Department of Plant Sciences, University of California Davis, One Shields Avenue, Davis, CA, 95616, USA.
BMC Genomics. 2013 Oct 1;14:668. doi: 10.1186/1471-2164-14-668.
Crown gall (CG) (Agrobacterium tumefaciens) and the root lesion nematodes (RLNs) (Pratylenchus vulnus) are major challenges faced by the California walnut industry, reducing productivity and increasing the cost of establishing and maintaining orchards. Current nematode control strategies include nematicides, crop rotation, and tolerant cultivars, but these methods have limits. Developing genetic resistance through novel approaches like RNA interference (RNAi) can address these problems. RNAi-mediated silencing of CG disease in walnut (Juglans regia L.) has been achieved previously. We sought to place both CG and nematode resistance into a single walnut rootstock genotype using co-transformation to stack the resistance genes. A. tumefaciens, carrying self-complimentary iaaM and ipt transgenes, and Agrobacterium rhizogenes, carrying a self-complimentary Pv010 gene from P. vulnus, were used as co-transformation vectors. RolABC genes were introduced by the resident T-DNA in the A. rhizogenes Ri-plasmid used as a vector for plant transformation. Pv010 and Pv194 (transgenic control) genes were also transferred separately using A. tumefaciens. To test for resistance, transformed walnut roots were challenged with P. vulnus and microshoots were challenged with a virulent strain of A. tumefaciens.
Combining the two bacterial strains at a 1:1 rather than 1:3 ratio increased the co-transformation efficiency. Although complete immunity to nematode infection was not observed, transgenic lines yielded up to 79% fewer nematodes per root following in vitro co-culture than untransformed controls. Transgenic line 33-3-1 exhibited complete crown gall control and 32% fewer nematodes. The transgenic plants had thicker, longer roots than untransformed controls possibly due to insertion of rolABC genes. When the Pv010 gene was present in roots with or without rolABC genes there was partial or complete control of RLNs. Transformation using only one vector showed 100% control in some lines.
CG and nematode resistance gene stacking controlled CG and RLNs simultaneously in walnuts. Silencing genes encoding iaaM, ipt, and Pv010 decrease CG formation and RLNs populations in walnut. Beneficial plant genotype and phenotype changes are caused by co-transformation using A. tumefaciens and A. rhizogenes strains. Viable resistance against root lesion nematodes in walnut plants may be accomplished in the future using this gene stacking technology.
冠瘿病（CG）（根癌农杆菌）和根结线虫（RLNs）（滑刃线虫属）是加利福尼亚核桃产业面临的主要挑战，降低了生产力并增加了建立和维护果园的成本。目前的线虫控制策略包括杀线虫剂、轮作和耐性品种，但这些方法都有局限性。通过 RNA 干扰（RNAi）等新方法开发遗传抗性可以解决这些问题。以前已经实现了通过 RNAi 介导的核桃（Juglans regia L.）CG 疾病沉默。我们试图通过共转化将 CG 抗性和线虫抗性基因置于单个核桃砧木基因型中，以实现抗性基因的叠加。携带自我互补的 iaaM 和 ipt 转基因的根癌农杆菌和携带滑刃线虫属 Pv010 基因的根癌农杆菌 rhizogenes 被用作共转化载体。 rolABC 基因由根癌农杆菌 Rhizogenes Ri 质粒中的驻留 T-DNA 引入，用作植物转化载体。 Pv010 和 Pv194（转基因对照）基因也分别使用根癌农杆菌进行转移。为了测试抗性，将转化的核桃根与滑刃线虫属进行了挑战，并用强毒力的根癌农杆菌菌株对微芽进行了挑战。
将两种细菌菌株以 1:1 而不是 1:3 的比例混合可以提高共转化效率。虽然未观察到对线虫感染的完全免疫，但与未转化对照相比，经过体外共培养的转基因系每根根中的线虫数量减少了高达 79%。转基因系 33-3-1 表现出完全的冠瘿病控制和 32%的线虫减少。转基因植物的根比未转化对照的根更厚、更长，这可能是由于 rolABC 基因的插入。当 Pv010 基因存在于有或没有 rolABC 基因的根中时，对 RLNs 有部分或完全的控制。仅使用一种载体进行转化，在一些系中可达到 100%的控制。
CG 和线虫抗性基因叠加在核桃中同时控制 CG 和 RLNs。沉默编码 iaaM、ipt 和 Pv010 的基因减少了核桃中的 CG 形成和 RLNs 种群。使用根癌农杆菌和根癌农杆菌 rhizogenes 菌株进行共转化会导致有益的植物基因型和表型变化。使用这种基因叠加技术，未来可能在核桃植物中实现对根结线虫的有效抗性。
