New Recombinant Antimicrobial Peptides Confer Resistance to Fungal Pathogens in Tobacco Plants

Abstract

Antimicrobial peptides have been long known to confer resistance to plant pathogens. In this study, new recombinant peptides constructed from a dermaseptin B1 (DrsB1) peptide fused to a chitin-binding domain (CBD) from Avr4 protein, were used for Agrobacterium tumefaciens-mediated transformation of tobacco plants. Polymerase chain reaction (PCR), semi-quantitative RT-PCR, and western blotting analysis demonstrated the incorporation and expression of transgenes in tobacco genome and transgenic plants, respectively. In vitro experiments with recombinant peptides extracted from transgenic plants demonstrated a significant (P<0.01) inhibitory effect on the growth and development of plant pathogens. The DrsB1-CBD recombinant peptide had the highest antifungal activity against fungal pathogens. The expression of the recombinant peptides greatly protected transgenic plants from Alternaria alternata, Alternaria solani, Fusarium oxysporum, and Fusarium solani fungi, in comparison to Pythium sp. and Pythium aphanidermatum. Expression of new recombinant peptides resulted in a delay in the colonization of fungi and appearance of fungal disease symptoms from 6 days to more than 7 weeks. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, cling together, and crushed following the antimicrobial activity of the recombinant peptides. Greenhouse bioassay analysis showed that transgenic plants were more resistant to Fusarium and Pythium infections as compared with the control plants. Due to the high antimicrobial activity of the recombinant peptides against plant pathogens and novelty of recombinant peptides, this report shows the feasibility of this approach to generate disease resistance transgenic plants.

Keywords: antifungal; chitin-binding domain; effector protein; expression; genetic engineering; transgenic plant.

Figure 1

Schematic representation of the three genetic constructs used for Agrobacterium-mediated transformation. MAS, mannopine synthase; npt II: neomycin phosphotransferase II; CaMV35S(3×) cauliflower mosaic virus 35S promoter; OSC, octopine synthase; SP, Avr4 signal peptide; HIS, RGS histidine tag; DrsB1, dermaseptin B1; CBD, carbohydrate binding domain from Avr4 effector protein; L1, helix-forming linker (EAAAK)4; L2, linker sequence from Caenorhabditis elegans chitinase (CCD73759) gene; RB and LB, right and left borders of Agrobacterium T-DNA region. Two CBDs are fused together by a linker sequence from Caenorhabditis elegans chitinase (CCD73759) gene.

Figure 2

Semi-quantitative RT-PCR (A) and western blotting (B) analysis of transgenic and control plants. The upper panel shows the PCR products of the CBD-DrsB1, (CBD)2-DrsB1, and DrsB1-CBD recombinant genes in the selected transgenic line and the lower panel shows the PCR products elf housekeeping gene. Mouse monoclonal anti-His antibody (Sigma-Aldrich, product No. A7058) was used for recombinant peptides detection.

Figure 3

The diagram showing the effect of recombinant peptides on fungal growth. P value of 0.01 was considered as significant; 50 µg/ml of transgenic and non-transgenic total protein was used. Percentage of inhibition was calculated relative to the phosphate buffer treatment. The antifungal activity data were statistically analyzed and the mean comparison was done by least significant differences (LSD) test (P ≤ 0.01) test in three replicates using SAS 9.1 (SAS, Inc., North Carolina, USA) software.

Figure 4

Hypha extension inhibition assay by mixing recombinant peptides with the culture medium. Fungi were cultured on potato dextrose agar (PDA); 50 μg/ml of total protein from the non-transgenic line (Ut) and sterilize phosphate buffer (Pb) were used as the negative controls.

Figure 5

Scanning electron microscopy images of fungal hyphae treated with the total protein (50 μg/ml). Scale bars are indicated in micrometer for each image. Ut, non-transgenic control line; Pb, treated with phosphate buffer as control.

Figure 6

Detached leaves assay for the fungal disease incidence in different transgenic lines and control tobacco plants challenge with different fungi. Dotted circles indicate the inoculation points with a 1-cm² agar block of the fungal culture, control and transgenic lines, 21 days post-incoulation with Alternaria, control and transgenic lines, control and transgenic lines, 11 days post-inoculation with Fusarium. 7 days post-inoculation with Pythium sp.

Figure 7

The diagram showing the disease severity of the detached leaves. The disease severity is calculated based on the ratio of the damaged area to the total leaf area. The antifungal activity data were statistically analyzed as a completely randomized design in a 6×7 factorial arrangement and the mean comparison was done by least significant differences (LSD) (p ≤ 0.01) test in three replicates using SAS 9.1 (SAS, Inc., North Carolina, USA) software. The p value of ≤0.01 was considered as significant.

Figure 8

Resistance of transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants expressing the recombinant peptides constructs in jars inoculated with different fungi.

Figure 9

Greenhouse evaluation of transgenic tobacco plants expressing recombinant peptides for resistance against different fungi. Symptoms of fungal disease on transgenic and non-transgenic plants 2 months after inoculation with three fungal species under greenhouse conditions are seen.