smAMPsTK: a toolkit to unravel the smORFome encoding AMPs of plant species

The pervasive repertoire of plant molecules with the potential to serve as a substitute for conventional antibiotics has led to obtaining better insights into plant-derived antimicrobial peptides (AMPs). The massive distribution of Small Open Reading Frames (smORFs) throughout eukaryotic genomes with proven extensive biological functions reflects their practicality as antimicrobials. Here, we have developed a pipeline named smAMPsTK to unveil the underlying hidden smORFs encoding AMPs for plant species. By applying this pipeline, we have elicited AMPs of various functional activity of lengths ranging from 5 to 100 aa by employing publicly available transcriptome data of five different angiosperms. Later, we studied the coding potential of AMPs-smORFs, the inclusion of diverse translation initiation start codons, and amino acid frequency. Codon usage study signifies no such codon usage biases for smORFs encoding AMPs. Majorly three start codons are prominent in generating AMPs. The evolutionary and conservational study proclaimed the widespread distribution of AMPs encoding genes throughout the plant kingdom. Domain analysis revealed that nearly all AMPs have chitin-binding ability, establishing their role as antifungal agents. The current study includes a developed methodology to characterize smORFs encoding AMPs, and their implications as antimicrobial, antibacterial, antifungal, or antiviral provided by SVM score and prediction status calculated by machine learning-based prediction models. The pipeline, complete package, and the results derived for five angiosperms are freely available at https://github.com/skbinfo/smAMPsTK. Communicated by Ramaswamy H. Sarma广泛存在的植物分子具备替代传统抗生素的潜力，这推动学界对植物源抗菌肽（antimicrobial peptides, AMPs）的研究认知进一步深化。小型开放阅读框（small open reading frames, smORFs）广泛分布于真核生物基因组中，且已被证实具备丰富的生物学功能，体现了其作为抗菌剂的应用价值。本研究开发了一款名为smAMPsTK的分析流程，用于挖掘植物物种中编码抗菌肽的潜在隐蔽型smORFs。借助该分析流程，我们利用5种不同被子植物的公开转录组数据，筛选获得了长度介于5至100个氨基酸、具备多样功能活性的抗菌肽。随后，我们针对编码抗菌肽的smORFs的编码潜力、所包含的多样翻译起始密码子类型以及氨基酸频率展开了系统分析。密码子使用偏好性分析结果显示，编码抗菌肽的smORFs不存在显著的密码子使用偏好。在抗菌肽的编码过程中，主要有三类起始密码子占据主导地位。进化与保守性分析表明，编码抗菌肽的基因广泛分布于整个植物界。结构域分析结果显示，几乎所有抗菌肽均具备几丁质结合能力，证实了其作为抗真菌剂的功能属性。本研究建立了一套用于鉴定编码抗菌肽的smORFs的方法体系，并结合基于机器学习预测模型计算得到的支持向量机（Support Vector Machine, SVM）评分与预测状态，对这些smORFs的抗菌、抗细菌、抗真菌及抗病毒功能潜力进行了评估。本分析流程、完整工具包以及针对5种被子植物的分析结果，均可通过https://github.com/skbinfo/smAMPsTK免费获取。本文由Ramaswamy H. Sarma转交刊发。