Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi

Abstract

The soil-borne oomycete Phytophthora cinnamomi is a highly destructive Phytophthora species associated with the decline of forest. This pathogen secretes a novel class of necrosis-inducing proteins known as Nep1-like proteins (NLPs). In this work, we report the sequencing and molecular characterization of one of these proteins, more specifically the necrosis-inducing Phytophthora protein 1 (NPP1). The ORF of the npp1 gene (EMBL database AM403130) has 768 bp encoding a putative peptide of 256 amino acids with a molecular weight of approximately 25 kD. In order to understand its function, in vitro gene expression was studied during growth in different carbon sources (glucose, cellulose, and sawdust), and at different times of infection, in vivo by RT-qPCR. The highest expression of the npp1 gene occurred in glucose medium followed by sawdust. In vivo infection of Castanea sativa roots with P. cinnamomi revealed a decrease in npp1 expression from 12 to 24 h; at 36 h its expression increased suggesting the existence of a complex mechanism of defense/attack interaction between the pathogen and the host. Expression of recombinant npp1 gene was achieved in Pichia pastoris and assessed by SDS-PAGE analysis of the protein secreted into the culture supernatant, revealing the presence of the NPP1 protein.

ROS and Oxidative Response Systems in Plants Under Biotic and Abiotic Stresses: Revisiting the Crucial Role of Phosphite Triggered Plants Defense Response

“…Plant defense responsive cutters are induced and secreted by Phytophthora to some extent; it can be said that pathogenesis relies on protein-targeting motifs ( Bengyella et al, 2019 ). A necrosis-inducing protein isolated from P. cinnamon facilitates the colonization of host tissues during the necrotrophic stage by stimulating plant cell death by facilitating nutrient uptake by pathogens ( Martins et al, 2019 ). Effects of fungal and oomycete proteins have been shown to play an important role in apoplasts for adaptation, with recent advances in understanding the role of these proteins in escaping chitin-induced immunity ( Rocafort et al, 2020 ).…”

Unraveling Plant Cell Death during Phytophthora Infection

“…A P. cinnamomi NPP1 has been reported in a study where the authors investigated the expression of the gene using RT-qPCR, both in vitro, using different carbon sources, and in vivo, during infection of Castanea sativa roots [139]. A decrease in NPP1 expression was noted between 12 and 24 h post-inoculation (hpi) with a significant increase at 36 hpi, suggesting a complex host-pathogen interaction.…”

Phytopathogenic oomycetes: a review focusing on Phytophthora cinnamomi and biotechnological approaches

“…Moreover, the same study revealed that β-cinnamomin is secreted at different life stages of P. cinnamomi, through the use of a β-cinnamomin immune-labeling [72]. Furthermore, recent studies using bioinformatics approaches, heterologous protein expression system and molecular biology techniques have characterized necrosis-inducing Phytophthora protein 1 (NPP1) elicitin and an endo-1,3-β-d-glucosidase of P. cinnamomi [73,74].…”