Project

Signal transduction by extracellular plant immune receptors

Resistance to the plant pathogenic fungi Verticillium dahliae and Cladosporium fulvum is conferred by extracellular trans-membrane immune receptors, so-called Receptor-Like Proteins (RLPs). These receptors recognize the effectors that these fungi secrete in the intercellular spaces (apoplast) of plant tissues. Effectors are virulence factors that are able to manipulate the host to induce susceptibility when the appropriate plant immune receptors are absent. As RLPs lack any other predicted internal signalling domain, it is likely they rely on other proteins, possibly containing a kinase domain, for the initiation of the signal transduction cascade.

Resistance of tomato to C. fulvum is conferred by RLPs known as Cf proteins, which recognize the effectors of this pathogen. To date, several Cf proteins and their corresponding secreted C. fulvum effectors have been identified. For instance, Cf-4 recognizes the effector Avr4. Upon recognition a rapid defence response is mounted to arrest fungal growth. This response is typically associated with a form of programmed cell death, known as the hypersensitive response (HR). We study how these Cf proteins function in Solanaceous plants.

Identification of a Cf-4-Binding Protein (Cf-4-BP) that is required for the Cf-4/Avr4-triggered HR and Cf-4-mediated resistance. A. A Cf-4-enhanced green fluorescence protein (eGFP) fusion localises mainly to the ER-network upon Agroinfiltration in N. benthamiana, as visualized by confocal microscopy. B. Immuno-precipitation of Cf-4-eGFP from such cells reveals several Cf-4-Binding Proteins (Cf-4-BPs), in addition to Cf-4-eGFP itself (arrows). A 50 kDa protein band also co-purified in the negative (GUS) control (asterisk). C. Virus-induced gene silencing (VIGS) of a Cf-4-BP-encoding gene results in compromised Avr4-triggered HR in N. benthamiana:Cf-4. Note that HR caused by auto-active Rx (RxD460V) and BAX is not affected. D. VIGS of Cf-4-BP in tomato:Cf-4 results in compromised Cf-4-mediated resistance to C. fulvum secreting Avr4. Plants were inoculated with C. fulvum expressing Avr4 and the GUS reporter gene. Fungal growth was visualized by GUS-staining and subsequent chlorophyll removal with EtOH. These results show that Cf-4-BP is required for Cf-4-mediated HR and resistance to C. fulvum.
Identification of a Cf-4-Binding Protein (Cf-4-BP) that is required for the Cf-4/Avr4-triggered HR and Cf-4-mediated resistance. A. A Cf-4-enhanced green fluorescence protein (eGFP) fusion localises mainly to the ER-network upon Agroinfiltration in N. benthamiana, as visualized by confocal microscopy. B. Immuno-precipitation of Cf-4-eGFP from such cells reveals several Cf-4-Binding Proteins (Cf-4-BPs), in addition to Cf-4-eGFP itself (arrows). A 50 kDa protein band also co-purified in the negative (GUS) control (asterisk). C. Virus-induced gene silencing (VIGS) of a Cf-4-BP-encoding gene results in compromised Avr4-triggered HR in N. benthamiana:Cf-4. Note that HR caused by auto-active Rx (RxD460V) and BAX is not affected. D. VIGS of Cf-4-BP in tomato:Cf-4 results in compromised Cf-4-mediated resistance to C. fulvum secreting Avr4. Plants were inoculated with C. fulvum expressing Avr4 and the GUS reporter gene. Fungal growth was visualized by GUS-staining and subsequent chlorophyll removal with EtOH. These results show that Cf-4-BP is required for Cf-4-mediated HR and resistance to C. fulvum.
Our current knowledge on how effector proteins are recognized and activate the defence signal transduction cascade within the cells by Cf proteins is very limited. For example, the host proteins that interact with Cf immune receptors before and after their activation by effectors are not known (the so-called ‘resistosome’ or perception complex). Furthermore, it is also not known whether receptor internalization (endocytosis) occurs and whether this process is required for effector recognition.
In this project we aim to identify and characterize proteins that are required for Cf-mediated signal transduction. We transiently express Cf proteins fused to eGFP in the Solanaceous plant Nicotiana benthamiana (a plant species in which Cf proteins are also functional) by Agroinfiltration. Subsequently, we immuno-precipitate the tagged Cf-protein and analyse co-purifying proteins by using mass-spectrometry. These co-purifying proteins will be studied for their role in the Cf-induced signal transduction cascade by various techniques, like VIGS, confocal microscopy, transient expression, co-immunoprecipitation etc..