Ss-Rhs1, a secretory Rhs repeat-containing protein, is required for the virulence of Sclerotinia sclerotiorum

Abstract

Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a worldwide distribution. Cell wall-degrading enzymes and oxalic acid are important to the virulence of this pathogen. Here, we report a novel secretory protein, Ss-Rhs1, which is essential for the virulence of S. sclerotiorum. Ss-Rhs1 is believed to contain a typical signal peptide at the N-terminal and eight rearrangement hotspot (Rhs) repeats. Ss-Rhs1 exhibited a high level of expression at the initial stage of sclerotial development, as well as during the hyphal infection process. Targeted silencing of Ss-Rhs1 resulted in abnormal colony morphology and reduced virulence on host plants. Microscopic observations indicated that Ss-Rhs1-silenced strains exhibited reduced efficiency in compound appressoria formation.

Keywords: Rhs repeat; Sclerotinia sclerotiorum; appressorium; sclerotia; secretory protein; virulence.

Figure 1

Functional validation of the predicted signal peptide of the Sclerotinia sclerotiorum Ss‐Rhs1 (Sclerotinia sclerotiorum Rearrangement hotspot repeat 1) protein. The 20 initial amino acids of Ss‐Rhs1 were fused into the invertase sequence of pSUC2 and the resulting vector pSURHS1 was transformed into the yeast strain YTK12. The transformed YTK12 cells were grown on CMD‐W and YPRAA media. The untransformed YTK12 strain and the YTK12 strain that transformed with pSUC2 were used as controls.

Figure 2

Secretion of the Ss‐Rhs1 (Sclerotinia sclerotiorum Rearrangement hotspot repeat 1) protein. The Ss‐Rhs1‐Flag‐engineered strain was cultured with shaking for 4 days and the proteins in the hyphae and culture filtrate were extracted. The proteins were then subjected to Western analysis using an anti‐Flag (top) or anti‐Actin (bottom) antibody.

Figure 3

Characterization of the Ss‐Rhs1 (Sclerotinia sclerotiorum Rearrangement hotspot repeat 1) protein. (A) Alignment of the repeat peptide sequences of Ss‐Rhs1. (B) Comparison of the peptide repeat consensus of Ss‐Rhs1 with the Rhs repeat consensus (Wang et al., 1998). (C) Domain organization of the Ss‐Rhs1 protein.

Figure 4

Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of the Ss‐Rhs1 (Sclerotinia sclerotiorum Rearrangement hotspot repeat 1) gene transcript during different sclerotial development stages (A) and after contact with Arabidopsis thaliana (dark columns) and growing on potato dextrose agar (PDA) plates (grey columns) (B). The quantity of Ss‐Rhs1 cDNA in each sample was normalized to that of tub1 cDNA. The relative abundance of Ss‐Rhs1 cDNA in the stage of hyphal growth or in mycelium inoculated on PDA or plants at 0 h was set as unity. Bars indicate the standard error. The analyses were repeated three times. Gene expression levels in different replicates showed similar trends. One replicate is shown.

Figure 5

Construction of the Ss‐Rhs1 (Sclerotinia sclerotiorum Rearrangement hotspot repeat 1) gene RNA interference (RNAi) vector and phenotype of Ss‐Rhs1‐silenced strains. (A) Construction of the Ss‐Rhs1 RNAi vector pSIRHS1. (B) Relative expression level of Ss‐Rhs1 in different isolates containing pSIRHS1, as well as in the wild‐type strain, as determined by real‐time reverse transcription‐polymerase chain reaction (RT‐PCR). The quantity of S. sclerotiorum tub1 cDNA normalized different samples. The relative expression level of Ss‐Rhs1 in the wild‐type strain was set to unity. Bars indicate the standard error. (C) Phenotypes of the wild‐type strain, Sirhs‐66, Sirhs‐68 and Sirhs‐93. The strains were grown on PDA medium for 15 days.

Figure 6

Radial growth rates of wild‐type and Ss‐Rhs1 (Sclerotinia sclerotiorum Rearrangement hotspot repeat 1)‐silenced strains. The experiment was repeated three times; one replicate is shown here. Bars indicate the standard error. *Significantly different from the wild‐type strain on potato dextrose agar (PDA) plates.

Figure 7

Virulence assays of Ss‐Rhs1 (Sclerotinia sclerotiorum Rearrangement hotspot repeat 1)‐silenced strains. (A) Detached leaves of rapeseed (Brassica napus) and Arabidopsis thaliana were inoculated with potato dextrose agar (PDA) plugs colonized with the wild‐type strain, Sirhs‐66 and Sirhs‐93. (B) Detached leaves of rapeseed were wounded with a dissecting needle and the wild‐type strain and Sirhs‐93 were placed over the wound. The experiment was repeated three times, and each strain was investigated with five rapeseed leaves or A. thaliana plants each time.

Figure 8

Compound appressoria formation phenotypes of wild‐type and Sirhs‐93 strain. (A) Compound appressoria formation on parafilm surrounding mycelia‐colonized agar plugs (8 h post‐inoculation, hpi). Scale bars correspond to 2 mm. (B) Compound appressoria formation on Arabidopsis thaliana leaves inoculated with mycelial plugs (6 hpi). Scale bars correspond to 100 μm.