Table_4_Silencing of the Wheat Protein Phosphatase 2A Catalytic Subunit TaPP2Ac Enhances Host Resistance to the Necrotrophic Pathogen Rhizoctonia cerealis.DOCX

Eukaryotic type 2A protein phosphatases (protein phosphatase 2A, PP2A) consist of a scaffold subunit A, a regulatory subunit B, and a catalytic subunit C. Little is known about the roles of PP2Ac proteins that are involved in plant responses to necrotrophic fungal pathogens. Sharp eyespot, caused by the necrotrophic fungus Rhizoctonia cerealis, is a destructive disease of wheat (Triticum aestivum), an important staple food crop. Here, we isolated TaPP2Ac-4D from wheat, which encodes a catalytic subunit of the heterotrimeric PP2A, and characterized its properties and role in plant defense response to R. cerealis. Based on the sequence alignment of TaPP2Ac-4D with the draft sequences of wheat chromosomes from the International Wheat Genome Sequencing Consortium (IWGSC), it was found that TaPP2Ac-4D gene is located on the long arm of the wheat chromosome 4D and has two homologs assigned on wheat chromosomes 4A and 4B. Sequence and phylogenetic tree analyses revealed that the TaPP2Ac protein is a typical member of the PP2Ac family and belongs to the subfamily II. TaPP2Ac-4B and TaPP2Ac-4D displayed higher transcriptional levels in the R. cerealis-susceptible wheat cultivar Wenmai 6 than those seen in the resistant wheat line CI12633. The transcriptional levels of TaPP2Ac-4B and TaPP2Ac-4D were significantly elevated in wheat R. cerealis after infection and upon H2O2 treatment. Virus-induced gene silencing results revealed that the transcriptional knockdown of TaPP2Ac-4D and TaPP2Ac-4B significantly increased wheat resistance to R. cerealis infection. Meanwhile, the transcriptional levels of certain pathogenesis-related (PR) and reactive oxygen species (ROS)-scavenging enzyme encoding genes were increased in TaPP2Ac-silenced wheat plants. These results suggest that TaPP2Ac-4B and TaPP2Ac-4D negatively regulate defense response to R. cerealis infection possibly through modulation of the expression of certain PR and ROS-scavenging enzyme genes in wheat. This study reveals a novel function of the plant PP2Ac genes in plant immune responses.

真核生物2A型蛋白磷酸酶（protein phosphatase 2A, PP2A）由支架亚基A、调节亚基B与催化亚基C构成。目前对于PP2Ac蛋白在植物应对坏死性真菌病原菌过程中所发挥的作用，尚缺乏深入了解。由坏死性真菌禾谷丝核菌（Rhizoctonia cerealis）引发的小麦纹枯病，是重要主粮作物小麦（Triticum aestivum）的毁灭性病害。本研究从小麦中分离得到TaPP2Ac-4D，其编码异源三聚体PP2A的催化亚基，并对其特性以及在植物抵御禾谷丝核菌的防御反应中的作用进行了系统表征。基于TaPP2Ac-4D与国际小麦基因组测序联盟（International Wheat Genome Sequencing Consortium, IWGSC）公布的小麦染色体草图序列的比对分析，发现TaPP2Ac-4D基因定位于小麦4D染色体长臂，且在4A与4B染色体上各存在一个同源基因。序列与系统发育树分析结果显示，TaPP2Ac蛋白属于典型的PP2Ac家族成员，归类于II亚家族。TaPP2Ac-4B与TaPP2Ac-4D在感病小麦品种温麦6号中的转录水平，显著高于其在抗病小麦品系CI12633中的转录水平。在小麦被禾谷丝核菌侵染以及经H₂O₂处理后，TaPP2Ac-4B与TaPP2Ac-4D的转录水平均显著上调。病毒诱导基因沉默（virus-induced gene silencing, VIGS）实验结果表明，敲低TaPP2Ac-4D与TaPP2Ac-4B的转录水平后，小麦对禾谷丝核菌侵染的抗性显著提升。同时，在TaPP2Ac沉默的小麦植株中，部分病程相关（pathogenesis-related, PR）基因以及活性氧清除酶编码基因的转录水平均有所升高。上述结果提示，TaPP2Ac-4B与TaPP2Ac-4D可能通过调控小麦中部分PR基因与活性氧清除酶编码基因的表达，负向调控小麦对禾谷丝核菌侵染的防御反应。本研究揭示了植物PP2Ac基因在植物免疫响应中的全新功能。