Comparative transcriptome analysis of Citrus macrophylla tree infected with Citrus tristeza virus stem pitting mutants provides new insight into the role of phloem regeneration in stem pitting disease

Abstract

Stem pitting is a complex and economically important virus-associated disease of perennial woody plants. Molecular mechanisms and pathways occurring during virus-plant interaction that result in this phenomenon are still obscure. Previous studies indicated that different Citrus tristeza virus (CTV) mutants induce defined stem pitting phenotypes ranging from mild (CTVΔp13) to severe (CTVΔp33) in Citrus macrophylla trees. In this study, we conducted comparative transcriptome analyses of C. macrophylla trees infected with CTV mutants (CTVΔp13 and CTVΔp33) and a full-length virus in comparison to healthy plants as control. The mild CTV stem pitting mutant had very few differentially expressed genes (DEGs) related to plant defense mechanism and plant growth and development. In contrast, substantial gene expression changes were observed in plants infected with the severe mutant and the full-length virus, indicating that both the p13 and p33 proteins of CTV acted as a regulator of symptom production by activating and modulating plant responses, respectively. The analysis of transcriptome data for CTVΔp33 and the full-length virus suggested that xylem specification has been blocked by detecting several genes encoding xylem, cell wall and lignin degradation, and cell wall loosening enzymes. Furthermore, stem pitting was accompanied by downregulation of transcription factors involved in regulation of xylem differentiation and downregulation of some genes involved in lignin biosynthesis, showing that the xylem differentiation and specification program has been shut off. Upregulation of genes encoding transcription factors associated with phloem and cambium development indicated the activation of this program in stem pitting disease. Furthermore, we detected the induction of several DEGs encoding proteins associated with cell cycle re-entry such as chromatin remodeling factors and cyclin, and histone modification. This kind of expression pattern of genes related to xylem differentiation and specification, phloem and cambium development, and cell cycle re-entry is demonstrated during secondary vascular tissue (SVT) regeneration. The microscopy analysis confirmed that the regeneration of new phloem is associated with stem pitting phenotypes. The findings of this study, thus, provide evidence for the association between stem pitting phenotypes and SVT regeneration, suggesting that the expression of these genes might play important roles in development of stem pitting symptoms. Overall, our findings suggest that phloem regeneration contributes to development of stem pitting symptoms.

Keywords: Citrus tristeza virus (CTV); secondary vascular tissue regeneration; stem-pitting; transcriptome (RNA-seq); virus-plant interaction.

Figure 1

Increase in the stem pitting symptoms of the CTV-infected C. macrophylla trees is associated with increase in the number of up- and downregulated genes. (A–C) Stem pitting symptoms in the experimental host at 8 months post-inoculation. (A) CTVΔp13 induced no visible stem pits. (B) The full-length virus-induced moderate stem pits. (C) CTVΔp33 induced severe stem pits in the main stem. (D) Number of up- and downregulated genes for each pairwise comparisons. Venn diagrams demonstrate numbers of unique and common (E) upregulated and (F) downregulated differentially expressed genes (DEGs) between Citrus macrophylla trees infected with CTVΔp13, full-length CTV, and Δp33 vs. not infected trees.

Figure 2

Validation of transcriptome results by reverse transcription quantitative PCR (RT-qPCR). Some genes involved in lignin monomer synthesis were selected for RT-qPCR verification. Expression levels of tested genes were normalized based on transcript levels of ubiquitin 10 (UBQ 10) gene. Different letters (a and b) represent a significant difference at p ≤ 0.05. Error bars represent standard deviations of the means (n = 3).

Figure 3

MapMan visualization of differential expressed genes related to biotic stress overview, (A) CTVΔp13 vs Healthy, (B) full-length CTV vs Healthy, and (C) CTVΔp33 vs Healthy. Blue and Red squares indicate up- and down regulation of genes respectively.

Figure 4

Functional annotation of differential expressed genes. (A) Full-length CTV infected vs not infected and (B) CTVΔp33 infected vs not infected Citrus macrophylla trees. The genes were divided into three categories: Cellular component, molecular function, and biological process genes.

Figure 5

Expression pattern of some genes associated with (A) Cell cycle re-entry, (B) xylem specification, and (C) phloem and cambium development in Citrus macrophylla infected with CTV mutants during stem-pitting symptom development. Complete description for each gene can be found in Supplementary Table 1. Gene identifier, functional grouping and gene description is based on the gene ontology in the MapMan program (Thimm et al., 2004).

Figure 6

Transverse thin hand-cut sections of young stems from the healthy and infected Citrus macrophylla trees with CTV mutants showing the association of stem-pitting symptom development with secondary phloem regeneration. The photographs were taken under light microscope. (A) Healthy, (B) infected trees with CTVΔp13, (C) full-length CTV, and (D) CTVΔp33 respectively (40 × objective lens). Abbreviation: Xy, xylem; Ph, phloem; PF, phloem fiber. Scale bar = 200 μm. (E) Phloem width measurements of young stems from the healthy and infected Citrus macrophylla trees with CTV mutants. Shaded bars represent the mean values of phloem thickness (n = 20). The different letters denote statistically differences as determined by analysis of variance of mean values (P = < 0.05).

Figure 7

Illustration of gene expression changes and DEGs related to secondary phloem regeneration in infected plants with CTV moderate and severe stem-pitting phenotype during stem-pitting symptom development.

Figure 8

Transcriptome response of Citrus macrophylla trees infected with CTV wild-type and CTV different mutants which induce defined stem-pitting (SP) phenotypes from mild to severe. Degrees of SP symptom is correlated with the number of differentially expressed genes. Blue and red arrows represent the up and downregulation of genes related to each category, respectively. Thin arrows indicate that there are few numbers of DEGs in putative category.