Identiﬁcation and Functional Characterization of CsMYCs in Cucumber Glandular Trichome Development

: Glandular trichomes (GTs), specialized structures formed by the differentiation of plant epidermal cells, are known to play important roles in the resistance of plants to external biotic and abiotic stresses. These structures are capable of storing and secreting secondary metabolites, which often have important agricultural and medicinal values. In order to better understand the molecular developmental mechanisms of GTs, studies have been conducted in a variety of crops, including tomato ( Solanum lycopersicum ), sweetworm ( Artemisia annua ), and cotton ( Gossypium hirsutum ). The MYC transcription factor of the basic helix-loop-helix (bHLH) transcription factor family has been found to play an important role in GT development. In this study, a total of 13 cucumber MYC transcription factors were identiﬁed in the cucumber ( Cucumis sativus L.) genome. After performing phylogenetic analyses and conserved motifs on the 13 CsMYCs in comparison to previously reported MYC transcription factors that regulate trichome development, seven candidate MYC transcription factors were selected. Through virus-induced gene silencing (VIGS), CsMYC2 is found to negatively regulate GT formation while CsMYC4 , CsMYC5 , CsMYC6 , CsMYC7 , and CsMYC8 are found to positively regulate GT formation. Furthermore, the two master effector genes, CsMYC2 and CsMYC7 , are observed to have similar expression patterns indicating that they co-regulate the balance of GT development in an antagonistic way.


Introduction
Trichomes on the aerial parts of plants are usually divided into two types: glandular tichomes (GTs) and non-glandular trichomes (NGTs) [1]. Known as the 'plant biofactory' due to their ability to synthesize and store specific secondary metabolites, the GTs structure and the products they synthesize and secrete are agriculturally important for plant adaptation and resistance to external stresses [2,3]. In addition, the metabolites secreted by some specific plant GTs can be used to make medicines and essential oils, such as artemisinin in the GTs of Artemisia annua and cannabinoid in the GTs of Cannabis sativa L., which have medicinal and economic value [4][5][6]. Therefore, the study of the developmental mechanism of GTs and the synthetic mechanism of their internal metabolites is important for the development of natural products metabolic engineering. Research on the development of GTs and their internal metabolite synthetic mechanism is relatively lagging behind due to the fact that the model plant Arabidopsis thaliana only has NGTs. Currently, researchers

