Genetics of variable disease expression conferred by inverse gene-for-gene interactions in the wheat-parastagonospora nodorum pathosystem

The wheat-Parastagonospora nodorum pathosystem involves the recognition of pathogen-secreted necrotrophic effectors (NEs) by corresponding wheat NE sensitivity genes. This inverse gene-for-gene recognition leads to necrotrophic effector-triggered susceptibility and ultimately septoria nodorum blotch disease. Here, we used multiple pathogen isolates to individually evaluate the effects of the host gene-NE interactions Tan spot necrosis1-Stagonospora nodorum ToxinA (Tsn1-SnToxA), Stagonospora nodorum necrosis1-Stagonospora nodorum Toxin1 (Snn1-SnTox1), and Stagonospora nodorum necrosis3-B genome homeolog1-Stagonospora nodorum Toxin3 (Snn3-B1-SnTox3), alone and in various combinations, to determine the relative importance of these interactions in causing disease. Genetic analysis of a recombinant inbred wheat population inoculated separately with three P. nodorum isolates, all of which produce all three NEs, indicated that the Tsn1-SnToxA and Snn3-B1-SnTox3 interactions contributed to disease caused by all four isolates, but their effects varied and ranged from epistatic to additive. The Snn1-SnTox1 interaction was associated with increased disease for one isolate, but for other isolates, there was evidence that this interaction inhibited the expression of other host gene-NE interactions. RNA sequencing analysis in planta showed that SnTox1 was differentially expressed between these three isolates after infection. Further analysis of NE gene-knockout isolates showed that the effect of some interactions could be masked or inhibited by other compatible interactions, and the regulation of this occurs at the level of NE gene transcription. Collectively, these results show that the inverse gene-for-gene interactions leading to necrotrophic effector-triggered susceptibility in the wheat-P. nodorum pathosystem vary in their effects depending on the genetic backgrounds of the pathogen and host, and interplay among the interactions is complex and intricately regulated

The Cysteine Rich Necrotrophic Effector SnTox1 Produced by Stagonospora nodorum Triggers Susceptibility of Wheat Lines Harboring Snn1

The wheat pathogen Stagonospora nodorum produces multiple necrotrophic effectors (also called host-selective toxins) that promote disease by interacting with corresponding host sensitivity gene products. SnTox1 was the first necrotrophic effector identified in S. nodorum, and was shown to induce necrosis on wheat lines carrying Snn1. Here, we report the molecular cloning and validation of SnTox1 as well as the preliminary characterization of the mechanism underlying the SnTox1-Snn1 interaction which leads to susceptibility. SnTox1 was identified using bioinformatics tools and verified by heterologous expression in Pichia pastoris. SnTox1 encodes a 117 amino acid protein with the first 17 amino acids predicted as a signal peptide, and strikingly, the mature protein contains 16 cysteine residues, a common feature for some avirulence effectors. The transformation of SnTox1 into an avirulent S. nodorum isolate was sufficient to make the strain pathogenic. Additionally, the deletion of SnTox1 in virulent isolates rendered the SnTox1 mutated strains avirulent on the Snn1 differential wheat line. SnTox1 was present in 85% of a global collection of S. nodorum isolates. We identified a total of 11 protein isoforms and found evidence for strong diversifying selection operating on SnTox1. The SnTox1-Snn1 interaction results in an oxidative burst, DNA laddering, and pathogenesis related (PR) gene expression, all hallmarks of a defense response. In the absence of light, the development of SnTox1-induced necrosis and disease symptoms were completely blocked. By comparing the infection processes of a GFP-tagged avirulent isolate and the same isolate transformed with SnTox1, we conclude that SnTox1 may play a critical role during fungal penetration. This research further demonstrates that necrotrophic fungal pathogens utilize small effector proteins to exploit plant resistance pathways for their colonization, which provides important insights into the molecular basis of the wheat-S. nodorum interaction, an emerging model for necrotrophic pathosystems

Characterization of the Interaction of a Novel Stagonospora nodorum Host-Selective Toxin with a Wheat Susceptibility Gene1[W]

Recent work suggests that the Stagonospora nodorum-wheat pathosystem is controlled by host-selective toxins (HSTs; SnToxA, SnTox1, and SnTox2) that interact directly or indirectly with dominant host genes (Tsn1, Snn1, and Snn2) to induce disease. Here we describe and characterize a novel HST designated SnTox3, and the corresponding wheat sensitivity/susceptibility gene identified on chromosome arm 5BS, which we designated as Snn3. SnTox3 is a proteinaceous necrosis-inducing toxin between 10 and 30 kD in size. The S. nodorum isolates Sn1501 (SnToxA−, SnTox2+, and SnTox3+), SN15 (SnToxA+, SnTox2+, and SnTox3+), and SN15KO18, a strain of SN15 with a disrupted form of SnToxA, were evaluated on a population of wheat recombinant inbred lines. A compatible Snn3-SnTox3 interaction played a significant role in the development of disease caused by isolates Sn1501 and SN15KO18, with Snn2 being epistatic to Snn3. Snn3 was not significantly associated with disease caused by SN15 presumably due to the major effects observed for Snn2 and Tsn1, which were largely additive. This work introduces a fourth HST produced by S. nodorum and builds on the notion that the S. nodorum-wheat pathosystem is largely based on multiple host-toxin interactions that follow an inverse gene-for-gene scenario

