Expression characteristics of CsESA1 in citrus and analysis of its interacting protein

doi:10.21203/rs.3.rs-4979104/v1

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Expression characteristics of CsESA1 in citrus and analysis of its interacting protein

https://doi.org/10.21203/rs.3.rs-4979104/v1

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The most damaging disease affecting citrus globally is Huanglongbing (HLB), primarily attributed to the infection by ‘Candidatus Liberibacter asiaticus’ (CaLas). Based on comparative transcriptome data, two cellulose synthase (CESA) genes responsive to CaLas infection induction were screened, and one gene cloned with higher differential expression level was selected and named CsCESA1. The yeast two-hybrid (Y2H) and Bimolecular Fluorescent Complementation (BiFC) experiments confirmed the interaction between CsCESA1 and citrus exopolysaccharide 2 (CsEPS2). Subcellular localization in tobacco indicated that both CsCESA1 and CsEPS2 proteins are primarily located in the nucleus and cytoplasm. RT-qPCR analysis indicated that the expression levels of CsCESA1 and CsEPS2 were associated with variety tolerance, tissue site, and symptom development. Furthermore, we employed the Virus-induced gene silencing (VIGS) system to generate CsCESA1 and CsEPS2 silencing plants. We established a stable transformation system mediated by Agrobacterium rhizogenes in citrus and obtained CsCESA1 and CsEPS2 silencing and overexpressing hairy roots. The analysis of hormone content and gene expression also showed that CsCESA1 and CsEPS2 are involved in transcriptional regulation of genes involved in systemic acquired resistance (SAR) response. In conclusion, our results suggested that CsCESA1 and CsEPS2 could serve as potential resistance genes for HLB disease, offering insights into the plant's defense mechanisms against HLB.

Citrus

CsCESA1

CsEPS2

VIGS

hairy roots

Huanglongbing (HLB) in citrus, represents a significant global threat to plant health (Soares et al., 2022). This phloem-restricted α-proteobacterium is Gram-negative and is known to cause chronic conditions in infected plants (Soares et al., 2022). Citrus trees infected with CaLas exhibit a wide range of symptoms, such as yellowing of leaves, fruit deformities, and yellowing of leaf veins (McCollum et al., 2016). However, so far, there is no effective method to prevent and control citrus HLB disease (Baldwin et al., 2018).

Cellulose is synthesized from plasma membrane complexes, which are believed to be composed of CESA proteins. CESA1 appears to be crucial for the production of primary wall cellulose (Paredez et al., 2006). The negative regulatory factor of CESA complex exocytosis is involved in cellulose synthesis, which is essential for plant resistance to bacterial and fungal pathogens (Benschop et al., 2007; Polko et al., 2018). EPS synthase is an enzyme of the shikimate pathway (Wang et al., 1991). EPS genes have been cloned from tobacco, Arabidopsis, cotton, and other plants, and related gene function studies have been conducted (Chen et al., 2016). In most plants, EPS coding genes belong to multi genes, encoded by the nuclear genome (Della-Cioppa et al., 1986). In recent years, some glyphosate resistant plants have been obtained through radiation mutagenesis, targeted screening, and transgenic research, and have been widely used in production (Herrmann et al., 1995).

During the long-term co evolution process between plants and pathogenic microorganisms, a two-level defense system is formed to resist pathogen infection (Boller et al., 2009). The immune system of plants has long been categorized primarily into two types: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) (Zhou et al., 2020). Through pattern recognition receptors (PRRs) found on their cell membranes, plants identify pathogen-associated molecular patterns (PAMPs), initiating the initial layer of their immune defense against pathogen invasion (Couto et al., 2016). Pathogens that successfully penetrate will release toxic proteins into plant cells, which subsequently undermine the plant's immune defenses, facilitating further infection. Plants have the capacity to recognize pathogen effectors directly or indirectly via NLR receptor proteins inside the cells, thus activating the second level of their immune system and generating a more robust immune response to combat pathogen invasion (Cui et al., 2015). Citrus HLB disease, caused by CaLas, represents a significant threat to citrus (Wang et al., 2019). The infection by CaLas can trigger chronic and systemic immune reactions within the phloem tissue, leading to events such as callose deposition, production of reactive oxygen species (ROS), and the activation of immune-related genes (Yuan et al., 2021). Studies indicate that HLB is a disease mediated by immune responses (Ma et al., 2022).

