Laboratory of Fruit Cell and Molecular Breeding, China Agricultural University, Beijing, 100193, China.
Jiangsu Polytechnic College of Agriculture and Forestry, Zhenjiang, Jiangsu, 212400, China.
Plant J. 2021 Dec;108(5):1522-1538. doi: 10.1111/tpj.15526. Epub 2021 Oct 28.
Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide-binding, and leucine-rich repeat domain CC -NB-LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB-LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1-inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB-ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad-spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple.
苹果叶斑病是由链格孢Alternaria alternata f. sp. mali 和其他真菌物种引起的一种病害，可导致严重落叶，给中国的苹果（Malus × domestica）产业带来巨大损失。我们之前鉴定了三个 RPW8、核苷酸结合和富含亮氨酸重复域 CC-NB-LRR 蛋白（RNLs），分别命名为 MdRNL1、MdRNL2 和 MdRNL3，它们有助于苹果对链格孢叶斑病（ALT1）的抗性。然而，NB-LRR 蛋白在苹果对真菌病害的抗性中的作用仍知之甚少。因此，我们使用 MdRNL1/2/3 作为诱饵，从 ALT1 接种叶片中筛选相互作用蛋白，仅鉴定到 MdRNL6（另一个 RNL）是 MdRNL2 的互作蛋白。蛋白互作实验表明，MdRNL2 和 MdRNL6 通过其 NB-ARC 结构域相互作用。在苹果中的瞬时表达实验表明，含有 MdRNL2 和 MdRNL6 的复合物是对链格孢叶斑病抗性所必需的。有趣的是，相同的复合物在瞬时表达实验中也需要赋予对球腔菌叶斑病和轮斑病菌叶斑病的抗性。此外，稳定转化的苹果植株中 MdRNL6 表达受到抑制后，对链格孢叶斑病、球腔菌叶斑病和轮斑病菌叶斑病表现出超敏反应；这些效应与在转基因苹果幼苗中抑制 MdRNL2 表达的效应相似。这些新型广谱真菌抗性基因的鉴定将有助于苹果真菌病抗性的培育。
