Suppression of <i>pinoid</i> mutant phenotypes by mutations in <i>PIN-FORMED 1</i> and PIN1-GFP fusion.

Abstract

Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the <i>PIN1</i> coding sequence using CRISPR/Cas9, and the resulting <i>pin1</i> mutant (<i>pin1-27</i>) was a strong allele. Surprisingly, heterozygous <i>pin1-27</i> suppressed two independent <i>pid</i> null mutants, whereas homozygous <i>pin1-27</i> enhanced the phenotypes of the <i>pid</i> mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous <i>pin1</i> mutants were also capable of rescuing <i>pid</i> nulls. Moreover, we inserted green fluorescent protein (GFP) into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the <i>PIN1-GFP_HDR</i> line are similar to those in the previously reported <i>PIN1-GFP</i> transgenic lines. Interestingly, the <i>PIN1-GFP_HDR</i> line also rescued various <i>pid</i> null mutant alleles in a semidominant fashion. We conclude that decreasing the number of functional <i>PIN1</i> copies is sufficient to suppress the <i>pid</i> mutant phenotype, suggesting that PIN1 is likely part of a larger protein complex required for organogenesis.