The Calcium-Dependent Protein Kinase TaCDPK7 Positively Regulates Wheat Resistance to Puccinia striiformis f. sp. tritici

Ca2+ plays a crucial role as a secondary messenger in plant development and response to abiotic/biotic stressors. Calcium-dependent protein kinases (CDPKs/CPKs) are essential Ca2+ sensors that can convert Ca2+ signals into downstream phosphorylation signals. However, there is limited research on the function of CDPKs in the context of wheat–Puccinia striiformis f. sp. tritici (Pst) interaction. In this study, we aimed to address this gap by identifying putative CDPK genes from the wheat reference genome and organizing them into four phylogenetic clusters (I-IV). To investigate the expression patterns of the TaCDPK family during the wheat–Pst interaction, we analyzed time series RNA-seq data and further validated the results through qRT-PCR assays. Among the TaCDPK genes, TaCDPK7 exhibited a significant induction during the wheat–Pst interaction, suggesting that it has a potential role in wheat resistance to Pst. To gain further insights into the function of TaCDPK7, we employed virus-induced gene silencing (VIGS) to knock down its expression which resulted in impaired wheat resistance to Pst, accompanied by decreased accumulation of hydrogen peroxide (H2O2), increased fungal biomass ratio, reduced expression of defense-related genes, and enhanced pathogen hyphal growth. These findings collectively suggest that TaCDPK7 plays an important role in wheat resistance to Pst. In summary, this study expands our understanding of wheat CDPKs and provides novel insights into their involvement in the wheat–Pst interaction.


Introduction
Calcium ions (Ca 2+ ) are important cellular ionic species that function as a second messenger in signaling transduction pathways when plants are encountered with a variety of developmental and environmental stimuli [1].In response to these stimuli, the cytosolic free Ca 2+ levels rise drastically [2], and these changes are sensed by Ca 2+ sensors.The Ca 2+ sensor proteins are divided into two categories: those that relay calcium signals, such as calcineurin B-like/CBL-interacting protein kinases (CBL/CIPKs) and calmodulin proteins (CaMs), while the others function as sensor protein kinases, including calcium-dependent protein kinases (CDPKs/CPKs) and calmodulin-dependent protein kinases (CaMKs) [3].In contrast to non-catalytic relay sensors like CaMs and CBLs, which transduce calcium signals to target proteins, CDPKs have the unique ability to directly communicate Ca 2+ signals.They achieve this by binding calcium to the EF hands located at their C-terminus and phosphorylating substrates through the catalytic kinase domain at their N-terminus.
Typical CDPKs have an N-terminal domain followed by a protein kinase domain, an autoinhibitory domain, and a C-terminal regulatory calmodulin-like domain that contains one to four EF-hand motifs responsible for binding Ca 2+ [4].Additionally, CDPKs possess myristylation and palmitoylation sites at the beginning of their N-terminal domains, which help them attach to or detach from membranes.The N-terminal domains of CDPKs are highly variable in length and amino acid content, contributing to their diverse functional roles [5].The protein kinase domain, located immediately upstream of the auto-inhibitory domain, comprises a catalytic Ser/Thr protein kinase domain with an ATP binding site.In the absence of Ca 2+ , the auto-inhibitory domain acts as a pseudo substrate of the kinase domain and interacts with its active sites to keep CDPKs inactive [6].Upon Ca 2+ binding, CDPKs undergo a conformational change that disrupts the inhibitory interaction between the auto-inhibitory domain and the active site, leading to the activation of the kinase domain.In addition to their significant roles in developmental processes such as pollen tube formation, root expansion, stem elongation, and cell division/differentiation, CDPKs and related kinases (CRKs) are also actively involved in responding to biotic/abiotic stresses and phytohormone-mediated signaling pathways [7,8].For instance, the AtCPK10 protein participates in Ca 2+ -and ABA-mediated stomatal movements during drought stress in Arabidopsis [9], while AtCPK1 is associated with the accumulation and constitutive biosynthesis of salicylic acid (SA) and regulates defense through modulation of disease resistance gene expression [10].
CDPKs play a crucial role in plant defense responses against pathogens, encompassing oxidative burst, alterations in phytohormone synthesis and signaling, and modulation of gene expression [11].They exhibit both positive and negative regulatory effects on plant defense mechanisms [12].In wheat, TaCPK2-A is required for resistance to powdery mildew, while its overexpression in rice enhances resistance to bacterial blight (caused by Xanthomonas oryzae pv.oryzae, Xoo) by modulating the expression of the transcription factor WRKY45-1 [13].In a recent study, silencing of TaCDPK27 improves wheat resistance to powdery mildew infection [14].Overexpression of AtCPK1 in Arabidopsis confers broadspectrum defense against fungi and bacteria [10].Moreover, overexpression of OsCPK4 in rice enhances resistance to blast disease caused by Magnaporthe oryzae [15].The CDPK2 and CDPK3 in Nicotiana species have been shown to play essential roles in the initiation of programmed cell death (PCD) triggered by the Avr4 or Avr9 elicitors, which are specific to particular races of Cladosporium fulvum [16].Interestingly, the stable expression of StCPK5-VK in transgenic potatoes resulted in enhanced resistance against the hemi-biotrophic oomycete Phytophthora infestans, while it led to reduced resistance against the necrotrophic fungus Alternaria solani [17].Recent research has identified Arabidopsis CPK3 as a key regulator in both pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) and effectors-triggered immunity (ETI) [18].Arabidopsis CPK4, 5, 6, and 11 collectively and redundantly contribute to PAMP-induced resistance against Pseudomonas syringae [19].On the other hand, CDPKs can also exhibit negative regulatory effects during pathogen interactions.For instance, overexpression of the OsCPK12 in rice enhances sensitivity to both avirulent and virulent strains of M. oryzae [7], while overexpression of an auto-active variant of HvCDPK3 in barley leads to increased susceptibility to powdery mildew [20].
CDPKs are plant-specific proteins that have been widely identified from algae to flowering plants [4].Despite extensive studies on CDPKs in other plants, our understanding about CDPKs in bread wheat, the third-largest cultivated crop in the world remains limited [21,22].Earlier, twenty wheat CDPK genes were identified when the whole genome sequence of wheat was not available [22].However, after the complete sequencing of the hexaploidy wheat genome in 2018 [23], a comprehensive investigation of CDPKs in wheat was needed.
Stripe rust, caused by the fungus Puccinia striiformis f. sp.tritici (Pst), is the most devastating fungal disease of wheat, leading to yield losses of up to 50% [24].The control of stripe rust in wheat involves various strategies, including chemical and biological methods.The most cost-effective and environmentally friendly approach is to utilize resistant genes.
However, the rapid evolution of Pst, and emergence of new races can breakdown the resistance provided by race-specific resistant gene(s) in wheat.For example, recently emerged Pst races, TSA-6 [25], and TRVR20-5 [26], have shown virulence to the wheat Yr5 gene, which has been known to convey resistance to Pst races worldwide and has been utilized in the international wheat breeding programs focused on strip rust resistance.Consequently, it is of utmost importance to investigate and discover novel genes that exhibit resistance to Pst.Notably, no TaCDPK genes have been reported to be involved in wheat resistance against stripe rust so far.Therefore, there is a need to investigate the functional roles of wheat CDPKs in combating this notorious pathogen.
In this study, we conducted a comprehensive genome-wide analysis of CDPK genes in the bread wheat reference genome, resulting in the identification of 79 putative wheat CDPK genes, which were categorized into four clusters based on phylogenetic analysis.We examined the synteny, analyzed gene structure, motif organization, and exon-intron composition.Expression analyses revealed that Pst infection regulated the expression of several of these 79 wheat genes.Furthermore, functional characterization using VIGS indicated a positive role of TaCDPK7 in the wheat-Pst interaction, possibly by inhibiting the expression of ROS-scavenging genes while promoting the expression of PR genes.Our study lays the basis for further characterization of the function of CDPKs in wheat during stress responses and enhances our understanding of the role of CDPKs in plant defense against pathogens.

