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Article

Wheat Susceptibility Genes TaCAMTA2 and TaCAMTA3 Negatively Regulate Post-Penetration Resistance against Blumeria graminis forma specialis tritici

by
Mengmeng Li
†,
Zige Yang
†,
Jiao Liu
and
Cheng Chang
*
College of Life Sciences, Qingdao University, Qingdao 266071, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(12), 10224; https://doi.org/10.3390/ijms241210224
Submission received: 22 May 2023 / Revised: 12 June 2023 / Accepted: 14 June 2023 / Published: 16 June 2023
(This article belongs to the Special Issue Host-Pathogen Interaction 4.0)
Browse Figures
Versions Notes

Abstract

:
Blumeria graminis forma specialis tritici (B.g. tritici) is the airborne fungal pathogen that causes powdery mildew disease on hexaploid bread wheat. Calmodulin-binding transcription activators (CAMTAs) regulate plant responses to environments, but their potential functions in the regulation of wheat–B.g. tritici interaction remain unknown. In this study, the wheat CAMTA transcription factors TaCAMTA2 and TaCAMTA3 were identified as suppressors of wheat post-penetration resistance against powdery mildew. Transient overexpression of TaCAMTA2 and TaCAMTA3 enhanced the post-penetration susceptibility of wheat to B.g. tritici, while knockdown of TaCAMTA2 and TaCAMTA3 expression using transient- or virus-induced gene silencing compromised wheat post-penetration susceptibility to B.g. tritici. In addition, TaSARD1 and TaEDS1 were characterized as positive regulators of wheat post-penetration resistance against powdery mildew. Overexpressing TaSARD1 and TaEDS1 confers wheat post-penetration resistance against B.g. tritici, while silencing TaSARD1 and TaEDS1 enhances wheat post-penetration susceptibility to B.g. tritici. Importantly, we showed that expressions of TaSARD1 and TaEDS1 were potentiated by silencing of TaCAMTA2 and TaCAMTA3. Collectively, these results implicated that the Susceptibility genes TaCAMTA2 and TaCAMTA3 contribute to the wheat–B.g. tritici compatibility might via negative regulation of TaSARD1 and TaEDS1 expression.

1. Introduction

As one of the most widely grown small-grain cereal crops, bread wheat (Triticum aestivum L.) has served as a major staple food for thousands of years and provided about 20% of the calories consumed by humans [1]. With the increase in the global population, the demand for wheat grains is rapidly growing [1]. However, wheat production is seriously threatened by attacks from adapted pathogens and pests [2]. Powdery mildew is a devastating disease of wheat that is caused by the obligate biotrophic fungal pathogen Blumeria graminis forma specialis tritici (B.g. tritici), leading to 5–50% yield losses [3,4]. To date, the safest, most economical, and most effective strategy to control this epidemic is breeding B.g. tritici-resistant wheat cultivars [3,4]. Therefore, it is critical to elucidate the molecular interaction between wheat and B.g. tritici and identify key regulators of wheat resistance against powdery mildew disease.
In general, plants employ two classes of immune receptors to detect adapted pathogens and initiate defense responses [5,6,7]. The pattern recognition receptors (PRRs) residing on the plant cell surface recognize the conserved pathogen-associated molecular pattern (PAMP) to initiate PAMP-triggered immunity (PTI) [8,9,10,11,12]. Upon detection of pathogen effectors, plant resistance proteins activate effector-triggered immunity (ETI) [13,14,15,16]. Although PTI and ETI are activated by distinct immune receptors and display different amplitudes and durations, they are both associated with massive transcriptomic reprogramming governed by transcription factors [17,18].
As Ca2+-loaded calmodulin binding (CaMB) transcription factors, calmodulin-binding transcription activators (CAMTAs) play important roles in regulating plant growth, development, and responses to environmental stresses [19,20,21]. For instance, expressions of six CAMTA genes differentially respond to environmental cues like drought, salinity, and extreme temperatures in the model plant Arabidopsis thaliana [22,23,24,25,26]. Arabidopsis mutant camta1 exhibited hypersensitivity to cold and drought stress, and AtCAMTA1 was shown to regulate the expression of cold and drought-responsive genes like AtRD26, AtERD7, AtCBF2, and AtRAB18 [22,23,24,25,26]. 4- to 11-day-old Arabidopsis mutant camta6 exhibited hypersensitivity to NaCl treatment, and AtCAMTA6 was demonstrated to regulate expression of salt resilience-related genes, including HIGH-AFFINITY K+ TRANSPORTER1, SALT OVERLY SENSITIVE1, and Na+/H+ ANTIPORTER [27]. In addition, CAMTA transcription factors get involved in the regulation of plant defense against pathogens. For instance, Arabidopsis AtCAMTA3 was shown to function in concert with AtCAMTA1 and AtCAMTA2 in suppressing plant defense responses [28,29,30,31,32]. However, whether and how CAMTA transcription factors regulate wheat disease resistance against B.g. tritici remains largely unknown.
In this research, two CAMTA transcription factor genes, TaCAMTA2 and TaCAMTA3, were characterized as Susceptibility (S) genes contributing to wheat–B.g. tritici compatibility. Transient overexpression of TaCAMTA2 and TaCAMTA3 resulted in enhanced wheat post-penetration susceptibility to B.g. tritici, while transient silencing of TaCAMTA2 and TaCAMTA3 led to attenuated wheat post-penetration susceptibility to B.g. tritici. Furthermore, overexpressing TaSARD1 and TaEDS1 could confer wheat post-penetration resistance against powdery mildew, while silencing TaSARD1 and TaEDS1 enhanced wheat post-penetration susceptibility to B.g. tritici. Moreover, TaCAMTA2 and TaCAMTA3 were demonstrated to negatively regulate the expression of the defense genes TaSARD1 and TaEDS1. These results strongly support that S genes TaCAMTA2 and TaCAMTA3 partially redundantly suppress wheat post-penetration resistance against B.g. tritici presumably via the negative regulation of expressions of defense genes TaSARD1 and TaEDS1.

