FvNST1b NAC Protein Induces Secondary Cell Wall Formation in Strawberry

Secondary cell wall thickening plays a crucial role in plant growth and development. Diploid woodland strawberry (Fragaria vesca) is an excellent model for studying fruit development, but its molecular control of secondary wall thickening is largely unknown. Previous studies have shown that Arabidopsis NAC secondary wall thickening promoting factor1 (AtNST1) and related proteins are master regulators of xylem fiber cell differentiation in multiple plant species. In this study, a NST1-like gene, FvNST1b, was isolated and characterized from strawberry. Sequence alignment and phylogenetic analysis showed that the FvNST1b protein contains a highly conserved NAC domain, and it belongs to the same family as AtNST1. Overexpression of FvNST1b in wild-type Arabidopsis caused extreme dwarfism, induced ectopic thickening of secondary walls in various tissues, and upregulated the expression of genes related to secondary cell wall synthesis. In addition, transient overexpression of FvNST1b in wild-type Fragaria vesca fruit produced cells resembling tracheary elements. These results suggest that FvNST1b positively regulates secondary cell wall formation as orthologous genes from other species.


Introduction
The secondary cell wall (SCW) is typically composed of lignin, cellulose, and hemicelluloses (xylan and glucomannan and galactoglucomannans). The SCW is formed inside the primary cell wall after the cell is fully expanded. SCW structures have large impacts on the characteristics of plant cells and organ development and play important roles in the dehiscence of anthers and silique pods, enhance mechanical support of organs, facilitate water transport, and provide a barrier against invasive pathogens [1][2][3][4]. The SCW is characteristically formed in xylem vessels and fibers and is crucial in the development of secondary xylem. The deposition of secondary walls reinforces stability of these cells, allowing them to provide structural support and protection [5]. The secondary walls of anther endothecium have striated patterns similar to those in tracheary elements. These secondary wall thickenings are necessary for anther dehiscence, and they generate the tensile force necessary for the rupture of the stomium [6]. Lignification in the endodermal layer of the valve margin of silique pods is necessary for their dehiscence, generating tension via desiccation and leading to pod shattering [7][8][9].Thus, SCW provides crucial biological roles in various organs, and unveiling mechanisms behind the regulation of SCW formation has been an important topic in the plant developmental research.
Extensive studies have been performed in multiple species from angiosperms such as Arabidopsis and Zinnia elegance to resolve the transcriptional network controlling xylem vessel differentiation and SCW formation. Several proteins in the plant-specific NAC (NAM, ATAF1/2 and CUC2) transcription factor (TF) family have been found to play a pectin lyase) and contributes to cell wall remodeling [46]. Although FcNAC1 was reported to be clustered with VND family members in the phylogenetic analysis, whether it has the ability to induce the SCW thickening as members from other species has not been tested yet. FaRIF regulates ABA biosynthesis/signaling and cell wall degradation/modification [45]. Lignin synthesis is an important pathway regulated by VNSs. VNSs may have additional significance in strawberry fruit development besides regulators of SCW development, since biosynthesis pathway of lignin and anthocyanin, an important factor of color formation in strawberry fruit, share common precursor molecules [47,48]. Balance between lignin synthesis and anthocyanin synthesis needs to be well-coordinated for proper strawberry fruit development, and regulation towards VNSs may have roles in this process.
In this study, we isolated and characterized a VNS subfamily gene in Fragaria vesca and named FvNST1b. The sequence information, subcellular localization, and expression pattern of FvNST1b were investigated. Transgenic Arabidopsis plants overexpressing FvNST1b showed abnormal SCW thickening and induction of SCW-associated genes. Ectopic xylem cells were also produced by transient overexpression of FvNST1b in strawberry fruits. Our work demonstrated that FvNST1b of the NAC transcription factor family in strawberry possess conserved activity to promote SCW development, and may play critical roles in SCW formation in fruit.

