Mining the Roles of Wheat (Triticum Aestivum) SnRK Gene Family in Biotic and Abiotic Responses


 BackgroundSucrose non-fermenting-1-related protein kinase (SnRK) is a class of Ser/Thr protein kinases and plays vital functions in the plant stress responses. However, little is known about the SnRK in Triticum aestivum (TaSnRK).ResultsIn this study, 149 TaSnRKs were identified from wheat and divided into three subfamilies, which may be due to the polyploidization induced gene duplication and high rate of homologous retention. A combination of public microarray datasets and quantitative real-time quantitative PCR (qRT-PCR) have further revealed the distinct expression patterns of TaSnRKs under specific abiotic/biotic stress responses. TaSnRK2.4-B, a member of SnRK2 subfamily, was located in the nucleus, cytoplasm, and cell membrane and showed ubiquitous expression in wheat life cycle, suggesting the possible response to polyethylene glycol (PEG), NaCl, heat, and cold stress, as well as the high concentrations of abscisic acid (ABA) application. Besides, transient Agro-infiltration assays showed that TaSnRK2.4-B was also involved in the resistance to pathogen.ConclusionsThese results imply that TaSnRK2.4-B may act as a multifunctional regulatory factor involved in multiple stress response pathways. Overall, our study provides new insights into the roles of TaSnRKs in biotic and abiotic responses.

genes in wheat was not consistent currently, with several genes having several synonymous names, we renamed all genes according to their subfamily (SnRK1, SnRK2, and SnRK3) and sub-genome location (A, B, or D) (Table S2). Gene's name started with an abbreviation for the acronym of species name Triticum aestivum (Ta), followed by subfamily (SnRK1, SnRK2, and SnRK3). Genes belonging to triads share the same gene number, but use su xes A, B, and D to distinguish subgenomes they located. Consecutive minuscules separated by a semicolon distinguished splice variants (e.g., TaSnRK1.3-B;a and TaSnRK1.3-B;b) [26].
Phylogenetic trees generated by SnRK protein sequences of Arabidopsis, rice, and T. aestivum showed that TaSnRKs belong to well-de ned subfamilies (Fig. 1). A previous study found that 94 proteins from 30 genes of the TaSnRK3 subfamily were identi ed [23]. This article matched all these genes except TaCIPK6 because we did not retrieve its gene or CDS (Coding Domain Sequence) sequence. The remaining 29 genes belonging to the SnRK3 subfamily were rstly identi ed in this paper ( Fig. 1; Table S2).
Chromosomal localization, gene duplication, synteny and Ka/Ks analysis TaSnRK genes are located on 21 chromosomes, except gene TaSnRK3.44-U in an unanchored contig ( Fig. 2a; Table S2). And their chromosome distributions are uneven. There are eight, seven, and eight genes on the 1A, 1B, and 1D chromosomes, respectively, and three, one, and two genes on the 7A, 7B, and 7D chromosomes, respectively. That may be related to the different sizes and structure of the chromosomes. Overall, TaSnRK genes tended to located in the more central segments of the chromosomes (R2a, R2b, and C; 73.3% of genes) were more than genes in the distal telomeric parts of the chromosomes (R1 and R3; 26.7% of genes) (Fig. 2a) [26].
