Apple scar skin viroid causes dapple and scar skin apple diseases by disrupting phenylpropanoid-mediated metabolic pathways

Apple scar skin viroid causes dapple and scar skin apple diseases by disrupting phenylpropanoid-mediated metabolic pathways | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 21 October 2025 V1 Latest version Share on Apple scar skin viroid causes dapple and scar skin apple diseases by disrupting phenylpropanoid-mediated metabolic pathways Authors : He Lingzhu , Xu Huiyuan , Xie Jipeng , Tao Zhou 0000-0001-7702-8472 , Yang Xiuling , Li Shifang , and Zhang Zhixiang 0000-0002-1171-6966 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176103045.51528753/v1 360 views 217 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Dapple apple and scar skin apple diseases, caused by apple scar skin viroid (ASSVd), pose serious threats to apple production in China by substantially reducing fruit yield and market value. However, the molecular mechanisms underlying fruit symptom induction remains poorly understood, primarily due to the long latency period required for perennial fruit trees to exhibit fruit-specific symptoms. Here, we investigated ASSVd accumulation and genetic diversity in symptomatic and asymptomatic fruit skins of dapple apples over three consecutive years. While no specific sequence variants were detected in symptomatic tissues, ASSVd accumulation was markedly higher in symptomatic skin during the early fruit-coloring stage. Genome-wide transcriptomic analyses of dapple and scar skin apples collected from the field revealed thousands of differentially expressed genes (DEGs) associated with plant–pathogen interactions, hormone signaling, and primary and secondary metabolite biosynthesis. Notably, anthocyanin biosynthesis pathways were downregulated, while suberin biosynthesis pathways were upregulated in both disease types, aligning with symptom development. Both pathways act as parallel branches of the core phenylpropanoid pathway, suggesting that ASSVd interferes with upstream metabolic steps. Key regulators of anthocyanin and suberin synthesis, as well as genes involved in hormone metabolism, such as brassinosteroids (BR) were dysregulated, indicating a complex regulatory network underlying disease manifestation. The repression of anthocyanin structural genes by ASSVd was further confirmed using established infection of in vitro apple seedlings and callus cultures. Collectively, these results identify the metabolic reprogramming and gene regulatory changes associated with dapple and scar skin apple diseases, providing a direct molecular link between ASSVd infection and altered phenylpropanoid metabolism. Apple scar skin viroid causes dapple and scar skin apple diseases by disrupting phenylpropanoid-mediated metabolic pathways He Lingzhu 1 , Xu Huiyuan 1 , Xie Jipeng 1 , Zhou Tao 2 , Yang Xiuling 1 , Li Shifang 1 , Zhang Zhixiang 1 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China 2 Key Laboratory for Pest Monitoring and Green Management of Ministry of Agriculture and Rural Affairs, and Department of Plant Pathology, China Agricultural University, Beijing 100193, China Correspondence Yang Xiuling, Li Shifang, and Zhang Zhixiang, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. Emails: [email protected] , [email protected] , and [email protected] . Funding information National Natural Science Foundation of China (Grant No. 32472532) the Central Public-Interest Scientific Institution Basal Research Fund (Y2025YC25) Abstract Dapple apple and scar skin apple diseases, caused by apple scar skin viroid (ASSVd), pose serious threats to apple production in China by substantially reducing fruit yield and market value. However, the molecular mechanisms underlying fruit symptom induction remains poorly understood, primarily due to the long latency period required for perennial fruit trees to exhibit fruit-specific symptoms. Here, we investigated ASSVd accumulation and genetic diversity in symptomatic and asymptomatic fruit skins of dapple apples over three consecutive years. While no specific sequence variants were detected in symptomatic tissues, ASSVd accumulation was markedly higher in symptomatic skin during the early fruit-coloring stage. Genome-wide transcriptomic analyses of dapple and scar skin apples collected from the field revealed thousands of differentially expressed genes (DEGs) associated with plant–pathogen interactions, hormone signaling, and primary and secondary metabolite biosynthesis. Notably, anthocyanin biosynthesis pathways were downregulated, while suberin biosynthesis pathways were upregulated in both disease types, aligning with symptom development. Both pathways act as parallel branches of the core phenylpropanoid pathway, suggesting that ASSVd interferes with upstream metabolic steps. Key regulators of anthocyanin and suberin synthesis, as well as genes involved in hormone metabolism, such as brassinosteroids (BR) were dysregulated, indicating a complex regulatory network underlying disease manifestation. The repression of anthocyanin structural genes by ASSVd was further confirmed using established infection of in vitro apple seedlings and callus cultures. Collectively, these results identify the metabolic reprogramming and gene regulatory changes associated with dapple and scar skin apple diseases, providing a direct molecular link between ASSVd infection and altered phenylpropanoid metabolism. Keywords : apple, viroid, transcriptome, metabolic pathway, phenylpropanoid 1 Introduction Viroids are small (246–434 nt), circular, single-stranded, non-coding RNA molecules capable of autonomous replication and systemic infection in higher plants (Navarro, Flores, and Di Serio 2021). As the smallest known plant pathogens, they can cause severe diseases in susceptible hosts, including apple (Flores et al. 2021). Apple scar skin viroid (ASSVd), a ~330-nt viroid that forms a characteristic rod-like secondary structure through extensive