Genome-Wide Identification and Analysis of the NPR1-Like Gene Family in Bread Wheat and Its Relatives

NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), and its paralogues NPR3 and NPR4, are bona fide salicylic acid (SA) receptors and play critical regulatory roles in plant immunity. However, comprehensive identification and analysis of the NPR1-like gene family had not been conducted so far in bread wheat and its relatives. Here, a total of 17 NPR genes in Triticum aestivum, five NPR genes in Triticum urartu, 12 NPR genes in Triticum dicoccoides, and six NPR genes in Aegilops tauschii were identified using bioinformatics approaches. Protein properties of these putative NPR1-like genes were also described. Phylogenetic analysis showed that the 40 NPR1-like proteins, together with 40 NPR1-related proteins from other plant species, were clustered into three major clades. The TaNPR1-like genes belonging to the same Arabidopsis subfamilies shared similar exon-intron patterns and protein domain compositions, as well as conserved motifs and amino acid residues. The cis-regulatory elements related to SA were identified in the promoter regions of TaNPR1-like genes. The TaNPR1-like genes were intensively mapped on the chromosomes of homoeologous groups 3, 4, and 5, except TaNPR2-D. Chromosomal distribution and collinearity analysis of NPR1-like genes among bread wheat and its relatives revealed that the evolution of this gene family was more conservative following formation of hexaploid wheat. Transcriptome data analysis indicated that TaNPR1-like genes exhibited tissue/organ-specific expression patterns and some members were induced under biotic stress. These findings lay the foundation for further functional characterization of NPR1-like proteins in bread wheat and its relatives.


Introduction
Bread wheat (Triticum aestivum L.), also known as common wheat, is a major grain crop with very high economic value globally. This allohexaploid bread wheat (T. aestivum, 2n = 6x = 42) is comprised of three closely homoeologous chromosome groups (AABBDD), originating from a series of natural hybridization events [1,2]. Firstly, diploid Triticum urartu (the A-genome donor) hybridized with an unknown diploid grass (considered probably as Aegilops speltoides, the B-genome donor) to produce tetraploid emmer wheat (Triticum dicoccoides, AABB). Subsequently, emmer wheat hybridized with diploid goat grass (Aegilops tauschii, the D-genome donor) to form hexaploid wheat around 8000 years ago. In field conditions, bread wheat is challenged by various fungal pathogens, such as Fusarium graminearum (Fusarium head blight, FHB), Puccinia striiformis (stripe rust), and Blumeria graminis (powdery mildew), which cause huge losses in yield and quality [3]. The salicylic acid (SA) signaling co-repressor activity on TGAs [9]. This enables TGAs to turn on defense-related gene expression and activates defense response. In addition to pathogen invasion, exogenous application of SA or its analogs (BTH; benzothiodiazole and INA; 2,6-dichloroisonicotinic acid) could also induce the resistance mechanism in plants [48,49]. Taken together, NPR1, and its paralogues NPR3 and NPR4, are all SA receptors through an antagonistic manner to finely regulate plant immune response dependent on distinct threshold levels of SA [9].
Arabidopsis NPR1 and its homologs have been proved to be involved in SA-mediated defense responses through genetic transformation in many plant species. For example, these NPR1-like genes likely function similar to AtNPR1, which acts as a positive regulator in the SA-mediated immunity. Transgenic rice (Oryza sativa) overexpressing OsNPR1 displayed enhanced resistance to bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) and fungal blast pathogen Magnaporthe oryzae (Mo), and knockdown of OsNPR1 exhibited increased susceptibility to Xoo and Mo [50][51][52]. Transgenic mustard (Brassica juncea) overexpressing BjNPR1 showed enhanced resistance to fungal pathogens Alternaria brassicae and Erysiphe cruciferarum [53]. Overexpression of mulberry (Morus multicaulis) MuNPR1 in Arabidopsis displayed enhanced resistance to Pseudomonas syringae pv. tomato DC3000 (PstDC3000) [54]. Knockdown of barley HvNPR1 [55,56], tobacco NtNPR1 [57], and tomato NPR1-like gene [58] showed elevated susceptibility to powdery mildew fungus, tobacco mosaic virus (TMV), and Ralstonia solanacearum, respectively. Moreover, these NPR1-like genes likely function similar to AtNPR3/4, which acts as a negative regulator in the SA-mediated resistance. Transgenic rice overexpressing OsNPR2 and OsNPR3 had no enhanced resistance to Xoo [51], and overexpression of OsNPR3 using its own promoter resulted in increased expression of several PR genes and enhanced resistance only after treatment with BTH [59]. Knockdown of Theobroma cacao TcNPR3 conferred resistance against Phytophthora tropicalis [60,61]. Overexpression of mulberry MuNPR4 [54] or strawberry FvNPRL-1 [62] in Arabidopsis both showed enhanced susceptibility to PstDC3000.
In emmer wheat, wNPR1 was isolated from Triticum turgidum ssp. durum by homology cloning strategy [63], and transgenic barley overexpressing wNPR1 displayed elevated resistance to Mo [64]. Overexpression of AtNPR1 or Secale cereale ScNPR1 in bread wheat both conferred enhanced FHB resistance [65][66][67][68]. Based on the above research, it is necessary to systematically identify and analyze the NPR1-like family in bread wheat (T. aestivum) and its relatives (T. urartu, T. dicoccoides, and Ae. tauschii). The recently published reference genome sequences provide an opportunity for this study [69][70][71][72]. Here, the NPR1-like family was identified from bread wheat and its relatives' genomes. These putative NPR1-like genes were analyzed in detail, including molecular characterization, chromosomal distributions, phylogenetic classification, gene structures, protein domain compositions, conserved motifs and amino acid residues, and cis-regulatory elements. The collinearity analysis for NPR1-like genes was performed among bread wheat and its relatives. Furthermore, the expression pattern of TaNPR1-like genes in various tissues/organs and under biotic stress conditions was also analyzed using publicly available bread wheat RNA-sequencing (RNA-seq) datasets. Overall, these results provide an invaluable resource for further functional study of NPR1-like genes in bread wheat and its relatives.