2. Results

2.1. Homology-Based Identification of TaCAMAT2 and TaCAMTA3 in Bread Wheat

Previous studies revealed that the Arabidopsis CAMTA transcription factor AtCAMTA3 plays a vital role in the regulation of plant immunity [29,30,31,32]. In this study, we are interested in exploring the function of the wheat homolog of AtCAMTA3 in the wheat–B.g. tritici interaction. To this end, we first searched the reference genome of the hexaploid bread wheat by using the amino acid sequence of Arabidopsis AtCAMTA3 (At2g22300) as a query and obtained TaCAMAT2 and TaCAMTA3, the most closely related homologs of AtCAMTA3, in bread wheat. Three highly homologous sequences of TaCAMAT2 genes separately located on chromosomes 4A, 4B, and 4D were obtained from the genome sequence of the hexaploid wheat and designated as TaCAMTA2-4A (TraesCS4A02G407100), TaCAMTA2-4B (TraesCS4B02G306300), and TaCAMTA2-4D (TraesCS4D02G304500). Similarly, three highly homologous sequences of TaCAMAT3 genes separately located on chromosomes 2A, 2B, and 2D were obtained from the genome sequence of the hexaploid wheat and designated as TaCAMTA3-2A (TraesCS2A02G163000), TaCAMTA3-2B (TraesCS2B02G188800), and TaCAMTA3-2D (TraesCS2D02G169900).
As shown in Figure 1A, these predicted TaCAMTA2-4A, TaCAMTA2-4B, TaCAMTA2-4D, TaCAMTA3-2A, TaCAMTA3-2B, and TaCAMTA3-2D proteins shared about 46% identity with Arabidopsis AtCAMTA3. In addition, TaCAMTA2-4A, TaCAMTA2-4B, TaCAMTA2-4D, TaCAMTA3-2A, TaCAMTA3-2B, and TaCAMTA3-2D proteins all contain a conserved CG-1 DNA-binding domain at their N-terminal parts, a transcription factor immunoglobulin-like (TIG) DNA-binding domain, several ankyrin repeats (ANK) in the middle parts, as well as two IQ CaMB motifs (IQXXXRGXXXR) at their C-termini (Figure 1B). The coding regions of these allelic TaCAMAT2 and TaCAMTA3 genomic sequences all contained 13 exons and 12 introns (Figure 1C).

2.2. TaCAMAT2 and TaCAMTA3 Contribute to the Wheat Susceptibility to B.g. tritici

To study the function of TaCAMAT2 and TaCAMTA3 in the wheat–B.g. tritici interaction, we first employed transient gene expression assays to overexpress these TaCAMTA2-4A, TaCAMTA2-4B, TaCAMTA2-4D, TaCAMTA3-2A, TaCAMTA3-2B, or TaCAMTA3-2D genes in the leaf epidermal cells of the B.g. tritici-susceptible wheat cultivar Yannong 999. After inoculation of conidia from the virulent B.g. tritici isolate E09, the formation of fungal haustoria in the transformed wheat cells was statistically analyzed. As shown in Figure 2A, the B.g. tritici haustorium index (HI%) increased from 56% for the empty vector (OE-EV) control to above 70% on wheat cells overexpressing TaCAMTA2 or TaCAMTA3 genes. These results suggested that overexpression of TaCAMAT2 and TaCAMTA3 could significantly enhance wheat post-penetration susceptibility to B.g. tritici.
To further verify the function of TaCAMAT2 and TaCAMTA3 in the regulation of wheat–B.g. tritici interaction, we employed transiently induced gene silencing (TIGS) assays to silence all endogenous TaCAMAT2 or TaCAMTA3 genes in the epidermal cell of the B.g. tritici-susceptible wheat cultivar Yannong 999. After inoculation of conidia from the virulent B.g. tritici isolate E09, the frequency of fungal haustorium formation in the transformed plant cells was scored. As shown in Figure 2B, the silencing of TaCAMAT2 or TaCAMTA3 genes resulted in a marked HI% decrease to about 27%, compared to 33% for empty vector (EV) controls. Significantly, simultaneous silencing of TaCAMAT2 and TaCAMTA3 could lead to a further decrease in HI% to approximately 13%, suggesting that TaCAMTA2 and TaCAMTA3 might partially redundantly suppress post-penetration resistance of wheat to B.g. tritici.
In addition, we performed barley stripe mosaic virus (BSMV)-induced gene silencing (BSMV-VIGS) to silence all endogenous TaCAMAT2 or TaCAMTA3 genes in the leaves of the B.g. tritici-susceptible wheat cultivar Yannong 999. qRT-PCR showed that the endogenous transcript level of TaCAMAT2 or TaCAMTA3 was substantially reduced in the indicated VIGS plants (Figure 2C). Thereafter, these VIGS plants were inoculated with conidia from the virulent B.g. tritici isolate E09, and the formation of microcolonies was analyzed to evaluate the wheat’s susceptibility to powdery mildew. B.g. tritici microcolony index (MI%) declined to approximate 40% on BSMV-TaCAMTA2as plants and 47% on BSMV-TaCAMTA3as plants, compared with 55% for the BSMV-γ plants (Figure 2D). Notably, simultaneous silencing of TaCAMAT2 and TaCAMTA3 could lead to a further MI% decrease to about 28%. These data clearly indicate that TaCAMAT2 and TaCAMTA3 partially redundantly contribute to the wheat susceptibility to B.g. tritici.

