Mechanistic roles of DNA polymerase delta subunit 1 in resistance to DNA geminiviruses

木薯是小农、中农、大农的重要作物。它是一种耐寒植物，可以在没有灌溉、化肥或杀虫剂的情况下生长，即使在边际土地上生长也是非常高产的。因此，面对全球气候变化，它很可能成为一种更重要的作物。木薯花叶病(CMD)是横跨非洲大陆的一种毁灭性的木薯病害，最近已蔓延到亚洲。一些木薯品种对CMD具有天然的遗传抗性，最近发现了导致这种抗性的基因。然而，目前还不清楚抵抗是如何起作用的。由于数以亿计的人依赖于这种对毁灭性病原体的抗药性来源，因此需要对抗药性背后的机制有更深入的了解。这项研究将揭示抗性的分子机制，并测试这种类型的抗性是否能够保护各种重要作物免受其他病毒病原体的侵染。如果成功，这项研究将为番茄和棉花等许多重要作物带来可持续和有效的疾病控制策略。在整个研究过程中，将为本科生和研究生提供培训，并将通过各种公开讲座、外联活动和YouTube视频向社会分享这项研究的重要性，解释这一针对非科学家受众的特定项目的重要性。CMD是由DNA双生病毒引起的。CMD抗性与DNA聚合酶增量亚单位1(POLD1)蛋白中特定氨基酸的变化有关。病毒在克服宿主抗性机制方面非常有效，但这种抗性特征几十年来一直是稳定的。此外，几个木薯抗性品种是倾周嵌合体，抗性等位基因只存在于特定的细胞层。为什么这种抗性如此稳定，以及它如何在倾斜嵌合体的背景下发挥作用，目前尚不清楚。虽然一些已发现的POLD1突变是新的，但其他突变已在酵母中观察到，并导致DNA复制保真度降低，这表明可能的作用机制。瞬变测试将被用来检验这一假设和其他候选的机械假说。这些包括表征不同POLD1等位基因的病毒复制率和保真度，与候选病毒和宿主辅助因子的相互作用，以及蛋白质结晶学。除了木薯，这项研究可能会为其他重要作物带来新的抗性策略。一个POLD1抗性等位基因将被转化到模式系统拟南芥中，以直接测试这种抗性机制是否在不同的病理系统中有效。此外，对公开可用的基因组数据的探索表明，在番茄、棉花和其他几种重要作物的种质收集中存在类似的等位基因。相关的种质已经获得，并将与各自的病毒病原体一起挑战。该奖项反映了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。

Cassava is an important crop for small, medium, and large-scale farmers. It is a hardy plant that can be grown without irrigation, fertilizer or pesticides and is highly productive even when grown on marginal land. As such, it is likely to become an even more important crop in the face of global climate change. Cassava Mosaic Disease (CMD) is a devastating disease of cassava across the African continent and has recently spread to Asia. Some varieties of cassava have natural genetic resistance to CMD and the gene responsible for this resistance was recently identified. However, it is not yet understood how the resistance works. With hundreds of millions of people depending on this one source of resistance to a devastating pathogen, a deeper understanding of the mechanism behind the resistance is desired. This research will reveal the molecular mechanisms of resistance and test whether this type of resistance can protect diverse and important crops from other virus pathogens. If successful, this research will lead to sustainable and effective disease control strategies for many important crops such as tomato and cotton. Throughout the research, training will be provided for undergraduate and graduate students and the importance of this research will be shared with society through a variety of public lectures, outreach events and YouTube videos explaining the importance of this specific project targeted at non-scientist audiences.CMD is caused by species of DNA geminiviruses. The CMD resistance was tracked to specific amino acid changes within the DNA polymerase delta subunit 1 (POLD1) protein. Viruses are fantastically effective at overcoming host resistance mechanisms and yet this resistance trait has been stable for decades. Further, several resistant cultivars of cassava are periclinal chimeras with the resistance allele present in only specific cell layers. Why this resistance is so stable and how it can function in the context of a periclinal chimera, is not yet clear. While some of the identified POLD1 mutations are novel, others have been observed in yeast and result in decreased DNA replication fidelity, pointing towards a possible functional mechanism. Transient assays will be used to test this and other candidate mechanistic hypotheses. These include characterizing viral replication rates and fidelity of the different POLD1 alleles, interaction with candidate viral and host co-factors and protein crystallography. Beyond cassava, this research may yield new resistance strategies for other important crops. A resistant POLD1 allele will be transformed into the model system Arabidopsis, to directly test if this resistance mechanism is effective in a distinct pathosystem. In addition, exploration of publicly available genomic data suggests that similar alleles exist in germplasm collections from tomato, cotton, and several other important crops. The relevant germplasm has been obtained and will be challenged with the respective viral pathogensThis 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.