Compared with the number of SnRK genes in Arabidopsis (47) and rice (38), the number in wheat was much higher (149) (Fig. 3a-c). This partially due to the hexaploid nature of wheat. However, even when corrected for ploidy-level, the number of SnRK genes in bread wheat was signi cantly higher than in Arabidopsis and rice (1.71-and 1.38-fold higher, respectively). Speci cally, genes in SnRK1 and SnRK3 subfamilies are largely more than expectation ( Fig. 3a-d). To better understand why SnRK genes are so abundant in the wheat, we analyzed homoeologous groups in detail (Table 1). At the genome-wide level, 35.8% of wheat genes were present triads [26]. By contrast, the triad proportion of TaSnRK genes is as high as 84.86%. If only "HC" SnRK genes (HC de nes genes with high con dence in the wheat genome) were considered, this ratio was even higher (85.42%, Table 1). Moreover, the loss of one homolog was less pronounced in SnRK genes (2.68% vs. 13.2%, Table 1). And only 19 genes were singletons (12.75%; 11.81% for HC only) because they could not be identi ed as groups. Therefore, the high homologous retention rate could partly explain the high number of TaSnRK genes. That is also evidence that genome-wide doubling events (WGD) contribute to the expansion of gene families. Furthermore, to investigate the gene duplication events in wheat, tandem and segmental duplication were also analyzed. Through screening sequence's similarity and matching rate, 49 paralogous pairs belonging to segmental copies were found, and there was no tandem repetition (triads were not considered) ( Fig. 2b; Table   S3). Combined with the phylogenetic tree ( Fig. 1), we found that the segment duplications occurred within each subfamily (SnRK1, 17 pairs; SnRK2, three pairs; SnRK3, 29 pairs). Additionally, synteny analysis between wheat, Arabidopsis, and rice were also compared to explore the evolutionary constraints acting on the SnRK gene family (Fig. 3e) Motif composition, exon-intron structure, protein feature and structure analysis The conserved domain and gene structure analysis provide information regarding gene duplication and their functional conservation during evolution [30]. We analyzed TaSnRKs according to the following criteria: (1) one gene of the triads was retained, (2) only the rst variant was kept, the 65 representative TaSnRKs were chosen (Fig. 4). First, we found six of 20 conserved motifs related to the functional domains in plant SnRK proteins ( Fig. 4b; Fig. S1-2,  4c). We also noted that the exon number of TaSnRKs varied from 1 to 13. In short, the difference of gene and protein structure within three subfamilies illustrates the possibility related to the existence of gene subfunctionalization or neofunctionalization in the TaSnRK family.
To further understand the features of TaSnRKs, protein properties of 186 TaSnRK proteins were analyzed (  (Fig. 5a). All the TaSnRKs belonged to hydrophilic proteins due to their grand average of hydropathicity (GRAVY) values are negative. The secondary and tertiary structure analyses revealed the prominence of helices and loops in TaSnRK proteins (> 73%) ( Fig. 5b; Table   S2). In addition, SnRK1 proteins matched models 6b1u.2 and 6c9h.1, while SnRK2s matched three different models (3zuu.1, 3ujg.1, and 5wax.1). Members of the SnRK3 were matched to ve models, two of which were shared with SnRK1s, and the remaining three were 5iso.1, 4czt.2, and 6c9d.1, respectively. Generally, protein properties were similar between the SnRK1s and SnRK3s, and SnRK2s were signi cantly different from them, which supports the functional differentiation of this gene family.

Promoter analysis
After analyzing the 1.5 kb upstream region of TaSnRK genes, we found that most of them have been broadly categorized into growth and development relate cis-acting elements. CAAT-box, a common element in promoter and enhancer regions that plays an essential role in transcription, is predominant among these genes (98.5%) [31]. Other cis-acting elements, such as A-box, AT-rich sequence, CAT-box, CCAAT-box, GCN4_motif, and O2-site, were also found in various genes and known for participating in multiple growth regulation processes ( Fig. 6a; Fig. S3, Table S5) [31][32][33]. Furthermore, many identi ed elements are related to hormone signaling pathways, namely auxin (AuxRR-core and TGA-element), abscisic acid (ABRE), gibberellin (TATC-box, GARE-motif, and P-box), salicylic acid (TCA-element and SARE), and methyl jasmonate (CGTCAmotif and TGACG-motif) [31,34]. In addition, a few elements were predicted to be involved in abiotic stresses, such as wounding (WUN-motif), cold (LTR), and light (Box 4, GATA-motif, G-Box, I-box, Sp1, and MRE) [31][32][33][34]. We also found that the number of cis-acting elements distributed in the TaSnRK genes varied from 2 (TaSnRK3.42-B and TaSnRK3.44-U) to 63 (TaSnRK3.11-A) (Fig. 6b). The different numbers and types of ciselements presenting indicated the diversi ed regulatory networks that the TaSnRK genes may involve.
