The amino acid transporter UmamiT20 confers susceptibility.

• Induction of SWEET sugar transporters by bacterial pathogens via transcription activator-like (TAL) effectors is necessary for successful blight infection of rice, cassava and cotton, - likely providing sugars for bacterial propagation. • Here, we show that infection of by the necrotrophic fungus causes increased accumulation of amino acid transporter UmamiT20 mRNA in leaves. UmamiT20 protein accumulates in leaf veins surrounding the lesions after infection.

Proteomic analysis of the pyrenoid-traversing membranes of reveals novel components.

Pyrenoids are algal CO-fixing organelles that mediate approximately one-third of global carbon fixation and hold the potential to enhance crop growth if engineered into land plants. Most pyrenoids are traversed by membranes that are thought to supply them with concentrated CO. Despite the critical nature of these membranes for pyrenoid function, they are poorly understood, with few protein components known in any species.

SAGA1 and MITH1 produce matrix-traversing membranes in the CO-fixing pyrenoid.

Approximately one-third of global CO assimilation is performed by the pyrenoid, a liquid-like organelle found in most algae and some plants. Specialized pyrenoid-traversing membranes are hypothesized to drive CO assimilation in the pyrenoid by delivering concentrated CO, but how these membranes are made to traverse the pyrenoid matrix remains unknown. Here we show that proteins SAGA1 and MITH1 cause membranes to traverse the pyrenoid matrix in the model alga Chlamydomonas reinhardtii.

Biogenesis, engineering and function of membranes in the CO -fixing pyrenoid.

Approximately one-third of global CO assimilation is performed by the pyrenoid , a liquid-like organelle found in most algae and some plants . Specialized membranes are hypothesized to drive CO assimilation in the pyrenoid by delivering concentrated CO , but their biogenesis and function have not been experimentally characterized. Here, we show that homologous proteins SAGA1 and MITH1 mediate the biogenesis of the pyrenoid membrane tubules in the model alga and are sufficient to reconstitute pyrenoid-traversing membranes in a heterologous system, the plant .

Multi-omics analysis of green lineage osmotic stress pathways unveils crucial roles of different cellular compartments.

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages.

Chloroplast Methyltransferase Homolog RMT2 is Involved in Photosystem I Biogenesis.

Oxygen (O), a dominant element in the atmosphere and essential for most life on Earth, is produced by the photosynthetic oxidation of water. However, metabolic activity can cause accumulation of reactive O species (ROS) and severe cell damage. To identify and characterize mechanisms enabling cells to cope with ROS, we performed a high-throughput O sensitivity screen on a genome-wide insertional mutant library of the unicellular alga .

Chloroplast phosphate transporter CrPHT4-7 regulates phosphate homeostasis and photosynthesis in Chlamydomonas.

In eukaryotic cells, phosphorus is assimilated and utilized primarily as phosphate (Pi). Pi homeostasis is mediated by transporters that have not yet been adequately characterized in green algae. This study reports on PHOSPHATE TRANSPORTER 4-7 (CrPHT4-7) from Chlamydomonas reinhardtii, a member of the PHT4 transporter family, which exhibits remarkable similarity to AtPHT4;4 from Arabidopsis (Arabidopsis thaliana), a chloroplastic ascorbate transporter.

A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways.

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii.

Effects of linker length on phase separation: lessons from the Rubisco-EPYC1 system of the algal pyrenoid.

Biomolecular condensates are membraneless organelles formed via phase separation of macromolecules, typically consisting of bond-forming "stickers" connected by flexible "linkers". Linkers have diverse roles, such as occupying space and facilitating interactions. To understand how linker length relative to other lengths affects condensation, we focus on the pyrenoid, which enhances photosynthesis in green algae.

The pyrenoid: the eukaryotic CO2-concentrating organelle.

The pyrenoid is a phase-separated organelle that enhances photosynthetic carbon assimilation in most eukaryotic algae and the land plant hornwort lineage. Pyrenoids mediate approximately one-third of global CO2 fixation, and engineering a pyrenoid into C3 crops is predicted to boost CO2 uptake and increase yields. Pyrenoids enhance the activity of the CO2-fixing enzyme Rubisco by supplying it with concentrated CO2.

