Functional characterization of the maize lateralrootless 1 gene

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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.