Mining of CDPK Members from Wheat Genome
To identify wheat CDPKs, we used the Arabidopsis and rice CDPK protein sequences as queries to search against the wheat genome database at http://plants.ensembl.org/(accessed on 6 September 2022) using the Local BLASTP program available.After eliminating sequence redundancies, we obtained a total of 79 putative TaCDPKs that contain the corresponding signature domain (SMART acc.No. SM000220 and InterPro acc.No. IPR000719).The length of the coding sequences (CDS) of TaCDPKs range from 1384 bp to 1885 bp.
Since rice is considered a close relative of wheat in the classification system, the 79 identified genes were named in ascending order according to their phylogenetic relationships with the rice CDPK family [27] (Table S1).All the identified TaCDPKs are located on the wheat chromosomes 1 to 7, except for TaCDPK4UN, which is located at an unknown locus.Among them, 24 genes are located on the 5th chromosome, followed by 17 genes on the 2nd chromosome, 11 genes on the 4th chromosome, 9 genes each on the 1st and 3rd chromosomes, 5 genes on the 6th chromosome, and 3 genes on the 7th chromosome.Therefore, chromosome 5 has the maximum number of TaCDPKs.

Phylogeny and Computational Characterization of Wheat CDPKs
To determine the evolutionary relationship of CDPKs in wheat, rice, and Arabidopsis, a phylogenetic tree was constructed using full-length protein sequences.Similar to rice and Arabidopsis, the wheat CDPKs were grouped into four clusters in the phylogenetic tree.Among these clusters, cluster I was the largest, containing 47 members, with 26 from wheat, 11 from rice, and 10 from Arabidopsis (Figure 1).Second largest is cluster II having 43 members in total, and cluster III contained 42 members.Cluster IV is the smallest one, comprising 10 members, 5 from wheat, 2 from rice, and 3 from Arabidopsis.Notably, all the wheat CDPK genes have complete triplet copies of their sub-genomes (BB, AA, DD) except for two genes, TaCDPK18 from cluster IV and TaCDPK27 from cluster I, which were found to lack their B and A sub-genome homologous triplets' copies, respectively.The phylogenetic analysis revealed that the wheat CDPK genes displayed closer relationships with the CDPK genes in rice (monocot) compared to those in Arabidopsis (dicot).In light of this, the naming of TaCDPK proteins was assigned based on their homologies with rice CDPKs rather than Arabidopsis.Furthermore, we investigated the putative orthologous similarity index between wheat and rice CDPKs based on the phylogenetic tree.The highest similarity index, 95%, was observed between TaCDPK16D and OsCDPK16, while the lowest index, 65%, was found between TaCDPK22D and OsCDPK22.Notably, nineteen members of the TaCDPK family exhibited a similarity index above 90% (Table S2).
except for two genes, TaCDPK18 from cluster IV and TaCDPK27 from cluster I, which were found to lack their B and A sub-genome homologous triplets' copies, respectively.The phylogenetic analysis revealed that the wheat CDPK genes displayed closer relationships with the CDPK genes in rice (monocot) compared to those in Arabidopsis (dicot).In light of this, the naming of TaCDPK proteins was assigned based on their homologies with rice CDPKs rather than Arabidopsis.Furthermore, we investigated the putative orthologous similarity index between wheat and rice CDPKs based on the phylogenetic tree.The highest similarity index, 95%, was observed between TaCDPK16D and OsCDPK16, while the lowest index, 65%, was found between TaCDPK22D and OsCDPK22.Notably, nineteen members of the TaCDPK family exhibited a similarity index above 90% (Table S2).To examine the gene structures of TaCDPKs, we analyzed the exon-intron structures by comparing their cDNA sequences with the corresponding genomic sequences.The number of exons in TaCDPKs ranged from three to twelve per gene, while the introns varied from two to eleven per gene (Figure S1).We used the TBtools kit to identify gene duplication events (Figure S2).Tandem duplication is the likely cause of having two or more homologous genes on the same chromosome within a 200 kb region [28].Since To examine the gene structures of TaCDPKs, we analyzed the exon-intron structures by comparing their cDNA sequences with the corresponding genomic sequences.The number of exons in TaCDPKs ranged from three to twelve per gene, while the introns varied from two to eleven per gene (Figure S1).We used the TBtools kit to identify gene duplication events (Figure S2).Tandem duplication is the likely cause of having two or more homologous genes on the same chromosome within a 200 kb region [28].Since chromosome 5 harbors the maximum number of TaCDPKs, it is likely that tandem duplications have occurred on this chromosome.Four possible tandem duplication events were identified for eight TaCDPK genes on chromosome 5 (Table S5), and five out of these eight genes belonged to cluster I. Furthermore, a considerable number of TaCDPKs seemed to have arisen from segmental duplication events (Figure S2), suggesting that both tandem and segmental duplications played a role in the expansion of the wheat CDPK family.In conclusion, our findings indicate that a significant number of TaCDPK genes may have undergone duplication events during evolution, shedding light on the evolutionary and functional potential of TaCDPK genes.
To gain further insights into the expression regulation patterns of wheat CDPK genes, we conducted in silico investigations of the possible cis-elements present in the promoters of each TaCDPK family member.The analysis revealed a diverse range of cis-elements in the promoters of TaCDPKs, including plant hormone stimulus-responsive elements (such as P-box, TGACG motif, ABRE, TCA element, CGTCA motif, and GARE motif) and biotic/abiotic stress response elements (such as MBS, TC-rich repeats, Circadian, and LTR) (Figure S3).Additionally, based on the predicted functions of all TaCDPKs, the ciselements were categorized into major groups, comprising hormone-responsive cis-elements, followed by stress-responsive cis-elements, with other categories represented to a lesser extent (Figure S4).The identification of a substantial number of cis-elements associated with hormone and stress responsiveness highlights the signaling role of the wheat CDPK gene family in response to hormonal stimuli and abiotic/biotic stress conditions.Domains and motifs play crucial roles in protein interactions, transcriptional activity, and DNA binding for transcription factors [29].In our study, we utilized the MEME suite to identify conserved motifs within the TaCDPK proteins.Among the identified motifs, motifs 1 and 2 corresponded to the protein kinase domain (IPR000719) were highly conserved across all members of the TaCDPK family (Figure S5).Additionally, motifs 5, 7, and 9, which corresponded to the EF-hand domain (IPR002048) and EF-hand domain pair (IPR011992), were also observed throughout the entire family.These conserved motifs provide further evidence of the functional importance of protein kinase and EF-hand domains in the TaCDPK family.

