Genome-wide identification of TGA transcription factors in wheat: Evolution, expression and verification

doi:10.21203/rs.3.rs-2693252/v1

Background: The TGA transcription factors have been considered one of the essential gene families for the physiological process of plants, and they participate in plant defence by sensing and modulating reactive oxygen species. In contrast, the study about the function of TGA transcription factors in hexaploid wheat and its progenitors has not been conducted.

Results: In this study, we identified 38, 12 and 26 TGA genes in hexaploid wheat and its two progenitors, respectively. The functional similarities between Arabidopsis thaliana, Oryza sativa, Triticum aestivum, T. dicoccoides and Aegilops tauschii were performed by the phylogenetic tree and classified into four groups. The chromosome location and the collinear pairs were relatively well conserved. Based on that, the gene structure and conserved motifs of the TaTGAs indicated a solid ability to integrate gene networks. The cis-element related to hormone and stress resistance was identified in the promoter region of TaTGAs. We retrieved the expression profile of TaTGAs' response to biotic stress. 12 TaTGAs with significant differential expression were confirmed by qRT-PCR. TaTGA13 was chosen for the function analysis as it was significantly differentially expressedafter stripe rust inoculation. We obtained the phenotype of the plants silenced by virus-induced gene silencing (VIGS) and mutation lines.

Conclusions: The TGA gene family were identified in hexaploidy wheat and its two progenitors. These results suggest TGAs were relatively well conserved in Gramineae and play an essential role in wheat stripe rust resistance.

Triticum aestivum

TGA transcription factor

stripe rust

gene family

VIGS

As the population increases, higher wheat yields are needed. Increased food security is closely related to economic and political stability[1, 2]. Stripe rust, caused by wheat Puccinia striiformis f. sp. tritici, leads to a devastating reduction in wheat production annually[3, 4]. Due to the genetic diversity and rapid virulence variation, the single gene resistance of the cultivar was quickly lost[5]. It is essential to constantly explore and screen new stripe rust resistance genes for wheat resistance breeding.

The TGA transcription factors are group D members of the bZIP family. They were isolated based on their ability to identify and bind to the core-binding motif TGACG related to the oxidative stress response [6, 7]. TGA transcription factor is one of the critical roles in the SA signalling pathway[8, 9]. The role of TGAs in stress response and the regulation of flower development has attracted widespread attention[10, 11]. Most knowledge of TGAs was from work on the model plant Arabidopsis thaliana. In Arabidopsis, TGAs have ten members and can be divided into five groups according to their sequence similarity[10]. AtTGA1 and AtTGA4, belonging to clade I, were considered to play a role in basal defence against the virulent bacterial pathogen but not SAR[12]. Clade II includes AtTGA2, AtTGA5 and AtTGA6, which showed a redundant function in resistance against pathogens and are described as co-activators of NPR1 to induce PR gene expression[13, 14]. Interestingly, clade II TGAs have been reported to have a complementary phenotype in mutant: compromised in SAR but not basal defence[15]. Clade III includes AtTGA3 and AtTGA7. AtTGA3 is a positive regulator of both basal defences and SAR[16]. AtTGA9 and AtTGA10 formed clade IV, and AtPAN was divided into clade V. these transcription factors were related to regulating the flower developmental processes[17].

In recent years, comprehensive analyses of the TGA transcription factor have been reported in soybean[18], potato[19], tomato[20, 21], and peach[22]. The TGA enhances the plant's resistance to the pathogen in tomato and peach. More profound functional studies in Arabidopsis have also been reported[23-25]. These studies indicate that TGA transcription factors have excellent research value and play different functions in different plants. But this work has not been conducted in wheat, especially on the defence response against pathogens.

In this study, we identified the TGA transcription factors in T. aestivum. Through a comprehensive analysis of this gene family, we got their evolution relationship and screened some of the differentially expressed TaTGAs related to wheat stripe rust resistance. One significantly differentially expressed gene was chosen to conduct a further experiment, and the phenotype of mutants finally verified its functions. In conclusion, the study suggested that TGAs play an essential role in wheat disease resistance. It helps to provide candidate genes for breeding resistant wheat varieties.

