Ss NEP2 Contributes to the Virulence of Sclerotinia sclerotiorum

Abstract

Sclerotinia sclerotiorum is a notorious soilborne fungal pathogen that causes serious economic losses globally. The necrosis and ethylene-inducible peptide 1 (NEP1)-like proteins (NLPs) were previously shown to play an important role in pathogenicity in fungal and oomycete pathogens. Here, we generated S. sclerotiorum necrosis and ethylene-inducible peptide 2 (SsNEP2) deletion mutant through homologous recombination and found that SsNEP2 contributes to the virulence of S. sclerotiorum without affecting the development of mycelia, the formation of appressoria, or the secretion of oxalic acid. Although knocking out SsNEP2 did not affect fungal sensitivity to oxidative stress, it did lead to decreased accumulation of reactive oxygen species (ROS) in S. sclerotiorum. Furthermore, Ssnlp24_SsNEP2 peptide derived from SsNEP2 triggered host mitogen-activated protein kinase (MAPK) activation, increased defense marker gene expression, and enhanced resistance to Hyaloperonospora arabidopsidis Noco2. Taken together, our data suggest that SsNEP2 is involved in fungal virulence by affecting ROS levels in S. sclerotiorum. It can serve as a pathogen-associated molecular pattern (PAMP) and trigger host pattern triggered immunity to promote the necrotrophic lifestyle of S. sclerotiorum.

Keywords: ROS; Sclerotinia sclerotiorum; SsNEP2; plant immunity; virulence.

Figure 1

Generation of knockout and complementation strain of SsNEP2 in S. sclerotiorum. (A) Split marker method technical process. Fragment 1 is composed of upstream of target gene and the first half of hygromycin-resistance gene (HY), and fragment 2 is composed of the second half of hygromycin (YG) and downstream of the target gene. Both fragments replace the target gene with hygromycin-resistance gene by homologous recombination. (B) Full-length SsNEP2 gene amplification in genomes of WT (1), ΔSsNEP2 (2), and SsNEP2-C (3). (C) Relative expression level of SsNEP2. β-tubulin was used as internal reference. Relative expression of SsNEP2 in WT strain was set as control. WT, wild type; ΔSsNEP2, knockout strain; SsNEP2-C, complementation strain.

Figure 2

SsNEP2 deletion does not affect growth phenotype. (A) Mycelial morphology of SsNEP2 knockout and complementation strain. Bar = 0.5 mm. (B) Mycelial growth of WT, ΔSsNEP2, and SsNEP2-C. Strains were grown on PDA medium and colony diameters were measured every 24 h. (C) Colony morphology of WT, ΔSsNEP2, and SsNEP2-C strains after 14 days. (D) Number of sclerotia per plate. (E) Sclerotial weight per plate. WT, wild-type strain; ΔSsNEP2, knockout strain; SsNEP2-C, complementation strain. Experiments were conducted three times. Error bars indicate standard deviation (SD). Statistical significance was analyzed using Student’s t-test between wild-type and knockout mutant or complementation strain.

Figure 3

SsNEP2 is associated with virulence. (A,C) Inoculated lesions and trypan blue staining of WT, SsNEP2 deletion, and complementation strains of detached and undetached leaves of A. thaliana and N. benthamiana. Data were recorded at 24 hpi. Bar = 10 mm. (B,D) Lesion areas of WT, ΔSsNEP2, and SsNEP2-C on leaves of A. thaliana and N. benthamiana. WT, wild-type strain; ΔSsNEP2, knockout strain; SsNEP2-C, complementation strain. ImageJ was used to analyze lesion areas. Experiments were conducted three times. Error bars represent SD. Statistical significance was analyzed using Student’s t-test between wild-type and knockout mutant or complementation strain (** p < 0.01).

Figure 4

SsNEP2 does not affect oxalate secretion and appressorium formation. (A) WT, ΔSsNEP2, and SsNEP2-C placed on glass slides and cultured for 16 h to observe formation and number of appressoria. (B) Penetration assay of WT, ΔSsNEP2, and SsNEP2-C on onion epidermis cells. Invasion hyphae were stained with trypan blue. Bar = 0.2 mm. (C) Mycelium of WT, ΔSsNEP2, and SsNEP2-C grown on PDA medium containing bromophenol blue. Experiments were conducted three times. WT, wild-type strain; ΔSsNEP2, knockout strain; SsNEP2-C, complementation strain.

Figure 5

SsNEP2 affects ROS accumulation. (A) DAB staining of WT, ΔSsNEP2, and SsNEP2-C. (B) Growth inhibition rate of WT, ΔSsNEP2, and SsNEP2-C. (C) Colonial morphology and mycelial growth of WT, ΔSsNEP2, and SsNEP2-C under different concentrations of H₂O₂. Bar = 10 mm. Experiments were conducted three times. WT, wild type; ΔSsNEP2, knockout strain; SsNEP2-C, complementation strain.

Figure 6

Ssnlp24 triggers MAPK activation in Arabidopsis. (A) Multiple sequence alignment of SsNEP2 (SS1G_11912), BcNEP1(XP_001555180), BcNEP2(XP_001551049), BsNPP1(WP_019714591.1). and HaNLP3(6QBE_A). Red box: conserved nlp24 region. (B) Ssnlp24_SsNEP2-induced MAPK activation. Seedlings were treated with 1 μM nlp24 or Ssnlp24_SsNEP2. “Control” refers to the untreated “0” time point. Western blot with anti-pERK antibody is used for analysis of MAPK activation. Equal loading confirmed by Ponceau staining of Rubisco. Experiments were conducted three times.

Figure 7

Ssnlp24_SsNEP2 enhances plant immunity. (A,B) Relative expression levels of PR1 and PR2 in Col-0 after treatment with 1 μM nlp24 or Ssnlp24_SsNEP2 for 4 or 24 h. Error bars represent SD. Statistical significance was analyzed using Student’s t-test between wild-type and knockout mutant or complementary strain (** p < 0.01). (C) Three-week-old soil-grown plants of indicated genotypes were pretreated with H₂O and 1 μM nlp20 or Ssnlp24_SsNEP2 and sprayed with H. a. Noco2 spores (30,000 spores/mL) 24 h later. Experiments were conducted three times. Error bars represent SD. Statistical significance was analyzed using Student’s t-test between ddH₂O, nlp24 and Ssnlp24_SsNEP2 (** p < 0.01).