Analysis of the Localization of Fluorescent PpROP1 and PpROP-GEF4 Fusion Proteins in Moss Protonemata Based on Genomic “Knock-In” and Estradiol-Titratable Expression

Tip growth of pollen tubes, root hairs, and apical cells of moss protonemata is controlled by ROP (Rho of plants) GTPases, which were shown to accumulate at the apical plasma membrane of these cells. However, most ROP localization patterns reported in the literature are based on fluorescent protein tagging and need to be interpreted with caution, as ROP fusion proteins were generally overexpressed at undefined levels, in many cases without assessing effects on tip growth. ROP-GEFs, important regulators of ROP activity, were also described to accumulate at the apical plasma membrane during tip growth. However, to date only the localization of fluorescent ROP-GEF fusion proteins strongly overexpressed using highly active promoters have been investigated. Here, the intracellular distributions of fluorescent PpROP1 and PpROP-GEF4 fusion proteins expressed at essentially endogenous levels in apical cells of Physcomitrella patens “knock-in” protonemata were analyzed. Whereas PpROP-GEF4 was found to associate with a small apical plasma membrane domain, PpROP1 expression was below the detection limit. Estradiol-titratable expression of a fluorescent PpROP1 fusion protein at the lowest detectable level, at which plant development was only marginally affected, was therefore employed to show that PpROP1 also accumulates at the apical plasma membrane, although within a substantially larger domain. Interestingly, RNA-Seq data indicated that the majority of all genes active in protonemata are expressed at lower levels than PpROP1, suggesting that estradiol-titratable expression may represent an important alternative to “knock-in” based analysis of the intracellular distribution of fluorescent fusion proteins in protonemal cells

Physcomitrium patens PpRIC, an ancestral CRIB-domain ROP effector, inhibits auxin-induced differentiation of apical initial cells

RHO guanosine triphosphatases are important eukaryotic regulators of cell differentiation and behavior. Plant ROP (RHO of plant) family members activate specific, incompletely characterized downstream signaling. The structurally simple land plant Physcomitrium patens is missing homologs of key animal and flowering plant RHO effectors but contains a single CRIB (CDC42/RAC interactive binding)-domain -contain-ing RIC (ROP-interacting CRIB-containing) protein (PpRIC). Protonemal P. patens filaments elongate based on regular division and PpROP-dependent tip growth of apical initial cells, which upon stimulation by the hor-mone auxin differentiate caulonemal characteristics. PpRIC interacts with active PpROP1, co-localizes with this protein at the plasma membrane at the tip of apical initial cells, and accumulates in the nucleus. Remark-ably, PpRIC is not required for tip growth but is targeted to the nucleus to block caulonema differentiation downstream of auxin-controlled gene expression. These observations establish functions of PpRIC in medi-ating crosstalk between ROP and auxin signaling, which contributes to the maintenance of apical initial cell identity