Expression Patterns of TaCDPKs during Wheat-Pst Interaction
To investigate the expression patterns of TaCDPKs during the wheat-Pst interaction, we analyzed their transcript levels using RNA-seq data obtained from wheat samples at 0, 18, 24, 48, 96, and 168 hpi (hours post inoculation).TaCDPK2A, TaCDPK3B, TaCDPK7D, TaCDPK9D, TaCDPK11B, TaCDPK14A,B,D, TaCDPK20A,B,D, TaCDPK21B,D, TaCDPK27D, and TaCDPK29A,B,D have been neglected due to their very low expression values (fragments per kilobase of exon per million mapped reads, FPKM < 0.1 or 0).Among the 79 members of TaCDPK family, TaCDPK5A, TaCDPK7A, TaCDPK7B, TaCDPK25A, TaCDPK25B, TaCDPK26A, TaCDPK26B, and TaCDPK27B showed higher FPKM value in most of the examined time points in the incompatible set of interaction (32R) compared to the compatible interaction (32S), suggesting their potential role in wheat resistance against Pst infection.Conversely, six members, TaCDPK3A, TaCDPK3D, TaCDPK11A, TaCDPK22A, TaCDPK22B, and TaCDPK22D from cluster I and cluster III, displayed the strongest induction during the compatible interaction, but were consistently downregulated at all examined time points during the incompatible interaction) (Figure 2), suggesting their possible negative roles in wheat defense against Pst.

Transcript Profile of TaCDPKs Challenged with Pst (qRT-PCR Analysis)
To validate the RNA-seq data by qRT-PCR analysis, fourteen members of the TaCDPK family have been selected based on their expression level during compatible or incompatible interactions.These selected members are TaCDPK3A, TaCDPK3D, TaCDPK5A, TaCDPK7A, TaCDPK7B, TaCDPK11A, TaCDPK22A, TaCDPK22B, TaCDPK22D, TaCDPK25A, TaCDPK25B, TaCDPK26A, TaCDPK26B, and TaCDPK27B.Due to the high sequence similarity among homologous genes from different sub-genomes, such as TaCDPK3A/TaCDPK3D (97.27%),TaCDPK7A/TaCDPK7B (92.35%),TaCDPK22A/TaCDPK22B/TaCDPK22D (95.00%),TaCDPK25A/TaCDPK25B (98.25%), and TaCDPK26A/TaCDPK26B (97.72%), we performed qRT-PCR analysis using one representative gene copy from each pair.TaCDPK3, member of cluster III, showed strong significant upregulation at the middle stage 12 hpi of the compatible interaction (CYR31) compared with the mock control, as indicated in the RNA-seq data (Figure 2).Similarly, TaCDPK5 from cluster I exhibited higher upregulation at 18 hpi during the compatible interaction (Figure 3), which is consistent with the RNA-seq data showing upregulation at the middle stage (24 hpi) of the compatible interaction (Figure 2).TaCDPK11 displayed peak expression at middle stages (12 and 18 hpi) during the incompatible interaction (CYR23), followed by decreased expression in both compatible and incompatible interactions.Transcript levels of TaCDPK22 showed significant upregulation at 9 and 12 hpi during the compatible interaction (Figure 3), which aligned with the RNA-seq data showing strong induction of TaCDPK22 expression during all the time points of compatible set.Similar to the RNA-seq data, the mRNA accumulation of TaCDPK25 showed significant upregulation at the middle stages (12 and 18 hpi) under the incompatible interaction (CYR23) (Figure 3), suggesting a positive role of TaCDPK25 in wheat resistance against Pst.TaCDPK26 displayed significantly higher expression during the compatible interaction at 9 and 12 hpi compared to the control (Figure 3).TaCDPK27, consistent with RNA-seq data showing strong expression during the compatible set, also showed significant upregulation at 6, 9, and 12 hpi during the compatible interaction (CYR31) (Figure 3) in qRT-PCR analysis, suggesting its possible role in wheat susceptibility to Pst.TaCDPK7 exhibited strong induction at most of the time points (3, 6, 9, 12, 18 hpi) in both compatible and incompatible interactions (Figure 3).Both the RNA-seq and qRT-PCR results suggest that TaCDPK7 may play a potential role in wheat-Pst interaction, either positively or negatively.Therefore, TaCDPK7 was selected for further investigation of its functional role in wheat defense against Pst.Overall, our results indicate the reliability of the RNA-seq data, and highlight the regulation of several TaCDPKs during the wheat-Pst interaction.

