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Phytophthora 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 Phytophthora. However, Phytophthora 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 Phytophthora 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 Phytophthora resistance was thus far unknown. The research described in this thesis was aimed at unravelling the role of LecRKs in plant immunity, in particular to Phytophthora pathogens.
Chapter I describes various Phytophthora diseases and the current understanding of the mechanisms underlying plant innate immunity with emphasis on disease resistance to Phytophthora pathogens.
In Chapter II, we describe the development of a new Arabidopsis-Phytophthora pathosystem. We demonstrated that Phytophthora capsici is capable to infect Arabidopsis. Inoculation assays and cytological analysis revealed variations among Arabidopsis accessions in response to different P. capsici isolates. Moreover, infection assays on Arabidopsis mutants with specific defects in defence showed that salicylic acid signaling, camalexin and indole glucosinolates biosynthesis pathways are required for P. capsici resistance (Chapter IIa). The importance of these pathways in Arabidopsis resistance was supported by the finding that the corresponding marker genes are induced upon infection by P. capsici (Chapter IIb). This model pathosystem can be used as an additional tool to pinpoint essential components of Phytophthora resistance.
We then exploited Arabidopsis-Phytophthora pathosystems to uncover the role of LecRKs in Phytophthora resistance. In Chapter III we describe a systematic phenotypic characterization of a large set of Arabidopsis LecRK T-DNA insertion lines. The T-DNA insertion lines were assembled and assayed for their response towards different Phytophthora pathogens. This revealed that next to LecRK-I.9, several other LecRKs function in Phytophthora resistance. We have also analysed whether the LecRKs are involved in response to other biotic and abiotic stimuli. Several T-DNA insertion lines showed altered responses to 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 LecRK expression profiles obtained from publicly available datasets revealed that LecRKs that are hardly induced or even suppressed upon infection, might still have a function in pathogen resistance. Computed co-expression analysis revealed that LecRKs with similar function display diverse expression patterns.
Arabidopsis LecRK clade IX comprises two members. T-DNA insertion mutants of both LecRK-IX.1 and LecRK-IX.2 showed gain of susceptibility to non-adapted Phytophthora isolates and therefore the role of these two LecRKs in Phytophthora resistance was further investigated. In Chapter IV we describe that overexpression of either LecRK-IX.1 or LecRK-IX.2 in Arabidopsis resulted in increased resistance to Phytophthora, 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 Phytophthora 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 LecRKs in Nicotiana benthamiana also resulted in increased Phytophthora resistance and induction of cell death, we used N. benthamiana to explore downstream components required for LecRK-IX.1- and LecRK-IX.2-mediated Phytophthora resistance and cell death. Virus-induced gene silencing of candidate signaling genes revealed that NbSIPK1/NPT4 is essential for LecRK-IX.1-mediated cell death but not for Phytophthora resistance. Collectively, these results illustrate that the Phytophthora 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 in planta with both LecRK-IX.1 and LecRK-IX.2. Similar to the LecRK mutants, Arabidopsis ABCG40 mutants showed compromised Phytophthora resistance, indicating that ABCG40 has a function in Phytophthora resistance.
In Chapter V, we describe the generation of stable transgenic N. benthamiana plants expressing Arabidopsis LecRK-I.9 or LecRK-IX.1. Multiple transgenic lines were obtained varying in transgene copy number and transgene expression level. Ectopic expression of LecRK-I.9 resulted in reduced plant sizes and aberrant leaf morphology. In addition, expression of LecRK-IX.1 induced plant cell death. Transgenic N. benthamiana lines expressing either LecRK-I.9 or LecRK-IX.1 showed increased resistance towards P. capsici or Phytophthora infestans. This demonstrated that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their role in Phytophthora resistance upon interfamily transfer.
Based on the results obtained on Arabidopsis LecRKs, we speculated that LecRKs in other plant species could play a similar role in Phytophthora resistance. In Chapter VI, we focus on LecRKs in two Solanaceous plants, i.e. N. benthamiana and tomato. By exploring genome databases, we identified 38 and 22 LecRKs in N. benthamiana and tomato, respectively. Phylogenetic analysis revealed that both N. benthamiana 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 LecRKs using virus-induced gene silencing followed by infection assays revealed that homologs of Arabidopsis LecRK-IX.1 and LecRK-IX.2 in N. benthamiana and tomato are implicated in Phytophthora resistance. These results indicate that the role of clade IX LecRKs in Phytophthora resistance is conserved across plant species.
In Chapter VII, 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 LecRKs can be exploited to improve plant resistance.
|Qualification||Doctor of Philosophy|
|Award date||24 Nov 2014|
|Place of Publication||Wageningen|
|Publication status||Published - 2014|
- phytophthora capsici
- plant pathogenic fungi
- plant-microbe interactions
- transgenic plants
- gene expression
- disease resistance