Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, Bundoora, Victoria, Australia.
Wellcome Sanger Institute, Hinxton, Cambridgeshire, United Kingdom.
Antimicrob Agents Chemother. 2019 Oct 22;63(11). doi: 10.1128/AAC.01097-19. Print 2019 Nov.
Plant defensins are a large family of proteins, most of which have antifungal activity against a broad spectrum of fungi. However, little is known about how they exert their activity. The mechanisms of action of only a few members of the family have been investigated and, in most cases, there are still a number of unknowns. To gain a better understanding of the antifungal mechanisms of a set of four defensins, NaD1, DmAMP1, NbD6, and SBI6, we screened a pooled collection of the nonessential gene deletion set of Strains with increased or decreased ability to survive defensin treatment were identified based on the relative abundance of the strain-specific barcode as determined by MiSeq next-generation sequencing. Analysis of the functions of genes that are deleted in strains with differential growth in the presence of defensin provides insight into the mechanism of action. The screen identified a novel role for the vacuole in the mechanisms of action for defensins NbD6 and SBI6. The effect of these defensins on vacuoles was further confirmed by using confocal microscopy in both and the cereal pathogen These results demonstrate the utility of this screening method to identify novel mechanisms of action for plant defensins.
植物防御素是一大类蛋白质，其中大多数对广谱真菌具有抗真菌活性。然而，关于它们如何发挥作用的知之甚少。该家族中只有少数成员的作用机制得到了研究，在大多数情况下，仍然有许多未知因素。为了更好地了解一组四种防御素（NaD1、DmAMP1、NbD6 和 SBI6）的抗真菌机制，我们筛选了非必需基因缺失集合的汇集池。基于 MiSeq 下一代测序确定的菌株特异性条形码的相对丰度，确定了对防御素处理具有增加或减少生存能力的菌株。对在防御素存在下生长差异的菌株中缺失基因的功能分析提供了对抗菌素作用机制的深入了解。该筛选确定了液泡在防御素 NbD6 和 SBI6 的作用机制中的新作用。通过共聚焦显微镜在 和谷类病原体 中进一步证实了这些防御素对液泡的影响。这些结果证明了这种筛选方法在鉴定植物防御素新的作用机制方面的有效性。
