Award details

RLP and RLK-mediated innate immune responses in Arabidopsis and tomato triggered by PAMPS and Avrs

Reference	BB/E024874/1
Principal Investigator / Supervisor	Dr Cyril Zipfel
Co-Investigators / Co-Supervisors
Institution	University of East Anglia
Department	Sainsbury Laboratory
Funding type	Research
Value (£)	279,974
Status	Completed
Type	Research Grant
Start date	11/05/2007
End date	10/09/2010
Duration	40 months

Abstract

Genetic methods will define genes required for pathogen recognition and PAMP-triggered immunity. EMS mutagenized M2 seeds of Col-0 and Ler-0 will be selected for elf18-insensitive (elfin) mutants. We will use standard methods to distinguish dominant and recessive mutants, and complementation analysis to reveal an allelic series at the elf18 receptor (EFR) locus. These alleles will be sequenced, and inferences drawn about key amino acids for EFR function. We already confirmed ~ 30 elfin mutants, one of which appears also compromised in flg22 responses. We will further characterize mutants from activation tagging lines; we have identified one mutant that is almost certainly not at EFR, and which confers enhanced susceptibility to Pseudomonas. We will also screen M2 seed generated in house from lines carrying 2 copies of EFR, to maximize recovery of mutants at loci other than EFR. Non-EFR mutants will be characterised for a range of defence phenotypes, including responsiveness to other elicitors, and enhanced susceptibility to various pathogen races and mutants. They will be classified into elf18 response-specific mutants, and mutants that affect other defence response to eg flg22, chitin or oligogalacturonides. These mutants will generate F2 seed from crosses to accession Ler-0, and those with interesting phenotypes will be prioritized for gene isolation by standard map-based cloning. Also, we will investigate some of the RLP and RLK-encoding genes in the recognition of oomycete pathogens including Hyaloperonospora parasitica, Albugo candida and Phytophthora infestans. Candidate genes identified previously will be studied via additional mutant alleles, and complementation by genetic transformation. Double mutants of some RLK and RLP genes will be generated to determine the additive affect of these genes. More mutant RLK-encoding genes RLK-encoding genes will be screened with several oomyces pathogens to identify further novel genes required for recogniton.

Summary

Plant resistance or susceptibility to disease primarily involves 3 components. Firstly, many microbes make Pathogen-Associated Molecular Patterns (PAMPs), such as bacterial flagellin and fungal chitin, which plants have evolved the capacity to recognize and respond to. This results in PAMP-triggered immunity (PTI). Secondly, many pathogens make and deliver to plant cells, effector proteins that interfere with PTI, lowering defence activation, resulting in susceptibility. Thirdly, plants have a repertoire of resistance (R) genes that encode sensor proteins that recognize specific pathogen effectors, either directly, or indirectly via effector action on a host component. R gene-dependent recognition results in effector-triggered immunity (ETI), restoring resistance. To understand resistance, we need to better understand PTI, not only because of its intrinsic interest and important role in crop protection, but also because host components involved in PTI are targets of pathogen effectors. We also need to know whether ETI and PTI involve the same mechanisms. PTI has been studied in Arabidopsis by investigating responses to flg22, a peptide surrogate of flagellin. Defence activation requires a transmembrane receptor FLS2 that binds flg22, and is correlated with protein kinase activation, reactive oxygen species production, cell wall fortification and gene induction. The main aim of this proposal is 1) to use forward genetics to understand PTI and 2) to use reverse genetics to understand the role of cell surface receptors in activating the immune system of plants. Genetic analysis provides a more stringent test of whether or not a correlated phenomenon is actually required for the process under study. In a forward genetic approach, mutants are sought in which the process of interest is inactivated; such mutations are in genes required for that process. Arabidopsis also responds to another bacterial PAMP protein called EF-Tu, and to its corresponding peptide surrogate elf18. Elf18 triggers, via another transmembrane receptor called EFR, a very similar response to flg22. However, elf18 can be used at lower doses, enabling an Arabidopsis mutant screen that involves 100s of thousands mutagenized seeds, in which every gene will have been 'hit' scores of times. This enables recovery of rare new alleles of genes with subtle effects, which often provide the most subtle and interesting new insights into biological mechanisms. In a reverse genetic approach, mutants will be used to investigate the role of receptor proteins in recognition of oomycete pathogens including downy mildew and white rusts of brasssicas and late blight pathogen of potato and tomato. Our initial data suggests that some of these cell surface receptor proteins may play a role. We anticipate that this research program will provide profound mechanistic insights into how the receptor proteins work. It will also identify genes encoding proteins required for signalling after recognition. It will be very interesting to examine whether these genes are required for PTI triggered by multiple elicitors or just elf18. Conceivably, such mutants will be compromised in disease resistance to many pathogens, caused by fungi as well as bacteria; this possibility will be tested. Many biochemical phenomena are correlated with PTI- these will be examined in the various mutants, to see if they differentially disable some but not all elf18 responses. Finally, genes required for signalling for either elf18 alone, or from multiple PAMPs, will be isolated by standard map-based cloning methods and then further examined.

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Crop Science, Microbiology, Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	European Research Area Network in Plant Genomics (ERA-PG) [2006-2009]
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/E02484X/1 RLP- and RLK-mediated innate immune responses in Arabidopsis and tomato triggered by PAMPs and avirulence factors

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