Genome-Wide Identification and Phylogenetic Analysis of Cucumber MYC Transcription Factors
To identify MYC transcription factors in the cucumber genome, we downloaded the genome protein sequences through the cucurbit genome website (http://cucurbitgenomics.org/ (accessed on 25 January 2023)) and searched for genes with conserved the bHLH-MYC_N structural domain (PF14215) of MYC transcription factors and obtained a total of 18 candidate genes (Table S1). Among the 18 candidate genes, a total of 13 MYC genes contain both bHLH-MYC_N domain and bHLH domain and were identified in cucumber through the NCBI-CDD database and SMART database, namely CsMYC1-13 ( Table 1). The physical features of these MYC TFs were predicted: The protein length varies from 322 (CsMYC6) to 959 (CsMYC13) amino acids; the pI (Isoelectric Point) varies from 5.11 (CsMYC2) to 8.66 (CsMYC4); and the molecular weight varies from 36.38 kDa (CsMYC6) to 104.48 kDa (CsMYC13) ( Table 1). To gain a preliminary understanding of these genes, we looked for their homologs on Arabidopsis Thaliana. The homolog of CsMYC1 is AtGL3, which regulates the development of unicellular non-glandular trichomes on Arabidopsis [35] ( Table 1). The homolog of CsMYC2, CsMYC3, and CsMYC6 is AtMYC2, the homolog of CsMYC4 and CsMYC5 is AtbHLH14, the homolog of CsMYC7 is AtbHLH13, and the homologue of CsMYC8 is AtbHLH3 (Table 1). AtMYC2 encodes a MYC-related transcriptional activator with a typical DNA binding domain of a basic helix-loop-helix leucine zipper motif regulating diverse JA-dependent functions [42]. AtbHLH13 functions redundantly with AtbHLH3 and AtbHLH14, interacting with JAZ proteins, and negatively regulating jasmonate responses [27,28]. The homolog of CsMYC9 is AtAMS, which is involved in tapetal cell development [43]. AtEMB1444 and AtLHW, the homolog of CsMYC10, CsMYC11, CsMYC12, and CsMYC13, which take part in the production of stele cells in root meristems, are required to establish and maintain the normal vascular cell number and pattern in primary and lateral roots [44]. To further investigate the function of these MYC genes in trichome development, we performed the phylogenetic clustering analysis by comparing them with the previously reported glandular trichome development-related MYC TFs SlMYC1 [38], GoPGF [40], GhCGF1 [41] and the non-glandular trichome development-related MYC TFs AtGL3 [35], AtEGL3 [36], AtMYC1 [33], AtTT8 [34], GhDEL61 [45], GhDEL65 [46] (Tables 2 and S2). The tree with the highest log likelihood (−8624.0776) is shown (Figures 1 and S1). The percentage of trees in which the associated taxa clustered together is shown next to the branches. The phylogenetic analysis grouped these genes into three evolutionary branches, CsMYC2-8 belonging to the evolutionary branch related to GT development, CsMYC1 and CsMYC9 belonging to the evolutionary branch related to NGT development, and the other four CsMYCs in a new evolutionary branch ( Figure 1). Besides, the MEME analysis and NCBI-CCD analysis were carried out to predict the conserved motifs protein sequence and conserved domain to better understand the conservation and diversification of these MYCs ( Figure 1). A total five motifs were identified through all the MYC genes. Motif 1, motif 2, and motif 3 are located in the bHLH-MYC_N domain, while motif 4 is located in the bHLH domain. Previous studies have shown that the bHLH-MYC_N domain functions in metabolites biosynthesis and responds to jasmonic acid, while the bHLH domain functions in DNA binding or the dimer interface. A single amino acid change on motif 3 located in the bHLH-MYC_N domain would cause AtGL3 to lose its function in regulating NGT development, and therefore it can be hypothesized that motif 3 plays an important role in regulating trichome development [47]. In addition, motif 5 is not on any of the conserved structural domains but is unique to genes in two evolutionary branches that regulate trichome development, so it can be hypothesized that motif 5 also plays an important role in the regulation of trichome development in plants ( Figure 1). CsMYC2-CsMYC8 are in the GT-development regulating evolutionary branch with the same conserved motifs and domains as SlMYC1, GoPGF, and GhCGF1, suggesting that they may play a part in cucumber GT development. However, CsMYC10-CsMYC13 are on a novel phylogenetic branch that has a conserved MYC structural domain but lacks several motifs compared to other genes.

Identification of Candidate CsMYCs in Cucumber GT Development
To further investigate the role of the CsMYCs in cucumber GT development, we cloned the 7 CsMYCs (CsMYC2-CsMYC8) belonging to the group that regulates the GT development. Most of these CsMYCs contain only one exon, except CsMYC4 (Figure 2A). According to the translation results based on the sequence of the cloned CDS, all of the candidate CsMYCs have a conserved bHLH-MYC_N domain and bHLH domain ( Figure 2B).
Co-linear fragments are large segments of homology within a single species resulting from genome duplication, chromosome duplication, or large segment duplication. Conserved gene sequencing within a homologous segment means that it may also be functionally conserved. Because of the high evolutionary homology among the seven candidates, we hypothesized that these genes might be subject to intraspecies gene dupli-cation and therefore performed a collinearity analysis. The results show that in all gene duplications of the cucumber genome (grey line), there is only one pair of duplicated genes in seven candidates, which are CsMYC2 and CsMYC3 (red line) ( Figure 3A). The 2000 bp upstream sequences of CsMYC2-CsMYC8 were downloaded for cis-acting elements analysis and the number of cis-acting elements was calculated (Table S3). We found that the enriched cis-acting elements of the CsMYC2-CsMYC8 promoter can be broadly classified into six categories: plant hormone response elements, plant metabolic pathway response elements, plant developmental response elements, plant stress response elements, plant light response elements, and other transcription factor binding elements (Table S3). Of these, CsMYC2-CsMYC8 all contain a large number of MYB and MYC transcription factor binding elements ( Figure 3B). Among the phytohormone response elements, CsMYC2-CsMYC8 can be seen to respond to the regulation of various hormones, such as abscisic acid (ABRE), jasmonic acid (CGTCA-motif/TGACG-motif), auxin (AuxRE), and gibberellin (P-box/TATC-box/GAREmotif). Of these, abscisic acid and jasmonic acid have the largest number of response elements on the promoters of CsMYC2-CsMYC8 ( Figure 3B). In addition, CsMYC2-CsMYC8 can respond to a variety of stress signals, such as drought (MBS) ( Figure 3B, Table S3). Notably, among all seven MYC transcription factors, only the promoter of CsMYC3 contains an element that responds to flavonoid metabolism (MBSI) ( Figure 3B).

