The Wheat Annexin TaAnn12 Plays Positive Roles in Plant Disease Resistance by Regulating the Accumulation of Reactive Oxygen Species and Callose

(1) Annexins are proteins that bind phospholipids and calcium ions in cell membranes and mediate signal transduction between Ca2+ and cell membranes. They play key roles in plant immunity. (2) In this study, virus mediated gene silencing and the heterologous overexpression of TaAnn12 in Arabidopsis thaliana Col-0 trials were used to determine whether the wheat annexin TaAnn12 plays a positive role in plant disease resistance. (3) During the incompatible interaction between wheat cv. Suwon 11 and the Puccinia striiformis f. sp. tritici (Pst) race CYR23, the expression of TaAnn12 was significantly upregulated at 24 h post inoculation (hpi). Silencing TaAnn12 in wheat enhanced the susceptibility to Pst. The salicylic acid hormone contents in the TaAnn12-silenced plants were significantly reduced. The overexpression of TaAnn12 in A. thaliana significantly increased resistance to Pseudomonas syringae pv. tomato DC3000, and the symptoms of the wild-type plants were more serious than those of the transgenic plants; the amounts of bacteria were significantly lower than those in the control group, the accumulation of Reactive Oxygen Species (ROS)and callose deposition increased, and the expression of resistance-related genes (AtPR1, AtPR2, and AtPR5) significantly increased. (4) Our results suggest that wheat TaAnn12 resisted the invasion of pathogens by inducing the production and accumulation of ROS and callose.


Introduction
Annexins, which bind phospholipids and calcium ions in cell membranes to mediate signal transduction between Ca 2+ and cell membranes, are ubiquitous in plants, animals, and fungi [1].After regulation via a range of local environments (e.g., Ca 2+ , pH, voltage, and lipids) or atypical ordering motifs, plant annexins work by inserting themselves into cytosolic membranes and are Ca 2+ -dependent members of the phospholipid-binding protein family, the functions of which include exocytosis, peroxidase, and ATPase/GTPase activities; binding actin; regulating Ca 2+ channel activity; and transportation [2].Annexins have been shown to be involved in H 2 O 2 -activated Ca 2+ fluxes in plants [3,4], act as Ca 2+ -permeable transporters, and exhibit peroxidase activity [5].Consequently, annexins link Ca 2+ , redox, and lipid signaling to coordinate the development of responses to both biotic [6][7][8] and abiotic stresses [9][10][11][12][13].
In response to abiotic stresses, such as NaCl stress [13], PEG stress (simulated drought) [11], abscisic acid (ABA) stress [12], and high [10] or low temperatures [9], the expression levels of annexins are regulated, illustrating the involvement of annexins in plant stress responses.Arabidopsis thaliana MYB30 regulates high-temperature stress responses via annexin-mediated calcium signaling, which binds MYB30 to the promoters of AnnAt1 and AnnAt4 to inhibit the expression of annexins [14].Under abiotic stresses, OsANN1 overexpression in plants promoted the activities of superoxide dismutase (SOD) and catalase (CAT) to regulate the H 2 O 2 content, indicating that OsANN1 is involved in a feedback mechanism in H 2 O 2 production, whereas OsANN1-knockdown plants were susceptible to heat and drought stresses [15].The chitin receptor CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) interacted with ANNEXIN 1 to function in chitin or salt signaling in [16].
The functions of annexins in plant immunity have been identified [6][7][8].The heterologous expression of AnnBj1 (Brassica juncea) in tobacco provides tolerance to Phytophthora parasitica var.nicotianae [17].Similarly, the CkANN of Cynanchum komarovii enhanced resistance to Fusarium oxysporum in transgenic cotton in [18].Additionally, after powdery mildew infection, the expression of A. thaliana AnnAt1 increased significantly [19].However, AtAnn8 negatively regulated the RPW8.1-mediatedresistance against powdery mildew and cell death [20].In another study, due to their Ca 2+ -binding domains, annexins reduced the activity of Ca 2+ on callose ((1→3)-β-glucan) synthase, and cotton annexin inhibited the activities of callose synthase [21].The callose deposition increased the CmbHLH18 resistance to the necrotrophic fungus Alternaria brassicicola, which kills epidermal cells by secreting toxic metabolites and proteins [22], indicating that the accumulation of callose affects plants' defense against biotic stress [23].Regarding nematode resistance, the MIFlike effector MiMIF-2 of Meloidogyne incognita protected nematodes from oxidative stress by regulating the function of the plant annexins AnnAt1 and AnnAt4, and plants with AnnAt1 or AnnAt4 overexpression were more resistant to M. incognita, as illustrated by a reduced number of galls and nematodes inside the roots of the plants [24].Hs4F01, a homologous protein of Arabidopsis AnnAt1, is an annexin-like effector that mimics plant annexins and regulates the defense response by interacting with 2OG-Fe(II) oxygenase (DMR6) [25].
An analysis of the wheat genome by Xu et al. [26] led to the identification of 25 putative annexin genes, which were assigned to subgenomes A, B, and D. Further analyses led to the finding that each haploid genome of wheat has 12 annexin gene family members (TaAnn1-12).Based on these sequences, increases in the expression of the annexin gene family in response to abiotic stresses were detected.Among them, the expression of TaAnn12 was significantly induced by salt, drought, cold, and ABA [26].However, it is unclear whether TaAnn12 is involved in plant defense responses, especially in wheat resistant to stripe rust.Wheat (Triticum spp.) is the second largest major crop in the world, with wheat planting areas occupying approximately 220 million hectares worldwide [27].Wheat stripe rust, caused by Puccinia striiformis f. sp.tritici (Pst), is one of the main fungal diseases affecting wheat.Due to the changing climate and environment, the races of wheat stripe rust have rapidly mutated, overcoming the resistance of wheat varieties currently protected by resistance genes [28].Therefore, finding new ways to carry out studies on wheat disease resistance and cultivating wheat varieties with relatively durable resistance will provide new development directions for the genetic breeding of wheat disease resistance.In recent years, combining molecular breeding with phenotypic selection has provided great hope for the genetic diversity of stripe-rust-resistant wheat [27].In this study, to analyze whether the wheat annexin TaAnn12 plays a role in wheat defense, we detected the accumulation of TaAnn12 in wheat leaves upon Pst infection, which was significantly upregulated during the incompatible interaction between wheat and the Pst race CYR23.Furthermore, barley stripe mosaic virus (BSMV)-induced gene silencing technology and heterologous transgenic overexpression in Arabidopsis assays were used to identify the function of TaAnn12 in plant disease resistance.The results of this study provide important information for understanding the different functions of plant annexins.

