| الوصف: |
Plants and animals both rely on nucleotide-binding, leucine-rich repeat proteins (NLRs) to provide disease resistance. Plant NLRs often co-operate as pairs or networks. A sensor NLR detects the pathogen and a helper NLR executes cell death and/or downstream signalling. NLRs are believed to localise to the cytoplasm, plasma membrane or nucleus. However, these localisation patterns were observed in the absence of infection, not during infection with relevant pathogens due to the HR triggered by the NLR making it impossible to perform microscopy. Many plant proteins re-locate within the cell upon infection by microbes. Therefore the localisation of NLRs during infection may be different to those without infection. Additionally, determining the localisation of NLRs can help us understand what function the NLR provides and how it detects effectors and elicits an immune response. Here I study the localisation of a solanaceous helper NLR, NRC4 during live cell imaging of Nicotiana benthamiana leaves with and without infection by the Irish potato famine pathogen, Phytophthora infestans. I use transient gene expression and confocal microscopy to show that during NRC4, accumulates from the cytoplasm to a membrane surrounding the pathogen finger-like appendages called haustoria. The extra-haustorial membrane is a newly formed plant membrane interface surrounding the pathogen haustoria. This reveals that NLRs can be motile and alter localisation during infection to accumulate at pathogen contact sites. I overcome the problem of NLRs triggering cell death when their cognate effector is present by either studying the helper NLR NRC4 in the absence of a sensor, or by disarming NRC4 by mutating the first alpha helix of the NLR's cell death eliciting coiled-coil domain. When activated, we found that NRC4 forms puncta which associate with the EHM or in the absence of infection, the plasma membrane. These puncta may represent inflammasome-like 'resistosomes' or groups of resistosomes. A phylogenetically and functionally related NLR, ZAR1, was recently shown to form a resistosome and is proposed to execute cell death by possibly forming pores in the host membrane. Further evidence is required in the future to determine if NRC4 is forming resistosomes and whether these are eliciting cell death directly. Intriguingly, efforts to prevent NRC4 accumulation at the EHM, by mutation, truncation or chimeric NLR generation, led to loss of NRC4's cell death function. As a whole, this thesis provides evidence that NRC4 accumulates to the host-pathogen interface to provide disease resistance against P. infestans. This thesis reveals that NLRs can alter their localisations upon infection and that they can even focally accumulate at the host-pathogen interface. Further understanding into how NRC4 focally accumulates during infection and exactly what it achieves by doing so may help us understand how resistance to fungi and oomycetes occurs. Ultimately, this may allow us to engineer disease resistant crops by, for instance, altering NRC4's specificity to recognise other EHM localised effectors, without altering NRC4's localisation. |
