Genome-Wide Identification and Analysis of the ABCF Gene Family in Triticum aestivum

Abstract

The ATP-binding cassette (ABC) superfamily of proteins is a group of evolutionarily conserved proteins. The ABCF subfamily is involved in ribosomal synthesis, antibiotic resistance, and transcriptional regulation. However, few studies have investigated the role of ABCF in wheat (Triticum aestivum) immunity. Here, we identified 18 TaABCFs and classified them into four categories based on their domain characteristics. Functional similarity between Arabidopsis and wheat ABCF genes was predicted using phylogenetic analysis. A comprehensive genome-wide analysis of gene structure, protein motifs, chromosomal location, and cis-acting elements was also performed. Tissue-specific analysis and expression profiling under temperature, hormonal, and viral stresses were performed using real-time quantitative reverse transcription polymerase chain reaction after randomly selecting one gene from each group. The results revealed that all TaABCF genes had the highest expression at 25 °C and responded to methyl jasmonate induction. Notably, TaABCF2 was highly expressed in all tissues except the roots, and silencing it significantly increased the accumulation of Chinese wheat mosaic virus or wheat yellow mosaic virus in wheat leaves. These results indicated that TaABCF may function in response to viral infection, laying the foundation for further studies on the mechanisms of this protein family in plant defence.

Keywords: ABCF gene family; CWMV; biotic stress; expression; genome-wide; wheat.

Figure 1

Conserved domain analysis of the TaABCF protein family. Based on the conserved domain analysis, the 18 TaABCFs could be divided into four groups: ABCF1, ABCF2, ABCF3, and ABCF4.

Figure 2

A phylogenetic tree of ABCF proteins in Arabidopsis, wheat, rice, and maize constructed using the neighbour-joining method in MEGA-X. The number at the node represents the guidance value after 1000 iterations. Each group is represented by a different colour. Stars represent Arabidopsis, triangles represent wheat, circles represent rice, and rectangles represent maize.

Figure 3

Predictive structure of ABCF proteins in Arabidopsis and wheat. Randomly selected protein models from each group: ABCF1 (AT1G60790, TraesCS7B02G072300.1); ABCF2 (AT5G64840, TraesCS4A02G156300.1); ABCF3 (AT1G64550, TraesCS6A02G409800.1); and ABCF4 (AT3G54540, TraesCS3B02G388600.1). SWISS-MODEL was used for structural prediction. Based on QMEAN and GMQE, the model with the optimum results was selected.

Figure 4

Gene structures and conserved motifs of TaABCF. (a) Distribution of all motifs identified by MEME. Differently coloured frames represent different protein motifs, and each motif has its own number. (b) Exon–intron structures of 18 TaABCF genes. Exons, introns, and untranslated regions are indicated by yellow frames, grey lines, and green frames on the right, respectively.

Figure 5

Synteny analysis and chromosomal localisation of ABCFs in wheat. Differently coloured lines indicate duplicated TaABCF pairs on different chromosomes: red lines indicate duplicated TaABCF pairs on chromosomes 1, orange lines indicate duplicated TaABCF pairs on chromosomes 2, yellow lines indicate duplicated TaABCF pairs on chromosomes 3, green lines indicate duplicated TaABCF pairs on chromosomes 4, blue lines indicate duplicated TaABCF pairs on chromosomes 6, purple lines indicate duplicated TaABCF pairs on chromosomes 7. Grey lines indicate synthesis results for the T. aestitum genome, and the position of TaABCF is marked directly on the chromosome.

Figure 6

Prediction of cis-acting elements of TaABCF. TaABCF genes are shown on the left. Distinct colours indicate different subfamilies, and differently coloured boxes indicate different cis-acting elements. Names of cis-acting elements are shown on the right.

Figure 7

Tissue-specific differential expression of TaABCFs. (a) Model for five tissue types of wheat. TL, top leaf; ML, middle leaf; BL, bottom leaf; ST, stem; RT, root. (b–e) Relative expression of TaABCFs. Three independent biological replicates were used to calculate the mean expression of TaABCFs in other tissues relative to that in roots. The asterisks indicate significant differences determined by Student’s t-test (* p < 0.05). ns, no significant difference. Error bars, results are shown as mean ± SD.

