Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the~120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, too small, or occur too rapidly to see clearly with existing tools. We crafted ultra-thin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at sub-second intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and complexity of living systems.

The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are well suited for the investigation of the evolution of multicellularity and development. We sequenced the 138–mega–base pair genome of V. carteri and compared its ~14,500 predicted proteins to those of its unicellular relative Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials and few species-specific protein-coding gene predictions. Volvox is enriched in volvocine-algal–specific proteins, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity.

Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.

P values and error bars help readers infer whether a reported difference would likely recur, with the sample size n used for statistical tests representing biological replicates, independent measurements of the population from separate experiments. We provide examples and practical tutorials for creating figures that communicate both the cell-level variability and the experimental reproducibility.

The tomato (Lycopersicon esculentum) Cf-9 resistance gene encodes the first characterized member of the plant receptor-like protein (RLP) family. Other RLPs such as CLAVATA2 and TOO MANY MOUTHS are known to regulate development. The domain structure of RLPs consists of extracellular leucine-rich repeats, a transmembrane helix, and a short cytoplasmic region. Here, we identify 90 RLPs in rice (Oryza sativa) and compare them with functionally characterized RLPs from different plant species and with 56 Arabidopsis (Arabidopsis thaliana) RLPs, including the downy mildew resistance protein RPP27. Many RLPs cluster into four distinct superclades, three of which include RLPs known to be involved in plant defense. Sequence comparisons reveal diagnostic amino acid residues that may specify different molecular functions in different RLP subtypes. This analysis of rice RLPs thus identified at least 73 candidate resistance genes and four genes potentially involved in development. Due to the synteny between rice and other Gramineae, this analysis should provide valuable tools for experimental studies in rice and other cereals.

Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.Giardia intestinalis is the most frequently identified protozoan cause of intestinal morbidity worldwide (59) and has a two-stage life cycle: a "trophozoite," or flagellate, form that attaches to the intestinal microvilli and a cyst form that can persist in the environment (1, 21). As in other eukaryotes, the giardial microtubule cytoskeleton creates a stable scaffold for intracellular trafficking, for organelle attachment, and for cell polarization (21). However, other important functions of the microtubule cytoskeleton are dynamic and rely upon both intrinsic dynamic instability-stochastic switches between microtubule growth and shrinkage phases (43)-and active regulation of microtubule assembly and/or disassembly. Microtubule dynamics, for example, are critical during cell division in Giardia where the two nuclei (30) undergo mitosis with extranuclear spindles that penetrate at polar nuclear openings (58), followed by the duplication and repositioning of eight flagella into the daughter cells (75). Beyond descriptions of cytoskeletal movements, we currently have little understanding of the molecular mechanism of active regulation of interphase and mitotic microtubule dynamics in Giardia.One class of conserved regulators of microtubule dynamics that mediate interactions between microtubule plus ends and other organelles are t...

Tomato (Lycopersicon esculentum) Cf genes confer resistance to the fungal pathogen Cladosporium fulvum through recognition of secreted avirulence (Avr) peptides. Plant defense responses, including rapid alterations in gene expression, are immediately activated upon perception of the pathogen. Previously, we identified a collection of Avr9/Cf-9 rapidly (15 to 30 min) elicited (ACRE) genes from tobacco (Nicotiana tabacum). Many of the ACRE genes encode putative signaling components and thus may play pivotal roles in the initial development of the defense response. To assess the requirement of 42 of these genes in the hypersensitive response (HR) induced by Cf-9/Avr9 or by Cf-4/Avr4, we used virus-induced gene silencing (VIGS) in N. benthamiana. Three genes were identified that when silenced compromised the Cf-mediated HR. We further characterized one of these genes, which encodes a Ser/Thr protein kinase called Avr9/Cf-9 induced kinase 1 (ACIK1). ACIK1 mRNA was rapidly upregulated in tobacco and tomato upon elicitation by Avr9 and by wounding. Silencing of ACIK1 in tobacco resulted in a reduced HR that correlated with loss of ACIK1 transcript. Importantly, ACIK1 was found to be required for Cf-9/Avr9- and Cf-4/Avr4-mediated HRs but not for the HR or resistance mediated by other resistance/Avr systems, such as Pto/AvrPto, Rx/Potato virus X, or N/Tobacco mosaic virus. Moreover, VIGS of LeACIK1 in tomato decreased Cf-9–mediated resistance to C. fulvum, showing the importance of ACIK1 in disease resistance.