between wheat and Pst (Puccinia striiformis f. sp. tritici) (Xia et al., 2010).
Wheat leaf rust is a fungal disease caused by the obligate, biotrophic plant pathogen Puccinia triticina (Pt), which cannot grow without living hosts. It is one of the most widespread and destructive diseases of wheat worldwide, causing significant yield losses in major wheat-growing regions (Kolmer, 1996). Frequent variations in virulence genes of Pt yield new virulent species that can evade detection by wheat resistance genes, leading to the loss of rust-resistant wheat cultivars (Kolmer et al., 2020), which is likely to result in the outbreak of leaf rust. Thus, the excavation, screening, and identification of wheat resistance genes is crucial to understanding the molecular mechanisms underlying resistance against the wheat leaf rust pathogen. Wheat leaf rust resistance gene, Lr19, originally identified in Agropyron elongatum, on the 7DL chromosome, provides effective resistance to most pathotypes of Pt throughout the growth period of wheat (Sarma and Knott, 1966;Ordoñez and Kolmer, 2008). Recently, 453 NAC proteins in polyploid wheat were identified (Borrill et al., 2017). However, the specific functions and characteristics of NAC transcription factors are unknown, especially those involved in wheat resistance against Pt. In our previous study, based on the analysis of RNA-seq data of wheat, TcLr19, a NAC transcription factor induced by Pt (Traes_5DL_D1D0CA79E), was screened by combining differential gene expression and functional annotation (Zhang et al., 2020). In this study, we cloned the NAC transcription factor, aimed to identify its function in wheat resistance to Pt using barley stripe mosaic virus (BSMV)-induced gene silencing, and screened for interacting protein targets by yeast two-hybrid and yeast one-hybrid technologies, laying a good foundation to analyze the molecular mechanism of this NAC transcription factor underlying wheat resistance to Pt.

Cloning of the TaNAC069 Gene and TaNAC069 Promoter and Bioinformatics Analysis
A pair of gene-specific primers, TaNAC069-F and TaNAC069-R (Supplementary Table 1), was designed based on conserved regions of the NAC sequence mapping in the RNA-seq data. A mixture of RNA isolated at different time points from wheat leaves infected by Pt was used as the template in reverse transcription polymerase chain reaction (RT-PCR) to obtain fragments of TaNAC069. The conserved domains of TaNAC069 gene were deduced using NCBI (http://www.ncbi. nlm.nih.gov/Structure/cdd/wrpsb.cgi). CLUSTALW was used to create multiple alignments. MEGA5 (http://www.megasoftware. net) was used to generate phylogenetic trees.
Searching sequences upstream of TaNAC069 genes in the wheat database (http://plants.ensembl.org/Taestivum/Info/ Index), an approximately 2.0-kb sequence of the homologous sequence of TaNAC069 on 5D was intercepted from each gene and used to design primers pTaNAC069-F and pTaNAC069-R (Supplementary Table 1). Genomic DNA of TcLr19 was used as template, and RT-PCR was performed to generate the promoter of the TaNAC069 gene. The cis-acting elements of the promoter were analyzed using the PlantCARE (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/) and PLACE (http://www. dna.affrc.go.jp/PLACE/index.html) databases (Sun et al., 2015).

Expression Analysis of TaNAC069 by qPCR
The specific primers qTaNAC069-F and qTaNAC069-R were used to investigate the expression level of TaNAC069. Wheat glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank: AF251217) was used as an endogenous control as previously described by Gao et al. (2015). Quantitative real-time PCR (qPCR) was performed using TransStart R Top Green qPCR SuperMix (TransGen Biotech, China) according to the manufacturer's instructions. All qPCR experiments were performed on a LightCycler R 96 Real-Time PCR System (Roche, Switzerland), and relative gene expression was calculated using the 2 − Ct method. Three independent biological replicates were performed per treatment.

