SnTox3 acts in effector triggered susceptibility to induce disease on wheat carrying the Snn3 gene

Abstract

The necrotrophic fungus Stagonospora nodorum produces multiple proteinaceous host-selective toxins (HSTs) which act in effector triggered susceptibility. Here, we report the molecular cloning and functional characterization of the SnTox3-encoding gene, designated SnTox3, as well as the initial characterization of the SnTox3 protein. SnTox3 is a 693 bp intron-free gene with little obvious homology to other known genes. The predicted immature SnTox3 protein is 25.8 kDa in size. A 20 amino acid signal sequence as well as a possible pro sequence are predicted. Six cysteine residues are predicted to form disulfide bonds and are shown to be important for SnTox3 activity. Using heterologous expression in Pichia pastoris and transformation into an avirulent S. nodorum isolate, we show that SnTox3 encodes the SnTox3 protein and that SnTox3 interacts with the wheat susceptibility gene Snn3. In addition, the avirulent S. nodorum isolate transformed with SnTox3 was virulent on host lines expressing the Snn3 gene. SnTox3-disrupted mutants were deficient in the production of SnTox3 and avirulent on the Snn3 differential wheat line BG220. An analysis of genetic diversity revealed that SnTox3 is present in 60.1% of a worldwide collection of 923 isolates and occurs as eleven nucleotide haplotypes resulting in four amino acid haplotypes. The cloning of SnTox3 provides a fundamental tool for the investigation of the S. nodorum-wheat interaction, as well as vital information for the general characterization of necrotroph-plant interactions.

Figure 1. SDS-PAGE image of a size exclusion chromatography fraction containing SnTox3 activity.

Lane 1. Genescript Smart His-tagged protein standard; Lane 2 and 3, duplicate protein samples of the size exclusion chromatography fraction 17-12 (Figure S1B); Lane 4. Bio-Rad pre-stained protein standard. The molecular weight (kDa) of each protein is labeled and the arrows indicate the two protein bands that were excised from the gel. The larger band is estimated at 18 kDa and contains SnTox3.

Figure 2. Genomic location, gene structure and nucleotide and amino acid sequence of SNOG_08981.

A. The genomic location and gene structure of SNOG_08981. Top: Supercontig14 was schematically drawn to show one end containing SNOG_08981 (Gene 1 - light blue-color filled arrow) as well as the four other SNOG genes (2, 3, 4 and 5, black color filled arrows) present in the flanking region. A large amount of AT rich sequence closely flanking SNOG_08981 was labeled that contains a polyprotein gene and a putative transposase gene. Below: A close up of a 1.6 kb genomic region (dash boxed) indicates a single exon, the 5′- and 3′- UTR, and the putative promoter region. B. Nucleotide sequence of a 1.6 kb genomic region of SNOG_08981 (SnTox3) and the deduced amino acid sequence of the SNOG_08981 protein (SnTox3). The underlined peptide sequence was detected in mass spectrometry. The amino acids highlighted in blue indicate the predicted signal peptide. The six cysteine residues highlighted in green are predicted to form 3 disulfide bonds. The bold DNA sequence indicates the start codon (ATG), stop codon (TAG) and the SalI restriction site. DNA sequence highlighted in yellow is UTR and the red highlighted region is a putative TATA box. Bold italicized sequence indicates the primer sites used to amplify the genomic region for transformation.

Figure 3. Southern analysis of SNOG_08981 in S. nodorum and related fungi.

Southern hybridization was performed using a probe from the full length SNOG_08981 ORF as well as λDNA. Size ladders include Hyperladder I and λDNA HindIII fragments. The isolate designations starting with Sn or SN are S. nodorum isolates, and Ptr86-124, St975, and Pt-15A, are isolates of Pyrenophora tritici-repentis, Mycosphaerella graminicola and P. teres, respectively.

Figure 4. Expression pattern of SnTox3 in planta.

The expression level of SnTox3 was examined and compared to that of the Act1 gene at 3, 5, 7 and 10 days after inoculation. The x axis shows the number of days post-infection. The y axis represents relative gene expression levels normalized to Act1. Standard error bars are shown.

Figure 5. Toxin bioassay of the Pichia pastoris X33 strain transformed with SNOG_08981.

Leaves of toxin differential and parental lines, including BR34, Grandin, BG261, BG220 and BG223 were infiltrated with culture filtrate from P. pastoris X33 transformed with SNOG_08981. Wheat lines containing Snn3 (Grandin and BG220) were sensitive whereas lines not containing Snn3 (e.g. containing snn3) (BR34, BG261, and BG223) were insensitive.

Figure 6. Molecular characterization of SnTox3 transformation in Sn79-1087 and disruption in Sn1501.