Molecular Characterization of the Major Wheat Domestication Gene Q

The Q gene is largely responsible for the widespread cultivation of wheat because it confers the free-threshing character. It also pleiotropically influences many other domestication-related traits such as glume shape and tenacity, rachis fragility, spike length, plant height, and spike emergence time. We isolated the Q gene and verified its identity by analysis of knockout mutants and transformation. The Q gene has a high degree of similarity to members of the AP2 family of transcription factors. The Q allele is more abundantly transcribed than q, and the two alleles differ for a single amino acid. An isoleucine at position 329 in the Q protein leads to an abundance of homodimer formation in yeast cells, whereas a valine in the q protein appears to limit homodimer formation. Ectopic expression analysis allowed us to observe both silencing and overexpression effects of Q. Rachis fragility, glume shape, and glume tenacity mimicked the q phenotype in transgenic plants exhibiting post-transcriptional silencing of the transgene and the endogenous Q gene. Variation in spike compactness and plant height were associated with the level of transgene transcription due to the dosage effects of Q. The q allele is the more primitive, and the mutation that gave rise to Q occurred only once leading to the world's cultivated wheats

Association mapping of resistance to tan spot in the Global Durum Panel

Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr), is an important disease of durum and common wheat worldwide. Compared to common wheat, less is known about the genetics and molecular basis of tan spot resistance in durum wheat. We evaluated 510 durum lines from the Global Durum wheat Panel (GDP) for sensitivity to the necrotrophic effectors (NEs) Ptr ToxA and Ptr ToxB, and for reaction to Ptr isolates representing races 1–5. Overall, susceptible durum lines were most prevalent in South Asia, the Middle East, and North Africa. Genome-wide association analysis showed the resistance locus Tsr7 was significantly associated with tan spot caused by races 2 and 3, but not races 1, 4, or 5. The NE sensitivity genes Tsc1 and Tsc2 were associated with susceptibility to Ptr ToxC- and Ptr ToxB-producing isolates, respectively, but Tsn1 was not associated with tan spot caused by Ptr ToxA-producing isolates, which further validates that the Tsn1-Ptr ToxA interaction does not play a significant role in tan spot development in durum. A unique locus on chromosome arm 2AS was associated with tan spot caused by race 4, a race once considered avirulent. A novel trait characterized by expanding chlorosis leading to increased disease severity caused by the Ptr ToxB-producing race 5 isolate DW5 was identified, and this trait was governed by a locus on chromosome 5B. We recommend that durum breeders select resistance alleles at the Tsr7, Tsc1, Tsc2, and the chromosome 2AS loci to obtain broad resistance to tan spot

Genomic location, structure and deduced amino acid sequence of <i>SnTox1</i>.

<p><b>A</b>. <i>SnTox1</i> genomic region. <i>SnTox1</i> is located in supercontig 10 of the assembled SN15 genome sequence and is surrounded by four other predicted genes (boxed arrows, SNOG7153 to SNOG7156). A short truncated molly-type retrotransposon sequence (gray rectangle) closely follows <i>SnTox1</i>. <b>B</b>. <i>SnTox1</i> gene structure. The full length transcript of <i>SnTox1</i> and contains three exons (black rectangles) and both 5′ and 3′ untranslated regions (white rectangles). <b>C</b>. SnTox1 amino acid sequence. SnTox1 protein contains 117 amino acids with the first 17 (in bold) being a predicted signal sequence. The 16 cysteine residues are underlined. <b>D</b>. The alignment of 11 different SnTox1 protein isoforms. Two regions of SnTox1, from 36 to 87 and 97 to 117 are shown to indicate the variable amino acid positions (arrows). The remaining amino acids, (except for the cysteine residues) are shown as stars, indicating they are conserved.</p

<i>SNOG_20078</i> encodes SnTox1.

<p>The reaction of wheat lines W-7984 (<i>Snn1</i>), Chinese Spring (CS) (<i>Snn1</i>), CS 1BS-18 (<i>snn1</i>) and CS ems237 (<i>snn1</i>) to culture filtrates of a <i>Pichia pastoris</i> strain transformed with <i>SNOG_20078</i>.</p

SnTox1-<i>Snn1</i> interaction induces increased defense gene expression.

<p><b>A</b>. Expression of three pathogenesis-related (PR) genes using RT-PCR. CS (<i>Snn1</i>) and CS ems237-1 (<i>snn1</i>) leaf samples were collected at 1, 2, 4, 8, 12, 24, 36, 48, 60, and 72 h after infiltration with SnTox1 or control yeast culture filtrates. RT-PCR was conducted to compare the expression of three PR-genes (<i>PR-1-A1</i>, <i>chitinase</i>, and <i>thaumatin</i>) among four different interactions including CS infiltrated with SnTox1 culture filtrates (CS/+SnTox1), CS infiltrated with control culture filtrates (CS/-SnTox1), CS ems237 infiltrated with SnTox1 (ems/+SnTox1), and CS ems237 infiltrated with control culture filtrates (ems/-SnTox1). The wheat 18S gene was used as an RNA quantity control. <b>B</b>. Expression of three PR genes using qPCR. Comparisons were made among the four different interactions described above. The same RNA samples from RT-PCR were used in qPCR analysis. The relative expression level for each time point was normalized to the wheat 18S gene.</p