Identification of new protein–protein and protein–DNA interactions linked with wood formation in Populus trichocarpa

First author: H. Earl Petzold; Affiliations: Virginia Tech (弗吉尼亚理工大学): Blacksburg, USA

Corresponding author: Amy M Brunner

Cellular processes, such as signal transduction and cell wall deposition, are organized by macromolecule (高分子) interactions. Experimentally determined protein–protein interactions (PPIs) and protein–DNA interactions (PDIs) relevant to (有关的) woody plant development are sparse (稀少的). To begin to develop a Populus trichocarpa Torr. & A. Gray wood interactome (互作组), we applied the yeast-two-hybrid (Y2H) assay in different ways to enable the discovery of novel PPIs and connected networks. We first cloned open reading frames (ORFs) for 361 genes markedly upregulated in secondary xylem compared with secondary phloem and performed a binary Y2H screen with these proteins. By screening a xylem cDNA library for interactors of a subset of these proteins and then recapitulating the process by using a subset of the interactors as baits (诱饵), we ultimately identified 165 PPIs involving 162 different ORFs. Thirty-eight transcription factors (TFs) included in our collection of P. trichocarpa wood ORFs were used in a Y1H screen for binding to promoter regions of three genes involved in lignin biosynthesis resulting in 40 PDIs involving 20 different TFs. The network incorporating both the PPIs and PDIs included 14 connected subnetworks, with the largest having 132 members. Protein–protein interactions and PDIs validated previous reports and also identified new candidate wood formation proteins and modules through their interactions with proteins and promoters known to be involved in secondary cell wall synthesis. Selected examples are discussed including a PPI between Mps one binder (MOB1) and a mitogen-activated protein kinase kinase kinase kinase (M4K) that was further characterized by assays confirming the PPI as well as its effect on subcellular localization (亚细胞定位). Mapping of published transcriptomic data showing developmentally detailed expression patterns across a secondary stem onto the network supported that the PPIs and PDIs are relevant to wood formation, and also illustrated that wood-associated interactions involve gene products that are not upregulated in secondary xylem.

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细胞过程，包括信号转导和细胞壁沉积，是由高分子间的互作所组织的。有关木本植物发育的蛋白-蛋白互作（PPI）和蛋白-DNA互作（PDI）的试验研究还是很少。本文为了研究毛果杨木材互作组，作者通过不同的方式进行酵母双杂（Y2H）来发现新的PPI及关联网络。作者首先克隆了相比于次生韧皮部在次生木质部中表达上调的361个基因的开放阅读框ORFs，并利用这些蛋白进行双向的Y2H试验。通过在木质部cDNA库中扫描这些蛋白互作，再利用互作作为受体重塑这些进程，作者最终鉴定了165个PPIs，涉及到162个不同的ORFs。作者进一步利用这其中所含的38个转录因子进行酵母单杂（Y1H）扫描其在木质素生物合成相关基因启动子区结合的情况，最终鉴定了40个PDIs，涉及20个不同的转录因子。结合PPIs和PDIs的网络共包括14个关联亚网络，其中最大亚网络的包含132个成员。PPIs和PDIs验证了之前的一些报道，同时也通过与已知次生细胞壁合成相关蛋白和启动子互作的情况鉴定了一些新的候选木材形成相关蛋白和分子。作者选择了一些例子进行了讨论，包括MOB1和M4K互作的一个PPI，后续的试验以及亚细胞定位也验证了这一PPI。通过已发表的转录组数据显示了一些在次生茎发育相关的表达模式，其处于支持与木材形成相关的PPI和PDI的网络上，同样也鉴定了一些在次生木质部并不上调表达但参与到木材相关互作的基因。

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通讯：Amy M Brunner (https://cnre.vt.edu/faculty/brunner/index.html)

个人简介：1982年，伍斯特学院，生物学学士；1984年，范德堡大学，分子生物学硕士；1998年，俄勒冈州立大学，森林学、遗传学博士；现为弗吉尼亚理工大学副教授。

研究方向：树木发育、生态生理及适应；树木成熟、开花及花冠分子遗传学；林木表观遗传和小RNA；林木长距离信号转导。

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doi: https://doi.org/10.1093/treephys/tpx121

Journal: Tree Physiology

Published data: October 10, 2017.

（P.S. 欢迎访问个人博客：http://bbs.sciencenet.cn/u/TickingClock）