Transcription Factor TaMYB30 Activates Wheat Wax Biosynthesis

The waxy cuticle covers a plant’s aerial surface and contributes to environmental adaptation in land plants. Although past decades have seen great advances in understanding wax biosynthesis in model plants, the mechanisms underlying wax biosynthesis in crop plants such as bread wheat remain to be elucidated. In this study, wheat MYB transcription factor TaMYB30 was identified as a transcriptional activator positively regulating wheat wax biosynthesis. The knockdown of TaMYB30 expression using virus-induced gene silencing led to attenuated wax accumulation, increased water loss rates, and enhanced chlorophyll leaching. Furthermore, TaKCS1 and TaECR were isolated as essential components of wax biosynthetic machinery in bread wheat. In addition, silencing TaKCS1 and TaECR resulted in compromised wax biosynthesis and potentiated cuticle permeability. Importantly, we showed that TaMYB30 could directly bind to the promoter regions of TaKCS1 and TaECR genes by recognizing the MBS and Motif 1 cis-elements, and activate their expressions. These results collectively demonstrated that TaMYB30 positively regulates wheat wax biosynthesis presumably via the transcriptional activation of TaKCS1 and TaECR.


Introduction
The lipophilic cuticle covers the above-ground portions of plants and represents a true interphase between plant aerial organs and surrounding environments [1][2][3]. The cuticle is mainly composed of cutin matrices filled and covered with wax mixtures [1][2][3]. Through preventing organ fusion and promoting root formation, the cuticle tightly regulates plant development [4][5][6]. In addition, the waxy cuticle greatly contributes to the plant's adaption to environmental challenges associated with terrestrialization such as water deficit (drought), high salinity, extreme temperatures (cold and heat), ultraviolet (UV) radiation, and attacks from pathogens and pests (P&Ps) [7][8][9][10][11]. Increasing studies suggested that cuticle-associated traits have been selected and/or manipulated for genetic improvement in important crop plants such as hexaploid bread wheat (Triticum aestivum) [12,13].
Accumulating evidence suggests that cuticular wax biosynthesis is tightly regulated at transcriptional levels, which has been overviewed recently [31]. The SHINE (SHN) clade of the AP2 domain transcription factors AtSHN1, AtSHN2, and AtSHN3 were first identified as transcriptional activators of cuticle lipid biosynthesis in the dicot model plant Arabidopsis thaliana [32][33][34]. Orthologs of AtSHN1, AtSHN2, and AtSHN3 have been characterized as key regulators of wax biosynthesis in other plant species such as Hordeum vulgare, T. aestivum, and Physcomitrium patens [35][36][37][38][39]. In addition, myeloblastosis (MYB)-type transcription factors AtMYB16, AtMYB30, AtMYB41, AtMYB94, AtMYB96, and AtMYB106 are revealed to become widely involved in the transcriptional regulation of wax biosynthesis in A. thaliana [1][2][3][40][41][42]. For instance, leaf cuticular wax components and the expressions of wax biosynthesis genes were altered in the Arabidopsis MYB30 knockout mutant and overexpressing lines [43]. Similarly, the knockdown of MdMYB30, an apple (Malus domestica) ortholog of the AtMYB30 gene, attenuated wax deposition in apples [44]. In addition, wax biosynthesis is demonstrated to be regulated by MYB transcription factors in bread wheat. For instance, wheat MYB transcription factor TaMYB96/TaEPBM1 is recently identified as a wheat ortholog of the AtMYB96 and demonstrated to activate wax biosynthesis genes and potentiate wheat wax biosynthesis [45,46].
In this study, three closely homologous MYB genes TaMYB30-5A, TaMYB30-5B, and TaMYB30-5D were demonstrated to positively regulate wheat wax biosynthesis. The silencing of all endogenous TaMYB30 genes resulted in attenuated wax accumulation and potentiated cuticle permeability in bread wheat. Wheat ortholog genes of KCS1 and ECR (TaKCS1-4A, TaKCS1-4B, TaKCS1-4D, TaECR-3A, TaECR-3B, and TaECR-3D) were found to be essential for the wheat wax biosynthesis. Knockdown expression levels of all endogenous TaKCS1 or TaECR genes led to reduced wax accumulation and enhanced cuticle permeability in bread wheat. In addition, TaMYB30 proteins were found to be transcriptional activators and could directly activate the expressions of wax biosynthesis genes TaKCS1 and TaECR. These results strongly suggest that the transcriptional activator TaMYB30 positively regulates wheat wax biosynthesis, possibly via the transcriptional activation of wax biosynthesis genes TaEDS1 and TaSRAD1.