intramolecular base pairing, is the causal agent of both dapple apple and scar skin apple diseases (Hadidi et al. 2017; Zhai et al. 2019). ASSVd infects several species in the genera Malus , Pyrus , Sorbus , Chaenomeles , Cydonia , and Pyronia (Desvignes et al. 1999). Dapple apple disease primarily affects red-skinned apple varieties and is prevalent across different apple production regions of China (Liang et al. 2022). Infected fruits develop numerous yellow or green spots, significantly diminishing commercial value. In contrast, scar skin apple disease occurs mainly in green-skinned varieties, producing smaller fruits with scarred or cracked surfaces, rendering them commercially unmarketable. More recently, a novel disease affecting small, immature ‘Saiwaihong’ apple fruits was also linked to ASSVd infection (Xu et al. 2024). Despite extensive studies, the molecular mechanisms underlying ASSVd-induced diseases remain largely unclear. Dapple apple symptoms manifest as irregular red pigmentation in the skin, corresponding to abnormal anthocyanin accumulation (Honda and Moriya 2018). Therefore, symptom development likely results from localized repression of anthocyanin biosynthesis. Indeed, the content of anthocyanin is significantly reduced in diseased fruits compared to healthy ones (Zhang et al. 2024). Transcriptomic analyses have further identified numerous DEGs within the phenylpropanoid metabolism pathway, which lies upstream of anthocyanin biosynthesis (Zheng et al. 2023), establishing a molecular correlation between ASSVd infection and disrupted anthocyanin synthesis. However, this relationship requires further direct experimental evidence, particularly regarding the viroid’s impact on key genes in the anthocyanin biosynthetic pathway. In many viroid pathosystems, symptom development is mediated by RNA silencing, where viroid-derived small RNAs modulate host gene expression (Flores et al. 2020). For example, a 17-nt small interfering RNA derived from apple dimple fruit viroid (ADFVd) was shown to regulate anthocyanin biosynthesis by targeting the mRNA of the bHLH transcription factor MdPIF1 (Zhang et al. 2024). Although ASSVd is closely related to ADFVd, no corresponding small RNA has been detected in the ASSVd genome, leaving the role of RNA silencing in dapple apple pathogenesis unresolved. To elucidate the effect of ASSVd infection on anthocyanin biosynthesis and to characterize host responses under both dapple apple and scar skin apple disease conditions, we conducted comparative transcriptome analyses of symptomatic and asymptomatic skins from infected fruits alongside healthy controls. The results revealed pronounced downregulation of anthocyanin biosynthesis genes in dapple apple, and upregulation of cutin, suberin, and wax biosynthesis genes in scar skin apple. These pathways are parallel branches of the core phenylpropanoid metabolism network. Furthermore, we established experimental systems, including ASSVd-infected apple seedlings, fruit callus cultures, and transgenic tomato plants expressing ASSVd, to validate the viroid’s regulatory influence on phenylpropanoid metabolism. Together, these approaches provide comprehensive molecular insight into how ASSVd infection disrupts phenylpropanoid-derived metabolic pathways, leading to the characteristic symptoms of dapple and scar skin apple diseases. 2 Materials and Methods 2.1 Plant materials Dapple apples ( cv. ‘Fuji’) were collected from an orchard in Changping District, Beijing. The trees were 4–5 years old at the start of a surveillance program conducted from 2021 to 2024. Fruits were harvested in September and October after removal of the paper coverings. Scar skin apples ( cv . ‘Venus Gold’) were collected from a three-year-old orchard in Pinggu District, Beijing. Symptom development began in 2022, and fruits were harvested in early June. Virus- and viroid-free apple seedlings ( cv . ‘Gala’, ‘Yanfu8’, ‘Orin’, and ‘Venus Gold’) were kindly provided by Professor Hu Guojun, Research Institute of Pomology, Chinese Academy of Agricultural Sciences. Red-fleshed apple ( cv . ‘Redlove’) calli were induced and subcultured on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 1-naphthylacetic acid (NAA) and 1.0 mg/L 6-benzylaminopurine (6-BA), and were maintained at 25 °C in darkness. 2.2 RT-PCR, cloning, and RT-qPCR Fruit skins from dapple and scar skin apples were scraped for total RNA extraction using the cetyltrimethylammonium bromide (CTAB) method (Zhang et al. 2014). Total RNA from tomato leaves was extracted using TRNzol reagent (TIANGEN, Beijing). Complementary DNA (cDNA) was synthesized using M-MLV reverse transcriptase (Promega, China) and random hexamer primers. Apple necrotic mosaic virus (ApNMV), apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), and apple scar skin viroid (ASSVd) were amplified using specific primers (Table S1), cloned into the pTOPO-Blunt vector (Aidlab Biotechnologies, Beijing), and verified by Sanger sequencing. Expression levels of anthocyanin biosynthesis genes were quantified via RT-qPCR using the Talent qPCR PreMix (SYBR Green) (TIANGEN, Beijing). Relative gene expression was calculated using the 2 −ΔΔCT method, with Mdactin and Slactin serving as internal controls for apple and tomato, respectively. The genes involved in RT-qPCR include following genes: 4CL , 4-coumarate-CoA ligase; CHS , chalcone synthase; CHI , chalcone isomerase; F3H , flavanone 3-hydroxylase; F3’H , flavonoid 3′-hydroxylase; DFR , dihydroflavonol 4-reductase; ANS , anthocyanidin synthase; UFGT , UDP-glucose:flavonoid 3-O-glucosyltransferase. 2.3 Northern blot hybridization ASSVd accumulation in apple fruits, seedlings, callus, and tomato plants was determined by northern blot analysis as described previously (Xia et al. 2017). Total RNA was resolved on a denaturing agarose gel, transferred to a nylon membrane, and crosslinked by UV irradiation. Membranes were prehybridized and hybridized with digoxigenin (DIG)-labeled RNA probes specific for ASSVd. 