The Expression of TaAnn12 Is Differently Induced during Pst Infection, Hormone Treatments, and Abiotic Stresses
Full-length primers were designed according to the known TaAnn12 sequence in the NCBI sequence database (GenBank accession number: AK331881.1)to obtain a targeting gene from wheat cv.Suwon 11 cDNA using PCR technology, with the ORF sequence length being 945 bp.To analyze the function of TaAnn12 in wheat defense, we detected the accumulation of TaAnn12 in wheat leaves upon Pst infection.As shown in Figure 1a, in the incompatible interaction, compared with samples at 0 hpi, the expression of TaAnn12 was significantly induced at 24 hpi, with a 6.3-fold increase, indicating that TaAnn12 provides tolerance to biotic stress.
the accumulation of TaAnn12 in wheat leaves upon Pst infection.As shown in Figure 1a, in the incompatible interaction, compared with samples at 0 hpi, the expression of TaAnn12 was significantly induced at 24 hpi, with a 6.3-fold increase, indicating that TaAnn12 provides tolerance to biotic stress.
Additionally, we analyzed how TaAnn12 responded to an exogenous hormone stimulus.Different hormones were sprayed on wheat, and the results are shown in Figure 1b.The expression of TaAnn12 mRNA increased significantly under the treatments of salicylic acid (SA), jasmonate (JA), and abscisic acid (ABA), suggesting that TaAnn12 may be involved in hormone signaling pathways.The expression of TaAnn12 responded to abiotic stresses (i.e., salt, drought, and cold) [26], and we also confirmed that the expression of TaAnn12 was significantly induced under salt, drought, cold, and heat treatments (Figure S1a).In addition, we found that the TaAnn12 mRNA accumulations were different in roots, stems, and leaves (Figure S1b).

Suppression of TaAnn12 Reduces Wheat Resistance to Pst
To examine the potential role of TaAnn12 in the process of wheat stripe rust infection, we used barley stripe mosaic virus (BSMV)-induced gene silencing technology to silence TaAnn12 during Pst infection.A specific 132 bp segment of TaAnn12 was inserted into the BSMV: γ vector.Ten days after inoculating with a recombinant virus (BSMV: γ, BSMV: TaPDS, and BSMV: TaAnn12), obvious photo-bleaching (BSMV: TaPDS) and virus mosaic symptoms (BSMV: γ and BSMV: TaAnn12) were observed on fourth Su11 leaves (Figure 2a).Then, the new fourth leaves were inoculated with the Pst races CYR23 and CYR31, with the infected wheat leaves being observed, photographed, and recorded at 15 dpi.Remarkably, as shown in Figure 2b, typical hypersensitive reactions (HRs) appeared on the mock and BSMV: γ leaves after infecting with CYR23 (incompatible interaction), while the necrosis area in TaAnn12-silenced plants increased, and small amounts of uredia were produced.On the other hand, on wheat leaves inoculated with CYR31 (compatible interaction), TaAnn12-silenced plants produced more uredia compared with the control plants Additionally, we analyzed how TaAnn12 responded to an exogenous hormone stimulus.Different hormones were sprayed on wheat, and the results are shown in Figure 1b.The expression of TaAnn12 mRNA increased significantly under the treatments of salicylic acid (SA), jasmonate (JA), and abscisic acid (ABA), suggesting that TaAnn12 may be involved in hormone signaling pathways.The expression of TaAnn12 responded to abiotic stresses (i.e., salt, drought, and cold) [26], and we also confirmed that the expression of TaAnn12 was significantly induced under salt, drought, cold, and heat treatments (Figure S1a).In addition, we found that the TaAnn12 mRNA accumulations were different in roots, stems, and leaves (Figure S1b).