Identification of Wheat TaMYB30 Based on Homology with Arabidopsis AtMYB30
The MYB transcription factor AtMYB30 was demonstrated to be a key regulator of wax biosynthesis in the dicot model plant Arabidopsis [43]. In this study, we are interested in characterizing the function of the wheat homolog of AtMYB30 in the wax biosynthesis of bread wheat. To this end, we first searched the reference genome of the hexaploid bread wheat by using the amino acid sequence of Arabidopsis AtMYB30 (At3g28910) as a query and obtained TaMYB30, the most closed homologs of AtMYB30, in bread wheat. Three highly homologous sequences of TaMYB30 genes separately located on chromosomes 5A, 5B, and 5D were obtained from the genome sequence of the hexaploid wheat and designated as TaMYB30-5A (TraesCS5A02G227400), TaMYB30-5B (TraesCS5B02G226100), and TaMYB30-5D (TraesCS5D02G234800), respectively. As shown in Figure 1A, these predicted TaMYB30-5A, TaMYB30-5B, and TaMYB30-5D proteins shared about 45% identities with Arabidopsis AtMYB30. The TaMYB30-5A, TaMYB30-5B, and TaMYB30-5D proteins all contain two conserved MYB motifs at their N-termini ( Figure 1B). The coding regions of these allelic TaMYB30 genomic sequences all contained three exons and two introns ( Figure 1C). contain two conserved MYB motifs at their N-termini ( Figure 1B). The coding regions of these allelic TaMYB30 genomic sequences all contained three exons and two introns (Figure 1C).

TaMYB30 Positively Regulates Wheat Wax Biosynthesis
To study the function of TaMYB30 in wheat wax biosynthesis, we performed barley stripe mosaic virus (BSMV)-induced gene silencing (BSMV-VIGS) to silence all endogenous TaMYB30 genes in the leaves of the wheat cultivar Yannong 999. BSMV-γ-infected wheat plants were employed as the negative controls. As revealed by the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay, the TaMYB30 expression level was significantly reduced in the BSMV-TaMYB30as-infected wheat leaves ( Figure  2A). We then conducted the gas chromatography-mass spectrometry (GC-MS) assay to measure cuticular wax constituents released from BSMV-γ and BSMV-TaMYB30as plants. As shown in Figure 2B, the amount of total cuticular wax was reduced from 12.1 µg cm −2 in the wheat leaves infected with BSMV-γ to 2.29 µg cm −2 in the BSMV-TaMYB30as-infected wheat plants ( Figure 2B). Further wax component analyses revealed that the most abundant wax constituent, C28-alcohol, decreased from 8.05 µg cm −2 in the BSMV-γ wheat leaves to 1.05 µg cm −2 in the BSMV-TaMYB30as plants ( Figure 2C). Likewise, other VLC fatty acids, alcohols, aldehydes, alkanes, and esters all showed an obvious reduction in the BSMV-TaMYB30as-infected wheat plants compared with BSMV-γ control plants (Figure 2C). These results indicated that the silencing of TaMYB30 significantly attenuated wax biosynthesis in wheat leaves and suggested that TaMYB30 positively regulates wheat wax biosynthesis.