Cloning and Sequence Analysis of FvNST1b
Characterization of VNS genes from Fragaria vesca has not been performed yet. We identified Fragaria vesca VNS candidate genes from the SGR: Strawberry Genomic Resources database (http://bioinformatics.towson.edu/strawberry/; accessed on 1st November 2018). We performed multiple sequence alignment of Arabidopsis VNS family members and Fragaria vesca VNDs and NSTs. These protein sequences contain a highly conserved region towards the N-terminal, corresponding to the NAC domain, which is divided into five subdomains, A to E (Figure 1). To investigate the relationship between FvVNSs and AtVNSs, a phylogenetic tree was constructed using their amino acid sequences. The phylogenetic tree indicated that all members are divided into VND, NST, and SMB subclades, with FvNST1b together with AtNST1 protein grouped into one cluster ( Figure 2). The FvNST1b is annotated to encode a protein of 365 amino acids with an estimated molecular mass of 40.8 kDa and an isoelectric point of 6.27. These results suggested that FvNST1b is the closest counterpart protein of AtNST1, and a plausible candidate for the regulator of secondary wall thickening.   Amino acid sequences alignment of FvVNS and NAC proteins from Arabidopsis including AtVNSs. The proteins were initially aligned using Clustal omega. The NAC domain was marked with solid lines.

Figure 2.
Phylogenetic tree of FvVNS and AtVNS proteins. The proteins were initially aligned using Clustal omega and then submitted for phylogenetic analysis using MEGA X software. The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replications. Numbers indicate bootstrap values for the clades that received support values of over 50%.

Subcellular Localization of FvNST1b Protein
As a transcription factor, FvNST1b is expected to function in the nucleus. In order to examine its subcellular localization in vivo, we generated a vector containing coding region of FvNST1b fused with GFP reporter gene. The fusion gene plasmid and GFP control plasmid were transiently transformed into Nicotiana Benthamiana (hereafter tobacco) leaves and strawberry fruits. At 3 days after injection, a strong fluorescence signal was Figure 2. Phylogenetic tree of FvVNS and AtVNS proteins. The proteins were initially aligned using Clustal omega and then submitted for phylogenetic analysis using MEGA X software. The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replications. Numbers indicate bootstrap values for the clades that received support values of over 50%.

Subcellular Localization of FvNST1b Protein
As a transcription factor, FvNST1b is expected to function in the nucleus. In order to examine its subcellular localization in vivo, we generated a vector containing coding region of FvNST1b fused with GFP reporter gene. The fusion gene plasmid and GFP control plasmid were transiently transformed into Nicotiana Benthamiana (hereafter tobacco) leaves and strawberry fruits. At 3 days after injection, a strong fluorescence signal was detected in the nucleus of tobacco leaf epidermal cells ( Figure 3A), and strong GFP signal in nucleus was detected in the strawberry fruits at 4 days after injection ( Figure 3B). In some cells, the GFP signal was also weakly detected in the surrounding area of the nucleus presumably in cytosol or ER that may represent FvNST1-GFP protein unsorted to the nucleus. At 7 days after induction, tobacco cells with ectopic striated cell walls are formed as in the overexpression of AtNST1 reported by others (Supplemental Figure S1) [2]. Transient overexpression of FvNST3-GFP also induced similar effects (Supplemental Figure S1). We also generated stably transformed Arabidopsis plants with FvNST1-GFP. Arabidopsis transgenic seedlings also showed strong nuclear-localized GFP signals and weak cytoplasmic/ER signals in roots ( Figure 3C).  The images of bright field, GFP and merged were shown. Scale bars represent 10 µm for zoom and 100 µm for root, respectively.

Expression Analysis of the FvNST1b Gene
Tissue-specific expression analysis of FvNST1b was performed by qRT-PCR using various tissues from strawberry plants. FvNST1b displayed a differential expression pattern in F. vesca ( Figure 4). FvNST1b transcripts were almost undetectable in leaf and white fruit, and its expression was detected in vegetative parts of strawberry at relatively low levels, including in roots and stems, whereas its expression in flowers and green fruits are significantly higher, with the highest level in green fruits. This observation suggested that FvNST1b mainly function in the fruits of strawberry at the earlier developmental stages.

Overexpression of FvNST1b Induces Ectopic Thickening of Secondary Walls in Various Tissues of A. thaliana
In order to test if FvNST1b has the ability to promote SCW deposition, we expressed FvNST1b-GFP ectopically under the control of the CaMV35S promoter (35S:FvNST1b-GFP) in transgenic Arabidopsis plants. The 35S:FvNST1-GFP b plants were usually smaller and grew more slowly than wild-type plants. Ectopic expression of FvNST1b-GFP induced ectopic lignified secondary wall thickening in various tissues, including anthers, stamens, ovules, stems, leaves, and root tissues as reported for the overexpression of Arabidopsis NST1 [2]. Epidermal cells with ectopic secondary wall thickening typically had a striated appearance similar to that of tracheary elements ( Figure 5).
These observations suggest that the abnormal appearance of leaves and floral organs of 35S:FvNST1b-GFP plants was due to the ectopic accumulation of lignified materials, reflecting previous reports that NSTs are regulators of secondary wall thickening in various tissues.