2.4 Library construction, transcriptome sequencing, and analysis Total RNA was extracted using the CTAB method (Zhang et al. 2014). Polyadenylated mRNA was enriched with Oligo(dT) magnetic beads. First-strand cDNA synthesis was performed using random hexamer primers, followed by second-strand synthesis with DNA polymerase. The resulting double-stranded cDNA was end-repaired, adaptor-ligated, and size-selected using the AMPure XP system (Beckman Coulter, USA). Libraries were amplified with Phusion high-fidelity DNA polymerase (New England Biolabs) using universal and index primers, purified, and assessed for quality using an Agilent 2100 Bioanalyzer. Sequencing was conducted on an Illumina NovaSeq platform (Novogene, Beijing) to generate 150-bp paired-end reads. Low-quality and non-informative reads were removed using Fastp, and clean reads were mapped to the apple reference genome (GCA_002114115.1) with HISAT2 (Kim, Langmead, and Salzberg 2015). Transcript assembly and gene annotation were performed with StringTie (Pertea et al. 2015). Gene expression levels were quantified as fragments per kilobase of exon per million mapped reads (FPKM). Differential expression analysis between sample groups was conducted using DESeq2 (Love, Huber, and Anders 2014), and P -values were adjusted via the Benjamini–Hochberg method (klipper-Aurbach et al. 1995). DEGs) were defined as those with |log₂ (fold change) | ≥ 1 and adjusted P ≤ 0.05. GO and KEGG enrichment analyses were performed using cluster Profiler. 2.5 Quantification of anthocyanin accumulation Anthocyanin quantification was based on its characteristic absorption behavior. At pH 1.0, anthocyanins exhibit a strong absorption peak at 530 nm, while at pH 4.5 they are converted into colorless chalcones with no such peak. Dried apple peel (0.1 g) was homogenized in 1 mL ethanol–HCl (99:1, v/v) and incubated at 60 °C for 30 minutes. After centrifugation, absorbance at 530 nm and 700 nm was measured using a spectrophotometer. For pH differential analysis, 20 μL of supernatant was diluted tenfold with potassium chloride buffer (pH 1.0) and incubated at 40 °C for 20 minutes. Absorbances at 530 nm and 700 nm were recorded as A₁ and A₂, respectively. Another 20 μL aliquot was diluted tenfold with sodium acetate buffer (pH 4.5) and incubated under identical conditions, with absorbances recorded as A₃ and A₄. The corrected absorbance (ΔA) was calculated as (A₁ – A₂) – (A₃ – A₄). Anthocyanin concentration (μg/g DW) was calculated using the formula. (μg/g dry weight) = [∆A×V÷(ε×d)×M×10×10 6 ]÷W = 16.7×∆A×10÷W where W is the dry sample weight (g). Constants include ε = 2.69 × 10⁴ L mol⁻¹ cm⁻¹ (molar extinction coefficient) and M = 449.2 g mol⁻¹ (molecular weight of anthocyanin). 2.6 ASSVd infection in apple callus, seedlings, and tomato plants A dimeric cDNA of ASSVd (DQ362906) was cloned into the pCass vector under control of the CaMV 35S promoter. The recombinant plasmid was introduced into Agrobacterium tumefaciens strain LBA4404 (Coolaber Science & Technology, Beijing) and used to infect apple callus, apple seedlings, and tomato plants. Apple callus was preconditioned under a 16-hour light/8 8-hour dark photoperiod (25 °C) for five days before infiltration. Callus tissues were cut into 2 mm³ pieces, immersed in the Agrobacterium suspension for 20 minutes in darkness, blotted dry, and incubated on subculture medium for five days in darkness. Subsequently, calli were transferred to selection medium (subculture medium containing 50 mg/L kanamycin and 100 mg/L timentin) and subcultured every 15 days. Transformed calli were then incubated under light for 10 days to monitor color changes; uninfected callus served as a control. For apple seedlings, the upper two to three young leaves were wounded (5–6 cuts of ~1 cm each), immersed in Agrobacterium suspension for 20 minutes in darkness, and blotted dry. Regeneration was induced on MS medium containing 0.15 mg/L NAA and 0.5 mg/L 6-BA, and maintained in darkness for 7 days before transfer to light (25 °C, 16 hours of white light/8 hours of dark). Tomato cotyledons were transformed via Agrobacterium-mediated transformation. After 2 days of co-cultivation in the dark, they were subjected to a differentiation culture for 27 to 34 days. Transformed tomato plants were subsequently rooted and acclimated under greenhouse conditions (Zhai et al. 2025). T1 generation plants were analyzed to assess gene expression levels and ASSVd accumulation. 3 Results 3.1 Dapple and scar skin apple symptoms are associated with ASSVd infection Dapple apples ( cv . ‘Fuji’) were collected from diseased trees, approximately two weeks after removing the paper fruit covers. These fruits displayed circular or irregular lime-green patches on the skin (Figure 1a). Asymptomatic apples were simultaneously collected from symptom-free trees in the same orchard. Both symptomatic and asymptomatic fruits were tested by RT-PCR using specific primers (Table S1) for the major apple viruses and viroids prevalent in China, including ApNMV, ACLSV, ASPV, ASGV, and ASSVd (Ni et al. 2023). Although ApNMV, ACLSV, and ASGV were detected in both groups, only ASSVd showed a strong association with dapple symptoms (Figure S1). All symptomatic apples were ASSVd-positive, whereas asymptomatic apples tested negative. Northern blot analysis further confirmed the correlation between ASSVd infection and dapple symptoms (Figure 1b). Scar skin apples ( cv. ’Venus Gold’) were collected from diseased trees in early June, showing irregular scar-like lesions on the skin (Figure 1c). These fruits were smaller than asymptomatic apples collected from healthy trees in the same orchard. RT-PCR and northern blot analyses confirmed that ASSVd infection was closely associated with scar symptoms (Figure 1d). 