Comparative and Functional Analysis of the Widely Occurring Family of Nep1-Like Proteins

Abstract

Nep1-like proteins (NLP) are best known for their cytotoxic activity in dicot plants. NLP are taxonomically widespread among microbes with very different lifestyles. To learn more about this enigmatic protein family, we analyzed more than 500 available NLP protein sequences from fungi, oomycetes, and bacteria. Phylogenetic clustering showed that, besides the previously documented two types, an additional, more divergent, third NLP type could be distinguished. By closely examining the three NLP types, we identified a noncytotoxic subgroup of type 1 NLP (designated type 1a), which have substitutions in amino acids making up a cation-binding pocket that is required for cytotoxicity. Type 2 NLP were found to contain a putative calcium-binding motif, which was shown to be required for cytotoxicity. Members of both type 1 and type 2 NLP were found to possess additional cysteine residues that, based on their predicted proximity, make up potential disulfide bridges that could provide additional stability to these secreted proteins. Type 1 and type 2 NLP, although both cytotoxic to plant cells, differ in their ability to induce necrosis when artificially targeted to different cellular compartments in planta, suggesting they have different mechanisms of cytotoxicity.

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

“…The second conserved region in the 28-aa fragment is the GHRHDWE heptapeptide that is characteristic for the NLP family, and that is conserved in both type 1 and type 2 NLPs. The weblogo is based on 378 type 1 NLP sequences and 122 type 2 NLP sequences (19). Ethylene production in Arabidopsis is induced in response to nlp24 peptides (1 μM) of HaNLP3, BcNEP2, and BsNPP1, but not to the nlp26 fragment of the type 2 NLP Pcc .…”

“…Type 2 NLPs differ from type 1 by an additional conserved second cysteine bridge and putative calcium-binding domain (19). In Arabidopsis, cytotoxic NLPs were found to activate immunity-related gene expression, which strongly overlapped with that induced by flg22 (21,22).…”

“…Two major NLP types are found in bacteria, fungi, and oomycetes (18,19) and are known to cause rapid necrosis and ethylene production in many dicot, but not in monocot plant…”

A Conserved Peptide Pattern from a Widespread Microbial Virulence Factor Triggers Pattern-Induced Immunity in Arabidopsis

“…NLPs form a superfamily of proteins that are produced and secreted by bacterial, fungal and oomycete species [24], [25], [26]. NLPs have initially been discovered as cytotoxic proteins triggering leaf necrosis and plant defenses in dicotyledonous, but not in monocotyledonous plants [27].…”

“…There is accumulating evidence that NLP effectors have diversified in function [26]. The fungal pathogen Mycosphaerella graminicola produces Mg NLP that is toxic on dicot plants, but not on its monocot host, wheat [31].…”