Genetic Interactions Between BOB1 And Multiple 26S Proteasome Subunits Suggest A Role For Proteostasis In Regulating Arabidopsis Development

Protein folding and degradation are both required for protein quality control, an essential cellular activity that underlies normal growth and development. We investigated how BOB1, an Arabidopsis thaliana small heat shock protein, maintains normal plant development. bob1 mutants exhibit organ polarity defects and have expanded domains of KNOX gene expression. Some of these phenotypes are ecotype specific suggesting that other genes function to modify them. Using a genetic approach we identified an interaction between BOB1 and FIL, a gene required for abaxial organ identity. We also performed an EMS enhancer screen using the bob1-3 allele to identify pathways that are sensitized by a loss of BOB1 function. This screen identified genetic, but not physical, interactions between BOB1 and the proteasome subunit RPT2a. Two other proteasome subunits, RPN1a and RPN8a, also interact genetically with BOB1. Both BOB1 and the BOB1-interacting proteasome subunits had previously been shown to interact genetically with the transcriptional enhancers AS1 and AS2, genes known to regulate both organ polarity and KNOX gene expression. Our results suggest a model in which BOB1 mediated protein folding and proteasome mediated protein degradation form a functional proteostasis module required for ensuring normal plant development

Characterizing the Relationship between a Small Heat Shock Protein with Chaperone Activity and the 26S Proteasome in Arabidopsis

Molecular chaperones and proteasomes are required for the normal development of plants via their role in the folding and degradation of cellular proteins. Using a genetic screen, we identified a genetic interaction between BOBBER1—a NudC domain containing Arabidopsis small heat shock protein with both developmental and thermotolerance functions—and RPT2a, an AAA-ATPase subunit of the 26S proteasome. This interaction suggests a connection between the pathways of protein folding and degradation in plant development. We used a series of genetic, molecular, and pharmacological approaches to better understand the relationship between these genes, and how they collectively contribute to development. The results of these experiments have shown that bom and bob1-3 do not interact physically and that knockouts of other 26SP subunits produce synergistic phenotypes like those observed in bom;bob1-3 mutants. This suggests that the genetic interaction observed between bom and bob1-3 is due to a general decrease in proteasome function and not a specific interaction with bom. Additionally, we demonstrate that born behaves like the null allele rpt2a-2 in response to proteasome inhibition even though it differs from the wild type allele by a single point mutation. We also show that no epistatic relationship exists between bom and bobl-3 in response to proteasome inhibition and suggests that these genes do not operate in a simple linear genetic pathway. Finally, we have identified and characterized a heritable mutant phenotype within the mutagenized AT87 family and have demonstrated that it is BOB1 dependent. Next-generation sequencing of the AT87 mapping population was unable to identify a causal genetic basis for the AT87 developmental phenotype