Prokaryotic expression, purification and activity analysis of necrosis and ethylene-inducing peptide 1 (Nep1)-like protein from <italic>Magnaporthe grisea</italic>

Abstract

<p indent="0mm">Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are kinds of secreted by a range of plant-associated microorganisms including bacteria, fungi, and oomycetes. The first member of NLP families is Nep1 protein isolated and purified from the culture filtrates of <italic>Fusarium oxysporum</italic> by Bailey in 1995. The protein induced ethylene and necrosis in leaves of a variety of dicotyledoneae but not in members of the monocotyledoneae tested. In addition, the Nep1 and some members of NLP family can also induce defense-related responses, including the expression of pathogenesis-related genes, callose deposition, accumulation of reactive oxygen species (ROS), and hypersensitive-like response in plants. Previous studies have shown that the Nep1 and some members of NLP family play dual roles in plant-pathogen interactions. They not only act as a cytolytic toxin that causes plasma membrane permeabilization and cytolysis in plant cells but also represent a class of microbial-associated molecular patterns (MAMPs) that trigger plant innate immune response. Many plant pathogen genomes contain different numbers and types of NLP family members. Different pathogens differ significantly in the number of NLP members, for example, the genome of <italic>Mycosphaerella graminicola</italic> contains only a single NLP homologue, whereas the number of NLP members in the genome of<italic> Phytophthora sojae</italic> and <italic>Phytophthora capsici</italic> up to 33 and 39, respectively. Recently, the NLP toxins are reported to target the plant cell-surface GIPC sphingolipids, and the difference in predominant GIPC series produced by the Eudicot and monocot determine host selectivity of microbial NLP cytolysins. However, recently, some NLP toxins produced by the pathogen of monocot were also proved to have cytotoxic activity on various monocot plant species and legitimize their presence in monocot-specific plant pathogens. The<italic> Magnaporthe grisea</italic>, causal agent of rice blast disease, contains four NPP1 domain-containing gene (MoNLP) in the fully sequenced genome. Although they are all highly induced during the infection of rice, the quadruple ΔMoNLP <italic>M</italic>.<italic>griseae</italic> mutant strains do not compromise its virulence. Therefore, the function and significance of their presence are still largely unknown. To further explore the potential function of these NLP members during the interactions between rice and <italic>Magnaporthe oryzae</italic> as well as the possibility that they may act as potential plant immunity inducers to modulate the plant immune response, the coding region sequences were cloned and ligated into the pGEX-6P-1 vector to prokaryotically express and purify these proteins by analyzing their sequence through bioinformatics methods. The results indicated that the solubility of prokaryotically expressed protein decreased obviously with the induction temperature increasing, but the expressed recombinant protein also increased. These corresponding purified and soluble proteins were then further infiltrated into the leaves of tobacco, indicated that the MoNLP1 and MoNLP4 fused with N-terminal GST tag were bioactive and induced the obvious leaf tissue necrosis of tobacco. However, the purified GST-MoNLP2 protein did not induce tissue necrosis in tobacco leaves, which may be due to expressed fusion protein with unexcised signal peptide or low protein concentration for injection. Our study laid a foundation for further investigating the function of this gene family in rice blast fungus and exploiting more potential protein elicitors for plants.