Genome-wide identification of TGAs in wheat and the two progenitors

As described in the method, a total of 38, 12, and 26 TGA transcription factors were identified in wheat (TaTGA1-TaTGA38) and its two progenitors-wild emmer (T. dicoccoides) (TdTGA1-TdTGA26) and Aegilops tauschii(AeTGA1-AeTGA12), respectively. According to the published papers, these identified genes were named based on their chromosome locations. In terms of quantity, these identified TGAs unevenly distributed in each chromosome of their genomes varies from 1 to 3 differently (Fig. 1). In wheat, chromosome group 3 possessed the most abundant TaTGAs, with each containing 3 TaTGA transcription factors, and group 1, 2 and 4 with each containing 2 TaTGA transcription factors in the chromosome. While groups 6 and 7 only contained 1 TaTGA transcription factor in each chromosome. It's different from the other cases in that A, B, and D chromosomes had the same TGA transcription numbers. There are two transcription factors on both chromosomes 5A and 5D, but one transcription factor on chromosome 5B. Compared to the sequence and location of TGAs in the wild emmer (T. dicoccoides) genome, which offered the genome B of wheat, the TaTGAs genes in the B sub-genome may be lost during evolution. This lost TGA gene in 5B may have had a redundant function with the TGA genes on 5A or 5D, which were eventually lost during evolution due to low purifying selection.

[Figure 1]

In the perspective of A, B and D sub-genomes, 13, 12 and 13 TaTGA transcription factors were identified, suggesting no significant variation in the TaTGA transcription factor abundance. The sequence characteristics of these proteins were investigated and compared (Table 1). The amino acid length ranged from 157 aa(TaTGA22) to 580 aa(TaTGA15), averaging 438 aa. In comparison, the size of TdTGAs ranged from 203 to 526 aa with an average of 371aa, and AeTGAs ranged from 111 to 477 aa with an average of 346 aa. Accordingly, the molecular weight (Mw) ranged from 32.75(TaTGA19) to 62.18(TaTGA25) kDa, 17.20 to 58.03 kDa, and 12.78 to 52.04 kDa for wheat, T. dicoccoides and A. tauschii, respectively.

[Table 1]

Tandem and segmental duplications are the most common driving forces for gene family expansions during evolution. From these identified proteins above, TaTGA proteins have longer amino acids than its two progenitors. The segmental duplications are the main event responsible for the evolution of the TaTGA gene family. Therefore, we speculate that the TGA gene families in higher plants expanded primarily because of segmental duplications.

Phylogenetic and molecular evolution analysis of TGA genes

The TGA transcription factors were well studied in the model plant Arabidopsis. To investigate the evolutionary relationships of TGA genes, we built a phylogenetic tree of these identified TGAs between species, including two model plants and two wheat progenitors. We aligned the protein sequences of all these TGAs, and a phylogenetic tree was constructed using their conserved domains. As shown in Fig. 2a, The evolutionary relationships of these TGA transcription factors were determined. The phylogenetic tree was classified into four groups, with each clade consisting of 21–59 members. 8, 3, 6, and 21 TaTGAs are clustered in clades I, II, III, and IV, respectively.

We examined the synteny between wheat and its two progenitors to determine the phylogenetic mechanisms of these TGA genes. A total of 28 and 53 orthologous gene pairs between hexaploid wheat (T. aestivum) and its two progenitors (Ae. tauschii, T. dicoccoides) were identified (Figure 2b). The collinear pairs were identified, suggesting these orthologous pairs were relatively well conserved during the evolution. Except for a few TGA genes, almost all TGA genes in its two progenitors showed synteny to A, B and D sub-genomes in hexaploid wheat. However, some orthologous gene pairs were only identified between chromosome 5B of T. dicoccoides and 5A of hexaploid wheat, but not 5B of hexaploid wheat. They combined the chromosome location and sequence similarity, possibly due to gene loss or chromosomal recombination during evolution and polyploidization.