Functional Analysis via Virus-Induced Gene Silencing Experiments
Target fragments for silencing (V1 and V2) that had no or low similarity with other genes and thus unlikely to cause offtarget effects were selected based on predictions made using si-Fi software (http://www.snowformatics.com/si-fi.html). The plasmids (γ-V1 and γ-V2) utilized for gene silencing were constructed according to the methods described by Holzberg et al. (2002). Plasmids α, β, γ, γ-PDS, γ-V1, and γ-V2 were each linearized. The linearized plasmids were used as templates for in vitro transcription using the mMESSAGE mMACHINE R Kit High Yield Capped RNA Transcription Kit (Ambion) according to the manufacturer's protocol. BSMV VIGS vectors harboring target gene sequences and Pt urediospores were co-inoculated into wheat cultivars TcLr19 and Thatcher according to previously described methods . Plants mock inoculated with sterile water and plants inoculated with a VIGS vector targeting the wheat phytoene desaturase (TaPDS) gene were used as negative and positive controls, respectively.
For GUS staining, the TaNAC069 promoter fragment was inserted into the pBI121 vector digested with HindIII and XbaI, using the primer pair pBI121-pTaNAC069-F/pBI121-pTaNAC069-R (Supplementary Table 1). The recombinant plasmid, pBI121-pTaNAC069, was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation and then agroinfiltrated into Nicotiana benthamiana plants (grown for 45 days) . At 72 hpi, inoculated leaves were collected and immediately treated with GUS staining solution. Under the naked eye or microscope, the small blue dots on the white background indicate GUS expression sites.
To generate the GFP constructs, the TaNAC069 promoter fragment was recombined into the vector pCamA-GFP digested with HindIII and KpnI, using the primer pair pCamA-pTaNAC069-F/pCamA-pTaNAC069-R (Supplementary Table 1). The recombinant plasmid pCamA-pTaNAC069-GFP was used for Agrobacterium-mediated transformation.

Yeast One-Hybrid and Yeast Two-Hybrid Assays
For yeast one-hybrid assays, the recombinant yeast strain Y1HGold (pAbAi-pTaNAC069) was identified via PCR using Matchmaker Insert Check PCR Mix 1 (Clontech, Palo Alto, CA, United States) as described by Yang et al. (2019). Yeast library plasmids (10 µg) were transformed into the recombinant yeast strain Y1HGold (pAbAi-pTaNAC069). Healthy single clones were selected for colony PCR using Matchmaker Insert Check PCR Mix 2 (Clontech, Palo Alto, CA, United States) to obtain the inserted fragment of the cDNA library. The amplified products (1-2 kb) were purified by agarose gel electrophoresis and sequenced at Sangon Biotech, Shanghai, China, using the T7 promoter sequence as primer. The sequencing results were analyzed using NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
For yeast two-hybrid assays, the bait vector pGBKT7-TaNAC069-N was transformed into Y2HGold yeast strain using the LiAc (Clontech, Palo Alto, CA, United States) and confirmed by toxicity and self-activation. Screening of interacting targets of TaNAC069 was done using the Matchmaker Gold Yeast Two-Hybrid (Y2H) System (Clontech, Palo Alto, CA, United States). Positive colonies were selected for colony PCR using Matchmaker Insert Check PCR Mix 2 (Clontech, Palo Alto, CA, United States) and sequenced at Sangon Biotech, Shanghai, China, using the T7 primer.

Data Availability Statement
The data presented in this study can be found in online repositories. The data have been deposited in NCBI. The SRA accession number is SRR13254555. The TSA accession number is GIXT00000000.

TaNAC069 Expression Was Significantly Induced in TcLr19 Plants Upon Pt Infection
qPCR was used to investigate the expression of TaNAC069 in response to Pt infection. The expression level of TaNAC069 during incompatible interactions increased from 0 to 24 hpi, peaking at 48 hpi (about 23.8 times higher than the 0 hpi control), and then decreased at 72 hpi. A second expression peak observed at 96 hpi was lower than the first but 20.1 times higher than the control (Figure 2A). During compatible interactions, TaNAC069 expression was up-regulated compared with the non-inoculated control at most time points (Figure 2A). It exhibited expression peaks at 48 hpi (8.9 times higher than the control) and 168 hpi (7.3 times higher than the control) and then declined. Overall, the expression level of TaNAC069 was higher during incompatible interactions than compatible ones at most time points, indicating that TaNAC069 expression was induced by Pt infection.
Additionally, the expression level of TaNAC069 was low in non-inoculated roots and stems and exhibited almost no expression in wheat leaves. After inoculation with Pt race PHNT, higher expression levels of TaNAC069 were observed in leaves and stems than in roots, with the expression in leaves being 3.0 times higher than in roots and 1.9 times than in stems ( Figure 2B). TaNAC069 expression showed a significant variation in different plant tissues before and after inoculation with PHNT, indicating that expression is tissue specific and induced by the leaf rust pathogen.