A. Verification of the SnTox3 transformation into Sn79-1087 and disruption in Sn1501 using Southern analysis. Genomic DNA from the avirulent isolate Sn79-1087 and its two SnTox3 transformed strains Sn79+SnTox3A and Sn79+SnTox3B were digested with XhoI. The virulent SnTox3 containing isolate Sn1501 and its two SnTox3-disrupted strains Sn1501ΔSnTox3A and Sn1501ΔSnTox3B as well as a strain with an ectopic integration, Sn1501Ect, were digested with EcoRI. The blot was probed with a full length SnTox3 cDNA. The two red dots indicate the bands of 7.5 kb and 3.0 kb specifically present in the SnTox3-disrupted strains (see Figure 7B). A 7.9 kb band is present in the wild type and ectopic strain. More ectopic insertions were detected by southern in Sn1501ΔSnTox3A and Sn1501Ect, compared to Sn1501ΔSnTox3B. B. SnTox3 disruption strategy in Sn1501. The SnTox3 gene in Sn1501 was disrupted by insertion of the hygromycin B resistance gene (orange bar) into a SalI restriction site located in the center of the SnTox3 gene. A ∼1.2 kb genomic region of SnTox3 (black bar) amplified with primers 8981g1F_XbaI and 8981g1R_XbaI was cloned and then linearized with SalI. The linearized vector was re-ligated with a cpc-1:HYG :tryptophan c cassette that was released from the pLP605KO vector using the restriction enzyme XhoI. The resulting vector was linearized via XbaI before transformation into Sn1501 protoplasts. The disruption of SnTox3 can be identified using primers 8981g0F with HY and verified by primers 8981g1F and 8981g1R. C. PCR screening and verification of SnTox3-disrupted mutants in Sn1501. The primer 8981g0F (g0F) located outside of the ∼1.2 kb region along with primer HY amplify a ∼2.9 kb fragment in strains which have SnTox3 disrupted by the insertion. No amplification is observed in wild type and ectopics. The primers 8981g1F1 (g1F) and 8981g1R (g1R) amplify a ∼1.2 kb fragment in the wild type and ectopic strains indicating the SnTox3 gene remains intact, while they amplify a ∼3.8 kb fragment (∼1.2 kb of SnTox3 region plus ∼2.6 kb of cpc-1:HYG :tryptophan c cassette) in the SnTox3-disrupted strains. D. RT-PCR verification of different mutated strains. The primers 8981cF and 8981cR which amplify the SnTox3 ORF region were used to test the presence of transcripts of SnTox3 in different isolates and genetically modified strains including Sn79-1087 and its two transformed SnTox3+ strains (Sn79+SnTox3A and Sn79+SnTox3B) and Sn1501 and its two SnTox3-disrupted strains Sn1501ΔSnTox3A and Sn1501ΔSnTox3B along with the ectopic transformant Sn1501Ect. The S. nodorum actin gene was used as an internal control and water was used as a PCR negative control.

Figure 7. Toxin bioassay and virulence analysis of the SnTox3 genetically modified strains.

A. Toxin bioassay of Sn79-1087 with its two transformed SnTox3+ strains Sn79+SnTox3A and Sn79+SnTox3B. Leaves of BG220 (SnTox3 differential line) were infiltrated with culture filtrates of Sn79-1087 wild type, Sn79+SnTox3A and Sn79+SnTox3B and photographed 3 days after infiltration showing culture filtrates from the wild type isolate did not induce necrosis while the strains transformed with SnTox3 (Sn79+SnTox3A and Sn79+SnTox3B) induced necrosis. B. Virulence of the SnTox3 transformed strain Sn79+SnTox3A. Conidia from Sn79+SnTox3A were inoculated onto BR34, Grandin, BG220, BG261 and BG223 showing this strain was virulent on Grandin and BG220 which harbors Snn3, while it remained avirulent on BR34, BG261, and BG223 which harbors snn3. C. Toxin bioassay of SnTox3 disrupted strains. Leaves of BG220 (Snn3) were infiltrated with culture filtrate of Sn1501 wild type, Sn1501ΔSnTox3A, Sn1501ΔSnTox3B, and the ectopic strain Sn1501Ect and photographed 3 days after infiltration. Culture filtrates from the wild type and ectopic strains were able to induce necrosis while those from the two SnTox3-disrupted strains were unable to induce necrosis. D. Virulence comparison of Sn1501 wild type and the SnTox3 disrupted strains on BG220 (Snn3). Inoculation of conidia from Sn1501 wild type, Sn1501ΔSnTox3A, Sn1501ΔSnTox3B and Sn1501Ect were inoculated onto BG220 (Snn3) showing the loss of virulence of SnTox3 disrupted strains on the SnTox3 differential line BG220.

Figure 8. Interval map of chromosome 2D (Top) and 5B (bottom).

The map shows QTL of susceptibility associated with Snn2 and Snn3 loci in the BG population after being inoculated with genetically modified fungal strains. Strains are depicted by different colors as indicated. A centiMorgan scale is on the left of the map and markers are shown in their relative position along the right. An LOD scale is shown along the x axis, and the critical LOD threshold of 3.0 is indicated by the dotted line.

Figure 9. SnTox3 activity is sensitive to Dithiothreitol (DTT).

Leaves of BG220 were infiltrated with culture filtrates of P. pastoris expressing SnTox3 that had been treated for 2 h with 0 mM, 5 mM and 10 mM DTT.

Figure 10. Calculation of nucleotide diversity (Pi) at the SnTox3 coding region.

The calculations were done using a sliding window of size 10 with a step size of 5. Pi values of 0 indicate conserved regions of the gene for all 245 sequenced isolates.

Figure 11. The four protein variants of SnTox3 and their biological activity.

The different haplotypes of SnTox3 were amplified from the corresponding representative isolates and expressed in P. pastoris. The unfilled bars represent the complete SnTox3 protein and the number under the bar shows the position of each sequence variation indicated by pairs of amino acids (the first is the protein variant 1 and the substitution is in parenthesis). The biological activity of yeast clones expressing each protein haplotype is shown to the right.