[Figure 2]

Gene structure and conserved motifs analysis

The genes in the same subfamily showed similar gene structures. The closely related homologous TaTGA genes in the A, B, or D sub-genomes also have similar structures and encoded motifs, suggesting TGA genes were highly conserved in wheat and its progenitor during evolution. Exon-intron structure is also an essential evolutionary feature of genes, and this kind of evolution can lead to the functional diversification of genes. A total of 9 conversed motifs were identified (motifs 1-9) (Fig. 3). Motif 1 is a conserved region in all TGA proteins located in the N-terminus. It covers the bZIP domain, which is responsible for DNA binding and contains nuclear localization signals (NLS). This motif is usually rich in glutamine (Q), traditionally associated with the dimerization of proteins. Motif-3 is the typical signature of the TGA members in the C terminal, Yx2RL[RQ]ALSS[LS]W. Motif-3 and Motif-4 were also rich in Q, and Motif-4 was the Q-richest motif. The TaTGAs possess Q-rich motifs, suggesting that these proteins can interact with a variety of other proteins, making these TaTGAs have the potential to integrate various biological pathways in wheat. Motif-6 and motif-9 were highly variable among the TaTGA proteins, suggesting a wide range of functional diversity for TaTGAs.

[Figure 3]

Cis-Element Analysis of TaTGAs

The 1500bp sequence upstream of the promoter was selected to analyze and search for the regulatory element upstream of TaTGA. A single element involves multiple processes of multiple genes. Therefore, we mainly focused on the most representative genes and functions here. The results (Fig. 4) showed that, in addition to some common regulatory elements (CAAT-box, TATA-box), The promoter part of TaTGAs mainly contain elements related to light response, hormone regulation, biological and abiotic stress, injury stress and other reaction. Among them, the kinds of light-responsive elements are the most.

We pay attention to hormone regulatory elements like abscisic acid (ABRE), methyl jasmonate (CGTCA-motif, TGACG-motif), gibberellin (GARE-motif, P-box), salicylic acid (as-1, TCA-box) and biotic and abiotic stress-related regulatory elements (DRE, STRE, MBS, CCAAT-box, W-box, F-box etc.). According to the reported results, TGA transcription factors can interact with WRKY proteins to regulate plant resistance in the MeJA pathway[26]. In addition, EMSA experiments have verified that TGAs can bind ROXY proteins to form a complex to the as-1-like element[27]. These results validated that the predicted cis-acting elements are valid, which helps to identify more upstream genes regulating TGA transcription factors.

[Figure 4]

Expression profiles of TaTGAs under biotic stress

TGA transcription factors play essential roles in stress response and floral development. To further evaluate the potential functions of TaTGAs in response to biotic stress, the TaTGAs expression patterns under biotic stress were analyzed from transcriptome data based on the fragment per kilobase of transcript per million reads mapped (FPKM). The heat map showed that most TaTGA genes were expressed differently after pathogen inoculation. Results showed that TaTGAs had differential expression under different treatments. The expression of TaTGA was induced significantly by the stripe rust pathogen CYR31 infection. TaTGAs 7/13/25 were suppressed, while the other, like TaTGA8, was up-regulated under the stripe rust infection(Figure 5). Homologous genes in A, B and D sub-genomes showed the same pattern of expression, which suggested that there may be functional redundancy between them. These results indicate that TaTGAs contribute to the stripe rust resistance in wheat.

[Figure 5]

Validation of the expression of wheat TGA genes using qRT-PCR

To validate the expression patterns of these selected TaTGAs in response to biotic stresses, we collected the samples treated with stripe rust at the specified time points and performed qRT-PCR(Figure 6). The TaTGAs genes, which were significantly induced by the stripe rust, were selected to validate. TaTGA7, TaTGA8, TaTGA13, and TaTGA16 significantly upgraded after stripe rust infection. They showed several types of trends of incompatible interaction, some reached a peak at 24h and were significantly inhibited at 72h post-inoculation (hpi), and then the expression level rose again. Some genes reached the peak at 48hpi or 120hpi. The expression of TaTGA24 significantly decreases at 12hpi. The other TaTGA genes showed no obvious response to stripe rust. These results matched the results of the RNA-seq results.

