Nep1-like Proteins from Valsa mali Differentially Regulate Pathogen Virulence and Response to Abiotic Stresses

Abstract

Necrosis and ethylene-inducing peptide 1(Nep1)-like protein (NLP) is well known for its cytotoxicity and immunogenicity on dicotyledonous, and it has attracted large attention due to its gene expansion and functional diversification in numerous phytopathogens. Here, two NLP family proteins, VmNLP1 and VmNLP2, were identified in the pathogenic fungus Valsa mali. We showed that VmNLP2 but not VmNLP1 induced cell death when transiently expressed in Nicotiana benthamiana. VmNLP2 was also shown to induce cell death in apple leaves via the treatment of the Escherichia coli-produced recombinant protein. VmNLP1 and VmNLP2 transcripts were drastically induced at the early stage of V. mali infection, whereas only VmNLP2 was shown to be essential for pathogen virulence. We also found that VmNLP1 and VmNLP2 are required for maintaining the integrity of cell membranes, and they differentially contribute to V. mali tolerance to salt- and osmo-stresses. Notably, multiple sequence alignment revealed that the second histidine (H) among the conserved heptapeptide (GHRHDWE) of VmNLP2 is mutated to tyrosine (Y). When this tyrosine (Y) was substituted by histidine (H), the variant displayed enhanced cytotoxicity in N. benthamiana, as well as enhanced virulence on apple leaves, suggesting that the virulence role of VmNLP2 probably correlates to its cytotoxicity activity. We further showed that the peptide among VmNLP2, called nlp25 (VmNLP2), triggered strong immune response in Arabidopsis thaliana. This work demonstrates that NLPs from V. mali involve multiple biological roles, and shed new light on how intricately complex the functions of NLP might be.

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

“…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 ). In the present study, we employed purified recombinant protein infiltration into the leaf apoplast to analyze cytotoxic activity of NLPs, and obtained consistent results after repeated experiments.…”

SsNEP2 Contributes to the Virulence of Sclerotinia sclerotiorum

“…Cytotoxic NLPs can cause H 2 O 2 accumulation in plants, leading to elevated ROS levels, which triggers programmed cell death (PCD) [31,36]. As a member of the NLPs, SsNEP2 may also have such a function.…”

“…As shown in Figure 5A, the color of ∆SsNEP2 was lighter than that of the wild type, whereas the color of the complemented strain SsNEP2-C was not significantly different, indicating that H 2 O 2 content in the mutant strains was lower than that in the wild-type strain, and SsNEP2 knockout did affect ROS accumulation (Figure 5A). In addition to acting as effectors, NLPs also affect the growth and development of pathogens and their responses to abiotic stresses [24,31]. To test the function of SsNEP2 in response to peroxide stress, we inoculated WT, ∆SsNEP2, and SsNEP2-C on PDA medium containing different gradient concentrations of H 2 O 2 .…”

“…NLPs also have functions beyond plant-pathogen interactions; they affect conidia formation and play an important role in maintaining cell membrane integrity [24,31]. Although NLPs have been studied in different fungal pathogens, their role and molecular mechanism in S. sclerotiorum are still unclear.…”

VdERG2 was involved in ergosterol biosynthesis, nutritional differentiation and virulence of Verticillium dahliae

“…For the determination of abiotic stress, 1 mol/L KCl, 1 mol/L NaCl, 1 mol/L Sorbitol, 0.004% SDS and 0.02% CR were added to PDA medium (Liu, et al 2021a, Zhang, et al 2022a). Normal PDA medium was used as a control.…”