Transcript Profile of TaCDPKs Challenged with Pst (qRT-PCR Analysis)
To validate the RNA-seq data by qRT-PCR analysis, fourteen members of the TaCDPK family have been selected based on their expression level during compatible or incompatible interactions.These selected members are TaCDPK3A, TaCDPK3D, TaCDPK5A, TaCDPK7A, TaCDPK7B, TaCDPK11A, TaCDPK22A, TaCDPK22B, TaCDPK22D, TaCDPK25A, TaCDPK25B, TaCDPK26A, TaCDPK26B, and TaCDPK27B.Due 9, 12, 18 hpi) in both compatible and incompatible interactions (Figure 3).Both the RNA-seq and qRT-PCR results suggest that TaCDPK7 may play a potential role in wheat-Pst interaction, either positively or negatively.Therefore, TaCDPK7 was selected for further investigation of its functional role in wheat defense against Pst.Overall, our results indicate the reliability of the RNA-seq data, and highlight the regulation of several TaCDPKs during the wheat-Pst interaction.

Silencing of TaCDPK7 Enhances Wheat Susceptibility to Pst
To investigate the functional role of TaCDPK7 during the wheat-Pst interaction, we employed BSMV-gene silencing, a widely used and effective tool in wheat-Pst interaction studies [30,31].Two specific fragments within the coding region of TaCDPK7 were selected to silence its transcripts.Wheat seedling leaves were inoculated with BSMV:TaPDS-as, BSMV:TaCDPK7-1as, BSMV:TaCDPK7-2as, and BSMV:γ.Leaves inoculated with BSMV:TaPDS exhibited strong photo-bleaching symptoms (Figure 4A), while mild chlorotic mosaic patterns were observed on leaves infected with BSMV:γ, TaCDPK7-1as, and TaCDPK7-2as, confirming the functionality of the gene silencing system.studies [30,31].Two specific fragments within the coding region of TaCDPK7 were selected to silence its transcripts.Wheat seedling leaves were inoculated with BSMV:TaPDS-as, BSMV:TaCDPK7-1as, BSMV:TaCDPK7-2as, and BSMV:γ.Leaves inoculated with BSMV:TaPDS exhibited strong photo-bleaching symptoms (Figure 4A), while mild chlorotic mosaic patterns were observed on leaves infected with BSMV:γ, TaCDPK7-1as, and TaCDPK7-2as, confirming the functionality of the gene silencing system.The fourth leaf of wheat plants pre-inoculated with BSMV:γ, TaCDPK7-1as, or TaCDPK7-2as was subsequently inoculated with the avirulent Pst isolate CYR23 or the virulent isolate CYR31 at 10 days post inoculation (dpi).At 14 days after inoculation with CYR23, clear hypersensitive responses (HRs) were observed on all the virus-pre-inoculated leaves (Figure 4A).Fungal uredia were produced near the necrotic areas on the leaves of TaCDPK7-silenced plants but not on leaves pre-inoculated with BSMV (Figure 4A), indicating that silencing of TaCDPK7 reduced the resistance of wheat to CYR23.Conversely, normal disease development was observed on CYR31-inoculated plants, and no significant difference was observed between the control (BSMV:γ-pre-inoculated) and the TaCDPK7-silenced plants (Figure 4A).
To confirm the effective silencing of TaCDPK7, the transcript levels of TaCDPK7 were analyzed via qRT-PCR in leaves inoculated with BSMV:TaCDPK7-1as and The fourth leaf of wheat plants pre-inoculated with BSMV:γ, TaCDPK7-1as, or TaCDPK7-2as was subsequently inoculated with the avirulent Pst isolate CYR23 or the virulent isolate CYR31 at 10 days post inoculation (dpi).At 14 days after inoculation with CYR23, clear hypersensitive responses (HRs) were observed on all the virus-pre-inoculated leaves (Figure 4A).Fungal uredia were produced near the necrotic areas on the leaves of TaCDPK7silenced plants but not on leaves pre-inoculated with BSMV (Figure 4A), indicating that silencing of TaCDPK7 reduced the resistance of wheat to CYR23.Conversely, normal disease development was observed on CYR31-inoculated plants, and no significant difference was observed between the control (BSMV:γ-pre-inoculated) and the TaCDPK7-silenced plants (Figure 4A).
To confirm the effective silencing of TaCDPK7, the transcript levels of TaCDPK7 were analyzed via qRT-PCR in leaves inoculated with BSMV:TaCDPK7-1as and BSMV:TaCDPK7-2as.The transcript levels in TaCDPK7-knockdown plants from both incompatible and compatible interactions were significantly reduced up to two-fold compared to those in control plants, indicating successful silencing of TaCDPK7 (Figure 4B,C).Furthermore, the TaCDPK7-knockdown plants exhibited a significantly increased fungal biomass compared to the control plants during the incompatible interaction, while no significant difference in fungal biomass ratio was detected between the silenced and control plants during the compatible interaction (Figure 4D).Collectively, these results demonstrated that silencing of TaCDPK7 compromises the resistance of wheat to the stripe rust isolate CYR23.

Knockdown of TaCDPK7 Declines ROS Accumulation
Since CDPKs have been implicated in ROS (reactive oxygen species) production in Arabidopsis and Solanum tuberosum, we further investigated the accumulation of H 2 O 2 , which is strongly associated with the host resistance response, in TaCDPK7-silenced plants during the wheat-Pst incompatible interaction.Interestingly, the production of H 2 O 2 in TaCDPK7-silenced plants was significantly reduced compared to control plants treated with BSMV:γ at 48 hpi, although there was no difference between them at 24 hpi (Figure 5A,B).These findings suggest that TaCDPK7 may also play a role in regulating H 2 O 2 production in wheat in response to Pst infection.
control plants during the compatible interaction (Figure 4D).Collectively, these results demonstrated that silencing of TaCDPK7 compromises the resistance of wheat to the stripe rust isolate CYR23.

Knockdown of TaCDPK7 Declines ROS Accumulation
Since CDPKs have been implicated in ROS (reactive oxygen species) production in Arabidopsis and Solanum tuberosum, we further investigated the accumulation of H2O2, which is strongly associated with the host resistance response, in TaCDPK7-silenced plants during the wheat-Pst incompatible interaction.Interestingly, the production of H2O2 in TaCDPK7-silenced plants was significantly reduced compared to control plants treated with BSMV:γ at 48 hpi, although there was no difference between them at 24 hpi (Figure 5A,B).These findings suggest that TaCDPK7 may also play a role in regulating H2O2 production in wheat in response to Pst infection.