[Figure 6]

Interaction analysis of TaTGAs

To better understand the biological function and the regulatory network of TaTGAs, we submitted the protein sequences to the STRING database to predict the protein-protein interaction (PPI) in the TaTGA genes family. A limited number of proteins have been reported to interact with TGA transcription factors. However, according to the extensive regulation of TGA transcription factors and the genetic structure of TaTGAs, we still found some interaction proteins worthy of attention in biotic resistance(Figure 7). As expected, some proteins interacting with TaTGAs were SA signalling pathway components, such as NPR1 or NPR1-like proteins. Combined with the previous cis-acting element analysis and published research, TaTGAs can interact with themselves to form a dimer or polymer. Different TaTGAs can also interact with each other. These kinds of binding can influence the ability of DNA binding and the affinity of other transcription factors to the promoter elements. The TaTGAs may interact with stress-related transcription factors such as WRKY. In addition, some research has already shown that different types of ROXY bind to TGA to regulate the activity of TGA transcription factors. The hexaploid wheat has a larger and more complex genome and possesses unique genes compared to other plants. Identification of new interacting proteins of TaTGAs requires the combination of Chip-seq or DAP-seq experiments, and the bioinformatics predictions alone are limited.

[Figure 7]

Transient Silencing of TaTGA13 Enhances Wheat resistance to Pst

TaTGA13, which significantly responds to stripe rust inoculation, was chosen to implement the known down experiment. Firstly, we used BSMV-VIGS (virus-induced gene silencing) system to analyze the function of the TaTGA13. At ten days post-inoculation, mosaic symptoms of the virus were observed in virus-inoculated plants, but further leaf growth was not affected. In contrast, γ-PDS inoculated plants gradually displayed photobleaching symptoms (Figure 8), suggesting that the virus-induced gene silencing assay was successful. qRT-PCR results revealed that the transcript levels of endogenous TaTGA13 of the transformants were significantly reduced during compatible interactions. After inoculation with the compatible race V26, fungal growth was slightly decreased on γ-TaTGA13 plants compared to the control γ.

[Figure 8]

As a plant hormone, salicylic acid (SA) plays an essential role in plant defence against biotrophic and hemibiotrophic pathogens[28, 29]. TGA gene family are critical members in the SA signal transduction pathway, playing a role in floral development and boosting plant stress resistance. In plant pathogen resistance, whether P/MTI, ETI, or SAR, it will cause ROS levels and accumulation of SA. The intracellular redox state changes, and cell metabolism and gene expression have been regulated. TGA factors were postulated as redox sensors[30] and were considered to participate in the plant defence[10, 31, 32]. Stripe rust of wheat has complex genetic diversity and fast mutation. After popularizing in a large area, the resistant varieties often lose resistance quickly because of the single resistance gene. Studying the TGA transcription factor can provide a theoretical basis for wheat disease resistance breeding.

Recently, the TGA gene family has been identified in many plant species, like soybean, Tripterygium wilfordii[33] etc. The function of the TGA transcription factors is also improving. Still, there is no comprehensive analysis of TGA transcription factors in wheat, especially concerning their participation in stripe rust resistance. In this study, 38 TGA members were identified in the wheat genome and 26 TdTGAs in T. dicoccoides and 12 AeTGAs in A. tauschii, respectively. The TaTGAs can be clustered into four clades with their two progenitors, which consisted of other previous research. The homologous TGA genes at the branch end of each clade of the A, B or D sub-genome suggest that the evolution of TGA genes was conserved in Gramineae. However, the TGA transcription factors in wheat evolved differently from Arabidopsis TGAs. The number of TGAs in wheat is expanded, suggesting that TGA transcription factors have more complex functions than in Arabidopsis thaliana. Gene structure analysis showed that the TaTGA genes within the same clade had a similar intron-exon organization. This speculated that the different statuses of intron and exon splicing could be meaningful to the evolution of TGA genes. All predicted TaTGA proteins possessed motif 1, a conserved region in all TGA proteins located in the N-terminus, which covers the bZIP domain. Motif 1 is responsible for DNA binding and contains nuclear localization signals (NLS). Except for two pseudogenes at the C terminal end, Motif 3 is reported to function as a transcriptional activation domain[34]. TGA proteins have a conserved bZIP-D box in addition to the common domains of bZIP proteins[35]. Although the bZIP-D box is a characteristic domain for the TGA family, there are currently no articles of which we are aware of reporting the association of this feature with any particular biological function.

