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. 2021 Oct 21;20(3):454–467. doi: 10.1111/pbi.13728

Thinopyrum intermedium TiAP1 interacts with a chitin deacetylase from Blumeria graminis f. sp. tritici and increases the resistance to Bgt in wheat

Yanlin Yang 1, † , Pan Fan 1, † , Jingxia Liu 1, † , Wenjun Xie 2, Na Liu 1, Zubiao Niu 1, Quanquan Li 1, Jing Song 1, Qiuju Tian 1, Yinguang Bao 1, Honggang Wang 1, Deshun Feng 1,✉
PMCID: PMC8882775  PMID: 34651397

Summary

The biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt) is a crucial factor causing reduction in global wheat production. Wild wheat relatives, for example Thinopyrum intermedium, is one of the wild‐used parents in wheat disease‐resistant breeding. From T. intermedium line, we identified the aspartic protease gene, TiAP1, which is involved in resistance against Bgt. TiAP1 is a secreted protein that accumulates in large amounts at the infection sites of Bgt and extends to the intercellular space. Yeast two‐hybrid, luciferase complementation imaging and bimolecular florescent complimentary analysis showed that TiAP1 interacted with the chitin deacetylase (BgtCDA1) of Bgt. The yeast expression, purification and in vitro test confirmed the chitin deacetylase activity of BgtCDA1. The bombardment and VIGS‐mediated host‐induced gene silencing showed that BgtCDA1 promotes the invasion of Bgt. Transcriptome analysis showed the cell wall xylan metabolism, lignin biosynthesis‐related and defence genes involved in the signal transduction were up‐regulated in the transgenic TiAP1 wheat induced by Bgt. The TiAP1 in wheat may inactivate the deacetylation function of BgtCDA1, cause chitin oligomers expose to wheat chitin receptor, then trigger the wheat immune response to inhibit the growth and penetration of Bgt, and thereby enhance the resistance of wheat to pathogens.

Keywords: Blumeria graminis f. sp. tritici , chitin deacetylase, disease resistance, Thinopyrum intermedium, TiAP1, wheat

Introduction

Wheat (Triticum aestivum L.) is one of the main crops in the world, therefore the production of wheat is closely related to food security. In recent years, pathogenic infections have seriously affected the yield of wheat. Wheat powdery mildew caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici (Bgt) can result in severe reductions in grain yield (Singh et al., 2016). When powdery mildew invades the plant, it releases proteinaceous exudates that contain the mechanical and molecular features required for the full virulence of the pathogen (Zhang et al., 2005). Fungal exudates function in the attachment of conidia and in the adherence of the penetrating appressoria (App) to invade plants (Deising and Werner, 2000). At the same time, the plant responds immediately to the pathogen invasion, and a number of secreted proteins, especially protease, from plant cells accumulate in the apoplastic space to carry an apoplastic battle between hosts and pathogen (Geziel, 2015; Wang and Wang, 2020).

In wheat, more than 66 powdery mildew resistance loci (Pm1‐Pm66) located on different chromosomes of the common wheat have been named (Li et al., 2020a). Most of them originated from wild relative species of wheat, such as Pm21 from Haynaldia villosa has been introduced into the common wheat using the translocation line T6VS·6AL (Chen et al., 2013). Li et al. (2020b) cloned Pm41, which was a powdery mildew resistance gene deriving from the wild emmer wheat. Thinopyrum intermedium had been hybridized extensively with wheat and proven a useful source of resistance to various diseases of hexaploid wheat, and several powdery mildew resistance genes, such as Pm40, Pm43 and PmL962 (He et al., 2009; Luo et al., 2009; Shen et al., 2015), have been identified and genetic mapped. The mining and utilization of these new resistance genes are vital means to improve current wheat varieties and increase their resistance to powdery mildew.

Aspartic proteases consist of four main proteolytic enzymes that are widely present in animals, plants, yeast, microorganisms and viruses (Rawlings and Barrett, 1995). Xia et al. (2004) reported that the peptide signal system of CDR1 was involved in the activation of the resistance mechanism, while Prasad et al. (2009, 2010) found that OsCDR1/OsAP5 could enhance the disease resistance of rice and Arabidopsis thaliana. The aspartic protease gene, AP13, from grape could promote the salicylic acid‐dependent signal transduction pathway (Guo et al., 2016). Moreover, Alam et al. (2014) found that the rice OsAP77 gene accumulated in the extracellular space and sieve tubes to defend against the pathogens. Hence, these studies have shown aspartic proteases participate in the biotic stresses response of plants to enhance the resistance.

To resist pathogens, plants have developed a complex immune system including the plasma membrane receptors that recognize pathogen‐related molecular patterns, such as chitin from the fungal cell walls that can trigger defence responses. Kamakura et al. (2002) found a new germination tube‐specific gene, CBP1, from the rice blast fungus Magnaporthe grisea, which encoded a chitin‐binding protein (CBP) having two similar chitin‐binding domains at both sides of the central domain, might play a vital role in the hydrophobic surface sensing of M. grisea during App differentiation. Kuroki et al. (2017) confirmed CBP1 encoded a chitin deacetylase and participated in differentiation stage of App in M. oryzae. These results proved the chitin deacetylase activity of CBP1 is necessary for the formation of App. In addition, Yang et al. (2019) identified the chitinase gene MoChia1 from M. oryzae. This gene can trigger a plant’s defence response to M. oryzae in rice under an inducible promoter. MoChia1 was also a functional chitinase that is required for the growth and development of M. oryzae. The MoChia1 binding to free chitin could inhibit the plant immune response, while another protein, OsTPR1, competitively binding with MoChia 1, would re‐establish the immune response (Yang et al., 2019). Han et al. (2019) also found the mechanism of MoChi1 that targeted the host lectin to inhibit rice immunity and promote colonization. Furthermore, they also found rice protein OsMBL1 could interact with MoChi1 to enhance the resistance against M. oryzae. Overexpressing of OsMBL1 can lead to the activation of defence response genes of rice and the burst of chitin‐induced reactive oxygen species. Moreover, Gao et al. (2019) identified a secreted polysaccharide deacetylase (PDA1) from soil‐borne Verticillium dahliae. PDA1 can promote the deacetylation of chitin oligomers, whose N‐acetyl group contributes to the host lysine motif (LysM)‐containing receptor that perceives ligand‐triggered immunity and facilitates virulence. Thus, the silencing of PDA1 allows the N‐acetyl group of the chitin‐triggered host immunity to occur.

In a previous study, we obtained an aspartic protease gene, TiAP1, from trititrigia SN6306 through a comparative transcriptome analysis. Further sequence analysis showed TiAP1 originated from T. intermedium (Tian et al., 2017). Through gene expression and Barley Stripe Mosaic Virus (BSMV) virus‐induced gene silencing (VIGS) analyses, we found the TiAP1 gene might be involved in the resistance to wheat powdery mildew. In addition, subcellular localization analysis showed that TiAP1 is a secretory protein. During the Bgt invasion, TiAP1 would assemble in large numbers at the infect site. The yeast two‐hybrid (Y2H) verification, luciferase (LUC) complementation imaging (LCI) and bimolecular florescent complimentary (BiFC) analysis show that TiAP1 interacts with chitin deacetylase (BgtCDA1) of Bgt, while BgtCDA1 could promote the invasion of Bgt. Therefore, we propose that TiAP1 induces the plant to resist the invasion of pathogens by interacting with BgtCDA1, inactivating the deacetylation function of BgtCDA1, then triggering the wheat immune response to inhibit the growth and penetration of Bgt.

Results

The expression of TiAP1 mediates the resistance in trititrigia SN6306 to Bgt invasion

Since Li et al. (2016) analysed the transcriptome of SN6306 and YN15, and obtained a gene that was up‐regulated by Bgt induction, which encoded an aspartic protease, while Tian et al. (2017) cloned the TiAP1 gene and found that it came from T. intermedium. Here, we found that the TiAP1 gene was expressed in SN6306 not in wheat YN15, and was up‐regulated by the induction at the early stages of the Bgt invasion (Figure 1a).

Figure 1.

Figure 1

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qRT‐PCR and VIGS analysis of the TiAP1 effect on the Bgt resistance, the TiAP1 prokaryotic expression and purification and the reduction in the powdery mildew severity (lesions area) on the wheat seedlings by a recombination protein treatment. (a) Expression levels of TiAP1 in YN15 and SN6306 that were induced by Bgt. Total RNA from the leaves of YN15 and SN6306 that were induced by Bgt at 0, 12, 24, 48, 72 and 96 h were extracted as the template to be detected by qRT‐PCR. Data represent mean ± SD of n = 3 technical replicates. (b) Relative quantification of expression levels of TiAP1. Total RNA from the leaves of barley stripe mosaic virus (BSMV):00 and BSMV: TiAP1as inoculated with E09 for 10 days were extracted as the template for the detection by qRT‐PCR. (c) Disease phenotype of the BSMV:00 and BSMV: TiAP1as inoculated with E09. Photographs were taken at 10 dpi. (d) Microscopic observation of the disease symptoms of BSMV:00 and BSMV: TiAP1as 10 dpi. Bar = 100 µm. (e) The TiAP1 protein expression detection by Western blotting before and after induction. M: Pre‐stained protein ladder; Lane1: Supernatant of non‐induced; Lane 2: Precipitation of non‐induced; Lane 3: Supernatant of induced; Lane 4: Precipitation of induced. (f) SDS‐PAGE analysis of the inclusion body purification. M: Low‐molecular weight protein ladder; Lane 1: Inclusions after dissolving; Lane 2: The effluent absorbed by nickel iminodiacetic acid (Ni‐IDA); Lane 3–6: The eluent by 50 mm imidazole elution; Lane 7–11: The eluent by 100 mm imidazole elution; Lane 12–13: The eluent by 300 mm imidazole elution; (G) TiAP1 quality assurance after renaturation. M: Pre‐stained protein ladder; Lane 1: TiAP1 protein (1.5 µg). (H) Bgt phenotype on YN15 leaves smeared with TiAP1 and the tag protein from pET28a (CK) and inoculated with E09 for 5 days. (I) Microscopic (Nikon Ni‐U, Tokyo, Japan) analysis of the Bgt in YN15 leaves smeared with TiAP1 and CK and inoculated with E09 for 5 days. Bar = 100 µm. (j) The proportion of lesions area on the leaf of YN15 that was pre‐treated with the recombinant TiAP1 and inoculated by Bgt E09 for 5 days. Control: the purified recombinant tag protein of pET28a construct. Different letters above the bars indicate statistically significant differences (P < 0.05) as obtained by one‐way analysis of variance with least significance difference and Duncan’s multiple range test method.