TaMYB30 Positively Regulates Wheat Wax Biosynthesis
To study the function of TaMYB30 in wheat wax biosynthesis, we performed barley stripe mosaic virus (BSMV)-induced gene silencing (BSMV-VIGS) to silence all endogenous TaMYB30 genes in the leaves of the wheat cultivar Yannong 999. BSMV-γ-infected wheat plants were employed as the negative controls. As revealed by the quantitative reversetranscription polymerase chain reaction (qRT-PCR) assay, the TaMYB30 expression level was significantly reduced in the BSMV-TaMYB30as-infected wheat leaves ( Figure 2A). We then conducted the gas chromatography-mass spectrometry (GC-MS) assay to measure cuticular wax constituents released from BSMV-γ and BSMV-TaMYB30as plants. As shown in Figure 2B, the amount of total cuticular wax was reduced from 12.1 µg cm −2 in the wheat leaves infected with BSMV-γ to 2.29 µg cm −2 in the BSMV-TaMYB30as-infected wheat plants ( Figure 2B). Further wax component analyses revealed that the most abundant wax constituent, C28-alcohol, decreased from 8.05 µg cm −2 in the BSMV-γ wheat leaves to 1.05 µg cm −2 in the BSMV-TaMYB30as plants ( Figure 2C). Likewise, other VLC fatty acids, alcohols, aldehydes, alkanes, and esters all showed an obvious reduction in the BSMV-TaMYB30as-infected wheat plants compared with BSMV-γ control plants ( Figure 2C). These results indicated that the silencing of TaMYB30 significantly attenuated wax biosynthesis in wheat leaves and suggested that TaMYB30 positively regulates wheat wax biosynthesis.
Changes in wax deposition usually resulted in altered cuticle permeability. Compared with the BSMV-γ controls, the leaves of the BSMV-TaMYB30as-infected wheat plants displayed higher water loss rates with a loss of approximately 70% of the total water after 12 h ( Figure 2D). Consistently, the chlorophyll leaching assay demonstrated that the BSMV-TaMYB30as-infected wheat plants released chlorophyll faster than the BSMV-γ-infected wheat plants ( Figure 2E). Collectively, these results suggested that the silencing of

Homology-Based Identification of TaKCS1 and TaECR in Bread Wheat
Previous studies revealed that the Arabidopsis MYB transcription factor AtMYB30 could regulate the expressions of wax biosynthesis genes AtKCS1 and AtECR [43]. We were interested in testing the potential regulation of TaMYB30 on these wax biosynthesis genes in bread wheat. To this end, we searched the reference genome of the hexaploid bread wheat by using the amino acid sequence of Arabidopsis AtKCS1 (At1g01120) and AtECR (At3g55360) as a query and obtained TaKCS1 and TaECR1, the most closed homologs of AtKCS1 and AtECR1, in bread wheat. Three highly homologous sequences of TaKCS1 genes separately located on chromosomes 4A, 4B, and 4D were obtained from the genome sequence of the hexaploid wheat and designated as TaKCS1-4A (TraesCS4A02G068400), TaKCS1-4B (TraesCS4B02G225500), and TaKCS1-4D (TraesCS4D02G226100), respectively. Similarly, three highly homologous sequences of TaECR genes separately located on chromosomes 3A, 3B, and 3D were obtained from the genome sequence of the hexaploid wheat and designated as TaECR-3A, TaECR-3B, and TaECR-3D, respectively [33].