Cis-acting elements analysis helps to find genes upstream of the TaTGAs. In addition to some regulatory elements (CAAT-box and TATA-box), the promoter part of TaTGA mainly contains several elements related to light response, hormones, biological and abiotic stresses, and injury stress. In the perspective of plant resistance, elements associated with methyl jasmonate(CGTCA-motif, TGACG-motif), salicylic acid(as-1, TCA-box), biotic and abiotic stresses(DRE, STRE, MBS, W-box, F-box) are worthy of attention. Most of the predictive results of interacting proteins were NPR1-like proteins. This also demonstrated the indispensable role of TaTGA in the wheat SA pathway. Combined with cis-acting element analysis and experimental validation in other crops, TaWRKY and TaROXY in wheat could interact with TGA proteins to regulate their function. Further experiments are needed to confirm this hypothesis and to identify more interacting proteins.

The TaTGAs expression patterns under biotic stress were analyzed from transcriptome data. It is evident that TaTGA genes likely played a role in stripe rust resistance. The qRT-PCR data confirmed that some TaTGA genes are involved in stripe rust resistance with different mechanisms. For example, the peaks in qRT-PCR follow the biological processes, which helps to speculate the function of the process of resistance. TaTGA11, TaTGA13 and TaTGA16 were up-regulated and reached a peak at 24hpi and were significantly inhibited at 72hpi. While TaTGA25 were down-regulated and significantly decreased transcript level at 12hpi. Similar stress response mechanisms were observed in bZIP family genes of rice and Brachypodium distachyon[36, 37]. These results are basically under the results of the RNA-seq results. Some differences may be caused by using different stripe rust races and wheat varieties, but they have the same resistance trend.

Through the VIGS assays, we further characterized the roles of TaTGA13 in stripe rust resistance. Our data showed that the silence of TaTGA13 can enhance resistance to stripe rust, so it negatively regulated the plant defence. The phenotype of the mutant exhibited enhanced stripe rust resistance. The above results suggest that TaTGA13 negatively modulates the resistance to stripe rust. This observation followed the fact that TGA genes were involved in response to pathogen attacks . However, additional experiments are needed to determine the specific functions of the TaTGA genes. In addition, the cross-reaction of TGA transcription factors to different signals requires further verification.

This study comprehensively reported the genome-wide identification, gene structure, interacting proteins, expression profiles and function verification of TGA genes in wheat. The identified 38 TGA genes were classified into four structurally evolutionarily conserved subfamilies. Segmental duplications during evolution were essential for expanding the TaTGA gene family. Gene structure and conserved motifs analysis suggested a wide range of functional diversity for TaTGAs. Expression analysis showed that some of the TaTGA genes participate in the responses to stripe rust infection. The phenotype of VIGS assay combines mutant suggest that TaTGA13 negatively modulates the resistance to stripe rust. In sum, wheat TGA transcription factors may act as essential components of biotic stress resistance in plants. These results provide candidate gene resources for breeding new wheat varieties with enhanced resistance.

Genome-wide identification of TGA genes in wheat and its two progenitors

Firstly, we downloaded the Arabidopsis and the rice TGA protein sequences from the Ensemble Plants database (https://plants.ensembl.org/index.html)[38]. Then we used the amino acid sequence of AtTGA and OsTGA as the queries to perform a BLASTP search against the local protein database of wheat (IWGSC_v1.1) with the expected value (E-value) of 1e-5, which was downloaded from URGI database (https://urgi.versailles.inra.fr/download/iwgsc/). The protein sequences identified by both model plants were combined and manually parsed to remove a redundant sequence. The remaining proteins were considered candidate wheat TGA proteins. Finally, we submitted these candidate genes to InterPro (http://www.ebi.ac.uk/interpro/) and NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd/) to verify the TGA conserved domain. The same method was used to identify the TGA genes in T. dicoccoides and Ae. tauschii. The molecular weight (Mw), isoelectric point (pI), instability index (II), aliphatic index (AI), and grand average of hydropathicity (GRAVY) were investigated through ExPASy (http://web.expasy.org/protparam/)[39].

