The interplay between a Phytophthora RXLR effector and an Arabidopsis lectin receptor kinase

Phytophthora infestans – the causal agent of potato late blight – secretes a plethora of effector proteins to facilitate plant infection. The central subject of this thesis is ipiO, one of the first cloned Phytophthora genes with a putative function in pathogenicity as was anticipated based on its in planta induced (ipi) expression, in particular during early stages of host infection. IPI-O contains two striking motifs: RXLR-dEER and RGD. RGD is a cell adhesion motif and was shown to be involved in binding to the extracellular lectin domain of LecRK-I.9, a lectin receptor kinase of Arabidopsis. The RXLR-dEER motif plays a role in effector trafficking into host cells and is shared by several secreted oomycete effector proteins which are known to function as race-specific avirulence (Avr) factors. In a previous study, that was aimed at identifying novel pairs of P. infestans Avr and host plant resistance (R) genes, a high-throughput effector genomics screen identified ipiO as Avr-blb1, the counterpart of the late blight R gene Rpi-blb1 which originates from the nightshade Solanum bulbocastanum. Often R genes exploited in late blight resistance breeding become rapidly ineffective as a result of adaptation of P. infestans. However, unlike most late blight R genes that interact in a gene-for-gene manner with Avr genes, Rpi-blb1 seemed to have the potential to remain its effectiveness. In section 2 we monitored the genetic variation and distribution of the ipiO family in an extensive isolate collection of P. infestans and closely related species. This resulted in the identification of 16 IPI-O variants that could be sub-divided in three distinct classes. Variants from class I and class II were shown to induce cell death when co-infiltrated with Rpi-blb1 in Nicotiana benthamiana. Class III consists solely of the highly divergent variant IPI-O4, that is not able to trigger Rpi-blb1-mediated cell death. Class I is highly diverse and represented in all P. infestans isolates analyzed so far, except in two Mexican P. infestans isolates. The latter two are capable to infect Rpi-blb1 plants, suggesting that the lack of class I variants in the genome of these strains allows them to escape recognition by Rpi-blb1 plants. We propose that profiling of the ipiO variants within P. infestans populations can predict the effectiveness of Rpi-blb1-mediated resistance in potato and, as such, can facilitate integrated disease management. Section 3 of this thesis deals with legume-like lectin receptor kinases (LecRKs), membrane-spanning proteins with potential roles in adaptive responses and cell wall integrity. We present an inventory and a phylogenetic analysis of the Arabidopsis LecRK gene family. The rationale behind this study was to gain better insight into the diversity of LecRKs and their potential roles in plant defense. A comprehensive expression analysis based on exploration of existing databases revealed that several LecRK genes are induced upon treatment with elicitors or during pathogen infection. Based on the phylogenetic analysis we have reclassified the LecRK genes and proposed a new nomenclature. LecRK-I.9, one of the clade I Arabidopsis LecRKs which binds the RGD cell adhesion motif of IPI-O, was shown to mediate adhesion between the cell wall (CW) and plasma membrane (PM). In contrast, IPI-O disrupts these adhesions by virtue of its RGD motif. We analyzed Arabidopsis LecRK-I.9 knock-out lines (lecrk-I.9) for their response to pathogen infection, in particular to Phytophthora brassicae. We also analyzed transgenic Arabidopsis lines expressing ipiO, and observed that both the ipiO-expressing lines and lecrk-I.9 lines are impaired in their resistance to oomycete pathogens. To unravel the mechanisms underlying this phenomenon we analysed callose deposition upon MAMP (i.e. flg22) treatment and investigated the strength of CW-PM adhesions under plasmolysis-inducing conditions. The results indicated that LecRK-I.9 is not only important for the maintenance of the CW-PM continuum, but also in MAMP-triggered immunity. Also here, both the ipiO-expressing lines and the lecrk-I.9 knock-outs displayed a destabilized CW-PM continuum and impaired callose deposition, and hence, they can be regarded as phenocopies. Arabidopsis plants that constitutively express LecRK-I.9 were smaller in size, and displayed increased levels of anthocyanin and lignin. Additionally, these lines were shown to exhibit enhanced resistance to P. brassicae. Furthermore, we studied transgenic potatoes that constitutively Arabidopsis LecRK-I.9. In comparison to the parental control potato line the transgenic lines were less susceptible to mild and moderately aggressive P. infestans isolates, but the increased tolerance was not sufficient to provide resistance to aggressive isolates. These results strongly suggest that LecRK-I.9 is a novel resistance component that plays a role in defense against Phytophthora. In Section 4 we describe a novel method for propagating P. brassicae zoospores on an intermediate host plant. This resulted in the production of high numbers of zoospores thereby facilitating highly reproducible small and large scale inoculation experiments. This thesis is completed with a general discussion (Section 5) addressing the current understanding of effector uptake by host cells, the subsequent recognition by cognate R proteins mediating effector-triggered immunity, and RXLR-dEER effector diversity. We also discuss the role of the RGD motif in effectors of both animal and plant pathogens, and the potential functions of LecRKs. Finally, we high-light the advantages of Arabidopsis-Phytophthora pathosystems as research object.