Significance: Plant miRNAs can move from cell to cell through cytoplasmic channels called plasmodesmata, transmitting positional information that is decoded to make developmental decisions; one well-studied example of this is xylem patterning in the root of the model plant Arabidopsis thaliana , which depends on the formation of opposing gradients of two miRNAs and their target transcripts. Here, we show that two plasma membrane- and plasmodesmata-localized proteins, BAM1 and BAM2, are required for maintaining appropriate levels and distribution of the miRNA target transcripts, and hence for proper root xylem patterning, uncovering additional players in the regulation of this developmental process and adding one more function, direct or indirect, to the ever-growing portfolio of BAM1 and BAM2.

Reversible phosphorylation is an essential mechanism regulating signal transduction during development and environmental stress responses. An important number of dephosphorylation events in the cell are catalyzed by type one protein phosphatases (PP1), which catalytic activity is driven by the binding of regulatory proteins that control their substrate specificity or subcellular localization. Plants harbor several PP1 isoforms accounting for large functional redundancies. While animal PP1s were reported to play relevant roles in controlling multiple cellular processes, plant orthologs remain poorly studied. To decipher the role of plant PP1s, we compared PP1 genes from three monocot species, Brachypodium, common wheat and rice at the genomic and transcriptomic levels. To gain more insight into the wheat PP1 proteins, we identified and characterized TdPP1a, the first wheat type one protein phosphatase from a Tunisian durum wheat variety Oum Rabiaa3. TdPP1a is highly conserved in sequence and structure when compared to mammalian, yeast and other plant PP1s. We demonstrate that TdPP1a is an active, metallo-dependent phosphatase in vitro and is able to interact with AtI2, a typical regulator of PP1 functions. Also, TdPP1a is capable to complement the heat stress sensitivity of the yeast mutant indicating that TdPP1a is functional also in vivo. Moreover, transient expression of TdPP1a::GFP in tobacco leaves revealed that it is ubiquitously distributed within the cell, with a strong accumulation in the nucleus. Finally, transcriptional analyses showed similar expression levels in roots and leaves of durum wheat seedlings. Interestingly, the expression in leaves is significantly induced following salinity stress, suggesting a potential role of TdPP1a in wheat salt stress response.

Brassinosteroids (BRs) play key roles in diverse plant growth processes through a complex signaling pathway. Components orchestrating the BR signaling pathway include receptors such as kinases, transcription factors, protein kinases and phosphatases. The proper functioning of the receptor kinase BRI1 and the transcription factors BES1/BZR1 depends on their dephosphorylation by type 2A protein phosphatases (PP2A). In this work, we report that an additional phosphatase family, type one protein phosphatases (PP1), contributes to the regulation of the BR signaling pathway. Co-immunoprecipitation and BiFC experiments performed in Arabidopsis plants overexpressing durum wheat TdPP1 showed that TdPP1 interacts with dephosphorylated BES1, but not with the BRI1 receptor. Higher levels of dephosphorylated, active BES1 were observed in these transgenic lines upon BR treatment, indicating that TdPP1 modifies the BR signaling pathway by activating BES1. Moreover, ectopic expression of durum wheat TdPP1 lead to an enhanced growth of primary roots in comparison to wild-type plants in presence of BR. This phenotype corroborates with a down-regulation of the BR-regulated genes CPD and DWF4. These data suggest a role of PP1 in fine-tuning BR-driven responses, most likely via the control of the phosphorylation status of BES1.

SUMMARY: Rho‐of‐plant small GTPases (ROPs) are regulators of plant polar growth and of plant–pathogen interactions. The barley ROP, RACB, is involved in susceptibility toward infection by the barley powdery mildew fungus Blumeria hordei (Bh) but little is known about the cellular pathways that connect RACB signaling to disease susceptibility. Here we identify novel RACB interaction partners of plant or fungal origin by untargeted co‐immunoprecipitation of constitutively active (CA) RACB tagged by green fluorescent protein from Bh‐infected barley epidermal layers and subsequent analysis by liquid chromatography‐coupled mass spectrometry. Three of the immunoprecipitated proteins, a plant phosphoinositide phosphatase, a plant phosphoinositide phospholipase, and a putative Bh‐effector protein, are involved in the barley‐Bh‐pathosystem and support disease resistance or susceptibility, respectively. RACB and its plant interactors bind to overlapping anionic phospholipid species in vitro, and in the case of RACB, this lipid interaction is mediated by its carboxy‐terminal polybasic region (PBR). Fluorescent markers for anionic phospholipids show altered subcellular distribution in barley cells during Bh attack and under expression of a RACB‐binding fungal effector. Phosphatidylinositol 4‐phosphate, phosphatidylinositol 3,5‐bisphosphate, and phosphatidylserine show a distinct enrichment at the haustorial neck region, suggesting a connection to subcellular targeting of RACB at this site. The interplay of ROPs with anionic phospholipids and phospholipid‐metabolizing enzymes may thus enable the subcellular enrichment of components pivotal for success or failure of fungal penetration.