Identification of Candidate CsMYCs in Cucumber GT Development
To further investigate the role of the CsMYCs in cucumber GT development, we cloned the 7 CsMYCs (CsMYC2-CsMYC8) belonging to the group that regulates the GT development. Most of these CsMYCs contain only one exon, except CsMYC4 ( Figure 2A). According to the translation results based on the sequence of the cloned CDS, all of the  Co-linear fragments are large segments of homology within a single species resulting from genome duplication, chromosome duplication, or large segment duplication. Conserved gene sequencing within a homologous segment means that it may also be functionally conserved. Because of the high evolutionary homology among the seven candidates, we hypothesized that these genes might be subject to intraspecies gene duplication and therefore performed a collinearity analysis. The results show that in all

Candidate CsMYCs May Regulate the Formation of Cucumber GTs
We evaluated the possible roles of CsMYC2-CsMYC8 in GT development through TRSV (tobacco ringspot virus)-based VIGS. For each of the seven genes, RT-qPCR reveals that the expression level in the VIGS plant is significantly lower than the control ( Figure 4A), suggesting these genes are effectively silenced. The results of the VIGS suggest that almost all candidate CsMYCs are involved in the formation of GTs. TRSV::CsMYC2 VIIGS plants show a significant increase in GT density ( Figure 4B,C). TRSV::CsMYC4, TRSV::CsMYC5, TRSV::CsMYC6, TRSV::CsMYC7, TRSV::CsMYC8 plants all show a significant decrease in GT density, in which TRSV::CsMYC7 has the greatest decrease in GT density ( Figure 4B,C). The GT density of TRSV::CsMYC3 shows no change ( Figure 4B,C). The phenotypes of VIGS imply that CsMYC2 negatively regulates the formation of cucumber GTs, whereas CsMYC4, CsMYC5, CsMYC6, CsMYC7, and CsMYC8 positively regulate the formation of cucumber GTs redundantly. VIGS of the cucumber phytoene desaturase gene (CsPDS) is used as the positive control (TRSV::CsPDS), which results in a photo-bleaching phenotype ( Figure S2).
FOR PEER REVIEW 7 of 17 broadly classified into six categories: plant hormone response elements, plant metabolic pathway response elements, plant developmental response elements, plant stress response elements, plant light response elements, and other transcription factor binding elements (Table S3). Of these, CsMYC2-CsMYC8 all contain a large number of MYB and MYC transcription factor binding elements ( Figure 3B). Among the phytohormone response elements, CsMYC2-CsMYC8 can be seen to respond to the regulation of various hormones, such as abscisic acid (ABRE), jasmonic acid (CGTCA-motif/TGACG-motif), auxin (AuxRE), and gibberellin (P-box/TATC-box/GARE-motif). Of these, abscisic acid and jasmonic acid have the largest number of response elements on the promoters of CsMYC2-CsMYC8 ( Figure 3B). In addition, CsMYC2-CsMYC8 can respond to a variety of stress signals, such as drought (MBS) ( Figure 3B, Table S3). Notably, among all seven MYC transcription factors, only the promoter of CsMYC3 contains an element that responds to flavonoid metabolism (MBSI) ( Figure 3B).

