Transcriptomic Analysis of the Host Response to Mild and Severe CTV Strains in Naturally Infected Citrus sinensis Orchards

Abstract

Citrus tristeza virus (CTV) is an important threat to the global citrus industry, causing severe economic losses worldwide. The disease management strategies are focused on vector control, tree culling, and the use of resistant varieties and rootstocks. Sweet orange (Citrus sinensis) trees showing either severe or mild CTV symptoms have been observed in orchards in Veracruz, Mexico, and were probably caused by different virus strains. To understand these symptomatic differences, transcriptomic analyses were conducted using asymptomatic trees. CTV was confirmed to be associated with infected plants, and mild and severe strains were successfully identified by a polymorphism in the coat protein (CP) encoding gene. RNA-Seq analysis revealed more than 900 significantly differentially expressed genes in response to mild and severe strains, with some overlapping genes. Importantly, multiple sequence reads corresponding to Citrus exocortis viroid and Hop stunt viroid were found in severe symptomatic and asymptomatic trees, but not in plants with mild symptoms. The differential gene expression profiling obtained in this work provides an overview of molecular behavior in naturally CTV-infected trees. This work may contribute to our understanding of citrus-virus interaction in more natural settings, which can help develop strategies for integrated crop management.

Keywords: Citrus sinensis; Citrus tristeza virus; RNA-Seq; differential gene expression; mild/severe strain.

Figure 1

Location of collecting sites of sweet orange trees showing CTV symptoms in orchards in Veracruz, Mexico. Upper, right inset shows Mexico with the state of Veracruz labeled red. Green indicates the agricultural area. Red circles with a black triangle show the sites where 21 productive trees were sampled.

Figure 2

Contrasting symptoms of CTV-infected sweet orange trees in Veracruz, Mexico. Citrus sinensis trees infected with the mild (A–D), and the severe strain (E–H). (A) Sweet orange tree infected with the mild CTV strain; (B) leaves with yellowing of central vein; (C) rounded, normal orange fruits; (D) trunk with normal corky surface; € sweet orange tree infected with the severe CTV strain; (F) yellowing leaves; (G) orange fruits with yellow color and smaller size; (H) trunk with pitting damage. Bars in (A,E) = 50 cm; bars in (B–D) and (F–H) = 5 cm.

Figure 3

Phylogenetic relationships of CTV isolates based on the CP gene sequences. The Maximum Likelihood method was used for the phylogenetic tree inference. Upper semicircle shows a clade of severe CTV strains. Left semicircle shows a second clade of severe strains, including the strain identified in this study MZ670758. Right semicircle shows the clade with mild strains, including MZ670756 and MZ670757 identified in the present study.

Figure 4

Gene expression levels in C. sinensis during CTV infection. (A) Heat map showing downregulated transcripts in violet, and upregulated transcripts in yellow. Transcripts from mildly infected plants (left column), asymptomatic plants (center) or severely infected plants (right) are shown. Genes with similar expression pattern are depicted in the left tree. (B) Venn diagram representing overlapped gene expression. Upper left: mild infection, upregulated transcripts; upper right: severe infection, downregulated transcripts; lower left: mild infection, downregulated transcripts; lower right: severe infection, upregulated transcripts. Numbers representing transcript hits and their proportion (percentage) are in parenthesis.

Figure 5

Comparison of differentially expressed transcripts in GO terms of biological functions. Left panels: responses to infection with mild strain; right panels: responses to infection with severe strain. (A,B) Defense response; (C,D) innate immune response; (E,F) cell to cell transport. Significance levels of enrichment are shown in red (upper levels) to yellow (lower levels).

Figure 6

Upregulated transcripts associated with plant–pathogen interactions in the C. sinensis plant infected with the mild CTV strain. Green boxes indicate upregulated genes. Annotation was calculated with KEGG Blast Koala. Note the induction of CDPK, Rboh, CaM/CML PR1, RPS2 and Rd19.

Figure 7

Upregulated transcripts associated with plant–pathogen interaction in the C. sinensis plant infected with the severe CTV strain. Green boxes indicate upregulated genes. Annotation was calculated with KEGG Blast Koala. Note the induction of CDPK and CaM/CML.

Figure 8

Validation of differential gene expression in CTV-infected trees. Expression levels of transcripts in C. sinensis infected with mild CTV strain (A) and severe CTV strain (B) were analyzed by quantitative RT-PCR. Detection was normalized with the endogenous transcript for actin. Transcript accumulation levels were compared to asymptomatic plants. Red: downregulated transcripts; blue: overexpressed transcripts. Mild CTV strain: downregulated genes (CHC1, ABI2, loricrin-like protein) and upregulated genes (PR-6, PDP6, DRP). Severe CTV strain: downregulated genes (STIPL1, SAG29, BAG2), and upregulated genes (EDR1, RIN13, SYN4). Three independent trees (Mild 1–3; Severe 1–3) were evaluated by triplicate. Bars represent standard deviation. Asterisks represent statistical significance (* p < 0.05).