Knockdown of TaCDPK7 Improves Pst Growth
To assess whether the reduced resistance in TaCDPK7-knockdown plants was due to the limited growth of Pst in the plants, we conducted histological observations of wheat leaves during the incompatible interaction.At 48 hpi, there was no significant difference in hyphal growth, as indicated by the number of haustoria mother cells per infection site, hyphal length, and infection unit area, between the leaves of the control plants and those of the silenced plants (Figure 6A-D).However, at 120 hpi, the infection unit area was significantly larger in the TaCDPK7-silenced leaves compared to the controls (Figure 6A,D), suggesting that silencing the transcription of TaCDPK7 enhanced fungal growth in wheat leaves.

Knockdown of TaCDPK7 Improves Pst Growth
To assess whether the reduced resistance in TaCDPK7-knockdown plants was due to the limited growth of Pst in the plants, we conducted histological observations of wheat leaves during the incompatible interaction.At 48 hpi, there was no significant difference in hyphal growth, as indicated by the number of haustoria mother cells per infection site, hyphal length, and infection unit area, between the leaves of the control plants and those of the silenced plants (Figure 6A-D).However, at 120 hpi, the infection unit area was significantly larger in the TaCDPK7-silenced leaves compared to the controls (Figure 6A,D), suggesting that silencing the transcription of TaCDPK7 enhanced fungal growth in wheat leaves.

Transcript Levels of PR and ROS-Related Genes Are Affected in TaCDPK7-Silenced Plants
To assess the impact of TaCDPK7 silencing on the expression of PR-related and ROSscavenging genes, we analyzed the transcript levels of three PR genes (TaPR1, TaPR2, and TaPR5) and one ROS-scavenging gene (TaCAT1, catalase) using qRT-PCR.The Arabidopsis CAT1, CAT2, and CAT3 are crucial for the elimination of H 2 O 2 , and play important roles in regulating ROS homeostasis.Notably, the AtCAT1 specifically targets the removal of H 2 O 2 generated under various environmental stress conditions [32].Therefore, TaCAT1, the wheat homolog of AtCAT1, was selected for examination in this study.In TaCDPK7silenced plants, the expression of PR genes (TaPR1, TaPR2, and TaPR5) were significantly downregulated compared to the control plants, both in incompatible and compatible interactions (Figures 7 and S6).Conversely, the transcripts of the ROS-scavenging gene (TaCAT1) were significantly increased in TaCDPK7-silenced plants compared to the control plants during both incompatible and compatible interactions (Figures 7 and S6).These findings indicate that TaCDPK7 acts as a positive regulator of certain defense-related genes during the wheat-Pst interaction.

Transcript Levels of PR and ROS-Related Genes Are Affected in TaCDPK7-Silenced Plants
To assess the impact of TaCDPK7 silencing on the expression of PR-related and ROS-scavenging genes, we analyzed the transcript levels of three PR genes (TaPR1, TaPR2, and TaPR5) and one ROS-scavenging gene (TaCAT1, catalase) using qRT-PCR.The Arabidopsis CAT1, CAT2, and CAT3 are crucial for the elimination of H2O2, and play important roles in regulating ROS homeostasis.Notably, the AtCAT1 specifically targets the removal of H2O2 generated under various environmental stress conditions [32].Therefore, TaCAT1, the wheat homolog of AtCAT1, was selected for examination in this study.In TaCDPK7-silenced plants, the expression of PR genes (TaPR1, TaPR2, and TaPR5) were significantly downregulated compared to the control plants, both in incompatible and compatible interactions (Figures 7 and S6).Conversely, the transcripts of the ROS-scavenging gene (TaCAT1) were significantly increased in TaCDPK7-silenced plants compared to the control plants during both incompatible and compatible interactions (Figures 7 and S6).These findings indicate that TaCDPK7 acts as a positive regulator of certain defense-related genes during the wheat-Pst interaction.

Discussion
CDPKs are essential calcium sensors with calcium binding and signal transducing capabilities, playing crucial roles in plant development and stress responses.While genome-wide studies on CDPK families have been conducted in several plants such as rice [27], Arabidopsis [3], and barley [33], limited research has focused on CDPKs in bread wheat, a vital food source for humans.Earlier studies identified 20 CDPKs in