Candidate CsMYCs May Regulate the Formation of Cucumber GTs
We evaluated the possible roles of CsMYC2-CsMYC8 in GT development through TRSV (tobacco ringspot virus)-based VIGS. For each of the seven genes, RT-qPCR reveals that the expression level in the VIGS plant is significantly lower than the control ( Figure  4A), suggesting these genes are effectively silenced. The results of the VIGS suggest that almost all candidate CsMYCs are involved in the formation of GTs. TRSV::CsMYC2 VIIGS plants show a significant increase in GT density ( Figure 4B,C). TRSV::CsMYC4, TRSV::CsMYC5, TRSV::CsMYC6, TRSV::CsMYC7, TRSV::CsMYC8 plants all show a significant decrease in GT density, in which TRSV::CsMYC7 has the greatest decrease in GT density ( Figure 4B,C). The GT density of TRSV::CsMYC3 shows no change ( Figure  4B,C). The phenotypes of VIGS imply that CsMYC2 negatively regulates the formation of cucumber GTs, whereas CsMYC4, CsMYC5, CsMYC6, CsMYC7, and CsMYC8 positively regulate the formation of cucumber GTs redundantly. VIGS of the cucumber phytoene

CsMYC2 and CsMYC7 Show Similar Expression Patterns in Cucumber
Based on the observed phenotypes, we further analyzed the detailed expression patterns of CsMYC2, which have a unique negative regulatory role, and CsMYC7, which has a drastic phenotype. Subcellular localization results show that both CsMYC2 and CsMYC7 are localized in the nucleus ( Figure 5A). Previous study has divided cucumber GT development into five stages, namely initiation stage (Stage I), first division stage (Stage II), glandular head transition stage (Stage III), glandular head formation stage (Stage IV), and active metabolism stage (Stage V). We next investigated the expression of these two genes in the transcriptomic data of the fifth stages and we found that they are both stably expressed during trichome development in cucumber ( Figure 5B) [10]. Since these transcriptome data were sampled from the entire cotyledon and it is still unknown whether these two genes are expressed in GTs, we performed in situ hybridization. The results show that CsMYC2 and CsMYC7 are both specifically expressed in GTs at the fourth stage ( Figure 5C). Expression analysis of CsMYC2 and CsMYC7 in different cucumber organs reveals that they are widely expressed in various tissue parts, with CsMYC2 being highly expressed in the pericarp, root, and ovary, and CsMYC7 being most highly expressed in the ovary, as well as in the pericarp, root, stem, and tendril ( Figure 5D,E). These results suggest that both CsMYC2 and CsMYC7 have a range of functions in all parts of cucumber. They show similar expression patterns but antagonistic functions in GTs. The cucumber GT has been marked with a red color. Bars represent 500 μm. Error bars represent SD from three biological repeats. ** indicates p-value < 0.01. *** indicates p-value < 0.001.

CsMYC2 and CsMYC7 Show Similar Expression Patterns in Cucumber
Based on the observed phenotypes, we further analyzed the detailed expression patterns of CsMYC2, which have a unique negative regulatory role, and CsMYC7, which has a drastic phenotype. Subcellular localization results show that both CsMYC2 and CsMYC7 are localized in the nucleus ( Figure 5A). Previous study has divided cucumber GT development into five stages, namely initiation stage (Stage I), first division stage (Stage II), glandular head transition stage (Stage III), glandular head formation stage (Stage IV), and active metabolism stage (Stage V). We next investigated the expression of these two genes in the transcriptomic data of the fifth stages and we found that they are both stably expressed during trichome development in cucumber ( Figure 5B) [10]. Since

