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

真菌病原体造成农作物产量的重大损失，是对粮食安全的严重生物威胁。由于病原菌群体中杀真菌剂抗性的发展和单基因抗性的缺乏，作物真菌病害的有效管理已成为一个主要挑战。因此，重要的是寻找对策。富含半胱氨酸的小抗真菌肽对经济上重要的真菌病原体表现出有效的抗真菌活性，为农场化学杀真菌剂提供了基于肽的生物杀真菌剂替代品。我们已经从豆科植物中鉴定出两种小的抗真菌肽，它们作为第二代安全和可持续的生物杀真菌剂具有巨大的开发潜力。为了有效地利用这些肽在农业作物保护中，重要的是要了解它们的结构-活性关系和抗真菌作用的机制。初步研究表明，这些肽表现出多方面的作用模式。在这项建议中，这些肽的活性位点，他们的抗真菌活性将被确定和广泛的高分辨率显微镜将被用来调查这些肽的亚细胞目标的真菌病原体，导致水果和蔬菜中的灰霉病。此外，将采用生物化学工具来鉴定真菌中这些肽的分子靶标。该项目为博士后学生和代表性不足的少数民族本科生提供跨学科培训。此外，它还为圣刘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经验。两个阳离子序列趋异的根瘤特异性富含半胱氨酸的肽，NCR 044和NCR 13，对植物真菌病原体表现出有效的抗真菌活性。我们假设这两种NCR肽具有独特的结构特征，并且由于其多方面的作用模式而非常有效。我们进一步提出NCR肽作为安全的喷雾生物杀真菌剂具有未开发的潜力。在本项目中，将确定NCR 13的三维结构。将确定NCR 044和NCR 13控制膜破坏和抗灰葡萄孢的抗真菌活性的序列基序。特别地，将阐明NCR 044的核仁定位所需的序列基序。生化实验将确定NCR 044和NCR 13是否通过结合核糖体RNA和/或核糖体蛋白质来抑制蛋白质翻译。完全完成的B的无间隙和注释的38.8 MB基因组序列。灰葡萄基因组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。将使用共聚焦和超分辨率显微镜以及相关电子显微镜，使用细胞器特异性荧光蛋白和活体染料对每种肽的真菌细胞进行广泛的高分辨率活细胞成像，以确定每种肽的内化、亚细胞动力学和迁移率机制。最后，喷雾施用NCR 044、NCR 13及其变体将确定它们是否赋予番茄植物对灰霉病的抗性。实验计划阐明肽施加的变化“孢子萌发转录组”在planta.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.