Identification, Phylogeny, and Characterization of NPR1-Like Genes in Bread Wheat and its Relatives
Utilizing bioinformatics tools that contain BLAST search and HMMER analysis, a total of 40 putative NPR1-like genes were identified from bread wheat and its relatives' genomes. Among them, there were 17 in T. aestivum, five in T. urartu, 12 in T. dicoccoides, and six in Ae. tauschii (Table 1). To study the phylogeny of the NPR1-like family, an unrooted phylogenetic tree was constructed using the sequences of the 40 NPRs, and 4 OsNPRs, 5 PaNPRs, and 31 NPRs obtained through molecular cloning techniques from eight monocots and 15 dicots in the published literature (Table S1). Although individual phylogenetic analysis does not adequately perform functional annotation, phylogenetic grouping perhaps provides a reference for understanding functional diversification of the NPR1-like family.
The results demonstrated that NPR1-like proteins were clustered into three major clades: clade I (AtNPR1/2 subfamily) containing OsNPR1 [50][51][52], BjNPR1 [53], MuNPR1 [54], HvNPR1 [55,56], and NtNPR1 [57], etc; clade II (AtNPR3/4 subfamily) containing OsNPR2 [51], OsNPR3 [59], TcNPR3 [60,61], and MuNPR4 [54], etc; and clade III (AtBOP1/2 subfamily) ( Figure 1). In tetraploid and hexaploid wheat, homoeologous genes located in the branch ends of each clade belonging to the A, B, or D subgenomes, were regarded as the homeoalleles of one NPR1-like gene arising from allpolyploidization in genome evolution ( Figure S1A,B). All of the 40 identified NPR1-like genes were named and classified based on the phylogenetic relationship of the Arabidopsis NPR1-like family. TcNPR3 [60,61], and MuNPR4 [54], etc; and clade III (AtBOP1/2 subfamily) ( Figure 1). In tetraploid and hexaploid wheat, homoeologous genes located in the branch ends of each clade belonging to the A, B, or D subgenomes, were regarded as the homeoalleles of one NPR1-like gene arising from allpolyploidization in genome evolution ( Figure S1A,B). All of the 40 identified NPR1-like genes were named and classified based on the phylogenetic relationship of the Arabidopsis NPR1-like family.    Table S1. a Gladiolus hybridus, b Gossypium hirsutum, T. aestivum (Ta), T. urartu (Tu), T. dicoccoides (Td), Ae. tauschii (Aet). Three major clades are distinguished with three colors, and NPRs from T. aestivum and Arabidopsis are labeled with red and green markers. For example, 17 bread wheat NPR1-like genes were identified and evenly distributed in the three clades, consistent with the NPR1-like genes in Arabidopsis (Table 1). Each branch end of the TaNPR1-like family was composed of three homeoalleles in A, B, and D subgenomes, except the TaNPR2 branch, which had two homeoalleles in A and D subgenomes. Clade I contained TaNPR1 The isoelectric points (pI) of TaNPR1-like members ranged from 5.18 (TaNPR2-A) to 6.12 (TaNPR5-A), with an average of 5.76, showing a weak acid. A single transcript was available in 10 of 17 TaNPR1-like genes, and the remaining seven genes had two splice variants.

