Novel antifungal symbiotic peptides: Modes of action and control of fungal pathogens

真菌病原菌对农作物产量造成重大损失，对粮食安全构成严重的生物威胁。由于病原菌种群对杀菌剂的抗性发展以及缺乏单基因抗性，有效地管理作物中的真菌病害已成为一项重大挑战。因此，重要的是寻找对策。富含半胱氨酸的小分子抗真菌多肽对经济上重要的真菌病原体表现出强大的抗真菌活性，为农场化学杀菌剂提供了多肽类生物杀菌剂的替代品。我们已经从豆科植物中鉴定出两个小的抗真菌多肽，它们具有作为第二代安全和可持续的生物杀菌剂的巨大潜力。为了有效地利用这些多肽在农业上保护作物，了解它们的构效关系和抗真菌作用机制是很重要的。初步研究表明，这些多肽具有多方面的作用方式。在这项提案中，将确定这些多肽控制其抗真菌活性的活性部位，并将使用广泛的高分辨率显微镜来研究这些多肽在一种引起水果和蔬菜灰霉病的真菌病原体中的亚细胞靶标。此外，将使用生化工具来确定真菌中这些多肽的分子靶标。该项目为博士后和代表不足的少数民族本科生提供跨学科培训。此外，它还为圣路易斯地区表现不佳的公立学校的教师和学生提供实践研究体验。两个阳离子序列发散的富含半胱氨酸的多肽NCR044和NCR13对植物病原菌具有很强的抗真菌活性。我们推测这两个NCR多肽因其多方面的作用方式而表现出独特的结构特征和高效的作用。我们进一步认为，NCR多肽作为安全的喷雾生物杀菌剂具有尚未开发的潜力。在本项目中，将确定NCR13的三维结构。将确定NCR044和NCR13控制膜破裂和对灰霉病菌的抗真菌活性的序列基序。特别是，核仁定位所需的序列基序(S)将被阐明。生化实验将确定NCR044和NCR13是否通过与核糖体RNA和/或核糖体蛋白结合来抑制蛋白质翻译。完整的无间隙和注释的38.8 Mb的灰霉菌基因组序列及其功能基因组学工具将为蛋白质-蛋白质相互作用的研究提供必要的资源。利用共聚焦和超分辨率显微镜和相关的电子显微镜，将对真菌细胞进行广泛的高分辨率活细胞成像，以确定每个肽的内化、亚细胞动力学和移动性的机制，使用细胞器特定的荧光蛋白和活性染料。最后，喷施NCR044、NCR13及其变异体将确定它们是否对番茄植株的灰霉病具有抗性。计划进行实验，以阐明在Plans中由多肽引起的“孢子萌发转录组”的变化。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Fungal pathogens cause significant losses of crop yield and are a serious biological threat to food security. Effective management of fungal diseases in crops has become a major challenge due to development of fungicide resistance in pathogen populations and lack of single gene resistance. Thus, it is important to look for countermeasures. Small cysteine-rich antifungal peptides that exhibit potent antifungal activity against economically important fungal pathogens offer peptide-based biofungicide alternatives to on-farm chemical fungicides. We have identified two small antifungal peptides from leguminous plants that offer significant potential for development as second-generation safe and sustainable biofungicides. For effective use of these peptides for crop protection in agriculture, it is important to understand their structure-activity relationships and mechanisms of antifungal action. Preliminary studies have revealed that these peptides exhibit multi-faceted modes of action. In this proposal, active sites of these peptides governing their antifungal activity will be determined and extensive high-resolution microscopy will be used to investigate subcellular targets of these peptides in a fungal pathogen that causes gray mold disease in fruits and vegetables. Further, biochemical tools will be employed to identify molecular targets of these peptides in the fungus. This project provides interdisciplinary training to a postdoctoral student and under-represented minority undergraduate students. In addition, it provides hands-on research experiences to teachers and students from low performing public schools in the St Louis area. Two cationic sequence-divergent nodule-specific cysteine-rich peptides, NCR044 and NCR13, exhibit potent antifungal activity against plant fungal pathogens. We hypothesize that these two NCR peptides exhibit unique structural characteristics and are highly effective due to their multi-faceted modes of action. We further propose that NCR peptides have untapped potential for development as safe spray-on biofungicides. In this project, 3D structure of NCR13 will be determined. Sequence motifs of NCR044 and NCR13 governing membrane disruption and antifungal activity against Botrytis cinerea will be determined. In particular, sequence motif(s) required for nucleolus-localization of NCR044 will be elucidated. Biochemical experiments will establish if NCR044 and NCR13 inhibit protein translation by binding to ribosomal RNA and/or ribosomal proteins. The fully completed gapless and annotated 38.8 Mb genome sequence of the B. cinerea genome and its functional genomics tools will provide essential resources for the protein-protein interaction studies. Extensive high-resolution live cell imaging of fungal cells with each peptide will be performed to identify mechanisms of internalization, subcellular dynamics and mobility of each peptide using organelle-specific fluorescent proteins and vital dyes with confocal and super-resolution microscopy and correlative electron microscopy. Finally, spray-application of NCR044, NCR13 and their variants will determine if they confer resistance to gray mold disease in tomato plants. Experiments are planned to elucidate peptide-imposed changes in “spore germination transcriptome” in planta.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.