Figure 8

Expression of TaABCFs under different stresses. (a) Relative expression of TaABCF was measured using RT-qPCR in plants grown at different temperatures for 14 d. Mean expression values were calculated from three independent biological replicates and three technical replicates. The 8 °C treatment was used as a control. (b,c) Relative expression of four TaABCF genes in the leaves of wheat seedlings at 2, 4, 6, and 8 h after ABA or MeJA hormone treatment. Treatment after 2 h was used as a control. Three biological replication experiments were performed for each treatment, and gene expression was detected via RT-qPCR and visualised using GraphPad Prism 9.5 software. The asterisks indicate significant differences determined using Student’s t-test (* p < 0.05). ns, no significant difference. Error bars, results are shown as mean ± SD.

Figure 9

Relative expression of TaABCF was detected by RT-qPCR in plants inoculated with different viruses. (a) Changes in gene expression of wheat seedlings 14 d after CWMV infection. Healthy wheat samples from the same lot were used as controls. (b) Changes in gene expression of wheat seedlings after 14 d of WYMV infection. Healthy wheat samples from the same lot were used as controls. (c) Detection of changes in gene expression in wheat seedlings after 14 d of BSMV infection; plants inoculated with 1 × FES buffer (Mock) were used as a negative control. All expression values are presented as mean ± SEM and were calculated using three independent biological replicates and three technical replicates. The asterisks indicate significant differences determined by Student’s t-test (* p < 0.05). ns, no significant difference. Abbreviations: BSMV, barley stripe mosaic virus; CWMV, Chinese wheat mosaic virus; RT-qPCR, real-time quantitative reverse transcription polymerase chain reaction; SEM, standard error of the mean; WYMV, wheat yellow mosaic virus.

Figure 10

TaABCF2 was involved in wheat resistance to CWMV or WYMV infection. (a) Mosaic symptoms on wheat leaves infected with CWMV; BSMV:TaPDS (phytoene desaturase gene, acting as a positive control); BSMV:00 + CWMV; or BSMV:TaABCF2 + CWMV. Mock leaves were inoculated with excess inoculation buffer as a control. Photographs were taken at 21 dpi. (b) Relative expression of TaABCF2 in CWMV-inoculated wheat plants was analysed using RT-qPCR. (c) The accumulation of CWMV RNA during TaABCF2 silencing was analysed via RT-qPCR at 21 dpi using CWMV CP-specific primers. (d) Mosaic symptoms in the wheat leaves infected with WYMV, BSMV:TaPDS, BSMV:00 + WYMV, or BSMV:TaABCF2 + WYMV. (e) Relative expression of TaABCF2 in WYMV-inoculated wheat plants was analysed using RT-qPCR. (f) The accumulation of WYMV RNA during TaABCF2 silencing was analysed using RT-qPCR at 21 dpi using WYMV CP-specific primers. (g) Yeast two-hybrid (Y2H) assay to verify the interaction of TaABCF2 with CWMV-related proteins. AD-TaABCF2 was co-expressed with BD-CRP, BD-CP, BD-P153, BD-Rd4, BD-MP, BD-NCP, and BD-CPRT in yeast cells, and the transformed cells were grown on SD/−Leu/−Trp medium and then on SD/−Trp/−Leu/−His/−Ade medium to determine protein–protein interactions. Yeast cells co-transformed with AD-T + BD-lam were used as a negative control, and yeast cells co-transformed with AD-T + BD-53 were used as a positive control. (h) Y2H assay to verify the interaction of TaABCF2 with WYMV-associated proteins. AD-TaABCF2 was co-expressed with BD-NIa, BD-NIb, BD-7K, BD-14K, BD-P1, BD-P2, BD-P3, BD-CI, BD-CP, and BD-VPg in yeast cells, and the transformed cells were grown on SD/−Leu/−Trp medium and then on SD/−Trp/−Leu/−His/−Ade medium to determine protein–protein interactions. Yeast cells co-transformed with AD-T + BD-lam were used as a negative control, and yeast cells co-transformed with AD-T + BD-53 were used as a positive control. All RT-qPCR data are presented as means ± SD, as determined using the Student’s t-test. Each treatment had three biological replicates (* p < 0.05). Abbreviations: BSMV, barley stripe mosaic virus; CWMV, Chinese wheat mosaic virus; RT-qPCR, real-time quantitative reverse transcription polymerase chain reaction; SD, standard deviation; Y2H, yeast two-hybrid; WYMV, wheat yellow mosaic virus.

Figure 11

A model of the function of TaABCFs during virus infection. TaABCF2 in wheat can promote infection by CWMV and WYMV. The arrows represent positive regulation, perpendicular lines represent negative regulation, and solid lines represent results by determination.