2.3. Homology-Based Identification of TaSARD1 and TaEDS1 in Bread Wheat

Previous studies revealed that AtCAMTA3 could regulate the expression of defense genes SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (AtSARD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (AtEDS1) in A. thaliana [29,30,31,32]. We are interested in examining the potential regulation of TaCAMAT2 and TaCAMTA3 on the wheat defense genes. To this end, we first searched the reference genome of the hexaploid bread wheat by using the amino acid sequences of Arabidopsis AtSARD1 (At1g73805) and AtEDS1 (At3g48090) as a query and obtained TaSARD1 and TaEDS1, the most closely related homologs of AtSARD1 and AtEDS1, in bread wheat. Five highly homologous sequences of TaSARD1 genes separately located on chromosomes 6A, 6B, and 6D were obtained from the genome sequence of the hexaploid wheat and designated as TaSARD1.1-6A (TraesCS6A02G091700), TaSARD1.1-6B (TraesCS6B02G119900), TaSARD1.1-6D (TraesCS6D02G080500), TaSARD1.2-6A (TraesCS6A02G296600), and TaSARD1.2-6D (TraesCS6D02G276800). Similarly, three highly homologous sequences of TaEDS1 genes separately located on chromosomes 5A, 5B, and 5D were obtained from the genome sequence of the hexaploid wheat and designated as TaEDS1-5A, TaEDS1-5B, and TaEDS1-5D [33].
As shown in Figure 3A, these predicted TaSARD1.1-6A, TaSARD1.1-6B, TaSARD1.1-6D, TaSARD1.2-6A, and TaSARD1.2-6D proteins shared about 43% identities with Arabidopsis AtSARD1. In addition, TaSARD1.1-6A, TaSARD1.1-6B, TaSARD1.1-6D, TaSARD1.2-6A, and TaSARD1.2-6D proteins all contain a CBP60-conserved domain (Figure 3B). The coding regions of these allelic TaSARD1 genomic sequences all contained seven exons and six introns (Figure 3C). The predicted TaEDS1-5A, TaEDS1-5B, and TaEDS1-5D proteins shared about 38% identity with Arabidopsis AtEDS1 (Figure 3D). In addition, TaEDS1-5A, TaEDS1-5B, and TaEDS1-5D proteins all contain an N-terminal lipase-like domain and a C-terminal EP (EDS1–PAD4) domain (Figure 3E). The coding regions of these allelic TaEDS1 genomic sequences all contained 3 exons and 2 introns (Figure 3F).

2.4. TaSARD1 and TaEDS1 Positively Contribute to the Wheat Post-Penetration Resistance to B.g. tritici

To characterize the function of TaSARD1 and TaEDS1 in the wheat–B.g. tritici interaction, we first employed transient gene expression assays to overexpress TaSARD1.1-6A, TaSARD1.1-6B, TaSARD1.1-6D, TaSARD1.2-6A, TaSARD1.2-6D, TaEDS1-5A, TaEDS1-5B, or TaEDS1-5D genes in the leaf epidermal cell of the B.g. tritici-susceptible wheat cultivar Yannong 999. As shown in Figure 4A, the B.g. tritici HI% decreased from 54% for the empty vector control to less than 41% on wheat cells overexpressing TaSARD1 or TaEDS1 genes. These results suggested that overexpression of TaSARD1 or TaEDS1 remarkably attenuated wheat post-penetration susceptibility to B.g. tritici.
To further examine the function of TaSARD1 and TaEDS1 in regulating wheat–B.g. tritici interaction, we employed the TIGS assays to silence all endogenous TaSARD1 or TaEDS1 genes in the leaf epidermal cell of the B.g. tritici-susceptible wheat cultivar Yannong 999. As shown in Figure 4B, silencing of TaSARD1 or TaEDS1 genes resulted in a notable HI% increase to above 42%, compared to 31% for empty vector controls. In addition, we employed BSMV-VIGS to silence all endogenous TaSARD1 or TaEDS1 genes in the leaves of the B.g. tritici-susceptible wheat cultivar Yannong 999. qRT-PCR showed that the endogenous transcript level of TaSARD1 or TaEDS1 was significantly reduced in the indicated VIGS plants (Figure 4C). Thereafter, these VIGS plants were inoculated with B.g. tritici conidia, and the formation of microcolonies was statistically analyzed. B.g. tritici MI% increased to approximately 65% on BSMV-TaSARD1as plants and 72% on BSMV-TaEDS1as plants, compared with 53% for the BSMV-γ plants (Figure 4D). These data support that TaSARD1 and TaEDS1 positively regulate the wheat post-penetration resistance to B.g. tritici.

