Exploration of Cell Cycle-Specific Essential Gene Functions in the Microbial Plant Chlamydomonas Reinhardtii

The cell cycle encompasses all the steps required for cell proliferation, and is normally tightly coupled to growth and division in all organisms. Much research has resulted in a well-supported model of eukaryotic cell cycle control. However, since most of this research has been carried out in yeast and animals (opisthokonts), it could in principle apply poorly to early-diverging groups of organisms, such as the green plants. Plant cell cycle research has largely followed a candidate strategy based on reverse genetics. These studies have a provided insights into plant cell cycle control, but are generally dependent upon sequence conservation between plant and opisthokont genes. This thesis presents work from an ongoing screen to identify critical components of the plant cell cycle by forward genetic methods that are independent of prior knowledge of specific mechanisms of cell cycle control. The screen was carried out in the unicellular green alga Chlamydomonas, a microbial member of the Viridiplantae, which has wellestablished experimental Mendelian genetics, and many features that might facilitate identification of loss-of-function mutations. We have developed semi-automatic techniques for isolation of temperature-sensitive lethal mutants that are capable of cell growth at a near-wild-type rate, but that exhibit first-cycle failure of cell division (div phenotype). We developed efficient methods for identification of causative mutations by next-generation sequencing of bulked segregant pools. The normal cell division cycle in Chlamydomonas is characterized by a long period of G1 growth, followed by a series of rapidly alternating rounds of S phases and mitoses (S/M phase). Analysis of more than 50 div mutants identified two main phenotypic classes. One class showed somewhat reduced growth and arrested in a G1-like state. This class included genes with diverse molecular functions based on gene annotations, including transcription, translation, and membrane biogenesis. The other class exhibited wild-type cell growth rate, and entered the S/M program on time; mutant cells then developed various S/M-specific defects. This class included genes directly involved in DNA replication and chromosome segregation. Other mutations identified genes likely involved in cell cycle control, including the cyclin-dependent kinases CDKA and CDKB, two anaphase-promoting complex subunits, and the mitotic kinases Aurora B and MPS1. The phenotype of the cdka-1 mutant suggested a specific role for CDKA in the transition from cell growth to initiation of the S/M cell division program. CDKB, in contrast, functions specifically after DNA replication, in entry into the first mitosis. Although most DIV genes had clear homologues involved in cell cycle progression in opisthokonts, some genes had clear homologues in Viridiplantae but not in opisthokonts, including the BSL1 phosphatase, which we demonstrate to have a role in mitotic entry similar to that of CDKB. The div mutants isolated in this screen provide an opportunity to study the plant cell cycle in a simple microbial setting. Since a large majority of the mutants alter genes with clear Arabidopsis sequelogues, the results also suggest targeted candidates for cell cycle experiments in Angiosperms

A pathogen effector co-opts a host RabGAP protein to remodel pathogen interface and subvert defense-related secretion

Pathogens have evolved sophisticated mechanisms to manipulate host cell membrane dynamics, a crucial adaptation to survive in hostile environments shaped by innate immune responses. Plant-derived membrane interfaces, engulfing invasive hyphal projections of fungal and oomycete pathogens, are prominent junctures dictating infection outcomes. Understanding how pathogens transform these host-pathogen interfaces to their advantage remains a key biological question. Here, we identified a conserved effector, secreted by plant pathogenic oomycetes, that co-opts a host Rab GTPase-activating protein (RabGAP), TOPGAP, to remodel the host-pathogen interface. The effector, PiE354, hijacks TOPGAP as a susceptibility factor to usurp its GAP activity on Rab8a, a key Rab GTPase crucial for defense-related secretion. By hijacking TOPGAP, PiE354 purges Rab8a from the plasma membrane, diverting Rab8a-mediated immune trafficking away from the pathogen interface. This mechanism signifies an uncanny evolutionary adaptation of a pathogen effector in co-opting a host regulatory component to subvert defense-related secretion, thereby providing unprecedented mechanistic insights into the reprogramming of host membrane dynamics by pathogens

Time-resolved dual transcriptomics reveal early induced Nicotiana benthamiana root genes and conserved infection-promoting Phytophthora palmivora effectors

BACKGROUND: Plant-pathogenic oomycetes are responsible for economically important losses in crops worldwide. Phytophthora palmivora, a tropical relative of the potato late blight pathogen, causes rotting diseases in many tropical crops including papaya, cocoa, oil palm, black pepper, rubber, coconut, durian, mango, cassava and citrus. Transcriptomics have helped to identify repertoires of host-translocated microbial effector proteins which counteract defenses and reprogram the host in support of infection. As such, these studies have helped in understanding how pathogens cause diseases. Despite the importance of P. palmivora diseases, genetic resources to allow for disease resistance breeding and identification of microbial effectors are scarce. RESULTS: We employed the model plant Nicotiana benthamiana to study the P. palmivora root infections at the cellular and molecular levels. Time-resolved dual transcriptomics revealed different pathogen and host transcriptome dynamics. De novo assembly of P. palmivora transcriptome and semi-automated prediction and annotation of the secretome enabled robust identification of conserved infection-promoting effectors. We show that one of them, REX3, suppresses plant secretion processes. In a survey for early transcriptionally activated plant genes we identified a N. benthamiana gene specifically induced at infected root tips that encodes a peptide with danger-associated molecular features. CONCLUSIONS: These results constitute a major advance in our understanding of P. palmivora diseases and establish extensive resources for P. palmivora pathogenomics, effector-aided resistance breeding and the generation of induced resistance to Phytophthora root infections. Furthermore, our approach to find infection-relevant secreted genes is transferable to other pathogen-host interactions and not restricted to plants.This work was supported by the Gatsby Charitable Foundation (RG62472), by the Royal Society (RG69135) and by the European Research Council (ERC-2014-STG, H2020, 637537)

Patching Holes in the Chlamydomonas Genome

The Chlamydomonas genome has been sequenced, assembled, and annotated to produce a rich resource for genetics and molecular biology in this well-studied model organism. However, the current reference genome contains ∼1000 blocks of unknown sequence (‘N-islands’), which are frequently placed in introns of annotated gene models. We developed a strategy to search for previously unknown exons hidden within such blocks, and determine the sequence, and exon/intron boundaries, of such exons. These methods are based on assembly and alignment of short cDNA and genomic DNA reads, completely independent of prior reference assembly or annotation. Our evidence indicates that a substantial proportion of the annotated intronic N-islands contain hidden exons. For most of these, our algorithm recovers full exonic sequence with associated splice junctions and exon-adjacent intronic sequence. These new exons represent de novo sequence generally present nowhere in the assembled genome, and the added sequence improves evolutionary conservation of the predicted encoded peptides