To investigate the effect of endogenous TiAP1 on SN6306 resistance to Bgt, we reduced the endogenous TiAP1 gene levels by suppressing TiAP1 expression using BSMV‐VIGS. The TiAP1 expression levels were significantly suppressed in the leaves of BMSV:TiAP1as SN6306 relative to the BMSV:00 negative control (Figure 1b). When the BMSV:TiAP1as SN6306 was inoculated with Bgt at the three‐leaf stage, the infection in BMSV:TiAP1as SN6306 was much more severe than in the BMSV:00 control at 10 days post‐inoculation (dpi) (Figure1c,d). This proved that the TiAP1 gene was resistant to Bgt invasion and could play a role in the resistance.

The TiAP1 protein enhances resistance to Bgt in vitro

Western blot analysis showed that TiAP1 was induced in the precipitation through prokaryotic expression (Figure 1e,f, Figure S1). After renaturation, the protein concentration was determined to be 0.437 mg/mL by the Bradford method (Figure 1g; Bradford, 1976). Moreover, regarding the TiAP1 protein effect on the Bgt invasion, the number of Bgt conidia on YN15 leaf after smearing TiAP1 for 5 days was significantly lower than that of YN15 with CK (pET28a). In addition, there was a smaller number of hyphae in the leaves smeared with TiAP1, less hyphal length and the diseased area than those of the leaves smeared with CK (Figure 1h,i). Lesion areas of the leaves treated with TiAP1 were also significantly less than that of the control (Figure 1j), suggesting that TiAP1 can resist the Bgt invasion in vitro.

TiAP1 gene transfer can elevate the resistance to Bgt of the wheat recipients

To investigate the function of TiAP1, the pMUbi‐TiAP1 vector and the screening marker Bar gene vector, were co‐transformed into the susceptible hexaploid wheat cultivar, Bobwhite. The obtained transgenic Bobwhite with the TiAP1 gene was subjected to PCR identification of the Bar and TiAP1 genes (Figure S2A,B) to obtain the positive transgenic plants. The qRT‐PCR analysis showed that the expression level of the TiAP1 gene was 20 and 110 times higher in OE1 and OE2 than that of SN6306 when infected by Bgt 2 days respectively (Figure 2a). After multiple comparisons, we found that the expression level of TiAP1 when infected by Bgt in OE2‐2 was significantly different from that of OE2‐0 (P < 0.05) (Figure 2a), indicating that the expression of TiAP1 could be involved in the response to Bgt. In addition, we found that the overexpression of TiAP1 gene in Bobwhite (OE1 or OE2) showed moderate resistance, while the leaf surface was still entirely green and had less conidia at 5 dpi than those of the control wheat Bobwhite (Figure 2b). A large number of Bgt in the OE1‐2, OE2‐2 and SN6306 were retained at App of 41.2%, 43.4% and 83.7%, and of 16.2%, 14.5% and 0.34% retained at hyphae stage, respectively, while in the control Bobwhite, 28.9% of the Bgt were present in the form of App, and 25.6% in the form of hyphae (Figure 2c,d). The HI in Bob‐2 was 68.6%, while in OE1‐2, OE2‐2 and SN6306 it was 53.5%, 48.8% and 6.7% respectively (Figure 2e). In addition, Figure 2d,e suggested that the transgenic TiAP1 gene could affect the formation of Bgt Hau, thereby resisting the invasion of Bgt.

Figure 2.

Figure 2

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Identifying the Bgt resistance of Bobwhite. SN6306, two transgenic TiAP1 wheat and the offspring of the hybrid. (a) Expression levels of TiAP1 in SN6306, Bobwhite and the transgenic Bobwhite with TiAP1 when inoculated with E09 for 2 days. The symbols ‘‐0’ and ‘‐2’ means treated o and 2 days respectively. (b) Phenotype of Bobwhite, SN6306, and two transgenic Bobwhite with TiAP1 (OE1 and OE2) when inoculated with E09 for 5 days. (c) Microscopic (Nikon Ni‐U, Tokyo, Japan) observation of the disease symptoms of Bobwhite, SN6306, and the transgenic Bobwhite with TiAP1 when inoculated with E09 for 2 days. Bar = 100 µm. (d) Count of Bgt E09 at different stages inoculated for 2 days in Bobwhite, SN6306, and the transgenic Bobwhite with TiAP1. (e) The HI statistics of Bgt E09 inoculated for 2 days in Bobwhite, SN6306, and the transgenic Bobwhite with TiAP1. Different letters above the bars indicate statistically significant differences (P < 0.05) as obtained by one‐way analysis of variance with least significance difference and Duncan’s multiple range test method. (f) Identifying the resistance of different wheat at the adult stage infected by Bgt E09 in the controlled glasshouse. (1) Bobwhite; (2) The transgenic Bobwhite with TiAP1 (OE1); (3) YN15; (4) F1 obtained from cross breeding between YN15 and homozygous transgenic TiAP1 wheat OE1.

Meanwhile, we crossbred between the homozygous transgenic TiAP1 Bobwhite and YN15 to obtain the hybrid F1. Gene amplification and phenotype investigation confirmed the inheritance of the TiAP1 gene and its function in resisting Bgt in the hybrid F1. A chi‐square test showed that the proportion of resistant‐susceptible separation in the F2 generation was 3:1. The subsequent F3 generation at the seedling stage (Figures S2C and S3; Table S1) also double confirmed TiAP1 endue the resistance to Bgt for wheat.

In the adult stage, through infection by Bgt E09 in the controlled glasshouse, the overexpression of TiAP1 gene in Bobwhite showed high resistance to Bgt (Figure 2f2, Figure S4; Table S2), whereas Bobwhite (Figure 2f1) and YN15 (Figure 2f3) showed high susceptibility to Bgt on the leaves with many lesions of more than 1 mm in diameter and more hyphae. At the same time, the hybrid F1 between the overexpression of TiAP1 gene in Bobwhite and YN15, showed high resistance to Bgt, where the diameter of the lesions was <1 mm, the amount of the lesions was little and the whole leaf surface was still green (Figure 2f4).

TiAP1 is a secretory protein that accumulates at the infection site

To determine the localization of TiAP1 in plant cells, we utilized the TiAP1‐GFP, Δsp‐TiAP1‐GFP and free GFP transient transformation in Nicotiana benthamiana. Through plasmolysis methods, we found that there was much fluorescent signal of TiAP1‐GFP outside the cell membrane, while that of Δsp‐TiAP1‐GFP (without signal peptide) was in the cytoplasm (Figure 3a,b,c, Figure S5). To further confirm whether TiAP1 is a secretory protein, the mCherry‐TaSYP51, which is a member of the syntaxin superfamily on plasma membrane, was co‐bombarded with TiAP1‐mYFP into the barley leaf epidermal cells. We found a large numbers of TiAP1 and TaSYP51 proteins accumulated at the infection site of the primary and App germination tube of Bgh, and TiAP1 extended to the barley intercellular space (Figure 3d; Video S1). Integrating with our observations of the Bgt on the leaves of TiAP1 gene transferred wheat, we speculated that TiAP1 restrains the powdery mildew from penetration, assisting the plants in resisting the invasion of Bgt.

Figure 3.

Figure 3

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Subcellular localization of the fusion protein through transient expression of agroinfiltrated GFP, TiAP‐GFP and Δsp‐TiAP‐GFP in the epidermal cells of N. benthamiana after plasmolysis, and co‐transient expression of TiAP1‐mYFP and mCherry‐TaSYP51 in the epidermal cells of barley through bombardment. (a) Confocal laser scanning microscopy (Leica SP5‐X, Wetzlar, Germany) observation of the leaf epidermal cells revealed that Δsp‐TiAP‐GFP appears to be localized in the cytoplasm, while TiAP1‐GFP is predominantly localized in the extracellular region, where white arrows indicate plasmolysis and the yellow dotted line shows the location of the cell membrane of the two adjacent cells. A GFP signal was used as the control. Bar = 20 µm. (b) The enlargement of the blue box in TiAP1‐GFP. (c) The enlargement of the blue box in Δsp‐TiAP1‐GFP. (d) B. graminis f. sp. hordei induced the accumulation of TiAP1‐mYFP and mCherry‐TaSYP51 at the infection site. This figure is part of Video S1. Bar = 20 µm.

The BgCDA1 interact with TiAP1

To clarify how TiAP1 can regulate wheat resistance to Bgt, CDS of TiAP1 without signal peptide was fused with the GAL4 DNA‐binding domain sequence to generate the bait vector. We performed a Y2H screen assay using Bgt cDNAs as a prey library. Interestingly, one of the obtained interactors was highly similar (95.06%) to the chitin deacetylase (EPQ66796.1) of Bgt. Furthermore, the analysis of the cDNA revealed that the related protein contains a polysaccharide deacetylation domain. Therefore, we named this gene chitin deacetylase (BgtCDA1) of Bgt (Figure S6A,B). The BgtCDA1 has an N‐terminal 19‐aminoacid signal peptide (http://www.cbs.dtu.dk/services/SignalP) and five conserved motifs that are required for hydrolysing the acetyl groups of the substrates (Figure S6C,D). Moreover, the point‐to‐point hybridization analysis verified the BgtCDA1 interacted with TiAP1 (Figure 4a).

Figure 4.

