Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins

Abstract

We developed a method for expression in Arabidopsis of a transgene encoding a cleavable chimeric polyprotein. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and RsAFP2 originating from Raphanus sativus seeds, which are linked by an intervening sequence ("linker peptide") originating from a natural polyprotein occurring in seed of Impatiens balsamina. The chimeric polyprotein was found to be cleaved in transgenic Arabidopsis plants and the individual AMPs were secreted into the extracellular space. Both AMPs were found to exert antifungal activity in vitro. It is surprising that the amount of AMPs produced in plants transformed with some of the polyprotein transgene constructs was significantly higher compared with the amount in plants transformed with a transgene encoding a single AMP, indicating that the polyprotein expression strategy may be a way to boost expression levels of small proteins.

Figure 1

A, Diagram of the expression cassettes in plant transformation vectors pFAJ3105, pFAJ3109, pFAJ3339, pFAJ3340, pFAJ3433, and pFAJ3375. p35S, Enhanced CaMV35S promoter (Kay et al., 1987); TMV, 5′ leader sequence of tobacco mosaic virus; SP, coding region of the leader peptide derived from the DmAMP1 precursor; DmAMP1, coding region of the mature DmAMP1; LP, coding region of the linker peptide; RsAFP2, coding region of the mature RsAFP2; tNos, 3′-untranslated terminator region of the Agrobacterium tumefaciens nopaline synthase gene (Bevan et al., 1983); [uOcs]pMas, chimeric promoter consisting of the enhancer of the A. tumefaciens octopine synthase gene and the promoter of the A. tumefaciens mannopine synthase gene (M.F.C. De Bolle, unpublished data); tMas, 3′-untranslated terminator region of the A. tumefaciens mannopine synthase gene (Barker et al., 1983). B, Amino acid sequence of the (poly)proteins encoded by constructs pFAJ3105, pFAJ3109, pFAJ3339, pFAJ3340, pFAJ3433, and pFAJ3375. The leader peptide of DmAMP1 is in normal font. The mature DmAMP1 domain is underlined. The linker peptide domain is in bold. The mature RsAFP2 domain is italicized.

Figure 2

Northern-blot analysis of DmAMP1 expression in leaves of transgenic T₁ generation lines transformed with constructs pFAJ3109 and pFAJ3105. All analyzed samples represent 15 μg of total RNA. NT, Non-transformed Arabidopsis plants; 3109, Arabidopsis plants transformed with the single-protein construct pFAJ3109 (transgenic line 1 and 2); 3105, Arabidopsis plants transformed with the double-protein construct pFAJ3105 (transgenic line 1 and 2). Similar northern-blot analyses were obtained for another eight lines of Arabidopsis plants transformed with construct pFAJ3105 and six lines of Arabidopsis plants transformed with construct pFAJ3109.

Figure 3

Box plots of the DmAMP1 expression levels of T₁ generation transgenic Arabidopsis plants transformed with constructs pFAJ3105 and pFAJ3109, respectively. DmAMP1 expression level is expressed as the ratio of the amount of DmAMP1 to the amount of total soluble protein in crude extracts from leaves of transgenic T₁ Arabidopsis plants. Gridlines depicted as vertical dotted lines represent the DmAMP1 expression level scale. Vertical lines in the box plots correspond to the 0th, 25th, 50th (or median), 75th, and 100th percentile, respectively.

Figure 4

A, Box plots of the DmAMP1 expression levels of T₁ generation Arabidopsis plants transformed with the polyprotein constructs pFAJ3339 and pFAJ3340 and the single-protein construct pFAJ3433, respectively. B, Box plots of the RsAFP2 expression levels of T₁ generation Arabidopsis plants transformed with the polyprotein constructs pFAJ3339 and pFAJ3340 and the single-protein construct pFAJ33775, respectively. Legend for both box plots as in Figure 3.

Figure 5

Chromatographic purification of DmAMP1-CRPs and RsAFP2-CRPs from extracellular fluid fraction of Arabidopsis plants transformed with the double-protein construct pFAJ3105. The extracellular fluid fraction was loaded on a C8 RP-HPLC column equilibrated in 15% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA). The column was eluted at a flow rate of 1 mL min⁻¹ for 15 min with 15% (v/v) acetonitrile in 0.1% (v/v) TFA followed by a linear gradient of acetonitrile in 0.1% (v/v) TFA from 15% (v/v) to 50% (v/v) acetonitrile in 35 min. The eluate was monitored by on-line measurement of the A₂₈₀ (A₂₈₀;—) and the acetonitrile gradient (---) was monitored with an on-line conductivity sensor. Fractions were collected and assessed for the presence of DmAMP1-CRPs and RsAFP2-CRPs by the respective ELISA assays. Dotted bars represent the presence of DmAMP1-CRPs (p3105EF1 and p3105EF2), whereas the black bar represents the presence of RsAFP2-CRPs (p3105EF3). Triangles indicate the elution position of native DmAMP1 (white triangle) and of native RsAFP2 (black triangle).

Figure 6

Schematic representation of the polyprotein precursor encoded by construct pFAJ3105 and pFAJ3340. The protein domain of the leader peptide of DmAMP1, DmAMP1, and RsAFP2 is represented by boxes containing the amino- and carboxy-terminal amino acids of the different subdomains. The protein domain of the linker peptide is represented by a box containing the complete amino acid sequence. Cleavage of the pFAJ3105 polyprotein precursor results in the release of a DmAMP1 derivative with an additional Ser at its carboxy terminus (DmAMP1+S) and an RsAFP2 derivative with an additional pentapeptide at its amino-terminus (RsAFP2+DVEPG). Cleavage of the pFAJ3340 polyprotein precursor results in the release of RsAFP2, an RsAFP2 derivative with an additional Ser at its carboxy terminus (DmAMP1+S) and a DmAMP1 derivative with an additional pentapeptide at its amino terminus (DVEPG+DmAMP1).