Sequence and Structural Analysis of TaNPR1-Like Genes and Proteins
To further understand the potential functions of TaNPR1-like genes, the structural feature and sequence composition were analyzed using GSDS, NCBI-CDD, and the Clustal Omega program. The exon-intron distributions of TaNPR1-like genes were consistent with the corresponding phylogenetic clade in Arabidopsis ( Figure 2B). However, only clade I, TaNPR1-A/-B/-D and TaNPR2-A/-D, and clade II, TaNPR3-A/-B/-D and TaNPR4-A/-B/-D, contained the NPR1-like C-terminal region that was essential for AtNPR1 activity [35,36]. The C-terminal region contained penta-amino acid motif (LENRV), NIMIN-binding region, and nuclear localization signal (NLS) ( Figure 2C) [45,73].

Analysis of cis-Regulatory Elements in the Promoter Regions of TaNPR1-like Genes
In plants, cis-regulatory elements control the expression of target genes by interacting with transcription factors [75]. Therefore, identifying potential cis-regulatory elements in the promoter regions of TaNPR1-like genes will provide useful information for understanding their regulatory expression. The 1000-bp upstream promoter sequences of TaNPR1-like genes were selected and submitted to the PlantCARE online service [76] for SA-responsive cis-regulatory identification.
The results showed that two hormone-related regulatory elements, CAAT-box and TATA-box, were overrepresented in all 17 TaNPR1-like gene promoters ( Figure 3). The activation sequence-1 (as-1) element (TGACG) involved in transcription activation of several SA-regulated PR genes was detected in all TaNPR1-like gene promoters, except TaNPR1-B and TaNPR5-A/-B/-D. Moreover, the W-box element (TTGACC) was known to be the DNA-binding site for the SA-induced WRKY transcription factors [46]. This W-box motif enriched in the AtNPR1 and OsNPR1 promoter was only found in the TaNPR1  Exons, UTRs, and introns are denoted by yellow boxes, blue boxes, and grey lines, respectively. Conserved domain organization and distribution (B) of the BTB/POZ, ANK, and NPR1-like Cterminal region in Arabidopsis and T. aestivum NPR1-like proteins. Multiple alignment of amino acid sequences (C) of T. aestivum NPR1-like proteins (TaNPR1 to TaNPR6) and other NPR1-related proteins with experimentally confirmed functions (AtNPR1 to AtNPR6, OsNPR1, and wNPR1). The position of point mutation sites (npr1-1, npr1-2, nim1-2, and nim1-4 in AtNPR1), and three of the conserved cysteines residues (C82, C216, and C156 in AtNPR1) are marked with arrows. The conserved domains, BTB/POZ and ANK, and important motifs, putative hinge region (LENRV), EARlike repression motif (VDLNETP), NIMIN-binding region, and nuclear localization signal (NLS), are highlighted with solid lines.

