Global analysis of the transcriptional response of whitefly to tomato yellow leaf curl China virus reveals the relationship of coevolved adaptations

Abstract

The begomoviruses are the largest and most economically important group of plant viruses transmitted exclusively by the whitefly Bemisia tabaci in a circulative, persistent manner. The circulation of the viruses within the insect vectors involves complex interactions between virus and vector components; however, the molecular mechanisms of these interactions remain largely unknown. Here we investigated the transcriptional response of the invasive B. tabaci Middle East-Asia Minor 1 species to Tomato yellow leaf curl China virus (TYLCCNV) using Illumina sequencing technology. Results showed that 1,606 genes involved in 157 biochemical pathways were differentially expressed in the viruliferous whiteflies. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that TYLCCNV can perturb the cell cycle and primary metabolism in the whitefly, which explains the negative effect of this virus on the longevity and fecundity of B. tabaci. Our data also demonstrated that TYLCCNV can activate whitefly immune responses, such as autophagy and antimicrobial peptide production, which might lead to a gradual decrease of viral particles within the body of the viruliferous whitefly. Furthermore, PCR results showed that TYLCCNV can invade the ovary and fat body tissues of the whitefly, and Lysotracker and Western blot analyses revealed that the invasion of TYLCCNV induced autophagy in both the ovary and fat body tissues. Surprisingly, TYLCCNV also suppressed the whitefly immune responses by downregulating the expression of genes involved in Toll-like signaling and mitogen-activated protein kinase (MAPK) pathways. Taken together, these results reveal the relationship of coevolved adaptations between begomoviruses and whiteflies and will provide a road map for future investigations into the complex interactions between plant viruses and their insect vectors.

FIG. 1.

Distribution of total tags and distinct tags over different tag abundance categories. Numbers in square brackets indicate the range of copy numbers for a specific category of tags. For example, “[2, 5]” means all the tags in this category have 2 to 5 copies. Numbers in parentheses show the total tag copy number for all the tags in that category.

FIG. 2.

The level of gene expression. The level of gene expression was determined by calculating the number of unambiguous tags for each gene and then normalizing to the number of transcripts per million tags (TPM). (A) The level of gene expression of the nonviruliferous whiteflies. (B) The level of gene expression of the viruliferous whiteflies.

FIG. 3.

Histogram presentations of GO classification of putative functions of genes from nonviruliferous and viruliferous whiteflies. The functions of genes identified cover three main categories: biological process, cellular component, and molecular function. The right y axis indicates the number of genes in a category. The left y axis indicates the percentage of a specific category of genes in that main category. GO analysis showed that the distributions of gene functions for the nonviruliferous and viruliferous whiteflies are similar.

FIG. 4.

Analysis of differentially expressed genes between two libraries. (A) Summary of the numbers of differentially expressed genes in the Tomato yellow leaf curl China virus viruliferous whiteflies. “FDR < 0.001 and the absolute value of log₂ ratio ≥ 1” were used as the threshold to judge the significance of gene expression difference. (B) Fold change distribution of differentially expressed genes. (C) Correlation analysis of two libraries. The Pearson correlation coefficient for two libraries is shown in the upper left corner of the plot. (D) Comparison of DGE data and qPCR results. “Concordant up” means that genes in the viruliferous whiteflies were upregulated for both DGE and qPCR analyses. “Concordant down” means that genes in the viruliferous whiteflies were downregulated for both DGE and qPCR analyses. “Different” means that, for DGE and qPCR analyses, the direction of change of gene expression in the viruliferous whiteflies was contrary.

FIG. 5.

Regulated genes involved in the cell cycle and primary metabolism in the viruliferous whiteflies. (A) Regulated genes related to the cell cycle in viruliferous whiteflies. (B) Regulated genes involved in primary metabolism in viruliferous whiteflies.

FIG. 6.

Detection of autophagy in adult whiteflies. Ovariole of ovary (A) and fat body (B) tissues stained with the lysosome-specific fluorescent dye Lysotracker Red (red) and the nuclear dye Hoechst 33342 (blue). Compared with the tissues from the nonviruliferous whitefly (left), there is increased punctate Lysotracker staining (white arrows) in the viruliferous whiteflies (right). Scale bar in panels A and B, 50 μm and 10 μm, respectively. Similar findings were observed in three independent experiments. (C) Western blotting of the Atg8 protein. Arabic numbers above each lane indicate the time points (in hours) after the nonviruliferous and viruliferous whiteflies were transferred to healthy cotton plants. A molecular size markers (in kilodaltons) is shown on the left of the panel. Atg8-I (∼16 kDa) is observed in all whiteflies, and Atg8-II (∼14 kDa) is induced only in the viruliferous whiteflies feeding on cotton plants for 120 h.

FIG. 7.

Percentages of viruliferous whiteflies (obtained by feeding for 24 h on infected tobacco plants) with detectable TYLCCNV DNA in the whole body, ovaries, and fat bodies after 0 h and 120 h of feeding on healthy cotton plants.