3.2 ASSVd accumulates at higher levels in symptomatic than in asymptomatic skin of dapple apples The lime-green patches on dapple apples were typically scattered, with areas of apparently healthy skin in between (Figure 1a). To determine whether the asymptomatic regions (AD) of dapple apples represented restricted ASSVd infection or lower viroid accumulation, ASSVd levels were quantified in symptomatic (SD) and asymptomatic (AD) skin from six dapple fruits over three weeks by northern blot analysis. Both SD and AD tissues were infected with ASSVd; however, ASSVd accumulation was significantly higher in SD tissues at the first two sampling points, while no difference was detected by the third week (Figure 1e). These results suggest that the symptomatic regions of dapple apples are associated with higher ASSVd accumulation. To examine whether sequence variation in ASSVd contributed to differential pathogenicity between SD and AD regions, ASSVd was amplified, cloned, and sequenced from both tissue types in six dapple apples. Approximately 20 clones were obtained per sample (Table S2). Sequence analysis revealed similar population structures between SD and AD regions, each comprising a dominant variant and several minor variants (Figure S2). The dominant variant was identical between symptomatic and asymptomatic skin from the same fruit (Table S2), indicating that the dapple phenotype is not caused by mixed infections with ASSVd variants of differing pathogenicity. 3.3 Genome-wide expression changes in dapple and scar skin apples To elucidate the molecular mechanisms underlying dapple and scar skin symptoms, we performed transcriptome analyses on dapple and scar skin apples. For dapple apples, three sample types were analyzed: asymptomatic apple skin (AA), symptomatic dapple skin (SD), and asymptomatic dapple skin (AD), each with six biological replicates. For scar skin apples, asymptomatic (A) and symptomatic (S) skins were collected, each with four biological replicates. 3.3.1 Differentially expressed genes of dapple apples Each transcriptome library generated over 6.3 Gb of high-quality reads, with a low error rate (0.03), high Q30 values (> 92.73%), and a high proportion of apple genome-mapped reads (> 91.72%) (Table S3). Gene expression levels were quantified as previously described (Mortazavi et al. 2008) and normalized by FPKM. Replicate consistency within each treatment group was high (Figure S3a), confirming sequencing reliability. Principal component analysis (PCA) revealed that one or two samples per treatment deviated slightly, with lower correlation coefficients (< 90%). To ensure biological reproducibility, four closely clustered replicates with higher correlation coefficients (Figure S3b, S3c) were selected for differential expression analysis. DEGs were identified using DESeq2 (Love, Huber, and Anders 2014) with thresholds of |log₂FoldChange| ≥ 1 and adjusted P -value ( P adj ) ≤ 0.05. Compared to AA, SD exhibited 3,917 DEGs (Table S4 and Figure 2a), about half the number previously reported between asymptomatic and dapple ‘Fuji’ apples (6,938 DEGs) (Zheng et al. 2023). In contrast, AD displayed only 857 DEGs (Table S4 and Figure 2a). Although both SD and AD were infected by ASSVd, they shared only 519 DEGs, while SD contained 3,398 unique DEGs (Figure 2b). Comparison between SD and AD revealed 2,300 DEGs, including 983 downregulated and 1,317 upregulated genes (Figure 2a). These results indicate that the manifestation of dapple symptoms is associated with extensive transcriptional reprogramming involving thousands of apple genes. 3.3.2 Differentially expressed genes in scar skin apples Transcriptome sequencing of scar skin apples yielded over 6.1 Gb of high-quality reads per sample, with Q30 values exceeding 93% (Table S5). More than 90% of reads were successfully mapped to the apple genome (Table S5). The four biological replicates within each treatment exhibited highly consistent gene expression profiles (Figure S4a) and strong correlations (r > 0.95) (Figure S4b). Principal component analysis (PCA) clearly separated symptomatic from asymptomatic samples along the first principal component (PC1) (Figure S4c). Compared with asymptomatic apples, 3,520 genes were significantly differentially expressed in scar skin apples (Figure 2c)—a number comparable to that observed between SD and AD in dapple apples. Of these, 2,654 were upregulated and 866 were downregulated, indicating a transcriptional reprogramming predominantly driven by gene activation. These results demonstrate that scar skin symptoms, like dapple symptoms, are closely associated with large-scale changes in gene expression affecting thousands of apple genes. 3.4 Downregulation of the anthocyanin biosynthesis pathway in dapple apples Gene Ontology (GO) enrichment analysis was performed for DEGs in dapple apples using P adj < 0.05 (Table S7). For DEGs between the symptomatic skin of dapple fruits (SD) and asymptomatic fruits (AA), 58 significantly enriched GO terms were identified across the biological process (BP), cellular component (CC), and molecular function (MF) categories. Many were functionally related or hierarchically connected. Notably, most CC terms were subcategories of thylakoid (GO:0009579) (Table S7), and all were linked to photosynthesis-related BP terms. The DEGs associated with these terms were uniformly downregulated, suggesting suppression of photosynthetic activity in dapple apples. Nine enriched MF terms were represented by transmembrane signaling receptor activity (GO: 0004888), encompassing 10 upregulated DEGs that were also annotated under signal transducer activity (GO: 0004871) and signaling receptor activity (GO: 0038023). These overlapping categories indicate the activation of signaling cascades, likely related to plant hormone signal transduction. Another notable GO cluster centered on pollination (GO: 0009856), comprising 23 shared DEGs, reflecting potential impacts of ASSVd infection on reproductive tissues such as seeds. In the comparison between asymptomatic dapple skin (AD) and asymptomatic fruit skin (AA), 18 GO terms were significantly enriched, with only