Phylogenetic relationship, gene structure and conserved motifs analysis

To understand the distance relationship of the TGA genes with other plants, the sequence alignments of wheat identified TGA proteins were performed using the ClustalW tool. The neighbour-joining (NJ) tree was constructed by MEGA 8.0 software with a bootstrap value of 1000[40]. These TGA genes' chromosomal location and exon-intron structures were extracted from the genome annotation files, which were downloaded from the Ensembl Plants database (http://plants.ensembl.org/index.html). The physical chromosome location of these TGA genes was displayed by MapGene2Chromosome v2.0 software (http://mg2c.iask.in/mg2c_v2.0/), and the exon-intron structure was displayed using the Gene Structure Display Server (GSDS2.0) (http://gsds.cbi.pku.edu.cn/)[41]. Furthermore, the orthologous and homoeologous gene pairs were identified based on the chromosome's physical position and sequence similarity. The genes with the dN and dS value of 0 as well as the dS value of more than 0.3 were discarded, as they might result from sequence saturation or misalignment, and then the PSGs (dN/dS > 1) and NSGs (dN/dS < 1) were obtained.

Cis-Element Analysis of TaTGAs

We extract 1500 bp upstream of the ATG start codon as the promoter sequences of each TaTGA gene from the local wheat genome database. The sequence was uploaded to the PlantCARE website[42] to predict their cis-elements.

Expression profiles and interaction networks analysis

To understand the spatial-temporal expression patterns of these identified TaTGAs, RNA-seq samples from 92R137 biotic stresses were from transcriptome data. RNA-seq data used in this work were listed in Supplementary Table S1, Some data is preserved, further inquiries can be directed to the corresponding author/s. The FPKM (fragments per kilobase of transcript per million fragments mapped reads) value of each TaTGA was calculated[43], and the differentially expressed genes were then identified. The heat map was constructed to visualize the expression through the pheatmap package in R software. The Ortho Venn tool (http://www.bioinfogenome.net/OrthoVenn/) was used to identify the orthologous pair between TGAs. Then, the interaction networks of TaTGA genes involved were identified based on the STRING (http://string-db.org/cgi) database, The relevant data were placed in the supplementary Table S4, and the predicted interaction network was displayed through Cytoscape software.

Plant materials and pathogens inoculation

Wheat (Triticum aestivum L.) line 92R137 and mutant material TaTGA13 were used in this study. They are both compatible interaction with Pst isolation V26. The isolation V26 was used for the inoculation assay.

RNA extraction and qRT-PCR validation

Plants were grown in the light incubator and maintained following the procedure described by[44]. The uredospore mixed with electron fluoride solution was sprayed onto ten-day-old seedlings at the 2-leaf stage. Simultaneously, plants were sprayed with electron fluoride as control. After inoculation, plants were kept for 24-h at 100% humidity and dark. Then the inoculated plants were transferred to a growth chamber with a 16-h photoperiod (light intensity, 2000 lx) at 16°C. Inoculated leaves control wheat leaf tissues were harvested at 0, 24, 48, 72 and 120 hpi, quickly frozen in liquid nitrogen and stored at −80°C until total RNA extraction.

The qRT-PCR reaction was conducted as the protocol described[45]. We carried out three biological replications, and the total RNA of the samples were isolated by Quick RNA isolation Kit (HUAYUEYANG, CHN) according to the product manual. A total of 12 significantly expressed TaTGAs were selected to investigate their expression levels under biological stress through qPCR analysis. The qRT-PCR was performed with the 7500 real-time PCR system (Applied Biosystems), and the data were analyzed through the ABI prism 7500 Software Tool (Applied Biosystems). The average expression ratio (Standard Curve method of relative gene quantification) was calculated. Threshold values (CT) generated from the ABI prism 7500 Software Tool (Applied Biosystems) was employed to quantify relative gene expression using the comparative 2−ΔΔCT method[46].