Figure 4

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Interaction of TiAP1 with BgtCDA1. (a) Yeast two‐hybrid analysis of the interaction between TiAP1 and BgtCDA1. Yeasts expressing the interacting combinations of bait and prey were spotted on the SD‐HAWL/X‐α‐Gal/ABA. Bait‐53 and AD‐T (prey) were the positive controls, while Bait‐Lam and AD‐T as well as Bait‐BD and AD‐BgtCDA1 were used as the negative controls. (b) Luciferase complementation imaging assay showing that TiAP1 interacted with and BgtCDA1. The ΔspTiAP1‐nLuc and cLuc‐ΔspBgtCDA1 were transiently co‐expressed in N. benthamiana. Co‐infiltration of cLuc/nLuc‐tagged ΔspTiAP1 protein with the nLuc/cLuc‐tagged ΔspBgtCDA1 or the empty vectors were used as negative controls, where the images were observed using chemiluminescence imaging 2 days later. (c) Bimolecular florescent complimentary assay showing TiAP1 interaction with BgtCDA1. The cGFP‐ΔspTiAP1 and nGFP‐ΔspBgtCDA1 were transiently co‐expressed in N. benthamiana. Co‐infiltrations of nGFP/cGFP‐ΔspTiAP1, cGFP/nGFP‐ΔspBgtCDA1 or the vectors group nGFP/cGFP were used as negative controls. The positive controls were nYFP‐14‐3‐3/14‐3‐3‐cCFP. The green fluorescent protein (GFP) signal was visualized using confocal microscopy (Leica SP5‐X). Bar = 20 µm.

Subsequently, we further confirmed the interaction through LCI, and BiFC. As expected, the LUC activity in the N. benthamiana leaves was high when co‐expressed using the ΔspTiAP1‐nLuc and ΔspBgtCDA1‐cLuc, while the other three combinations were used as negative controls (Figure 4b). The BiFC analysis also confirmed that the co‐expression of nGFP‐ΔspBgtCDA1 and cGFP‐ΔspTiAP1 resulted in a clear GFP signal in the cytoplasm. In contrast, no visible signal was detected in any of the corresponding negative controls (Figure 4c). Therefore, these observations indicate that there is an in vivo interaction between TiAP1 and BgtCDA1.

HIGS analysis showed that the silencing of BgtCDA1 inhibits the penetration and Hau formation of Bgt

To confirm the role of BgtCDA1 in the growth and development of Bgt, we used a transient transformation system based on the particle bombardment of RNAi constructs (Douchkov et al., 2005). The HI of the empty vector control was set to 100%, then the HI of the positive control, Mlo‐RNAi, was 47.44%, while that of BgtCDA1 RNAi was 55.36% (Figure 5a,b), Bgt haustorial formation rate was significantly decreased during silencing of BgtCDA1. And through BSMV‐VIGS, we also found that the lesions area in the leaves of BSMV: BgtCDA1as wheat infected with powdery mildew for 5 days and the hyphal density score of Bgt infection for 3 days were significantly lower than those of BSMV:00 wheat (Figure 5c–f). This indicated that BgtCDA1 gene silencing suppressed the growth of Bgt, and that is the BgtCDA1 gene promoted the invasion of Bgt.

Figure 5.

Figure 5

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The silencing of BgtCDA1 reduced Bgt infection and the BgtCDA1‐mediated deacetylation of chitohexaose (A6). (a) The haustorial index of wheat YN15 leaves, bombarded different RNAi together with GUS reporter constructs, and infected with Bgt. The haustorial index was calculated as the ratio of haustoria‐containing transformed cells (GUS expressing cells) divided by the total number of transformed cells. Data shown are mean values, n = 4, and a minimum of 150 transformed cells was counted in each repeat. The relative haustorial index was computed relative to the empty vector pIPKTA30N control of each experiment, which was set to 100%. The Mlo‐RNAi was another control. Different letters above the bars indicate statistically significant differences (P < 0.05) as obtained by one‐way analysis of variance with least significance difference and Duncan’s multiple range test method. (b) Wheat YN15 leaves epidermal cells, bombarded with RNAi and GUS reporter constructs, and infected with Bgt. Arrow indicates haustoria. Bar = 20 μm. (c) Disease phenotype of the BSMV:00 and BSMV: BgtCDA1as inoculated with E09. Photographs were taken at 5 dpi. (d) Microscopic observation of the disease symptoms of BSMV:00 and BSMV: BgtCDA1as 3 dpi using E09. Bar = 100 µm. (e) The proportion of lesions area on the leaves of BSMV:00 and BSMV: BgtCDA1as inoculated with E09 for 5 days. (f) The hyphal density score of BSMV:00 and BSMV: BgtCDA1as 3 dpi using E09. Different letters above the bars indicate statistically significant differences (P < 0.05) as obtained by one‐way analysis of variance with least significance difference and Duncan’s multiple range test method. (g) The deacetylation function of purified BgtCDA1. The partially deacetylated product A5D1 was detected after incubated for 5, or 30 min. A, N‐acetyl‐D‐glucosamine; D, D‐glucosamine.

The chitin deacetylase activity of BgtCDA1

We examined the chitin deacetylase activity of BgtCDA1. After Pichi yeast expression, the purified BgtCDA1‐His (2 μm) was incubated with 1 mm Chitooctaose with six GlcNAc moieties (A6) at 37 °C in 50 mm Tris‐HCl buffer (pH8.0), followed by detection of the enzymatic reaction products using MALDI‐TOF‐MS. As shown in Figure 5g, the deacetylated intermediate products A5D1 (where A is N‐acetyl‐D‐glucosamine and D is D‐glucosamine) were detected after 5‐ or 30‐min incubation. It showed that BgtCDA1 has the deacetylase activity.

TiAP1 and BgtCDA1 co‐regulate the expression of wheat pathogen‐responsive genes

To evaluate the impact of TiAP1 and BgtCDA1 on the pathogen‐responsive gene expression on a genome‐wide scale, we performed RNA‐Seq experiments using Bobwhite and OE2 seedlings inoculated with E09 for 2 days (Figure S7A). Genes with more than twofold change in expression (P < 0.01) were considered to be differentially expressed. Total 274 differentially expressed genes were detected in the OE2‐vs‐Bobwhite at 2 dpi, of which 116 genes were up‐regulated in the OE2‐2 (Figure S7B,C; Table S3). Gene ontology (GO) analysis indicated that these differentially expressed genes were enriched in the chitin catabolic process, cell wall macromolecule catabolic process, defence response to bacterium and fungus, chitinase activity and chitin‐binding (Figure S7D; Table S4). Defence‐related genes were also up‐regulated in OE2‐2 relative to Bob‐2 (Figure 6a; Table S5), such as traesCS1A02G410500, traesCS2B02G187500, traesCS2A02G161500, traesCS3B02G379200 and traesCS1B02G440300 that encode proteins containing WRKY domains; traesCS5A02G049600 encodes phytochrome‐interacting bHLH; traesCS7A02G201100 encodes TIFY family gene; traesCS5D02G188600 encodes protein phosphatase 2C (PP2C); traesCS6D02G217800 and traesCS3A02G404400 encode ERF transcription factors; traesCS6B02G018700 encodes LigB domain‐containing protein, and may involve in betaine metabolism; traesCS7D02G201600 and traesCS6D02G237900 are ubiquitin E3 ligase RING‐type related genes and traesCS6A02G266100 that encodes the xyloglucan glycosyltransferase/hydrolase and may be involved in cell wall xylan metabolism. By qRT‐PCR, we further analysed 12 genes which were differential expression induced by Bgt for 2 days. Among these 12 genes, the expression levels of TraesCS1B02G440300, TraesCS2A02G161500, TraesCS2A02G199300, TraesCS3B02G379200, TraesCS5D02G188600 and TraesCS5A02G049600 which related to plant hormone signal transduction were all up‐regulated in OE2‐2 relative to Bob‐2, beside TraesCS5D02G18860.0 which was no difference in OE2‐2 relative to Bob‐2 (Figure 6b). The TraesCS3B02G259500, TraesCS2B02G224300, TraesCS6A02G266100 and TraesCS2D02G099900 involved in cell wall anabolism, in which TraesCS3B02G259500 and TraesCS6A02G266100 were consistent with transcriptome were up‐regulated in OE2‐2 relative to Bob‐2, TraesCS2B02G224300 and TraesCS2D02G099900 were no difference in OE2‐2 relative to Bob‐2, but the expression in OE2‐2 was higher than Bob‐2, and TraesCS6B02G018700 and TraesCS2B02G529400 genes expression pattern were similar with TraesCS2B02G224300 and TraesCS2D02G099900 (Figure 6b). These quantitative results were slightly different from those at the transcriptome, possibly because of the differences between individuals. In summary, the up‐regulated expression of these genes provided favourable evidence to understand the disease‐resistant mechanism of the TiAP1 gene.

Figure 6.

Figure 6

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The transcriptome and qRT‐PCR analysis of the transgenic wheat with TiAP1. (a) Heat map of selected pathogen‐responsive genes in Bobwhite without and with induction of Bgt for 2 days (Bob‐0 and Bob‐2 respectively), and overexpression line OE2 as the same treatment (OE2‐0 and OE2‐2 respectively). (b) qRT‐PCR analysis of 12 selected genes induced by Bgt for 2 days.