Analysis of cis-Regulatory Elements in the Promoter Regions of TaNPR1-like Genes
In plants, cis-regulatory elements control the expression of target genes by interacting with transcription factors [75]. Therefore, identifying potential cis-regulatory elements in the promoter regions of TaNPR1-like genes will provide useful information for understanding their regulatory expression. The 1000-bp upstream promoter sequences of TaNPR1-like genes were selected and submitted to the PlantCARE online service [76] for SA-responsive cis-regulatory identification.
The results showed that two hormone-related regulatory elements, CAAT-box and TATA-box, were overrepresented in all 17 TaNPR1-like gene promoters (Figure 3). The activation sequence-1 (as-1) element (TGACG) involved in transcription activation of several SA-regulated PR genes was detected in all TaNPR1-like gene promoters, except TaNPR1-B and TaNPR5-A/-B/-D. Moreover, the W-box element (TTGACC) was known to be the DNA-binding site for the SA-induced WRKY transcription factors [46]. This W-box motif enriched in the AtNPR1 and OsNPR1 promoter was only found in the TaNPR1

Chromosomal Distribution and Collinearity Analysis of NPR1-like Genes among Bread Wheat and Its Relatives
To explore the orthologous relationships of NPR1-like genes among bread wheat and its relatives, 6 Ae. tauschii-NPRs and 6 T. aestivum-NPRs in the D subgenome, 12 T.dicoccoides-NPRs and 11 T. aestivum-NPRs in the AB subgenome, and 5 T.urartu-NPRs and 7 T.dicoccoides-NPRs in the A subgenome protein sequences were used to generate phylogenetic trees (Figures S1C-S1E). Pairs of The promoter regions (1000-bp upstream of the ATG translation start codon) of TaNPR1-like genes were used to analyze four specific SA-responsive cis-acting regulatory elements, including CAAT-box (yellow blocks), TATA-box (blue blocks), W-box (red blocks), and as-1 element (green blocks).

Chromosomal Distribution and Collinearity Analysis of NPR1-like Genes among Bread Wheat and Its Relatives
To explore the orthologous relationships of NPR1-like genes among bread wheat and its relatives, 6 Ae. tauschii-NPRs and 6 T. aestivum-NPRs in the D subgenome, 12 T.dicoccoides-NPRs and 11 T. aestivum-NPRs in the AB subgenome, and 5 T.urartu-NPRs and 7 T.dicoccoides-NPRs in the A subgenome protein sequences were used to generate phylogenetic trees ( Figure S1C-E). Pairs of six Aet/Ta-D, 11 Td/Ta-AB, and four Tu/Td-A orthologs were identified (Table S2) (Table S2) and mapped to genome chromosomes (Figure 4). The collinearity analysis illustrated that six pairs of Aet/Ta-D orthologs were located on the same chromosomes with three on 3D, one on 4D, one on 5D, and one on 7D (Figure 4). Moreover, 10 Td-NPRs could be mapped to bread wheat AB subgenomes on the same chromosomes with three on 3A, one on 4A, one on 5A, three on 3B, one on 4B, and one on 5B, except TdNPR2-A-2/TaNPR2-A orthologous gene pairs (TdNPR2-A-2 on 7AS, TaNPR2-A on 4AL). Furthermore, four Tu/Td-A orthologous gene pairs were located on the same chromosomes, with three on 3A and one on 4A. However, orthologous TuNPR4 was located on T.urartu chromosome 4AS, corresponding to TdNPR4-A on T.dicoccoides chromosome 4AL. The orthologous gene pairs on different chromosomal positions indicated that chromosomal recombination events (chiasmata and crossing-over) had occurred during the evolutionary process. On the whole, however, the NPR1-like genes had an intact collinearity among bread wheat and its relatives, suggesting that the evolution of this gene family was more conservative following formation of hexaploid wheat. The collinearity analysis illustrated that six pairs of Aet/Ta-D orthologs were located on the same chromosomes with three on 3D, one on 4D, one on 5D, and one on 7D (Figure 4). Moreover, 10 Td-NPRs could be mapped to bread wheat AB subgenomes on the same chromosomes with three on 3A, one on 4A, one on 5A, three on 3B, one on 4B, and one on 5B, except TdNPR2-A-2/TaNPR2-A orthologous gene pairs (TdNPR2-A-2 on 7AS, TaNPR2-A on 4AL). Furthermore, four Tu/Td-A orthologous gene pairs were located on the same chromosomes, with three on 3A and one on 4A. However, orthologous TuNPR4 was located on T.urartu chromosome 4AS, corresponding to TdNPR4-A on T.dicoccoides chromosome 4AL. The orthologous gene pairs on different chromosomal positions indicated that chromosomal recombination events (chiasmata and crossing-over) had occurred during the evolutionary process. On the whole, however, the NPR1-like genes had an intact collinearity among bread wheat and its relatives, suggesting that the evolution of this gene family was more conservative following formation of hexaploid wheat.