TaKCS1 and TaECR Positively Contribute to Wheat Wax Biosynthesis
To study the function of TaKCS1 and TaECR in wheat wax biosynthesis, we employed the BSMV-VIGS to silence all endogenous TaKCS1 or TaECR genes in the leaves of the wheat cultivar Yannong 999. As shown in Figure 4A, the expression levels of TaKCS1 or TaECR genes are significantly reduced in the BSMV-TaKCS1as-or BSMV-TaECRas-infected

TaKCS1 and TaECR Positively Contribute to Wheat Wax Biosynthesis
To study the function of TaKCS1 and TaECR in wheat wax biosynthesis, we employed the BSMV-VIGS to silence all endogenous TaKCS1 or TaECR genes in the leaves of the wheat cultivar Yannong 999. As shown in Figure 4A, the expression levels of TaKCS1 or TaECR genes are significantly reduced in the BSMV-TaKCS1as-or BSMV-TaECRas-infected wheat plants ( Figure 4A). We then measured cuticular wax constituents released from BSMVγ, BSMV-TaKCS1as, or BSMV-TaECRas plants by GC-MS assay. As shown in Figure 4B, the amount of total cuticular wax decreased from 11.68 µg cm −2 in the BSMV-γ leaves to 2.89 µg cm −2 and 2.51 µg cm −2 in the wheat leaves infected with BSMV-TaKCS1as and BSMV-TaECRas, respectively ( Figure 4B). Wax component analyses revealed that the amounts of VLC fatty acids, alcohols, aldehydes, alkanes, and esters were reduced significantly in the BSMV-TaKCS1as and BSMV-TaECRas leaves compared with the BSMV-γ controls ( Figure 4C). These results collectively showed that the knockdown of TaKCS1 and TaECR expression levels remarkably attenuated wax biosynthesis in wheat leaves and confirmed that TaKCS1 and TaECR positively contribute to wheat wax biosynthesis.    Thereafter, we analyzed the cuticle permeability in the TaKCS1or TaECR-silenced wheat plants by measuring the water loss rate and chlorophyll extracted. As shown in Figure 4D, the leaves of the BSMV-TaKCS1as-or BSMV-TaECRas-infected wheat plants displayed higher water loss rates than the BSMV-γ controls. Consistent with this, the chlorophyll leaching assay showed that the wheat leaves with silenced TaKCS1 or TaECR genes released chlorophyll faster than the BSMV-γ controls ( Figure 4E). Overall, these results suggested that the knockdown of the TaKCS1 and TaECR expression levels led to compromised attenuated wax biosynthesis and enhanced surface barrier properties in wheat leaves.

