EAGER: Enhancing plant immunity and growth with cell-penetrating peptides for organic agriculture

为了养活不断增长的世界人口，农业部门需要增加粮食生产，同时注意新战略对生态系统和人类健康的影响。虽然化肥和农药在现代农业中发挥着至关重要的作用，但它们对环境和动物/人类健康的不利影响最近促使可持续作物生产迅速增加。一个有希望的环境友好的替代方法是使用生物制剂。然而，目前在可持续农业中使用的许多植物防御激活剂、生物刺激剂和生物农药是进入植物细胞的效率低的蛋白质和肽，因此需要使用高浓度并因此需要高成本。为了解决这一限制，该项目将利用强大的膜易位结构域（MTD）技术来改善生物制剂的递送。HrpZ是一种经过验证的防御激活剂，将与MTD 4融合，MTD 4是目前最有效的MTD之一，并在番茄中测试其在引发对细菌和真菌病原体的免疫反应中的功效。这种方法应该降低防御激活剂的有效浓度，从而提高植物生物制剂的经济可行性，以促进可持续农业和农民收入。此外，该团队还将探索推广活动，以展示生物防御分子如何帮助植物抵御病原体。尽管细胞穿透肽（CPP）已被广泛探索用于提供治疗剂来治疗人类疾病/病症，但它们在植物中的应用较为有限。该项目的目标是研究新发现的一类CPP，MTD的效用，以有效地将已知的生物防御激活剂/生物刺激剂HrpZ递送到作物植物中，以保护它们免受病原体感染和昆虫侵染。该项目的三个具体目标是：[1]提高MTD 4的细胞进入效率和代谢稳定性; [2]确定MTD 4-HrpZ和MTD 4-N21对番茄和水稻防御，生长，产量和耐旱性的影响;[3]优化和开发低成本的重组蛋白表达和无色谱纯化策略，用于大规模田间应用。在完成这三个目标后，该项目将深入了解CPP如何进入植物细胞以及它们的不同细胞结构（例如，膜中的脂质组成）影响CPP的细胞进入机制和效率。重要的是，使用CPP促进植物免疫可能会对可持续农业中的植物病害控制产生变革性影响。我们的重组蛋白的过表达和无色谱纯化的新策略也可能引起广泛的兴趣。此外，鉴定出能够有效地将肽/蛋白质递送到植物细胞中的强大CPP将为生物学研究提供新的工具，这些研究旨在揭示植物中不同蛋白质（包括病原体效应物）之间的相互作用。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

To feed the growing world population, the agricultural sector needs to increase food production even while being attentive to the impact of new strategies on ecosystems and human health. Although fertilizers and pesticides play a crucial role in modern agriculture, their adverse impact on the environment and animal/human health have recently inspired a rapid rise in sustainable crop production. A promising environment-friendly alternative is the use of biologics. However, many of the plant defense activators, biostimulants, and biopesticides currently used in sustainable agriculture are proteins and peptides that have low entry efficiency into plant cells, thus necessitating the use of high concentrations and consequently high costs. To address this limitation, this project will leverage a powerful membrane translocation domain (MTD) technology to improve the delivery of biological agents. HrpZ, a proven defense activator, will be fused to MTD4, one of the most potent MTDs in hand, and tested in tomato for its efficacy in eliciting immune responses to bacterial and fungal pathogens. This approach should decrease the effective concentration of defense activators and thereby increase the economic viability of plant biologics for sustainable agriculture and farmers’ income. In addition, the team will explore outreach activities to demonstrate how biodefense molecules can help plants ward off pathogens.Although cell-penetrating peptides (CPPs) have been widely explored for delivering therapeutic agents to treat human diseases/conditions, their applications in plants have been more limited. The goal of this project is to examine the utility of a newly discovered class of CPPs, MTDs, to efficiently deliver HrpZ, a known biodefense activator/biostimulant, into crop plants to protect them from pathogen infection and insect infestation. The three specific aims of this project are: [1] improve the efficiency of cellular entry and metabolic stability of MTD4; [2] determine the effects of MTD4-HrpZ and MTD4-N21 on tomato and rice defense, growth, yield, and drought tolerance; and [3] optimize and develop low-cost recombinant protein expression and chromatography-free purification strategies for large-scale field applications. After completion of the three objectives, this project will provide insights into how CPPs enter plant cells and how their different cellular structures (e.g., lipid composition in membranes) affect the cellular entry mechanism and efficiency of CPPs. Importantly, the use of CPPs to promote plant immunity is likely to be transformative for plant disease control in sustainable agriculture. Our new strategies for overexpression and chromatography-free purification of recombinant proteins are also likely to be of broad interest. In addition, identification of powerful CPPs that can efficiently deliver peptides/proteins into plant cells will provide new tools for biological studies that seek to uncover in planta interactions between different proteins, including pathogen effectors.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.