Expression Analysis of TaNPR1-Like Genes in Various Tissues/Organs
To analyze the tissue/organ-specific expression patterns of TaNPR1-like genes, the expression data in eight tissues/organs (roots, stem axis, first leaf blade, shoot apical meristem, flag leaf, internode, glumes, and lemma) during the seedling and reproductive stages [77] were downloaded from the wheat URGI website.
The results revealed that TaNPR1-like genes were constitutively expressed in various tissues/organs, although the level of expression varied greatly ( Figure 5). The expression level of TaNPR1-A/-B/-D was moderate and was relatively high in root and flag leaf tissues. The expression of TaNPR2-A/-D was much lower in most tissues/organs with a similar pattern as AtNPR2 in Arabidopsis tissues [78]. TaNPR4

Expression Analysis of TaNPR1-Like Genes in Various Tissues/Organs
To analyze the tissue/organ-specific expression patterns of TaNPR1-like genes, the expression data in eight tissues/organs (roots, stem axis, first leaf blade, shoot apical meristem, flag leaf, internode, glumes, and lemma) during the seedling and reproductive stages [77] were downloaded from the wheat URGI website.
The results revealed that TaNPR1-like genes were constitutively expressed in various tissues/organs, although the level of expression varied greatly ( Figure 5). The expression level of TaNPR1-A/-B/-D was moderate and was relatively high in root and flag leaf tissues. The expression of TaNPR2-A/-D was much lower in most tissues/organs with a similar pattern as AtNPR2 in Arabidopsis tissues [78]. TaNPR3

Expression Analysis of TaNPR1-Like Genes in Response to Biotic Stress
To determine the potential functions of TaNPR1-like genes in response to biotic stress, the expression data under PAMP (chitin and flg22) treatment [77] and inoculation with F. graminearum [79] were obtained from the wheat URGI website.
In leaf tissue treated with fresh water/mock or PAMPs (chitin and flg22), the expression levels of TaNPR2 Figure 6B). Taken together, these putative candidates could be used as the preferred genes to prove their biological functions through molecular experiments in development and defense.

Identification and Phylogenic Analysis of NPR1-Like Family
In this study, we isolated five NPRs in diploid T. urartu, six in diploid Ae. tauschii, 12 in tetraploid emmer wheat (T. dicoccoides), and 17 in hexaploid wheat (T. aestivum) by genome-wide approaches. The NPR1-like family is a small community in triticeae crops similar to other plant species, such as four members in Oryza sativa [51], six in Arabidopsis [40], five in Persea Americana [79], six in Populus trichocarpa [80], three in Vitis vinifera [80], and four in Medicago truncatula [80]. The phylogenetic tree showed that the 40 identified NPR1-like genes in bread wheat and its relatives clustered into three major clades and distributed evenly among the clades (Figure 1). For example, NPR1 homologs in Ae.tauschii and T.aestivum divide into three clades and each clade contains two family members. Furthermore, the phylogenetic analysis of NPR1-like family further supports the previously evolutionary hypothesis [78]. It reveals that an ancestral gene of NPR1-like family through duplication differentiates into two main clades, NPR and BOP. Then, the progenitor NPR gene could undergo a second round of duplication event leading to the NPR1/2 and the NPR3/4 clades. Since the above-mentioned five monocots and five dicots have at least one member in each of the three clades ( Figure 1) [78], the ancient duplication events resulting in functional divergence of NPR1-like genes likely occurred prior to the monocot-dicot split. After the monocot-dicot split, members of the three clades may have experienced another round of gene duplication events, leading to the current state of each clade with two related genes, such as six NPRs (AtNPR1/AtNPR2, AtNPR3/AtNPR4, and AtBOP1/AtBOP2) in Arabidopsis and six NPRs (AetNPR1/AetNPR2, AetNPR3/AetNPR4, and AetNPR5/AetNPR6) in Ae. tauschii (Figure 1).