Induction of TaNAC069 by Chemical Reagents
To better understand the signal pathways of TaNAC069, the expression profiles of TaNAC069 induced by ABA, SA, ETH, and MeJA were analyzed. The expression level of TaNAC069 increased from 0 to 6 h after ABA treatment and was markedly high at 6 hpt, being nearly 4.7 times higher than that of the control, followed by a slight decrease. During SA treatment, the TaNAC069 expression levels peaked at 48 hpt, which was nearly 11.2 times higher than those of the control, followed by a sharp decrease from 48 to 96 hpt and a subsequent increase at 96 hpt, and peaked again at 120 hpt. The expression level of TaNAC069 was significantly increased, peaking at 72 hpt, after ETH treatment, which was nearly 10.1 times higher than in the control, then followed by a decrease (Figure 3). MeJA treatment had no obvious effect on TaNAC069 expression. The above-mentioned results show that TaNAC069 expression was induced by ABA and SA, experiencing an earlier induction by ABA together with ETH; however, it was not significantly induced by MeJA.

TaNAC069 Knockdown Decreased the Resistance of Wheat Against Pt
To confirm the function of TaNAC069 in wheat resistance to Pt, the gene was knocked down using the VIGS system. Knockdown of the PDS gene in plants produces a visible photobleaching phenotype that can be used as a positive control to confirm the proper functioning of the VIGS system. Mock (buffer inoculated without BSMV) and BSMV:00 (empty vector)inoculated plants displayed an immune phenotype (scale 0) and no other obvious defects at 14 days post-inoculation with Pt pathotype PHNT compared to Thatcher (scale 4), which indicated that virus inoculation did not affect the wheat-Pt incompatible interaction. BSMV:V1 and BSMV:V2 inoculated with PHNT exhibited more Pt uredinia (scale 3) compared with the mock and BSMV:00 treatments. Thus, knocking down the expression of the TaNAC069 gene reduced the resistance of wheat against PHNT ( Figure 4A).
qPCR was used to determine the expression level of TaNAC069 in each treatment. Without inoculation, the expression level of TaNAC069 in BSMV:V1 and BSMV:V2 was reduced significantly,  The assays for each treatment consisted of three biological replicates. Data are means ± standard errors of three independent experiments. Differences between time-course points were assessed using SSPS. * p < 0.05, n = 3. and the silencing efficiency was more than 50% compared to BSMV:00 ( Figure 4B). After inoculation with PHNT, compared to BSMV:00, the expression of TaNAC069 was significantly reduced to 56%, 51%, and 60% in BSMV:V1 at 24, 48, and 120 h, respectively, and for the BSMV:V2 treatment expression, it was reduced to 69%, 64%, and 70% at 24, 48, and 120 h, respectively ( Figure 4B).
To determine whether TaNAC069 activates or represses the expression of downstream defense-related genes, several pathogenesis-related (PR) genes and reactive oxygen species (ROS)-related genes were selected and analyzed via qPCR. The expression of TaPR1, TaPR2, TaTLP1, and ROS synthesisrelated genes (TaNOX) was significantly lower in the TaNAC069knockdown plants compared with the control, and the expression of ROS clearance-related genes (TaCAT and TaSOD) was significantly higher (Figure 5).
H 2 O 2 accumulation by DAB staining and related enzyme activity (CAT and SOD) were measured during the wheat-Pt interaction. As shown in Figure 6, H 2 O 2 accumulation was significantly reduced and the activities of SOD and CAT enzymes were significantly increased in TaNAC069-knockdown plants compared with the control. These results indicate that the TaNAC069 gene plays a positive regulatory role in wheat resistance to Pt.