Overexpression of FvNST1b Induces SCW Formation in Strawberry Fruits
To further confirm that FvNST1b has the ability to promote SCW deposition in strawberry fruit, we transiently overexpressed the FvNST1b gene by using the agrobacterium infiltration into S7 fruits of Fragaria vesca ( Figure 6), which is at the green stage and make the transition to ripening [49]. After four days from infiltration, the 35S:FvNST1b-GFP infiltrated strawberry fruits exhibited GFP signals ( Figure 6A-D). After five days, the 35S:FvNST1b-GFP infiltrated strawberry fruits exhibited enhanced lignification phenotypes, along with many cells having striated appearance similar to that of tracheary elements ( Figure 6E,F,J-L). The 35S:FvNST1b-GFP infiltrated fruits tended to be more pliable and the space among seeds closer than that of 5d after the injection of PGWB505 vector-control infiltrated fruits ( Figure 6G-I,M-O). Longitudinal sections of strawberry fruits stained for lignin and cell wall structure confirmed that overexpression of FvNST1b-GFP resulted in excessive SCW deposition in strawberry fruit cells, indicating their ability to promote SCW formation in strawberry fruits.

Enhanced Gene Expression of SCW Related Genes in 35S:FvNST1b Transgenic Arabidopsis Plants
To further prove the ability of FvNST1b to promote SCW development as in the orthologous genes from other species such as AtNST1, we examined the effect of FvNST1b-GFP overexpression in Arabidopsis on gene expression of known downstream genes for AtNST1 (Figure 7). We examined the expression of IRREGULAR XYLEM3 (IRX3;

Enhanced Gene Expression of SCW Related Genes in 35S:FvNST1b Transgenic Arabidopsis Plants
To further prove the ability of FvNST1b to promote SCW development as in the orthologous genes from other species such as AtNST1, we examined the effect of FvNST1b-GFP overexpression in Arabidopsis on gene expression of known downstream genes for AtNST1 (Figure 7). We examined the expression of IRREGULAR XYLEM3 (IRX3; encodes a cellulose synthase), IRX4 (encodes a cinnamoyl CoA reductase), IRX12 (encodes a putative laccase) as genes known to be upregulated by AtNST1 overexpression [50][51][52], and HOMEOBOX GENE8 (ATHB-8), which is involved in the vascular developmental process upstream of VNDs/NSTs [53]. encodes a cellulose synthase), IRX4 (encodes a cinnamoyl CoA reductase), IRX12 (encodes a putative laccase) as genes known to be upregulated by AtNST1 overexpression [50][51][52], and HOMEOBOX GENE8 (ATHB-8), which is involved in the vascular developmental process upstream of VNDs/NSTs [53].
The expression of the IRX3, IRX4, and IRX12 genes was enhanced 1-to 10-fold in all four of the independent 35S:FvNST1b-GFP transgenic lines examined, as compared with the wild type. In contrast, ATHB-8 was not upregulated in 35S:FvNST1b-GFP plants. Our results suggest that FvNST1b has the ability to function as a crucial regulator for secondary wall thickening by inducing key downstream genes similar to their counterpart transcription factors in other plant species. Expression of genes related to the differentiation of tracheary elements were analyzed in 2 WT plants as controls and 4 independent transgenic Arabidopsis lines overexpressing FvNST1b-GFP by quantitative RT-PCR. Each bar represents the amount of the transcript of a gene relative to that of the internal control. Error bars represent ± SD (n = 3). Asterisk indicate a significant difference compared to the WT1 by t-test (****, p < 0.0001; ***, p < 0.001).

Discussion
Strawberry is one of the most economically important fruit crops and has been considered a genuine example of a plant showing non-climacteric fruit ripening [54,55]. The ripe strawberry fruits undergo continual softening and easily become rotten. Thus, improvement of strawberry shelf life has become an important factor in current breeding programs, even when these quality attributes are controlled by a complex genetic background [56]. Secondary wall thickening provides mechanical support for various plant tissues, and thus the SCW formation may contribute to fruit firmness [25,57]. NSTs/VNDs are master transcriptional switches regulating the developmental program of SCW biosynthesis by activating downstream transcription factors [25,58]. Although NAC transcription factors and the lignin biosynthesis have been studied in strawberry fruit development recently [45,46], their contribution regarding the molecular control of secondary The expression of the IRX3, IRX4, and IRX12 genes was enhanced 1-to 10-fold in all four of the independent 35S:FvNST1b-GFP transgenic lines examined, as compared with the wild type. In contrast, ATHB-8 was not upregulated in 35S:FvNST1b-GFP plants.
Our results suggest that FvNST1b has the ability to function as a crucial regulator for secondary wall thickening by inducing key downstream genes similar to their counterpart transcription factors in other plant species.