Sequence and Structural Features of TaNPR1-Like Genes and Proteins
The sequence and structural features of TaNPR1-like genes further support the phylogenetic analysis. NPR1 homologs on the same clade of bread wheat and Arabidopsis share similar exon-intron structures (Figure 2A). Moreover, the structural organization of NPR1-like genes in clade I and II consisted of four exons and three introns, which were also conserved in other plant species, such as rice OsNPR1, OsNPR2, and OsNPR3 [51], barley HvNPR1 [55], Theobroma cacao TcNPR1 and TcNPR3 [61], and Persea americana PaNPR1, PaNPR2, and PaNPR4 [80]. This conservation across different species showed that the NPR1-like family was also conserved in the genomic structure.
On the other hand, all of the 17 TaNPR1-like proteins harbor the BTB/POZ and ANK repeat domains ( Figure 2B). Only TaNPR1-A/-B/-D and TaNPR2-A/-D in clade I, and TaNPR3-A/-B/-D and TaNPR4-A/-B/-D in clade II, contain an NPR1-like C-terminal region. In particular, the Arg432 residues in AtNPR1, Arg428 in AtNPR3, and Arg419 in AtNPR4 required for binding SA were highly conserved in TaNPR1

Evolution and Expansion of NPR1-Like Family among Bread Wheat and Its Relatives
The gene duplication events deriving from tandem duplication, segmental duplication, and genome-wide polyploidization cause gene family expansion in plant genome evolution [81]. Chromosome distribution and collinearity analysis indicated that allopolyploid events were the main reason for the expansion of NPR1-like family in hexaploid wheat ( Figure 4). The first polyploidy event of hybridization between T. urartu and A. speltoides resulted in the NPR1-like family being duplicated in T. turgidum. Subsequently, the second polyploidy event, which crossed tetraploid emmer wheat and Ae. tauschii, led to the 17 NPR1-like sequences in bread wheat, including six, five, and six genes from the A, B, and D subgenomes, respectively.
Although the NPR1-like family was conservative among bread wheat and its relatives, intrachromosomal serial replication and gene-loss events also occurred during the evolution process. For example, replication events appeared in emmer wheat TdNPR2-A and T. urartu TuNPR5, resulting in TdNPR2-A-1/TdNPR2-A-2 and TuNPR5-1/TuNPR5-2. Gene-loss events occurred in NPR2 on chromosome 7B of bread wheat and emmer wheat, and on chromosome 7A of T. urartu, and in NPR6 on chromosome 5A of T. urartu. To find the residual sequences of the missing NPR1-like genes, putative homologous chromosomal regions were identified between emmer wheat and T. urartu using MCScanX [82]. In the collinear chromosomal region containing TdNPR2-A, we manually found a gene (TuG1812G0700000282.01) on chromosome 7AL of T. urartu (Figure 4). The protein length (396 amino acids) translated by the gene is smaller than that of TdNPR2-A (570 amino acids). Moreover, this protein also contains ANK repeats and an NPR1-like C-terminal domain similar to TdNPR2-A, but its N-terminal BTB/POZ domain is unintegrated. Furthermore, TaNPR2-A/D have very low levels of expression both in normal tissues/organs and in stress-treated samples ( Figures 5 and 6), and the rice NPR1-like family has only one member (OsNPR1) in clade I (Figure 1). It is supposed that these functionally redundant genes may have been subjected to gradual pseudogenization or gene deletion during evolution.