Expression patterns of tissue developmental stages and stress conditions To determine TaSnRK genes' expression patterns, we analyzed 209 RNA-seq samples under nonstress conditions of 'Azhurnaya' (a hexaploid wheat variety) ( Fig. 7a; Fig. S4, showed a de cient expression with a TPM < 1 and were considered not expressed (SnRK1s, six genes; SnRK2s, three genes; SnRK2s, 26 genes). TaSnRK genes were expressed in multiple tissues, including leaves, roots, sheaths, spikelets, anthers, and grains ( Fig. 7a; Table S8), which was consistent with previous reports in Arabidopsis [4][5]35], tomato [11], potato [36], pea [37], and maize [38][39]. To verify the expression patterns in wheat, we made hierarchical clustering according to expression similarity, and then group the data into 15 different expression modules ( Fig. 7b; Fig. S4). The result showed that only four expression patterns in SnRK1, while the SnRK3 contained the most types of expression patterns (up to 12). Moreover, the expression modules I and V were the most widely distributed, with multiple gene anastomoses in all three subfamilies ( Fig. 7b-c). In general, the genes of TaSnRK1s and TaSnRK2s were both ubiquitously expressed during the plant life cycle (Fig. 7c). In addition to being generally expressed (39/127, 30.7%), the TaSnRK3 genes were also explicitly highly expressed in reproductive organ spikelets (modules XIII (66/127, 60.0%) and XIV (2/127, 1.6%)). Interestingly, not or low-expressed genes (module VIII) were also concentrated in the SnRK3 subfamily (20/127, 15.7%). That may be related to a large number of genes and functional redundancy in this subfamily. The multi-tissue and multi-stage expression suggest the TaSnRK gene family's critical role in wheat growth and development.
To verify the response pattern of TaSnRKs to abiotic and biotic stresses in wheat, nine genes belonging to different subfamilies with universally expression patterns were randomly selected. Their expression patterns under various stresses were further illustrated by heatmap and analyzed by qRT-PCR.
At low temperature, TaSnRK2.4-B' expression was decreased (Fig. 8a), which was similar to the expression pattern of ZmSnRK2.10 in maize [40]. In the SnRK3 subfamily, two genes were responsive to cold stress. One is TaSnRK3.35-A, whose expression was notably increased, may form a complex with CBL1 to regulate cold stress like homologous gene AtSnRK3.10 [41]. Another is TaSnRK3.16-D, which was drastically downregulated than control, implying that TaSnRK3 genes participate in cold response in various ways. Also, TaSnRK1.1-A, TaSnRK2.4-B, and TaSnRK2.7-A responded to phosphorus de ciency only in the root. Under drought treatment (Fig. 8a), the expression of TaSnRK2.4-B, TaSnRK2.7-A, and TaSnRK3.35-A were increased signi cantly. In heat stress, except for TaSnRK3.37-D, the remaining eight genes all reached a peak at six hours. When subjected dual stress of drought and heat, TaSnRK2.7-A and TaSnRK3.37-D showed a gradual increase, and the remaining seven genes were down-regulated rst and then up-regulated. In the qRT-PCR result ( Fig. 8b), these nine genes also respond to drought stress. Under osmotic stress, the expression levels For biotic treatment, the response patterns of the nine genes were also varied (Fig. 8a)

Gene Ontology (GO), Kyoto Encyclopedia of Gene and Genome (KEGG) and Protein-protein interaction(PPI)network Analysis
To understand the functions of the biological processes, all TaSnRKs were searched for GO and KEGG databases. In total, 86.5% (129/149) TaSnRKs were assigned to one or more GO terms in the biological process (129 genes), molecular function (129 genes), and cellular component (102 genes) categories Table S10). We also mapped the TaSnRKs in the KEGG pathway database (Fig. 9b). The result revealed that most genes were enriched in signal transduction of environmental information processing pathways, supporting the previous studies [1][2][4][5]. By grouping comparison, we found that the members from the TaSnRK1 and the TaSnRK3 subfamilies may perform similar functions on plant physiological regulation (Fig. S6). The reason is they shared eight identical pathways, except the pathways of "Folding, sorting and degradation" and "Membrane transport", which were only annotated with the SnRK3 subfamily. By contrast, the TaSnRK2 members were only enriched on the "signal transduction pathway" of environmental information processing. That signals functional differentiation among the TaSnRK subfamilies and supports the SnRK genes regulation network drawn by Cramer et al. [42].
