Functional Characterization of the Nep1-Like Protein Effectors of the Necrotrophic Pathogen – Alternaria brassicae

Abstract

Alternaria brassicae is an important necrotrophic pathogen that infects the Brassicaceae family. A. brassicae, like other necrotrophs, also secretes various proteinaceous effectors and metabolites that cause cell death to establish itself in the host. However, there has been no systematic study of A. brassicae effectors and their roles in pathogenesis. The availability of the genome sequence of A. brassicae in public domain has enabled the search for effectors and their functional characterization. Nep1-like proteins (NLPs) are a superfamily of proteins that induce necrosis and ethylene biosynthesis. They have been reported from a variety of microbes including bacteria, fungi, and oomycetes. In this study, we identified two NLPs from A. brassicae viz. AbrNLP1 and AbrNLP2 and functionally characterized them. Although both AbrNLPs were found to be secretory in nature, they localized differentially inside the plant. AbrNLP2 was found to induce necrosis in both host and non-host species, while AbrNLP1 could not induce necrosis in both species. Additionally, AbrNLP2 was shown to induce pathogen-associated molecular pattern (PAMP)-triggered immunity in both host and non-host species. Overall, our study indicates that AbrNLPs are functionally and spatially (subcellular location) distinct and may play different but important roles during the pathogenesis of A. brassicae.

The Necrosis- and Ethylene-inducing peptide 1-like protein (NLP) gene family of the plant pathogen Corynespora cassiicola

“…The low, but increasing levels of gene expression of the Cc_NLP1.2B gene may be due to a time-increasing gene expression cascade. It agrees with the hypothesis of redundancy of NLP copies as a strategy of functional diversification to overcome the host surveillance in a time-dependent manner (Seidl et al 2015; Duhan et al 2021; Dal’Sasso et al 2022). The NLP encoding-genes are usually expressed in the early hours of host colonization but their expression could follow up to days, depending on the time needed by the pathogen to penetrate the host tissue (Levin et al 2019; Pour et al 2020; Duhan et al 2021).…”

“…It agrees with the hypothesis of redundancy of NLP copies as a strategy of functional diversification to overcome the host surveillance in a time-dependent manner (Seidl et al 2015; Duhan et al 2021; Dal’Sasso et al 2022). The NLP encoding-genes are usually expressed in the early hours of host colonization but their expression could follow up to days, depending on the time needed by the pathogen to penetrate the host tissue (Levin et al 2019; Pour et al 2020; Duhan et al 2021). Taken together, our gene expression analyses suggested that the expression of the NLP effector genes may occur in different levels during host colonization; likely, not all NLP effector genes are essential for pathogenicity of C. cassiicola to soybean.…”

“…The likely role of NLPs as virulence factors during the early stages of infection and disease development (Fellbrich et al 2002; Gijzen and Nürnberger, 2006; Levin et al 2019; Pour et al 2020; Duhan et al 2021), the previous finding that the NLPs comprised a small gene family (Dal’Sasso et al 2022), and the worldwide emergence of target spot in economically important crops (Lopez et al 2018; Sumabat et al 2018; Banguela-Castillo et al 2020; Gao et al 2020;) instigated us into exploring the diversity of NLPs in the plant pathogen C. cassiicola .…”

Characterization and necrosis-inducing activity of necrosis- and ethylene-inducing peptide 1-like proteins from Colletotrichum australisinense, the causative agent of rubber tree anthracnose

“…In this study, we found that the non-cytolytic activity of CaNLPs resulted from substitution of the KD motif and the heptapeptide motif GHRHDWE, and changes of structure characteristics lead to significant differences in the strength of necrosis-inducing activity between CaNLPs. Similar results have been reported in the necrotrophic pathogen Alternaria brassicae ( Duhan et al, 2021 ). Additionally, the responses of plants to NLPs not only depend on the type of NLP, but also the plant species in which the responses are tested.…”

“…Several different approaches have been implemented to explore the cell death-inducing activity of purified or recombinantly produced NLPs, among which include infiltration of purified proteins into the leaf apoplast, transpiration streams upon placing leaf petioles in protein solution, spraying protein solutions as a surfactant, and Agrobacterium-mediated transient gene expression. The Agrobacterium-mediated transient gene expression method is widely used in the literature, but this method is mainly applied to model plants, such as Nicotiana species ( Chen et al, 2021 ; Duhan et al, 2021 ). The infiltration of purified proteins method has also been used for the activity evaluation of NLPs in some forest plants ( Cobos et al, 2019 ; Hunziker et al, 2021 ; Liu et al, 2021 ).…”