A triple threat: the Parastagonospora nodorum SnTox267 effector exploits three distinct host genetic factors to cause disease in wheat

Abstract

Parastagonospora nodorum is a fungal pathogen of wheat. As a necrotrophic specialist, it deploys effector proteins that target dominant host susceptibility genes to elicit programmed cell death (PCD). Here we identify and functionally validate the effector targeting the host susceptibility genes Snn2, Snn6 and Snn7. We utilized whole-genome sequencing, association mapping, gene-disrupted mutants, gain-of-function transformants, virulence assays, bioinformatics and quantitative PCR to characterize these interactions. A single proteinaceous effector, SnTox267, targeted Snn2, Snn6 and Snn7 to trigger PCD. Snn2 and Snn6 functioned cooperatively to trigger PCD in a light-dependent pathway, whereas Snn7-mediated PCD functioned in a light-independent pathway. Isolates harboring 20 SnTox267 protein isoforms quantitatively varied in virulence. The diversity and distribution of isoforms varied between populations, indicating adaptation to local selection pressures. SnTox267 deletion resulted in the upregulation of effector genes SnToxA, SnTox1 and SnTox3. We validated a novel effector operating in an inverse-gene-for-gene manner to target three genetically distinct host susceptibility genes and elicit PCD. The discovery of the complementary gene action of Snn2 and Snn6 indicates their potential function in a guard or decoy model. Additionally, differences in light dependency in the elicited pathways and upregulation of unlinked effectors sheds new light onto a complex fungal necrotroph-host interaction.

Keywords: Parastagonospora nodorum; association mapping; fungi; genomics; necrotrophic effector; programmed cell death; wheat.

Fig. 1

Identification of markers associated with virulence on wheat lines BG223 (Snn2) and ITMI37 (Snn6) (BG, BR34 × Grandin recombinant inbred line population; ITMI, International Triticeae Mapping Initiative). (a) Histogram showing the distribution of disease scores for 197 Parastagonospora nodorum isolates on BG223 (left) and ITMI37 (right). x‐axis, disease score; y‐axis, frequency. (b) Manhattan plot illustrating markers associated with virulence on wheat line BG223. x‐axis, P. nodorum chromosomes; y‐axis, marker significance. The red line indicates a Bonferroni‐corrected P‐value threshold of 0.01. (c) Manhattan plot illustrating markers associated with virulence on wheat line ITMI37. x‐axis, P. nodorum chromosomes; y‐axis, marker significance. The red line indicates a Bonferroni corrected P‐value threshold of 0.01. (d) Distribution of virulence phenotypes on BG223 (left) and ITMI37 (right) for isolates that harbor the avirulent (A) and virulent (G) alleles of SNP_66420. Boxplots illustrate the distribution of virulence phenotypes on wheat lines BG223 (pink) and ITMI37 (teal). Thick horizontal lines within boxes denote the median. The height of each box represents the interquartile range. Dots represent the disease phenotype from an individual isolate. x‐axis, allele categories; y‐axis, disease score.

Fig. 2

Disruption of CJJ_13380 results in the loss of virulence on lines harboring Snn2. (a) Wheat line BG296 (Snn2 differential line) inoculated with Sn4 wild‐type (WT), Sn4 ectopic transformant, and two independent mutants with a disrupted CJJ_13380 gene. (b) Quantitative trait locus (QTL) analysis of the BR34 × Grandin (BG) population with the Sn4 WT, Sn4 ectopic and two CJJ_13380‐disrupted isolates. x‐axes, genetic positions along wheat chromosome 2D (in cM); y‐axis, logarithm of the odds (LOD). The red dotted line corresponds to a LOD threshold of P = 0.05 as determined by 1000 permutations conducted for each analysis separately.

Fig. 3

Inoculation of gain‐of‐function transformant Sn79+CJJ13380 and infiltration of culture filtrates result in virulence or cell death on lines harboring Snn2. (a) Inoculation of BG296 (Snn2 differential line) with gain‐of‐function transformant Sn79+CJJ13380 and Sn79 WT (BG, BR34 × Grandin). (b) Infiltration of Sn79+CJJ13380 culture filtrates on wheat lines Grandin (sensitive) and BR34 (insensitive). (c) Quantitative trait locus (QTL) analysis of the BG population with the Sn79+CJJ13380 isolate (left) and infiltration of Sn79+CJJ13380 culture filtrates (right). x‐axes, genetic positions along wheat chromosome 2D (in cM); y‐axis, logarithm of the odds (LOD). The red dotted line corresponds to a LOD threshold of P = 0.05 as determined by 1000 permutations conducted for each analysis separately.

