Jia Hongge, Orbovic Vladimir, Jones Jeffrey B, Wang Nian
Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences (IFAS), University of Florida, Lake Alfred, FL, USA.
Citrus Research and Education Center, Institute of Food and Agricultural Sciences (IFAS), University of Florida, Lake Alfred, FL, USA.
Plant Biotechnol J. 2016 May;14(5):1291-301. doi: 10.1111/pbi.12495. Epub 2015 Nov 2.
Citrus canker caused by Xanthomonas citri subspecies citri (Xcc) is a severe disease for most commercial citrus cultivars and responsible for significant economic losses worldwide. Generating canker-resistant citrus varieties will provide an efficient and sustainable solution to control citrus canker. Here, we report our progress in generating canker-resistant grapefruit by modifying the PthA4 effector binding elements (EBEs) in the CsLOB1 Promoter (EBEPthA4 -CsLOBP) of the CsLOB1 (Citrus sinensis Lateral Organ Boundaries) gene. CsLOB1 is a susceptibility gene for citrus canker and is induced by the pathogenicity factor PthA4, which binds to the EBEPthA4 -CsLOBP to induce CsLOB1 gene expression. There are two alleles, Type I and Type II, of CsLOB1 in Duncan grapefruit. Here, a binary vector was designed to disrupt the PthA4 EBEs in Type I CsLOB1 Promoter (TI CsLOBP) via epicotyl transformation of Duncan grapefruit. Four transgenic Duncan plants with targeted modification of EBEPthA4 -T1 CsLOBP were successfully created. As for Type I CsLOB1 promoter, the mutation rate was 15.63% (#D13), 14.29% (#D17), 54.54% (#D18) and 81.25% (#D22). In the presence of wild-type Xcc, transgenic Duncan grapefruit developed canker symptoms similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not the mutated Type I CsLOBP or Type II CsLOBP, was developed to infect Duncan transformants. Consequently, #D18 had weakened canker symptoms and #D22 had no visible canker symptoms in the presence of XccΔpthA4:dCsLOB1.3. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by PthA4 is sufficient to induce citrus canker disease, and mutation in the promoters of both alleles of CsLOB1 is probably required to generate citrus canker-resistant plants. This work lays the groundwork to generate canker-resistant citrus varieties via Cas9/sgRNA in the future.
由柑橘溃疡病菌（Xanthomonas citri subspecies citri，Xcc）引起的柑橘溃疡病对大多数商业柑橘品种来说都是一种严重病害，在全球范围内造成重大经济损失。培育抗溃疡病的柑橘品种将为防治柑橘溃疡病提供一种高效且可持续的解决方案。在此，我们报告了通过修饰CsLOB1（柑橘侧生器官边界，Citrus sinensis Lateral Organ Boundaries）基因的CsLOB1启动子中的PthA4效应子结合元件（EBEs）（EBEPthA4 -CsLOBP）来培育抗溃疡病葡萄柚的进展。CsLOB1是柑橘溃疡病的一个感病基因，受致病因子PthA4诱导，PthA4与EBEPthA4 -CsLOBP结合以诱导CsLOB1基因表达。邓肯葡萄柚中存在CsLOB1的两个等位基因，即I型和II型。在此，设计了一个二元载体，通过邓肯葡萄柚的上胚轴转化来破坏I型CsLOB1启动子（TI CsLOBP）中的PthA4 EBEs。成功培育出了4株对EBEPthA4 -T1 CsLOBP进行了靶向修饰的转基因邓肯植株。对于I型CsLOB1启动子，突变率分别为15.63%（#D13）、14.29%（#D17）、54.54%（#D18）和81.25%（#D22）。在野生型Xcc存在的情况下，转基因邓肯葡萄柚出现的溃疡病症状与野生型类似。开发了一种人工设计的dTALE dCsLOB1.3，它能特异性识别I型CsLOBP，但不能识别突变的I型CsLOBP或II型CsLOBP，用于感染邓肯转化体。结果，在存在XccΔpthA4:dCsLOB1.3的情况下，#D18的溃疡病症状减轻，#D22没有可见的溃疡病症状。我们的数据表明，PthA4激活感病基因CsLOB1的单个等位基因就足以诱发柑橘溃疡病，可能需要对CsLOB1两个等位基因的启动子进行突变才能培育出抗柑橘溃疡病的植株。这项工作为未来通过Cas9/sgRNA培育抗溃疡病柑橘品种奠定了基础。
