The role of heterotrimeric G-proteins in development and virulence of Phytophthora infestans

Ever since the 1840s, when the disease first appeared in Europe and the United States, the threat of new outbreaks of late blight has terrified potato growers. In years when cool and wet weather conditions are prevailing, the disease is most destructive and nearly impossible to control. Decades of breeding efforts failed to yield resistant cultivars able to satisfy potato producers and consumers. In addition, many of the present-day fungicides are either ineffective or hazardous to human health or the natural environment. Phytophthora infestans , the organism causing late blight, is an oomycete. The class oomycetes encompasses eukaryotic pathogens and saprophytes with morphological similarities to fungi. Since fungi and oomycetes are evolutionary unrelated organisms, the ability to infect plants also evolved independently in both classes of organisms.Heterotrimeric G-proteins are ubiquitously occurring signaling components that function in the translation of extracellular cues into intracellular signals and are indispensable for cells to adapt to alterations in environmental conditions. Animals, plants, fungi and lower eukaryotes such as the slime molds, all possess heterotrimeric G-proteins. In fungal plant pathogens, heterotrimeric G-proteins have been shown to control developmental processes such as mating, sporulation and formation of infection structures, including appressoria. Moreover, the virulence of plant pathogenic fungal species, in which genes encoding G-protein subunits were disrupted, was often reduced. To gain more insight in the regulation of development and virulence in P. infestans , we aimed at elucidating the role of conserved signaling pathways in this pathogen. We set off by studying the role of heterotrimeric G-proteins and the results are described in this thesis.Heterotrimeric G-proteins consist of three subunits (a,bandg) of which theaandbsubunits are highly conserved. This high level of conservation was utilized to isolate P. infestans genes encoding these two subunits ( chapter 2 ). Degenerate primers corresponding to conserved regions in Gawere used in a PCR resulting in the amplification of a fragment of a Phytophthora Gasubunit gene. The gene was named Pigpa1 . A Gbgene, Pigpb1 , was isolated based on sequence information from a P. infestans mycelium EST database with high homology to Gbgenes from other organisms. Pigpa1 and Pigpb1 expression was developmentally regulated. Both genes were transcribed at the highest level in sporangia, the asexual spores of P. infestans . Both genes displayed lower levels of expression in zoospores and cysts. In mycelium, Pigpb1 was only lowly expressed while now expression of Pigpa1 could be detected.Introduction of extra copies of Pigpa1 in the P. infestans genome triggered gene-silencing in a subset of the transformants, resulting in mutants that no longer transcribed the Pigpa1 gene and failed to synthesize the PiGPA1 protein ( chapter 3 ). Sporangial cytoplasmic cleavage and zoospore release was less efficient in these Pigpa1 -silenced mutants compared with the wildtype strain. Wildtype zoospores generally swim in straight lines and form aggregates at the liquid surface. The zoospores of the Pigpa1 -silenced mutants turned much more frequently than wildtype and showed no preference to move to the liquid surface. Neither did they aggregate and chemotaxis towards amino acids was absent in mutant zoospores. This indicates that PiGPA1 is involved in the regulation of flagellar movements, perception of chemotactic compounds, or both. The Pigpa1 -silenced mutants were severely reduced in virulence. In contrast, PiGPA1 gain-of-function mutants expressing a constitutively active form of PiGPA1, showed increased virulence in three out of four of these mutants. These observations point to a role for PiGPA1 in virulence of P. infestans .Pigpb1 -silenced mutants were generated using a similar procedure as used for obtaining Pigpa1 -silenced mutants ( chapter 4 ). Two transformation methods were used to introduce copies of the Pigpb1 gene. The traditional PEG-mediated protoplast transformation resulted in a higher frequency of gene silencing than transformation by electroporation of zoospores. Pigpb1 -silenced mutants formed only very few sporangia when cultured on rye sucrose agar. Sporulation in the completely Pigpb1 -silenced mutants was even below 1% of wildtype sporulation. Instead, the mutants produced a denser mat of aerial mycelium than wildtype on the same growth medium. Pigpb1 expression in mycelium was induced under starvation conditions. PiGPB1 seems to represent an important link between starvation and sporulation.Activation of G-proteins triggers a signaling cascade, which eventually results in changes in expression of particular genes. In chapter 5, experiments are described that aimed at finding genes, the expression of which is under control of the G subunit PiGPA1. Gene expression in sporangia of the wildtype strain, one of the PiGPA1 gain-of-function mutants and three Pigpa1 -silenced mutants was profiled using cDNA amplified fragment length polymorphism (cDNA-AFLP) analysis. Gene expression in wildtype mycelium was used as an internal control. Seventy-seven transcript-derived fragments (TDFs) were generated that were present in the wildtype profile and absent in the profile of the Pigpa1 -silenced mutants. Vice versa , 11 TDFs were present in the Pigpa1 -silenced mutants but absent in the wildtype strain. These two groups of TDFs are highly interesting as they are potentially derived from genes the expression of which is normally up- or downregulated by PiGPA1, respectively. The expression patterns of a subset of the genes from which those TDFs are derived were confirmed by RT-PCR and northern blot analysis. Twenty-seven of the differentially expressed TDFs were cloned and sequenced. A subset of these 27 clones showed homology with sequences present in P. infestans EST databases. Further analysis of these genes will reveal their possible functions in signaling and virulence.Chapter 6 describes the first steps in the characterization of phospholipase D (PLD) signaling in P. infestans . PLD hydrolyzes phospholipids that are abundant in membranes, such as phosphatidylcholine or phosphatidylserine. The products are phosphatidic acid (PA) and the respective headgroup. PLD and PA are involved in vesicle trafficking in animals and possibly also in plants. PLD is activated by a variety of stress conditions in plants and conversion of PA to diacylglycerolpyrophosphate (DGPP) is often observed, presumably as a means to quench the PA signal. Treatment of P. infestans sporangia, zoospores, cysts and mycelium with the G-protein-activating peptide mastoparan resulted in increases in the levels of both PA and DGPP. This can be due to activation by mastoparan of the phospholipases PLD, PLC, PLC, or both. If available, PLD favors alcohols over water as the 'acceptor' of the phosphatidyl moiety, producing phosphatidylalcohol instead of PA. This feature was exploited to monitor PLD activity in P. infestans . Mastoparan treatment in the presence of butanol resulted in an increase in the level of phosphatidylbutanol, evidencing activation of PLD by mastoparan. Similarly, treatment with primary and secondary alcohols also activated PLD. Furthermore, it was demonstrated that mastoparan did not activate PLC in sporangia of P. infestans . PLD was activated during zoospore encystment. All PLD activators tested, as well as the product of PLD, PA, induced encystment. Taken together, we consider it likely that PLD plays a role in encystment.Chapter 7 is a treatise on differences and similarities between strategies of fungal and oomycete pathogens to colonize plants. These groups are taxonomically unrelated. The comparison of the infection strategies of these two groups based on published data led us to conclude that they are more similar than anticipated. There are clear resemblances in infection structures, virulence factors and signal transduction pathways that govern development and virulence. Of course, there are also differences and we mention in this chapter the presence of certain classes of virulence factors that are described in fungi but have not (yet) been detected in oomycetes. We concluded that convergent evolution has had a large impact on the development of infection of plants in the two groups.