Suppression of TaAnn12 Reduces Wheat Resistance to Pst
To examine the potential role of TaAnn12 in the process of wheat stripe rust infection, we used barley stripe mosaic virus (BSMV)-induced gene silencing technology to silence TaAnn12 during Pst infection.A specific 132 bp segment of TaAnn12 was inserted into the BSMV: γ vector.Ten days after inoculating with a recombinant virus (BSMV: γ, BSMV: TaPDS, and BSMV: TaAnn12), obvious photo-bleaching (BSMV: TaPDS) and virus mosaic symptoms (BSMV: γ and BSMV: TaAnn12) were observed on fourth Su11 leaves (Figure 2a).Then, the new fourth leaves were inoculated with the Pst races CYR23 and CYR31, with the infected wheat leaves being observed, photographed, and recorded at 15 dpi.Remarkably, as shown in Figure 2b, typical hypersensitive reactions (HRs) appeared on the mock and BSMV: γ leaves after infecting with CYR23 (incompatible interaction), while the necrosis area in TaAnn12-silenced plants increased, and small amounts of uredia were produced.On the other hand, on wheat leaves inoculated with CYR31 (compatible interaction), TaAnn12silenced plants produced more uredia compared with the control plants (Figure 2c).In addition, the silencing efficiency of TaAnn12 was confirmed via RT-qPCR.Compared with BSMV: γ, the expression of TaAnn12 reached 61-78% when inoculated with the Pst race CYR23, while it reached 54-70% when inoculated with the Pst race CYR31 in the TaAnn12-knockdown plants at 0, 24, 48, and 120 hpi (Figure 2d).
affected, the expression of the SA-pathway-related gene (TaNPR1), H2O2-pathway-related genes (TaCAT and TaSOD) and resistance-related genes (TaPR1 and TaPR2) was quantified with RT-qPCR.The transcript levels of TaNPR1, TaPR1, and TaPR2 were significantly reduced in the BSMV: TaAnn12 leaves compared to BSMV: γ after inoculating with CYR23, which indicated that TaAnn12 may affect wheat resistance to Pst by involving the SA signaling pathway.In contrast, the expression levels of TaCAT and TaSOD were found to be significantly upregulated in TaAnn12-gene-silenced plants (Figure 2e).Additionally, histological observations (i.e., branches, hyphal length, and colony area) were made on BSMV: TaAnn12 leaves at 24 h, 48 h, and 120 h after inoculating with CYR23 to further clarify the function of TaAnn12 in wheat resistance to stripe rust infection.The growth of Pst was expanded in BSMV: TaAnn12 plants under a fluorescence microscope (Figure 3a).The hyphal length of gene-silenced plants was significantly increased compared with the control at 48 hpi.And the hyphal length and colony area of infected BSMV: TaAnn12 plants increased significantly at 120 hpi (Figure 3b-d).In summary, these results demonstrated that Pst infection was enhanced in gene-silenced wheat, suggesting that TaAnn12 plays an important role in attenuating stripe rust.To determine whether the resistance-related genes in TaAnn12-silenced plants were affected, the expression of the SA-pathway-related gene (TaNPR1), H 2 O 2 -pathway-related genes (TaCAT and TaSOD) and resistance-related genes (TaPR1 and TaPR2) was quantified with RT-qPCR.The transcript levels of TaNPR1, TaPR1, and TaPR2 were significantly reduced in the BSMV: TaAnn12 leaves compared to BSMV: γ after inoculating with CYR23, which indicated that TaAnn12 may affect wheat resistance to Pst by involving the SA signaling pathway.In contrast, the expression levels of TaCAT and TaSOD were found to be significantly upregulated in TaAnn12-gene-silenced plants (Figure 2e).Additionally, histological observations (i.e., branches, hyphal length, and colony area) were made on BSMV: TaAnn12 leaves at 24 h, 48 h, and 120 h after inoculating with CYR23 to further clarify the function of TaAnn12 in wheat resistance to stripe rust infection.The growth of Pst was expanded in BSMV: TaAnn12 plants under a fluorescence microscope (Figure 3a).The hyphal length of gene-silenced plants was significantly increased compared with the control at 48 hpi.And the hyphal length and colony area of infected BSMV: TaAnn12 plants increased significantly at 120 hpi (Figure 3b-d).In summary, these results demonstrated that Pst infection was enhanced in gene-silenced wheat, suggesting that TaAnn12 plays an important role in attenuating stripe rust.

