A secretory protein of necrotrophic fungus Sclerotinia sclerotiorum that suppresses host resistance

Abstract

SSITL (SS1G_14133) of Sclerotinia sclerotiorum encodes a protein with 302 amino acid residues including a signal peptide, its secretion property was confirmed with immunolocalization and immunofluorescence techniques. SSITL was classified in the integrin alpha N-terminal domain superfamily, and its 3D structure is similar to those of human integrin α4-subunit and a fungal integrin-like protein. When S. sclerotiorum was inoculated to its host, high expression of SSITL was detected during the initial stages of infection (1.5-3.0 hpi). Targeted silencing of SSITL resulted in a significant reduction in virulence; on the other hand, inoculation of SSITL silenced transformant A10 initiated strong and rapid defense response in Arabidopsis, the highest expressions of defense genes PDF1.2 and PR-1 appeared at 3 hpi which was 9 hr earlier than that time when plants were inoculated with the wild-type strain of S. sclerotiorum. Systemic resistance induced by A10 was detected by analysis of the expression of PDF1.2 and PR-1, and confirmed following inoculation with Botrytis cinerea. A10 induced much larger lesions on Arabidopsis mutant ein2 and jar1, and slightly larger lesions on mutant pad4 and NahG in comparison with the wild-type plants. Furthermore, both transient and constitutive expression of SSITL in Arabidopsis suppressed the expression of PDF1.2 and led to be more susceptible to A10 and the wild-type strain of S. sclerotiorum and B. cinerea. Our results suggested that SSITL is an effector possibly and plays significant role in the suppression of jasmonic/ethylene (JA/ET) signal pathway mediated resistance at the early stage of infection.

Figure 1. Characterization of the S. sclerotiorum SS1G_14133 gene.

(A) Alignment of the amino acid sequences of SS1G_14133 protein of S. sclerotiorum and other organisms using MCOFFEE and ClustalX program. AF: Aspergillus fumigates (XP_750162.2); NF: Neosartorya fischeri (XP_001265249.1); AO: A. oryzae (EIT81778.1). (B) Alignment of the repeat peptides sequences and prediction of secondary structure of SS1G_14133 protein. These alignments were obtained using the MCOFFEE and ClustalX program and the default color scheme for ClustalW alignment in the Jalview program was used. The secondary structure prediction was completed with Jnetpred program–beta strands as green arrows. Quality (yellow) is the quality level for the multiple alignments. (C) The comparison of the 3D structural models of SS1G_14133 protein, Integrin α4-subunit (from 35 aa to 478 aa) (UniProt Id: P13612) and Psathyrella velutina Integrin-like protein (UniProt Id: Q309D1) . The images were obtained from the top (upper) and side (lower) of these proteins. The 3D structural models were generated with Phyre2 program.

Figure 2. Gene expression analysis of SSITL gene in the wild-type strain Ep-1PNA367 of S. sclerotiorum.

(A) The Northern blot analysis shows gene expression levels of SSITL grown on PDA from 24 hr to 120 hr, respectively. The rRNA levels on the nylon membrane transferred from the ethidium bromide (EtBr)-staining of the gel (lower) were used as sample loading marker. (B) The relative transcript accumulation patterns of SSITL gene detected with Real-time RT-PCR amplification after contacting with Arabidopsis plants (dark columns) or growing on minimal medium (red columns) for 0–12 hr. The relative levels of transcript were calculated by the comparative Ct method. The SSITL gene expression of S. sclerotiorum inoculated on plants or in plate at 0 hr was set as level one. The levels of β-tubulin transcript were used to normalize different samples. Bars represent means and standard deviations (three replications).

Figure 3. SSITL silenced transformants showing abnormal phenotypes.