Identification of TaNAC069 and pTaNAC069 Activity
In order to examine the transcriptional activity and selfinteraction of TaNAC069, the yeast-transformed system was used. All transformed yeast strains grew well on SD/-Trp medium, indicating that these vectors were successfully  transformed into yeast cells. Yeast cells containing pBD-TaNAC069 or TaNAC069-C grew well and turned blue on SD/-Trp/-His/-Ade/X-α-Gal, whereas cells harboring pGBKT7 or pBD-TaNAC069-N did not ( Figure 7A). Yeast cells containing pBD-TaNAC069-N and pAD-TaNAC069 in combination and the positive controls grew well and turned blue on SD/-Trp/-Leu/-His/-Ade/X. In contrast, replacement of either the pBD-TaNAC069-N or the pAD-TaNAC069 with vectors expressing only the AD or BD yeast proteins resulted in no production of blue coloration (Figure 7B). These results suggest that TaNAC069 exhibits transactivation activity, that the C-terminus can be attributed to transcriptional activation, and that TaNAC069 forms NAC protein homomeric dimers which possess unique biological functions.
To further understand how TaNAC069 is regulated, the promoter with a length of 2,103 bp (upstream of the translational initiation codon ATG) was cloned by RT-PCR (Supplementary  Figure 2). The sequence shared 98% identity compared with the wheat genome; the sequence was given the label pTaNAC069. Analysis of the pTaNAC069 sequence using PlantCARE software FIGURE 5 | Relative transcript levels of TaPR1, TaPR2, TaTLP1, TaSOD, TaCAT, and TaNOX in TaNAC069-knockdown plants inoculated with PHNT. The y-axis indicates the amounts of gene transcripts normalized to the GAPDH gene and expressed relative to that of mock-inoculated control plants treated with 0.1% (v/v) ethanol solution. The x-axis indicates different sampling timepoints. The reference gene is GAPDH. The assays for each gene consisted of at least three biological replicates. Data are means ± standard errors of three independent experiments. Differences between time-course sampling points were assessed using SSPS. * p < 0.05, n = 3.
and PLACE database revealed that two classical promoter cisacting elements, TATA-box (-1,977, -385, and -201 bp upstream from the transcription start site) and CAAT-box (-1435, -1136, and -878 bp upstream from the transcription start site), were present. cis-Acting motifs of the pTaNAC069 sequence were categorized into five major groups as follows: conserved motifs, abiotic stress, pathogens, phytohormones, and tissuespecific expression-responsive cis-regulatory motifs ( Table 1 and Supplementary Figure 3) according to Tiwari et al. (2019). The CaMV35S promoter in the pBI121 vector was replaced by pTaNAC069 using the seamless cloning method to form the recombinant plant expression vector pBI121-pTaNAC069 (Supplementary Figure 4) to verify the promoter activity of pTaNAC069. As shown in Figure 7C, the results of GUS staining revealed a strong blue signal in N. benthamiana leaves injected with pBI121 and pBI121-pTaNAC069 that was not likewise observed for the control (normal leaves). Additionally, the promoter region of pCamA-GFP was replaced by pTaNAC069. As shown in Figure 7, GFP fluorescence was uniformly distributed throughout the cells of leaves injected with pCamA-GFP and pCamA-pTaNAC069-GFP, respectively, indicating the functional promoter activity of pTaNAC069.

