Table_1_The Armadillo BTB Protein ABAP1 Is a Crucial Player in DNA Replication and Transcription of Nematode-Induced Galls.XLSX

The biogenesis of root-knot nematode (Meloidogyne spp.)-induced galls requires the hyperactivation of the cell cycle with controlled balance of mitotic and endocycle programs to keep its homeostasis. To better understand gall functioning and to develop new control strategies for this pest, it is essential to find out how the plant host cell cycle programs are responding and integrated during the nematode-induced gall formation. This work investigated the spatial localization of a number of gene transcripts involved in the pre-replication complex during DNA replication in galls and report their akin colocation with the cell cycle S-phase regulator Armadillo BTB Arabidopsis Protein 1 (ABAP1). ABAP1 is a negative regulator of pre-replication complex controlling DNA replication of genes involved in control of cell division and proliferation; therefore, its function has been investigated during gall ontogenesis. Functional analysis was performed upon ABAP1 knockdown and overexpression in Arabidopsis thaliana. We detected ABAP1 promoter activity and localized ABAP1 protein in galls during development, and its overexpression displayed significantly reduced gall sizes containing atypical giant cells. Profuse ABAP1 expression also impaired gall induction and hindered nematode reproduction. Remarkably, ABAP1 knockdown likewise negatively affected gall and nematode development, suggesting its involvement in the feeding site homeostasis. Microscopy analysis of cleared and nuclei-stained whole galls revealed that ABAP1 accumulation resulted in aberrant giant cells displaying interconnected nuclei filled with enlarged heterochromatic regions. Also, imbalanced ABAP1 expression caused changes in expression patterns of genes involved in the cell division control as demonstrated by qRT-PCR. CDT1a, CDT1b, CDKA;1, and CYCB1;1 mRNA levels were significantly increased in galls upon ABAP1 overexpression, possibly contributing to the structural changes in galls during nematode infection. Overall, data obtained in galls reinforced the role of ABAP1 controlling DNA replication and mitosis and, consequently, cell proliferation. ABAP1 expression might likely take part of a highly ordered mechanism balancing of cell cycle control to prevent gall expansion. ABAP1 expression might prevent galls to further expand, limiting excessive mitotic activity. Our data strongly suggest that ABAP1 as a unique plant gene is an essential component for cell cycle regulation throughout gall development during nematode infection and is required for feeding site homeostasis.

根结线虫（Meloidogyne spp.）诱导形成根结的生物发生过程，需要细胞周期过度激活，并维持有丝分裂与核内周期程序的可控平衡，以维持其稳态。为更好地解析根结的功能，并开发针对该害虫的新型防控策略，明确植物宿主的细胞周期程序在线虫诱导根结形成过程中的响应与整合机制至关重要。本研究对根结内DNA复制过程中涉及前复制复合物（pre-replication complex）的多种基因转录本的空间定位进行了探究，并发现其与细胞周期S期调控因子臂重复BTB结构域拟南芥蛋白1（Armadillo BTB Arabidopsis Protein 1, ABAP1）存在相似的共定位现象。ABAP1是前复制复合物的负调控因子，可通过调控参与细胞分裂与增殖的基因的DNA复制过程发挥功能，因此本研究对其在根结发生过程中的作用展开了分析。我们在拟南芥（Arabidopsis thaliana）中通过ABAP1基因敲低与过表达开展了功能验证实验，检测发现ABAP1的启动子活性与蛋白定位均存在于发育中的根结内；而过表达ABAP1可导致根结体积显著减小，且其中存在形态异常的巨型细胞，ABAP1的过度表达还会阻碍根结的诱导过程，并抑制线虫的繁殖。值得注意的是，ABAP1基因敲低同样会对根结与线虫的发育产生负面影响，这表明ABAP1参与了取食位点的稳态维持。对经透明化处理与细胞核染色的完整根结进行显微镜分析后发现，ABAP1的积累会导致巨型细胞出现异常，表现为相互连接的细胞核内充满了膨大的异染色质区域。此外，实时定量荧光PCR（qRT-PCR）结果显示，ABAP1表达失衡会改变细胞分裂调控相关基因的表达模式：在ABAP1过表达的根结中，CDT1a、CDT1b、CDKA;1及CYCB1;1的mRNA水平显著升高，这可能与线虫侵染过程中根结的结构变化密切相关。综上，根结内的相关研究结果进一步证实了ABAP1通过调控DNA复制与有丝分裂，进而调控细胞增殖的功能。ABAP1的表达可能参与了高度有序的细胞周期平衡调控机制，以阻止根结过度扩张，ABAP1的表达可抑制根结进一步膨大，限制过度的有丝分裂活性。本研究结果强烈表明，ABAP1作为一种独特的植物基因，是线虫侵染过程中根结发育全程细胞周期调控的必需组分，同时也是取食位点稳态维持所必需的。