The broad host range pathogen Sclerotinia sclerotiorum produces multiple effector proteins that induce host cell death intracellularly

Abstract

Sclerotinia sclerotiorum is a broad host range necrotrophic fungal pathogen, which causes disease on many economically important crop species. S. sclerotiorum has been shown to secrete small effector proteins to kill host cells and acquire nutrients. We set out to discover novel necrosis-inducing effectors and characterize their activity using transient expression in Nicotiana benthamiana leaves. Five intracellular necrosis-inducing effectors were identified with differing host subcellular localization patterns, which were named intracellular necrosis-inducing effector 1-5 (SsINE1-5). We show for the first time a broad host range pathogen effector, SsINE1, that uses an RxLR-like motif to enter host cells. Furthermore, we provide preliminary evidence that SsINE5 induces necrosis via an NLR protein. All five of the identified effectors are highly conserved in globally sourced S. sclerotiorum isolates. Taken together, these results advance our understanding of the virulence mechanisms employed by S. sclerotiorum and reveal potential avenues for enhancing genetic resistance to this damaging fungal pathogen.

Keywords: NLR protein; RxLR motif; Sclerotinia sclerotiorum (white mould); effector; localization; necrotrophic fungus; plant pathogen.

FIGURE 1

Necrosis‐inducing activity of SsINE proteins in Nicotiana benthamiana leaf tissue. (a) Macroscopic cell death symptoms induced by agroinfiltration of mature effector proteins SsINE1–5. Green fluorescent protein (GFP) was included as a negative control. PpNLP2 was included as a positive control. Photographs were taken at 7 days postinfiltration. A red border indicates cell death symptoms; a black border indicates no cell death symptoms. (b) Relative ion leakage induced by agroinfiltration of SsINE1–5. GFP was included as a negative control. PpNLP2 was included as a positive control. Different letters above the boxes indicate significant differences, as determined by analysis of variance with a post hoc Tukey honestly significant difference test.

FIGURE 2

Subcellular localization of SsINE proteins expressed without a signal peptide in Nicotiana benthamiana epidermal cells. Panels on the left‐hand side labelled “GFP” show green fluorescent protein (GFP) fluorescence. Panels on the right‐hand side labelled “Merged” show GFP fluorescence, chloroplast autofluorescence, and bright field images. The white arrows indicate nuclei. Images are z‐projections. The scale bars are 50 μm.

FIGURE 3

Amino acid conservation and predicted features of SsINEs. (a) Alignment of two alleles of SsINE1 and SsINE3 within 26 globally sourced Sclerotinia sclerotiorum isolates. The number in brackets indicates the number of isolates that harbour the corresponding allele. The line above the amino acids shows the predicted signal peptide sequence. (b) Length and predicted features of SsINE proteins. Predicted domains and motifs are represented by the different shades and patterns.

FIGURE 4

Investigation into the RxLR‐like and dEER‐like motifs of SsINE1. (a) Amino acid sequence of the CU8.24 SsINE1 protein. The SP annotation indicates the predicted signal peptide. Amino acid letters above the RxLR‐like and dEER‐like motifs indicate the sequence of the mutated variants (RTLT‐AAAA and DFDVTEKR‐AFAVTAKA). (b) Macroscopic cell death symptoms induced by agroinfiltration of SsINE1 variants with and without a signal peptide. Green fluorescent protein (GFP) was included as a negative control. A red border indicates cell death symptoms; a dashed red border indicates attenuated cell death; a black border indicates no cell death symptoms. (c) The left and right panels are boxplots showing GFP fluorescence and red light fluorescence, respectively, of agroinfiltrated Nicotiana benthamiana leaf sections. Representative fluorescence images of the “SP” leaves in panel (b) are shown above the boxplot. Black diamonds represent the mean values. The three asterisks (***) indicate a significant difference from SP‐SsINE1 (p ≤ 0.001), as determined by a Student's t test.

FIGURE 5

Subcellular localization of SsINE1 variants expressed with a signal peptide (SP) in Nicotiana benthamiana epidermal cells. Green fluorescent protein (GFP) was included as a control. The leaf samples were plasmolysed prior to mounting on microscope slides. Panels on the left‐hand side labelled “GFP” show GFP fluorescence. Panels on the right‐hand side labelled “Merged” show GFP fluorescence, chloroplast autofluorescence, and bright field images. The white arrows indicate nuclei; the red arrows indicate apoplastic localization. Images are z‐projections. The scale bars are 100 μm.

FIGURE 6

NLR virus‐induced gene silencing (VIGS) screen of SsINE‐induced cell death. (a) Macroscopic cell death symptoms induced by agroinfiltration of SsINE2, SsINE3, and SsINE5 in com5 VIGS Nicotiana benthamiana plants. Photographs were taken at 7 days postinfiltration. A red border indicates cell death symptoms; a dashed red border indicates attenuated cell death. The number of replicates with cell death is indicated in the top right corner of each image. (b) Quantification of SsINE3‐ and SsINE5‐induced cell death in NLR gene‐silenced N. benthamiana plants. Boxplot showing red light fluorescence of agroinfiltrated N. benthamiana leaf sections. Black diamonds represent mean values. Coloured dots represent individual biological replicates. The asterisk (*) indicates a significant difference from the empty vector (EV) negative control (p ≤ 0.05), as determined by a Student's t test. The photographs and fluorescence images on the left‐hand side are representative images of leaves with fluorescence intensity in approximate increments of 5000. A red border indicates cell death symptoms; a dashed red border indicates weak cell death; a black border indicates no cell death symptoms. (c) Section of a phylogenetic tree showing the relationship between the NLR proteins targeted by the six com5 cassettes and 121 known NLR proteins. Alignment of full‐length amino acid sequences was performed with ClustalW using the Jukes–Cantor model. The neighbour‐joining method was used to construct the phylogenetic tree. The NLR targeted by com5‐2 is labelled as “NbNLR 061‐1.”