Confirmation of TaNAC069 Interaction
With TaCP5 by Y2H Assay Zhang et al. (2019) reported that TaCP3, a cysteine protease family gene and a type of papain protease, plays a role in drought stress, so the papain family cysteine protease was selected for further investigation. BLASTp analysis showed that the papain family cysteine protease, labeled as screened by TaNAC069, shared 100% homology with published sequences (TraesCS5B02G208100.1) and was given the label TaCP5. To test if TaNAC069 can interact with TaCP5, pGADT7-TaCP5 plasmids were co-transformed into the yeast strain Y2HGold and cultured on the selection medium SD/-Trp/-Leu/-His/-Ade and X-α-gal together with construct pBD-TaNAC069-N. The results showed that the yeast transformant co-expressing the pBD-TaNAC069-N and pGADT7-TaCP5 plasmids produced  blue coloration (Figure 8A, rows 5) similar to the positive control ( Figure 8A, rows 1). In contrast, replacement of either the pBD-TaNAC069-N prey or the pGADT7-TaCP5 bait with vectors expressing either the AD or BD yeast proteins produced no blue coloration (rows 2-4).
To further explore their potential molecular functions in regulation relationship between wheat and Pt, the co-expression patterns of TaCP5 were tested in the same manner as TaNAC069. We found that the expression level of the TaNAC069 gene in TcLr19 plants at different sampling time points after inoculation with Pt were similar to TaCP5 (Figure 8B), suggesting that the expression of both TaNAC069 and TaCP5 is induced by leaf rust infection and shares similar expression profiles, supporting the hypothesis that TaNAC069 and TaCP5 are likely to interact with each other.