2.5. TaCAMAT2 and TaCAMTA3 Negatively Regulate Expression of TaSARD1 and TaEDS1

To determine the potential regulation of TaCAMAT2 and TaCAMTA3 on the expression of TaSARD1 and TaEDS1 in bread wheat, we employed BSMV-VIGS to silence all endogenous TaCAMAT2 or TaCAMTA3 genes in the leaves of the B.g. tritici-susceptible wheat cultivar Yannong 999. Thereafter, these VIGS plants were inoculated with B.g. tritici conidia, and expression levels of TaSARD1 and TaEDS1 were analyzed. As shown in Figure 5, the silencing of TaCAMAT2 or TaCAMTA3 genes resulted in a marked increase in the expression levels of TaSARD1 and TaEDS1. Significantly, simultaneous silencing of TaCAMAT2 and TaCAMTA3 could lead to a further increase in the expression levels of TaSARD1 and TaEDS1, suggesting that partially redundant TaCAMTA2 and TaCAMTA3 negatively regulate the expressions of TaSARD1 and TaEDS1.
Since PR expressions are usually activated in the plant defense responses to biotrophic pathogens like B.g. tritici, we compared the transcript levels of TaPR1, TaPR2, and TaPR5 among BSMV-TaCAMTA2as, BSMV-TaCAMTA3as, BSMV-TaSARD1as, BSMV-TaEDS1as, and BSMV-γ infected plants. As shown in Figure 6A, the expressions of TaPR1, TaPR2, and TaPR5 were remarkably reduced by silencing of TaSARD1 or TaEDS1, further confirming the fact that TaSARD1 and TaEDS1 positively regulate the wheat defense against B.g. tritici. In contrast, the expressions of TaPR1, TaPR2, and TaPR5 were significantly affected by the silencing of TaCAMAT2 or TaCAMTA3 genes (Figure 6B). Notably, simultaneous silencing of TaCAMAT2 and TaCAMTA3 could lead to a further increase in the activation of TaPR1, TaPR2, and TaPR5 (Figure 6B), which is consistent with the fact that partially redundant TaCAMTA2 and TaCAMTA3 negatively regulate expressions of the wheat defense genes TaSARD1 and TaEDS1.

3. Discussion

3.1. TaCAMAT2 and TaCAMTA3 Are Wheat S Genes Suppressing Post-Penetration Resistance against B.g. tritici

Powdery mildew, caused by the adapted fungal pathogen B.g. tritici, seriously threatens global wheat production [3,4]. To improve wheat resistance against powdery mildew, it is vital to identify the important genes involved in the regulation of the wheat–B.g. tritici interaction [3,4]. Powdery mildew (Pm) resistance genes and quantitative trait loci (QTL) contributed to wheat resistance to B.g. tritici and have been employed in wheat breeding for powdery mildew resistance [3,4]. Compatibility between wheat and B.g. tritici underlies wheat’s susceptibility to powdery mildew. A plethora of wheat S genes have been identified to facilitate compatibility by inducing B.g. tritici (pre)penetration, suppressing wheat immunity, and supporting the sustenance of B.g. tritici [34,35]. For instance, wheat S genes TaWIN1, TaKCS6, and TaECR were revealed to facilitate the conidial germination of B.g. tritici by promoting the biosynthesis of wheat cuticular wax, whereas wheat S gene TaSTP13 encodes a sugar transporter facilitating wheat hexose accumulation for B.g. tritici acquisition [36,37,38,39,40,41]. TaMLO, TaEDR1, and TaPOD70 genes contribute to wheat susceptibility to powdery mildew by suppressing plant defense responses [42,43,44,45,46,47]. In addition, S factors TaMED25, TaHDA6, TaHOS15, and TaHDT701 positively contribute to wheat susceptibility to B.g. tritici by suppressing defense-related transcriptional reprogramming in bread wheat [48,49,50,51,52,53].
Through homology-based searching, TaCAMAT2 and TaCAMTA3 were identified as the most closely related homologs of AtCAMTA3, which is consistent with the reported phylogenetic analysis of the CAMTA homologs in different species [19]. TaCAMAT2 and TaCAMTA3 are characterized as wheat S genes contributing to the wheat post-penetration susceptibility to B.g. tritici in this study. Overexpression of TaCAMTA2 and TaCAMTA3 in the leaf epidermal cell by transient gene expression assays led to enhanced wheat susceptibility to B.g. tritici, while knockdown of TaCAMTA2 and TaCAMTA3 expression using transient- or virus-induced gene silencing resulted in compromised wheat post-penetration susceptibility to B.g. tritici. Interestingly, a gain-of-function mutation in SIGNAL RESPONSIVE1 (SR1), which encodes the Arabidopsis homologs of wheat TaCAMTA2 and TaCAMTA3, could suppress the edr2-associated powdery mildew resistance [29]. The sr1-4D single mutant is more susceptible to Arabidopsis powdery mildew (Golovinomyces cichoracearum), whereas the sr1-1 null mutant plants displayed enhanced post-penetration resistance against G. cichoracearum [29]. In addition, Arabidopsis AtCAMTA1 was revealed to function partially redundantly with AtCAMTA2 and AtCAMTA3 in suppressing plant immunity [30,31,32]. In this study, simultaneous silencing of TaCAMAT2 and TaCAMTA3 could lead to a further decrease in the HI% and MI% compared with single silencing of TaCAMAT2 or TaCAMTA3, supporting the fact that TaCAMTA3 functions partially redundantly with TaCAMAT2 in suppressing wheat post-penetration resistance against B.g. tritici. In Arabidopsis, CAMTA transcription factors AtCAMTA1, AtCAMTA2, and AtCAMTA3 partially redundantly suppress the biosynthesis of salicylic acid (SA) and N-hydroxypipecolic acid (NHP), a metabolite duo essential for systemic acquired resistance (SAR) [30,31,32]. Therefore, it is intriguing to examine the potential roles of the S genes TaCAMAT2 and TaCAMTA3 in the regulation of SA and NHP biosynthesis, as well as SAR establishment, in bread wheat in future research.