TaMYB30 Activates Expression of TaKCS1 and TaECR
To determine the potential regulation of TaMYB30 on the expression of TaKCS1 and TaECR in bread wheat, we employed BSMV-VIGS to silence all endogenous TaMYB30 genes in the leaves of wheat cultivar Yannong 999. Thereafter, expression levels of TaKCS1 or TaECR genes were analyzed by qRT-PCR assay. As shown in Figure 5A, the silencing of TaMYB30 genes resulted in above 70% decreases in the expression levels of TaKCS1 and TaECR, suggesting that the MYB transcription factor TaMYB30 positively regulates the expressions of TaKCS1 and TaECR.
To quantify the transcriptional activity of TaMYB30 proteins, we performed the Arabidopsis leaf protoplast transfection assay, in which a luciferase (LUC) reporter was cotransfected with effector constructs expressing TaMYB30-5A, TaMYB30-5B, or TaMYB30-5D protein, and a control plasmid, to analyze the reporter luciferase activity (LucA). The Gal4 DNA-binding domain (DBD) was used to determine the basal LUC activity. As shown in Figure 5B, the LucA ratio has increased from 1 for the DBD control to above 2.2 under the expression of DBD-TaMYB30-5A, DBD-TaMYB30-5B, or DBD-TaMYB30-5D, indicating that TaMYB30 proteins exhibit transcriptional activator activity.
To determine the potential regulation of TaMYB30 on the expression of TaKCS1 and TaECR in bread wheat, we employed BSMV-VIGS to silence all endogenous TaMYB30 genes in the leaves of wheat cultivar Yannong 999. Thereafter, expression levels of TaKCS1 or TaECR genes were analyzed by qRT-PCR assay. As shown in Figure 5A, the silencing of TaMYB30 genes resulted in above 70% decreases in the expression levels of TaKCS1 and TaECR, suggesting that the MYB transcription factor TaMYB30 positively regulates the expressions of TaKCS1 and TaECR.  It has been demonstrated that wheat MYB transcription factor TaMYB96/TaEPBM1, a homolog of TaMYB30, could directly bind to the cis-elements MBS and Motif 1 [45,46]. Since MBS and Motif 1 cis-elements were found in the promoter regions of TaKCS1 and TaECR genes, we examined whether TaMYB30 proteins could directly bind to the TaKCS1 and TaECR promoters and activate their expressions. In the Arabidopsis leaf protoplast transfection assay, LUC reporters containing promoter regions of TaKCS1-4A, TaKCS1-4B, TaKCS1-4D, TaECR-3A, TaECR-3B, or TaECR-3D genes were co-transfected with effector constructs overexpressing TaMYB30-5A, TaMYB30-5B, or TaMYB30-5D proteins, and a control plasmid. Wild-type promoter regions of TaKCS1-4A, TaKCS1-4B, TaKCS1-4D, TaECR-3A, TaECR-3B, or TaECR-3D genes harboring the cis-elements MBS and Motif 1, and TaKCS1-4A, TaKCS1-4B, TaKCS1-4D, TaECR-3A, TaECR-3B, or TaECR-3D promoter mutants containing mutated MBS and Motif 1 cis-elements, were employed. As shown in Figure 5C,D, the LucA ratio obtained from LUC reporters containing wild-type TaKCS1 and TaECR promoters increased to above 2.07 in the presence of over-accumulated TaMYB30 proteins, compared with 1 for the Gal4 DNA-binding domain (DBD) proteins and with less than 0.3 for the empty vector (EV) control. In contrast, the LucA ratio obtained from mutant TaKCS1 and TaECR promoters was not significantly affected by over-accumulated TaMYB30 proteins ( Figure 5C,D). This result implies that the transcriptional activators TaMYB30-5A, TaMYB30-5B, and TaMYB30-5D could directly bind to the promoter regions of TaKCS1 and TaECR genes by recognizing the MBS and Motif 1 cis-elements, and activate their expression in plant cells.

TaMYB30 Is a Positive Regulator of Wax Biosynthesis in Bread Wheat
In this study, TaMYB30 is characterized as a positive regulator of wheat wax biosynthesis. The knockdown of TaMYB30 expression using BSMV-VIGS resulted in attenuated wax biosynthesis and enhanced cuticle permeability. Increasing evidence revealed that wheat wax biosynthesis is governed by transcription factors and their interactors. For instance, the knockout or knockdown expression of the transcription factor gene TaSHN1/WIN1 attenuated wheat wax biosynthesis, whereas the transgenic overexpression of the TaSHN1/WIN1 gene resulted in enhanced wax accumulation in bread wheat [38,39,47]. The mediator component CYCLIN-DEPENDENT KINASE8 (TaCDK8) was demonstrated to interact and phosphorylate the transcription factor TaSHN1/WIN1 [38,[48][49][50]. The silencing of TaCDK8 by BSMV-VIGS assay resulted in attenuated wax accumulation in wheat leaves, indicating that TaCDK8 positively regulates wheat wax biosynthesis [38]. Interestingly, AtCDK8 interacts with AtSHN1 to potentiate wax biosynthesis in the dicot model plant A. thaliana, implying that the SHN1-CDK8 interacting module regulates wax biosynthesis in dicot and monocot plants [51]. The wheat R2R3-type MYB transcription factor TaMYB96/TaEPBM1 was revealed to trigger cuticular wax biosynthesis [45,46]. The TaGCN5-TaADA2 histone acetyltransferase module was shown to associate with TaMYB96/TaEPBM1 and mediate histone acetylation at the promoter regions of TaMYB96/TaEPBM1 target genes [45]. The knockdown of TaGCN5 or TaADA2 expression levels led to reduced wax accumulation, indicating that the TaGCN5-TaADA2 histone acetyltransferase module interacts with TaMYB96/TaEPBM1 to trigger wax biosynthesis [45]. Similarly, the CHD3-type chromatin remodeling factor TaCHR729 could interact with the basic helix-loop-helix (bHLH) transcription factor TaKPAB1 and mediate the deposition of the permissive epigenetic mark H3K4me3 at TaKCS6 promoters [52,53]. These studies demonstrated that transcription factors interact with other regulators such as mediator components and epigenetic components to fine-tune wheat wax biosynthesis. Therefore, it is intriguing to identify TaMYB30-interacting partners and characterize their potential roles in the regulation of wheat wax biosynthesis in future research.

