Investigation of plant immunity activated by enforced nuclear localization of EDS1 (Enhanced Disease Susceptibility1) in Arabidopsis
Plant Genetics and Genomics
Final Report Abstract
Plant nucleotide-binding domain—leucine-rich repeat-type receptors (NLRs) constitute an importantbarrier to microbial infection. NLRs recognize effector proteins, microbial virulence factors secretedinto plant cells during infection, either by direct binding or indirect detection of effector activity.Effector recognition induces a rapid immune response, which efficiently restricts microbialcolonization. However, NLRs can also become activated erroneously, then driving plants into anautoimmune state that is detrimental for plant fitness. NLR genes are highly variable betweenspecies and even isolates of the same species. Some NLR genes, that occur in large and complex geneclusters and evolve particularly fast, are also particularly prone to induction of autoimmunity. One ofthese clusters is the Arabidopsis thaliana DANGEROUS MIX2 (DM2)/ RECOGNITION OFPERONOSPORA PARASITICA1 (RPP1) locus. DM2/RPP1 was first identified for its function in racespecificresistance to the oomycete Hyaloperonospora arabidopsidis (formerly Peronosporaparasitica), and more recently emerged as a major hotspot for induction of autoimmunity. InArabidopsis accession Landsberg erecta (Ler), the DM2 cluster encompasses seven to eight full lengthor truncated NLR genes (DM2a – DM2h). Functions in natural pathogen resistance are not known, but DM2Ler is involved in autoimmunity in three different genetic interactions. In one of these scenarios, DM2Ler-dependent autoimmunity is induced by expression of a fusionprotein of ENHANCED DISEASE SUSCEPTIBILITY1 with YFP and a strong nuclear localization signal(EDS1-YFPNLS). EDS1 is an immune regulator that is strictly required for signaling downstream of onesubclass of plant NLRs. However, EDS1 molecular functions remain unknown. Also, what mightdifferentiate NLRs prone to autoimmune induction from classical sensor NLRs operating in naturalpathogen resistance, and how they are activated in autoimmunity, remains unknown. In previous work, a screen for suppressors of EDS1-YFPNLS-induced autoimmunity was conducted, andmutations in DM2h were identified in a first set of near death experience (nde) mutant lines. In thisproject, we aimed to characterize additional nde lines, and to analyze roles of DM2 genes inautoimmunity and pathogen resistance. Surprisingly, analysis of remaining nde lines revealed thatDM2h was the only locus targeted by our suppressor screen. We showed, by crosses and/or dm2mutant derivatives generated by genome editing, that DM2h is necessary and sufficient for inductionof autoimmunity not only in interaction with EDS1-YFPNLS, but also in the two additional describedcases of DM2Ler-dependent autoimmunity induction. This highlighted DM2h as the risk-NLR containedin the DM2 cluster. To reveal how the risk-NLR is activated in EDS1-YFPNLS-induced autoimmunity, DM2h was functionallycharacterized using heterologous reconstitution assays and stable transgenic Arabidopsis lines. Ourdata suggest that there are no conceptual or mechanistic differences between NLR activation inautoimmunity or effector-triggered immunity, and that DM2h thus functions similar to welldescribedsensor NLRs with assigned resistance functions. Furthermore, we showed that activation ofDM2h is specific to the EDS1-YFPNLS fusion: Indeed, a comparable fusion protein containing an alteredlinker connecting the EDS1 and YFP moieties was functional in the context of immune signaling, butdid not induce DM2h-mediated autoimmunity. Also, the activation of DM2h by EDS1-YFPNLS isunrelated to EDS1 immune functions, as a signaling-deficient EDS1(F419E) variant, in the context ofthe EDS1-YFPNLS fusion protein, retains the capacity to induce autoimmunity. We propose thatstochastic interactions of novel or foreign proteins with NLRs may, in many cases, underlie inductionof autoimmunity. This is a provocative model contradicting the common notion that autoimmuneinduction suggests resistance functions and/or guard-guardee relationships of a protein and arespective NLR.
Publications
- An EDS1-SAG101 complex is essential for TNL-mediated immunity in Nicotiana benthamiana. Plant Cell (2019)
Gantner, Ordon, Kretschmer, Guerois, Stuttmann J
(See online at https://doi.org/10.1105/tpc.19.00099) - Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system. PLoS ONE (2018)
Gantner, Ordon, Ilse, Kretschmer, Gruetzner, Löfke, Dagdas, Bürstenbinder, Marillonnet, Stuttmann
(See online at https://doi.org/10.1371/journal.pone.0197185) - Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation. Functional & Integrative Genomics (2019)
Ordon, Bressan, Kretschmer, Dall’Osto, Marillonnet, Bassi, Stuttmann J
(See online at https://doi.org/10.1007/s10142-019-00665-4) - Disentangling cause and consequence: Genetic dissection of the DANGEROUS MIX2 risk locus, and activation of the DM2h NLR in autoimmunity. The Plant Journal (2021)
Ordon, Martin, Erickson, Ferik, Balcke, Bonas, Stuttmann
(See online at https://doi.org/10.1111/tpj.15215) - Highly efficient multiplex editing: One-shot generation of 8x Nicotiana benthamiana and 12x Arabidopsis mutants. The Plant Journal (2021)
Stuttmann, Barthel, Martin, Ordon, Erickson, Herr, Ferik, Kretschmer, Berner, Keilwagen, Marillonnet, Bonas
(See online at https://doi.org/10.1111/tpj.15228)