Discussion

Aspartic proteases play a vital regulatory role in plant growth and may be involved in the senescence process (Chen et al., 2015), plant reproduction (Niu et al., 2013), programmed cell death (Chen and Foolad, 1997), resistance to pathogens (Prasad et al., 2010), response to stress (Guo et al., 2013) and endoproteases in plants (Rawlings and Barrett, 2016). If the endoproteases encoding genes were deleted or inhibited, the plant’s sensitivity to pathogens increases (Díaz et al., 2018; Jashni et al., 2015). Through multiple sequence comparisons, we found that the TiAP1 gene is 98% similar to scaffold 208 assembled from the T. intermedium genome (https://jgi.doe.gov/), and Yang and Feng (2020) analysed the aspartic protease gene family and their response under powdery mildew stress in wheat. Among those family genes, the AP gene which was most similar to TiAP1, TRIAE_CS42_5DS_TGACv1_456657_AA1475640.1, was located on the chromosome 5DS with 70% identity. The qRT‐PCR analysis showed that the TiAP1 gene was rapidly expressed under the induction of Bgt in SN6306 with no expression in YN15 (Figure 1a). The silencing of TiAP1 significantly destroyed the resistance to Bgt in the leaves of BMSV:TiAP1as SN6306 (Figure 1b–d). The lesion area on the leaves that smeared with the TiAP1 protein was remarkably decreased when compared with the control (Figure 1h–j). These results indicated that TiAP1 might resist the invasion of powdery mildew.

Furthermore, the identification of the Bgt resistance phenotype at the seedling stage showed that the overexpression of TiAP1 gene in wheat was moderately resistant to powdery mildew (Figure 2b), while the life cycle of Bgt in the 2‐day‐infected leaves of the overexpression of TiAP1 gene in Bobwhite was primarily retained at the App stage. In contrast, the Bgt on the Bobwhite leaves were mainly at Hau stage. Hau was wrapped in the extrahaustorial membrane (EHM), which was usually used for signal exchange and nutrient absorption (Panstruga, 2013), where the development of the Hau reflected the growth and metabolism of the entire pathogen to a certain extent. It was a vital trait for evaluating host cell resistance (Bracket, 1968).

The HI of Bgt in Bobwhite was significantly higher than that of the transgenic TiAP1 gene lines, indicating that overexpression of TiAP1 gene in Bobwhite was resistant to the Bgt invasion (Figure 2e). This resistance may be because the transgenic wheat inhibited the penetration of the Bgt, thereby affecting their infection rate. Subsequently, we found that the transgenic TiAP1 Bobwhite and the F1 generation showed high resistance to powdery mildew in the adult stage (Figure 2f2, f4, Figure S4). Many studies refer to this trait, which showed less significant resistance to the disease at the seedling stage than that of the adult stage (Hautea et al., 1987; Griffey and Das 1994), or of a chronic disease‐resistant type (Shaner, 1973). Moreover, Shaner (1973) found condia of Konx, a typical slow‐powdery mildew variety, could not successfully invade. Therefore, we speculated that the transgenic wheat line with the TiAP1 gene might be used as a chronic powdery mildew‐resistant variety to prevent the invasion of Bgt. Syntaxins were members of the SNARE superfamily of proteins that mediated membrane fusion events (Collins et al., 2003). Since TiAP1 was a secreted protein (Figure 3a–c) which expressed in large quantities at intercellular space of the infection site (Figure 3D, Video S1), we speculate that syntaxin TaSYP51 may help to transport the TiAP1 protein to the outside of the cell, where the detailed mechanisms need to be further elucidated.

Through the Y2H, LCI and BiFC in vivo verification, we determined TiAP1 interacts with BgtCDA1 (Figure 4). Subsequently, we found that when the BgtCDA1 gene was silenced, the HI, lesions area and hyphal density score were significantly reduced (Figure 5). Zhao and Park (2010) found that chitin deacetylase was secreted when it interacted with other substances. The chitin deacetylase of C. lindemuthianum was secreted during the process of fungal hyphae invading plants to modify the chitin that can be recognized by the plant’s resistance system (Tsigos and Bouriotis, 1995). Geoghegan and Gurr (2017) also found that CDA1 and CDA4 were necessary for chitin deacetylation in the cell wall of the mycelium in M. oryzae. During infection, the ability of the fungal cells to convert chitin to chitosan was critical for masking the cell wall chitin from being recognized by the host chitinases, thereby promoting the fungal attack (Upadhya et al., 2018). Chitin oligomers can also act as ligands that were recognized by LysM receptors on the cell surface and triggered pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) (Gao et al., 2019; Hurlburt et al., 2018). We found the silencing of BgtCDA1 can inhibit the invasion of Bgt and speculated that the interaction between BgtCDA1 and TiAP1 made BgtCDA1 be degraded by TiAP1 which inhibits the deacetylation function of BgtCDA1. The chitinase of the wheat was secreted to release the chitin oligomers, which were recognized by the wheat chitin receptor, such as LysM. This led to PTI and caused the transgenic wheat with the TiAP1 gene to be resistant to powdery mildew (Figure 7). When the pathogen invades the host, an extremely complex and precise arms race of ‘attack and defence’ occurs in the apoplastic space. Xia et al. (2020) found that soybean could secret an apoplastic aspartic protease, GmAP5, to bind and degrade a pathogen‐secreted apoplastic endoglucanase PsXEG1 to destroy its virulence in Phytophthora sojae invasion, and proposed a ‘multi‐layered immune model’ of the plants against pathogens in the extracellular region. Therefore, this study revealed the complexity of the interaction between pathogens and hosts as well as provided a theoretical basis to further study the role of TiAP1 in resistance mechanisms.

Figure 7.

Figure 7

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A proposed model of the TiAP1 interaction with chitin deacetylase to increase the resistance to Bgt in wheat. (a) The Bgt cell walls were targeted by wheat‐secreted chitinases to release the chitin fragments that can further activate the host immune system. (b) Bgt would secrete BgtCDA1 to convert the cell wall chitin into chitosan to protect the hyphae of the pathogenic fungi from being hydrolysed by the extracellular plant chitinases, thereby protecting the integrity of the fungal cell wall and inhibiting the resistance response mechanism of the plant. (c) Interaction between TiAP1 and BgtCDA1 causing BgtCDA1 to inactivate its deacetylation function, therefore the chitin in fungal cell walls would be targeted by the wheat‐secreted chitinases that liberate chitin the fragments that can further activate the host immune system. When the transgenic TiAP1 wheat was infected by Bgt, the hormone signals related gene expression in vivo would interact to activate plant disease resistance and up‐regulate the expression of phenylalanine ammonia lyases in the lignin biosynthesis and XTH30 in the xylan metabolism of the transgenic TiAP1 wheat. Hence, it enhances the ability of the plant cell walls to resist the penetration by pathogens.

When chitin receptors recognize the chitin, the plant can active defence genes and release some metabolites that initiate the signal transduction in plant cells to trigger an immune response. Many defence‐related genes were up‐regulated in OE2‐2 relative to Bob‐2 (Figure 6A; Table S5). WRKY TFs are key nodes in abscisic acid (ABA)‐responsive signalling networks (Rushton et al., 2012) and Zhang et al. (2015) found the OsWRKY24, ‐53 and ‐70 are involved in gibberellic acid. Therefore, we speculate that these genes may be involved in the ABA or gibberellic acid (GA) signalling pathways. Phytochrome‐interacting bHLH. which are known to play important roles in red light‐mediated (phyA and/or phyB‐dependent) signal transduction pathways at immediate positions downstream of the photoreceptor in A. thaliana (Nakamura et al., 2007). In A. thaliana, the TIFY family may be involved in jasmonic acid (JA) pathway (Chung and Howe, 2009). PP2Cs are vitally involved in ABA signalling (Park et al., 2009). The ERF transcription factor family is involved in ethylene‐activated signalling pathway (Xu et al., 2008). The metabolites include ABA, ethylene, JA, GA and ubiquitination that caused the plants to respond to the invasion of pathogens (Figure 7; Table S3). Moreover, RNA‐Seq and qRT‐PCR analysis revealed that there was a cell wall xylan metabolism gene, traesCS6A02G266100, whose expression in Bobwhite was sharply reduced after being induced by powdery mildew, while the gene in OE2‐2 was significantly up‐regulated relative to that of Bob‐2 (Figure 6).

Since xylan was a type of hemicellulose that was closely related to functional proteins in the cell wall, it had a great influence on the ductility, pore elasticity, flow and rheological properties of plant cells during growth (Zhou et al., 2017). Therefore, we infer that the penetration during the powdery mildew infection of the transgenic wheat with the TiAP1 gene was blocked. We also found that the TraesCS2B02G224300 gene encoded a phenylalanine ammonia‐lyase (PAL), which was up‐regulated and significant difference in OE2‐2 relative to Bob‐2 in transcriptome, (Figure 6a; Table S5) and in qRT‐PCR the expression of TraesCS2B02G224300 in OE2‐2 was twice higher than Bob‐2 (Figure 6b) and the PAL was pivotal for the enzymes related to the biosynthesis of lignin, salicylic acid and other phenylalanine‐derived metabolites (You et al., 2020). As a main component of the plant cell wall, lignin contributes to the formation of the physical and chemical barriers in plant immunity. Zhou et al. (2018) also found that OsPAL1‐mediated resistance to rice blast might be due to the accumulation of lignin, while the broad‐spectrum blast‐resistant rice varieties Pigm‐ and PIBP1‐mediated resistance might be related to lignin content (Zhai et al., 2019). Similarly, the overexpression of OsPAL8 in rice can lead to increased lignin content and enhanced brown planthopper resistance (He et al., 2020). Thus, these results demonstrate that the transgenic wheat with the TiAP1 gene could enhance resistance to Bgt.

In summary, we speculate the fungal chitin deacetylase BgtCDA1 can block the PAMP processes that the fungal‐derived chitin oligoes are recognized by wheat chitin receptor protein and then trigger an immune response, while TiAP1 competitively interacting with BgtCDA1 is the causal reason to re‐establish resistance to fungal pathogen Bgt. Moreover, the overexpression of TiAP1 up‐regulates the cell wall‐related genes in transgenic TiAP1 wheat thereby blocks the penetration of powdery mildew and enhances the resistance to pathogens. Furthermore, we conclude many other plants may share a similar mechanism and this mechanism can be used for future genetic improvement of crops. However, the mechanism of BgtCDA1 degraded by TiAP1, and the resulting detailed disease resistance pathway remains to be studied.

