Plants optimize carbon assimilation while limiting water loss by adjusting stomatal aperture. In grasses, a developmental innovation-the addition of subsidiary cells (SCs) flanking two dumbbell-shaped guard cells (GCs)-is linked to improved stomatal physiology. Here, we identify a transcription factor necessary and sufficient for SC formation in the wheat relative Unexpectedly, the transcription factor is an ortholog of the stomatal regulator, which defines GC precursor fate in The novel role of in specifying lateral SCs appears linked to its acquisition of cell-to-cell mobility in Physiological analyses on SC-less plants experimentally support classic hypotheses that SCs permit greater stomatal responsiveness and larger range of pore apertures. Manipulation of SC formation and function in crops, therefore, may be an effective approach to enhance plant performance.

Stomata, epidermal valves facilitating plant-atmosphere gas exchange, represent a powerful model for understanding cell fate and pattern in plants. Core basic helix-loop-helix (bHLH) transcription factors regulating stomatal development were identified in Arabidopsis, but this dicot's developmental pattern and stomatal morphology represent only one of many possibilities in nature. Here, using unbiased forward genetic screens, followed by analysis of reporters and engineered mutants, we show that stomatal initiation in the grass Brachypodium distachyon uses orthologs of stomatal regulators known from Arabidopsis but that the function and behavior of individual genes, the relationships among genes, and the regulation of their protein products have diverged. Our results highlight ways in which a kernel of conserved genes may be alternatively wired to produce diversity in patterning and morphology and suggest that the stomatal transcription factor module is a prime target for breeding or genome modification to improve plant productivity.stomatal development | bHLH transcription factor | Brachypodium | grass S tomata are valves on the surface of plants with central roles in gas exchange and biosphere productivity. Stomata are both ancientthey appear on 400 million-year-old fossils-and nearly ubiquitously found in extant land plants. The diversity of stomatal morphologies and patterned distributions across different plant families coupled with rapidly advancing functional genomic resources offers a powerful opportunity to follow morphological innovation and gene regulatory network evolution simultaneously. In most plants, stomata consist of two kidney-shaped epidermal guard cells (GCs) surrounding a pore (Fig. 1A). Grass stomatal morphology is unique, featuring dumbbell-shaped GCs flanked by subsidiary cells (SCs) (Fig. 1A), and physiological measurements suggest this derived form is more efficient (1). The distribution of stomata on leaves is also species specific. Dicots such as Arabidopsis display a scattered distribution, with avoidance of direct contact being the most basic patterning rule; dispersed stem cell-like stomatal precursors divide throughout the leaf to produce this pattern and promote the typical "broadleaf" or radial growth characteristic of these plants (Fig. 1A). Grasses, in contrast, generate stomata, which are always oriented in the same direction, from specific cell files. These stomatal lineage files are established in a single zone at the leaf base with differentiation proceeding in a linear gradient toward the tip (Fig. 1A).Our understanding of the genetic underpinnings of stomatal fate and pattern is derived mostly from studies in the dicot Arabidopsis where the group Ia basic helix-loop-helix (bHLH) transcription factors SPEECHLESS (AtSPCH), AtMUTE, and AtFAMA establish stomatal lineage identity, regulate the transition to terminal precursor fate, and promote the differentiation of GCs, respectively (2-4). The function of these stage-specific factors requires heterodimerization with one of ...

Trogocytosis is part of an emerging, exciting theme of cell-cell interactions both within and between species, and it is relevant to host-pathogen interactions in many different contexts. Trogocytosis is a process in which one cell physically extracts and ingests “bites” of cellular material from another cell. It was first described in eukaryotic microbes, where it was uncovered as a mechanism by which amoebae kill cells. Trogocytosis is potentially a fundamental form of eukaryotic cell-cell interaction, since it also occurs in multicellular organisms, where it has functions in the immune system, in the central nervous system, and during development. There are numerous scenarios in which trogocytosis occurs and an ever-evolving list of functions associated with this process. Many aspects of trogocytosis are relevant to microbial pathogenesis. It was recently discovered that immune cells perform trogocytosis to kill Trichomonas vaginalis parasites. Additionally, through trogocytosis, Entamoeba histolytica acquires and displays human cell membrane proteins, enabling immune evasion. Intracellular bacteria seem to exploit host cell trogocytosis, since they can use it to spread from cell to cell. Thus, a picture is emerging in which trogocytosis plays critical roles in normal physiology, infection, and disease.