Discussion
MYC transcription factors are key components of the bHLH transcription factor family and have been extensively studied in maize, Arabidopsis, rice, and other crops [21,22,48]. However, comparatively little research has been conducted on MYC transcription factors in cucumber. In this study, we identified a total of 13 MYC transcription factors in cucumber, significantly more than in other crops, belonging to three distinct evolutionary branches, indicating a high degree of redundancy in MYC functions in cucumber (Table 1). Previous studies have demonstrated that MYC transcription factors play a role in the morphogenesis of both glandular and non-glandular trichomes [33,[39][40][41]49]. Based on the important function of GTs, we focused our research on the role of MYC transcription factors in the regulation of GT development. Through a phylogenetic clustering analysis of previously reported MYC transcription factors, we found that the MYC transcription factors regulating the morphogenesis of GTs are on the same evolutionary branch, and seven of the 13 MYC transcription factors in cucumber belonging to this branch (Figure 1). This leads us to speculate that these seven genes are highly likely to regulate the development of GTs in cucumber. Previous studies have shown that a single amino acid change in the bHLH-MYC_N structural domain can alter the function of genes in regulating NGT development [47]. It remains to be seen if a similar change exists in the new branch that regulates GT development.
To further investigate the role of the CsMYCs in cucumber GT development, we cloned the seven CsMYCs (CsMYC2-CsMYC8) belonging to the group that regulates the GT development. Most of these CsMYCs contain only one exon, except CsMYC4 (Figure 2A). Since CsMYC4 is the most homologous gene for GoPGF in cotton and the cotton gland is a different lumenal structure from those of cucumber and tomato, we hypothesized that there may be some unique regulatory mechanism for CsMYC4 [40].
Although the seven genes are highly homologous, gene duplication research found that there are only one possible pair of duplicated genes, which are CsMYC2 and CsMYC3 ( Figure 3A). However, VIGS results suggest that CsMYC2 can negatively regulate GT formation, while no function is observed for CsMYC3 in cucumber GT development ( Figure 4B,C). A similar homologous pair of genes is also present in tomato. In tomato, SlMYC2, a homolog of SlMYC1 that regulates GT development, does not play a role in GT development but is involved in methyl jasmonate-induced tomato fruit resistance to pathogens [38,50]. Both CsMYC2 and CsMYC3 are the most homologous genes for SlMYC1. However, CsMYC3 is the only one with a cis-acting element for responding to the biosynthesis pathway of flavonoids on its promoter ( Figure 3B). GTs are the sites of secondary metabolite synthesis in plants, and CsMYC3, as the most homologous gene of CsMYC2, may be involved in the synthesis of secondary metabolites in GTs, which requires further investigation.
All seven candidate genes that potentially regulate the development of cucumber GTs were analyzed for cis-acting elements, and most of the candidate genes contain cis-acting elements in response to stress as well as plant hormones ( Figure 3B). MYC transcription factors are known to play an important role in the jasmonic acid-induced stress response pathway [32,42,51,52]. The regulation of GT development by jasmonic acid has also been reported in Artemisia annua and tomato [53,54]. In cucumber, the role of MYC between GT development and the jasmonic acid response pathway needs to be further investigated. The genes that regulate the formation of GTs in response to stress signals also suggest that GTs, as ubiquitous structures on the plant epidermis, serve as the first line of defense for the plant. In addition, candidate CsMYCs have a large number of binding elements for MYC transcription factors on their promoters, suggesting that they may be regulated by homologous genes (Figure 3B).
Our results showed that CsMYC2 negatively regulates the formation of GTs in cucumber, while CsMYC4-CsMYC8 redundantly and positively regulate the formation of GTs, with CsMYC7 being the primary effector gene (Figure 4). CsMYC2 is the first MYC transcription factor reported to negatively regulate GT formation, and its function deserves further exploration. In the previous transcriptome data, both CsMYC2 and CsMYC7 are stably expressed at five stages of multicellular trichome development without temporal variability ( Figure 5B). However, the transcriptome sampling is not GTs-specific, the expression pattern was not representative of their function in cucumber GTs. Thus, in situ hybridization was performed and found that they are highly expressed in GTs at the fourth stage ( Figure 5C). Combined with the VIGS results, it suggests that they are involved in different stages of glandular development, including not only the initiation differentiation from epidermal cells to trichome cells, but also the differentiation from trichome cells to glandular trichomes. Although we observed that CsMYC2 and CsMYC7 function in the leaf to regulate GT development, further expression patterns of these two genes reveal that they are expressed in various parts of the plant tissue, and the expression of them in leaf are relatively low. This result suggests that MYC transcription factors have powerful functions that are not limited to the regulation of GT formation. In addition, MYC transcription factors can form homopolymers or be regulated by each other, and we speculated that their expression may be influenced by each other (Figure 5D,E).
In Arabidopsis, the homologues of CsMYC2 and CsMYC7 are AtMYC2 and AtJAM2/AtbHLH13, respectively. Previous studies have shown that AtJAM2/AtbHLH13 are transcriptional repressors and AtMYC2 is a transcriptional activator functioning antagonistically to bind the G-box of downstream genes to regulate the JA signaling pathway [27]. This study also seems to be consistent with our finding that CsMYC2 and CsMYC7 exhibit similar expression patterns but antagonistic functions, with CsMYC2 inhibiting GT formation while CsMYC7 positively regulating it (Figures 4 and 5). The combined action of CsMYC2 and CsMYC7 helps to maintain the balance of GT formation, while the functional redundancy of CsMYC7, CsMYC4, CsMYC5, CsMYC6, and CsMYC8 may ensure the stable formation of GTs. However, the VIGS phenotypes only represent the result of transient silencing. To gain a more comprehensive understanding of gene function, we need to construct stable, genetically modified knockout plants.