Phase-separating pyrenoid proteins form complexes in the dilute phase.

While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1.

Modelling the pyrenoid-based CO-concentrating mechanism provides insights into its operating principles and a roadmap for its engineering into crops.

Many eukaryotic photosynthetic organisms enhance their carbon uptake by supplying concentrated CO to the CO-fixing enzyme Rubisco in an organelle called the pyrenoid. Ongoing efforts seek to engineer this pyrenoid-based CO-concentrating mechanism (PCCM) into crops to increase yields. Here we develop a computational model for a PCCM on the basis of the postulated mechanism in the green alga Chlamydomonas reinhardtii.

Systematic characterization of gene function in the photosynthetic alga Chlamydomonas reinhardtii.

Most genes in photosynthetic organisms remain functionally uncharacterized. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. A total of 59% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes.

Coexpressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts.

In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas () to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled.

The structural basis of Rubisco phase separation in the pyrenoid.

Approximately one-third of global CO fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii.

Assembly of the algal CO-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif.

Approximately one-third of the Earth's photosynthetic CO assimilation occurs in a pyrenoid, an organelle containing the CO-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid and how the organelle's subcompartments-membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath-are held together is unknown. Using the model alga , we found that pyrenoid proteins share a sequence motif.

The pyrenoid.

Wang and Jonikas take a look at an unconventional organelle, the pyrenoid.

Rigidity enhances a magic-number effect in polymer phase separation.

Cells possess non-membrane-bound bodies, many of which are now understood as phase-separated condensates. One class of such condensates is composed of two polymer species, where each consists of repeated binding sites that interact in a one-to-one fashion with the binding sites of the other polymer. Biologically-motivated modeling revealed that phase separation is suppressed by a "magic-number effect" which occurs if the two polymers can form fully-bonded small oligomers by virtue of the number of binding sites in one polymer being an integer multiple of the number of binding sites of the other.

Prospects for Engineering Biophysical CO Concentrating Mechanisms into Land Plants to Enhance Yields.

Although cyanobacteria and algae represent a small fraction of the biomass of all primary producers, their photosynthetic activity accounts for roughly half of the daily CO fixation that occurs on Earth. These microorganisms are able to accomplish this feat by enhancing the activity of the CO-fixing enzyme Rubisco using biophysical CO concentrating mechanisms (CCMs). Biophysical CCMs operate by concentrating bicarbonate and converting it into CO in a compartment that houses Rubisco (in contrast with other CCMs that concentrate CO via an organic intermediate, such as malate in the case of C CCMs).

The Mars1 kinase confers photoprotection through signaling in the chloroplast unfolded protein response.

In response to proteotoxic stress, chloroplasts communicate with the nuclear gene expression system through a chloroplast unfolded protein response (cpUPR). We isolated mutants that disrupt cpUPR signaling and identified a gene encoding a previously uncharacterized cytoplasmic protein kinase, termed Mars1-for utant ffected in chloroplast-to-nucleus etrograde ignaling-as the first known component in cpUPR signal transmission. Lack of cpUPR induction in mutant cells impaired their ability to cope with chloroplast stress, including exposure to excessive light.

A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology in .

A phase-separated, liquid-like organelle called the pyrenoid mediates CO fixation in the chloroplasts of nearly all eukaryotic algae. While most algae have 1 pyrenoid per chloroplast, here we describe a mutant in the model alga that has on average 10 pyrenoids per chloroplast. Characterization of the mutant leads us to propose a model where multiple pyrenoids are favored by an increase in the surface area of the starch sheath that surrounds and binds to the liquid-like pyrenoid matrix.

A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis.

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii.

Effects of microcompartmentation on flux distribution and metabolic pools in chloroplasts.

Cells and organelles are not homogeneous but include microcompartments that alter the spatiotemporal characteristics of cellular processes. The effects of microcompartmentation on metabolic pathways are however difficult to study experimentally. The pyrenoid is a microcompartment that is essential for a carbon concentrating mechanism (CCM) that improves the photosynthetic performance of eukaryotic algae.

The Eukaryotic CO-Concentrating Organelle Is Liquid-like and Exhibits Dynamic Reorganization.

Approximately 30%-40% of global CO fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division.