Understanding the role of L-type lectin receptor kinases in Phytophthora resistance

<strong>Abstract</strong> <em>Phytophthora</em> pathogens are notorious for causing severe damage to many agriculturally and ornamentally important plants. Effective plant resistance depends largely on the capacity to perceive pathogens and to activate rapid defence. Cytoplasmic resistance (R) proteins are well-known for activation of plant immunity upon recognition of matching effectors secreted by <em>Phytophthora</em>. However, <em>Phytophthora</em> pathogens are notoriously difficult to control due to their rapid adaptation to evade R protein-mediated recognition. Hence, exploring novel resistance components is instrumental for developing durable resistance. Receptor-like kinases (RLKs) function as important sentinels in sensing exogenous and endogenous stimuli to initiate plant defence. One RLK that was previously identified as a novel <em>Phytophthora</em> resistance component is the Arabidopsis L-type lectin receptor kinase LecRK-I.9. This RLK belongs to a multigene family consisting of 45 members in Arabidopsis but whether or not the other members function in <em>Phytophthora</em> resistance was thus far unknown. The research described in <strong>this thesis</strong> was aimed at unravelling the role of LecRKs in plant immunity, in particular to <em>Phytophthora</em> pathogens. <strong>Chapter I</strong> describes various <em>Phytophthora</em> diseases and the current understanding of the mechanisms underlying plant innate immunity with emphasis on disease resistance to <em>Phytophthora</em> pathogens. In <strong>Chapter II</strong>, we describe the development of a new Arabidopsis-<em>Phytophthora</em> pathosystem. We demonstrated that <em>Phytophthora</em> <em>capsici </em>is capable to infect Arabidopsis. Inoculation assays and cytological analysis revealed variations among Arabidopsis accessions in response to different <em>P. capsici</em> isolates. Moreover, infection assays on Arabidopsis mutants with specific defects in defence showed that salicylic acid signaling, camalexin and <em>indole glucosinolates</em> biosynthesis pathways are required for <em>P. capsici</em> resistance (<strong>Chapter IIa</strong>)<em>.</em><em> The importance of these pathways in Arabidopsis resistance was supported by the finding that the corresponding marker genes are induced upon infection by </em><em>P. capsici </em><em>(</em><strong>Chapter IIb</strong><em>). This model pathosystem can be used as an additional tool to pinpoint essential components of </em><em>Phytophthora</em><em> resistance.</em> <em>We then exploited Arabidopsis-</em><em>Phytophthora</em><em> pathosystems to uncover the role of LecRKs in </em><em>Phytophthora</em><em> resistance. In <strong>Chapter III</strong></em> <em>we describe a systematic phenotypic characterization of a large set of Arabidopsis </em><em>LecRK </em><em>T-DNA insertion lines. The T-DNA insertion lines were assembled and assayed for their response towards different </em><em>Phytophthora</em><em> pathogens. This revealed that next to </em><em>LecRK-I.9</em><em>, several other </em><em>LecRK</em><em>s function in </em><em>Phytophthora</em><em> resistance. We have also analysed whether the </em><em>LecRK</em><em>s are involved in response to other biotic and abiotic stimuli. Several T-DNA insertion lines showed altered responses to</em> <em>bacterial or fungal pathogens, but none of the lines showed visible developmental changes under normal conditions or upon abiotic stress treatment. Combining these phenotypic data with </em><em>LecRK</em><em> expression profiles obtained from publicly available datasets revealed that </em><em>LecRK</em><em>s that are hardly induced or even suppressed upon infection, might still have a function in pathogen resistance. Computed co-expression analysis revealed that </em><em>LecRK</em><em>s with similar function display diverse expression patterns.