Identification of Endogenous Genes for Normalizing Titer Variation of Citrus Tristeza Virus in Aphids at Different Post-acquisition Feeding Times

Abstract

Citrus tristeza virus (CTV) is efficiently transmitted in a semi-persistent manner by the brown citrus aphid (Toxoptera citricida (Kirkaldy)). Currently, the most sensitive method for detecting plant viruses in insect vectors is reverse-transcription quantitative polymerase chain reaction (RT-qPCR). In this study, the elongation factor-1 alpha (EF-1α) gene and acidic p0 ribosomal protein (RPAP0) gene were confirmed to be suitable reference genes for RT-qPCR normalization in viruliferous T. citricida aphids using the geNorm, NormFinder, and BestKeeper tools. Then the relative CTV titer in aphids (T. citricida) at different post-acquisition feeding times on healthy plants was quantified by RT-qPCR using EF-1α and RPAP0 as reference genes. The relative CTV titer retained in the aphids gradually decreased with increasing feeding time. During the first 0.5 h of feeding time on healthy plants, the remaining CTV titer in aphids showed about 80% rapid loss for the highly transmissible isolate CT11A and 40% loss for the poorly transmissible isolate CTLJ. The relative CTV titer in aphids during more than 12 h post-acquisition times for CT11A was significantly lower than at the other feeding times, which is similar to the trend found for CTLJ. To our knowledge, this is the first report about the relative titer variation of CTV remaining in T. citricida at different post-acquisition feeding times on healthy plants.

Keywords: Toxoptera citricida; citrus tristeza virus; relative titer variation.

Fig. 1

The melting curve for RPAP0 (A) and EF-1α (B) with single peaks obtained from three technical replicates. Real-time polymerase chain reaction (PCR) was performed in a 20-μl reaction volume that contained 1× SYBR Premix ExTaq II enzyme, 0.6 μM of each gene-specific primer, and 2 μl of the first strand of cDNA. The PCR reaction procedure was as follows: denaturation at 95°C for 30 s, 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 15 s, and extension at 72°C for 20 s. Once PCR was finished, the PCR reaction product was incubated at 95°C for 15 s, annealed at 65°C for 30 s, and gradually increased to 95°C again for melting curve analysis. Data acquisition and analysis were performed using the iCycleriQ Real-Time PCR Detection System.

Fig. 2

Average expression stability value of candidate reference genes in viruliferous Toxoptera citricida analyzed by geNorm. Based on the average pairwise variation of each candidate reference gene compared to the other four genes, the geNorm assigns a stability index M to the gene, and then all the studied genes are ranked. The least stable gene with the highest M-value is excluded sequentially (from left to right) until the most stable gene pair remains.

Fig. 3

Optimal number of reference genes for normalization in viruliferous Toxoptera citricida by geNorm. The pairwise variation (V_n/n+1) analysis showing the normalization factor (NF) variability (V) between the NF_n and NF_n+1 that results from including an additional gene in the NF_n calculation. Pairwise variations (v_n/n+1) under 0.15 indicate that no additional genes are required for normalization.

Fig. 4

Citrus tristeza virus (CTV) titer variation in Toxoptera citricida after different post-acquisition feeding times on healthy plants. Thousands of non-viruliferous aphids, which had been apterous adults of 1–2 days old, were fed on CTV-infected ‘Jincheng’ sweet orange seedlings. After a 6 h acquisition access period on CTV-infected ‘Jincheng’ sweet orange plants, 45 adult aphids were collected for virus detection, and 450 adult aphids were used for post-acquisition feeding on healthy ‘Jincheng’ sweet orange seedling for each replication. Ten sampling events (0.5, 1, 2, 3, 4, 6, 9, 12, 24, and 48 h) were performed during feeding time on healthy sweet orange seedlings. This experiment was repeated three times. At the completion of each feeding period, total RNA was extracted from three groups of 15 aphids, and CTV was quantified by reverse-transcription quantitative polymerase chain reaction in T. citricida. Individuals in each group were randomly collected from several sweet orange plants during each feeding period. EF-1α and RPAP0 were used as reference genes in viruliferous T. citricida. Duncan’s multiple comparison was explored. The same letter indicates no significant difference at P < 0.05.