Transcriptome sequencing analysis of differentially expressed genes involved in the formation of dapple symptoms in apple fruits

Abstract: 【Objective】Apple (Malus domestica) is one of the most important fruit crops worldwide, especially in China. As perennial woody plants, apple trees are vulnerable to multiple pathogens, including fungi, bacteria, viruses, and viroids. Among them, apple scar skin disease is a common apple disease induced by apple scar skin viroid (ASSVd). Apple scar skin disease has two types of symptoms that may be related to the apple cultivar. One type is scar skin symptom, which usually appears on cultivars such as Golden Delicious, Indo, and Ralls Janet. The other type is dapple symptom that commonly appears on red-skin cultivars, such as Red Fuji, Red Gold, and so on. The red-skin Red Fuji is the major apple cultivar in China, the disease has caused severe losses due to the unmarketable fruits and attenuated yield. ASSVd, the major causal agent of the disease, is mainly transmitted by grafting among apple trees. The disease with dapple symptom has been reported in some of the apple cultivated area aroundthe world, such as China, Japan, the republic of Korea, the United States, Canada, and etc. Although this disease has been identified many years ago, the mechanism of the dapple symptom formation is still unclear. In this sutdy, we used high-throughput sequencing technology to determine the differentially expressed genes that are related to the dapple symptom to investigate the behind mechanism of how the symptom is formed in apple fruits.【Methods】The apple peels from the apple fruits without symptom or with dapple symptom were obtained and stored in freezer (-80 ℃). The total RNA was subsequently extracted using an RNA extraction kit, and cDNA was synthesized by reverse transcriptase. The specific primers were designed to detect the presence of ASSVd and other possible viruses and viroids by RT- PCR. The full-length ASSVd genome was amplified by two rounds RT- PCR. Then, the RNA samples were delivered to company for high-throughput sequencing. The GO analysis and KEGG analysis were performed to analyze the annotation the differentially expressed genes. Moreover, differentially expressed genes involved in plant defence-related hormone signaling and transcription factors were also analyzed through the transcriptome data. Finally, qRT-PCRexperiments were utilized to verify the reliability of the transcriptome data.【Results】After RT-PCR analysis with specific primers, we found that ASSVd indeed present in the apple peel with dapple symptom, but not in the healthy apple fruit. Moreover, other viruses and viroids, including apple necrosis mosaic virus (ApNMV), apple chlorotic leafspot virus (ACLSV), apple stem grooving virus (ASGV), and apple dimple fruit viroid (ADFVd), that are commonly infectious to apple were not detected when performing RT-PCR assay using specific primers. We next obtained the full-length genome sequence of ASSVd via two round RT- PCR assay. Specifically, a new pair of primers were designed based on the previously obtained sequence, and the resulted sequences of the two-round PCR were assembled. We finally got two distinct isolates from the apple fruits with dapple symptom. The length of the two isolates were 331 nt and 330 nt, which are identical to the sequences of ASSVd Shandong Yantai apple isolate SDYT- 1 (MW302328.1) and SDYT- 3 (MW315909.1), respectively. The transcriptome sequencing assay was performed using Illumina platform, and the high quality of transcriptome sequencing data were obtained. By |log2 Fold Change|＞1 & p＜0.05 screening standard, we screened out 6938 differentially expressed genes. Among them, 3331 were significantly up-regulated and 3607 were significantly down-regulated in dapple apple peels compared with those of healthy apple. GO analysis revealed that the differentially expressed genes were mainly concentrated in the functions of cellular processes, metabolic processes, cellular components, binding and catalytic activities. The KEGG analysis showed that the differentially expressed genes were mainly enriched in plant hormone signal transduction, phenylpropanoid biosynthesis, photosynthesis-antenna proteins, photosynthesis, and other pathways. Furthermore, the analysis of the hormonal pathways showed thatmetabolism and signal transduction of jasmonic acid (JA), salicylic acid (SA), auxin, and other hormones might be involved in the dapple symptom appearance. The expression levels of the SArelated gene NPR4a (MD05G1256300), the JA-related gene TIFYs (MD13G1127100, MD09G1178600, MD02G1096100, MD16G1127400, and MD17G1164400), and the ethylene- related gene ERS1 (MD03G1292200) were down-regulated, while the expression level of EIN3 (MD08G1245800) was upregulated. In addition, a large number of genes involved in auxin pathway-relatedwere were also downregulated in dapple symptom fruits. Among the auxin- related genes, most of them were key enzymes and receptor proteins encoding IAA. The multiple transcription factor- encoding genes, including the WRKY, MYB, ERF and NAC were also identified in the differentially expressed genes. Specifically, the MdWRKY18-like, MdWRKY71, MdNAC29, and MdWRKY70 were up- regulated in dapple apple fruits, while genes such as the MdERF61 were down-regulated. Finally, quantitative real-time PCR assay wasutilized to confirm the reliability of the transcriptome data.【Conclusion】Transcriptome analysis showed that multiple cellular pathways, especially phenylpropanoid biosynthesis, and hormonal pathways were involved in the dapple symptom formation in apple fruits. Moreover, defensive transcription factor-encoding genes might also be related to the plant defense responses during the course of interactions between ASSVd and apple plants.