Furthermore, we used the model plant Arabidopsis protein database as a reference to constructed the interaction network of the three TaSnRK subfamilies, respectively. At rst, 26 protein pairs were predicted with con dence to interact (score > 0.47) between 21 TaSnRK1s and 12 other proteins in the SnRK1 subfamily ( Fig. 9c; Table S11). Among these protein pairs, AT4G16360, AKINBETA1, SEX4, and SNF4 code subunit β, and SNF4 code subunit γ, together with subunits α to form TaSnRK1 heterotrimers (Table S11) [7,10]. And, TaSnRK1s maybe regulate sugar signals and control plant energy transfer through interacting with B3domain transcription factor FUSCA3 (FUS3), which have been veri ed by experiments in Arabidopsis [35,43]. Simultaneously, TaSnRK1s also bind to a myristoylated 2C-type protein phosphatase ABI1, which contributes to balance carbon and nitrogen and ABA-free signaling pathways [44]. Another PP2C protein that binds TaSnRK1s is PP2C74, which involved the early development in the plant as one of the substrates of AtNMT1 in Arabidopsis [45]. Secondly, 52 protein pairs associated with TaSnRK2s were identi ed. Of these, four pairs occurred within the TaSnRK2 subfamily, and 48 pairs were associated with 38 TaSnRK2s and other ve functional proteins ( Fig. 9c; Table S11). TaSnRK2s and their interaction proteins are all concentrated in the PYR/PRL-PP2C-SNRK-ABF signaling pathway [8, 13-15, 22, 24-25, 35, 43-44]. When ABA content is low, the activities of SnRK2s were inhibited by clade A PP2C phosphatases (ABI1, ABI2, and HAB1) [46][47][48]. After the concentration increases, ABA is bound to receptor PYR/PRL and PP2C phosphatases to form regulating complexes and release the inhibition of SnRK2s and downstream stress signaling [46][47][48]. Meanwhile, uninhibited SnRK2s [49][50][51] activate downstream bZIP-like transcription factor (ABF), induces ABA response gene expression, and regulates plant growth and development [52]. Ultimately, the signal transduction process is inhibited by SnRK-Calcium-binging Sensor (OZS1) and scaffold protein ABA Terminator (Fig. 9c,   10a) [53][54]. Finally, the SnRK3 subfamily has been shown to participate in complex networks of interactions. We identi ed 135 protein pairs involving 24 SnRK3 proteins and ten other proteins. Among them,  (Fig. 9b) [59]. To investigate whether TaSnRK2.4-B indeed responds to ABA signaling, we quanti ed the expression levels of several key genes involved in the ABA pathways concerning to SnRK2 subfamily ( Fig. 10a; Fig. S7) [8,22,24,44,60]. On the one hand, TaPYR1 (homologous to AtPYR1) was weakly expressed after ABA treatment, and TaPYR4 (homologous to AtPYR4) and TaPP2C (homologous to AtPP2CA and AtABI2) was continuously decreased with stress time. Moreover, TaSnRK2.4-B and TaABF (homologous to AtABF) both increased and reached a peak at 12 h and then decreased continuously. On the other hand, in the SCS-SnRK2 signaling pathway [60], TaSCS (homologous to AtSCS-A and AtSCS-B) was highly expressed at all time points, especially at 12 h, whose expression level was 731 times compared to control. Furthermore, its expression trend is utterly consistent with TaSnRK2.4-B. That suggests that ABA signaling may cause an outbreak of ROS (Reactive Oxygen Species), which induce SCS overexpression in a calcium-or non-calciumdependent manner [60]. After that, TaSCS may inhibit the expression of TaSnRK2.4-B by directly interacting with it, as similarly reported in Arabidopsis [60].
