A Conserved Hypothetical Gene Is Required but Not Sufficient for Ptr ToxC Production inPyrenophora tritici-repentis

Abstract

The fungus Pyrenophora tritici-repentis (Ptr) causes tan spot, an important foliar disease of wheat worldwide. The fungal pathogen produce three necrotrophic effectors, namely Ptr ToxA, Ptr ToxB and Ptr ToxC to induce necrosis or chlorosis in wheat. Both Ptr ToxA and Ptr ToxB are proteins, and their encoding genes have been cloned. Ptr ToxC was characterized as a low-molecular-weight molecule 20 years ago but the gene(s) controlling its production in Ptr are unknown. Here, we report the genetic mapping, molecular cloning and functional analysis of a fungal gene that is required for Ptr ToxC production. The genetic locus controlling the production of Ptr ToxC, termed as ToxC, was mapped to a subtelomeric region using segregating bi-parental populations, genome sequencing, and association analysis. Additional marker analysis further delimited ToxC to a 173 kb region. The predicted genes in the region were examined for presence/absence polymorphism in different races/isolates leading to the identification of a single candidate gene. Functional validation showed that this gene was required but not sufficient for Ptr ToxC production, thus it is designated as ToxC1. ToxC1 encoded a conserved hypothetical protein likely located on the vacuole membrane. The gene was highly expressed during infection, and only one haplotype was identified among 120 isolates sequenced. Our work suggests that Ptr ToxC is not a protein and is likely produced through a cascade of biosynthetic pathway. The identification of ToxC1 is a major first step towards revealing the Ptr ToxC biosynthetic pathway and studying its molecular interactions with host factors.

A global pangenome for the wheat fungal pathogen Pyrenophora tritici-repentis and prediction of effector protein structural homology

“…When all sequenced isolates were considered, only a single locus for a transmembrane protein, an integral membrane component, was identified core to all ToxC-producing isolates, represented by the M4 protein (KAF7575752) on chr2 position 5 052 985–5 053 659 bp (). This gene was recently identified as ToxC1 , a gene required but not sufficient for ToxC production in Ptr [78]. A less stringent search for ToxC1 in all isolates detected the presence of ToxC1 in the race 2 isolate Biotrigo9-1 genome, which was disrupted by a large insertion of 5 348 bp, positioned at 45 946–51 292 bp on contig 12, which disrupted the ToxC1 protein coding region in the 582–583 bp position.…”

A global pangenome for the wheat fungal pathogen Pyrenophora tritici-repentis and prediction of effector protein structural homology

“…1D). This gene was recently identified as ToxC1 , a gene required but not sufficient for ToxC production in Ptr (Shi, Kariyawasam et al 2022). A less stringent search for ToxC1 in all isolates detected the presence of ToxC1 in the race 2 isolate Biotrigo9-1 genome, which was disrupted by a large insertion of 5,348 bp, positioned at 45,946 to 51,292 bp on contig 12, which disrupted the ToxC1 protein coding region in the 582-583 bp position.…”

“…Interestingly, our analysis found no putative effectors that were core to all isolates, again indicating a large variability within Ptr for this type of gene. Recently, ToxC1 was functionally validated using a gene knockout approach (Shi, Kariyawasam et al 2022), where it was found to be required, but not sufficient, for ToxC production. In our study, no clear gene cluster for a secondary metabolite or Ribosomally synthesized and post-translationally modified peptides (RiPPs) was identified, in part due to the positioning of the ToxC locus within the complex subtelomeric region of chromosome 2 (Shi, Kariyawasam et al 2022), which despite long read sequencing still remains a problematic region to resolve.…”

“…Recently, ToxC1 was functionally validated using a gene knockout approach (Shi, Kariyawasam et al 2022), where it was found to be required, but not sufficient, for ToxC production. In our study, no clear gene cluster for a secondary metabolite or Ribosomally synthesized and post-translationally modified peptides (RiPPs) was identified, in part due to the positioning of the ToxC locus within the complex subtelomeric region of chromosome 2 (Shi, Kariyawasam et al 2022), which despite long read sequencing still remains a problematic region to resolve. The presence of ToxC1 in a non-ToxC producing isolate (Biotrigo9-1) was surprising, and raises more questions regarding the evolution and/or origin of ToxC production.…”

Dissecting thePyrenophora tritici-repentis(tan spot of wheat) pangenome

“…ToxA and ToxB are ribosomally-synthesized proteins, and while ToxA is encoded by a single copy ToxA gene, ToxB is encoded by the multi-copy ToxB genes (3). Understanding of ToxC-coding gene(s) and its synthesis awaits further characterization (5, 6). In Ptr, the ToxA gene is present only in isolates that secrete the ToxA effector, and no homolog has been identified in non-producing isolates of Ptr.…”