Reduced Accumulation of SA in TaAnn12-Silenced Plants
SA and JA are indispensable hormones in plant immune response signaling pathways [29].The exogenous hormone treatment experiment showed that MeJA and SA induced the expression of TaAnn12 (Figure 1b).In addition, the BSMV-VIGS system showed that the expression of the SA signaling pathway marker genes TaNPR1, TaPR1, and TaPR2 were significantly downregulated in BSMV: TaAnn12 plants (Figure 2e).In order to confirm the specific signaling pathway affecting TaAnn12 under Pst infection, we detected (using liquid chromatography-mass spectrometry (LC/MS)) the accumulation of SA and JA in BSMV: TaAnn12 and BSMV: γ at 18 h and 24 h after inoculating with the Pst race CYR23.As shown in Figure 4, the SA content of TaAnn12-silenced plants decreased by 24% compared to the control and was significantly decreased at 24 hpi (Figure 4a), while the JA concentration had no significant change (Figure 4b).These results indicated that TaAnn12 regulates wheat resistance to stripe rust via the SA signaling pathway.

Reduced Accumulation of SA in TaAnn12-Silenced Plants
SA and JA are indispensable hormones in plant immune response signaling pathways [29].The exogenous hormone treatment experiment showed that MeJA and SA induced the expression of TaAnn12 (Figure 1b).In addition, the BSMV-VIGS system showed that the expression of the SA signaling pathway marker genes TaNPR1, TaPR1, and TaPR2 were significantly downregulated in BSMV: TaAnn12 plants (Figure 2e).In order to confirm the specific signaling pathway affecting TaAnn12 under Pst infection, we detected (using liquid chromatography-mass spectrometry (LC/MS)) the accumulation of SA and JA in BSMV: TaAnn12 and BSMV: γ at 18 h and 24 h after inoculating with the Pst race CYR23.As shown in Figure 4, the SA content of TaAnn12-silenced plants decreased by 24% compared to the control and was significantly decreased at 24 hpi (Figure 4a), while the JA concentration had no significant change (Figure 4b).These results indicated that TaAnn12 regulates wheat resistance to stripe rust via the SA signaling pathway.

Overexpressing TaAnn12 in Arabidopsis Enhances Plant Defense
A transgenic overexpression of TaAnn12 in Arabidopsis thaliana Col-0 materials was created to investigate the results obtained in the transient gene silencing experiments of TaAnn12 in disease resistance, and we screened two T1-generation lines (TaAnn12-OE1 and -OE2).After the expansion and PCR identification of the T2 generation (TaAnn12-T2-OE1: 1, 4-7, 9, 10, 12, 13, and 15-18; TaAnn12-T2-OE2: 1-7, 8-11, and 13) (Figure S2), the T3-generation lines were injected with Pseudomonas syringae pv.tomato (Pto) DC3000, and the symptoms were observed at 4 dpi.The yellowing and wilting of wild-type plants were more serious than in transgenic plants (Figure 5a).Incidentally, the amounts of Pto DC3000 were isolated and detected, with the colony densities of TaAnn12-T3-OE1 and TaAnn12-T3-OE2 being significantly lower than Col-0 at 24 h (10 5.08 CFU/cm 2 and 10 5.08 CFU/cm 2 ) and 48 h (10 5.73 CFU/cm 2 and 10 5.40 CFU/cm 2 ) (Figure 5b).In addition, trypan blue was used to stain the necrotic area of Pto DC3000 at 24 hpi and 48 hpi, and the plants overexpressing TaAnn12 showed more intense cell necrosis (Figure 5c).These results indicated that TaAnn12-overexpressing Arabidopsis plants were more resistant to Pto DC3000 than wild-type plants and that TaAnn12 had a positive regulatory role in the process of resistance to Pto DC3000.
Subsequently, the expression of defense-related genes was detected via RT-qPCR.Samples were taken at 0, 3, 8, and 24 h after inoculating with Pto DC3000, and the expression levels of AtPR1, AtPR2, AtPR5, and AtPDF1.2 were detected.The results were as follows (Figure 6): compared with Col-0, the expression of AtPR1, AtPR2, and AtPR5 in TaAnn12-T3-OE1 and TaAnn12-T3-OE2 plants was significantly increased at 8 and 24 hpi, while the JA signaling pathway marker gene AtPDF1.2had no significant change, which further confirmed that the overexpression of TaAnn12 significantly increased the resistance of Arabidopsis to Pto DC3000.