By comparing the findings of genomic differential expression analysis, this experiment identified the CsCESA1 gene that is responsive to CaLas infection. Additionally, we have confirmed the interaction between the CsCESA1 and CsEPS2 proteins in this study. We selected CsCESA1 and CsEPS2 genes as target genes and established a transient transformation system and a stable transformation system for hairy roots in citrus using the VIGS system. The results of this study provided technical support for the functional identification of citrus genes, so as to explore potential candidate genes for molecular breeding against HLB disease.

2.1 Plant materials, microbial strains and growth conditions

Following the transformation, the citrus seedlings were cultivated in a greenhouse maintained at 26 ℃. Leaves of Nicotiana benthamiana were grown under conditions of 18 hours of light and 6 hours of darkness for a period of 5 to 6 weeks at 26°C. E. coli DH5α was cultured on Luria-Bertani (LB) medium at a temperature of 37 ℃. Both Agrobacterium tumefaciens strains EHA105 and GV3101 were propagated on LB medium containing 50 µg/mL of kanamycin at 28 ℃.

2.2 Y2H

In the directed Y2H assay, CsCESA1 was introduced into the pGBKT7 vector to serve as bait, while CsEPS2 was inserted into the pGADT7 vector to act as prey. The primers employed can be found in Supplementary Table 1, with the vectors confirmed through Sanger sequencing. The chosen pairs of bait and prey constructs were co-transformed into Y2H Gold yeast and subsequently plated on SD-Leu-Trp (DDO) medium, where they were cultured in a 30 ℃ thermostatic incubator. Yeast clones containing the paired constructs that were able to grow on DDO were resuspended in sterile water, diluted between 10 and 10,000 times, and then 10 µL droplets of these culture dilutions were transferred onto SD-Leu-Trp (DDO), SD-Leu-Trp-His (TDO), and SD/-Leu/-Trp/-Ade/-His (QDO) plates.

2.3 BIFC

To perform protein-protein interaction assays, cultures of Agrobacterium GV3101 containing CsCESA1-nYFP and CsEPS2-cYFP were co-infiltrated into the leaves of N. benthamiana. At 3 days post-infiltration (dpi), YFP fluorescence signals were analyzed using confocal microscopy. Detection of YFP occurred following excitation with a laser at a wavelength of 488 nm, with emissions captured in the range of 500 to 550 nm.

2.4 Subcellular localization

CsCESA1 and CsEPS2 genes were inserted into the pCAMBIA1300-35S-GFP vector (Liu et al., 2019). At 3 dpi, leaves from N. benthamiana were harvested. The localization of CsCESA1 and CsEPS2 at the subcellular level was examined using an FV3000 confocal microscope equipped with a UV light source (Olympus, Tokyo, Japan). In this study, the green fluorescent protein (GFP) served as a marker for the nucleus, while the red fluorescent protein (RFP) was utilized for the cytoplasmic membrane. The test was repeated three times.

2.5 DNA/RNA extraction, cDNA synthesis, and quantitative PCR analysis

The samples of Carrizo citrange leaves collected were preserved at − 80°C. The method for extracting citrus genomic DNA adhered to the guidelines provided by the EASYspin Plus kit (Aidlab, Beijing, China). A PCR reaction was conducted in a total volume of 20 µL, which included 1 µg of DNA. The conditions for PCR amplification were set as follows: an initial treatment at 98°C for 3 minutes, followed by 35 cycles of amplification (30 seconds at 98°C, 30 seconds at 58°C, and 30 seconds at 72°C), concluding with a 3-minute extension at 72°C. Total RNA was extracted from Carrizo citrange using an RNA extraction kit (Aidlab, Beijing, China). The Prime ScriptTM RT Master Mix (TaKaRa, Ojin, Japan) was employed to reverse transcribe RNA into cDNA, utilizing 1 µg of total RNA in a 20 µL reaction. The resulting cDNA was diluted fivefold, and a qPCR assay was conducted in a 10 µL reaction mixture that included 6 µL of the SYBRPRIME qPCR Kit, 4.4 µL of DNase/RNase-free water, 0.3 µL of both forward and reverse real-time primers, and 1 µL of the diluted cDNA template. The PCR conditions were as follows: an initial step at 95°C for 3 minutes, succeeded by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Utilizing the citrus GAPDH gene (Mafra et al., 2012) as the internal control, the relative expressions of CsCESA1 and CsEPS2 were assessed using the 2^−ΔΔCt method (Livak and Schmittgen, 2001). The gene expression analysis was conducted with three biological and technical replicates. Detailed information on the primers used can be found in Supplementary Table 1.

