Expression of a novel antimicrobial peptide Penaeidin4-1 in creeping bentgrass (Agrostis stolonifera L.) enhances plant fungal disease resistance

Abstract

Background: Turfgrass species are agriculturally and economically important perennial crops. Turfgrass species are highly susceptible to a wide range of fungal pathogens. Dollar spot and brown patch, two important diseases caused by fungal pathogens Sclerotinia homoecarpa and Rhizoctonia solani, respectively, are among the most severe turfgrass diseases. Currently, turf fungal disease control mainly relies on fungicide treatments, which raises many concerns for human health and the environment. Antimicrobial peptides found in various organisms play an important role in innate immune response.

Methodology/principal findings: The antimicrobial peptide - Penaeidin4-1 (Pen4-1) from the shrimp, Litopenaeus setiferus has been reported to possess in vitro antifungal and antibacterial activities against various economically important fungal and bacterial pathogens. In this study, we have studied the feasibility of using this novel peptide for engineering enhanced disease resistance into creeping bentgrass plants (Agrostis stolonifera L., cv. Penn A-4). Two DNA constructs were prepared containing either the coding sequence of a single peptide, Pen4-1 or the DNA sequence coding for the transit signal peptide of the secreted tobacco AP24 protein translationally fused to the Pen4-1 coding sequence. A maize ubiquitin promoter was used in both constructs to drive gene expression. Transgenic turfgrass plants containing different DNA constructs were generated by Agrobacterium-mediated transformation and analyzed for transgene insertion and expression. In replicated in vitro and in vivo experiments under controlled environments, transgenic plants exhibited significantly enhanced resistance to dollar spot and brown patch, the two major fungal diseases in turfgrass. The targeting of Pen4-1 to endoplasmic reticulum by the transit peptide of AP24 protein did not significantly impact disease resistance in transgenic plants.

Conclusion/significance: Our results demonstrate the effectiveness of Pen4-1 in a perennial species against fungal pathogens and suggest a potential strategy for engineering broad-spectrum fungal disease resistance in crop species.

Figure 1. Generation and molecular analysis of the transgenic lines expressing Pen4-1.

(a) Schematic diagram of the Pen4-1 expression chimeric gene construct, p35S-bar/Ubi-Pen4-1. Pen4-1 gene is under the control of the maize ubiquitin promoter (Ubi) and linked to the herbicide resistance gene, bar, driven by the CaMV 35S promoter. (b) Schematic diagram of the AP24::Pen4-1 expression chimeric gene construct, p35S-bar/Ubi-AP24::Pen4-1, in which the AP24::Pen4-1 fusion gene is under the control of the maize Ubi promoter. The CaMV35S promoter-driven bar gene is included for herbicide resistance. (c) Example of Southern blot analysis of Pen4-1 expression transgenics. Twenty micrograms of the genomic DNA extracted from young leaves and digested with BamHI that cuts once within the T-DNA region was probed by a 440 bp ³²P-labelled bar gene fragment. Hybridization signals revealed were indication of copy numbers of transgene insertion. Lanes 1–6 were DNAs from representative transgenic creeping bentgrass plants. The negative control (WT) was BamHI-digested genomic DNA from a non-transformed wild-type plant. (d) Example of Northern blot analysis of Pen4-1 expression transgenics. Lanes 1-6 were total RNA from the same representative transgenic creeping bentgrass plants used for Southern analysis in (c). Twenty micrograms of the total RNA extracted from young leaves and probed with a ³²P-labelled Pen4-1 gene fragment. The negative control (WT) was total RNA from a non-transformed wild-type plant.

Figure 2. Nucleotide and deduced amino acid sequences of the Pen4-1 gene.

The original nucleotide sequences of Pen4-1 were modified for plant-optimized codon usage. The predicted single-letter amino acids are shown above the coding sequence. The added translation stop codon is also indicated.

Figure 3. Response of transgenic creeping bentgrass plants expressing Pen4-1 to R. solani infection - in vitro plant leaf inoculation assay.

(a) The detached second expanded leaves from the top of plant stolons were used for pathogen inoculation test. The image shows example of representative leaves from all tested Pen4-1-expressing transgenic plants with a single transgene insertion (TG, on the right) and wild-type control plants (WT, on the left) 14 days post-inoculation (DPI). Transgenic plants exhibited significant resistance to R. solani in comparison to wild-type controls. (b) The development of brown patch disease was rated by measuring the lesion length of the infected leaves 2, 8 and 14 DPI. Statistical analysis of R. solani inoculation test was conducted on wild-type control plants (WT) and various transgenic lines harboring either p35S-bar/Ubi-Pen4-1 (TG1 and TG2) or p35S-bar/Ubi-AP24::Pen4-1 (TG3 and TG4). Data are presented as means ± SE (n = 10), and error bars represent standard error. Asterisks (** or *) indicate a significant difference between Pen4-1-expressing transgenic and control plants at P<0.01 or P<0.05 by Tukey's HSD test using JMP 9.0.0. The P values are listed in Table S1.

Figure 4. Response of transgenic creeping bentgrass plants expressing Pen 4-1 to R. solani infection - in vivo direct plant inoculation bioassays with lower dose of R. solani.