Discussion
CDPKs are essential calcium sensors with calcium binding and signal transducing capabilities, playing crucial roles in plant development and stress responses.While genomewide studies on CDPK families have been conducted in several plants such as rice [27], Arabidopsis [3], and barley [33], limited research has focused on CDPKs in bread wheat, a vital food source for humans.Earlier studies identified 20 CDPKs in wheat and observed their expression patterns under various stresses in 2008, when the wheat genome was not fully sequenced [22].The hexaploid wheat originated through complex evolutionary processes approximately 8000-10,000 years ago [34].It underwent two rounds of genome duplication, leading to the formation of a complex genome comprising three linked sub-genomes (BB, AA, DD).These sub-genomes originated from three distinct diploid species [34].
Therefore, it is expected that hexaploid wheat possesses a larger number of CDPK genes due to its complex genome resulting from the combination of three distinct subgenomes.Consistently, we identified 79 CDPK genes from the hexaploid wheat genome, which is a higher number compared to the number of CDPKs in rice and Arabidopsis.This observation further supports the expectation of a larger repertoire of CDPK genes in hexaploid wheat due to its complex genome structure.Phylogenetic analysis grouped wheat CDPKs into four clusters (I-IV), similar to rice CDPKs [27].All identified CDPK genes (26 + 26 + 26) are equally distributed across sub-genomes A, B, D, except for TaCDPK4UN, which is located on an unknown locus (Table S1).All the TaCDPKs have counterparts in the other two sub-genomes, except for TaCDPK18 and TaCDPK27, they lack copies in subgenome B and A, respectively, which is possibly due to genetic erosion.Moreover, possible tandem duplication and segmental duplication events were observed among TaCDPKs.These findings suggest that the variations in the number of CDPK family members among different plants are likely a result of gene loss and gene duplication, which are key factors in the evolution of gene families.Such evolutionary processes can lead to the emergence of new genes with distinct roles [35].Consistent with the phylogenetic relationships among species, the wheat CDPKs exhibit a closer phylogenetic association with that of rice CDPKs in comparison with that of Arabidopsis, and most Arabidopsis CDPK members exhibited a higher degree of divergence from the other two taxa (wheat and rice).This observation might be attributed to the fact that both wheat and rice (monocot) belong to the same Poaceae family in contrast to Arabidopsis, which is a dicot belonging to the family Brassicaceae.
CDPKs are membrane-embedded proteins, and their second signal's reversibility as calcium sensors may allow CDPKs to shuttle between membranes and the cytosol or the nucleus.Here, we also predicted a diverse range of cellular localizations of CDPKs, including the cytosol, chloroplasts, plasma membrane, nucleus, endoplasmic reticulum, mitochondria, and peroxisomes (Table S4), suggesting that TaCDPKs have access to a large range of possible substrates throughout the cell and have a wide variety of functions.CDPKs are evolved in perceiving changes in Ca 2+ levels within plant cells and play a crucial role in disease resistance and stress-responsive signaling pathways.These proteins decode and interpret Ca 2+ flux signals through phosphorylation processes, which are crucial events for signal transduction pathways.Numerous functional and expression studies of different CDPKs confirmed their versatile roles in drought, salt, cold stress tolerance, as well as calcium homeostasis in plant cells.Additionally, CDPK family members from diverse plant species have been reported to be implicated in disease defense mechanism.The Arabidopsis CDPKs AtCPK4/5/6/11 phosphorylate the transcription factors WRKY8/28/48, thereby modulating the expression of downstream biotic-responsive genes [36,37].In particular, AtCPK4/5/6/11 from group I of Arabidopsis CDPK family classification induced flg22-mediated gene expression and production of ROS [19,38].They target RBOHD on phosphosites S148/S163/S347 that are PAMP stimulated and required for RBOHD activity [38][39][40][41].Interestingly, a fifth isoform from subgroup IV, AtCPK28, negatively regulates PAMP-induced oxidative burst by reducing the stability of another RBOHD-stimulating enzyme Botrytis-induced kinase1 (BIK1) [42].AtCPK5/6 also regulate ethylene production in response to wounding and Botrytis.cinerea infection, through the modulation of ethylene biosynthesis enzyme ACC synthase (ACS), either at the transcriptional level or by stabilizing the proteins [43,44].CPKs from subgroups II and III have also been involved in plant immunity.In particular, AtCPK3 was implicated in various defense pathways.It was required for phytosphingosine-induced PCD in response to the fungus Fusarium moniliforme [45].There are few reports of CDPKs role against plant viruses resistance, in which AtCPK3 could limit cell-to-cell propagation of potato virus X by phosphorylating the remorin StRem1.3[46].In a recent study, TaTHI2 interacts with TaCPK5 to suppress Chinese wheat mosaic virus (CWMV) infection [47].Despite previous evidence showcasing the positive role of wheat TaCPK7-D, assigned to subgroup III based on previous wheat CDPK family classification, in enhancing resistance against Rhizoctonia cerealis [48], there is limited research on the activities of wheat CDPKs during biotic stress.In this study, we showed that several TaCDPKs were either upregulated or downregulated during the compatible and incompatible interactions between wheat and Pst, as shown in Figure 2, suggesting that certain TaCDPKs may potentially contribute to wheat defense against Pst.Specifically, TaCDPK7, a member of cluster I, displayed strong induction in both compatible and incompatible interactions (Figure 3), highlighting its potential importance in the defense response of wheat against Pst.
In barley and wheat, BSMV-mediated VIGS has been established as a quick and effective reverse genetic technique for studying gene functions [49].Silencing of TaCDPK7 by VIGS caused increased susceptibility to Pst accompanied by an enlarged infection area within leaf tissues in TaCDPK7-silenced plants (Figure 6), strongly suggesting that TaCDPK7 plays a crucial role in conferring wheat resistance to Pst.The ROS burst, one of the earliest signaling events in plants, happens in the initial phases during plant-pathogen interactions [50].As demonstrated in this study, the ROS accumulation was significantly reduced in TaCDPK7-knockdown plants.Furthermore, the transcript levels of the ROS-scavenging gene (TaCAT1) were significantly induced in TaCDPK7-knockdown plants related to control plants in incompatible and compatible interactions (Figure 7 and Figure S6).These findings suggest that TaCDPK7 plays a role in wheat's resistance against Pst by inhibiting the expression of the ROS-scavenging gene TaCAT1, thereby leading to increased ROS content in wheat.Plant defense responses to pathogens are accomplished through transcriptional activation of defense-related genes.The overexpression of OsCDPK1 has been demonstrated to upregulate the rice PATHOGEN-RELATED 1/4/10a (PR1, PR4, and PR10a) genes, and thereby positively affecting rice resistance to Xoo infection [51].In alignment with these findings, our study observed a downregulation of transcript levels for PR genes TaPR1, TaPR2, and TaPR5 in TaCDPK7-knockdown plants during both incompatible and compatible interactions (Figure 7 and Figure S6).Overall, our data strongly indicate that TaCDPK7 plays a positive role during wheat resistance to Pst by promoting ROS accumulation and enhancing the expression of PR genes.However, to further strengthen and solidify this conclusion, it would be valuable to conduct future investigations focusing on the effects of TaCDPK7 overexpression during the wheat-Pst interaction, which would provide a complementary approach to validate and expand upon the findings presented in this study, ultimately enhancing our understanding of the precise role of TaCDPK7 in wheat resistance to Pst.

Plant Material and Fungal Isolates
Wheat cultivar Suwon 11 (Su11) and Pst isolates CYR23 (avirulent), and CYR31/CYR32 (virulent) were used in this study.Wheat cultivar Su11 and Pst isolates, CYR23, CYR31, and CYR32 were obtained from the Institute of Plant Pathology, Northwest A&F University.Su11 is reported to be resistant to CYR23 but susceptible to CYR31 and CYR32 [52].The wheat cultivars NIL_R (Yr26) and NIL_S (yr26) were generated as described in [53].All the plants in this study were grown in a growth chamber (Percival, Des Moines, IA, USA) under a controlled environment at 14-16 • C, half lamps, with a 16 h photoperiod.

