Fig 1.
Development of transgenic tomato plants expressing the TYLCV C4 gene.
A) A schematic model for TYLCV (upper panel) showing its genome organization as a typical monopartite begomovirus and yellow leaf curl symptoms on tomato plants naturally infected by TYLCV (lower panel). B) A schematic model of the T-DNA region between the right border (RB) and Left border (LB) depicting the TYLCV C4 gene under 35S promoter control and a NOS terminator (top panel) used to develop transgenic tomato plants. A side-by-side comparison of the phenotypes (middle panel) displayed on a Gfp-transgenic plant (left side) and a TYLCV C4-transgenic tomato plant (right side). A close-up view of the yellow leaf curl disease-like phenotypes (yellowing and upward cupping leaves) displayed on a TYLCV C4-transgenic plant (lower panel).
Fig 2.
Biological and molecular characterization of TYLCV C4 gene expression in transgenic tomato plants.
A) A control Gfp-transgenic plant with normal phenotypes in plant growth and fruit development. B) Transgenic tomato plants expressing the TYLCV C4 gene developed upward leaf cupping phenotypes resembling TYLCV infection on tomato. The T0 line ‘C4-C1’ (with a close-up view on a leaflet) was able to generate fruits, albeit of a smaller size, which allowed us to evaluate the T1 plants. C). Another independent line, ‘C4-C5,’ expressed a similar leaf curl phenotype but bearing no fruit. D) Molecular characterization of the transgene C4 expression in transgenic tomato plants using their respective DNA preparations with gene-specific primers (top panel) in polymerase chain reaction (PCR) (left panel) or RNA preparations by reverse transcription PCR (RT-PCR) (right panel). Two TYLCV C4-transgenic tomato plants (C4-C1 and C4-C5) along with a control Gfp-transgenic plant (GFP) were used. “+” and “-” were plasmid DNA with or without TYLCV C4 sequence, respectively. In the bottom panels, an endogenous host gene “Actin” was used as internal quality control for DNA or RNA preparations used for their respective reactions.
Table 1.
Transgene expression analysis of RNA-seq reads mapped to the C4 or Gfp transgene.
Fig 3.
Comparative transcriptome analysis on differential gene expression between the TYLCV C4-transgenic tomato plants and those control transgenic plants expressing the Gfp gene under the same genetic background.
A) A volcano plot showed a distribution pattern of differentially expressed genes (DEGs) with number of up-regulated (in green) or down-regulated (in red) genes in the C4-transgenic tomato plants over that of the Gfp-transgenic plants. X-axis represents -log10 (p-value) and y-axis represents log2 (fold change). Black horizontal dotted lines show the p-value cut off at 0.05. Black vertical dotted lines were drawn using log2 (fold change) cut off at -1.5 and 1.5. B) Gene Ontology (GO) enrichment analysis revealed 13 enriched categories of the identified DEGs, with category in the y-axis and–log10 (p-value) in the x-axis.
Table 2.
Classification of differentially expressed genes to prominent annotation groups.
Table 3.
Glutaredoxin genes differentially expressed between the transgenic C4 plants and the transgenic Gfp control plants.
Table 4.
Differentially expressed genes representing transcription factors between transgenic C4 plants and the Gfp control plants.
Table 5.
Differentially expressed genes in protein kinase families between the transgenic C4 plants and the control Gfp plants.
Table 6.
Summary of qRT-PCR validation of selected differentially expressed genes.
Fig 4.
A schematic flow chart depicts the potential functional interference of the TYLCV C4 protein to a series of plant developmental genes, especially those involving in transcriptional regulation, protein kinase, glutaredoxin and gene silencing pathways.
The top panel shows a natural field infection of tomato plants by TYLCV through transmission by viruliferous whiteflies. The middle and lower panels showed key steps in the development of transgenic tomato plants expressing the TYLCV C4 gene, transcriptome analysis and predicted functional interference on host genes that are regulating plant development, resulting in yellow leaf curl disease-like phenotypes.