To validate the subcellular localization of TaSnRK2.4-B, we successfully constructed the part27-35S:TaSnRK2.4-B-GFP fusion expression vector into Agrobacterium GV3101 and transferred it to tobacco leaves. The results showed that TaSnRK2.4-B was located in the cell membrane, cytoplasm, and nucleus (Fig. 10b), which was consistent with the localization results of its homologous gene TaSnRKs [22,60].
In this study, we have demonstrated that TaSnRK2.4-B responds to many biotic stresses, and we wanted to examine whether this response exists in Phytophthora infestans (P. infestans) challenge. Therefore, the mature protein-coding regions of TaSnRK2.4-B were cloned and transiently expressed in N. benthamiana using Agrobacterium-mediated expression followed by a P. infestans infection. After six days post inoculation (dpi), signi cantly smaller lesions were observed in areas expressing TaSnRK2.4-B compared to that of free GFP (Fig. 10c-d; P < 0.05), suggesting that the expression of TaSnRK2.4-B inhibited P. infestans invasion to the host. This inhibition is likely to enhance plants' ability to resist stress by responding to signal transduction pathways of hormones such as salicylic acid or gibberellin [61][62]. However, the speci c mechanism of action still needs further experimental investigation.

Discussion
SnRK family was highly conserved in eukaryotes, working as a sensor in the cellular energy metabolism [10-12, 35, 43, 62-63]. PKABA1, the primary member of the wheat SnRK family, was discovered and cloned from ABA-induced wheat embryo cDNA [19]. After that, ve PKABA1-like protein kinase genes (TaPK3, W55a, W55b, W55c, and TaSRK2C1), activated by multiple stresses related to ABA signals, were excavated from wheat [21,[64][65][66]. However, compared with Arabidopsis and rice, the research on SnRKs in wheat is limited. So far, there has been no genome-wide analysis of the SnRK family in wheat. We have thereby developed a comprehensively evolutionary analysis and functional classi cation of the three SnRK subfamilies in wheat.
Based on the BLAST and HMM algorithm, 149 non-redundant SnRK genes were identi ed at the whole genome level of wheat ( Fig. 1; Table S2). The number of TaSnRKs was 3.92 and 3.17 times higher than in Arabidopsis and rice, which could generally be explained by its hexaploidy and fragment replication (Fig. 3ad). The Ka/Ks ratio test explains that the wheat SnRK family has experienced positive selection, but displayed different evolutionary branch selection pressures ( Fig. 3f; Table S2). Meanwhile, we calculated that 77.9% (116/149) SnRK members had collinear relationships with rice, and these pairs were all distributed in the three subfamilies ( Fig. 3e; Table S4). More interestingly, the same TaSnRK gene co-linear with multiple rice genes and vice versa. However, only 10.1% (15/149) of the SnRK members had collinearity pairs with Arabidopsis ( Fig. 3e; Table S4). In general, the rice SnRK genes showed a more intimate genetic relationship with wheat than Arabidopsis, consistent with the evolution tendency. Three branches of the SnRKs existent in Arabidopsis and rice (Fig. 3f), re ecting that the evolution of SnRK2 and SnRK3 from SnRK1 has already been completed before the arose of monocot and eudicot plants.