RNA, DNA Extraction, and cDNA Synthesis
The wheat cultivar Suwon 11 was infected with Pst race CYR23 (avirulent), CYR31 (virulent), or CYR32 (virulent).For RNA extraction, Pst inoculated samples were collected at 0, 6, 12, 24, 48, 72, and 120 hpi (hours post inoculation) [54,55].Then, total RNA was extracted with the Quick RNA isolation Kit (Huayueyang Biotechnology, Beijing, China) following the manufacturer's instructions and contaminating DNA was removed by treatment with DNase I enzyme.First-strand cDNA was synthesized using 2 µg of the extracted total RNA with the Promega Reverse Transcription System (Promega, Madison, WI, USA) and Oligo (dT) 18 primer.To assess the fungal/wheat biomass ratio, samples were collected at 7 dpi (days post inoculation) for genomic DNA extraction.Quantitative PCR of the DNA was conducted relative to the internal reference genes PstEF-1 and TaEF-1a, respectively, according to the previous study [56].

Mining of CDPKs from Wheat Genome
To identify the putative CDPKs in wheat, a local BLASTP search in the wheat database (http://plants.ensembl.org/(accessed on 6 September 2022)) was performed with evalue of ≤e −10 .Two BLAST approaches were implemented to search the wheat CDPK proteins.First, all known Arabidopsis CDPK protein sequences retrieved from TAIR (www.arabidopsis.org(accessed on 17 July 2022) were used as query to search their corresponding potential homologs in the wheat genome.Secondly, the similar method was performed using all known rice CDPK sequences retrieved with NCBI (https://www.ncbi.nlm.nih.gov/(accessed on 3 August 2022)).All the matching sequences were further validated with the SMART program and InterPro, an online catalogue for protein classification (https://www.ebi.ac.uk/interpro/ (accessed on 15 October 2022)), with default settings.The protein sequences lacking the signature domains were excluded from further analysis.As a result, 79 identified CDPK candidates with the conserved domains were designated as TaCDPK proteins.

Phylogeny and Gene Structure Analysis
Maximum likelihood mid-rooted phylogenetic tree was built using the mega 6 tool to investigate the evolutionary relationships between CDPKs in wheat, Arabidopsis, and rice.Afterwards, the data of the corresponding tree were uploaded at Interactive Tree of Life (IToL) v. 6 (https://itol.embl.de/(accessed on 30 September 2022)), an online program and a phylogenetic tree of wheat CDPKs was drawn with scale bars corresponding to 0.1 substitution, as previously reported [57,58].The exon-intron assemblies of TaCDPKs were analyzed with Gene Structure Display Server v. 2.0 as described in [59].

The Conserved Motif, Cis-Acting Regulatory Elements, and Synteny Analysis
The conserved motifs of TaCDPKs were analyzed using MEME (multiple expectation maximization for motif elicitation) v. 5.3 with default parameters as described by [60].Amino acid sequences of TaCDPKs ranging from 6 to 100 residues in length were used for identifying conserved motifs.The subcellular localization of TaCDPK proteins was predicted using the online program WOLFPSORT II, with default options and selecting plants as the organism type, following the method by Waterhouse et al. [61].Biochemical characteristics of TaCDPK proteins, such as molecular weight (MW), amino acid composition, instability index, theoretical pI, aliphatic index, and grand average of hydropathicity (GRAVY), were determined using the ExPASy bioinformatics resource portal.Default options were selected for the analysis, following the protocol described by Artimo et al. [62].
To explore the potential regulatory mechanisms of TaCDPK genes, the 1.5 kb upstream promoter region of each gene was extracted from the wheat genome reference database.The cis-elements of TaCDPK promoters were assessed using the plant cis-acting regulatory DNA elements (PLACEs) and plant cis-acting regulatory element (PlantCARE) tools, following the methods previously employed [63,64].For synteny illustration, TBtools data managing toolkit by Chen et al. [65] was utilized.

Expression Profiling of TaCDPK Family Challenged with Pst
The transcript levels of all the TaCDPK genes during wheat Pst compatible (NIL_R vs. CYR32) and incompatible (NIL_S vs. CYR32) interactions were examined using RNA-seq data obtained from wheat samples collected at 0, 18, 24, 48, 96, and 168 hpi as previously described [66].Each sample was sequenced with HiSeq2500 (PE125) as described in [67].Corresponding heat maps demonstrating the transcript levels of TaCDPKs were generated using log2 FC by an online program (https://software.broadinstitute.org/morpheus/(accessed on 20 February 2023)).

Real-Time PCR (qRT-PCR) Analysis
Total RNAs were extracted with the Trizol reagent according to the manufacturer's protocols (Invitrogen, Carlsbad, CA, USA), and then treated with DNase I (Promega, Madison, WI, USA) to remove DNA contamination.cDNA was synthesized with GoScript Reverse Transcription System (Promega, USA) and an oligo (dT18) primer (Invitrogen, USA).Primers specific to the examined TaCDPKs were used to perform qRT-PCR using the synthesized cDNA as a template (Table S3).A 7500 Real-Time PCR System (Applied Biosystems, San Francisco, CA, USA) was used to quantify the transcripts.TaEF-1α (GenBank Q03033) was used as the internal reference gene.The relative expression of TaCDPKs was determined with comparative method 2 −∆∆CT [68], qRT-PCR experiments were carried out three times.

BSMV-Mediated Gene Silencing of TaCDPK7
Silencing of TaCDPK7 was achieved using the barley stripe mosaic virus (BSMV)mediated gene silencing method, as previously described [49].Two cDNA fragments of TaCDPK7 were selected and incorporated into the BSMV-γ-vector, resulting in the construction of BSMV-γ:TaCDPK7-1as/2as constructs.Capped in vitro transcripts were generated using the RiboMAXTM (LargeScale RNA Production System-T7, Promega, Madison, WI, USA) and Ribom7G Cape Analog (Promega, Madison, WI, USA) according to the manufacturer's protocol.
The barley stripe mosaic virus constructs were inoculated onto the second leaf of the wheat plants, following the previously reported method [49].The plants were then kept in darkness at a temperature of 25 ± 2 • C for 24 h with adequate humidity.Control plants were inoculated with 1 × Fes buffer (0.06 M K2HPO4, 0.1 M glycine, 1% w/v tetrasodium pyrophosphate, 1% w/v celite, 1% w/v bentonite, pH 8.5).After 10 days, fresh urediniospores of Pst race CYR23 or CYR31 were collected and inoculated onto the fourth leaf.Leaf samples treated with Pst were collected at 0, 24, 48, and 120 hpi for histological investigation and RNA extraction.The silencing efficacy of the TaCDPK7-silenced plants and the relative expression of pathogenesis (PR) and ROS-related genes were quantified using qRT-PCR analysis.After 14 days, the phenotypic expression of Pst-inoculated leaves was assessed, and infection phenotypes were documented through photography.The entire experiment was repeated three times.