Fig. 4

Disruption of CJJ_13380 results in the loss of virulence on lines harboring Snn6. (a) Wheat line ITMI37 (Snn6 differential line) inoculated with Sn4 wild‐type (WT), Sn4 ectopic transformant and two independent mutants with a disrupted CJJ_13380 gene. (b) Quantitative trait locus (QTL) analysis of the (ITMI, International Triticeae Mapping Initiative) population with the Sn4 WT, Sn4 ectopic and two CJJ_13380‐disrupted isolates. x‐axes, genetic positions along wheat chromosome 6A (in cM); y‐axis, logarithm of the odds (LOD). The red dotted line corresponds to a LOD threshold of P = 0.05 as determined by 1000 permutations conducted for each analysis separately.

Fig. 5

Inoculation of gain‐of‐function transformant Sn79+CJJ13380 and infiltration of culture filtrates result in virulence or cell death on lines harboring Snn6. (a) Inoculation of ITMI37 (Snn6 differential line) with gain‐of‐function transformant Sn79+CJJ13380 and Sn79 wild‐type (WT) (ITMI, International Triticeae Mapping Initiative). (b) Infiltration of Sn79+CJJ13380 culture filtrates on wheat lines Opata 85 (sensitive) and W7984 (insensitive). (c) Quantitative trait locus (QTL) analysis of the ITMI population with the Sn79+CJJ13380 isolate (left) and infiltration of Sn79+CJJ13380 culture filtrates (right). x‐axes, genetic positions along wheat chromosome 6A (in cM); y‐axis, logarithm of the odds (LOD). The red dotted line corresponds to a LOD threshold of P = 0.05 as determined by 1000 permutations conducted for each analysis separately.

Fig. 6

Snn2 and Snn6 are both required to produce SnTox267‐mediated cell death. Culture filtrates of the gain‐of‐function transformant Sn79+CJJ13380 were infiltrated in wheat lines BR34 (snn2/Snn6), F₁ plants (n = 3) derived from a cross of BR34 and W7984, W7984 (Snn2/snn6), and F₂ (n = 49) plants derived from a cross of BR34 and W7984. ‘I’, insensitive reactions; ‘S’, sensitive reactions.

Fig. 7

Sequence diversity of SnTox267 in a natural population (n = 197) of Parastagonospora nodorum. (a) Gene model of SnTox267 located on chromosome 14. The arrow corresponds to the single exon structure of SnTox267. Colored boxes illustrate the locations of polymorphisms (annotated in legend). (b) Nucleotide diversity within the SnTox267 coding region. x‐axis, position (bp); y‐axis, nucleotide diversity (π). (c) Prevalence of protein isoforms in discrete P. nodorum populations. x‐axis, specific isoforms; y‐axis, frequency of each isoform. Colors shown in the legend (upper right) correspond to discrete populations previously determined by Richards et al., (2019). (d) Quantitative virulence profiles of protein isoform groups with significant representation (n > 10). Boxplots illustrate the distribution of virulence phenotypes for isolates with a specific SnTox267 protein isoform. Thick horizontal lines within boxes denote the median. The height of each box represents the interquartile range. Dots represent outlier values. x‐axis, specific isoforms; y‐axis, disease ratings. Letter codes signify significantly different groups as determined by a Dunn’s multiple comparisons test (Bonferroni‐adjusted P‐value < 0.10).

Fig. 8

Disruption of SnTox267 resulted in the upregulation of unrelated effectors, leading to detection of corresponding susceptibility loci. (a) Quantitative trait locus (QTL) analysis of the International Triticeae Mapping Initiative (ITMI) population inoculated with Sn4 wild‐type (WT), Sn4 ectopic transformant and two Sn4∆CJJ1380 mutants. QTL corresponding to Snn1 on wheat chromosome 1B (left) and Snn3 on chromosome 5B (right) were only detected following inoculation with the gene‐disrupted mutants. x‐axes, genetic positions (cM); y‐axes, logarithm of the odds (LOD). The red dotted line corresponds to a LOD threshold of P = 0.05 as determined by 1000 permutations conducted for each analysis separately. (b) A quantitative PCR assay to evaluate differences in SnTox1, SnTox3 and SnToxA expression at five in planta time points (24, 48, 72, 96 and 120 h postinoculation (hpi)). x‐axes, time points; y‐axis, relative expression values. Bar colors correspond to either the WT or knockout strain Sn4∆CJJ13380‐5 (legend on the right). Error bars, SEM. ‘*’ represents significant upregulation in the knockout compared to the WT at the same time point (Student’s t‐test, P < 0.05).