half overlapping those found between SD and AA. This partial overlap suggests that DEGs enriched in shared terms such as photosystem (GO: 0009521) are not directly associated with dapple symptom development. For DEGs between SD and AD, 18 terms were also enriched, though none in the CC category; all were included among the SD vs. AA terms, mainly associated with oxidoreductase activity, tetrapyrrole binding, transferase activity, sequence-specific DNA binding, and defense response. Collectively, these results indicate that ASSVd infection perturbs multiple hierarchically linked biological processes in dapple apples. Pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified significantly enriched pathways ( P < 0.05) (Table S8 and Figure 3a). Two upregulated pathways, plant–pathogen interaction and plant hormone signal transduction, were consistently enriched across all three comparisons (SD vs . AA, SD vs . AD, and AD vs . AA). The magnitude of enrichment was greater in SD, indicating a stronger activation of these pathways in symptomatic tissue. Ten additional pathways were enriched in both SD vs . AA and SD vs . AD comparisons, including fatty acid metabolism, biosynthesis, and elongation, all belonging to the fatty acid metabolic network. Fatty acid biosynthesis provides long-chain acyl–acyl carrier proteins (ACPs) required for biotin metabolism, linking these pathways metabolically. Several other downregulated pathways—flavonoid biosynthesis and stilbenoid, diarylheptanoid, and gingerol biosynthesis—share the common precursor acetyl-CoA, connecting them metabolically to fatty acid metabolism. In the SD vs . AD comparison, phenylpropanoid biosynthesis and cutin, suberin, and wax biosynthesis were also enriched. These pathways share a common upstream phenylpropanoid core that channels acetyl-CoA into divergent metabolic branches. Importantly, the flavonoid biosynthesis pathway, a downstream branch of phenylpropanoid metabolism that produces anthocyanins (Ma et al. 2021; An et al. 2020; El-Sharkawy, Liang, and Xu 2015), was strongly downregulated. Most genes in these pathways were transcriptionally repressed (Figure 4), suggesting that anthocyanin-associated metabolism is specifically inhibited in the symptomatic skin of dapple apples. To validate the transcriptome findings, RT-qPCR was used to quantify the expression of five key genes in the anthocyanin biosynthetic pathway. Consistent with the RNA-seq data, five key genes ( Md4Cl , MdCHS , MdCHI , Md3′H , MdDFR , MdANS ) showed significant downregulation in SD in comparison to AA (Figure 3b). Anthocyanin content was further quantified spectrophotometrically in the SD, AD, and AA groups ( n = 6 per group). Compared with AA, SD showed a significantly lower anthocyanin concentration (Figure 3c), corroborating previous findings (Li et al. 2023). 3.5 Suberin biosynthesis is upregulated in scar skin apples DEGs in scar skin apples were categorized and annotated through GO analysis, with enrichment assessed using adjusted P- values ( P adj < 0.05), yielding 58 significantly enriched terms (Table S9). Among these, 38 belonged to the biological process (BP) category. Compared to the enriched terms identified in dapple apples, only two were shared: multi-organism process (GO: 0051704) under BP and DNA-binding transcription factor activity (GO: 0003700) under molecular function (MF). This discrepancy may reflect differences in cultivar background and developmental stages between the two experimental sample sets. Nonetheless, several functionally related GO terms were identified. For instance, in scar skin apples, 15 BP terms were associated with responses to biotic and abiotic stimuli and plant hormones, such as responses to fungi (GO: 0050832), absence of light (GO: 0009646), and gibberellin (GO: 0009739), which are closely related to the defense response term (GO: 0006952) enriched in dapple apples (Table S7). Moreover, multiple BP terms were linked to phenylpropanoid metabolism, including biosynthetic processes related to phenylpropanoids, flavonoids, lignin, wax, cutin, and suberin, as well as fatty acid metabolism (Table S9). These findings suggest a strong association between the phenylpropanoid pathway and scar skin disease development, which was further supported by KEGG pathway enrichment. KEGG analysis identified 23 significantly enriched biological pathways ( P adj < 0.05) in scar skin apples (Figure 5a and Table S10), encompassing nearly all pathways enriched in dapple apples. Common responses to pathogen attack included plant hormone signal transduction and plant–pathogen interaction. Brassinosteroid biosynthesis was upregulated in both scar skin and dapple apples, implicating brassinosteroids in disease development. Importantly, phenylpropanoid-related pathways were consistently enriched in both diseases, including those for flavonoid, stilbenoid, diarylheptanoid, and gingerol biosynthesis, as well as phenylpropanoid, cutin, suberin, and wax biosynthesis, fatty acid biosynthesis, elongation, and degradation, and phenylalanine metabolism (Figure 5a). The characteristic scar skin symptoms primarily reflect dysregulation of cutin, suberin, and wax biosynthesis, particularly excessive suberin accumulation in apple skins. Consistently, most DEGs involved in phenylpropanoid and suberin biosynthetic pathways (Ma et al. 2021; An et al. 2020; El-Sharkawy, Liang, and Xu 2015) were significantly upregulated (Figure 5b). These included nearly all structural and functional genes responsible for producing phenolic acid precursors of suberin (Serra and Geldner 2022), such as phenylanaline ammonia-lyase ( PAL , LOC103440652), 4-coumarate-CoA ligase ( 4CL , LOC103427406), and caffeoyl-CoA O-methyltransferase ( COMT , LOC103448267) (Table S6). Collectively, these results demonstrate that scar skin disease is characterized by the upregulation of the suberin biosynthetic pathway. 