Materials and methods

Plant material and pathogen infection

The trititrigia SN6306 was highly resistant to the powdery mildew and obtained by hybridization between the highly susceptible to powdery mildew wheat (Triticum aestivum L.) cultivar Yannong 15 (YN15) and T. intermedium (Li et al., 2016). Wheat Bobwhite was highly susceptible to the powdery mildew. The transgenic overexpression TiAP1 in Bobwhite line 1 and 2 were named as OE1 and OE2 respectively. The untreated with Bgt wheat lines Bobwhite, OE1 and OE2 were named as Bob‐0, OE1‐0 and OE2‐0 respectively. The Bobwhite, OE1 and OE2 wheat inoculated with E09 for 2 days were named as Bob‐2, OE1‐2 and OE2‐2 respectively. The seeds of these materials above and N. benthamiana were sown in a mixed soil (loess, matrix and vermiculite of 1 : 1 : 1, v/v/v) at 25°C, 70% humidity and a long‐term photoperiod of a 16 h/8 h light/dark cycle in a glasshouse with a light intensity of 600 mmol/m2/s. The virulent isolate E09 of the wheat powdery mildew fungus (Bgt) was propagated on the wheat variety YN15 via a weekly inoculum transfer. The barley (Hordeum vulgare L.) line Ingrid ror2 (Collins et al., 2003) was highly susceptible to Blumeria graminis f. sp. hordei (Bgh) and used for the localization and expression profiling of TiAP1. The Bgh isolate B103 was propagated on barley P‐10 via a weekly inoculum transfer, while the Escherichia coli (E. coli) strain BL21 (DE3) was used for the prokaryotic expression of TiAP1. The prevailing Bgt isolate E09 was kindly provided by Dr. Hongjie Li, Institute of crop science, Chinese Academy of Agricultural Science.

VIGS, BSMV and host‐induced gene silencing (HIGS)

Virus‐induced gene silencing analysis was conducted according to Yuan et al. (2011) to insert the conserved sequence of the target TiAP1 or BgtCDA1 gene (316 or 300 bp respectively) into the BSMV vector to create BSMV:TiAP1as and BSMV:BgtCDA1as and transform the Agrobacterium. The detailed method was described in Methods S1. Ten days after inoculation with BSMV, six leaves were harvested to test the expression level of TiAP1 or BgtCDA1 gene, while the other six leaves were inoculated with Bgt race E09 and were observed and used to evaluate the resistance to the powdery mildew using a biological microscope (Nikon Ni‐U, Tokyo, Japan) after the Coomassie Brilliant Blue staining and the statistics of hyphal density score refers to the method of Norriss and Frost‐Smith (2007) and three biological replicates.

BgtCDA1 gene silencing in Bgt was performed using the HIGS method as described by Nowara et al. (2010). The RNA interference (RNAi) construct BgtCDA1 RNAi and a β‐glucuronidase (GUS) reporter gene construct were co‐transformed into the leaf epidermal cells of the wheat YN15 that grew for 7 days using particle bombardment. The fungal haustorial formation was examined for the transformed (GUS expressing) blue cells using a biological microscope (Nicon Ni‐U). The empty vectors pIPKTA30N, and the Mlo‐RNAi (pIPKTA36) constructs, were used as negative and positive controls, respectively, and four biological replicates were conducted per analysis with three technical replicates. The detailed method was described in Methods S1 according to Ahmed et al. (2015).

Constructing the TiAP1 prokaryotic expression plasmid and the protein expression

To improve the expression of TiAP1, the gene sequence was optimized according to E. coli bias, and constructed into the pET28a vector (containing 6xHis tags). Recombinant plasmids were transformed into BL21 (DE3) chemically competent cells where a single‐colony transformant was cultured and inoculated at 15 °C overnight induced expression with 0.25 mm isopropyl β‐ d‐1‐thiogalactopyranoside (IPTG). The cells were harvested and subjected to a sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) to detect the protein expression. Then, the recombinant TiAP1 was purified according to the method described in Methods S1.

Determination of the antifungal activity of TiAP1

To confirm the effect of TiAP1 protein on Bgt, we used the renatured TiAP1 and pET28a tag protein to add 0.025% Tween‐20 to smear the leaves of YN15 and inoculate with E09. After 5 days, the infectivity of Bgt was observed and the leaves inoculated for 2 days were stained with the Coomassie Brilliant Blue stain, as described previously (Göllner et al., 2008) and three biological replicates were conducted per analysis with three technical replicates. Lesion areas on the leaf surface were calculated according to Goodwin and Hsiang (2010) to analyse the pixels of the Bgt infection area relative to the normal leaf area.

Constructing the particle bombardment genetic transformation vector to transform wheat and identify the disease resistance phenotype

The coding sequence (CDS) of TiAP1 (KJ513672) was cloned into the Ubi‐gene‐Tnos vector with a ubiquitin (Ubi) promoter, yielding a recombinant pMUbi‐TiAP1 vector. The resulting plasmid pMUbi‐TiAP1 and the vector harbouring the Bar gene were mixed and co‐bombarded with a particle bombardment of 1100 psi, 1 μg per gun and 60 μg gold powder into the callus that was induced from the mature embryos of the Bobwhite wheat, which was highly susceptible to Bgt. During the induction and regeneration, the transformed wheat was screened using the herbicide Basta, while the presence of the transgenes was determined by amplifying the target gene using the pUbi‐ASPF/R primers (Table S6).

Next, the disease responses to powdery mildew of the transgenic plants were tested. When the Bobwhite and the transgenic Bobwhite with the TiAP1 gene grew to the three‐leaf stage, the first leaves of them were spread out on an acrylic plate with the conidium of the Bgt race E09 sprayed evenly. The leaves were harvested at 48 h post‐inoculation with E09 and stained with Coomassie Brilliant Blue as described previously (Göllner et al., 2008) and three biological replicates were conducted per stains with three technical replicates. Furthermore, to evaluate the resistance to Bgt, of the Bobwhite, SN6306, the transgenic Bobwhite with the TiAP1 gene, and the F1 obtained from cross breeding between YN15 and homozygous transgenic TiAP1 Bobwhite, they were evaluated under controlled greenhouse conditions. The infection type (IT) was recorded 15 days after inoculation and reconfirmed after 20 days with a scale of 0–4, according to Wang et al. (2015). The values 0–2 were classified as resistant, and 3–4 were as susceptible.

DNA and RNA extraction and qRT‐PCR

Total DNA was extracted from the plant leaves using the cetyltrimethylammonium bromide method (Allen et al., 2006). While total RNA (500 ng) was extracted using the EasyPure® Plant RNA kit (TransGen Biotech Co., Ltd, Beijing, China), reverse‐transcribed (RT) using a RevertAid First Strand cDNA synthesis kit (TransGen Biotech Co. Ltd) and quantified on an ABI Quantitative PCR Q6 Detection System (Thermo Fisher Scientific, Waltham, USA) with the SYBR Premix Ex Taq kit (TransGene Biotech Co., Ltd). The wheat actin gene was used as the reference gene, in which three independent biological replicates were conducted per analysis with at least three technical replicates. The primers have been listed in Table S6.

Subcellular localization

The CDS of the TiAP1 fragments was fused green fluorescent protein (GFP) in the vector pCAMBIA1300. Then the constructed TiAP1‐GFP or Δsp‐TiAP1‐GFP (with or without signal peptide of TiAP1 respectively) were transferred into the Agrobacterium tumefaciens GV3101 and infiltrated into N. benthamiana leaves. Two days after transiently expression, they were observed using a confocal laser scanning microscope (Leica SP5‐X, Germany), 10 N. benthamiana leaves were analysed in each of the three experiments and the detailed method was described in Methods S1.

Transient expression of the TiAP1 protein induced by Bgh in barley leaves

The expression vector of the TiAP1 protein was constructed using the gateway system (Thermo Fisher Scientific, Waltham, USA). The two recombinant plasmids, TiAP1‐mYFP and mCherry‐TaSYP51, were co‐bombarded onto the leaves of the 7‐day‐old barley by particle bombardment at a helium pressure of 1100 psi and placed on the 1% PhytagelTM Petri dishes containing 40 mg/mL of benzimidazole (Sigma‐Aldrich, St Louis MO, USA) to culture for 24 h, and then inoculated with Bgh spores. After an additional 24 h of inoculation, the expression of TiAP1 and TaSYP51 proteins were observed and analysed using a confocal laser‐scanning microscope (Leica SP5‐X) according to the methodology of Smigielski et al. (2019). And three biological replicates were conducted per co‐bombarded with six leaves.

Protein interaction assay

We sampled the leaves of YN15 and SN6306 every day, which were infected with Bgt from 0 to 5 days, and then construct a primary cDNA library using the BP reaction of the CloneMiner II kit (Invitrogen, Thermo Fisher Scientific) by Qingdao Oebiotech Co. Ltd. (China). The quality of the cDNA library was identified. Next, we used the nuclear system Y2H mating standard operating procedure method as described in the manufacturer’s instructions (Clontech Laboratories, Inc., Palo Alto, USA) to screen the protein that interacts with TiAP1 and further verified the obtained interaction protein, and three biological replicates were conducted per Y2H verify. The structure, amino acid sequence domain and the signal peptide of the obtained protein BgCDA1 were analysed using the Basic Local Alignment Search Tool (PBLAST; Altschul et al., 1997), Pfam (Finn et al., 2014) and SignalP 5.0 (Almagro Armenteros et al., 2019) software respectively.

For LCI assay, by amplifying the sequence of TiAP1 and BgtCDA1 and applying homologous recombination with the nLuc and cLuc, as previously described (Chen et al., 2008), the fusion protein and one of the corresponding co‐injected empty vectors, nLuc/cLuc, was co‐expressed in N. benthamiana as a negative control. At 48 h post‐infiltration, the LUC activity of the leaves was observed using an in vivo imaging system (Berthold LB985 NightSHADE, Bad Wildbad, Germany). Ten N. benthamiana leaves were analysed in each of the three experiments.

