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In plants, cell polarity plays key roles in coordinating developmental processes. Despite the characterization of several polarly localized plasma membrane proteins, the mechanisms connecting protein dynamics with cellular functions often remain unclear. Here, we introduce a polarized receptor, KOIN, that restricts cell divisions in the Arabidopsis root meristem. In the endodermis, KOIN polarity is opposite to IRK, a receptor that represses endodermal cell divisions. Their contra-polar localization facilitates dissection of polarity mechanisms and the links between polarity and function. We find that IRK and KOIN are recognized, sorted, and secreted through distinct pathways. IRK extracellular domains determine its polarity and partially rescue the mutant phenotype, whereas KOIN’s extracellular domains are insufficient for polar sorting and function. Endodermal expression of an IRK/KOIN chimera generates non-cell-autonomous misregulation of root cell divisions that impacts patterning. Altogether, we reveal two contrasting mechanisms determining these receptors’ polarity and link their polarity to cell divisions in root tissue patterning.

 

ABSTRACT Gene-editing tools such as CRISPR-Cas9 have created unprecedented opportunities for genetic studies in plants and animals. We designed a course-based undergraduate research experience (CURE) to train introductory biology students in the concepts and implementation of gene-editing technology as well as develop their soft skills in data management and scientific communication. We present two versions of the course that can be implemented with twice-weekly meetings over a 5-week period. In the remote-learning version, students performed homology searches, designed guide RNAs (gRNAs) and primers, and learned the principles of molecular cloning. This version is appropriate when access to laboratory equipment or in-person instruction is limited, such as during closures that have occurred in response to the COVID-19 pandemic. In person, students designed gRNAs, cloned CRISPR-Cas9 constructs, and performed genetic transformation of Arabidopsis thaliana . Students learned how to design effective gRNA pairs targeting their assigned gene with an 86% success rate. Final exams tested students’ ability to apply knowledge of an unfamiliar genome database to characterize gene structure and to properly design gRNAs. Average final exam scores of ∼73% and ∼84% for in-person and remote-learning CUREs, respectively, indicated that students met learning outcomes. The highly parallel nature of the CURE makes it possible to target dozens to hundreds of genes, depending on the number of sections. Applying this approach in a sensitized mutant background enables focused reverse genetic screens for genetic suppressors or enhancers. The course can be adapted readily to other organisms or projects that employ gene editing. 

In plants, coordination of cell division and differentiation is critical for tissue patterning and organ development. Directional cell signaling and cell polarity have been proposed to participate in coordination of these developmental processes. For instance, a leucine-rich repeat receptor-like kinase (LRR-RLK) named INFLORESCENCE AND ROOT APICES KINASE (IRK) functions to restrict stele area and inhibit longitudinal anticlinal divisions (LADs) in the endodermis where it is polarly localized. The LRR-RLK most closely related to IRK is PXY/TDR CORRELATED 2 (PXC2) and we find that PXC2 shows similar polarized accumulation as IRK in root cell types. To further understand how these proteins operate in directional cell-cell signaling and root development we explored PXC2 function. pxc2 roots have an increase in stele area, indicating that PXC2 also functions to restrict stele size. Additionally, compared to either single mutant, irk pxc2 roots have an enhanced phenotype with further increases in endodermal LADs and stele area indicating redundant activities of these receptors. The double mutant also exhibits abnormal root growth, suggesting broader functions of PXC2 and IRK in the root. However, PXC2 is not functionally equivalent to IRK, as endodermal misexpression of PXC2 did not fully rescue irk. We propose that PXC2 is at least partially redundant to IRK with a more predominant role for IRK in repression of endodermal LADs. Our results are consistent with the hypothesis that repression of specific endodermal cell divisions and stele area through a PXC2/IRK-mediated directional signaling pathway is required for coordinated root growth and development.