3.6 Establishment of ASSVd infection systems Although both dapple and scar skin symptoms are associated with ASSVd infection, field-collected diseased fruits are often co-infected by insects and latent apple viruses such as ACLSV, ASGV, and ASPV, which can confound host gene expression analysis. To verify the specific impact of ASSVd infection on gene expression within phenylpropanoid-related pathways, we established artificial ASSVd infection systems using red-fleshed apple callus ( cv . ‘Redlove’), tissue-cultured apple seedlings, and transgenic tomato plants. 3.6.1 ASSVd-infected apple callus Due to the long growth cycle and environmental variability of field-grown fruits, in vitro apple fruit callus provided a practical alternative for infection studies. Calli were generated and co-cultured with Agrobacterium tumefaciens harboring recombinant plasmids containing dimeric ASSVd cDNA. Control calli, co-cultured with Agrobacterium containing an empty vector, served as mock treatments. After 10 days under a 16 h light/8 h dark photoperiod, control calli developed a red coloration, whereas ASSVd-infected calli did not (Figure 6a), indicating that ASSVd infection suppresses anthocyanin synthesis in apple callus tissue. Northern blot analysis confirmed successful ASSVd infection (Figure 6b). 3.6.2 ASSVd-infected tissue-cultured apple seedlings Previous studies showed that ASSVd could infect tissue-cultured apple seedlings via slashing inoculation using crude extracts from infected trees (Xi et al. 2020), although infection efficiency was low (~30%). To enhance infection rates, both dimeric ASSVd transcripts and synthetic circular ASSVd RNA were used as inocula for slashing inoculation under sterile culture conditions. Of 16 seedlings inoculated with dimeric transcripts and 27 with circular RNA, 1 (6.25%) and 12 (44.44%), respectively, tested positive for ASSVd by RT-PCR one-month post-inoculation (Figure S5a). In addition, an Agrobacterium -mediated agroinfiltration method was developed for young apple plantlets. The upper 2–3 fully expanded leaves were excised, infiltrated with Agrobacterium carrying the dimeric ASSVd plasmid, and cultured on regeneration medium (Figure 6c). ASSVd was detected in all regenerated plantlets at 42 days post-inoculation and reached levels comparable to field-collected apple epidermis by 72 days (Figure 6d). 3.6.3 ASSVd-expressing transgenic tomato Although ASSVd has been reported to infect herbaceous plants such as cucumber, common bean, Nicotiana benthamiana , and tomato through mechanical inoculation (Walia et al. 2014), attempts to infect cucumber, tomato, and N. benthamiana under our experimental conditions were unsuccessful (Du 2022). Therefore, dimeric ASSVd cDNA was introduced into tomato ( cv . ‘Micro-Tom’) via Agrobacterium -mediated transformation. In the resulting transgenic plants, ASSVd was detectable by RT-PCR but not by northern blot, indicating low accumulation levels. RT-qPCR quantification confirmed that ASSVd levels in transgenic tomato were significantly lower than those in infected apple trees (Figure S5d). 3.7 ASSVd suppresses the expression of genes involved in anthocyanin biosynthesis The expression of six key structural genes in the anthocyanin biosynthetic pathway was quantified by RT-qPCR in four ASSVd-infected and three control samples. In callus tissues, five ( MdC4H , MdANS , MdDFR , MdCHS , MdCHI ) of the six ( MdC4H , MdF3H , MdANS , MdDFR , MdCHS , MdCHI ) genes were significantly downregulated (Figure 6e). Similarly, all six genes ( MdC4H , MdF3H , MdANS , MdCHS , MdUFGT , MdCHI ) exhibited significant downregulation in infected apple seedlings (Figure 6f), confirming the inhibitory effect of ASSVd on anthocyanin biosynthesis. This suppression was further validated in transgenic tomato plants expressing ASSVd. RT-qPCR quantification showed that three ( SlF3H , SlCHS , SlCHI ) of the six ( SlC4H , SlF3H , SlANS , SlDFR , SlCHS , SlCHI ) genes were significantly downregulated (Figure S5e). 4 Discussion Elucidating the molecular mechanisms underlying ASSVd-induced dapple and scar skin diseases in apple remains challenging, primarily due to the long developmental period of this woody host. In this study, through transcriptome sequencing and the establishment of ASSVd-infected apple and tomato systems, we revealed that dapple symptoms are associated with suppression of the anthocyanin biosynthesis pathway, while scar skin symptoms are linked to the upregulation of suberin biosynthesis. These findings provide a comprehensive understanding of how ASSVd alters secondary metabolism and gene expression, highlighting distinct yet related mechanisms underlying the two disease phenotypes. Dapple apple symptoms are characterized by reduced anthocyanin content in localized skin areas. Consistent with previous findings (Wu 2015), our analyses confirmed significantly lower anthocyanin accumulation in symptomatic regions compared with both asymptomatic areas of the same fruit and asymptomatic apples (Figure 3c). The reduced pigment levels likely result from suppression of the anthocyanin biosynthetic pathway, as most of its structural genes were downregulated in symptomatic tissues (Table S4). This aligns with the transcriptome data from 11-year-old dapple apples ( cv . ‘Hirosaki Fuji’) (Zheng et al., 2023). Furthermore, pathways associated with anthocyanin metabolism, including fatty acid and biotin metabolism, as well as cutin, suberin, and wax biosynthesis, were significantly enriched, suggesting altered metabolic flux toward anthocyanin biosynthesis in dapple apples. It has been hypothesized that apple skin-specific genes could be targeted by ASSVd-derived small RNAs (sRNAs), initiating dapple symptom development (Flores et al., 2020). Although such sRNAs have not yet been identified for ASSVd, apple dapple fruit viroid (ADFVd), a closely related viroid, generates vsiR693, which targets the bHLH transcription factor MdPIF1 , a key regulator of anthocyanin biosynthesis (Zhang et al. 2024). MdPIF1 binds to G-box motifs in the promoters