2.6 Citrus transformation

The primer sets FVIGS-CsCESA1/RVIGS-CsCESA1 and FVIGS-CsEPS2/RVIGS-CsEPS2 were utilized for amplifying VIGS fragments (Supplementary Table 1), which were subsequently inserted into the TRV2 vector to create TRV2-CsCESA1 and TRV2-CsEPS2. VIGS transformation, following infiltration with A. tumefaciens, was performed as previously outlined. After a period of 30 days, plants exhibiting green fluorescence under UV illumination were selected for additional analysis. The primers designed for detecting TRV1 were TRV1-F/R, while TRV2 detection employed TRV2-F/R primers (Supplementary Table 1). PCR analysis was conducted to identify the silenced plants. The expression levels of the CsCESA1 and CsEPS2 genes in the silenced specimens were assessed through RT-qPCR analysis.

To generate transgenic hairy roots, pCAMBIA1300, pCAMBIA1300-CsCESA1, pCAMBIA1300- CsEPS2, pCAMBIA1300RNAi, pCAMBIA1300-CsCESA1RNAi, and pCAMBIA1300-CsEPS2RNAi vectors were transformed into a K599 strain, utilizing the stem segment of Carrizo citrange for A. rhizogenes-mediated transformation (He et al., 2024). PCR analysis was conducted to identify the successful transgenic plants. RT-qPCR analysis was employed to determine the expression levels of the CsCESA1 and CsEPS2 genes in the transgenic hairy roots.

2.7 Measurement of hormone content

0.1g of freshly harvested transgenic hairy roots (including wild-type) was frozen and ground into a fine powder using liquid nitrogen. To this powder, 0.9mL of PBS buffer at a pH of 7.4 was added, and the mixture was then centrifuged at 8000 rpm for 30 minutes. The supernatant obtained was stored at -20 ℃ for future analysis. The methods for determining the contents of salicylic acid (SA), methyl salicylate (MeSA), jasmonic acid (JA), and reactive oxygen species (ROS) followed the instructions provided by the kit (TaKaRa, Ojin, Japan). All hormone content tests were repeated three times.

2.8 Statistical analyses

Data were analyzed with Excel 365, and significance assessment was conducted using IBM SPSS Statistics 23.0. The Student's t-test was utilized to evaluate the differences between the control group and the samples at a significance level of 5%.

3.1 Interaction between CsCESA1 and CsEPS2

Y2H was used to analysis putative interactions between CsCESA1 and CsEPS2. The results showed that the yeast transformants carrying pGBKT7-CsCESA1 and pGADT7-CsEPS2 grew normally on the QDO media (Fig. 1A). pGBKT7-53 and pGADT7-T served as positive controls, whereas pGBKT7-Lam and pGADT7-T functioned as negative controls.

BiFC showed that strong fluorescence signals were observed in the cytomembrane of tobacco cells when the CsEPS2 tagged with the C-terminal fragment of YFP (CsEPS2-cYFP) and CsCESA1 tagged with the N-terminal YFP-fragment (CsCESA1-nYFP) were co-expressed in N. benthamiana. No signals were detected when the negative controls combinations of CsEPS2-cYFP with nYFP, CsCESA1-nYFP with cYFP, and nYFP with cYFP were co-expressed (Fig. 1B). These results indicated that CsCESA1 interacts with CsEPS2.

3.2 Subcellular localization of the CsCESA1 and CsEPS2 proteins

CsCESA1 and CsEPS2 were fused with GFP at the BamHI and SalI sites of the 35sGFP vector to obtain p35s-CsCESA1-GFP and p35s-CsEPS2-GFP (Fig. 2A). Using p35S: GFP as the control and H2B: RFP as the nuclear marker gene, tobacco epidermal cells were transiently transformed by using Agrobacterium-mediated method. Confocal laser scanning microscopy showed that CsCESA1 and CsEPS2 were located in the nucleus and cytoplasm (Fig. 2B).