(a) The fully developed transgenic (independent events TG1 to TG4) and wild-type (WT) plants clonally propagated from individual stolons were grown and maintained in pots (15 cm×10.5 cm) and inoculated with 3 g of rye seeds colonized by R. solani. The image on the upper panel shows plants before pathogen infection. Example of plants from wild-type (WT) and representative transgenic lines harboring either p35S-bar/Ubi-Pen4-1 (TG1, TG2) or p35S-bar/Ubi-AP24::Pen4-1 (TG3 and TG4) two weeks after pathogen inoculation (14 DPI) are shown on the bottom panel. Transgenic plants exhibited less sever disease symptom than wild-type controls. (b) A closer look of infected plants showing the different lesion size of WT and TG. (c) The development of brown patch disease was rated by measuring the lesion diameters of the infected leaves 14 DPI. Statistical analysis of R. solani inoculation test was conducted on WT and various TG lines. Data are presented as means ± SE (n = 6), and error bars represent standard error. Asterisks (** or *) indicate a significant difference between transgenic plants and wild-type controls at P<0.01 or P<0.05 by Wilcoxon test using JMP 9.0.0. The P values are listed in Table S2.

Figure 5. Response of transgenic creeping bentgrass plants expressing Pen 4-1 to R. solani infection - in vivo direct plant inoculation bioassays with higher dose of R. solani.

(a) Transgenic (TG) and wild-type (WT) plants were inoculated with a second dose of R. solani (3g of rye seeds colonized by the pathogen) 14 days after the first inoculation with 3 g of rye seeds colonized by R. solani. The image shows example of plants from wild-type (WT) and representative transgenic lines harboring either p35S-bar/Ubi-Pen4-1 (TG1) or p35S-bar/Ubi-AP24::Pen4-1 (TG3 and TG4) two weeks after the second pathogen inoculation. Transgenic plants exhibited much less sever disease symptom than wild-type controls. (b) A closer look of infected plants showing the different lesion size of WT and TG. (c) The development of brown patch disease was rated by visual estimation of the lesion percentage of the infected leaves 14 DPI using the Horsfall/Barrett scale. Statistical analysis of R. solani inoculation test was conducted on WT and various TG lines. Data are presented as means ± SE (n = 6), and error bars represent standard error. Asterisks (** or *) indicate a significant difference between transgenic plants and wild-type controls at P<0.01 or P<0.05 by Wilcoxon test JMP 9.0.0. The P values are listed in Table S3.

Figure 6. Response of transgenic creeping bentgrass plants expressing Pen 4-1 to S. homoeocarpa infection - in vitro plant leaf inoculation assay.

(a) The detached second expanded leaves from the top of plant stolons were used for pathogen inoculation test. The image shows example of representative leaves from all tested Pen 4-1-expressing transgenic plants with a single transgene insertion (TG, on the right) and wild-type control plants (WT, on the left) 7 days post-inoculation (DPI). Transgenic plants exhibited significant resistance to S. homoeocarpa in comparison to wild-type controls. (b) The development of dollar spot disease was rated by measuring the lesion length of the infected leaves 2, 4 and 7 DPI. Significant resistance to S. homoeocarpa by transgenic plants was observed 7 DPI when compared to wild-type controls. Statistical analysis of S. homoeocarpa inoculation test was conducted on wild-type control plants (WT) and various transgenic lines harboring either p35S-bar/Ubi-Pen4-1 (TG1 and TG2) or p35S-bar/Ubi-AP24::Pen4-1 (TG3 and TG4). Data are presented as means ± SE (n = 10), and error bars represent standard error. Asterisks (** or *) indicate a significant difference between transgenic plants and wild-type controls at P<0.01 or P<0.05 by Tukey's HSD test using JMP 9.0.0. The P values are listed in Table S4.

Figure 7. Response of transgenic creeping bentgrass plants expressing Pen 4-1 to S. homoeocarpa infection - in vivo direct plant inoculation bioassays with higher dose of S. homoeocarpa.

(a) The fully developed transgenic (TG) and wild-type (WT) plants clonally propagated from individual stolons were grown and maintained in pots (15 cm×10.5 cm) and inoculated with 0.5 g of rye seeds colonized by S. homoeocarpa. The image shows example of plants from wild-type (WT) and representative transgenic lines harboring either p35S-bar/Ubi-Pen4-1 (TG1) or p35S-bar/Ubi-AP24::Pen4-1 (TG3) 9 days after pathogen inoculation (9 DPI). The plants in the front row are uninfected controls. Transgenic plants exhibited significant disease resistance compared to wild-type controls. (b) The development of dollar spot disease was rated by visual estimation of the lesion percentage of the infected leaves 3, 5, 7, 9 DPI, and 21 days post-recovery (DPR) using the Horsfall/Barrett scale. Statistical analysis of S. homoeocarpa inoculation test was conducted on WT and various TG lines. Data are presented as means ± SE (n = 6), and error bars represent standard error. Asterisks (** or *) indicate a significant difference between transgenic plants and wild-type controls at P<0.01 or P<0.05 by Wilcoxon test using JMP 9.0.0. The P values are listed in Table S5.