Discussion
Strawberry is one of the most economically important fruit crops and has been considered a genuine example of a plant showing non-climacteric fruit ripening [54,55]. The ripe strawberry fruits undergo continual softening and easily become rotten. Thus, improvement of strawberry shelf life has become an important factor in current breeding programs, even when these quality attributes are controlled by a complex genetic background [56]. Secondary wall thickening provides mechanical support for various plant tissues, and thus the SCW formation may contribute to fruit firmness [25,57]. NSTs/VNDs are master transcriptional switches regulating the developmental program of SCW biosynthesis by activating downstream transcription factors [25,58]. Although NAC transcription factors and the lignin biosynthesis have been studied in strawberry fruit development recently [45,46], their contribution regarding the molecular control of secondary wall thickening is largely unknown. In the present study, we described the isolation and characterization of FvNST1b, an NST1-like homolog from Fragaria vesca.
Amino acid sequence alignment and phylogenetic analysis suggested that FvNST1b is a member of the NST class of NACs. Plant NAC domain proteins are one of the largest groups of plant-specific transcriptional factors and have been reported to participate in many developmental processes, including SCW formation and biotic and abiotic stress responses [59,60]. Amino acid sequences of NAC proteins typically contain an NAC domain; five highly conserved subdomains at the N-terminal. The five highly conserved subdomains are also present in the FvNST1b sequence. Our phylogenetic analysis placed FvNST1a and FvNST1b as the closest homolog of Arabidopsis NST1 and NST2. Indeed, results of our overexpression experiments suggested that FvNST1b and FvNST3 have the ability to promote SCW development as in Arabidopsis NSTs. In our phylogenetic analysis, we could not find counterparts for some members of the VND-related Arabidopsis proteins, namely SOMBRERO (SMB), BEARSKIN1 (BRN1), and BRN2 among strawberry VNS candidates [35]. Those proteins still have the ability to induce ectopic SCW deposition when overexpressed, but they are involved in root cap development in Arabidopsis [34,35]. It is speculated that there may be other unidentified members of the VND family or other transcription factors in strawberry to regulate the development of strawberry root caps.
FvNST1b is expressed preferentially in strawberry green fruit, suggesting that it has important roles in the regulation of strawberry fruit development. Overexpression of FvNST1b in Arabidopsis caused ectopic deposition of SCW ( Figure 5), in agreement with previous studies. In addition, overexpression of FvNST1b in strawberry fruits also caused ectopic deposition of SCW along with lignin accumulation, fruit shrinkage, and fruit color change. Anthocyanin contributes to the fruit color in strawberry. Biosynthetic pathways for lignin and flavonoids, including anthocyanin, share common precursors from the general phenylpropanoid pathway [48]. Several TF genes in the MYB family are reported to corepress or co-activate genes involved in the biosynthesis of lignin and flavonoids. There are also cases of regulation towards lignin or flavonoid synthesis to achieve proper balance of carbon flow between lignin and flavonoids. Some of those members such as AtMYB20 are reported to be under regulation by VNSs [61]. Thus, VNSs in strawberry including FvNST1b may contribute to co-regulate and/or balance lignin deposition and anthocyanin synthesis, which is a crucial factor for strawberry fruit quality and commercial value. Future analysis of contribution of VNSs on the regulation of flavonoid-synthesis-related genes will be important to further prove this idea.
A nuclear localization signal was predicted within FvNST1b amino acid sequences, and the transient transformation of tobacco and strawberry fruit cells with FvNST1b fused to a reporter gene showed that FvNST1-GFP localizes to the nucleus. Furthermore, nuclear localization was detected in 35S:FvNST1b-GFP transgenic Arabidopsis plants. Other members of this NAC family also exhibited nuclear localization: an NAC from S. lycopersicum was shown to be located in the nucleus when ectopically expressed in onion epidermal cells, as was also the case for AtNAC2 [62]. MtNST1 from alfalfa, described as a SCW master switch, was also identified in the nucleus of epidermal tobacco cells [63]. The tomato SlNAC3 has been localized in the nucleus of onion epidermal cells by transient expression analysis [62]. Seven GhSWN proteins from cotton all located in the nucleus and were consistent with their functions as transcription factors [27]. In order to test whether FvNST1b has ability to activate SCW-related genes as in AtNSTs, the expression of IRX3, IRX4, IRX12, and ATHB-8 were examined in 35S:FvNST1b transgenic plants, which are involved in the differentiation of tracheary elements upstream or downstream of AtNSTs [2]. IRX3, IRX4, and IRX12 were upregulated in 35S:FvNST1b transgenic plants. In contrast, ATHB-8 was not regulated in 35S:FvNST1b plants, which is in line with previous reports in Arabidopsis. These results show that FvNST1b is a positive regulator of secondary wall thickening. Induction of downstream genes of AtNST1 in transgenic 35S:FvNST1b Arabidopsis plants further support the idea that FvNST1b acts as a transcriptional factor to regulate downstream processes of SCW development.
In summary, an NST1-like gene, FvNST1b, was isolated and characterized from strawberry. FvNST1b has high sequence similarity to other NSTs homologs and contained the well-conserved NAC domain. The FvNST1b protein mainly localizes in the nucleus. FvNST1b is highly expressed in young fruit. In addition, overexpression of FvNST1b caused ectopic deposition of SCW and upregulated the expression of genes related to the differentiation of tracheary elements such as IRX3, IRX4, and IRX12 in transgenic Arabidopsis. Moreover, overexpression of FvNST1b in strawberry fruits also caused ectopic deposition of SCW along with lignin accumulation and fruit shrinkage. These results suggest that FvNST1b is a transcription factor promoting SCW thickening in strawberry. Although we were unable to detect any expression of FvNST1a in strawberry fruit, we also showed that at least another closely related member, FvNST3, which is expressed in fruit, has a similar function. Functional redundancy as well as their specialization will be explored in the future. The evidence provided will contribute to understanding the regulatory network that takes place during the development and ripening of strawberry fruit.