The protein property and structure of the wheat SnRK family were analyzed concerning the interrelationship with the protein function. It was found in our study that the N-terminus of the SnRK protein was more conservative in each subfamily in the full-length sequence alignment ( Fig. 4; Fig. S2). By contrast, TaSnRKs shared a lower similarity in C-terminal sequences among the three subfamilies. The C-terminal of SnRK sequences generally includes KA1, SnRK2-speci c box, and NAF domain [7][8][9], which is common in Arabidopsis [6,10,24], Oryza sativa [24], Zea mays [40], and Brachypodium distachyon [25]. However in wheat, motif 17 with the KA1 domain is distributed only in the SnRK1 subfamily, and motif 8 containing the NAF domain is distributed in the SnRK3 subfamily ( Fig. 4; Fig. S2, Table S5). Motif 15 containing the SnRK2speci c box is conserved in the TaSnRK2 family and distributed in each member sequence. This result indicated that the wheat SnRK protein sequences were conserved in each subfamily. The tertiary structure prediction result further revealed the same subfamily members share similar protein models, suggesting that members of the same subfamily may also share similar functions (Fig. 5b). Nevertheless, the differences between the TaSnRK subfamilies cannot be ignored, which may relate to one or more domains at the Cterminal end.
At present, most of the SnRK proteins reported are found to be located in the cell membrane, cytoplasm, and nucleus as the regulation of SnRK kinase activity at different levels of transcription, post-transcription, protein translation, and post-translational modi cation [3,10,19,46,48,52,55,58,64]. For example, wheat SnRK2 member PKABA1 was not regulated by ABA in the protein level but was regulated by ABA in the mRNA leve1 [19]. This multilevel regulatory ability has determined that the SnRK family plays a vital role in the plant regulatory network as an essential protein kinase [4,[6][7]43]. For a better understanding of the plant SnRK families, we drew a schematic diagram of the regulatory network of plant SnRK family (Fig. 10e) [1,6,13,16,52], but also the stomatal closure [6,13], abiotic stress [6,67], growth and development regulation [11,[35][36] are involved.
In this research, we have identi ed multiple cis-acting elements that responded to wheat growth and development regulation, as well as the hormone and stress response in the promoter regions of the three TaSnRK subfamily members (Fig. 6). Simultaneously, TaSnRKs were annotated to the three types of GO items and enriched to multiple KEGG pathways ( Fig. 9a-b). The nine genes focused on were ubiquitously expressed during the wheat life cycle, except for TaSnRK3.16-D, whose expression level was low in general, but relatively higher in the reproductive organs. Overall, the TaSnRKs may play a role in various aspects of vegetative growth and reproductive development, and is not just limited to regulating root development as previously reported [22,59].
For biotic stress, all nine genes responded to the infection of Zymoseptoria triticipowdery and stripe rust, according to public microarray datasets. In the powdery mildew and Fusarium graminearum treatment, there were discrepancies showed between the public data and our qRT-PCR results, which may be possibly due to the difference in experimental materials. But generally, some members of the three TaSnRK subfamilies responded to the invasion of powdery mildew and Fusarium graminearum, which was consistent with the former reports to a certain extent [21,62]. Still, further studies are needed for the veri cation.
Under the abiotic stress, TaSnRK2.4-B, TaSnRK3.16-D, and TaSnRK3.35-A both demonstrated the responses to cold stress. Except for TaSnRK3.37-D, the remaining eight genes were induced to express after high temperature treatment for 1 hour (Fig. 8a). Besides, these nine genes all responded to PEG, NaCl, ABA (100 uM), and phosphorus de ciency variously. In previous studies, the SnRK2 family was divided into three groups mainly based on the gene responding pattern to ABA signals (Fig. S8) [22,24,48,59,67]. SnRK2s in Group I cannot activated by ABA treatment and Group II SnRK2s responded weakly to ABA, whereas SnRK2s in Group III were strongly induced by the ABA signal. In 2011, Zhang et al. [59] identi ed a Group I gene, TaSnRK2.7 (renamed as TaSnRK2.4-D), that did not respond to ABA (5 uM). However, in our study, it was found the TaSnRK2.4-B, a triad gene of TaSnRK2.7, responded to ABA (100 uM) signal signi cantly (Fig. 8b). By adjusting the ABA application concentration in the experiment, we concluded that the Group I genes could be more sensitive to high concentration of ABA. Also, from the signaling pathway, TaSCS were signi cantly induced after the ABA application. We speculated that it might interact with and inhibit the expression of TaSnRK2.4-B through Ca 2+ dependent or independent means (Fig. 10e) [60]. Furthermore, TaSnRK2.4-B was found to be located in both the cell nucleus, cytoplasm, and membrane, and its overexpression in tobacco has enhanced tolerance to P. infestans invasion (Fig. 10c-d). Consequently, it could be reasoned that TaSnRK2.4-B has the very potential to improve the crop tolerance to stresses, underlying a practical breeding utility in the wheat production.