3.2. TaSARD1 and TaEDS1 Confer Wheat Post-Penetration Resistance against B.g. tritici

TaSARD1 and TaEDS1 are identified as positive regulators of wheat resistance against B.g. tritici in this study. Overexpression of TaSARD1 or TaEDS1 in the leaf epidermal cell by transient gene expression assays led to enhanced wheat post-penetration resistance to B.g. tritici, while knockdown of TaSARD1 or TaEDS1 expression using transient- or virus-induced gene silencing resulted in increased wheat post-penetration susceptibility to B.g. tritici. In Arabidopsis, transcription factor AtSARD1 functions in concert with AtCBP60g to activate the expression of SID2 (SA INDUCTION DEFICIENT 2), which encodes isochorismate synthase 1 (ICS1), essential for pathogen-induced SA biosynthesis [54,55,56]. Arabidopsis AtEDS1 was shown to heterodimerize with its partners, phytoalexin deficient 4 (PAD4) or senescence-associated gene 101 (SAG101), to play signaling roles in ETI as well as SA-dependent and SA-independent PTI pathways [57,58,59,60,61,62,63,64]. Consistent with this, expressions of SA defense marker genes TaPR1, TaPR2, and TaPR5 induced by B.g. tritici infection were attenuated by silencing of TaSARD1 or TaEDS1, suggesting that the SARD1-EDS1-SA defense axis might be partially conserved between model plant Arabidopsis and crop plant bread wheat. Therefore, it is intriguing to examine the potential regulation of wheat SA biosynthesis and signaling by TaSARD1 and TaEDS1 in future research.

3.3. TaCAMAT2 and TaCAMTA3 Negatively Regulate the Expression of TaSARD1 and TaEDS1 to Suppress Wheat Post-Penetration Resistance against B.g. tritici

In this study, expression levels of TaSARD1 and TaEDS1 were significantly enhanced by silencing TaCAMTA2 and TaCAMTA3. Notably, simultaneous silencing TaCAMAT2 and TaCAMTA3 could lead to a further increase in the expression levels of TaSARD1 and TaEDS1 compared with single silencing TaCAMAT2 or TaCAMTA3, indicating that TaCAMTA2 and TaCAMTA3 partially redundantly suppress expressions of TaSARD1 and TaEDS1. In Arabidopsis, AtCAMTA3 could bind to the promoter region of AtEDS1 by recognizing the CGCG box, thereby directly repressing the expression of AtEDS1 [28,29,30,31]. In addition, the expression of AtSARD1 was demonstrated to be negatively regulated by partially redundant AtCAMTA1, AtCAMTA2, and AtCAMTA3, presumably via an indirect effect [28,29,30,31]. These results indicate that negative regulation of the expressions of defense genes SARD1 and EDS1 by partially redundant CAMTA3 and its homologs might be partly conserved between the model plant Arabidopsis and the important crop bread wheat. Indeed, the expressions of SA defense marker genes TaPR1, TaPR2, and TaPR5 induced by B.g. tritici infection were found to be potentiated by silencing TaCAMAT2 or TaCAMTA3 in this study. However, binding sites for TaCAMAT2 and TaCAMTA3 in the promoter regions of TaSARD1 and TaEDS1 genes remain to be identified.
Herein, TaCAMAT2 and TaCAMTA3 are identified as wheat S genes partially redundantly suppressing post-penetration resistance against powdery mildew, presumably via negative regulation of the expressions of defense genes TaSARD1 and TaEDS1. Genetic manipulation of S genes TaMLO and TaEDR1 via targeting induced local lesions in genomes (TILLING) and genome editing techniques like transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) 9 systems compromised wheat compatibility with B.g. tritici and conferred wheat resistance against powdery mildew [65,66,67,68,69,70,71,72,73]. Therefore, it is intriguing to examine the potential of manipulating the S genes TaCAMAT2 and TaCAMTA3 in wheat breeding for powdery mildew resistance in future research.

4. Materials and Methods

4.1. Plant and Fungal Materials

The seedlings of bread wheat cultivar Yannong999 used in this study were grown in a growth chamber under a 16-h/8-h, 20 °C/18 °C day/night cycle with 70% relative humidity. The B.g. tritici strain E09 was maintained on the leaves of Jing411 plants. Conidia of B.g. tritici strain E09 were used for the inoculation of Jing411 leaves in the study of wheat–powdery mildew interaction. Arabidopsis thaliana used in this study was grown in the greenhouse under a 16 h/8 h light period at 23 ± 1 °C with 70% relative humidity.

4.2. Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from the wheat leaves using the EasyPure Plant RNA kit (Transgenbiotech, Beijing, China) and 2 μg of RNA was used to synthesize the cDNA template using the TransScript one-step gDNA removal and cDNA synthesis supermix (Transgenbiotech, Beijing, China) according to the manufacturer’s instructions. The real-time PCR assay was performed using the ABI real-time PCR system with the qPCR Master Mix (Invitrogen, Carlsbad, CA, USA). The expression of traditional housekeeping gene GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (TaGAPDH) was set as the internal control and expressions of TaGAPDH, TaCAMTA2, TaCAMTA3, TaSARD1, TaEDS1, TaPR1, TaPR2 and TaPR5 were analyzed using the primers 5′-TTAGACTTGCGAAGCCAGCA-3′/5′-AAATGCCCTTGAGGTTTCCC-3′, 5′-TACAGAAGTTGCAACAG-3′/5′-ATCTCCGTCGACTCCTCA-3′, 5′-CCTGACAAACAACTTGA-3′/5′-CGCCAGCTGCA TCGCTT-3′, 5′-GCGAGTAATGAAAGCAT-3′/5′-TTAATCAACTTGATCCC-3′, 5′-TGAAAGACAGGGTGGGT-3′/5′-CGAAGGCACAAGTCTCG-3′, 5′-GAGAATGCAGACGCCCAAGC-3′/5′-CTGGAGCTTGCAGTCGTTGATC-3′, 5′-AGGATGTTGCTTCCATGTTTGCCG-3′/5′-AAGTAGATGCGCATGCCGTTGATG-3′, and 5′-CTTCTACATCAAGA ACAACTG-3′/5′-CAGTCGCCGGTCTGGCAG-3′.