of MdPAL and MdF3H , promoting their expression and enhancing anthocyanin accumulation. However, in our study, these two genes were neither predicted targets of ASSVd-sRNAs nor significantly altered in dapple fruits, suggesting distinct regulatory mechanisms. Moreover, sequencing revealed no distinct ASSVd variants between symptomatic and asymptomatic tissues (Figure S2). Notably, DEGs identified in dapple apples also included regulatory genes of anthocyanin biosynthesis, such as MYB113 (Zhu et al. 2024), and several genes within the plant hormone signal transduction pathway, indicating a complex and multilayered regulatory network governing anthocyanin synthesis in response to viroid infection. Dapple-like symptoms have also been reported in sweet cherry fruits infected by hop stunt viroid (HSVd) (Xu et al. 2017; Xu et al. 2019). Transcriptomic analyses of dappled cherry fruits identified enrichment of plant hormone signaling, phenylpropanoid, and flavonoid biosynthetic pathways (Xu et al. 2020), mirroring our observations in apples. Although ASSVd and HSVd belong to different genera within the family Pospiviroidae and share low sequence similarity (Di Serio et al. 2021), the similar pathway responses suggest that suppression of anthocyanin biosynthesis may stem from shared upstream perturbations—particularly in plant hormone signaling and innate immune responses—rather than from direct targeting of structural genes by viroid-derived sRNAs. Notably, the brassinosteroid (BR) biosynthetic pathway was enriched and upregulated in both dapple apples and cherries. Since BRs are known inhibitors of anthocyanin biosynthesis (Wang et al. 2023), their increased activity may contribute to the pigment reduction observed in ASSVd-infected fruits (Table S7). In contrast, scar skin apples displayed significant enrichment of the cutin, suberin, and wax biosynthesis pathway, with most DEGs in this pathway markedly upregulated (Table S6 and Figure 5b), suggesting enhanced suberin accumulation that contributes to the roughened skin phenotype. Both anthocyanin and suberin biosynthetic routes are downstream branches of the phenylpropanoid pathway. While anthocyanins derive from flavonoid intermediates, suberin precursors are phenolic acids, indicating that differential regulation of this upstream pathway could drive the contrasting metabolic outcomes in dapple and scar skin apples. Notably, the BR biosynthesis pathway was also enriched in scar skin apples (Table S10), suggesting that ASSVd infection may activate BR-mediated signaling cascades, which in turn lead to divergent downstream outcomes, including suppression of anthocyanin biosynthesis and induction of suberin biosynthesis. This supports the proposed model of pospiviroid pathogenesis, in which viroid infection perturbs host hormone signaling and transcriptional networks to induce disease symptoms (Flores et al., 2020). Acknowledgements We gratefully acknowledge Professor Hu Guojun from the Research Institute of Pomology of Chinese Academy of Agricultural Sciences for providing the viroids/viruses-free apple seedlings and Professor Li Tianzhong from the College of Horticulture of China Agricultural University for providing apple callus. This work was supported by the National Natural Science Foundation of China (Grant No. 32472532) and the Central Public-Interest Scientific Institution Basal Research Fund (Y2025YC25) . Conflicts of Interest The authors declare no conflicts of interest. 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FIGURE 1. Association of ASSVd infection with dapple and scar skin apples. (a) Asymptomatic and dapple apples ( cv. ‘Fuji’) collected from the field. The skin of asymptomatic fruit (AA) and the symptomatic (SD) and asymptomatic (AD) regions of dapple apples were collected for transcriptome analysis. (b) Northern blot detection of ASSVd in dapple (D) and asymptomatic (A) apples. (c) Asymptomatic and scar skin apples ( cv . ‘Venus Gold’); scar skin (S) and asymptomatic (A) skin were collected for transcriptome analysis. (d) Northern blot detection of ASSVd in scar skin (S) and asymptomatic apples. (e) Quantification of ASSVd accumulation in SD and AD at weekly intervals using northern blot hybridization. Ribosomal RNA (rRNA) was used as the loading control. ASSVd-infected and healthy fruits served as positive (PC) and negative (NC) controls, respectively. FIGURE 2 Differentially expressed genes (DEGs) identified by transcriptome sequencing. (a) Numbers of DEGs in comparisons between SD and AD, SD and AA, and AD and AA in dapple apples. (b) Venn diagram showing overlapping and unique DEGs identified in SD and AD compared with AA. (c) DEGs between asymptomatic (A) and scar skin (S) apples. Green dots represent downregulated genes, and purple dots represent upregulated genes. FIGURE 3 Anthocyanin synthesis is repressed in dapple apples. (a) Significantly enriched biological pathways across comparisons. Anthocyanin biosynthesis forms a branch of the flavonoid biosynthesis pathway. (b) Expression levels of anthocyanin biosynthesis genes in dapple apple skins determined by RT-qPCR. Five of six genes were significantly downregulated in SD compared with AA. (c) Quantification of anthocyanin content in asymptomatic (AA), symptomatic (SD), and asymptomatic regions (AD) of dapple apples. Student’s t -test: ** P < 0.01, * P < 0.05; ns, not significant. Md, Malus domestica ; 4CL , 4-coumarate-CoA ligase; CHS , chalcone synthase; CHI , chalcone isomerase; F3’H , flavonoid 3′-hydroxylase; DFR , dihydroflavonol 4-reductase; ANS , anthocyanidin synthase. FIGURE 4 The anthocyanin biosynthesis pathway is downregulated in dapple apples. Gene abbreviations are indicated along the arrows, and the accompanying number strings represent gene IDs (upregulation in red, downregulation in green). PAL, phenylalanine ammonia-lyase; C4H, trans-cinnamate 4-monooxygenase; 4CL, 4-coumarate–CoA ligase; HCT, hydroxycinnamoyl-CoA shikimate; C3H, p-coumarate 