Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, United States of America.
PLoS One. 2011;6(9):e24677. doi: 10.1371/journal.pone.0024677. Epub 2011 Sep 12.
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.
草坪草种是具有重要经济价值的农业多年生作物。草坪草种极易受到多种真菌病原体的侵害。由真菌病原体核盘菌和立枯丝核菌分别引起的褐斑病和币斑病是最严重的草坪病害之一。目前，草坪真菌病害的防治主要依赖于杀菌剂处理，这引起了人们对人类健康和环境的诸多关注。各种生物体中发现的抗菌肽在先天免疫反应中起着重要作用。
方法/主要发现：来自虾类凡纳滨对虾的抗菌肽-Penaeidin4-1（Pen4-1）已被报道具有体外抗真菌和抗细菌活性，可对抗各种具有经济重要性的真菌和细菌病原体。在这项研究中，我们研究了使用这种新型肽将增强的抗病性工程导入匍匐翦股颖植物（Agrostis stolonifera L.，cv.Penn A-4）的可行性。制备了两个 DNA 构建体，一个含有单个肽 Pen4-1 的编码序列，另一个含有翻译融合到 Pen4-1 编码序列的分泌烟草 AP24 蛋白的转导信号肽的编码序列。两个构建体都使用玉米泛素启动子来驱动基因表达。通过农杆菌介导的转化生成含有不同 DNA 构建体的转基因草坪植物，并分析其转基因插入和表达。在受控环境下进行的重复体外和体内实验中，转基因植物表现出对褐斑病和币斑病这两种草坪主要真菌病害的显著增强的抗性。AP24 蛋白的转导肽将 Pen4-1 靶向内质网并没有显著影响转基因植物的抗病性。
结论/意义：我们的结果证明了 Pen4-1 在多年生植物中对真菌病原体的有效性，并提出了一种在作物物种中工程广谱真菌病抗性的潜在策略。