Wheat TaKCS1 and TaECR Are Essential Components of Wax Biosynthetic Machinery
TaKCS1 is identified as an important component of wax biosynthetic machinery in bread wheat. In A. thaliana, AtKCS1 encodes a 3-ketoacyl-CoA synthase involved in wax biosynthesis [54]. The T-DNA-tagged kcs1-1 mutant displayed a decrease of up to 80% in the levels of VLC wax alcohols and aldehydes, but much smaller effects on the major Arabidopsis wax components the C29 alkanes and on the total wax load, suggesting the potential redundancy in the KCS activities involved in Arabidopsis wax synthesis [54]. In contrast, the barley eceriferum-zh (cer-zh) mutant displays an obvious glossy and wax-deficient phenotype [55]. CER-ZH encodes the β-ketoacyl-CoA synthase HvKCS1, a barley ortholog of AtKCS1. In this study, the silencing of TaKCS1 by BSMV-VIGS led to compromised wax biosynthesis, increased water loss rates, and enhanced chlorophyll leaching [55]. These results indicate the potential functional conservation of KCS1 in monocot and dicot plants, and that the distinct contribution of KCS1 to total wax load in Arabidopsis, barley, and wheat might result from different wax compositions among these plant species.
As an obligate biotrophic fungal pathogen, Blumeria graminis forma specialis tritici (B.g. tritici) has evolved sophisticated mechanisms to infect the host wheat plants. Increasing evidence suggests that wax signals from wheat epidermis could be harnessed by the host-adapted B.g. tritici fungus to promote pre-penetration infection processes [38,45,52]. For instance, the silencing of wheat wax biosynthesis genes TaWIN1, TaKCS6 and TaECR resulted in the attenuated conidial germination of B.g. tritici. In this study, the knockdown of TaMYB30 or TaKCS1 expressions led to reduced cuticular wax biosynthesis. It is therefore intriguing to examine whether and how TaMYB30 and TaKCS1 affect pre-penetration events such as conidial germination in the infection of B.g. tritici in future studies.
The Enoyl-CoA Reductase (ECR) gene encodes the enoyl-CoA reductase that catalyzes the biosynthesis of VLC acyl-CoAs in the fatty acid elongase complex [56]. Recent phylogenetic analysis showed that ECR genes are highly conserved among land plants. The knockout of ECR genes could significantly attenuate wax biosynthesis in Arabidopsis, whereas the ectopic overexpression of CsECR cloned from the "Newhall" navel orange (Citrus sinensis [L.] Osbeck cv. Newhall) increased cuticular wax accumulation in the tomato (Solanum lycopersicum) and enhanced its drought resistance [57,58]. In this and previous studies, the silencing of TaECR by BSMV-VIGS resulted in reduced wax biosynthesis and enhanced cuticle permeability, as well as the attenuated germination of B.g. tritici conidia [45]. These data suggested that ECR plays a key role in the wax biosynthesis among dicot and monocot plants, and has potential values in crop breeding for improving stress resilience and disease resistance.