Overexpressing TaAnn12 in Arabidopsis Enhances Plant Defense
A transgenic overexpression of TaAnn12 in Arabidopsis thaliana Col-0 materials was created to investigate the results obtained in the transient gene silencing experiments of TaAnn12 in disease resistance, and we screened two T1-generation lines (TaAnn12-OE1 and -OE2).After the expansion and PCR identification of the T2 generation (TaAnn12-T2-OE1: 1, 4-7, 9, 10, 12, 13, and 15-18; TaAnn12-T2-OE2: 1-7, 8-11, and 13) (Figure S2), the T3-generation lines were injected with Pseudomonas syringae pv.tomato (Pto) DC3000, and the symptoms were observed at 4 dpi.The yellowing and wilting of wild-type plants were more serious than in transgenic plants (Figure 5a).Incidentally, the amounts of Pto DC3000 were isolated and detected, with the colony densities of TaAnn12-T3-OE1 and TaAnn12-T3-OE2 being significantly lower than Col-0 at 24 h (10 5.08 CFU/cm 2 and 10 5.08 CFU/cm 2 ) and 48 h (10 5.73 CFU/cm 2 and 10 5.40 CFU/cm 2 ) (Figure 5b).In addition, trypan blue was used to stain the necrotic area of Pto DC3000 at 24 hpi and 48 hpi, and the plants overexpressing TaAnn12 showed more intense cell necrosis (Figure 5c).These results indicated that TaAnn12-overexpressing Arabidopsis plants were more resistant to Pto DC3000 than wild-type plants and that TaAnn12 had a positive regulatory role in the process of resistance to Pto DC3000.
Subsequently, the expression of defense-related genes was detected via RT-qPCR.Samples were taken at 0, 3, 8, and 24 h after inoculating with Pto DC3000, and the expression levels of AtPR1, AtPR2, AtPR5, and AtPDF1.2 were detected.The results were as follows (Figure 6): compared with Col-0, the expression of AtPR1, AtPR2, and AtPR5 in TaAnn12-T3-OE1 and TaAnn12-T3-OE2 plants was significantly increased at 8 and 24 hpi, while the JA signaling pathway marker gene AtPDF1.2had no significant change, which further confirmed that the overexpression of TaAnn12 significantly increased the resistance of Arabidopsis to Pto DC3000.

TaAnn12 Affects Plant Resistance by Regulating ROS and Callose Accumulation
Reactive oxygen species (ROS; e.g., O 2− , H 2 O 2 , OH•, and O 2 ) signaling is related to plant immunity [30].Herein, nitro-blue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB) staining were used as indicators of the superoxide anion (O 2− ) and hydrogen peroxide (H 2 O 2 ) to detect the ROS in Arabidopsis leaves inoculated with Pto DC3000.The NBT staining showed that Col-0 leaves had a small number of blue spots, while the TaAnn12-T3-OE1 and TaAnn12-T3-OE2 plants showed navy blue at 3 and 8 hpi, indicating that the overexpression of TaAnn12 promoted O 2− accumulation (Figure 7a).Similarly, H 2 O 2 accumulation was higher in overexpressing plants (Figure 7b).These results suggest that TaAnn12 may affect plant immunity by regulating the production of ROS.(DAB) staining were used as indicators of the superoxide anion (O ) and hydrogen per-oxide (H2O2) to detect the ROS in Arabidopsis leaves inoculated with Pto DC3000.The NBT staining showed that Col-0 leaves had a small number of blue spots, while the TaAnn12-T3-OE1 and TaAnn12-T3-OE2 plants showed navy blue at 3 and 8 hpi, indicating that the overexpression of TaAnn12 promoted O 2− accumulation (Figure 7a).Similarly, H2O2 accumulation was higher in overexpressing plants (Figure 7b).These results suggest that TaAnn12 may affect plant immunity by regulating the production of ROS.Plant annexins are involved in the regulation of callose synthesis [21].To determine whether TaAnn12 affects callose accumulation, TaAnn12-T3-OEs were stained with aniline blue in Arabidopsis leaves inoculated with Pto DC3000.Fluorescent signals were generated and observed under a fluorescence microscope (Figure 8a).The callose accumulation of TaAnn12-T3-OE1 and TaAnn12-T3-OE2 was increased compared to the control, and the amount of callose deposition at 24 hpi was higher than at 12 hpi, indicating that overexpression of TaAnn12 promotes the accumulation of callose and that the accumulation of callose gradually increases with time (Figure 8b).Plant annexins are involved in the regulation of callose synthesis [21].To determine whether TaAnn12 affects callose accumulation, TaAnn12-T3-OEs were stained with aniline blue in Arabidopsis leaves inoculated with Pto DC3000.Fluorescent signals were generated and observed under a fluorescence microscope (Figure 8a).The callose accumulation of TaAnn12-T3-OE1 and TaAnn12-T3-OE2 was increased compared to the control, and the amount of callose deposition at 24 hpi was higher than at 12 hpi, indicating that overexpression of TaAnn12 promotes the accumulation of callose and that the accumulation of callose gradually increases with time (Figure 8b).

Discussion
Annexins respond to biotic and abiotic stresses and are involved in signal transduction between calcium ions and cell membranes.They link Ca 2+ and lipid signaling to participate in callose synthesis and ion transport [1].According to the bioinformatics analysis of the wheat annexin gene family and its responses to abiotic stress, TaAnn12 is significantly induced by salt, drought, cold, and ABA and plays key roles in responding to abiotic stresses.However, it has been unclear whether TaAnn12 is involved in plant defense responses, especially in wheat resistant to stripe rust.In this study, we found that TaAnn12 played positive roles in plant disease resistance.

