Nep1-like proteins from three kingdoms of life act as a microbe-associated molecular pattern in Arabidopsis

Significance Peptide fragments of Nep1-like proteins (NLPs), occurring in diverse microorganisms of three different kingdoms of life, were found to trigger immunity in the model plant Arabidopsis , indicating that they act as a microbe-associated molecular pattern (MAMP). A synthetic peptide of 24 aa from the central part of the downy mildew HaNLP3 protein was found to activate the plant immune system and trigger resistance to this pathogen. Strikingly, not only peptides of oomycete NLPs, but also those of bacteria and fungi were shown to act as a MAMP. This unprecedented broad taxonomic distribution demonstrates that the occurrence of a MAMP is not necessarily restricted to a class of microorganisms but can occur in a wide range of species from the tree of life. Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by a wide range of plant-associated microorganisms. They are best known for their cytotoxicity in dicot plants that leads to the induction of rapid tissue necrosis and plant immune responses. The biotrophic downy mildew pathogen Hyaloperonospora arabidopsidis encodes 10 different noncytotoxic NLPs (HaNLPs) that do not cause necrosis. We discovered that these noncytotoxic NLPs, however, act as potent activators of the plant immune system in Arabidopsis thaliana . Ectopic expression of HaNLP3 in Arabidopsis triggered resistance to H. arabidopsidis , activated the expression of a large set of defense-related genes, and caused a reduction of plant growth that is typically associated with strongly enhanced immunity. N- and C-terminal deletions of HaNLP3, as well as amino acid substitutions, pinpointed to a small central region of the protein that is required to trigger immunity, indicating the protein acts as a microbe-associated molecular pattern (MAMP). This was confirmed in experiments with a synthetic peptide of 24 aa, derived from the central part of HaNLP3 and corresponding to a conserved region in type 1 NLPs that induces ethylene production, a well-known MAMP response. Strikingly, corresponding 24-aa peptides of fungal and bacterial type 1 NLPs were also able to trigger immunity in Arabidopsis . The widespread phylogenetic distribution of type 1 NLPs makes this protein family (to our knowledge) the first proteinaceous MAMP identified in three different kingdoms of life.