TaMYB30 Activates Expression of TaKCS1 and TaECR1 to Positively Regulate Wheat Wax
Three closely homologous TaMYB30-5A, TaMYB30-5B, and TaMYB30-5D proteins were identified as transcriptional activators in this study. Importantly, TaMYB30-5A, TaMYB30-5B, or TaMYB30-5D could directly bind to the promoter regions of TaKCS1 and TaECR genes by recognizing the MBS and Motif 1 cis-elements, and stimulate their expression in plant cells. These data collectively suggested that TaMYB30 positively regulates wheat wax biosynthesis presumably via the transcriptional activation of TaKCS1 and TaECR ( Figure 5E). It was previously reported that the expressions of Arabidopsis wax biosynthesis genes such as AtKCS1, AtECR, etc. were reduced in the AtMYB30-knockout mutant but enhanced in the AtMYB30-overexpressing lines, indicating that the up-regulation of wax biosynthesis genes by MYB30 and its orthologs is conserved among dicots and monocots [43].
It was previously demonstrated that another R2R3-type MYB transcription factor TaMYB96/TaEPBM1 could activate the expression of TaECR genes and positively regulate wheat wax biosynthesis [45]. In this study, the transcriptional activator TaMYB30 was shown to directly recognize the promoter regions of TaECR-3A, TaECR-3B, and TaECR-3D genes, and to activate their expression, suggesting that a single wax biosynthesis gene might be targeted by different MYB transcription factors. Accumulating studies revealed that plant wax biosynthesis is tightly regulated at the transcriptional level and affected by developmental stages and environmental conditions. It is intriguing to examine the interplays among different MYB transcription factors in the regulation of wax biosynthesis in response to developmental and environmental cues in future research.

Plant Materials
The plant seedlings of bread wheat cultivar Yannong 999 were grown in a growth chamber under a 16-h/8-h, 20 • C/18 • C day/night cycle with 70% relative humidity. A. thaliana Col-0 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.

Water Loss Rate and Chlorophyll Leaching Tests
The water loss rate tests were performed as described previously [59,60]. Briefly, the leaves (n = 5) with BSMV virus symptoms about 2 weeks post-BSMV-infection were dipped in ultrapure water under dark conditions for 1 h to maintain the stomatal closure, and leaf weights were then measured per 1 h for 12 h. The water loss rate tests were performed as described [59,61]. Wheat plants (n = 5) with BSMV virus symptoms about 2 weeks post-BSMV-infection were kept in the dark and immersed in ultrapure water for 1 h. Then leaves were detached and dipped in 80% ethanol, and the amount of chlorophyll extracted was measured at 647 and 664 nm with a spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA, USA) every 60 min for 12 h.

Wax Composition Analysis
The cuticular wax composition analysis was performed as described [38]. The leaves (n = 5) with BSMV virus symptoms about 2 weeks post-BSMV-infection were dipped into chloroform (Merck, Rahway, NJ, USA). The extracts dried under N 2 were derivatized at 70 • C for 30 min through reaction with bis-N,O-trimethylsilyl trifluoroacetamide and analyzed with a capillary GC (5890 Series II, Agilent Technologies) and a flame ionization detector (6890 N, Agilent Technologies) with a mass spectrometer (MSD 5973, Agilent Technologies) as previously described [38]. Quantification was based on FID peak areas relative to the internal standard n-Tetracosane (Merck) and the dry, delipidated tissue weights.

Arabidopsis Protoplast Transactivation Assay
The isolation and transformation of the Arabidopsis protoplast were conducted as described previously by Zhi et al. [62]. The vector pRT-DBD and pRT were employed to generate construct expression proteins with and without DBD fusion, respectively. The effector constructs, including pRT, pRT-BD, and derivatives, were mixed with the reporter gene and co-delivered into the Arabidopsis protoplasts by transfection. At 48 h after transfection, the LUC activity was analyzed using a Promega dual-luciferase reporter assay system (Promega, Madison, WI, USA, E1910) following the manufacturer's manual.
Author Contributions: L.L. and C.C. planned and designed the research; L.L. and X.W. performed most of the experiments with help from X.W.; L.L., H.L. and C.C. analyzed data and wrote the manuscript with contribution from X.W. 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.