Phylogenetic Analysis
Multiple sequence alignment of all listed MYCs (Table S1) was carried out using ClustalW. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [60]. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The analysis involved 22 amino acid sequences. All positions containing gaps and missing data were eliminated. There was a total of 226 positions in the final dataset. Evolutionary analyses were conducted in MEGA 7.0 [61]. The tree was further visualized by TBtools [62].

Gene Cloning
The CDS and genome reference sequence of seven candidate CsMYCs were downloaded from cucurbit genomics database (http://cucurbitgenomics.org/ (accessed on 25 January 2023)). Cloning primers designed by PrimerPremier 5.0 software. Total DNA was extracted using a DNA extraction kit (Huayueyang, Beijing, China) and cDNA was reverse transcribed using a PrimeScript reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan) from total RNA extracted using an RNA extraction kit (Huayueyang, Beijing, China).

Gene Structure and Protein Conserved Domain Alignment Analysis
The gene structure was visualized by the Gene Structure Display Server (http://gsds. gao-lab.org/index.php (accessed on 25 January 2023)) [64]. Multiple sequence alignment was carried out using ClustalW and the output data were saved in FASTA format. Protein conserved domain alignment was analyzed by Genedoc 2.6.002 software.

Collinearity Analysis
Homologous gene pairs relationships of candidate MYC genes in cucumber were identified using Multiple Collinearity Scan toolkit (MCScanX) software with default parameters [65]. The results were visualized using advanced circus plot in TBtools [62].

Cis-Regulatory Elements Analysis
The promoter sequences (2000 bp upstream of ATG) of seven candidates were extracted from genome sequences. The cis-regulatory elements in promoter region were analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/ html/ (accessed on 25 January 2023)) [66]. The heatmap of cis-regulatory elements analysis were generated by TBtools [62].

Plant Materials
The North China type (Chinese Long) cucumber inbred line Xintaimici (XTMC) was grown in a greenhouse of the China Agricultural University in Beijing. Pest control and water control followed standard practices. Nicotiana benthamiana plants were grown in a growth chamber set at 24 • C in a long-day condition (16-h light/8-h dark).

