SsNEP2 Plays a Role in the Interaction Between Sclerotinia sclerotiorum and Coniothyrium minitans

Abstract

Sclerotinia sclerotiorum, a fungal pathogen that is spread worldwide and causes serious diseases on crops, can be parasitized specifically by the mycoparasite Coniothyrium minitans. SsNEP2, encoding a necrosis-inducing protein in S. sclerotiorum, was previously inferred to play a role in the virulence to host plants. In this study, silencing of SsNEP2 in S. sclerotiorum had no significant (p < 0.01) influence on mycelial morphology, while overexpression led to lower mycelial growth and more branches. When amended with the fermentation broth of the SsNEP2 silencing mutants, conidial germination of C. minitans was promoted, while conidial production decreased. When parasitized by C. minitans, enhanced resistance of the SsNEP2 silencing mutants and weaker resistance of the overexpressed transformants were observed compared to the wild-type S. sclerotiorum strain 1980. In addition, the expression of SsNEP2 in C. minitans enhanced mycelial parasitism on S. sclerotiorum and restored the effect of silencing SsNEP2 in S. sclerotiorum on mycoparasitism. Thus, we highlight the role of SsNEP2 as a PAMP-like protein in the mycoparasitism between C. minitans and its host fungus S. sclerotiorum. SsNEP2 can be used to promote the biological potential of C. minitans.

Keywords: Sclerotinia sclerotiorum; SsNEP2; mycoparasitism; necrosis- and ethylene-inducing peptide 1.

Figure 1

Expression of SsNEP1 and SsNEP2 in S. sclerotiorum. (a) RT-PCR detection of the gene expression of SsNEP1 and SsNEP2 in S. sclerotiorum cultured on PDA. S48, S52, S60, S72, S96, S120, and S144 represent the incubating stage of S. sclerotiorum on PDA for 48 h, 52 h, 60 h, 72 h, 96 h, 120 h, and 144 h, respectively. β-tubulin was used as a reference. H₂O was used as the negative control. M: DNA ladder DL2000 (TaKaRa, Dalian, China). The primer pairs were Rt-SsNEP2-F/R for SsNEP2 and Rt-SsNEP1-F/R for SsNEP1. The primer sequences are listed in Table 1. (b) Fluorescence quantitative PCR detection of gene expression of SsNEP1 and SsNEP2 in S. sclerotiorum cultured on PDA. The normalized gene expression at 48 h was set as 1. The expression of β-tubulin was used to normalize different samples. (c) The expression of SsNEP1 and SsNEP2 in S. sclerotiorum induced by C. minitans at different stages. SC0, SC4, SC12, SC24, SC48, SC72, and SC96 represent the interaction stages of S. sclerotiorum with C. minitans for 0 h, 4 h, 12 h, 24 h, 48 h, 72 h, and 96 h, respectively. (d) The transcript level of SsNEP1 and SsNEP2 in S. sclerotiorum induced by C. minitans at different stages. The normalized gene expression at 0 h was set as 1. (e) Secretory identification of SsNEP2 using the yeast secretion trap system. SP^Avrb is the positive control. pSUC2 is the empty vector control. YTK12 is the yeast control. (f) Color identification of the samples from (e) by TTC. TTC is 2,3,5-triphenyltetrazolium chloride. TTC changed from colorless to red, indicating a responsive activity.

Figure 2

SsNEP2 mutants of S. sclerotiorum. (a) The transcript level of SsNEP2 cultured for 36 h on PDA. Three silencing mutants are represented as SsNEP2-sl-4, SsNEP2-sl-9, and SsNEP2-sl-19. OESsNep2-Ss-3, OESsNep2-Ss-5, and OESsNep2-Ss-10 represent the overexpressed transformants of S. sclerotiorum. (b) Radial growth of all the strains on PDA. The significance level is p < 0.01 and the significant differences are distinguished by capital letters of A, B and C at the top of bar chart. (c) Colony morphology of all the strains cultured on PDA for 7 days at 20 °C. (d) Hyphal tips of all strains growing on PDA for 36 h.

Figure 3

C. minitans affected by SsNEP2. (a) Colony morphology of C. minitans strain ZS-1 incubated at 20 °C for 15 days on PDA-FPDB (PDA amended with an equal volume of filter fermentation broth of S. sclerotiorum shaken in PDB for four days). (b) Colony morphology of C. minitans strain ZS-1 incubated at 20 °C for 15 days on WA-FPDB (water agar culture amended with filter fermentation broth of S. sclerotiorum). (c) Mycelial growth of C. minitans on PDA-FPDB and WA-FPDB. The significance level is p < 0.01 and the significant differences are distinguished by capital letters of A, B and C at the top of bar chart. (d) Conidiation of C. minitans cultured on PDA-FPDB or WA-FPDB with 15 days. The significance level is p < 0.01 and the significant differences are distinguished by a capital letter at the top of bar chart.