Virus-induced gene silencing (VIGS)

The specific primers were designed to amplify at the 5' and 3' regions of TaTGA13, respectively. The upstream and downstream primers introduced Pac I, and Not I was used to amplify the fragments. The target fragment replaced the PDS fragment in the γ-PDS positive control vector, and two vectors, γ-TaTGA13-1 and γ-TaTGA13-2, were constructed. The BSMV-α and BSMV-γ vectors were cut by Mlul enzyme, BSMV-β vectors were cut by Spel enzyme, γ-PDS, γ-TaTGA13-1 and γ -TatGA13-2 vectors were cut by BssH II enzyme, and the gel was recovered and purified[47]. The linearized product was used as a template for in vitro transcription using RiboMAX TM Large Scale RNA Production System-T7 kit (Promega, US).

We took 0.5ul α and β mixed with 0.5ul γ, γ-PDS, γ -TaTGA13-1 and γ-TaTGA13-2 in a ratio of 1:1:1, respectively. Then 8.5ul FES buffer was added. After the mixture, friction inoculation was carried out on the second leaf in the 3-leaf stage. Specific virus inoculation methods referred by Yin et al. Friction FES buffer was used as the negative control and γ-PDS as the positive control. After inoculation, the circumstance was carried out at 25℃ and 16/8 h light-dark cycle. The bleaching of γ-PDS wheat leaves and virus symptoms were observed. Once photobleaching was observed (about ten days post viral inoculation), the fourth leaf of wheat inoculation with Pst. The inoculated leaves were taken at 0, 24, and 120 hpi for silencing efficiency detection. Phenotypes were observed at about 14 dpi.

Ethics approval and consent to participate

All methods were in compliance with relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable.

Availability of data and material

All data generated or analysed during this study are included in this article and its supplementary material. Further inquiries can be directed to the corresponding authors.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by National Key R&D Program of China (2021YFD1401000).

Authors' contributions

JJL wrote the Paper and did the experiments; QDZ conducted data analysis and drew partial figures; QDZ and DJH checked and revised the Paper. All authors have read and approved the manuscript.

Acknowledgements

Not applicable.

Author details

¹State Key Laboratory of Crop Stress Biology for Arid Areas, College of Agronomy, Northwest A&F University, Yangling, Xianyang 712100, China. ²State Key Laboratory of Crop Stress Biology for Arid Areas, Plant Protection, Northwest A&F University, Yangling, Xianyang 712100, China.

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Table 1. Basic information of TGA genes identified in wheat.