DISCUSSION
Many studies have shown that NAC transcription factors have a variety of biological functions and play important roles in plant resistance to disease. Xue et al. (2006) isolated 14 TaNAC69 clones which were grouped into three highly homologous TaNAC69 gene categories (TaNAC69-1, TaNAC69-2, TaNAC69-3). TaNAC69 genes are likely to have some homologous genes due to the allopolyploid nature of hexaploid wheat. By analyzing Affymetrix expression data, TaNAC69 was found to be upregulated in rust-infected wheat leaves and involved in regulating wheat adaptation to drought stress (Xue et al., 2011). The expression of TaNAC4 isolated from wheat (T. aestivum L.) leaves was significantly induced by P. striiformis f. sp. tritici (Pst) (Xia et al., 2010), and the expression of BnNAC56 isolated from oilseed rape (Brassica napus) was rapidly and significantly induced by Sclerotinia sclerotiorum (Chen et al., 2017). In this study, TaNAC069, which is highly homologous to TaNAC69, was cloned and induced by Pt infection. To assess the role of TaNAC069 in wheat-Pt interactions, the TaNAC069 gene was knocked down using a BSMV-VIGS system, which has been successfully used to identify gene function in plants (Yang et al., 2018). The TaNAC069-silenced wheat plants exhibited reduced resistance against Pt, suggesting that TaNAC069 plays a positive role in wheat resistance against Pt. NAC transcription factors could activate defense-related genes and enhance plant disease resistance against various stress conditions. For example, MaNAC5 interacts with MaWRKY1/2 to regulate the expression of specific PR genes in banana (Musa acuminata) against the fungus Colletotrichum musae (Shan et al., 2016). OsNAC4, a transcription factor in rice, positively regulates the HR (Kaneda et al., 2009). H 2 O 2 accumulation is observed in a variety of defense responses, including those induced by wounding, insect feeding, and pathogen infection (Orozco-Cardenas and Ryan, 1999). H 2 O 2 is a reactive oxygen species, which is relatively stable and participates in the HR. Balazadeh et al. (2010) reported that some NACs are involved in H 2 O 2 -mediated signaling in Arabidopsis. TaNAC2 is likely to participate in resistance responses through the generation of H 2 O 2 during wheat-Pst compatible interactions . In the present study, we found that the expression of PR genes, ROSrelated genes, and H 2 O 2 accumulation were significantly induced in TaNAC069-knockdown plants compared with controls. Based on these results, we believe that TaNAC069 enhances wheat resistance to the leaf rust pathogen through transcriptional regulation of specific downstream disease-related resistance genes.
Yeast one-hybrid assay is widely recognized as a useful technique for detecting physical interactions between DNA and DNA-binding proteins. In this study, several potential transcription factors were successfully screened by yeast onehybrid assay, including WCOR410C, CBSX3, and eEF1A. WCOR410C is a member of the dehydrin (dehydration) multi-protein family and encodes a peripheral protein that is responsive to numerous abiotic and ABA stresses (Kiani et al., 2020). Dehydration-responsive element-binding proteins are a large family of transcription factors that induce the expression of a large number of functional genes responsible for stress endurance in plants (Agarwal et al., 2006). The dehydration-responsive element (DRE), a cis-acting element of the promoter regions, was identified (Shinozaki and Yamaguchi-Shinozaki, 2007). ABRE (ABA-responsive element) and DRE1 are present in the TaNAC069 gene promoter region. CBSX, FIGURE 8 | Interaction between TaNAC069 and TaCP5. (A) Yeast two-hybrid analysis of the interaction between TaNAC069 and TaCP5 in yeast. Yeasts expressing the indicated combinations of bait and prey proteins were spotted onto synthetic dropout medium lacking leucine and tryptophan (SD-WL) and SD medium lacking leucine, tryptophan, histidine, and adenine supplemented with X-α-Gal and Aureobasidin A (SD-HAWL + X-α-Gal). Only yeast strains co-expressing TaNAC069 and TaCP5 grew on SD-HAWL plates and developed blue coloration, indicative of an interaction between both proteins. TaPR1 and TaTLP1 are the positive controls; TaNAC069-N and pGADT7, TaCP5 and pGBKT7, and pGADT7 and pGBKT7 were used as negative controls. (B) Gene expression profiles of TaNAC069 and TaCP5 in wheat infected with Pt at different timepoints post-inoculation. The y-axis indicates the amounts of gene transcripts normalized to the GAPDH gene and expressed relative to plants at 0 hpi. The x-axis indicates different sampling timepoints. The reference gene is GAPDH. The assays for each treatment included three biological replicates. Data are means ± standard errors of three independent experiments. Differences between time-course sampling points were assessed using SSPS. *p < 0.05, n = 3.
a protein having only one pair of CBS domains without any other protein domains, binds to specific signal molecules to regulate the activity of various downstream partner proteins. In rice, OsCBSX3 was identified and up-regulated significantly by the inoculation of Magnaporthe oryzae and exogenous application of SA and MeJA (Mou et al., 2015). The TGACG motif of pTaNAC069 is involved in MeJA responsiveness, and TaNAC069 was induced by SA. Further experiments, such as Y2H and the electrophoretic mobility shift assay, are needed to determine whether WCOR410C is involved in the transcriptional regulation of pTaNAC069, and CBSX3 may regulate the expression of the TaNAC069 gene by binding to MeJA and SA.
Several potential target proteins were screened by yeast two-hybrid assay, including the salicylic acid-binding protein 2-like, papain family cysteine protease, Di19, and eEF1A proteins. SABP2 activates host SA-mediated defense responses to biotic stress (Park et al., 2007). Sweet potato papain-like cysteine protease, SPCP2, is a functional senescence-associated gene (Chen et al., 2010). The expression of Brassica oleracea BoCP4 (papain-like cysteine proteases) and A. thaliana RD19 (a dehydrationresponsive cysteine protease) was dehydration responsive and repressed by water and sucrose (Coupe et al., 2003). In our study, TaNAC069 was found to interact with TaCP5 as confirmed by Y2H. Further experiments such as Y2H, bimolecular fluorescence complementation, and co-immunoprecipitation are needed to determine whether these potential target proteins are involved in interactions with TaNAC069.
Taken together, TaNAC069 enhances wheat resistance to Pt by activating PR genes or inhibiting ROS clearance-related genes.
Our findings will broaden our understanding of the potentially versatile functions of NACs and provide valuable insight into the molecular mechanisms underlying plant immunity. Further experiments using stable genetic transformation of the TaNAC069 gene for reliable functional characterization are being carried out, and more than 10 T 0 lines were obtained to further confirm the function of TaNAC069.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The data presented in this study can be found in online repositories. The data has deposited in NCBI. The SRA accession number is SRR13254555. The TSA accession number is GIXT00000000.

AUTHOR CONTRIBUTIONS
HW conceived the research plans. HW and DL supervised the experiments. YZ performed most of the experiments. HG and ZC provided technical assistance to YZ. HW and YZ designed the experiments and analyzed the data. HW conceived the project. YZ wrote the manuscript with contributions of all the authors. HW and DL supervised and complemented the writing. All authors contributed to the article and approved the submitted version.