Phytophthora infestans enzymes and their role in the interaction with its hosts

Pathogens use a huge arsenal of proteins and metabolites in order to infect plants and establish disease. They function in facilitating pathogen entry or suppressing plant defence triggered upon pathogen attack. The proteins that pathogens employ to infect the host include enzymes such as cell wall degrading enzymes, phospholipid modifying enzymes and proteases, but also effectors that target and disable the host cell machinery. To facilitate disease these proteins function at several levels. First of all inside the pathogen, for example in processing or maturation of pathogenicity or virulence factors. Secondly in the apoplast, for example in cell wall degradation or receptor modification. And lastly, inside the host cell where they can have a role in a variety of processes such as disrupting vesicle transport, inhibiting transcription factors or deregulating the hormone balance. The oomycete Phytophthora infestans is the causal agent of late blight, the most important disease in potato crops worldwide. Since its appearance in Europe in the mid-nineteenth century, when it destroyed the potato crop all over Western Europe and gave rise to the Irish potato famine, this pathogen has been studied extensively. Since the early 1990’s, when the first molecular tools became available to study oomycete plant pathogens, attempts have been made to identify genes in P. infestans that play a role in pathogenicity or virulence and to unravel in depth the molecular and cellular mechanisms underlying pathogenicity. Despite rapid progress and the information provided by Phytophthora genome sequencing projects, these mechanisms are still poorly understood. The research described in this thesis deals with three different groups of enzymes in P. infestans that are anticipated to have a role in host-pathogen interactions and have not been studied previously. In Chapter 1 we first introduce potato and potato late blight and give a glimpse of the history of the disease and its societal impact. We then describe the life cycle of P. infestans and the molecular toolbox that is currently available to study this pathogen and the interaction with its host. Subsequently, we address the current practices of late blight control with emphasis on resistance genes and the ability of the pathogen to rapidly escape recognition. This is followed by an overview of enzymes and enzyme inhibitors that have been shown to play a role in virulence in a variety of pathogens and finally we present the scope of this thesis. In Chapter 2 we present an inventory of metalloproteases (MPs) in P. infestans. MPs are a very diverse group of proteases that function by virtue of a divalent metal cation positioned at their catalytic site. Several MPs have been shown to be involved in the pathogenicity of mammalian and plant pathogens. Thorough genome mining and gene model corrections based on RNA-Seq data, revealed 99 MPs, divided over 20 families. Searches for homologs in other oomycetes and Stramenopiles species showed that some MPs are expanded in Phytophthora species. Analyses of the domain compositions of MPs revealed a few MPs with a novel domain architecture exclusively found in Phytophthora species. Gene expression analyses showed that several MPs are highly expressed in infectious propagules. To our knowledge, this is the first systematic inventory of MPs in an oomycete. Based on these results MP genes can be selected as candidates for future functional studies in P. infestans and other oomycetes. Chapters 3 and 4 deal with aspartic proteases (APs). In other pathogens APs were found to be involved in effector modification and virulence. The Plasmodium falciparum AP PlasmepsinV (PMV) has been shown to modify PEXEL effectors of Plasmodium prior to their translocation into the host cell. Of the twelve P. infestans APs three are highly homologous to PMV Due to this homology as well as the homology between the host translocation motifs in Plasmodium PEXEL effectors and Phytophthora RXLR effectors, it has been suggested that these three APs, PiAP10, PiAP11 and PiAP12, play a similar role in effector modification as PMV. In order to test their involvement in virulence we generated P. infestans transformants in which the PiAPs genes are silenced or overexpressed (Chapter 3). PiAP11 transformants showed no obvious changes in virulence but transformants silenced for PiAP10 and PiAP12, showed reduced infection efficiency and smaller lesions upon inoculation on potato leaves. Overexpression of these two genes also resulted in transformants with compromised virulence, while in several cases, they triggered cell death upon inoculation. A small reduction in colony growth and sporulation of the transformants was also observed. In-gel zymography assays with gelatin showed that all PiAPs have enzymatic activity that was inhibited by pepstatin. Moreover, PiAP10 and PiAP12 showed proteolytic activity against the RXLR effector AVR4 with its authentic RXLR motif, but not against AVR4 with a mutated RXLR motif showing that the modification only occurs when the RXLR motif is intact. PiAP11 had no proteolytic activity against AVR4 effector, suggesting a different function for the particular PiAP. These results show that PiAP10 and PiAP12 are involved in virulence and suggest their involvement in effector modification. One of the P. infestans APs named PiAP5, is an AP with a unique domain architecture. It has a G-protein coupled receptor (GPCR) domain adjacent to the AP domain and its topology suggests that PiAP5, with its seven transmembrane spanning regions, is integrated in the membrane with the N-terminal AP domain as extracellular domain. Such an AP-GPCR is exclusively found in oomycetes. In Chapter 4 we describe that alteration of PiAP5 expression in P. infestans strongly reduced growth and sporulation and resulted in malformed germinating sporangia, especially in P. infestans transformants overexpressing the gene. Protease activity assays with general substrates and AVR4 did not show any activity of PiAP5 as a protease. These results show that PiAP5 is involved in fitness and sporulation of the pathogen, and consequently affects its virulence. Whether or not PiAP5 is active as a protease is unknown. Chapter 5 focuses on the function of a subclass of phospholipase D’s (PLDs). PLDs are enzymes that are involved in the hydrolysis of phospholipids, the main structural components of cell membranes, and in the production of the second messenger phosphatidic acid (PA). Three small PLD genes were selected for functional analysis by means of transient expression in Nicotiana benthamiana leaves The presence of PLD-like-1, sPLD-like-1 or sPLD-like-12 in the leaves gave rise to calcium-dependent cell death and, when the leaves were inoculated with P. infestans it enhanced lesion growth. Mutations in the catalytic HKD motifs of the PLDs or removal of the signal peptide strongly reduced the cell death responses and abolished the virulence promotion demonstrating that the enzymatic activity of the PLDs is the major determinant and that the PLDs likely function outside the cell. These results show that that PLD-likes play a role in virulence, either by modifying the host membranes or through PA signalling. To gain more insight in the detailed mechanisms underlying Phytophthora-plant interactions, it is desirable to have experimental systems that enable high quality data generation. The establishment of model systems that fit that purpose, yet resembling the natural infection process are necessary. In Chapter 6 we describe the development of a new infection system using the tomato cell line MsK8 as host for Phytophthora pathogens. The infection system was optimized by studying the interaction of MsK8 cells with several Phytophthora species over time. The experiments included infection assays, microscopy, gene expression profiling and ROS production measurements. The results show that the MsK8 infection system offers a versatile platform that can be used in studies ranging from analysing a single gene, testing chemical compounds, to large -omics studies. Chapter 7 addresses the main findings of this thesis, and puts them into a broader perspective. The function and potential involvement of MPs, APs and PLDs in fitness or virulence of P. infestans are discussed, as well as the possible effects of the enzymatic activity in relation to virulence. Overall, this thesis highlights the importance of enzymes in growth and virulence of P. infestans and gives insights in three different types of enzymes. The potential roles of these enzymes in Phytophthora-host interactions could serve as food for thought for further studies.