Gene name	EnsemblPlants ID	Gene Location	Deduced Protein			Splice Variants	bZIP domain	Subcellular location
Gene name	EnsemblPlants ID	Gene Location	Size (aa)	MW(KDa)	pI	Splice Variants	bZIP domain	Subcellular location
TaTGA1	TraesCS1A02G096300	1A:91602861:91604969	335	36.67	7.83	1	47-95	Nucleus
TaTGA2	TraesCS1A02G276600	1A:471695008:471701095	525	58.02	8.1	4	212-250	Nucleus
TaTGA3	TraesCS1B02G127400	1B:156565523:156567675	340	37.26	7.17	5	46-93	Nucleus
TaTGA4	TraesCS1B02G285800	1B:497124690:497131575	526	58.06	8.68	4	213-257	Nucleus
TaTGA5	TraesCS1D02G105300	1D:97214357:97216835	339	37.11	6.7	2	46-94	Nucleus
TaTGA6	TraesCS1D02G276100	1D:372147878:372155789	525	58.06	7.52	5	212-256	Nucleus
TaTGA7	TraesCS2A02G097500	2A:51493493:51497387	333	37.33	8.68	3	46-94	Nucleus
TaTGA8	TraesCS2A02G526300	2A:746682082:746688211	419	46.27	7.52	1	119-165	Nucleus
TaTGA9	TraesCS2B02G113200	2B:76816411:76820668	333	37.29	7.14	3	45-89	Nucleus
TaTGA10	TraesCS2B02G556600	2B:751317909:751324133	435	48.38	8.11	1	130-176	Nucleus
TaTGA11	TraesCS2D02G096800	2D:49195871:49199822	333	37.35	6.52	3	46-89	Nucleus
TaTGA12	TraesCS2D02G529000	2D:616525223:616530794	425	46.84	6.71	1	121-167	Nucleus
TaTGA13	TraesCS3A02G190700	3A:238676584:238691290	325	36.10	7.09	3	43-86	Nucleus
TaTGA14	TraesCS3A02G334100	3A:579377243:579383014	475	51.77	6.94	1	186-232	Nucleus
TaTGA15	TraesCS3A02G372400	3A:623047081:623054386	570	61.79	7.01	3	255-299	Nucleus
TaTGA16	TraesCS3B02G220400	3B:269247185:269257631	327	36.38	5.78	3	43-92	Nucleus
TaTGA17	TraesCS3B02G365100	3B:576449738:576455465	477	51.92	6.31	1	188-234	Nucleus
TaTGA18	TraesCS3B02G404800	3B:640060963:640067490	526	57.07	9.44	4	211-255	Nucleus
TaTGA19	TraesCS3D02G194800	3D:187831578:187841477	284	32.75	7.83	4	42-88	Nucleus
TaTGA20	TraesCS3D02G327600	3D:439677492:439683178	477	51.98	6.12	1	189-238	Nucleus
TaTGA21	TraesCS3D02G365200	3D:479612897:479620687	530	57.56	6.92	5	216-264	Nucleus
TaTGA22	TraesCS4A02G126300	4A:162423186:162427249	298	33.34	7.33	3	215-259	Nucleus
TaTGA23	TraesCS4A02G183400	4A:460896761:460903389	540	59.00	6.9	3	47-92	Nucleus
TaTGA24	TraesCS4B02G135000	4B:176260221:176265796	566	62.18	6.2	1	230-274	Nucleus
TaTGA25	TraesCS4B02G178600	4B:391537582:391541691	334	37.27	7.54	1	256-300	Nucleus
TaTGA26	TraesCS4D02G129900	4D:115934039:115940363	538	58.83	7.03	1	47-96	Nucleus
TaTGA27	TraesCS4D02G180200	4D:314063385:314067508	298	33.34	8.46	4	230-278	Nucleus
TaTGA28	TraesCS5A02G174200	5A:367556709:367565775	467	51.25	8.23	1	47-96	Nucleus
TaTGA29	TraesCS5A02G265600	5A:477392155:477401214	518	57.86	7.05	1	176-214	Nucleus
TaTGA30	TraesCS5B02G265300	5B:449612423:449621819	526	58.69	7.5	2	206-245	Nucleus
TaTGA31	TraesCS5D02G178800	5D:278260567:278270134	466	51.05	7.03	1	213-252	Nucleus
TaTGA32	TraesCS5D02G273500	5D:376437850:376444197	518	57.83	6.68	3	175-213	Nucleus
TaTGA33	TraesCS6A02G165800	6A:166982263:166991094	466	50.95	7.01	3	205-244	Nucleus
TaTGA34	TraesCS6B02G193200	6B:227641866:227651596	457	49.91	9.98	2	157-200	Nucleus
TaTGA35	TraesCS6D02G154400	6D:129486855:129495855	466	50.98	9.44	2	148-185	Nucleus
TaTGA36	TraesCS7A02G207100	7A:169397681:169401164	443	48.31	7.06	1	157-206	Nucleus
TaTGA37	TraesCS7B02G114300	7B:132168398:132171701	448	48.46	8.68	1	155-201	Nucleus
TaTGA38	TraesCS7D02G209800	7D:167584688:167588228	448	48.44	8.23	1	160-206	Nucleus

No competing interests reported.

DataSheet1.xlsx

Genome-wide identification of TGA transcription factors in wheat: Evolution, expression and verification

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