4.3. BSMV-Mediated Gene Silencing and B.g. tritici Infection

The antisense fragment of TaCAMTA2, TaCAMTA3, TaSARD1, and TaEDS1 was cloned into the pCa-γbLIC vector to create the BSMV-TaCAMTA2as, BSMV-TaCAMTA3as, BSMV-TaSARD1as, and BSMV-TaEDS1as constructs using the primer pair 5′-AAGGAAGTTTATACCATCATTAGCACTTGG-3′/5′-AACCACCACCACCGTCACTTTTGGAATTACATTC-3′, 5′-AAGGAAGTTTACATTATGCACCTGCGAGGA-3′/5′-AACCACCACCACCGTTCAGTGCACTTTGGTGAGC-3′, 5′-AAGGAAGTTTATGGTTCTAGTATCTATAAG-3′/5′-AACCACCACCACCGTGTTTGGAACCAGTTATTCG-3′, and 5′-AAGGAAGTTTAAGCGAATTCCCAACAGGTG-3′/5′-AACCACCACCACCGTAGACGGGGAAGTGTCAATC-3′. The BSMV-mediated gene silencing in wheat leaves was performed as described by Zhi et al. (2020) [52]. About 15 days after BSMV infection, the newly grown upper leaves with virus symptoms were collected and subjected to inoculation with B.g. tritici strain E09 conidia. About 72 h post-B.g. tritici inoculation, leaf segments were fixed with ethanol: acetic acid solution (1:1, v/v) and kept in the destaining solution (lactic acid: glycerol: water, 1:1:1, v/v/v). Before mounting for microscopy, B.g. tritici-infected leaves were stained with 0.1% (w/v) Coomassie Brilliant Blue R250 to visualize the fungal epiphytic structure, as reported previously [52].

4.4. Single-Cell Transient Gene Silencing and Overexpression Assay

Antisense fragments of TaCAMTA2, TaCAMTA3, TaSARD1, and TaEDS1 were, respectively, amplified using the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTACCATCATTAGCACTTGG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCACTTTTGGAATTACATTC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCATTATGCACCTGCGAGGA-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGTGCACTTTGGTGAGC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTGGTTCTAGTATCTATAAG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCGTTTGGAACCAGTTATTCG-3′, and 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAGCGAATTCCCAACAGGTG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGACGGGGAAGTGTCAATC-3′, and cloned into the pIPKb007 vector using a Gateway cloning system to create the TIGS-TaCAMTA2, TIGS-TaCAMTA3, TIGS-TaSARD1, and TIGS-TaEDS1 constructs. The coding regions of TaCAMTA2-4A, TaCAMTA2-4B, TaCAMTA2-4D, TaCAMTA3-2A, TaCAMTA3-2B, TaCAMTA3-2D, TaSARD1.1-6A, TaSARD1.1-6B, TaSARD1.1-6D, TaSARD1.2-6A, TaSARD1.2-6B, TaSARD1.2-6D, TaEDS1-5A, TaEDS1-5B, and TaEDS1-5D were, respectively, amplified using the primers 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGAAATAGCCCGGCAACG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCT GGGTCCTAGAAATAGCCAGGCAACG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGAGGGCCGGCGCTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGAAATAGCCCGGCAACG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAATATTGGACATCG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAACAGTGGACATCG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGAGATGCACAAGTAC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAAAATAGTGGACATCG-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGTGCGAAGGCCGCG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCGGTGCGAAGGCCCCG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCGGTGCGAAGGCCACG-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAATCAACTTGATCCCAAC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3′, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3′, and 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCCGATGGACACCCCGCC-3′/5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACGAAGGCACAAGTCTCGC-3′, and cloned into the pIPKb001 vector. The single-cell transient gene silencing and expression were conducted essentially as described (Zhi et al., 2020) [52]. Briefly, the GUS reporter vector was co-delivered (1:1 molar ratio) with pIPKb001 or pIPKb007 constructs into the wheat epidermal cell through the particle inflow gun (Bio-Rad). After inoculation with B.g. tritici strain E09 conidia, the leaf segments were stained for GUS activity 48 h post-B.g. tritici inoculation. Before mounting for microscopic analysis, the leaves were stained with 0.1% (w/v) Coomassie Brilliant Blue R250 to visualize the fungal epiphytic structure.

Author Contributions

C.C. and M.L. planned and designed the research; M.L. and Z.Y. performed most of the experiments with help from J.L.; C.C., M.L. and Z.Y. analyzed the data and wrote the manuscript with contributions from J.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Shandong Province (ZR2022MC008, ZR2017BC109), the Qingdao Science and Technology Bureau Fund (17-1-1-50-jch), and the Qingdao University Fund (DC1900005385).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data presented here are available on request from correspondence.