Functional Divergence of TaNPR1-Like Genes
The tissue/organ-specific expression patterns usually reflect their corresponding biological functions. From in silico assessment of RNA-seq experiments, TaNPR5-A/-B/-D and TaNPR6-A/-B/-D in clade III exhibited specific expression in the shoot apical meristem, internode, glume, and lemma tissues ( Figure 5), suggesting they may participate in growth and development.
The expression profiles of TaNPR1-like genes upon biotic stresses were also investigated to examine TaNPR1-like gene functions ( Figure 6). Functional receptors on the plant cell membranes recognize PAMPs, which trigger the first line of defense, called PTI. SA binds to its receptors (NPR1, NPR3, and NPR4) to induce the expression of PR genes, which contribute to trigger immune responses [5]. Under mock or PAMP (chitin and flg22) treatments, the up-regulation of TaNPR1-A/-B/-D, TaNPR3-A/-B/-D, and TaNPR4-A/-B/-D in 17 NPR1-like members was significant at 30 min between mock and PAMP treatment groups, and there was no significant difference at 180 min ( Figure 6A). Moreover, past studies have shown that overexpression of AtNPR1 or ScNPR1 in the transgenic bread wheat line enhances defense against F. graminearum [65][66][67][68]. Under mock or F. graminearum treatments, only TaNPR1-A/-B/-D was up-regulated at 30 and 50 hai ( Figure 6B). Taken together, these putative candidates could be used as the preferred genes to prove their biological functions through molecular experiments in development and defense.

Sources of Sequence Data
The genome sequences of T. aestivum (Ta), T. dicoccoides (Td), and Ae. tauschii

Identification of NPR1-Like Genes
To identify NPR1-like genes in bread wheat and its relatives (T. urartu, T. dicoccoides, and Ae. tauschii), three bioinformatics methods were executed, namely, BLASTP search, HMMER analysis, and validation of conservative domains. Firstly, a local protein database of bread wheat was established using the basic local alignment search tool (BLAST, ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/). All the Arabidopsis and rice NPR1-like protein sequences were used as queries to perform BLASTP searches against the local protein database (E-value < 1 × 10 −5 ). Secondly, the hidden markov model (HMM) profile of six AtNPRs and four OsNPRs was constructed to search the protein database using HMMER3.0 (http://hmmer.org/). After removing the redundant sequences of the above two search results, a total of 120 candidate sequences were retrieved and verified for conserved domains using the NCBI conserved domain database (CDD, https://www.ncbi.nlm.nih.gov/cdd), Pfam database(https: //pfam.xfam.org/), and InterPro database (http://www.ebi.ac.uk/interpro/). The candidate proteins without N-terminal BTB/POZ domain and ANK repeats in the central region were removed. Finally, a total of 20 putative NPR1-like protein sequences translated from 17 genes were identified from bread wheat genome. Using the same method, the NPR1-like genes of T.urartu, Ae. tauschii, and T.dicoccoides were retrieved from their genomic databases.

Analysis of NPR1-Like Gene Characteristics
Protein properties of identified NPR1-like genes, including protein length, molecular weights, and isoelectric points, were estimated using ExPASy (https://web.expasy.org/protparam/). Exon-intron structures of NPR1-like genes in bread wheat and Arabidopsis were displayed using the Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn/) [83]. NCBI-CDD was used to identify the conservative domain compositions of 17 TaNPRs and 6 AtNPRs proteins, and the results were visualized by the TBtools software [84]. Multiple sequence alignments of TaNPRs and known-function NPRs were generated using the Clustal Omega program (https://www.ebi.ac.uk/Tools/msa/clustalo/) and visualized in Jalview Version 2 [85]. The promoter sequences (1000-bp upstream of the ATG translation start codon) of TaNPR1-like genes were extracted from the bread wheat genome sequence. cis-Regulatory elements were predicted in the PlantCARE database (http://bioinformatics.psb.ugent. be/webtools/plantcare/html/) [76]. The promoter sequences are listed in Table S4.