Histology of Fungal Infection and Host Response
Wheat leaves were collected at 24, 48, and 120 hpi for the examination of H 2 O 2 accumulation using 3,3 ′ -diaminobenzidine (DAB) staining, following the method reported by Bai et al. [54].To investigate fungal growth in planta, leaves collected at the same time points were stained with wheat germ agglutinin (WGA) conjugated to Alexa (Invitrogen, Carlsbad, CA, USA).The presence of a vesicle under a stoma was used as an indicator of infection sites and at least 30 infection sites per samples were examined for both H 2 O 2 accumulation and fungal growth investigation.The H 2 O 2 accumulation and infection area were visualized using an Olympus BX-53 microscope (Olympus Corporation, Tokyo, Japan) and quantified using DP-BSW Ver.03.03 software.

Statistical Analysis and Graphical Presentation
All data were analyzed with GraphPad Prism v. 7 software.Student's t-test was employed to determine the statistical differences between treatments.

Conclusions
In summary, our genome-wide investigation identified a total of 79 CDPK genes in hexaploid wheat.Among these genes, several displayed differential expressions during the wheat-Pst interaction, with TaCDPK7 exhibiting the strong significant induction.Further functional characterization revealed that TaCDPK7 plays a crucial role in conferring wheat resistance against Pst by curbing fungal growth in leaf tissues.Gene expression analysis indicated that TaCDPK7 contributes to wheat defense against Pst infection by promoting the expression of PR genes and downregulating the transcription of the ROS-scavenging gene TaCAT1.The findings of this study not only provide a promising candidate gene for enhancing wheat resistance to Pst, but also shed light on the role of the CDPK family during biotic stress.Additionally, this study serves as a valuable reference for identifying key genes that can be targeted for breeding purposes to improve pathogen resistance in wheat.

Figure 2 .
Figure 2. Transcriptional levels of TaCDPKs in compatible and incompatible interactions between wheat and Puccinia striiformis f. sp.tritici (Pst).The expression patterns of TaCDPKs analyzed using logFC [log2 (foldchange) ] at 0, 18, 24, 48, 96, and 168 hpi in compatible and incompatible interactions in time series dual RNA-seq data.(A) The incompatible combination 32R, (B) the compatible combination 32S.Downregulation and upregulation are indicated with blue and red colors, respectively.The white color shows expression patterns that are comparable to those after mock treatment.

Figure 2 .
Figure 2. Transcriptional levels of TaCDPKs in compatible and incompatible interactions between wheat and Puccinia striiformis f. sp.tritici (Pst).The expression patterns of TaCDPKs analyzed using logFC [log 2 (foldchange) ] at 0, 18, 24, 48, 96, and 168 hpi in compatible and incompatible interactions in time series dual RNA-seq data.(A) The incompatible combination 32R, (B) the compatible combination 32S.Downregulation and upregulation are indicated with blue and red colors, respectively.The white color shows expression patterns that are comparable to those after mock treatment.

Figure 4 .
Figure 4. Silencing of TaCDPK7 enhances wheat susceptibility to Pst. (A) Mild chlorotic mosaic symptoms on viral-inoculated wheat plants.Mock: wheat leaves treated with 1 × Fes buffer.The leaves infected with the avirulent race CYR23 or the virulent race CYR31 were photographed at 14 days post infection (dpi).(B,C) Transcripts of TaCDPK7 in TaCDPK7-knockdown or control plants (plants inoculated with BSMV:γ) challenged with the avirulent CYR23 and virulent CYR31.(D) The fungal/wheat biomass ratio of TaCDPK7-knockdown and control plants at 7 dpi (*, p < 0.05).

Figure 4 .
Figure 4. Silencing of TaCDPK7 enhances wheat susceptibility to Pst. (A) Mild chlorotic mosaic symptoms on viral-inoculated wheat plants.Mock: wheat leaves treated with 1 × Fes buffer.The leaves infected with the avirulent race CYR23 or the virulent race CYR31 were photographed at 14 days post infection (dpi).(B,C) Transcripts of TaCDPK7 in TaCDPK7-knockdown or control plants (plants inoculated with BSMV:γ) challenged with the avirulent CYR23 and virulent CYR31.(D) The fungal/wheat biomass ratio of TaCDPK7-knockdown and control plants at 7 dpi (*, p < 0.05).

Figure 6 .
Figure 6.Knockdown of TaCDPK7 enhanced pathogen growth.(A) Pathogen advancement per infection site.Number of (B) haustorium mother cells (HMCs), (C) infection hypha (IH), and (D) infection area per site were measured in silenced and control plants.Asterisks show significant differences between TaCDPK7-knockdown and control plants, as determined by a student's t-test (*, p < 0.05).

Figure 6 .
Figure 6.Knockdown of TaCDPK7 enhanced pathogen growth.(A) Pathogen advancement per infection site.Number of (B) haustorium mother cells (HMCs), (C) infection hypha (IH), and (D) infection area per site were measured in silenced and control plants.Asterisks show significant differences between TaCDPK7-knockdown and control plants, as determined by a student's t-test (*, p < 0.05).Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 11 of 19

Figure 7 .
Figure 7. Transcriptional changes in pathogenesis-related (PR) genes and ROS-scavenging gene TaCAT1 in TaCDPK7-silenced and control plants challenged with avirulent Pst race CYR23.Relative expressions of (A) TaPR1, (B) TaPR2, (C) TaPR5, and (D) TaCAT1 were analyzed by qRT-PCR and the comparative threshold (2 −ΔΔCT ) method.Asterisks show significant differences between TaCDPK7-knockdown and control plants as assessed by a student's t-test (*, p < 0.05).All data were attained from three biological replicates.

Figure 7 .
Figure 7. Transcriptional changes in pathogenesis-related (PR) genes and ROS-scavenging gene TaCAT1 in TaCDPK7-silenced and control plants challenged with avirulent Pst race CYR23.Relative expressions of (A) TaPR1, (B) TaPR2, (C) TaPR5, and (D) TaCAT1 were analyzed by qRT-PCR and the comparative threshold (2 −∆∆CT ) method.Asterisks show significant differences between TaCDPK7knockdown and control plants as assessed by a student's t-test (*, p < 0.05).All data were attained from three biological replicates.