3.3 Expression of CsCESA1 and CsEPS2 in citrus

The expression levels of CsCESA1 and CsEPS2 in symptomatic leaves were considerably greater compared to those in asymptomatic leaves, suggesting a role for CsCESA1 and CsEPS2 in the development of citrus HLB symptoms (Fig. 3A-B). RT-qPCR was employed to investigate the expression characteristics of CsCESA1 and CsEPS2 in both the vein and root of Carrizo citranges. The data revealed that the expression of the CsCESA1 and CsEPS2 genes in the root was elevated by approximately 1.9 and 3.2 times, respectively (Fig. 3C-D). When healthy Carrizo citranges served as a control, the results indicated that the expression levels of CsCESA1 and CsEPS2 in CaLas-infected samples were significantly higher than those of the WT control (Fig. 3E-F). Further analysis of the expression characteristics of CsCESA1 and CsEPS2 in different citrus varieties. Using Pomelo and Carrizo citranges as the materials and the results showed that the CsCESA1 and CsEPS2 genes expression level in the Carrizo citrange leaves was up-regulated by about 3.1 and 2.5 times, respectively, compared to Pomelo (Fig. 3G-H).

3.4 Generation of TRV-CsCESA1 and TRV-CsEPS2 plants

TRV-CsCESA1 and TRV-CsEPS2 vectors were constructed (Fig. 4A). A total of 7 TRV-CsCESA1 plants and 8 TRV-CsEPS2 plants were generated through PCR verification (Fig. 4B). There was no significant difference in phenotype between TRV-CsCESA1 and TRV-CsEPS2 plants and control plants (Fig. 4C). RNA was isolated from the positive plants, and RT-qPCR was utilized to measure the relative expression levels in the silenced plants. The results indicated that the expression levels of the genes TRV-CsCESA1 and TRV-CsEPS2 in the experimental plants were markedly reduced compared to the control group (Fig. 4D and E).

3.5 Generation of transgenic citrus hairy roots overexpressing CsCESA1 and CsEPS2

pCAMBIA1300-CsCESA1 and pCAMBIA1300-CsEPS2 vectors were constructed (Fig. 5A). 9 CsCESA1 and 7 CsEPS2 transgenic hairy roots were identified by PCR amplification (Fig. 5B). The root rate of CsCESA1 and CsEPS2 transgenic hairy roots was 90% and 70%, respectively (Fig. 5C). Comparing transgenic hairy roots to wild-type (WT) hairy roots, no notable variation in phenotype was observed. The hairy roots were generally white or yellow in color, with a length ranging from 6cm to 15cm (Fig. 5D). Extracting RNA from the positive CsCESA1 and CsEPS2 transgenic hairy roots. Using RT-qPCR, we assessed the relative expression levels of CsCESA1 and CsEPS2 in the transgenic hairy roots. The results indicated that the expression levels of CsCESA1 and CsEPS2 genes in the transgenic hairy roots were considerably higher than those found in the control group (Fig. 5E and F).

3.6 Generation of transgenic citrus hairy roots silencing CsCESA1 and CsEPS2

pCAMBIA1300-CsCESA1RNAi and pCAMBIA1300-CsEPS2RNAi vectors were created (Fig. 6A). A total of 9 CsCESA1 and 6 CsEPS2 transgenic hairy roots were detected (Fig. 6B). The root rate of CsCESA1 and CsEPS2 transgenic hairy roots were 45% and 60%, respectively (Fig. 6C). Comparing with wild-type (WT) hairy roots, no significant differences in phenotype were noted. These hairy roots appeared either white or yellow and varied in length from 4 cm to 15 cm (Fig. 6D). RNA extraction was performed on CsCESA1 and CsEPS2 transgenic hairy roots identified as positive. Subsequently, we employed RT-qPCR to evaluate the relative expression levels of CsCESA1 and CsEPS2. The results indicated that the expression levels of CsCESA1 and CsEPS2 genes in the transgenic hairy roots were considerably lower than those found in the control group (Fig. 6E and F).

3.7 Characteristics of changes in SA, MeSA, JA and ROS content in CsCESA1 and CsEPS2 silencing hairy roots

SAR in plants is crucial for citrus plants' ability to tolerate HLB (Zou et al., 2021). SA, MeSA, and JA serve as significant signaling molecules in the SAR response of plants (Park et al., 2007; Zhu et al., 2014). To examine how the silencing of CsCESA1 and CsEPS2 affects the SAR response, we evaluated the alterations in the concentrations of SA, MeSA, and JA in these silenced hairy roots. Comparing these to the wild-type (WT) hairy roots, we observed a downregulation of SA, MeSA, and JA levels in all silenced hairy roots (Fig. 7A-F). CaLas infection promoted the accumulation of H₂O₂ in plants (Li et al. 2019). Therefore, this study detected H₂O₂ content in silencing hairy roots. In the CsCESA1 and CsEPS2 silencing hairy roots, the H₂O₂ content was significantly lower than that of the WT hairy roots (Fig. 7G-H). The results showed that silencing of CsCESA1 and CsEPS2 promoted the accumulation of H₂O₂.