For BiFC assay, the sequences of TiAP1 and BgtCDA1 were amplified and fused with the N and C terminals of the GFP, respectively, using the gateway system (Thermo Fisher Scientific, Waltham, USA). As mentioned previously (Bracha‐Drori et al., 2004), the fusion protein and one of the corresponding empty GFP‐N/GFP‐C vectors were co‐expressed in N. benthamiana as a negative control. The GFP signal was observed 48 h after infiltration using a confocal laser‐scanning microscope (Leica SP5‐X, Wetzlar, Germany). Ten N. benthamiana leaves were analysed in each of the three experiments.

The yeast expression, purification and chitin deacetylase activity assay of BgtCDA1

For protein expression and purification in yeast, BgtCDA1 coding sequences with a 6xHis tag were amplified and ligated into pPIC9K‐HIS vector (Invitrogen), and then transformed into Pichia pastoris strain GS115 (Invitrogen). The positive transformants were induced protein expression using methanol. The target proteins were purified using Ni‐column affinity chromatography. Purified protein was used to detect the enzyme activity of BgtCDA1 by measuring the amount of released acetate using ion chromatography and qualitative analysis of deacetylated products by MALDI‐TOF by Hoogen Biotech, Shanghai, China according to Gao et al. (2019). MALDI‐TOF MS analysis was performed on an Ultraflex Extreme MALDI‐TOF‐TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The standard reaction mixture (200 μl) containing 2 μm BgtCDA1, 50 mm Tris‐HCl (pH8.0) and 1 mm chitin oligomers with 6 GlcNAc moieties (A6) as the substrate were incubated at 37 °C for 5, 30 min followed by heating at 100 °C for 10 min. The mixture without BgtCDA1 was the control. The yeast expression, purification and enzyme activity assay of BgtCDA1 were detailed performed described in Methods S1.

RNA‐Seq and data analysis

RNA‐Seq analysis was performed using the three‐leaf stage Bobwhite (Bob‐0) and the transgenic Bobwhite with the TiAP1 gene (OE2‐0) as well as a Bobwhite inoculated with E09 2 days later (Bob‐2) and the corresponding transgenic Bobwhite with the TiAP1 gene (OE2‐2). Three biological replicates were used for the RNA extraction, where total RNA was extracted and treated with DNase I, in which the quality test was performed, and then used to construct the library. The quality of the library was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, USA), while the libraries were sequenced on an Illumina HiSeq 2500 platform (Illumina, Inc., San Diego, USA). The RNA‐Seq reads were aligned to the wheat genome of the International Wheat Genome Sequencing Consortium with the RefSeq v1.1 annotation (https://wheat‐urgi.versailles.inra.fr/Seq‐Repository/Annotations). Differentially expressed genes across the samples were identified using the DESeq2 package (Love and Huber, 2014) using the standard parameters with an adjusted false‐discovery rate of P‐value <0.01 and fold change >2.

Statistical analysis

The statistical significance of the results was calculated using a one‐way analysis of variance followed by least significant difference and Duncan’s new multiple range test at a significant difference (P < 0.05) using IBM SPSS software version 19.0 (Chicago, IL). All experiments reported include three to six biological replicates and a minimum of three technical replicates with similar results. All primers have been listed in Table S6.

Accession numbers

The cDNA and coded protein sequence of TiAP1 sequence in this research has been deposited in NCBI under the accession number KJ513672 and AJC64141.1; Blumeria graminis f. sp. tritici BgtCDA1 was coded_by =" join (KE374986.1:122515..122759, KE374986.1:122809..123142, KE374986.1:123188..123296, KE374986.1:123342..123381, KE374986.1:123432..123502, KE374986.1:123547..123801, KE374986.1:123919..123923, KE374986.1:123978..124142, KE374986.1:124319..124375), and had one and two additional alternative splicings compared with EPQ66796 between KE374986.1:123143 and KE374986.1:123187, KE374986.1:123802 and KE374986.1:123977 respectively; Other protein sequence data from this article can be found in the NCBI under the following accession numbers: Saccharomyces cerevisiae CDA1 (NP_013410) and Saccharomyces cerevisiae CDA2 (NP_013411). TaSYP51 was coded by sequence in chr5A of wheat between 67321475 and 67319313.

Conflict of interests

The authors have no competing interests to declare.

Author contributions

D.F., H.W. and Y.Y. conceived and designed the experiments. Y.Y., P.F., J.L., W.X., N.L., Z.N., Q.L., J.S., Q.T. and Y.B. performed the experiments and analysed the data; Y.Y. and D.F. wrote the paper; D.F. and H.W. conceived, directed and coordinated the project.

Supporting information

Figures S1. The prokaryotic expression and purification of TiAP1.

Figures S2. Identification of the transgenic TiAP1 gene Bobwhite and the subsequent hybrid generation.

Figures S3. Validation of Bgt resistance of the representative hybrid subsequent generation of F3, which showed resistance to E09 at 7 dpi at the seedling stage.

Figures S4. Validation of the wheat powdery mildew resistance in seven transgenic wheat Bobwhite with the TiAP1 gene at the adult stage, which showed high resistance to Bgt. Bobwhite was used as the susceptible control.

Figures S5.Subcellular localization of the fusion protein in the N. benthamiana leaf epidermal cells.

Figures S6. The BgtCDA1 protein sequence analysis.

Figures S7. Transcriptome analysis of the TiAP1 and BgtCDA1 co‐regulated genes.

PBI-20-454-s002.pdf (3MB, pdf)

Tables S1. Evaluation of the resistance to E09 in YN15, wheat lines overexpressing TiAP1, and the hybrid subsequent generation of F3 in the seedling stage.

Tables S2.Evaluation of the resistance to powdery mildew in Bobwhite and wheat lines overexpressing TiAP1 at the adult stage.

Tables S3. Differential gene expression in the transgenic lines overexpression OE2 and Bobwhite when inoculated with E09 for 2 days.

Tables S4. Gene ontology (GO) analysis of the differentially expressed genes in OE2‐2 and Bob‐2.

Tables S5.Expression of selected pathogen‐responsive genes in Bob‐0, Bob‐2, OE2‐0 and OE2‐2 in the RNA‐Seq experiments.

Tables S6. The primers used in this study.

PBI-20-454-s001.pdf (112.4KB, pdf)

Supplementary Material. Additional description of methods.

PBI-20-454-s003.docx (21KB, docx)

Video S1. Confocal microscopy (Leica SP5‐X, Germany) z‐scan image series of TiAP‐mYFP/mCherry‐TaSYP51 on barley leaf epidermal cell under the infection of Bgh.

Download video file (27MB, avi)

Acknowledgements

This work was supported by the grants from the National Natural Science Foundation of China (31771777); Shandong ‘Double Tops’ Program; the Major Basic Research Project of Shandong Natural Science Foundation (2017C03) and the Overseas Visiting Programme for Graduate Mentors of Shandong Province. We are very grateful to Prof. Hans Thordal‐Christensen’s group, University of Copenhagen, Denmark, for providing experimental assistance, to Dr. Patrick Schweizer, IPK, Gatersleben, Germany, for supplying the RNAi vector and the Mlo‐RNAi construct, to Prof. Pinghua Li, Shandong Agricultural University, for providing the LCI system, and to Prof. Dawei Li, China Agriculture University, for providing the BSMV‐RNAi system. Confocal image data were collected at the Center for Advanced Bioimaging (CAB), University of Copenhagen, Denmark.

Yang, Y. , Fan, P. , Liu, J. , Xie, W. , Liu, N. , Niu, Z. , Li, Q. , Song, J. , Tian, Q. , Bao, Y. , Wang, H. and Feng, D. (2022) Thinopyrum intermedium TiAP1 interacts with a chitin deacetylase from Blumeria graminis f. sp. tritici and increases the resistance to Bgt in wheat. Plant Biotechnol. J., 10.1111/pbi.13728

