Functional characterization of the maize lateralrootless 1 gene

侧根显著地增加植物根系的吸收表面。在玉米中，侧根起源于所有根类型的韧皮极周壁细胞。玉米突变体lateral rootless 1（lrt 1）在胚性初生根和种子根的侧根发生中有缺陷，但在胚后茎生根系统中没有缺陷。为了准备这项提案，我们通过精细定位和BSA-seq的结合鉴定了lrt 1候选基因。该候选基因编码DCAF 1（DDB 1-CUL 4相关因子1）蛋白，该蛋白是植物CULLIN 4-基E3连接酶（CRL 4）复合物的一部分。这将通过两条实验线来实现。首先，博士生（3年）将通过基因组编辑产生新的突变等位基因来验证lrt 1编码DCAF同系物。平行地，将通过基因组编辑产生旁系同源基因lrt 1样突变体和这两个基因的双突变体。然后对这些突变体的根系进行表型和细胞学分析。在对LRT 1和LRT 1-LIKE蛋白的功能结构域进行注释并进行系统发育分析后，将通过qRT-PCR和原位杂交实验详细研究lrt 1和lrt 1-LIKE在不同根类型、组织和突变体中的基因表达。为了证明LRT 1和LRT 1-LIKE是典型的DCAF蛋白，将进行亚细胞定位和稳定性实验以及与DDB 1的两种玉米同源物的相互作用研究。最后，LRT 1的新型底物蛋白将在酵母双杂交和随后的BiFC（双分子荧光互补）验证实验中鉴定。这些新型底物蛋白的一个子集将进行初步表征，并在第二个资助期内进行突变分析。第二，一名博士后研究员（2年）将研究野生型和lrt 1根中韧皮极和木质极周壁细胞在三个发育阶段的细胞学和组织化学特征，这三个发育阶段对应于野生型根的早期启动、周壁创始细胞建立和侧根起始。随后，将探讨韧皮部极和木质部极周细胞及其在发育过程中的重编程的lrt 1依赖的转录组网络。这将通过激光捕获显微切割分离玉米木质部极和韧皮部极周壁细胞并在上述三个发育阶段的RNA-seq实验后调查野生型和lrt 1根中这些细胞类型的表达来实现。

Lateral roots substantially increase the absorbing surface of plant root systems. In maize, lateral roots are initiated from phloem pole pericycle cells of all root types. The maize mutant lateral rootless 1 (lrt1) is defective in the initiation of lateral roots in embryonic primary and seminal roots but not in the postembryonic shoot-borne root system. In preparation for this proposal we have identified a lrt1 candidate gene by a combination of fine mapping and BSA-seq. This candidate gene encodes a DCAF1 (DDB1-CUL4 ASSOCIATED FACTOR 1) protein which is part of plant CULLIN4-based E3 ligase (CRL4) complexes.The overall goal of this proposal is to understand the molecular processes involved in lateral root initiation in maize. This will be achieved by two lines of experiments. First, a PhD student (3 years) will validate that lrt1 encodes a DCAF homolog by generating novel mutant alleles by genome editing. In parallel, mutants of the paralogous gene lrt1-like and double mutants of these two genes will be generated by genome editing. The root systems of these mutants will then be analyzed phenotypically and cytologically. After annotation of functional domains of the LRT1 and LRT1-LIKE proteins and phylogenetic analyses, gene expression of lrt1 and lrt1-like will be studied in detail in different root types, tissues and mutants by qRT-PCR and in situ hybridization experiments. To demonstrate that LRT1 and LRT1-LIKE are canonical DCAF proteins, subcellular localization and stability experiments and interaction studies with the two maize homologs of DDB1 will be performed. Finally, novel substrate proteins of LRT1 will be identified in yeast-two hybrid and subsequent BiFC (Bimolecular Fluorescence Complementation) validation experiments. A subset of these novel substrate proteins will be subjected to an initial characterization which will be complemented by mutant analyses in the second funding period. Second, a postdoctoral researcher (2 years) will study the cytological and histochemical characteristics of phloem pole and xylem pole pericycle cells in wild type and lrt1 roots during three developmental stages which correspond in wild type roots to early priming, pericycle founder cell establishment and lateral root initiation. Subsequently, lrt1-dependent transcriptomic networks of phloem pole and xylem pole pericycle cells and their reprogramming during development will be explored. This will be accomplished by isolating maize xylem pole and phloem pole pericycle cells by laser capture microdissection and surveying the expression of these cell types in wild type and lrt1 roots after RNA-seq experiments at the three developmental stages described above.