Plant Material and Growth Conditions
Diploid strawberry plants (Fragaria vesca), Yellow Wonder 5AF7 (YW5AF7) [40], planted in pots (90 mm × 90 mm × 90 mm) were used in this study. The seedlings were grown and maintained in a growth room with the following conditions: 22 • C, 60% humidity, and a 16-h photoperiod. Hand pollination was performed by using downy water bird feather to obtain pollinated fruit. Samples of root, stem, leaf, flower, and fruit were collected for tissue-specific expression assays. For Arabidopsis transformation, Arabidopsis thaliana (ecotype Columbia) was used and grown in soil at 22 • C with 16 h of light daily.

DNA Preparation and Gene Cloning
Genomic DNA (gDNA) of strawberry samples was isolated by the CTAB method. To clone the FvNST1b gene, the AtNST1 protein was used for a BLAST search in the strawberry genome GBrowse (http://www.strawberrygenome.org/ (accessed on 19 October 2022)), and a high homology protein with the gene locus101309102 was found. Then, the specific primers for full-length of DNA cloning were designed for FvNST1b (forward, 5 -attB1-ATG ACT GAA AAC GTG AGC AT-3 ; reverse, 5 -attB2-TTA TAT ATG ACC ATT CGA CAC GTG-3 ) and FvNST3 (forward, 5 -attB1-ATG TCT GCA GAG GAT CAA ATG-3 ; reverse, 5 -attB2-TTA TAC CGA CAG GTG GCA TAA TG-3 ) using the SnapGene program (https://www.snapgene.com/ (accessed on 19 October 2022)). PCR was performed using Primer Start Max Enzyme (TaKaRa Biotech, Dalian, China) under the following conditions: 98 • C for 30 s, followed by 34 cycles at 98 • C for 10 s, 55 • C for 15 s, and 72 • C for 30 s.

Construction of Plasmid DNA
The GATEWAY™ conversion technology (Invitrogen, Gaithersburg, MD, USA) was used in the experiment. To generate the FvNST1b overexpression vector, full-length FvNST1b DNA (1541 bp) was amplified and inserted into the PDONR221 vector under the treatment of BP Enzyme (Invitrogen). The entry vector DNAs were transformed into Escherichia coli DH5α cells and sequenced. The PDONR221-FvNST1b was treated with LR Enzyme (Invitrogen) and cloned into the PGWB505 vector containing the GFP reporter gene to generate PGWB505-FvNST1b-GFP.