Acknowledgments

We thank Andreas Burkovski for the kind invitation to submit this work to the Special Issue ‘Host-Pathogen Interaction 4.0’. We are also grateful to the anonymous reviewers for their very helpful comments on this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Identification of wheat TaCAMTA2 and TaCAMTA3 based on homology with Arabidopsis AtCAMTA3. (A) Protein sequence comparison of wheat TaCAMTA2, TaCAMTA3, and Arabidopsis AtCAMTA3. Residues conserved in at least 4 of the 7 proteins are shaded in gray, while identical residues among 7 protein sequences are shaded in dark. (B) Domain structure of wheat TaCAMTA2 and TaCAMTA3 proteins. (C) Gene architectures of the wheat TaCAMTA2 and TaCAMTA3 genes.
Figure 1. Identification of wheat TaCAMTA2 and TaCAMTA3 based on homology with Arabidopsis AtCAMTA3. (A) Protein sequence comparison of wheat TaCAMTA2, TaCAMTA3, and Arabidopsis AtCAMTA3. Residues conserved in at least 4 of the 7 proteins are shaded in gray, while identical residues among 7 protein sequences are shaded in dark. (B) Domain structure of wheat TaCAMTA2 and TaCAMTA3 proteins. (C) Gene architectures of the wheat TaCAMTA2 and TaCAMTA3 genes.
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Figure 2. Functional analyses of wheat TaCAMTA2 and TaCAMTA3 under B.g. tritici infection. (A) Haustorial index analysis in wheat epidermal cells transiently overexpressing TaCAMTA2 (OE-TaCAMTA2) and TaCAMTA3 (OE-TaCAMTA3). Haustorial formation on wheat epidermal cells bombarded with empty vector (OE-EV) was statistically analyzed as a control. At least 100 wheat cells were analyzed in each experiment. (B) Haustorial index analysis in wheat epidermal cells transiently silencing TaCAMTA2 (TIGS-TaCAMTA2), TaCAMTA3 (TIGS-TaCAMTA3), or co-silencing TaCAMTA2 and TaCAMTA3 (TIGS-TaCAMTA2 + TIGS-TaCAMTA3). Haustorial formation on wheat epidermal cells bombarded with an empty vector (TIGS-EV) was statistically analyzed as a control. (C) qRT-PCR analysis of TaCAMTA2 and TaCAMTA3 expression in wheat leaves infected with the indicated BSMV vectors. BSMV-γ empty vector was employed as the negative control. (D) B.g. tritici microcolony index analysis on wheat leaves silencing TaCAMTA2 (BSMV-TaCAMTA2as), TaCAMTA3 (BSMV-TaCAMTA3as), or co-silencing TaCAMTA2 and TaCAMTA3 (BSMV-TaCAMTA2as + BSMV-TaCAMTA3as). At least 1000 wheat–B.g. tritici interaction sites were counted in one experiment for each treatment. For (A–D), three independent biological replicates were statistically analyzed for each treatment (t-test; * p < 0.05, ** p < 0.01).
Figure 2. Functional analyses of wheat TaCAMTA2 and TaCAMTA3 under B.g. tritici infection. (A) Haustorial index analysis in wheat epidermal cells transiently overexpressing TaCAMTA2 (OE-TaCAMTA2) and TaCAMTA3 (OE-TaCAMTA3). Haustorial formation on wheat epidermal cells bombarded with empty vector (OE-EV) was statistically analyzed as a control. At least 100 wheat cells were analyzed in each experiment. (B) Haustorial index analysis in wheat epidermal cells transiently silencing TaCAMTA2 (TIGS-TaCAMTA2), TaCAMTA3 (TIGS-TaCAMTA3), or co-silencing TaCAMTA2 and TaCAMTA3 (TIGS-TaCAMTA2 + TIGS-TaCAMTA3). Haustorial formation on wheat epidermal cells bombarded with an empty vector (TIGS-EV) was statistically analyzed as a control. (C) qRT-PCR analysis of TaCAMTA2 and TaCAMTA3 expression in wheat leaves infected with the indicated BSMV vectors. BSMV-γ empty vector was employed as the negative control. (D) B.g. tritici microcolony index analysis on wheat leaves silencing TaCAMTA2 (BSMV-TaCAMTA2as), TaCAMTA3 (BSMV-TaCAMTA3as), or co-silencing TaCAMTA2 and TaCAMTA3 (BSMV-TaCAMTA2as + BSMV-TaCAMTA3as). At least 1000 wheat–B.g. tritici interaction sites were counted in one experiment for each treatment. For (A–D), three independent biological replicates were statistically analyzed for each treatment (t-test; * p < 0.05, ** p < 0.01).
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Figure 3. Identification of wheat TaSARD1 and TaEDS1 based on homology with Arabidopsis AtSARD1 and AtEDS1. (A) Protein sequence comparison of wheat TaSARD1 and Arabidopsis AtSARD1. Residues conserved in at least 3 of the 6 proteins are shaded in gray, while identical residues among 6 protein sequences are shaded in dark. (B) Domain structure of wheat TaSARD1 proteins. (C) Gene architectures of wheat TaSARD1 genes. (D) Protein sequence comparison of wheat TaEDS1 and Arabidopsis AtEDS1. Residues conserved in at least 2 of the 4 proteins are shaded in gray, while identical residues among 4 protein sequences are shaded in dark. (E) Domain structure of wheat TaEDS1 proteins. (F) Gene architectures of wheat TaEDS1 genes.
Figure 3. Identification of wheat TaSARD1 and TaEDS1 based on homology with Arabidopsis AtSARD1 and AtEDS1. (A) Protein sequence comparison of wheat TaSARD1 and Arabidopsis AtSARD1. Residues conserved in at least 3 of the 6 proteins are shaded in gray, while identical residues among 6 protein sequences are shaded in dark. (B) Domain structure of wheat TaSARD1 proteins. (C) Gene architectures of wheat TaSARD1 genes. (D) Protein sequence comparison of wheat TaEDS1 and Arabidopsis AtEDS1. Residues conserved in at least 2 of the 4 proteins are shaded in gray, while identical residues among 4 protein sequences are shaded in dark. (E) Domain structure of wheat TaEDS1 proteins. (F) Gene architectures of wheat TaEDS1 genes.
