Dissecting cellular signaling in Phytophthora

Many oomycetes are economically important pathogens, causing enormous yield losses in crop plants. Others threaten natural vegetation, while some species can cause harmful diseases in animals. Oomycetes, also known as water molds, are morphologically similar to fungi and also occupy similar environmental niches, but during evolution the two groups evolved independently (Chapter l). They show many differences, in particular at the subcellular level, and this often has consequences for the efficacy of chemical control agents and hence, the efficient control of oomycete disesases. The research described in this thesis is aimed at enhancing our basic knowledge of plant pathogenic oomycetes in the genus Phytophthora and to gain more insight in their remarkable biology. It focusses on two processes, cellular signaling and cytoskeleton dynamics. Uncovering mechanisms that govern these processes may help in designing novel, oomycete-specific control strategies. Cellular signaling is crucial for every living organism. It controls important processes, allows for communication, and enables organisms to respond to environmental cues. Two important eukaryotic signal transduction pathways that are usually interconnected through intermediate signaling components, are G-protein signaling and phospholipid signaling. Oomycetes, however, possess a unique class of G-protein coupled receptors (GPCRs) that have a phosphatidylinositol kinase (PIPK) as an accessory domain pointing to a more direct connection between the two major signaling pathways. When first discov- ered, these so-called GPCR-PIPKs were thought to be restricted to oomycetes. In Chapter 2, we show the sporadic occurrence of these so-called GPCR-PIPKs in a diverse but limited group of unicellular microorganisms, divided over nearly all eukaryotic supergroups. Our analyses revealed that nearly all GPCR-PIPKs contain a unique, conserved motif located in between the GPCR domain and the PIPK domain. GPCR-PIPKs are likely ancestral to eukaryotes and significantly expanded in the last common ancestor of oomycetes. We further identified five hitherto unknown classes of GPCRs with accessory domains, GPCR- bigrams. All classes of GPCR-bigrams are shared by oomycetes, and except for three, some classes are sparsely present in organisms from other taxa. Most accessory domains of GPCR-bigrams are universal players in signal transduction. Our findings point to an an- cestral signaling system in eukaryotic microorganisms where GPCR-mediated sensing is directly linked to downstream responses. In classical G-protein signaling, a GPCR senses extracellular signals and changes confor- mation upon ligand binding, thereby activating the associated heterotrimeric G-protein complex, consisting of a G-protein α (Gα), β (Gβ), and γ (Gγ) subunit. In turn, the acti- vated G-protein complex dissociates from the receptor and its subunits stimulate down- stream effector proteins. In Chapter 3, we investigated the function of the Gγ subunit of Phytophthora infestans. The overall similarity of this Gγ subunit with non-oomycete Gγ subunits is low, but the similarity with its homologs in other oomycetes is high. The Gγ- encoding gene, Pigpg1, is expressed in all life stages and peaks in spores. To elucidate the function of the P. infestans Gγ subunit, we generated Pigpg1-silenced and overexpression transformants and analyzed their phenotypes. However, many transformed lines had severe growth defects and were not viable. The few that could be maintained produced less sporangia, that were malformed. These findings demonstrate that the Gγ subunit has an important role in P. infestans. It is crucial for proper sporangia development, and likely forms a dimer with the P. infestans Gβ subunit, thereby mediating signaling. The microtubule (MT) cytoskeleton is a system of intracellular filaments, that is able to quickly adapt different configurations. This process is regulated by microtubular dynam- ics and MT-associated proteins (MAPs). The MT cytoskeleton has a myriad of roles, for example in processes that provide structural rigidity to cells or allow for polarized cell growth and cell movement. Chapter 4 focuses on the MT cytoskeleton in Phytophthora. Live cell imaging of transgenic Phytophthora palmivora lines carrying an ectopically inte- grated GFP-α-tubulin fusion gene provided insight in the spatio-temporal organization of the MT cytoskeleton in Phytophthora. In addition, we provide an inventory of putative MT-associated proteins in P. infestans. Unique types of the motor proteins dynein and kinesin were found, including some members with accessory domains not found else- where in combination with a motor protein domain. This study provides a basis for future research on MTs and MAPs in Phytophthora and a first glimpse of the dynamics of the MT cytoskeleton in an oomycete. The low rate of homologous recombination in oomycetes makes that transgenes are integrated randomly and until recently genome editing was unattainable. The implemen- tation of a CRISPR/Cas9 system in Phytophthora sojae is a significant asset for the molecular toolbox of oomycetes. So far, genome editing using CRISPR/Cas9 has been successfully applied in only a few Pyhytophthora species. In Chapter 5 we explore the effectuation of CRISPR/Cas9 for targeted genome editing in P. infestans. With the original constructs that were developed for P. sojae, we did not obtain any transformants in which the target gene was mutagenized. In an effort to pinpoint the reason for failure, we tailored the constructs for P. infestans and implemented several modifications in the CRISPR/Cas9 system but without success. We also explored the delivery of pre-assembled ribonucleoprotein com- plexes. We describe an extensive effort in optimization of the system and outline possible causes for failure. In Chapter 6, the main results of this thesis are integrated and discussed. Remarkable features of oomycetes and their cellular signaling systems are outlined. Possible modes of action of GPCR-bigrams are proposed as well as future directions for research on cellular signaling in Phytophthora. More knowledge on the elementary processes addressed in this thesis will expose new strategies for the design of novel, oomycete-specific control agents to mitigate damage caused by these devastating pathogens.