3-hydroxylase; CHS, chalcone synthase; CHI, chalcone–flavonone isomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; UFGTs, UDP-glucose: flavonoid 3-O-glucosyltransferase; OMTs, O-methyltransferases; BAHD-ATs, BAHD family acyltransferases. FIGURE 5 Suberin biosynthesis is upregulated in scar skin apples. (a) Significantly enriched biological pathways identified in scar skin apples. (b) Diagram of the suberin biosynthetic pathway, with upregulated genes shown in red and downregulated genes shown in green. Gene abbreviations and IDs are annotated along the arrows. PAL, phenylalanine ammonia-lyase; C4H, trans-cinnamate 4-monooxygenase; 4CL, 4-coumarate-CoA ligase; HCT, hydroxycinnamoyl-CoA shikimate; C3H, p-coumarate 3-hydroxylase; COMT, caffeic acid O-methyltransferase; F5H, ferulate 5-hydroxylase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase; LACS, long-chain acyl-CoA synthetase; KCS, β-ketoacyl-CoA synthase; KCR, β-ketoacyl-CoA reductase; HCD, hydroxyacyl-CoA dehydratase; ECR, enoyl-CoA reductase; FAR, fatty acyl-CoA reductase; CYP86, cytochrome P450 family 86; ASFT, sinapoylglucose:choline sinapoyltransferase; GPAT, glycerol-3-phosphate acyltransferase; GDSL, GDSL esterase. FIGURE 6 Establishment of ASSVd-infected apple callus and seedlings and quantification of anthocyanin synthase genes. (a) Healthy callus (H) and ASSVd-infected callus (D) after 10 days of light treatment exhibited distinct coloration. (b) Northern blot detection of ASSVd in apple fruit callus. (c) Healthy and ASSVd-infected apple seedlings. (d) Northern blot detection of ASSVd in tissue-cultured apple seedlings. (e) Quantification of anthocyanin biosynthesis genes in apple callus by RT-qPCR. (f) Quantification of anthocyanin biosynthesis genes in tissue-cultured apple seedlings by RT-qPCR. (Student’s t -test, *** P < 0.001, ** P < 0.01, * P < 0.05). ns , not significant. Md, Malus domestica . C4H , trans-cinnamate 4-monooxygenase; F3H , flavanone 3-hydroxylase; ANS , anthocyanidin synthase; DFR , dihydroflavonol 4-reductase; CHS , chalcone synthase; CHI , chalcone isomerase; UFGT , UDP-glucose:flavonoid 3-O-glucosyltransferase. FIGURE S1 RT-PCR detection of four viruses in dapple apples. (a) Apple necrosis mosaic virus (ApNMV); (b) Apple chlorotic leaf spot virus (ACLSV); (c) Apple stem pitting virus (ASPV); (d) Apple stem grooving virus (ASGV). AA, asymptomatic apples; AD, asymptomatic skin of dapple apples; SD, symptomatic skin of dapple apples. FIGURE S2 Population structure of ASSVd in the symptomatic (SD) and asymptomatic (AD) skins of dapple apples. Each color represents a distinct sequence variant. FIGURE S3 Gene expression analysis of dapple apple samples used for transcriptome sequencing. (a) Global gene expression patterns. (b) Principal component analysis (PCA). (c) Correlation coefficient analysis. AA, skin of asymptomatic apples; AD, asymptomatic skin of dapple apples; SD, symptomatic skin of dapple apples. FIGURE S4 Gene expression analysis of scar skin apple samples used for transcriptome sequencing. (a) Global gene expression patterns. (b) Correlation coefficient analysis. (c) Principal component analysis (PCA). A, asymptomatic apples; S, scar skin apples. FIGURE S5 Establishment of ASSVd-infected apple fruit callus, tissue-cultured apple seedlings, and transgenic tomato plants. (a) RT-PCR detection of ASSVd in inoculated tissue-cultured apple seedlings using dimeric RNA transcripts (ASSVd-dimer) and synthesized circular RNA (ASSVd-circ). (b) Empty vector-transformed callus (RZ) and ASSVd-infected callus (D) exhibited distinct pigmentation after 10 days of light treatment. (c) Wild-type (WT) tomato plants and ASSVd-transgenic tomato plants (D). (d) Quantification of ASSVd levels in transgenic tomato lines by RT-qPCR. The symptomatic skin of dapple apple (DD3) was used as the reference. (e) Quantification of anthocyanin biosynthesis gene expression in transgenic tomato plants by RT-qPCR. (Student’s t-test, *** P < 0.001, ** P < 0.01, * P < 0.05; ns, not significant). Sl, Solanum lycopersicum . C4H , trans-cinnamate 4-monooxygenase; F3H , flavanone 3-hydroxylase; ANS , anthocyanidin synthase; DFR , dihydroflavonol 4-reductase; CHS , chalcone synthase; CHI , chalcone isomerase. Information & Authors Information Version history V1 Version 1 21 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords apple phenylpropanoid secondary metabolism transcriptome viroid Authors Affiliations He Lingzhu Chinese Academy of Agricultural Sciences State Key Laboratory for Biology of Plant Diseases and Insect Pests View all articles by this author Xu Huiyuan Chinese Academy of Agricultural Sciences State Key Laboratory for Biology of Plant Diseases and Insect Pests View all articles by this author Xie Jipeng Chinese Academy of Agricultural Sciences State Key Laboratory for Biology of Plant Diseases and Insect Pests View all articles by this author Tao Zhou 0000-0001-7702-8472 China Agricultural University Department of Plant Pathology View all articles by this author Yang Xiuling Chinese Academy of Agricultural Sciences State Key Laboratory for Biology of Plant Diseases and Insect Pests View all articles by this author Li Shifang Chinese Academy of Agricultural Sciences State Key Laboratory for Biology of Plant Diseases and Insect Pests View all articles by this author Zhang Zhixiang 0000-0002-1171-6966 [email protected] Chinese Academy of Agricultural Sciences State Key Laboratory for Biology of Plant Diseases and Insect Pests View all articles by this author Metrics & Citations Metrics Article Usage 360 views 217 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation He Lingzhu, Xu Huiyuan, Xie Jipeng, et al. Apple scar skin viroid causes dapple and scar skin apple diseases by disrupting phenylpropanoid-mediated metabolic pathways. Authorea . 21 October 2025. DOI: https://doi.org/10.22541/au.176103045.51528753/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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