Discussion
Annexins respond to biotic and abiotic stresses and are involved in signal transduction between calcium ions and cell membranes.They link Ca 2+ and lipid signaling to participate in callose synthesis and ion transport [1].According to the bioinformatics analysis of the wheat annexin gene family and its responses to abiotic stress, TaAnn12 is significantly induced by salt, drought, cold, and ABA and plays key roles in responding to abiotic stresses.However, it has been unclear whether TaAnn12 is involved in plant defense responses, especially in wheat resistant to stripe rust.In this study, we found that TaAnn12 played positive roles in plant disease resistance.
Annexins have been identified in numerous studies of plant defenses [7,8].Overexpression of AnnBj1 in tobacco improves resistance to Phytophthora parasitica var.nicotianae [17].AnnAt1-or AnnAt4-overexpressing plants were more resistant to M. incognita [24].AtAnn1 and AtAnn2 double-mutant ann1ann2 showed more susceptibility to Botrytis cinerea [31], implicating the positive regulation effect of AtAnn1 and AtAnn2 in plant resistance against B. cinerea.Furthermore, the expression of AnnAt1 increased significantly during osmotic stress and powdery mildew infection [19,32,33].In addition, AtAnn8 was a negative regulator of RPW8.1-mediated resistance to powdery mildew and cell death [20].In order to determine whether TaAnn12 is involved in the interaction between plants and pathogens, in this study the expression levels of TaAnn12 in stripe-rust-infected wheat were measured.Among them, in the incompatible interaction, TaAnn12 was upregulated at 24 hpi, indicating that TaAnn12 positively responded to Pst CYR23, which was further improved by BSMV-VIGS technology.Afterwards, the TaAnn12 gene was heterologously overexpressed in Arabidopsis.The transgenic plants enhanced A. thaliana defense and further clarified that TaAnn12 played a positive regulatory role in disease resistance.
The incompatible interaction between a plant and a pathogen causes a hypersensitive reaction (HR) to limit the growth of the pathogen [34], which is a local defense, caused primarily by the production and accumulation of ROS [35].Overexpression of OsANN1 promoted SOD and CAT activity to regulate the accumulation of H 2 O 2 [15].In this study, the expression of TaCAT and TaSOD, which are H 2 O 2 -pathway-related genes, was increased in TaAnn12-silenced plants, accelerating the removal of ROS and leading to a decrease in the disease resistance level of silent plants.In addition, the accumulation areas of superoxide (O 2− ) and hydrogen peroxide (H 2 O 2 ) in TaAnn12 transgenic Arabidopsis were larger than in the control, and the necrotic area increased, indicating that TaAnn12 induced the HR and the accumulation of ROS to exert disease resistance.
The accumulation of callose affects plant defense against biotic stress [23], and plant annexins are involved in regulating callose ((1→3)-β-glucan) synthase synthesis [21].Oomycete annexin stimulated the (1→3)-β-d-glucan synthase activator [36], suggesting that specific annexins may have antagonistic effects in regulating callose enzyme activity [1].To determine the functions of TaAnn12 in callose accumulation, aniline blue staining was used to detect the callose content after inoculation with Pto DC3000, and the callose accumulation of TaAnn12-overexpressing Arabidopsis was increased compared to the control, showing that TaAnn12 was involved in the accumulation of callose.
Plants have evolved complex signaling and defense mechanisms to defend against pathogen infections.Fungi have diverse lifestyles in which they deploy distinct strategies to interact with their host plants, including necrotrophic, biotrophic, and hemibiotrophic strategies [22].For instance, the defense strategies of plants produce different stress signaling based on the type of invading pathogen (e.g., fungi or bacteria) or pathogenic lifestyle (biotroph, hemibiotroph, or necrotroph).The induction of defense genes is orchestrated by signaling networks that are directed by plant hormones.Salicylic acid (SA) and jasmonic acid (JA) are the major players, and SA plays an important role in plant responses to biotrophs and hemibiotrophs [29].After a pathogen infects a plant, the host recognizes signaling generated by the pathogen to initiate a defense response in the plant, which promotes the synthesis of SA and activates the expression of downstream disease-associated protein (PR) genes, so that the plant can prevent the pathogen infection [37].The CkANN enhanced the tolerance of transgenic cotton to Fusarium oxysporum, and the transcription level of PRs in CkANN transgenic cotton increased, indicating that CkANN is involved in PR protein-mediated SA-dependent defense responses [18].In order to clarify the signaling pathway of TaAnn12 in wheat resistance to stripe rust, wheat was treated with the exogenous hormones SA, MeJA, ET, and ABA, all of which induced the expression of TaAnn12.Furthermore, we measured the contents of SA and JA in TaAnn12-silenced wheat, and the results showed that the SA content in TaAnn12-silenced plants decreased significantly compared with the control, indicating that TaAnn12 participated in the SA signaling pathway of plant immunity.On the other hand, after infection with Pto DC3000 in TaAnn12-overexpressing Arabidopsis, the expression of the defense-related genes AtPR1, AtPR2, and AtPR5 was significantly increased, while the JA signaling pathway marker gene AtPDF1.2did not change significantly, further indicating that TaAnn12 was involved in the SA signaling pathway.TaAnn12 plays a positive role in regulating plant disease resistance through the SA signaling pathway.