(A) The construction of SSITL silenced vector. (B) Northern blots analysis of the SSITL gene transcript accumulation in SSITL silenced transformants. Expression of SSITL in the wild-type Ep-1PNA367 served as control. Hyphae mass from 3-day-old colonies on PDA was collected for gene expression analysis. RNA samples were monitored by Northern hybridization analysis of 18S rRNA on the nylon membrane. (C) Abnormal colony morphology produced by SSLTL silenced transformants. Colonies were grown on PDA for 30 days at 20°C. (D) Excessive branching of hyphal tips of SSITL silenced transformants. (E) Hyphal growth rates of SSITL silenced transformants. Growth rates were examined on PDA at 20°C. Different letters in the graph indicate statistical differences, P = 0.01. (F) Virulence decreases in SSITL silenced transformants. Virulence was evaluated on detached leaves of rapeseed (Brassica napus) measured by the lesions diameter at 20°C for 72 h. Different letters in the graph indicate statistical differences, P = 0.01. (G) Sclerotial sizes of SSITL silenced transformants. Sclerotia were produced on the autoclaved carrot rods in 250 ml flasks at 20°C for 30 days.

Figure 4. Immunolocalization of SSITL of S. sclerotiorum during hyphal growth and infection.

(A) Hyphae for ultrathin sections were collected from 3-day-old colony grown on PDA at 20°C. Ep-1PNA367 and the SSITL silenced transformants A10 and B6 were incubated with the antiserum raised by immunizing rabbits with SSITL, respectively; the hyphae of Ep-1PNA367 which was treated with the pre-immune serum were used as control. (B) Immunolocalization of SSITL (the arrow point) in A. thaliana leaf cells infected by Ep-1PNA367 at 12 hpi. Left: Treated with antiserum; Right: Control sections treated with pre-immune serum. Hyphal agar discs were cut from colony margins and inoculated to the leaves of Arabidopsis for 12 hr before the lesion margin was collected for ultrathin sectioning analysis.

Figure 5. Immunofluorescence detection of SSITL during S. sclerotiorum infecting on onion bulb epidermis.

A transgenic strain of S. sclerotiorum in which an SSITL:Flag tag fusion protein was expressed using PtrpC. Onion bulb epidermis was inoculated with strains for 12–24 hr at 20°C, and was used for immunofluorescence observations under a Nikon Eclipse 80i fluorescent microscope (Nikon, Japan).

Figure 6. Expression of PDF1.2 and PR-1 induced by transformants of S. sclerotiorum at locally inoculated leaves of A. thaliana at the early stage of infection.

The expression of PDF1.2 (A) and PR-1 (B) on leaves inoculated with silenced transformant A10 or with the wild-type strain Ep-1PNA367. The relative levels of transcript were calculated by the comparative Ct method. Expression on leaves of A. thaliana inoculated with pathogen for 0 hr was set as one. Transcript levels of GAPDH of Arabidopsis were used to normalize different samples. Bars represent means and standard deviations (three replications). (C) Lesions induced by transformant A10 and the wild-type strain Ep-1PNA367 on leaves observed at 20°C for 36 hr. Asterisks indicate statistical differences between the lesions diameter induced by A10 and Ep-1PNA367 (P<0.05).

Figure 7. Strong systemic resistance induced by SSITL silenced transformants of S. sclerotiorum.

(A) The lesions induced by B. cinerea with the lower leaves being pretreated with SSITL silence transformant A10, Ep-1PNA367 and the water agar plugs (CK), respectively. Leaves were inoculated with A10 or the wild-type S. sclerotiorum or water agar for two days before inoculated leaves were cut and then inoculated with B. cinerea at 20°C for 72 h. (B) Expression of PDF1.2 on upper leaves of inoculated plants pretreated with A10. Expression in un-inoculated leaves of A. thaliana was set as level 1. At 2 dpi, leaves of Arabidopsis were inoculated with A10 or the wild-type strain S. sclerotiorum or water agar and, at 0 dpi, and upper healthy leaves were inoculated with B. cinerea. Expression of GAPDH was used to normalize. Bars represent means and standard deviations (three replications).

Figure 8. Enhanced susceptibility to A10 produced by disruption of JA/ET and SA signal pathway of Arabidopsis.

(A, B) A10 induced larger lesions on the leaves of Arabidopsis mutant jar 1 and ein2, and mutant pad 4 and transgenic line NahG were more susceptible than the wild-type of A. thaliana. Different letters in the graph indicate statistical differences, P = 0.01. (C, D) The relative expression of PDF1.2 and PR-1 gene in Arabidopsis mutants and transgenic line NahG inoculated with A10. Plants were incubated at 20°C for 36 hr after being inoculated with active mycelial agar discs of A10. Bars represent means and standard deviations (three replications).