Phylogenetic Tree Construction, Chromosomal Location, and Homologous Relationships
To investigate the phylogenetic relationship of NPR1-like genes, the full-length protein sequences were aligned using ClustalW. Subsequently, an unrooted phylogenetic tree was constructed by MEGA v7.0 using the neighbor-joining (NJ) algorithm with the following parameters: bootstrap method (1000 replicates), Poisson model, and complete deletion. Chromosomal locations of NPR1-like genes in bread wheat and its relatives were obtained from general feature format (GFF3) files. MapChart software was used to map the distribution of TaNPR1-like genes. Phylogenetic trees for the T. aestivum AB subgenome-T. dicoccoides, T. aestivum D subgenome-Ae. tauschii, and T. dicoccoides A subgenome-T. urartu NPR1-like proteins were constructed in MEGA v7.0. In the phylogenetic trees, two genes from distinct species located in the same branch were defined as orthologs [86]. The orthologous gene pairs among bread wheat and its relatives were identified based on the homologous relationships (Table S2). Subsequently, the Circos tool [87] was used to display the chromosomal locations and homologous relationships of NPR1-like family among bread wheat and its relatives.

Expression Profiles of TaNPR1-Like Genes in RNA-Seq
To study the expression profiling of TaNPR1-like genes in different tissues/organs and under stress conditions, three sets of publicly available transcriptome data were downloaded from the wheat URGI (https://wheat-urgi.versailles.inra.fr/Seq-Repository/Expression). Raw data of the three datasets was also deposited in the NCBI Short Read Archive (SRA) database under accession numbers PRJEB25639 [77], ERS1978239 [77], and ERP003465 [79].
The PRJEB25639 data were collected from various tissues/organs of spring wheat cultivar Azhurnaya, such as roots, stem axis, first leaf blade, and shoot apical meristem at the seeding stage; and flag leaf blade, internode, glumes, and lemma at the reproductive stage. The ERS1978239 data were generated from leaf samples of three-week-old Chinese Spring (CS) after fresh water/mock or PAMP (chitin and flg22) treatments for 30 min and 180 min. The ERP003465 data were obtained from mature inflorescences of the FHB-resistant spring wheat line CM-82036 after mock or F. graminearum treatments for 30 and 50 h. For all the aforementioned transcriptome datasets, three biological replicates were in each treated sample. The expression levels of TaNPR1-like genes were quantified as transcripts per kilobase million (TPM).

Conclusions
In summary, a total of 40 NPR1-like genes were identified from bread wheat and its relatives' (T. urartu, T. dicoccoides, and Ae. tauschii) genomes. The TaNPR1-like genes were analyzed in depth, comprising molecular characterization, chromosomal distributions, phylogenetic classification, gene structures, protein domain compositions, conserved motifs, and amino acid residues, as well as cis-regulatory elements. A good collinearity of NPR1-like genes was present among bread wheat and its relatives. Based on RNA-seq data, TaNPR1-like genes exhibited distinct tissue/organ specific expression patterns and TaNPR1-A/-B/-D, TaNPR3-A/-B/-D, and TaNPR4-A/-B/-D were induced under biotic stress conditions. These results will be helpful in designing experiments to determine the biological functions and understanding the evolutionary relationship of the NPR1-like gene family in bread wheat and its relatives.
Supplementary Materials: It can be found at: http://www.mdpi.com/1422-0067/20/23/5974/s1. Table S1: Information about the NPR1-like genes in various plants. Table S2: Orthologous gene pairs and chromosomal location of NPR1-like genes in bread wheat and its relatives. Table S3: The data of genome sequences used in this study. Table S4: The promoter sequences of TaNPR1-like genes. Figure S1: Analysis of NPR1-like orthologous and paralogous groups among bread wheat and its relatives. Figure S2: Physical location of TaNPR1-like genes on bread wheat chromosomes.
Funding: This study is funded by the National Key Research and Development Program of China (2016YFD0100602) and the Foundation of Shandong Province Modern Agricultural Technology System Innovation Team (SDAIT-25-02 to L.Y.).

Conflicts of Interest:
The authors declare no conflicts of interest.