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

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.


Introduction
In eukaryotes, the ubiquitin/26S proteasome system (UPS) is responsible for the selective degradation     TaAFR   139 The wheat FBK gene, TaFBK19, was selected for further analysis. This gene is similar to the Kelch 140 containing F-box AFR genes from other species and is therefore described here as TaAFR. 141 Plant material, fungal strains and inoculum preparation 142 A near-isogenic wheat line of Thatcher for leaf rust resistance, TcLr15, and leaf rust strains 143 05-5-137③ and 05-19-43② were used in the present study. Unless otherwise specified, plants were 144 grown in a greenhouse as described in Yu et al. [20]. Urediniospore and inoculum preparation of leaf 145 rust pathogens were carried out as previously described [20]. 146 TaAFR cloning 147 Total RNA extraction and first strand cDNA synthesis were performed as previously described [20]. A 148 pair of gene specific primers TaAFR-F and TaAFR-R (S1 Table) and Tks Gflex™ DNA Polymerase 149 (TaKaRa, Japan) were used to amplify the full-length coding sequences CDS amplified with Tks 150 Gflex™ DNA Polymerase (TaKaRa, Japan) according to manufacturer's directions with an annealing 151 temperature of 56.4°C. The purity of the amplicon was verified by 1.2 % agarose gel electrophoresis 152 and the product was sequenced to confirm the identity of the clone.

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The TaAFR sequence was used to pull out related sequences from the NCBI transcript database 154 using the BLASTp tool, and sequences with an expect threshold of <0.05 were aligned together with 155 TaAFR in MEGA 7.0 and a phylogenetic tree was constructed, as described in the section on 156 phylogenetic analysis. The TaAFR protein sequence was also analyzed using various bioinformatics 157 tools to predict presence of signal peptides (SignalP-4.1, www.cbs.dtu.dk/services/SignalP/), 158 transmembrane domains (TMHMM Server v. 2.0, www.cbs.dtu.dk/services/TMHMM/), and 159 subcellular localization (cropPAL2020 dataset). The 3D structure was predicted in Phyre2 160 (www.sbg.bio.ic.ac.uk/phyre2/).

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Wheat treatments and sampling for quantitative real-time PCR 162 Sampling of wheat for gene expression analysis was carried out in different tissues (for tissue-specific 163 analysis) and in response to three different types of abiotic stresses and three hormone treatments, as 164 described below. For each experiment, samples were collected from three replicates, unless otherwise 165 specified, 3-5 samples were harvested for each replicate. Samples were flash frozen in liquid nitrogen 166 and stored at −80 °C prior to RNA extraction.

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Samples were also collected at the same time points from untreated negative control plants in 180 Hoagland's solution.  Quantitative real-time PCR (qRT-PCR) 188 Total RNA was extracted from the TcLr15 samples collected in the previous section for gene 189 expression analysis, using Biozol reagent (BioFlux, Japan) according to manufacturer's instructions.

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To eliminate gDNA contamination, 2 ug of each RNA sample was treated with 1 uL gDNA Remover 191 (TransGen, China). cDNA synthesis was carried out as described by Yu et al. [20]. qRT-PCR was 192 performed on a Bio-Rad CFX Manager qRT-PCR instrument (Bio-Rad, America). cDNA was diluted qRT-PCR-TaAFR-R (S1 Table), and the reaction carried out with an annealing temperature of 58.3°C.

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A similar reaction was carried out using primers for the wheat reference gene GAPDH (GenBank: 197 AF251217) (primers qRT-PCR-GAPDH-F and qRT-PCR-GAPDH-R, annealing temperature of 198 58.3°C) (S1 Table). Three technical replicates were conducted for each of three biological replicates 199 per sample. The relative expression of TaAFR was evaluated as described by Yu et al. [20]. For    assessment. Compared to the other protein sequences, TaFBK65 had 3 additional residues (PVP) at the 287 N-terminus of the F-box motif; these 3 residues were removed in order to prepare the Kelch WebLogo.

