An Easy and Rapid Transformation Protocol for Transient Expression in Cotton Fiber

Abstract

Cotton fiber is the most important natural textile material in the world. Identification and functional characterization of genes regulating fiber development are fundamental for improving fiber quality and yield. However, stable cotton transformation is time-consuming, low in efficiency, and technically complex. Moreover, heterologous systems, such as Arabidopsis and tobacco, did not always work to elucidate the function of cotton fiber specifically expressed genes or their promoters. For these reasons, constructing a rapid transformation system using cotton fibers is necessary to study fiber's specifically expressed genes. In this study, we developed an easy and rapid Agrobacterium-mediated method for the transient transformation of genes and promoters in cotton fibers. First, we found that exogenous genes could be expressed in cotton fibers via using β-glucuronidase (GUS) and green fluorescence protein (GFP) as reporters. Second, parameters affecting transformation efficiency, including LBA4404 Agrobacterium strain, 3 h infection time, and 2-day incubation time, were determined. Third, four different cotton genes that are specifically expressed in fibers were transiently transformed in cotton fibers, and the transcripts of these genes were detected ten to thousand times increase over the control. Fourth, GUS staining and activity analysis demonstrated that the activity profiles of GhMYB212 and GhFSN1 promoters in transformed fibers are similar to their native activity in developmental fibers. Furthermore, the transient transformation method was confirmed to be suitable for subcellular localization studies. In summary, the presented Agrobacterium-mediated transient transformation method is a fast, simple, and effective system for promoter characterization and protein expression in cotton fibers.

Keywords: Agrobacterium; cotton fiber; promoter activity; subcellular localization; transient transformation.

FIGURE 1

Schematic procedures for Agrobacterium-mediated transient transformation of cotton fibers.

FIGURE 2

Transient expression of enhanced green fluorescence protein (GFP) gene (eGFP) and β-glucuronidase (GUS) protein in cotton fibers incubated with different Agrobacterium strains. (A,B) eGFP signal observation in cotton fibers transiently expressing 35S::eGFP construct using GV3101 (A) or LBA4404 (B). (C) Fibers without Agrobacterium infection were used as control. The red line and white line indicate 100 and 25 μm, respectively. (D,E) GUS staining (D) and activity determination (E) of cotton fibers transiently expressing 35S::GUS construct using GV3101, LBA4404, or fibers without Agrobacterium infection. Fibers on one seed were used as one biological replicate, and at least, three independent biological replicates were detected. The black line indicates 2 mm. Different upper case letters in (E) indicate significant differences at P < 0.01 (Turkey’s test). Error bars indicate the SD of three biological replicates. Fibers at 20-day post-anthesis (DPA) were used for transformation.

FIGURE 3

GUS expression analyses of 35S::GUS construct in cotton fibers under different transformation conditions. (A) GUS staining of fibers under different infection times with Agrobacterium. (B) GUS activity of cotton fibers infected with Agrobacterium containing 35S::GUS construct under different infection times. (C) GUS staining of fibers under different incubation times after Agrobacterium infection. (D) GUS activity of cotton fibers under different incubation times following Agrobacterium infection. The black line indicates 2 mm in (A,C). Error bars indicate SD. Different letters indicate significant differences (Turkey’s test; P < 0.01). h, hour; d, day. Fibers at 20 DPA were used for transformation.

FIGURE 4

Transient overexpression of four cotton genes in fibers. (A) The expression pattern of four genes used for transient overexpression experiment was investigated in Gossypium hirsutum TM-1, including two genes, GhCFE and GhMYB212, which are preferentially expressed in elongating fibers, and two genes, GhSusC and GhFSN1, which are preferentially expressed in secondary cell wall depositing fibers. (B) The expression levels of GhCFE1, GhMYB212, GhSusC, and GhFSN1 were significantly higher in transiently transformed fibers than that in the control, which was transformed with empty pBI121 binary expression vector, respectively. Data above each column indicate the relative expression of each gene compared with the internal reference gene, GhUBQ7. Fibers at 20 DPA were used for transformation. Error bars indicate SD.

FIGURE 5

Activities of promoters of GhFSN1 and GhMYB212 at different fiber development stages were analyzed via Agrobacterium-mediated transient transformation. (A) GUS staining results showed that the GhFSN1 promoter had high activity at 20 DPA fibers, while the GhMYB212 promoter had high activity at 10 DPA fibers. Black line indicates 2 mm. (B) GUS activity analysis of cotton fibers infected with Agrobacterium containing pGhFSN1::GUS or pGhMYB212::GUS constructs, respectively. Error bars indicate SD. ** indicates a significant difference using the t-test with a P-value of 0.01.

FIGURE 6

Subcellular localization of mCherry-tagged proteins in fiber cells. (A) A strong fluorescence signal was observed in the nucleus of fiber cells transformed with Agrobacterium harboring 35S::AtHTB2-mCherry construct. (B) A strong fluorescence signal was observed in the plasma membrane of fiber cells transformed with Agrobacterium harboring 35S::AtPIP2A-mCherry construct. White lines indicate 50 μm. AtHTB2, Arabidopsis thaliana histone B2; AtPIP2A, an aquaporin, short for Arabidopsis thaliana plasma membrane intrinsic protein. Fibers at 20 DPA were used for transformation.