Figure 4

Mycelial parasitism of C. minitans against the SsNEP2 mutants of S. sclerotiorum. (a) Dual culture of C. minitans and the SsNEP2 silencing mutants for 30 days. ZS-1 means C. minitans. (b) Dual culture of C. minitans and the SsNEP2 overexpressed transformants for 20 days. (c) Schematic diagram showing the number of agar disks that gave rise to either C. minitans (black circle), S. sclerotiorum (hollow circle), or both (gray circle). Each circle represents a colony developed from a mycelial agar disk sampled from zones I, II, III, or IV between the inoculation sites of C. minitans and S. sclerotiorum in a dual culture. The appearance of S. sclerotiorum colonies indicates that C. minitans has not yet parasitized S. sclerotiorum in this region. Similarly, the presence of C. minitans colonies suggests that S. sclerotiorum was eliminated by C. minitans. The occurrence of colonies of both C. minitans and S. sclerotiorum indicates that C. minitans has not yet completely destroyed the hypha of S. sclerotiorum.

Figure 5

Parasitism of C. minitans to the sclerotia of S. sclerotiorum. (a) Sclerotia of SsNEP2 silencing mutants were covered with mycelia of C. minitans after inoculation with C. minitans conidia for 5 days at 20 °C. Strain 1980 is S. sclerotiorum strain 1980 (b) The conidial germination rate of C. minitans cultured in sclerotial soaked for 24 h at 20 °C. (c) Sclerotia of S. sclerotiorum parasitized by C. minitans 30 days after inoculation. Pycnidia formed on the surface of sclerotia (red arrows). (d) Rot index of sclerotia of the silencing SsNEP2 mutants parasitized by C. minitans. The surface-sterilized sclerotia of mutants and strain 1980 of S. sclerotiorum were submerged into the conidial suspension of C. minitans for 30 min, half-buried in sterilized wet sand in plates, and incubated for 30 days at 20 °C. Twenty sclerotia with five repeats were used for each strain, and the experiment was repeated twice independently. The rot index of sclerotia was evaluated according to the described method [41]. The significance level is p < 0.01 and the significant differences are distinguished by a capital letter at the top of bar chart.

Figure 6

SsNEP2 overexpressed mutants of C. minitans. (a) Colony morphology of SsNEP2 overexpressed mutants of C. minitans OESsNep2-Cm-2, OESsNep2-Cm-3, and OESsNep2-Cm-4. (b) qPCR detection of the expression of SsNEP2 in C. minitans. (c) Conidial production of SsNEP2 overexpressed mutants of C. minitans. (d) Growth rate of the SsNEP2 overexpressed mutants of C. minitans. (e,f) Sclerotial parasitism of the SsNEP2 overexpressed mutants of C. minitans. Sclerotia are from the S. sclerotiorum strain 1980. ZS-1 represents the C. minitans strain ZS-1. (g,h) Mycelium parasitism of SsNEP2 overexpressed mutants of C. minitans on PDA. Each circle represents a colony developed from a mycelial agar disk sampled from zones I, II, III, or IV between the inoculation sites of C. minitans and S. sclerotiorum in a dual culture. (i) Mycelial parasitism of SsNEP2 overexpressed mutants of C. minitans on water agar. (j) The colony diameter of C. minitans from the samples of (e). (k) The conidial production of C. minitans from the samples of (e). The significance level is p < 0.01 and the significant differences are distinguished by a capital letter at the top of bar chart.

Figure 7

Mycelial parasitism between SsNEP2 silencing transformants of S. sclerotiorum and expression mutants of C. minitans. (a) Dual culture of C. minitans and S. sclerotiorum for 20 days under 20 °C. ZS-1 represents the C. minitans strain ZS-1. (b) Results of the dual culture of (a). Schematic diagram showing the number of agar disks that gave rise to either C. minitans (black circle), S. sclerotiorum (hollow circle), or both (gray circle). Each circle represents a colony developed from a mycelial agar disk sampled from zones I, II, III, or IV between the inoculation sites of C. minitans and S. sclerotiorum in a dual culture.