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen.

Entamoeba histolytica is a microbial eukaryote and causative agent of the diarrheal disease amoebiasis. Pathogenesis is associated with profound damage to human tissues, and treatment options are limited. We discovered that amoebae attack and kill human cells through a cell-nibbling process that we named trogocytosis (trogo-: nibble). Trogocytosis is likely to underlie tissue damage during infection and it represents a potential target for therapeutic intervention, although the mechanism is still unknown. Assays in current use to analyze trogocytosis by amoebae have not been amenable to studying different types of human cells, or to high-throughput analysis. Here, we developed two complementary assays to measure trogocytosis by quantifying human cell viability, both of which can be used for suspension and adherent cells. The first assay uses CellTiterGlo, a luminescent readout for cellular ATP levels, as a proxy for cell viability. We found that the CellTiterGlo signal is proportional to the quantity of viable cells, and can be used to detect death of human cells after co-incubation with amoebae.We established a second assay that is microscopy-based and uses two fluorescent stains to directly differentiate live and dead human cells. Both assays are simple and inexpensive, can be used with suspension and adherent human cell types, and are amenable to high-throughput approaches. These new assays are tools to improve understanding of amoebiasis pathogenesis.

Entamoeba histolytica is a globally important pathogen that is dramatically understudied. Its challenging genome has limited tractability. To enable forward genetics, we constructed the first genome-wide E. histolytica RNAi knockdown mutant library. The library is designed to enable deep sequencing analysis for quantitative identification of mutants after selection. We developed a novel analysis pipeline to precisely define and quantify full-length gene fragments inferred from read mapping. We performed the first E. histolytica RNAi screen and identified slow growth mutants. Growth phenotypes were reproducible in independently generated mutants. Some of the genes targeted in slow growth mutants had annotated functions consistent with roles in growth or metabolism. Some targeted genes lacked annotation, supporting the power of forward genetics in uncovering gene function. This work opens up the possibility of applying genetics to improve understanding of this important pathogen. Moreover, the strategies behind this RNAi library, and its analysis, are novel, and can be applied to other organisms.

1Entamoeba histolytica is a microbial eukaryote and causative agent of the diarrheal 2 disease amoebiasis. Pathogenesis is associated with profound damage to human tissues, and 3 treatment options are limited. We discovered that amoebae attack and kill human cells through a 4 cell-nibbling process that we named trogocytosis (trogo-: nibble). Trogocytosis is likely to 5 underlie tissue damage during infection and it represents a potential target for therapeutic 6 intervention, although the mechanism is still unknown. Assays in current use to analyze 7 trogocytosis by amoebae have not been amenable to studying different types of human cells, or 8 to high-throughput analysis. Here, we developed two complementary assays to measure 9 trogocytosis by quantifying human cell viability, both of which can be used for suspension and 10 adherent cells. The first assay uses CellTiterGlo, a luminescent readout for cellular ATP levels, 11 as a proxy for cell viability. We found that the CellTiterGlo signal is proportional to the quantity 12 of viable cells, and can be used to detect death of human cells after co-incubation with amoebae. 13We established a second assay that is microscopy-based and uses two fluorescent stains to 14 directly differentiate live and dead human cells. Both assays are simple and inexpensive, can be 15 used with suspension and adherent human cell types, and are amenable to high-throughput 16 approaches. These new assays are tools to improve understanding of amoebiasis pathogenesis. 17