Genome-wide survey of the F-box/Kelch (FBK) members and molecular identification of a novel FBK gene TaAFR in wheat.

Abstract

F-box proteins play critical roles in plant responses to biotic/abiotic stresses. In the present study, a total of 68 wheat F-box/Kelch (TaFBK) genes, unevenly distributed across 21 chromosomes and encoding 74 proteins, were identified in EnsemblPlants. Protein sequences were compared with those of Arabidopsis and three cereal species by phylogenetic and domain analyses, where the wheat sequences were resolved into 6 clades. In silico analysis of a digital PCR dataset revealed that TaFBKs were expressed at multiple developmental stages and tissues, and in response to drought and/or heat stresses. The TaFBK19 gene, a homolog of the Attenuated Far-Red Response (AFR) genes in other plant species, and hence named TaAFR, was selected for further analysis. Reverse-transcription quantitative real-time PCR (RT-qPCR) was carried out to determine tissue-specific, hormone and stress (abiotic/biotic) responsive expression patterns. Of interest, TaAFR was expressed most abundantly in the leaves, and its expression in response to leaf rust variants suggests a potential role in compatible vs incompatible rust responses. The protein was predicted to localize in cytosol, but it was shown experimentally to localize in both the cytosol and the nucleus of tobacco. A series of protein interaction studies, starting with a yeast-2-hybrid (Y2H) library screen (wheat leaf infected with incompatible leaf rust pathogens), led to the identification of three TaAFR interacting proteins. Skp1/ASK1-like protein (Skp1) was found to interact with the F-box domain of TaAFR, while ADP-ribosylation factor 2-like isoform X1 (ARL2) and phenylalanine ammonia-lyase (PAL) were shown to interact with its Kelch domain. The data presented herein provides a solid foundation from which the function and metabolic network of TaAFR and other wheat FBKs can be further explored.