References

  1. Ahmed, A.A. , Pedersen, C. , Schultz‐Larsen, T. , Kwaaitaal, M. , Jørgensen, H.J.L. and Thordal‐Christensen, H. (2015) The barley powdery mildew candidate secreted effector protein CSEP0105 inhibits the chaperone activity of a small heat shock protein. Plant Physiol. 168, 321–333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alam, M.M. , Nakamura, H. , Ichikawa, H. , Miyao, A. , Hirochika, H. , Kobayashi, K. , Yamaoka, N. et al. (2014) Response of anaspartic protease gene OsAP77 to fungal, bacterial and viral infections in rice. Rice, 7, 1–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Allen, G.C. , Flores‐Vergara, M.A. , Krasynanski, S. , Kumar, S. and Thompson, W.F. (2006) A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat. Protoc. 1, 2320–2325. [DOI] [PubMed] [Google Scholar]
  4. Almagro Armenteros, J.J. , Tsirigos, K.D. , Sønderby, C.K. , Petersen, T.N. , Winther, O. , Brunak, S. , von Heijne, G. et al. (2019) SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 37, 420–423. [DOI] [PubMed] [Google Scholar]
  5. Altschul, S.F. , Madden, T.L. , Schäffer, A.A. , Zhang, J. , Zhang, Z. , Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI‐BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bracha‐Drori, K. , Shichrur, K. , Katz, A. , Oliva, M. , Angelovici, R. , Yalovsky, S. and Ohad, N. (2004) Detection of protein–protein interactions in plants using bimolecular fluorescence complementation. Plant J. 40, 419–427. [DOI] [PubMed] [Google Scholar]
  7. Bracket, C.E. (1968) The ultrastructure of the haustorial apparatus of Erysiphe graminis and its relationship to the epidermal cell of barley. Phytopathology, 58, 12–30. [Google Scholar]
  8. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein‐dye binding. Anal. Biochem. 72, 248–254. [DOI] [PubMed] [Google Scholar]
  9. Chen, F. and Foolad, M.R. (1997) Molecular organization of a gene in barley which encodes a protein similar to aspartic protease and its specific expression in nucellar cells during degeneration. Plant Mol. Biol. 35, 821–831. [DOI] [PubMed] [Google Scholar]
  10. Chen, H.J. , Huang, Y.H. , Huang, G.J. , Huang, S.S. , Chow, T.J. and Lin, Y.H. (2015) Sweet potato SPAP1 is a typical aspartic protease and participates in ethephon‐mediated leaf senescence. J. Plant Physiol. 180, 1–17. [DOI] [PubMed] [Google Scholar]
  11. Chen, H. , Zou, Y. , Shang, Y. , Lin, H. , Wang, Y. , Cai, R. , Tang, X. et al. (2008) Firefly luciferase complementation imaging assay for protein‐protein interactions in plants. Plant Physiol. 146, 368–376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chen, P. , You, C. , Hu, Y. , Chen, S. , Zhou, B. , Cao, A. and Wang, X. (2013) Radiation‐induced translocations with reduced Haynaldia villosa chromatin at the Pm21 locus for powdery mildew resistance in wheat. Mol. Breeding, 31, 477–484. [Google Scholar]
  13. Chung, H.S. and Howe, G.A. (2009) A critical role for the TIFY motif in repression of jasmonate signaling by a stabilized splice variant of the JASMONATE ZIM‐domain protein JAZ10 in Arabidopsis. Plant Cell, 21, 131–145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Collins, N.C. , Thordal‐Christensen, H. , Lipka, V. , Bau, S. , Kombrink, E. , Qiu, J.‐L. , Hückelhoven, R. et al. (2003) SNARE‐protein‐mediated disease resistance at the plant cell wall. Nature, 425, 973–977. [DOI] [PubMed] [Google Scholar]
  15. Deising, H.B. , Werner, S. and Wernitz, M. (2000) The role of fungal appressoria in plant infection. Microbes Infect. 2, 1631–1641. [DOI] [PubMed] [Google Scholar]
  16. Díaz, M.E. , Rocha, G.F. , Kise, F. , Rosso, A.M. , Guevara, M.G. and Parisi, M.G. (2018) Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits. Lett. Appl. Microbiol. 67, 168–174. [DOI] [PubMed] [Google Scholar]
  17. Douchkov, D. , Nowara, D. , Zierold, U. and Schweizer, P. (2005) A high‐throughput gene‐silencing system for the functional assessment of defense‐related genes in barley epidermal cells. Mol. Plant Microbe Interact. 18, 755–761. [DOI] [PubMed] [Google Scholar]
  18. Finn, R.D. , Bateman, A. , Clements, J. , Coggill, P. , Eberhardt, R.Y. , Eddy, S.R. , Heger, A. et al. (2014) Pfam: the protein families database. Nucleic Acids Res. 42, D222–D230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gao, F. , Zhang, B.S. , Zhao, J.H. , Huang, J.F. , Jia, P.S. , Wang, S. , Zhang, J. et al. (2019) Deacetylation of chitin oligomers increases virulence in soil‐borne fungal pathogens. Nat. Plants, 5, 1167–1176. [DOI] [PubMed] [Google Scholar]
  20. Geoghegan, I.A. and Gurr, S.J. (2017) Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae . Cell Microbiol. 19, e12743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Geziel, B.A. (2015) On the mechanism of action of selected effector candidates from the barley powdery mildew fungus. PhD thesis, University of Copenhagen, Copenhagen, Denmark. [Google Scholar]
  22. Göllner, K. , Schweizer, P. , Bai, Y. and Panstruga, R. (2008) Natural genetic resources of Arabidopsis thaliana reveal a high prevalence and unexpected phenotypic plasticity of RPW8‐mediated powdery mildew resistance. New Phytol. 177, 725–742. [DOI] [PubMed] [Google Scholar]
  23. Goodwin, P.H. and Hsiang, T. (2010) Quantification of fungal infection of leaves with digital images and scion image software. In Molecular and Cell Biology Methods for Fungi, Methods in Molecular Biology, Vol. 638 ( Sharon, A. , ed.), pp. 125–134;New York, NY:Springer Science+Business Media, LLC. [DOI] [PubMed] [Google Scholar]
  24. Griffey, C. and Das, M. (1994) Inheritance of adult‐plant resistance to powdery mildew in Knox62 and Massey winter wheats. Crop Sci. 34, 641–646. [Google Scholar]
  25. Guo, R. , Tu, M. , Wang, X. , Zhao, J. , Wan, R. , Li, Z. , Wang, Y. et al. (2016) Ectopic expression of a grape aspartic protease gene, AP13, in Arabidopsis thaliana improves resistance to powdery mildew but increases susceptibility to Botrytis cinerea . Plant Sci. 248, 17–27. [DOI] [PubMed] [Google Scholar]
  26. Guo, R. , Xu, X. , Carole, B. , Li, X. , Gao, M. , Zheng, Y. and Wang, X. (2013) Genome‐wide identification, evolutionary and expression analysis of the aspartic protease gene superfamily in grape. BMC Genom. 14, 554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Han, Y. , Song, L. , Peng, C. , Liu, X. , Liu, L. , Zhang, Y. , Wang, W. et al. (2019) A Magnaporthe chitinase interacts with a rice jacalin‐related lectin to promote host colonization. Plant Physiol. 179, 1416–1430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hautea, R.A. , Coffman, W.R. , Sorrells, M.E. and Bergstrom, G.C. (1987) Inheritance of partial resistance to powdery mildew in spring wheat. Theor. Appl. Genet. 73, 609–615. [DOI] [PubMed] [Google Scholar]
  29. He, J. , Liu, Y. , Yuan, D. , Duan, M. , Liu, Y. , Shen, Z. , Yang, C. et al. (2020) An R2R3 MYB transcription factor confers brown planthopper resistance by regulating the phenylalanine ammonia‐lyase pathway in rice. Proc. Natl Acad. Sci. USA, 117, 271–277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. He, R. , Chang, Z. , Yang, Z. , Yuan, Z. , Zhan, H. , Zhang, X. and Liu, J. (2009) Inheritance and mapping of powdery mildew resistance gene Pm43 introgressed from Thinopyrum intermedium into wheat. Theor. Appl. Genet. 118, 1173–1180. [DOI] [PubMed] [Google Scholar]
  31. Hurlburt, N.K. , Chen, L.H. , Stergiopoulos, I. and Fisher, A.J. (2018) Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)6 reveals the ligand‐binding mechanism and uncouples its intrinsic function from recognition by the Cf‐4 resistance protein. PLoS Pathog. 14, e1007263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Jashni, M.K. , Mehrabi, R. , Collemare, J. , Mesarich, C.H. and de Wit, P.J.G.M. (2015) The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant‐pathogen interactions. Front. Plant Sci. 6, 584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kamakura, T. , Yamaguchi, S. , Saitoh, K.I. , Teraoka, T. and Yamaguchi, I. (2002) A novel gene, CBP1, encoding a putative extracellular chitin‐binding protein, may play an important role in the hydrophobic surface sensing of Magnaporthe grisea during appressorium differentiation. Mol. Plant Microbe Interact. 15, 437–444. [DOI] [PubMed] [Google Scholar]
  34. Kuroki, M. , Okauchi, K. , Yoshida, S. , Ohno, Y. , Murata, S. , Nakajima, Y. , Nozaka, A. et al. (2017) Chitin‐deacetylase activity induces appressorium differentiation in the rice blast fungus Magnaporthe oryzae . Sci. Rep. 7, 9697. https://pubmed.ncbi.nlm.nih.gov/28852173/ [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Li, H. , Dong, Z. , Ma, C. , Xia, Q. , Tian, X. , Sehgal, S. , Koo, D.H. et al. (2020a) A spontaneous wheat‐Aegilops longissima translocation carrying Pm66 confers resistance to powdery mildew. Theor. Appl. Genet., 133, 1149–1159. [DOI] [PubMed] [Google Scholar]
  36. Li, M. , Dong, L. , Li, B. , Wang, Z. , Xie, J. , Qiu, D. , Li, Y. et al. (2020b) A CNL protein in wild emmer wheat confers powdery mildew resistance. New Phytol. 228, 1027–1037. [DOI] [PubMed] [Google Scholar]
  37. Li, Q. , Niu, Z. , Bao, Y. , Tian, Q. , Wang, H. , Kong, L. and Feng, D. (2016) Transcriptome analysis of genes related to resistance against powdery mildew in wheat‐Thinopyrum alien addition disomic line germplasm SN6306. Gene, 590, 5–17. [DOI] [PubMed] [Google Scholar]
  38. Love, M.I. , Huber, W. and Anders, S. (2014) Moderated estimation of fold change and dispersion for RNA‐seq data with DESeq2. Genome Biol. 15, 550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Luo, P. , Luo, H. , Chang, Z. , Zhan, H. , Zhang, M. and Ren, Z. (2009) Characterization and chromosomal location of Pm40 in common wheat: a new gene for resistance to powdery mildew derived from Elytrigia intermedium . Theor. Appl. Genet. 118, 1059–1064. [DOI] [PubMed] [Google Scholar]
  40. Nakamura, Y. , Kato, T. , Yamashino, T. , Murakami, M. and Mizuno, T. (2007) Characterization of a set of phytochrome‐interacting factor‐like bHLH proteins in Oryza sativa . Biosci. Biotechnol. Biochem. 71, 1183–1191. [DOI] [PubMed] [Google Scholar]
  41. Niu, N. , Liang, W. , Yang, X. , Jin, W. , Wilson, Z.A. , Hu, J. and Zhang, D. (2013) EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice. Nat. Commun. 4, 1445. [DOI] [PubMed] [Google Scholar]
  42. Norriss, M.G. , Frost‐Smith, T.J. and Sutherland, J.I.M. (2007) Increasing endophyte alkaloid expression in tall fescue by selecting for increased endophyte hyphal density. Proc. N Z Grassland Assoc. 69, 197–200. [Google Scholar]
  43. Nowara, D. , Gay, A. , Lacomme, C. , Shaw, J. , Ridout, C. , Douchkov, D. , Hensel, G. et al. (2010) HIGS: host‐induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis . Plant Cell, 22, 3130–3141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Panstruga, R. (2013) Establishing compatibility between plants and obligate biotrophic pathogens. Curr. Opin. Plant Biol. 6, 320–326. [DOI] [PubMed] [Google Scholar]
  45. Park, S.Y. , Fung, P. , Nishimura, N. , Jensen, D.R. , Fujii, H. , Zhao, Y. , Lumba, S. et al. (2009) Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science, 324, 1068–1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Prasad, B.D. , Creissen, G. , Lamb, C. and Chattool, B.B. (2009) Overexpression of rice (Oryza sativa L.) OsCDR1 leads to constitutive activation of defense responses in rice and Arabidopsis. Mol. Plant Microbe Interact. 22, 1635–1644. [DOI] [PubMed] [Google Scholar]
  47. Prasad, B.D. , Creissen, G. , Lamb, C. and Chattoo, B.B. (2010) Heterologous expression and characterization of recombinant OsCDR1, a rice aspartic proteinase involved in disease resistance. Protein Expr. Purif. 72, 169–174. [DOI] [PubMed] [Google Scholar]
  48. Rawlings, N.D. and Barrett, A.J. (1995) Families of aspartic peptidases, and those of unknown catalytic mechanism. Methods Enzymol. 248, 105–120. [DOI] [PubMed] [Google Scholar]
  49. Rawlings, N.D. , Barrett, A.J. and Finn, R. (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 44, 343–350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rushton, D.L. , Tripathi, P. , Rabara, R.C. , Lin, J. , Ringler, P. , Boken, A.K. , Langum, T.J. et al. (2012) WRKY transcription factors: key components in abscisic acid signalling. Plant Biotechnol. J. 10, 2–11. [DOI] [PubMed] [Google Scholar]
  51. Shaner, G. (1973) Evaluation of slow‐mildewing resistance of Knox wheat in the field. Phytopathology, 63, 867–872. [Google Scholar]
  52. Shen, X.K. , Ma, L.X. , Zhong, S.F. , Liu, N. , Zhang, M. , Chen, W.Q. , Zhou, Y.L. et al. (2015) Identification and genetic mapping of the putative Thinopyrum intermedium‐derived dominant powdery mildew resistance gene PmL962 on wheat chromosome arm 2BS. Theor. Appl. Genet. 128, 517–528. [DOI] [PubMed] [Google Scholar]
  53. Singh, R.P. , Singh, P.K. , Rutkoski, J. , Hodson, D.P. , He, X. , Jørgensen, L.N. , Hovmøller, M.S. et al. (2016) Disease impact on wheat yield potential and prospects of genetic control. Annu. Rev. Phytopathol. 54, 303–322. [DOI] [PubMed] [Google Scholar]
  54. Smigielski, L. , Aguilar, G.B. , Kwaaitaal, M. , Zhang, W.J. and Thordal‐Christensen, H. (2019) The isoelectric point of proteins influences their translocation to the extrahaustorial matrix of the barley powdery mildew fungus. Cell. Microbiol. 21, e13091. [DOI] [PubMed] [Google Scholar]
  55. Tian, Q.J. , Song, J. , Yang, Y.L. , Xu, T. , Wang, H.G. and Feng, D.S. (2017) Cloning and Bioinformatics of TiAP1 Gene from Thinopyrum intermedium . Shandong Agric. Sci. 49, 1–9. [Google Scholar]
  56. Tsigos, I. and Bouriotis, V. (1995) Purification and characterization of chitin deacetylase from Colletotrichum lindemuthianum . J. Biol. Chem. 270, 26286–26291. [DOI] [PubMed] [Google Scholar]
  57. Upadhya, R. , Baker, L.G. , Lam, W.C. , Specht, C.A. , Donlin, M.J. and Lodge, J.K. (2018) Cryptococcus neoformans Cda1 and its chitin deacetylase activity are required for fungal pathogenesis. mBio, 9(6), e02087–e2118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wang, Y. , Wang, Y. and Wang, Y. (2020) Apoplastic proteases: powerful weapons against pathogen infection in plants. Plant Commun. 1, 100085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wang, Z. , Li, H. , Zhang, D. , Guo, L. , Chen, J. , Chen, Y. , Wu, Q. et al. (2015) Genetic and physical mapping of powdery mildew resistance gene MlHLT in Chinese wheat landrace Hulutou. Theor. Appl. Genet. 128, 365–373. [DOI] [PubMed] [Google Scholar]
  60. Xia, Y.Q. , Ma, Z.C. , Qiu, M. , Guo, B.D. , Zhang, Q. , Jiang, H.B. , Zhang, B.Y. et al. (2020) N‐glycosylation shields Phytophthora sojae apoplastic effector PsXEG1 from a specific host aspartic protease. Proc. Natl Acad. Sci. USA, 117, 27685–27693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Xia, Y. , Suzuki, H. , Borevitz, J. , Blount, J. , Guo, Z. , Patel, K. , Dixon, R.A. et al. (2004) An extracellular aspartic protease functions in Arabidopsis disease resistance signaling. EMBO J. 23, 980–988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Xu, Z.S. , Chen, M. , Li, L.C. and Ma, Y.Z. (2008) Functions of the ERF transcription factor family in plants. Botany, 86, 969–977. [Google Scholar]
  63. Yang, C. , Yu, Y. , Huang, J. , Meng, F. , Pang, J. , Zhao, Q. , Islam, M.A. et al. (2019) Binding of the Magnaporthe oryzae chitinase MoChia1 by a rice tetratricopeptide repeat protein allows free chitin to trigger immune responses. Plant Cell, 31, 172–188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yang, Y.L. and Feng, D.S. (2020) Genome‐wide identification of the aspartic protease gene family and their response under powdery mildew stress in wheat. Mol. Biol. Rep. 47, 8949–8961. [DOI] [PubMed] [Google Scholar]
  65. You, X.M. , Fang, H. , Wang, R.Y. , Wang, G.L. and Ning, Y.S. (2020) Phenylalanine ammonia lyases mediate broad‐spectrum resistance to pathogens and insect pests in plants. Sci. Bull. 65, 1425–1427. [DOI] [PubMed] [Google Scholar]
  66. Yuan, C. , Li, C. , Yan, L. , Jackson, A.O. , Liu, Z. , Han, C. , Yu, J. et al. (2011) A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots. PLoS One, 6, e26468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Zhai, K. , Deng, Y. , Liang, D.I. , Tang, J. , Liu, J. , Yan, B. , Yin, X. et al. (2019) RRM transcription factors interact with NLRs and regulate broad‐spectrum blast resistance in rice. Mol. Cell, 74, 996–1009. [DOI] [PubMed] [Google Scholar]
  68. Zhang, L. , Gu, L. , Ringler, P. , Smith, S. , Rushton, P.J. and Shen, Q.J. (2015) Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells. Plant Sci. 236, 214–222. [DOI] [PubMed] [Google Scholar]
  69. Zhang, Z. , Henderson, C. , Perfect, E. , Carver, T.L.W. , Thomas, B.J. , Skamnioti, P. and Gurr, S.J. (2005) Of genes and genomes, needles and haystacks: Blumeria graminis and functionality. Mol. Plant Pathol. 6, 561–575. [DOI] [PubMed] [Google Scholar]
  70. Zhao, Y. , Park, R.D. and Muzzarelli, R.A. (2010) Chitin deacetylases: properties and applications. Mar. Drug. 8, 24–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Zhou, S. , Miao, Q.X. , Huang, L.L. and Chen, L.H. (2017) Research progress in hemicellulose‐based functional materials. Chem. Indus. for. Prod. 37, 10–18. [Google Scholar]
  72. Zhou, X. , Liao, H. , Chern, M. , Yin, J. , Chen, Y. , Wang, J. , Zhu, X. et al. (2018) Loss of function of a rice TPR‐domain RNA‐binding protein confers broad‐spectrum disease resistance. Proc. Natl Acad. Sci. USA, 115, 3174–3179. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figures S1. The prokaryotic expression and purification of TiAP1.