Transient Expression of FvNST1b in Nicotiana Benthamiana Leaves and Sub-Cellular Localization Analysis
PGWB505-FvNST1b vector was introduced into Agrobacterium tumefaciens strain GV3101 by thermal shock in liquid nitrogen. Transformed bacteria were plated on a selective medium yeast mold agar containing kanamycin, hygromycin, and rifampicin at a final concentration of 100 µg/mL each. Resistant colonies were analyzed by PCR for the presence of full-length FvNST1b gene using the primers mentioned above. A positive colony was cultured in selective LB liquid medium and incubated at 28 • C until an O.D.600 between 0.8 and 1.0, and then the cells were resuspended in infection buffer and shaken for 2 h at 28 • C. A 1-mL syringe was used to inject the agrobacterium suspension into the abaxial face of young tobacco leaves (two weeks old), and samples were analyzed after two days of infiltration. Subcellular localization of FvNST1b in transient-transformed leaf samples was analyzed through visualization of the tissue under a confocal fluorescence microscope (Leica Confocal microscope SP8X; Leica Microsystems GmbH, Wetzlar, Germany ) with a 10× objective lens, a 488 nm laser from tunable white light laser for excitation, and a 499 nm to 551 nm bandwidth for detection.

Gene Expression Level Analysis
Total RNA from the strawberry samples was extracted using the polysaccharide and polyphenolics-rich RNAprep Pure Kit (Tiangen, Beijing, China); cDNA was synthesized from total RNA using the PrimeScript RT reagent Kit (Perfect Real Time) (Takara). Total RNA of Arabidopsis was extracted using the PLANT RNA Kit (omega). The cDNA samples were diluted 1:5 with water; 2 µL of the diluted cDNA was used as a template for quantitative real-time PCR (qRT-PCR) analysis. Real-time quantitative PCR was performed in the ABI 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using SYBR Premix Ex Taq II (Takara). The PCR program included an initial denaturation step at 95 • C for 3 min, followed by 40 cycles of 10 s at 95 • C, and 30 s at 57 • C. Each sample represented three biological replicates; each of them included four technical replicates. The relative expression levels of target genes were calculated with the formula 2 −∆∆CT in strawberry and 2 −∆CT in Arabidopsis.

Arabidopsis Transformation
Agrobacterium tumefaciens strain mentioned earlier were transformed into wild-type Arabidopsis plants using the floral dip method. Transgenic seedlings were selected on half-strength Murashige and Skoog (MS) agar plates containing 50 mg/L hygromycin and 200 mg/L Timentin; antibiotic-resistant plants were then tested by GFP signal and separation ratio to confirm the presence of the transgene. Four independent lines of the T3 generation were randomly chosen for further analysis.

Transient Overexpression of FvNST1b in Strawberry Fruit
Agrobacterium tumefaciens strain GV3101 mentioned above was used to perform transient expression analyses in strawberry fruits [64]. For Agrobacterium infection, the Agrobacterium suspension was injected into the fruit using a syringe of 1 mL capacity. To do this, the needle tip was inserted into the fruit center from the top, and then the Agrobacterium suspension was slowly and evenly injected into the fruits until the strawberry fruit was completely infected. After the infection, the fruits were incubated under the conditions required for the different experimental aims. The effect of overexpression was evaluated by examining the changes in both reporter gene expression and related phenotypes after Agrobacterium infection.

Fruit Sections and Staining
The infected strawberry fruits were embedded in 10% agarose gel at 7 days after, and 200 µm thick sections were cut with a vibratome. Strawberry fruit sections and Arabidopsis seedlings were fixed with 4% PFA for 60-120 min at 23-25 • C temperature with vacuum treatment. After fixation, the materials were washed twice for 1 min in 1 × PBS and moved to the clearing solution. After rinsing in 1 × PBS, the plant material was transferred to the ClearSee solution [65] and cleared overnight at room temperature. We prepared 0.1% Auramine O in ClearSee solution and the materials were stained overnight. Then, the materials were washed for at least 1 h with gentle shaking. The materials were transferred to 0.1% Calcofluor White in ClearSee solution and stained for 30 min; the materials were washed in ClearSee for 30 min with gentle shaking. Materials were analyzed with Leica TCS SP8X inverted confocal microscope. Imaging Calcofluor White was performed by a 405-nm diode laser for excitation and detection band width at 425-475 nm. Imaging Auramine O was performed with 488 nm from a tunable white light laser and band width detected at 505-530 nm.