Phylogenetic analyses of the SnRK gene family
To further explore the evolutionary relationship of plant SnRKs, multiple sequence alignments and phylogenetic analysis of the SnRK proteins were performed with Clustalw [68]. And neighbor-Joining (NJ) was used to perform phylogenetic analyses of the SnRK proteins with 1000 replicated-bootstraps in MEGA 7.0 [69][70]. The midpoint rooted base tree was drawn and cleaned up via Interactive Tree of Life (ITOL, version 3.2.317, http://itol.embl.de).
GO annotation, KEGG annotation, and PPI network prediction GO annotations of TaSnRK proteins was performed by Blast2GO [74]. KEGG pathways analysis of TaSnRKs was performed using the KEGG web server (http://www.kegg.jp/). The PPI network was constructed using the STRING database (https://string-db.org/), and the genes with con dence score ≥ 0.4 were reserved.
Plant materials, treatments, and qRT-PCR analysis Seeds of Yangmai 20, a hexaploid common wheat cultivar, were surface-sterilized with 1% hydrogen peroxide, rinsed thoroughly with distilled water, and germinated in an incubator at 20 ℃ for two days then placed in 1/2 strength Hoagland nutrient solution [75]. When wheat grew to one heart and one leaf, the plants were treated with 150 mM sodium chloride (NaCl), 20% polyethylene glycol 6000 (PEG 6000), and 100 uM ABA. photoperiod. When wheat seedlings'coleoptiles reached 0.5 cm, they were point inoculated with either water (control) or Fusarium graminearum (strain PH-1). Plant growth, inoculation and infection conditions were described in detail previously [76]. The Blumeria graminis f. sp. Tritici (Bgt strain E09) was maintained on susceptible wheat 'MingXian 169' [77]. The 7-day-old seedlings were inoculated with Bgt conidia from 'MingXian 169' seedlings infected 10 days previously. The inoculated leaves of Yangmai 20 were harvested at one, three and ve dpi, frozen immediately in liquid nitrogen and stored at −80 °C. The test was carried out with three biological replications.
Total RNA was isolated from 100 mg tissues by using TRizol reagent (Invitrogen, U.S.A) and digested with DNaseI Agro-in ltration experiments were implemented on leaves of 6-to 8-week-old N. benthamiana. After 24 h, in ltrated N. benthamiana leaves were excised and transferred into sealed moist plastic trays and inoculated with 10 µl P. infestans strains (88069) zoospore suspension (100 zoospores/µl) at the in ltration range [78]. Photographing under ultraviolet light and recording lesion diameters of the inoculated leaves at six dpi. Assay consisted of three biological replications, and each biological replication contained ten technical duplications.   Table S3. There are no tandem duplication pairs predicted in TaSnRKs.   Table S2.   Table S9.  TaSnRKs annotation. KEGG terms were summarized in ve main categories, pink for genetic information processing, azure for environmental information processing, green for cellular processes, light gray for organismal systems, and light orange for human diseases. (c): Interaction network of AtSnRK proteins and related proteins. From left to right are subfamilies SnRK1, SnRK2, SnRK3. The line's colors indicated the relationship of the interaction between the two proteins, of which sky blue and rose red indicated known interactions from curated databases and experimentally determined respectively. The blue line indicated predicted interactions from gene co-occurrence. Other interactions, like textmining, co-expression, and protein homology, indicated by peak green, black, and bluish violet line, respectively.  osmotic stress have previously been reported in plant species [1,4,[6][7][10][11][24][25]42,[46][47]54,60]. Connections represent positive (arrow) and negative (block) regulation.