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Discovering the role of AVRblb2 in suppression of chitin triggered plant immunity

Final Report Summary - EFFECTOR FUNCTION (Discovering the role of AVRblb2 in suppression of chitin triggered plant immunity)

Due to their nutritious nature, plants attract many parasitic and symbiotic organisms. Some of the plant parasitic or symbiotic microbes are accommodated inside the host cells in specialised compartments. A well-known example is the oomycete pathogen Phytophthora infestans, which constitutes a major global threat to current food security causing late blight disease on several solanaceous plants such as potato and tomato. Upon entry inside the host tissue, P. infestans shows extensive hyphael growth in the host apoplast and penetrates multiple cells forming finger shaped structures called haustoria, which are believed function to deliver effector proteins and acquire nutrients. The haustorium is surrounded by a host-derived membrane called the extrahaustorial membrane (EHM), which differs from plasma membrane in various aspects. In a recent study we showed that while plasma membrane localised proteins are selectively excluded from EHM, some plasma membrane associated proteins and proteins mediating vesicle trafficking are still localised around the EHM. However the composition of the EHM and the mechanisms underlining its biogenesis are still poorly understood.

Interestingly, a small GTPase Rabg3c, that mediates vesicle traffic between the late endosomes and vacuole is not excluded from perihaustorial compartments suggesting potential vacuolar route towards EHM biogenesis. However, whether or not RabG3c localises or traffics to the EHM is yet unclear. Recently we discovered that a host-translocated RXLR-type effector protein AVRblb2 of P. infestans focally accumulates at the EHM, while another RXLR effector HaRXL17 secreted by Hyaloperonospora arabidopsidis localises to the tonoplast surrounding the EHM in N. benthamiana infected by P. infestans. Therefore, AVRblb2 and HaRXL17 can be used, as molecular probes to better understand the composition of the EHM.

Our overall objective in this work was to identify the pathways that lead to EHM formation and to gain insights about mechanisms for its biogenesis. Our specific objective was to investigate the potential intracellular traffic between the vacuole and the EHM.

We hypothesised that AVRblb2 and HaRXL17 as molecular probes to investigate the potential vacuolar route towards EHM biogenesis. For this, we undertook a cell biology approach utilising confocal laser scanning microscopy (CLSM) and exploited P. infestans Nicotiana benthamiana patho-system using fluorescently tagged effector proteins and various subcellular markers.

Our studies revealed that RabG3c localised both to the EHM and the perihaustorial tonoplast and occasionally showed punctate distribution around EHM in P. infestans infected plant cells highlighting the presence of intracellular traffic between the vacuole and the EHM. Furthermore, this transport was a selective process since a tonoplast localised sucrose transporter was still excluded from the EHM. Interestingly, unlike plasma membrane resident pattern recognition receptors (PRRs) FLS2 and EFR, their co-receptor BAK1 ortholog we cloned from potato (StSerk3a) labelled both EHM and the peri-haustorial tonoplast. Moreover, we occasionally detected StSerk3a in endomembrane compartments across EHM, which are marked by RabG3c suggesting its transport to the EHM is mediated by late endosomes. In summary, using effectors such as AVRblb2 and HaRXL17 as molecular markers we identified a selective vacuolar route to the EHM for protein transport and some immune receptors might traffic to the EHM via late endosomes.

Our data points to a previously unknown plant endomembrane pathway for EHM biogenesis. Effectors are great reagents for finding novel aspects of immunity; here AVRblb2 and HaRXL17 led us to identify an intracellular transport route for the EHM formation. A better understanding of EHM biogenesis might help developing new strategies to fight against filamentous plant pathogens such as manipulation of macromolecule transport at the EHM in favour of the plants.