Project

Identification and validation of immune signaling components in Cladosporium fulvum-resistant tomato

The project aim is to uncover which defense signaling proteins are involved in downstream signaling by the Cf-4 resistance protein in tomato, by taking advantage of CRISPR/Cas-mediated gene editing and proximity labelling methods.

Background

Unlike vertebrates, plants only have an innate immune system to defend themselves against diseases. The plant immune system is triggered upon the specific recognition of immunogenic patterns (IPs), causing either extracellularly-triggered immunity (ExTI) or intracellularly-triggered immunity (InTI).

The latter is based on the recognition of an intracellular IP (InIP), for instance of a protein, referred to as effector, of the invading pathogen in the cytoplasm of the plant cell, in most cases by nucleotide-binding (NB) and leucine-rich repeat (LRR)-containing immune receptors, whereas ExTI is triggered upon recognition of an Extracellular IP (ExIP) at the plasma membrane, by transmembrane receptor proteins.

Former studies on the C. fulvum-tomato interaction showed that the resistance response against C. fulvum is triggered extracellularly by ExIPs that can be regarded as avirulence factors (Avr), as instead of facilitating colonization, they trigger resistance in tomato carrying the matching Cf resistance protein. As the gene-for-gene model proposes, Avr proteins are recognized by Cf resistance proteins of tomato, either directly or indirectly.

Project description

The research has the following objectives:

  • Optimization of Agrobacterium Transient Transformation Assays (ATTAs) in tomato to increase protein expression levels and decrease the necrotic background
  • Screening of a pool of mutant tomato lines (obtained through the Boyce Thompson Institute, USA), by recombinant potato virus X (PVX) to select candidate immune signaling genes
  • Studying the interaction of potential candidate signaling proteins with SOBIR1, BAK1, and Cf-4 by proximity labelling to detect downstream signaling proteins in tomato
  • Studying the roles of candidate immune signaling genes in Cf-4-mediated immunity by the generation of single/multiplex mutant lines of such genes in Cf-4 tomato by CRISPR/Cas technology

Results

ATTA is optimized in tomato. For this, a special plasmid to increase the expression in tomato was used (Figure 1).
The albino phenotype was generated by CRISPR targeting of the PDS gene in tomato, that protects against photobleaching. Thus, we showed that our CRISPR protocol works in tomato (Figure 2).

Figure 1
Figure 1

Figure 2
Figure 2