Deciphering the role of a taxonomically restricted orphan gene TaFROG, in wheat resistance to Fusarium Head Blight disease

Fusarium head blight (FHB) is a major disease of wheat and other small-grain crops, causing economically important losses for farmers due to the associated yield loss and contamination of grains with mycotoxins. Fusarium graminearum, the major causal agent of FHB, is a hemibiotrophic fungus capable of producing trichothecenes mycotoxins such as deoxynivalenol (DON) that facilitate its’ spread through wheat spikes. The Pooideae-specific Triticum aestivum Fusarium Resistance Orphan Gene protein (TaFROG) and its interacting proteins Triticum aestivum NAC-like D1 (TaNACL-D1) and Triticum aestivum sucrose non-fermenting-1-related protein kinase 1 catalytic subunit a (TaSnRK1a) are among the genes known to enhance wheat resistance to FHB disease. The main objective of this study was to determine the pathways under the direct or indirect control of TaFROG. Using RNA sequencing, we investigated the transcriptome of a transgenic wheat line overexpressing TaFROG before and during F. graminearum infection of spikes. The transcriptomic data analysis revealed that, in the absence of the pathogen, defence-related genes involved in pathogen-recognition and fungal cell wall degradation were up-regulated in TaFROG overexpression line spikes, as compared to WT. In addition, 24 hours after Fusarium inoculation, a higher number of transcripts was found to be Fusarium-responsive in the line overexpressing TaFROG (180 transcripts) as compared to WT spikes (61 transcripts). These results indicated that TaFROG primes the plant to mount a greater/faster response to F. graminearum. From this transcriptomic analysis, we identified Triticum aestivum C2 domain-containing protein kinase chromosome 2A copy (TaC2Kin-2A) as a potential FHB resistance gene associated with TaFROG activity and used virus-induced gene silencing studies to assess its contribution to FHB resistance in wheat spikes. However, we failed to induce gene silencing and therefore cannot draw any conclusions regarding its role in FHB resistance. Phytohormones such as of jasmonic (JA) and salicylic acid (SA) play an important role in resistance against environmental stresses such as FHB disease. Previous results obtained in our lab suggested that TaFROG may promote the induction of the JA signalling pathway. To test that, we developed wheat marker genes for JA and SA signalling pathways using knowledge from the model plant Arabidopsis thaliana combined with in silico analysis of the wheat genome and gene expression data. We identified a allene oxide synthase (TaAOS1-5B) and a MYB transcription factor (TaJAMyb-4A) genes as JA-responsive markers, and a WRKY transcription factor (TaWRKY45-B) as a SA-responsive markers in cvs. Remus and CM82036 leaves. Using those markers, we confirmed that TaFROG overexpression leads to up-regulation of the JA-responsive marker TaJAMyb-4A in wheat spikes during anthesis. These results indicated that JA signalling is promoted by TaFROG overexpression in the absence of Fusarium. In addition to being involved in FHB resistance, JA is also involved in abiotic stress response, and OsJAMyb the rice ortholog of the TaFROG-induced gene TaJAMyb-4A was found to enhance tolerance to high salinity when overexpressed in A. thaliana. Hence, TaFROG warrants further study to determine its potential to enhance resistance against other stresses. Taken together, our results demonstrate that an orphan gene can increase FHB resistance in wheat by priming defences. Orphan genes, while often difficult to study due to their lack of known domains, warrant more in-depth studies to determine their potential as disease resistance genes.