RNA Extraction and Real-Time Quantitative PCR (RT-qPCR) Analysis
Total RNA was extracted using an RNA extraction kit (Huayueyang, Beijing, China) and then reverse transcribed using a PrimeScript reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). Subsequently, RT-qPCR was performed in 96-well plates with an ABI 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using SYBR Premix Ex Taq (TaKaRa, Shiga, Japan). For each sample, three biological and three technical replicates were conducted, with cucumber CsTUA (α-tubulin) as the reference. The gene-specific primers used for qPCR are listed in Table S4.

VIGS Assay and Phenotypic Observation
A modified tobacco ringspot virus (TRSV)-based virus induced gene silencing (VIGS) assay was performed to analyze the potential roles of candidate seven CsMYCs in cucumber [67]. In brief, a unique 300-to 500-bp CDS sequence for each candidate gene (primers are provided in Supplemental Table S4) was inserted into the SnaBI restriction site of pTRSV2 and then transformed into Agrobacterium tumefaciens GV3101. When the primary roots of germinating cucumber seeds reached~1 cm, the seeds were infected with mixed pTRSV1 and pTRSV2 (containing different target fragments) by vacuum-infiltration under −900 kPa for 5 min. The seeds were then put on half MS solid medium with 100 µM acetosyringone until the agrobacterium was visible around the seeds. The seedlings were transplanted in half Hoagland solution for 15 d.
A total 10-12 VIGS positive plants were developed for each gene. The leaf blade with main vein of the first true leaf was then collected from each seedling. We sampled all VIGS plants to observe the phenotype under SEM. Three areas of each VIGS plant were sampled and three technical replicates of density observations were counted for each area, and finally the nine data were averaged to obtain the mean glandular trichome density of a single plant. Half of each sample was used for RT-qPCR to verify the gene expression level of gene transient silencing.

Subcellular Localization
The full-length CDS without the stop codon of CsMYC2 and CsMYC7 was cloned into the pSUPER1300 vector and fused with the green fluorescent protein (GFP) to produce the CsMYC2-GFP and CsMYC7-GFP fusion protein. The empty pSUPER1300 vector was served as a control. The resultant constructs were introduced into the Agrobacterium tumefaciens strain GV3101. Nicotiana benthamiana leaves were co-transformed with the GFP-fusion construct and the nuclear location marker (mCherry). After 48 h infiltration, fluorescence signals were visualized at excitation/emission wavelength of 488/510 nm (GFP), 552/610 nm (mCherry), using a confocal laser scanning microscope (Leica SP8, Germany). The primer information is listed in Table S4.

In Situ Hybridization
The sampling method of multicellular trichome during development using cotyledon as material is described in a previous study [10]. Samples were fixed with 3.7% formalinacetic acid-alcohol. In situ sense and antisense probes were transcribed with T7 RNA polymerase, respectively. Sample fixation, embedding, sectioning, and hybridization were conducted as previously described [68]. The primer information is listed in Table S4.

Conclusions
In conclusion, we conducted a genome-wide identification and analysis of MYC transcription factors in cucumber. Thirteen MYCs were identified in the C. sativus genome and their key structural features were compared to the previously reported MYCs from Arabidopsis and tomato to identify the MYCs involved in cucumber GT development. Seven candidate MYCs were analyzed and functionally characterized by VIGS, and it is found that CsMYC2 is able to negatively regulate GT formation while CsMYC4-CsMYC8 could positively regulate GT formation. The data obtained from this study provides new insights into the potential roles of CsMYCs in cucumber trichome development and establishes a basis for further research on cucumber GTs.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ijms24076435/s1. Author Contributions: X.L. and H.R. configured and supervised research, and revised the manuscript; Z.F., L.S. and M.D. performed the research; Z.F. drafted of manuscript; S.F., K.S., Y.Q., L.Z., W.W., Y.L., J.S. and X.C. contributed to experimental design, data interpretation, and manuscript editing. Y.W. participated in data analysis, and manuscript writing. All authors have read and agreed to the published version of the manuscript.