Figure 9. Failure of A10 to induce systemic resistance in mutants of Arabidopsis disrupted in the JA/ET signal pathway.

(A) The lesions caused by B. cinerea on A. thaliana mutations and transgenic line NahG for 72 hr at 20°C after being pretreated with A10. (B) The lesions caused by B. cinerea on A. thaliana for 72 hr at 20°C after being pretreated with A10 (blue), Ep-1PNA367 (red) and water agar plugs (black). Asterisks indicate statistical differences from the A10 pretreated (P<0.05). (C, D) The expression of PDF1.2 and PR-1 during the infection of B. cinerea on A. thaliana mutations and transgenic line NahG after being pretreated with A10. Bars represent means and standard deviations (three replications).

Figure 10. Enhanced susceptibility to SSITL silenced transformant A10 induced by the transient expression of SSITL in the host plants.

(A, B) The lesions induced by silenced transformant A10 and the wild-type strain Ep-1PNA367 on the leaves of tobacco (Nicotiana benthamiana) for 48 hr at 20°C. (C, D) The lesions induced by silenced transformant A10 and the wild-type strain Ep-1PNA367 on the leaves of A. thaliana Col-0 for 48 hr and 24 hr at 20°C, respectively. SSITL was expressed transiently in plants leaves by infiltrating with Agrobacterium GV3101 strain carrying SSITL expression vector. The leaves infiltrated with GV3101 carrying empty vector were selected as control (CK). Asterisks indicate statistical differences from the control (P<0.05). (E, F) Transcript levels of PDF1.2 and PR-1 in the SSITL transiently expressed A. thaliana leaves (black) after being respectively inoculated with silenced transformant A10 and the wild-type strain Ep-1PNA367 for 3, 6, 9 and 12 hr, with the plant leaves infiltrated with the GV3101 carrying empty vector being sampled for control (red). (G) Analysis the SSITL expression in the transiently expressed leaves of A. thaliana and tobacco (N. benthamiana) with RT-PCR. The leaves infiltrated with the GV3101 carrying empty vector were selected as control (CK). The A. thaliana GAPDH and N. benthamiana actin genes (see primers in Table 1) were used to normalize different samples.

Figure 11. Suppression of the systemic resistance induced by SSITL silenced transformant A10 in the Arabidopsis plants of SSITL transient expression.

(A) The lesions induced by B. cinerea on upper leaves of plants after the SSITL transiently expressed leaves were inoculated with A10 for 48 hr. The lesions were induced by B. cinerea at 20°C and were measured at 72 hpi. Asterisks indicate statistical differences from the control (P<0.05). (B) The relative transcript levels of PDF1.2 and PR-1 in the upper leaves of plant after the SSITL transiently expressed leaves were inoculated with A10 for 48 hr. Plants infiltrated with GV3101 carrying empty vector only, served as control.

Figure 12. Suppression of resistance induced by A10 in SSITL transgenic lines of A. thaliana.

(A) Lesions on the leaves of SSITL transgenic lines (line 1 and line 2) and the wild-type A. thaliana Col-0 induced by A10. Lesions were measured at 72 hpi. Asterisks indicate statistical differences from the wild-type Col-0 (P<0.05). (B) PDF1.2 and PR-1 expression in the wild-type Col-0 (black), SSITL transgenic line 1 (red) and line 2 (blue) after inoculated with A10. (C) Lesions induced by Ep-1PNA367 on the wild-type A. thaliana Col-0, SSITL transgenic line 1 and line 2 at 20°C for 24 hr. Asterisks indicate statistical differences from the wild-type Col-0 (P<0.05). (D) PDF1.2 and PR-1 expression in the wild-type Col-0 (black), SSITL transgenic line 1 (red) and line 2 (blue) after inoculated with Ep-1PNA367. (E) Lesions induced by B. cinerea on the wild-type A. thaliana Col-0, SSITL transgenic line 1 and line 2 at 20°C for 24 hr. Asterisks indicate statistical differences from the wild-type Col-0 (P<0.05). (F) The analysis of SSITL expression in transgenic lines (line 1 and line 2) of A. thaliana with RT-PCR. A. thaliana GAPDH (see primers in Table 1) was used to normalize different samples.