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In general, the amino acid sequences within the Kelch motif were more divergent than that observed 289 within the F-box domain.       Table) and were used to construct a heat map using the zero to one normalized scale 338 method. Tissue-specific expression data (cultivar Chinese Spring) was available for 47 TaFBKs (Fig   339   2). In general, TaFBK genes exhibited differential expression in all five wheat tissues, suggesting that 340 these genes may be involved in the developmental regulation of multiple tissues. There were two 341 conditions where tissue-specific expression at specific developmental stages showed significantly less 342 transcript accumulation; these are leaf (z10) and grain (z75). Meanwhile, most TaFBK genes were 343 generally more abundantly expressed in the spikes (z32, z39, z65) and grains (z71, z85). TaFBK3  (Fig 4A). 385 Phylogenetic analysis indicated that the TaFBK19  (Nelumbo nucifera, Dendrobium catenatum and A. thaliana) were grouped in different clades (Fig 4C), 390 which indicates that these FBKs were conserved in monocots. Based on the similarities between 391 TaFBK19 and AFR genes from the cereal and monocot, TaFBK19 will henceforth be described as 392 TaAFR. Sequence analyses of TaAFR did not reveal any predicted signal peptide or transmembrane 393 domains, and the protein is predicted to localize to the cytosol. The predicted 3D structure showed 394 three distinct α-helices at the N-terminus and β-sheets at the C-terminal end. The β-sheets are 395 predicted to form 6 triangles, which further cluster to a regular hexagonal arrangement. These 396 secondary and ultra-secondary structures indicated that the protein folds into chair-like configuration 397 (Fig 4B).  TaAFR is primarily expressed in wheat leaves 406 Six tissues were sampled from wheat TcLr15 seedlings (root, leaf and stem) and adult plants (pistil, 407 stamen, flag leaf) to analyze the tissue-specific expression of TaAFR. The young leaf was used as a 408 control (the expression value was set 1.0) to measure it's relative expression to other tissues. TaAFR 409 was mainly expressed in young leaf, with lower expression in the flag leaf and extremely low 410 expression was detected in young root, pistil, and stamen ( Fig 5A).     physically interact with TaSkp1, TaSLY1, TaARL2, TaCYP51, TaPAL and TaNADH (Fig 7). constructs. If the gene products of the two constructs interact, a fluorescence signal will be emitted.

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Among the six combinations, three were found to emit fluorescent signals (Fig 8). pSPY CE-TaAFR proteins TaSkp1, TaARL2, TaPAL and negtive control GFP were successfully detected in the whole 512 cell lysates (WCL). After IP by HA-magnetic beads, the eluted proteins were subjected to immunoblot 513 analysis with anti-FLAG antibody, we found that TaSkp1, TaARL2 and TaPAL were 514 immunoprecipitated with TaAFR since a single band appeared at their corresponding MW sites, but no 515 band except HC (IgG heavy chain) was detected in the combination of TaAFR and GFP (Fig 9). Taken 516 together, these observations support that TaAFR interact with TaSkp1, TaARL2 and TaPAL in vivo.  example, the F-box protein containing two Kelch repeats in sugar beet, homologous to Arabidopsis 577 FBK AT1G74510, was found to interact with the beet necrotic yellow vein virus pathogenicity factor 578 P25, and it was speculated that P25 could affect the formation of SCF complex [44].

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Biotic and abiotic stress responses are often regulated by plant signaling hormones and exposure to 580 such stresses can activate these pathways [45]. It is therefore interesting that the expression of TaAFR interactions. First, using a leaf rust pathogen treated TcLr15 wheat leaf cDNA library, a Y2H library 589 screen was utilized as a broad scale approach to fish for candidate interacting proteins. A total of 13 590 candidates were identified, and 11 of these were cloned and re-screened by Y2H for interactions with 591 TaAFR. Additionally, a PAL gene, which was not identified in the pool, but has been shown to be 592 involved in regulation process of FBKs, was added to the list. Among these, a total of 6 interactions, 593 including the TaAFR-TaPAL interaction, were confirmed positives. However, since Y2H assays can 594 pick up false positives, these 6 genes were then validated using the BiFC and Co-IP methods, and 595 finally three partner proteins interacting with TaAFR were confirmed: TaSkp1, TaARL2, and TaPAL.

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To further characterize their detailed interacting domain, another Y2H assay was carried out   abiotic/biotic stresses [11,12]. Meanwhile, the work presented in this manuscript provides a glimpse 630 into their potential function, and opens the door for future studies to further characterize these genes.