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Figure 4. Functional analyses of wheat TaSARD1 and TaEDS1 under B.g. tritici infection. (A) Haustorial index analysis in wheat epidermal cells transiently overexpressing TaSARD1 (OE-TaSARD1) and TaEDS1 (OE-TaEDS1). Haustorial formation on wheat epidermal cells bombarded with empty vector (OE-EV) was statistically analyzed as a control. At least 100 wheat cells were analyzed in each experiment. (B) Haustorial index analysis in wheat epidermal cells transiently silencing TaSARD1 (TIGS-TaSARD1) or TaEDS1 (TIGS-TaEDS1). Haustorial formation on wheat epidermal cells bombarded with an empty vector (TIGS-EV) was statistically analyzed as a control. (C) qRT-PCR analysis of TaSARD1 and TaEDS1 expression in wheat leaves infected with the indicated BSMV vectors. The BSMV-γ empty vector was employed as the negative control. (D) B.g. tritici microcolony index analysis on wheat leaves silencing TaSARD1 (BSMV-TaSARD1as) or TaEDS1 (BSMV-TaEDS1as). At least 1000 wheat–B.g. tritici interaction sites were counted in one experiment for each treatment. For (A–D), three independent biological replicates were statistically analyzed for each treatment (t-test; ** p < 0.01).
Figure 4. Functional analyses of wheat TaSARD1 and TaEDS1 under B.g. tritici infection. (A) Haustorial index analysis in wheat epidermal cells transiently overexpressing TaSARD1 (OE-TaSARD1) and TaEDS1 (OE-TaEDS1). Haustorial formation on wheat epidermal cells bombarded with empty vector (OE-EV) was statistically analyzed as a control. At least 100 wheat cells were analyzed in each experiment. (B) Haustorial index analysis in wheat epidermal cells transiently silencing TaSARD1 (TIGS-TaSARD1) or TaEDS1 (TIGS-TaEDS1). Haustorial formation on wheat epidermal cells bombarded with an empty vector (TIGS-EV) was statistically analyzed as a control. (C) qRT-PCR analysis of TaSARD1 and TaEDS1 expression in wheat leaves infected with the indicated BSMV vectors. The BSMV-γ empty vector was employed as the negative control. (D) B.g. tritici microcolony index analysis on wheat leaves silencing TaSARD1 (BSMV-TaSARD1as) or TaEDS1 (BSMV-TaEDS1as). At least 1000 wheat–B.g. tritici interaction sites were counted in one experiment for each treatment. For (A–D), three independent biological replicates were statistically analyzed for each treatment (t-test; ** p < 0.01).
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Figure 5. qRT-PCR analysis of TaSARD1 and TaEDS1 expression levels in TaCAMTA2 and TaCAMTA3 silenced wheat leaves under B.g. tritici infection. The data are shown as means ± SEs (t-test; ** p < 0.01) from three independent biological replicates. hpi is the abbreviation for hours post B.g. tritici inoculation.
Figure 5. qRT-PCR analysis of TaSARD1 and TaEDS1 expression levels in TaCAMTA2 and TaCAMTA3 silenced wheat leaves under B.g. tritici infection. The data are shown as means ± SEs (t-test; ** p < 0.01) from three independent biological replicates. hpi is the abbreviation for hours post B.g. tritici inoculation.
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Figure 6. TaPR1, TaPR2, and TaPR5 expression levels in BSMV-VIGS wheat leaves. (A) qRT-PCR analysis of TaPR1, TaPR2, and TaPR5 expression levels in TaSARD1 and TaEDS1 silenced wheat leaves under B.g. tritici infection. (B) RT-PCR analysis of TaPR1, TaPR2, and TaPR5 expression levels in TaCAMTA2 and TaCAMTA3 silenced wheat leaves under B.g. tritici infection. The data are shown as means ± SEs (t-test; ** p < 0.01) from three independent biological replicates.
Figure 6. TaPR1, TaPR2, and TaPR5 expression levels in BSMV-VIGS wheat leaves. (A) qRT-PCR analysis of TaPR1, TaPR2, and TaPR5 expression levels in TaSARD1 and TaEDS1 silenced wheat leaves under B.g. tritici infection. (B) RT-PCR analysis of TaPR1, TaPR2, and TaPR5 expression levels in TaCAMTA2 and TaCAMTA3 silenced wheat leaves under B.g. tritici infection. The data are shown as means ± SEs (t-test; ** p < 0.01) from three independent biological replicates.
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MDPI and ACS Style

Li, M.; Yang, Z.; Liu, J.; Chang, C. Wheat Susceptibility Genes TaCAMTA2 and TaCAMTA3 Negatively Regulate Post-Penetration Resistance against Blumeria graminis forma specialis tritici. Int. J. Mol. Sci. 2023, 24, 10224. https://doi.org/10.3390/ijms241210224

AMA Style

Li M, Yang Z, Liu J, Chang C. Wheat Susceptibility Genes TaCAMTA2 and TaCAMTA3 Negatively Regulate Post-Penetration Resistance against Blumeria graminis forma specialis tritici. International Journal of Molecular Sciences. 2023; 24(12):10224. https://doi.org/10.3390/ijms241210224

Chicago/Turabian Style

Li, Mengmeng, Zige Yang, Jiao Liu, and Cheng Chang. 2023. "Wheat Susceptibility Genes TaCAMTA2 and TaCAMTA3 Negatively Regulate Post-Penetration Resistance against Blumeria graminis forma specialis tritici" International Journal of Molecular Sciences 24, no. 12: 10224. https://doi.org/10.3390/ijms241210224

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