Genome-wide analysis of bHLH transcription factor and involvement in the infection by yellow leaf curl virus in tomato (Solanum lycopersicum)

Abstract

Background: The basic helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that can bind to specific DNA target sites. They have been well characterized in model plants such as Arabidopsis and rice and have been shown to be important regulatory components in many different biological processes. However, no systemic analysis of the bHLH transcription factor family has yet been reported in tomatoes. Tomato yellow leaf curl virus (TYLCV) threatens tomato production worldwide by causing leaf yellowing, leaf curling, plant stunting and flower abscission.

Results: A total of 152 bHLH transcription factors were identified from the entire tomato genome. Phylogenetic analysis of bHLH domain sequences from Arabidopsis and tomato facilitated classification of these genes into 26 subfamilies. The evolutionary and possible functional relationships revealed during this analysis are supported by other criteria, including the chromosomal distribution of these genes, the conservation of motifs and exon/intron structural patterns, and the predicted DNA binding activities within subfamilies. Distribution mapping results showed bHLH genes were localized on the 12 tomato chromosomes. Among the 152 bHLH genes from the tomato genome, 96 bHLH genes were detected in the TYLCV-susceptible and resistant tomato breeding line before (0 dpi) and after TYLCV (357 dpi) infection. As anticipated, gene ontology (GO) analysis indicated that most bHLH genes are related to the regulation of macromolecule metabolic processes and gene expression. Only four bHLH genes were differentially expressed between 0 and 357 dpi. Virus-induced gene silencing (VIGS) of one bHLH genes SlybHLH131 in resistant lines can lead to the cell death.

Conclusion: In the present study, 152 bHLH transcription factor genes were identified. One of which bHLH genes, SlybHLH131, was found to be involved in the TYLCV infection through qRT-PCR expression analysis and VIGS validation. The isolation and identification of these bHLH transcription factors facilitated clarification of the molecular genetic basis for the genetic improvement of tomatoes and the development of functional gene resources for transgenic research. In addition, these findings may aid in uncovering an unexplored mechanism during the TYLCV infection in tomatoes.

Figure 1

The characterization and distribution of the bHLH domains. A. Sequence logo of the SlybHLH domain by MEME. The H5, E9 and R12 amino acids in the basic domain that are important for DNA binding are indicated by stars. Amino acids important for dimerization of the helix-loop-helix domain are indicated by arrows. B. Distribution of amino acids in the bHLH consensus motif among tomatoes. The numbers of horizontal ordinate refer to the positions of the residues in the alignments of the studies. C. Predicted DNA-binding characteristics of the bHLH domain of SlybHLH proteins.

Figure 2

Phylogenetic tree constructed from the neighbor-joining method using the bHLH transcription factor domain in tomato. The phylogenetic tree was constructed using MEGA6 software. The numbers are bootstrap values based on 1000 iterations. Only bootstrap values larger than 50% support are indicated.

Figure 3

Comparative analysis of synteny and expansion of bHLH genes. A. Twelve tomato chromosomes (Sl01-Sl12) and 12 of potato chromosomes (St01-St12) maps were based on the orthologous and paralogous pair positions and demonstrate highly conserved synteny. The red lines represent the orthologous bHLH genes between tomato and potato. The blue and yellow lines represent the paralogous bHLH genes in potato and tomato, respectively. B. Twelve tomato (Sl01-Sl12) and Arabidopsis chromosome (A01-A05) maps were based on the orthologous and paralogous pair positions and demonstrate highly conserved synteny. The red lines represent the orthologous bHLH genes between tomato and Arabidopsis. The blue and yellow lines represent the paralogous bHLH genes in tomato and Arabidopsis, respectively.

Figure 4

Distribution of 152 bHLH genes on the twelve tomato chromosomes using MapInspect software. Only SlybHLH001 could not be anchored onto a specific chromosome. The scale is in megabases (Mb). The duplicate bHLH genes are connected with blue lines.

Figure 5

The expression levels and related functions of bHLH genes. A. Venn’s diagram of the cross comparison of expressed genes based on RNA-seq data. B. Gene ontology (GO) enrichment analysis of expressed bHLH genes involved in the biological processes. C. Subcellular location of bHLH genes.

Figure 6

Quantitative RT-PCR was used to measure the relative expression levels of five pathogen resistance related genes in the R (TY-2) and S (4840) lines, with tomato α-Tubulin (Solyc04g077020.2) as an internal reference. A. SlybHLH077. B. SlybHLH079. C. SlybHLH131. D. SlybHLH132.

Figure 7

Validation of the bHLH gene ( SlybHLH131 ) with virus-induced gene silencing. A. Cotyledon agroinfiltration of TRV vectors was carried out in the S plantlets at the cotyledon stage. R plants treated with Phytoene desaturase (PDS) gene silencing constructs pTRV1 and pTRV2 - (PDS) showed bleached areas in leaflets (left). S plants treated with pTRV1 and pTRV2 vectors showed the normal phenotype (middle). S plantlets treated with SlybHLH131 gene silencing constructs pTRV1 and pTRV2 - (SlybHLH131) showed a cell death phenotype (right). Arrow indicates cell death. B. Trypan blue staining of VIGS and empty vector lines leaves. C. DAB staining of VIGS and empty vector lines leaves. D. Relative expression of SlybHLH131 transcript using real-time RT-PCR analysis in the VIGS-treated lines 20 days after agroinfiltration with TRV vectors. Tomato α-Tubulin (Solyc04g077020.2) was used as an internal reference. Error bars represented SE of three biological replicates and asterisks indicate significant differences by Student’s t test for P < 0.05.