Plants, Pathogen Material, and Treatment
Wheat (Triticum aestivum) cv.Suwon 11 (Su11) and Mingxian 169 were used.Fresh urediospores of Puccinia striiformis f. sp.tritici (Pst) were propagated on the Mingxian 169.The Pst race CYR23 had an interaction with Su11, triggering a typical hypersensitive reaction (HR) after inoculation, and the Pst race CYR31 had a compatible interaction with Su11, during which a large number of uredia were attached.Wheat seedlings were grown at 23 • C, with day and night periods of 16 h and 8 h, respectively.After inoculating with fresh urediospores of Pst, the wheat was placed in a dark incubator with a temperature of 10 • C and a humidity of 90% for 24 h and then placed in a 16 • C incubator (day/night cycle of 16 h/8 h) [38].
Wild-type Arabidopsis thaliana Columbia-0 (Col-0) was used as a transgenic Arabidopsis.Col-0 seeds were sterilized in 75% ethanol for 5 min and then immediately washed with sterile water three times.After surface sterilization, seeds were cultivated and germinated in a 1/2 MS medium (pH 5.5) and cultured at 4 • C for 2 days.After the emergence of the cotyledon, seedlings were transplanted into vermiculite seedling trays with a temperature of 23 • C and a day/night cycle of 10/14 h.Pseudomonas syringae pv.tomato (Pto) DC3000 was grown on King's B (KB) medium with 25 mg/L rifampicin at 28 • C. Leaves of 4-week-old Arabidopsis were injected with Pto using a syringe (without a needle) [39].
The Pto DC3000 was grown on King's B (KB) medium at 28 • C, and resuspended in 10 mM MgCl 2 to OD600 = 0.002.Leaves of 4-week-old plants were infected with the bacterial suspension by pressing a 1 mL syringe (without a needle) against the abaxial side of the leaves and forcing the suspension through the stomata into the intercellular spaces, as described in the previous study [40].Plants were sampled at 0, 3, 8, and 12 h and then used to extract RNA.

Real-Time Quantitative PCR (RT-qPCR) Analysis and Statistical Analysis
The specific primers TaAnn12-Q-F/TaAnn12-Q-R were used for RT-qPCR, and TaEF-1a was used as the control gene (Table S1).Total RNA was extracted according to the total RNA extraction kit (Invitrogen, Carlsbad, CA, USA).First-strand cDNA was prepared according to the FastKing RT Kit (Tiangen, Beijing, WI, China).The RT-qPCR reaction system and procedures referred to the ChamQTM SYBR qPCR Master Mix kit (Vazyme, Nanjing, China).The TaEF-1a gene (GenBank accession number: M90077.1) was used as the control gene of wheat, and the AteIF4A gene was used as the control gene of Arabidopsis [39].The 2 −∆∆t method was used to calculate the relative expression of genes.Each sample had three independent biological replicates.SPSS 23.0 was used to determine the statistical significance (p < 0.05) [42].

Barley Stripe Mosaic Virus (BSMV)-Mediated Gene Silencing
A specific fragment of TaAnn12 was cloned into the BSMV: γ vector, which was used in SNG-VIGS (https://vigs.solgenomics.net/,accessed on 6 September 2021) to determine the specificity.After linearizing the BSMV: α, BSMV: β, BSMV: γ, BSMV: γ-TaAnn12, and BSMV: γ-TaPDS plasmid vectors with restriction endonuclease, a RiboMAX TM Large Scale RNA Production Systems-T7 Kit (Promega, Austin, TX, USA) was used to reverse-transcribe BSMV RNAs in vitro.The BSMV: α, β, and γ genes were mixed proportionally with FES buffer and inoculated by rubbing leaves on the second leaf of a ten-day-old Su11 plant, where FES buffer was used as a blank control, BSMV:γ was used as a negative control, and BSMV: γ-TaPDS was used as a positive control.The sample was placed in a dark incubator with a temperature of 25 • C and a humidity of 90% for 24 h and then placed in a 25 • C incubator (day/night cycle of 16 h/8 h) [43].Ten days after inoculation, photobleaching and mild chlorotic mosaic symptoms were detected on the fourth leaves of wheat plants inoculated with the Pst races CYR23 and CYR31, respectively.The phenotypes were observed and photographed at 15 dpi.Samples were taken at 0, 24, 48, and 120 hpi to extract RNA and at 24, 48, and 120 hpi for histological observation [41].The expression level of TaAnn12 in the leaves with BSMV:γ at each time point was standardized as 1.To observe Pst development, wheat leaves were decolorized with 100% ethanol and then stained with WGA AF488.They were observed under a NIKON-80i fluorescence microscope, with 30 infection points being calculated at each time point, including the number of hyphal branches, hyphal length, and colony area [44].SPSS 23.0 was used to determine the statistical significance (p < 0.05).

Detection of the Accumulation of Plant Hormones in TaAnn12-Silenced Plants
First, 200 mg of BSMV: γ and BSMV: γ-TaAnn12 wheat leaves were sampled at 18 and 24 h after inoculating with CYR23.After grinding with liquid nitrogen, 1 mL of a precooled extraction liquid (methanol/water/glacial acetic acid = 90:9:1) was added and centrifuged to collect the supernatant.After repeating twice, samples were dried in nitrogen, and 200 µL of methanol was added to dissolve the samples.In order to detect the contents of salicylic acid (SA) and jasmonic acid (JA), SA and JA chemical molecular standard samples (Sigma, Shanghai, China) were prepared according to the concentration gradient.The standard samples and a sample of wheat leaves were tested using liquid chromatography-tandem mass spectrometry (LC/MS).Based on the standard curve, the accumulations of SA and JA were analyzed [41].