3.8 The expression level of the defense-related genes were remarkably increased in CsCESA1 and CsEPS2 transgenic hairy roots

The investigation of the expression levels of 6 SAR-associated genes, which include three CsNPR3 genes (Ciclev10017873m, Ciclev10031115m, Ciclev10031749m) as reported by Wang et al. (2016), along with CsPR1, CsPR2, and CsPR5 from Zou et al. (2021), was conducted using qPCR in transgenic hairy roots. The results indicated a significant elevation in the expression of these genes in CsCESA1 and CsEPS2 transgenic hairy roots (Fig. 8A-F and Fig. 9A-F).

To explore the role of CsCESA1 and CsEPS2 in plant defense mechanisms, we analyzed the expression levels of five marker genes associated with PTI and ETI (CsFRK1, CsGST1, CsWRKY22, CsWRKY92, and PR1) as described by Nguyen et al. (2021). The results indicated a notable increase in these genes within CsCESA1 and CsEPS2 transgenic hairy roots (Fig. 8G-K and Fig. 9G-K).

This study used transcriptome data to screen and clone a CsCESA1 gene, and predicted and validated its interacting CsEPS2 protein. Analysis of subcellular localization in tobacco suggested that CsCESA1 and CsEPS2 proteins may play roles in the nucleus and cytomembrane of plants.

In the process of interaction between pathogens and plants, transcriptional expression analysis of endogenous genes helps to analyze defense responses during pathogen infection. The expression levels of endogenous genes in citrus at varying times and locations might be vital for pathogen invasion and colonization, alongside host tolerance and survival (Shi et al., 2019). Findings indicated that the CsCESA1 and CsEPS2 expression levels were markedly greater in symptomatic leaves compared to asymptomatic ones, and also significantly elevated in susceptible leaves relative to healthy leaves. Further investigation into the expression levels of CsCESA1 and CsEPS2 was performed in the leaves of pomelo and Carrizo citrange, which revealed a significant upregulation of these genes in pomelo leaves. Moreover, the expression of CsCESA1 and CsEPS2 genes in the roots of Carrizo citrange was notably higher than in the leaf veins, implying that these genes could have a crucial role in pathogen colonization within the roots. In summary, RT-qRCR studies have shown that the expression of CsCESA1 and CsEPS2 was associated with variety tolerance, tissue location, and symptom development.

Genetic alterations affecting cellulose production and levels grant plants improved resistance against necrotrophic fungi. The genes encoding CESA have a transcriptional reprogramming mechanism, which occurs quickly after the attack of Botrytis cinerea, causing some CESA genes to temporarily shut down (Ramírez et al., 2011). In plants, the infection caused by pathogens triggers a temporary reduction in the expression of the CESA gene. This system detects the presence of pathogens and converts this signal into a swift transcriptional reprogramming of the affected cells (Ikeda et al., 2009). At present, research on EPS mainly focuses on herbaceous plants and herbicide resistance. EPS is a key enzyme in the synthesis of aromatic amino acids, but there are few reports on its research in plant stress physiology and secondary metabolism. The study of EPS expression regulation will provide theoretical basis for plant herbicide resistance and stress resistance mechanisms (Herrmann et al., 1995).

The genetic transformation of citrus mediated by Agrobacterium tumefaciens has drawbacks such as long cycle, high difficulty, false positivity, and easy contamination, which seriously hinders the research of citrus genetic breeding. In this study, citrus hypocotyls were used as materials to obtain silencing CsCESA1 and CsEPS2 transgenic hairy roots after vacuum infiltration with Agrobacterium rhizogenes. The method of using Agrobacterium rhizogenes mediated citrus hypocotyls to produce hairy roots was used to avoid bacterial contamination, efficiently produced transgenic hairy roots, and quickly performed preliminary functional identification and analysis of genes, significantly shortened the cycle of citrus genetic transformation and resistance evaluation, provided a good foundation for citrus genetic breeding. The Agrobacterium rhizogenes-mediated gene silencing system established in this study can be widely used for rapid gene function research in most herbaceous and woody plants.