Figures S2. Identification of the transgenic TiAP1 gene Bobwhite and the subsequent hybrid generation.

Figures S3. Validation of Bgt resistance of the representative hybrid subsequent generation of F3, which showed resistance to E09 at 7 dpi at the seedling stage.

Figures S4. Validation of the wheat powdery mildew resistance in seven transgenic wheat Bobwhite with the TiAP1 gene at the adult stage, which showed high resistance to Bgt. Bobwhite was used as the susceptible control.

Figures S5.Subcellular localization of the fusion protein in the N. benthamiana leaf epidermal cells.

Figures S6. The BgtCDA1 protein sequence analysis.

Figures S7. Transcriptome analysis of the TiAP1 and BgtCDA1 co‐regulated genes.

PBI-20-454-s002.pdf (3MB, pdf)

Tables S1. Evaluation of the resistance to E09 in YN15, wheat lines overexpressing TiAP1, and the hybrid subsequent generation of F3 in the seedling stage.

Tables S2.Evaluation of the resistance to powdery mildew in Bobwhite and wheat lines overexpressing TiAP1 at the adult stage.

Tables S3. Differential gene expression in the transgenic lines overexpression OE2 and Bobwhite when inoculated with E09 for 2 days.

Tables S4. Gene ontology (GO) analysis of the differentially expressed genes in OE2‐2 and Bob‐2.

Tables S5.Expression of selected pathogen‐responsive genes in Bob‐0, Bob‐2, OE2‐0 and OE2‐2 in the RNA‐Seq experiments.

Tables S6. The primers used in this study.

PBI-20-454-s001.pdf (112.4KB, pdf)

Supplementary Material. Additional description of methods.

PBI-20-454-s003.docx (21KB, docx)

Video S1. Confocal microscopy (Leica SP5‐X, Germany) z‐scan image series of TiAP‐mYFP/mCherry‐TaSYP51 on barley leaf epidermal cell under the infection of Bgh.

Download video file (27MB, avi)

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