Production of Transgenic Arabidopsis
The full-length ORF of TaAnn12 was constructed in a pCMBIA3301 (controlled by CaMV 35S promoter) vector, which was transformed into the Agrobacterium tumefaciens strain GV3101.Using a vacuum infiltration method, the overexpression vector was transformed into Col-0.Transgenic T1-generation seeds were selected with Kanamycin on a 1/2 MS medium.The seedlings were grown until they had four dark-green leaves and were transplanted into vermiculite seedling trays.After amplifying the insertion of the transgene in the genomic DNA using a PCR assay, transgenic lines were obtained in the T2 generation, and the T3-generation plants were tested for disease resistance [39].

Identification of Disease Resistance in Transgenic TaAnn12 Arabidopsis
Pseudomonas syringae pv.tomato (Pto) DC3000 was resuspended in 10 mM MgCl 2 to OD 600 = 0.002 (approximately 1 × 10 6 CFU/mL).Leaf phenotypes were observed at 4 dpi, and samples were collected at 0, 3, 8, and 24 hpi for RNA extraction.To isolate and detect the amount of Pto DC3000, leaf samples were taken at 0, 24, and 48 hpi with a punch (diameter of 5 mm) and 0.1 mL of sterile water was added.After gradient dilution, single colonies were counted on KB medium with 25 mg/L rifampicin.For 3,3-diaminobenzidine (DAB) staining, samples were taken at 3, 8, and 24 hpi and treated with 1 mg/mL DAB (pH 3.8) for 30 min, which decolorized after dark staining for 8 h.The samples at each timepoint (3, 8, and 24 hpi) were stained using 1 mg/mL nitro-blue tetrazolium (NBT) for 30 min, which stained in the dark for 2 h before decolorization [45].For trypan blue staining, samples were taken at 24 and 48 hpi and were boiled with a trypan blue solution for 3 min.Samples were collected at 12 and 24 hpi for callose staining.After decolorization, leaves were pretreated with 10% KOH and stained with 0.01% aniline blue in 67 mM K 2 HPO 4 for 6 h [46].

Conclusions
In conclusion, our results revealed that wheat TaAnn12 is a positive regulator of plant immunity that resists the invasion of a pathogen by inducing the production and accumulation of ROS and callose and regulates the disease resistance response of wheat through the SA signaling pathway.Thus, the expression of downstream disease resistance proteins is activated.

Figure 1 .
Figure 1.Real-time quantitative PCR (RT-qPCR) analysis of TaAnn12 in response to Pst and exogenous hormone treatments.(a) Accumulation of TaAnn12 was significantly induced in incompatible interaction.Su11 leaves were inoculated with CYR23 (incompatible interaction) or CYR31 (compatible interaction).(b) The transcription of TaAnn12 responded to exogenous hormone treatments.The 2 −ΔΔt method was used to calculate the relative expression of genes.Error bars represent ±SDs of three biological replications.Asterisks (*) indicate significant differences (p < 0.05) from 0 hpi.

Figure 1 .
Figure 1.Real-time quantitative PCR (RT-qPCR) analysis of TaAnn12 in response to Pst and exogenous hormone treatments.(a) Accumulation of TaAnn12 was significantly induced in incompatible interaction.Su11 leaves were inoculated with CYR23 (incompatible interaction) or CYR31 (compatible interaction).(b) The transcription of TaAnn12 responded to exogenous hormone treatments.The 2 −∆∆t method was used to calculate the relative expression of genes.Error bars represent ±SDs of three biological replications.Asterisks (*) indicate significant differences (p < 0.05) from 0 hpi.

Figure 2 .
Figure 2. TaAnn12 functions in response to Pst as indicated by the barley stripe mosaic virus (BSMV)induced gene silencing.(a) At 10 d after inoculating with BSMV RNAs, the photo-bleaching and mild chlorotic mosaic symptoms were detected and photographed on wheat leaves.Wheat leaves were inoculated with Pst races CYR23 (b) and CYR31 (c), and the typical leaves were photographed at 15 dpi.(d) The expression level of TaAnn12.(e) Relative expression levels of resistance-related genes in TaAnn12-silenced leaves inoculated with CYR23.TaNPR1: non-expression of PR1, TaPR1: pathogenesis-related protein, TaPR2: beta-1,3-glucanase, TaSOD: superoxide dismutase, TaCAT: catalase.Error bars represent ±SDs of three biological replications.Asterisks (*) indicate significant differences (p < 0.05).

Figure 7 .
Figure 7.The accumulation of superoxide (a) and peroxide (b) in Arabidopsis leaves.

Figure 7 .
Figure 7.The accumulation of superoxide (a) and peroxide (b) in Arabidopsis leaves.