Previous studies have shown that when plants are invaded by pathogens, a series of complex disease resistance mechanisms are formed, usually through the transmission pathways of disease resistance signals such as SA, JA, and ROS, thereby activating the plant's disease resistance and defense response pathways (Shi et al., 2019). CESA family genes are also involved in plant immune defense processes mediated by signals such as SA, JA, and ROS (Nuruzzaman et al., 2013). Existing studies have shown that SA, JA, and ROS are involved in the regulation of citrus response to HLB. For example, high levels of SA and JA content in plants are closely related to citrus HLB tolerance (Peng et al., 2021). In this study, the hormone contents in CsCESA1 and CsEPS2 transgenic hairy roots were detected, and the results showed that the hormone content of SA, JA, and ROS was significantly upregulated. Based on this speculation, CsCESA1 and CsEPS2 may be related to citrus disease tolerance and may be involved in the process of citrus resistance to CaLas infection.

SAR is an active defense mechanism mediated by SA in response to foreign pathogen invasion in plants, with broad-spectrum characteristics. In normal plant tissues, SA is at a lower level (Zhong et al., 2016). When plants are infected by foreign pathogens, the plant hormone signaling pathway is activated, promoting accelerated SA synthesis in the plant body, regulating the transcription of downstream related genes, and enabling local plant resistance (Park et al., 2007). Furthermore, the synthesis and expression of PRs proteins are induced through signal transduction pathways such as MAPK (Peng et al., 2021). In this research, the overexpression of CsCESA1 and CsEPS2 had a significant impact on the concentrations of SA, JA, MeSA, and ROS, leading to a marked increase in the expression of genes related to immunity in transgenic hairy roots, which are associated to the tolerance of citrus plants to HLB (Zou et al., 2019). To summarize, the aforementioned studies suggested that CsCESA1 and CsEPS2 might influence the defense responses in citrus by modulating the transcriptional activity of SAR-related genes in these plants. The molecular mechanism through which CsCESA1 and CsEPS2 collaborate to modulate plant immunity merits further investigation.

This experiment verified the interaction between CsCESA1 and CsEPS2, and analyzed their expression characteristics in citrus. Subsequently, CsCESA1 and CsEPS2 transgenic plants overexpressing and gene editing will be obtained through genetic transformation technology. An assessment of resistance and observation of symptoms in the transgenic plants will be carried out to further investigate the function of CsCESA1 and CsEPS2 during the infection process of CaLas.

In summary, our research indicated the interaction between CsCESA1 and CsEPS2, obtained CsCESA1 and CsEPS2 silencing and overexpressing hairy roots. The research results provided theoretical basis and genetic resources for molecular breeding of citrus resistant to HLB in the future.

Acknowledgements

We greatly appreciate the valuable contributions of our community advisory committee members. We would also like to thank every team member who took the time to participate in this study.

Authors Contributions

Xiao He: Designing the experiments, Writing and Revising the manuscript, Funding acquisition. Huiying Wang: Analyzing the data, Performing the experiments, Writing the manuscript. Wei Wei: Performing the experiments, Writing the manuscript. Ziyue Han: Performing the experiments, Writing the manuscript. Jiaqi Zuo: Writing the manuscript. Qing He: Designing the experiments, Writing and Revising the manuscript, Funding acquisition. All the authors read and approved the final version of the manuscript.

Funding Statement

This work was supported by the Chongqing Key Laboratory of Development and Utilization of Genuine Medicinal Materials in Three Gorges Reservoir Area Nursery project (XJ2023003503).

Data availability

The data supporting this study’s findings are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Ethics Approval:

Not applicable.

Conﬂicts of Interest

The authors declare no conflicts of interest.

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Expression characteristics of CsESA1 in citrus and analysis of its interacting protein

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Abstract

Figures

1 INTRODUCTION

2 MATERIALS AND METHODS

2.1 Plant materials, microbial strains and growth conditions

2.2 Y2H

2.3 BIFC

2.4 Subcellular localization

2.5 DNA/RNA extraction, cDNA synthesis, and quantitative PCR analysis

2.6 Citrus transformation

2.7 Measurement of hormone content

2.8 Statistical analyses

3 Results

3.1 Interaction between CsCESA1 and CsEPS2

3.2 Subcellular localization of the CsCESA1 and CsEPS2 proteins

3.3 Expression of CsCESA1 and CsEPS2 in citrus

3.4 Generation of TRV-CsCESA1 and TRV-CsEPS2 plants

3.5 Generation of transgenic citrus hairy roots overexpressing CsCESA1 and CsEPS2

3.6 Generation of transgenic citrus hairy roots silencing CsCESA1 and CsEPS2

4 DISCUSSION

5 Conclusions

Declarations

References

Supplementary Files

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