</em> <em>Arabidopsis LecRK clade IX comprises two members. T-DNA insertion mutants of both </em><em>LecRK-IX.1</em><em> and </em><em>LecRK-IX.2</em><em> showed gain of susceptibility to non-adapted </em><em>Phytophthora</em><em> isolates and therefore the role of these two </em><em>LecRK</em><em>s in </em><em>Phytophthora </em><em>resistance was further investigated. In <strong>Chapter IV </strong>we describe that overexpression of either </em><em>LecRK-IX.1</em><em> or </em><em>LecRK-IX.2</em><em> in Arabidopsis resulted in increased resistance to </em><em>Phytophthora</em><em>, but also induced plant cell death. A mutation in the kinase domain abolished the ability of LecRK-IX.1 and LecRK-IX.2 to induce </em><em>Phytophthora</em><em> resistance as did deletion of the lectin domain. Cell death induction however, only required the kinase, not the lectin domain. Since transient expression of both </em><em>LecRK</em><em>s in </em><em>Nicotiana benthamiana </em><em>also resulted in increased </em><em>Phytophthora </em><em>resistance and induction of cell death, we used </em><em>N. benthamiana</em><em> to explore downstream components required for LecRK-IX.1- and LecRK-IX.2-mediated </em><em>Phytophthora</em><em> resistance and cell death. Virus-induced gene silencing of candidate signaling genes revealed that </em><em>NbSIPK1/NPT4</em><em> is essential for LecRK-IX.1-mediated cell death but not for </em><em>Phytophthora</em><em> resistance. Collectively, these results illustrate that the </em><em>Phytophthora</em><em> resistance mediated by LecRK-IX.1 and LecRK-IX.2 is independent of the cell death phenotype. By co-immunoprecipitation we identified putative interacting proteins, one of which was an ATP-binding cassette (ABC) transporter. A homolog in Arabidopsis, the ABC transporter ABCG40, was found to interact </em><em>in planta</em><em> with both LecRK-IX.1 and LecRK-IX.2. Similar to the </em><em>LecRK</em><em> mutants, Arabidopsis </em><em>ABCG40</em><em> mutants showed compromised </em><em>Phytophthora</em><em> resistance, indicating that </em><em>ABCG40 </em><em>has a function in </em><em>Phytophthora</em><em> resistance.</em> <em>In <strong>Chapter V</strong>, we describe the generation of stable transgenic </em><em>N. benthamiana</em><em> plants expressing Arabidopsis </em><em>LecRK-I.9 </em><em>or </em><em>LecRK-IX.1</em><em>. Multiple transgenic lines were obtained varying in transgene copy number and transgene expression level. Ectopic expression of </em><em>LecRK-I.9</em><em> resulted in reduced plant sizes and aberrant leaf morphology. In addition, expression of </em><em>LecRK-IX.1</em><em> induced plant cell death. Transgenic </em><em>N. benthamiana</em><em> lines expressing either </em><em>LecRK-I.9</em><em> or </em><em>LecRK-IX.1</em><em> showed increased resistance towards </em><em>P. capsici</em><em> or </em><em>Phytophthora infestans</em><em>. This demonstrated that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their role in </em><em>Phytophthora</em><em> resistance upon interfamily transfer.</em> <em>Based on the results obtained on Arabidopsis LecRKs, we speculated that LecRKs in other plant species could play a similar role in </em><em>Phytophthora</em><em> resistance. In <strong>Chapter VI</strong>, we focus on LecRKs in two Solanaceous plants, i.e. </em><em>N</em><em>.</em><em> benthamiana</em><em> and tomato. By exploring genome databases, we identified 38 and 22 LecRKs in </em><em>N</em><em>.</em><em> benthamiana</em><em> and tomato, respectively. Phylogenetic analysis revealed that both </em><em>N. benthamiana</em><em> and tomato lack LecRKs homologous to Arabidopsis LecRKs of clades I, II, III and V, but contain a Solanaceous-specific clade of LecRKs. Functional analysis of various Solanaceous </em><em>LecRK</em><em>s using virus-induced gene silencing followed by infection assays revealed that homologs of Arabidopsis </em><em>LecRK-IX.1</em><em> and </em><em>LecRK-IX.2</em><em> in </em><em>N</em><em>.</em><em> benthamiana</em><em> and tomato are implicated in </em><em>Phytophthora</em><em> resistance. These results indicate that the role of clade IX LecRKs in </em><em>Phytophthora</em><em> resistance is conserved across plant species.</em> <em>In<strong> Chapter VII, </strong>the experimental data presented in this thesis are summarized and discussed in a broader context. We present an overview of the current understanding of LecRKs in plant immunity and discuss how </em><em>LecRK</em><em>s can be exploited to improve plant resistance.</em>