{"title":"Genetic and physiological characteristics of <i>CsNPR3</i> edited citrus and their impact on HLB tolerance.","authors":"Trishna Tiwari, Cecile Robertson, Choaa El-Mohtar, Jude Grosser, Tripti Vashisth, Zhonglin Mou, Manjul Dutt","doi":"10.3389/fgeed.2024.1485529","DOIUrl":null,"url":null,"abstract":"<p><p>Huanglongbing (HLB) disease, caused by <i>Candidatus</i> Liberibacte<i>r</i> asiaticus (<i>Ca</i>Las), severely impacts citrus production, and currently, there is no cure. Developing HLB-resistant or tolerant cultivars is crucial, with modifying defense-related genes being a promising approach to managing HLB. NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) is a positive regulator of systemic acquired resistance (SAR), which enhances resistance to pathogens, whereas NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 3 (NPR3) is a negative regulator of SAR. To unambiguously address the role of <i>CsNPR3</i> in HLB, we introduced mutations into the <i>CsNPR3</i> gene in sweet orange (<i>Citrus sinensis</i> L. Osbeck) through genome editing and assessed their effects on morphology, physiology, and resistance/tolerance to HLB. Several genome-edited 'Hamlin' sweet orange trees harboring frameshift-inducing insertions or deletions were identified. After confirming the genome editing using Sanger sequencing, selected lines were grafted onto C-146 trifoliate hybrid rootstocks for clonal propagation. The progenies were then infected with <i>Ca</i>Las using a no-choice Asian Citrus Psyllid (ACP) feeding assay. Evaluation of the genetic and physiological characteristics of <i>CsNPR3</i>-edited citrus trees under greenhouse conditions revealed that the edited trees exhibited greater vigor than the wild-type trees, despite the lack of significant differences in <i>Ca</i>Las titers. Although further field evaluation is needed, our findings indicate that <i>CsNPR3</i> contributes to HLB-caused tree deterioration and demonstrate that editing <i>CsNPR3</i> can enhance tolerance to HLB.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"6 ","pages":"1485529"},"PeriodicalIF":4.9000,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11652141/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in genome editing","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3389/fgeed.2024.1485529","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Huanglongbing (HLB) disease, caused by Candidatus Liberibacter asiaticus (CaLas), severely impacts citrus production, and currently, there is no cure. Developing HLB-resistant or tolerant cultivars is crucial, with modifying defense-related genes being a promising approach to managing HLB. NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) is a positive regulator of systemic acquired resistance (SAR), which enhances resistance to pathogens, whereas NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 3 (NPR3) is a negative regulator of SAR. To unambiguously address the role of CsNPR3 in HLB, we introduced mutations into the CsNPR3 gene in sweet orange (Citrus sinensis L. Osbeck) through genome editing and assessed their effects on morphology, physiology, and resistance/tolerance to HLB. Several genome-edited 'Hamlin' sweet orange trees harboring frameshift-inducing insertions or deletions were identified. After confirming the genome editing using Sanger sequencing, selected lines were grafted onto C-146 trifoliate hybrid rootstocks for clonal propagation. The progenies were then infected with CaLas using a no-choice Asian Citrus Psyllid (ACP) feeding assay. Evaluation of the genetic and physiological characteristics of CsNPR3-edited citrus trees under greenhouse conditions revealed that the edited trees exhibited greater vigor than the wild-type trees, despite the lack of significant differences in CaLas titers. Although further field evaluation is needed, our findings indicate that CsNPR3 contributes to HLB-caused tree deterioration and demonstrate that editing CsNPR3 can enhance tolerance to HLB.
黄龙冰(HLB)病是由亚洲游离念珠菌(Candidatus Liberibacter asiaticus, CaLas)引起的一种严重影响柑橘生产的病害,目前尚无根治方法。培育抗HLB或耐HLB的品种至关重要,修改防御相关基因是一种很有前途的管理HLB的方法。NONEXPRESSOR OF pathogenesisrelesgenes 1 (NPR1)是系统性获得性耐药(SAR)的正调控因子,增强了对病原体的抗性,而NONEXPRESSOR OF pathogenesreles3 (NPR3)是系统性获得性耐药的负调控因子。为了明确CsNPR3在HLB中的作用,我们通过基因组编辑将CsNPR3基因引入甜橙(Citrus sinensis L. Osbeck),并评估了它们对HLB形态学、生理学和抗性/耐受性的影响。发现了几种基因组编辑的“哈姆林”甜橙树,其中包含诱发帧移位的插入或缺失。通过Sanger测序确认基因组编辑后,将选择的品系嫁接到C-146三叶杂交砧木上进行无性系繁殖。然后用无选择亚洲柑橘木虱(ACP)饲养试验感染CaLas。在温室条件下对csnpr3编辑柑橘树的遗传和生理特性进行了评估,结果表明,尽管CaLas滴度没有显著差异,但编辑后的柑橘树比野生型柑橘树表现出更强的活力。虽然需要进一步的实地评估,但我们的研究结果表明,CsNPR3参与了HLB引起的树木退化,并表明编辑CsNPR3可以增强对HLB的耐受性。