Mealybugs (F. virgata) were originally isolated from betel palm and were reared on pumpkin fruit in nylon net cages (75 × 75 × 75 cm). One-year old betel palm seedlings and mealybugs were cultivated in chamber as previous report (Zhang et al., 2022). First instars of F. virgata were transferred from pumpkin fruit to APV1 infected plants for 48-h acquisition access period (AAP) then further transferred onto test seedlings for 7 days inoculation access period (IAP). The seedlings were sprayed with insecticide to kill the mealybugs after inoculation. A mock inoculation was performed using non-viruliferous mealybugs. The inoculated seedlings were detected by RT-PCR using APV1 specific primers 2 months after inoculation.

Three groups of betel palm seedlings were chosen for host choosing experiment, including APV1 positive seedlings with yellowing symptom (APV1+ yellow), APV1 positive seedling without yellowing symptom (APV1+ green), and the healthy seedlings (healthy control). After 4 h starvation, 100 adult F. virgate mealybugs were released from the cage laying between the two types of A. catechu seedlings, the number of mealybugs freely distributed onto each type of seedlings was calculated at 6 h after releasing, respectively.

Leaves from APV1 infected plants and control plants were collected and ground in liquid nitrogen for Western-blot analysis. Total proteins were isolated and separated by SDS-PAGE. The protein was transferred from gel onto PVDF membrane using a Mini Trans-Blot Electrophoretic Transfer system (Bio-Rad, #1703930). APV1 was detected using a mouse anti-APV1 CP monoclonal antibody (1:5,000 dilution) and horseradish peroxidase-conjugated goat anti-mouse IgG (1:2,000 dilution) as the secondary antibody (Solarbio, catalog no. SE131). The blot was visualized using a chemiluminescence film (Thermo Fisher, catalog no. 34577).

Leaf samples from the APV1-inoculated and mock-inoculated betel palm seedlings were collected for RNA-Seq and de novo assembly were conducted as previously described (Wang et al., 2020). All differential expressed genes (DEGs) were subjected to gene ontology (GO) and KEGG enrichment analysis using GOseq and KOBAS software, respectively (Li et al., 2020). qRT-PCR was carried out as previously described (Livak and Schmittgen, 2001; Zhang et al., 2022). Briefly, total RNA was purified from leave samples using a plant RNA extraction kit (Tiangen Biotech, Beijing, China), and cDNA was synthesized with a one-step reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). Actin gene was chosen for the normalization. The primer sequences used in this experiment are listed in Supplementary Table S1.

The penultimate fully unfolded leaves of YLD betel palm and healthy betel palm were collected, and 0.2 g of each leaf sample was cut into small pieces. These pieces were then homogenized in 25 ml of 95% ethanol and kept in darkness for 24 h. The supernatants were analyzed at 470, 649, and 665 nm, respectively. Chlorophyll was calculated according to Arnon's equation. The grand means of the values were subjected to analysis of variance (ANOVA) by SPSS version 19.0 (Li et al., 2020).

The penultimate fully expanded leaves of the YLD betel palm and healthy betel palm were measured using a portable open gas exchange system with a 3 × 3 cm leaf chamber (LI6800-12A, Li-COR Inc., Lincoln, NE, USA). Measurements were conducted between 9:00 and 11:00 in the morning on clear days with natural fluctuations in air temperature and vapor pressure deficit. The temperature in the climate chamber was set at 28°C and 55% relative humidity. The CO₂ level was ~400 μmol mol⁻¹. Each sample was measured five times (Tseliou et al., 2021).

Chlorophyll fluorescence of the YLD betel palm and healthy betel palm were analyzed using a MINI-PAM-II fluorometer (Walz, Germany). The analyses were repeated three times. Induction curves were performed using a MINI-PAM-II fluorometer following the manufacturer's protocol. The fluorescence parameters of the light response curve and induction curve were converted using WinControl-3 software (Walz, Germany), and the graphs were drawn using Excel software (Ivanov and Bernards, 2016).

Transmission electron microscopy (TEM) was conducted as previously described (Jin et al., 2018). Ultrathin sections were cut to 70–90 nm thickness using a Leica EM UC7 ultramicrotome. The sections were sequentially stained with uranyl acetate for 20 min and Reynolds' lead citrate for 5 min. The sections were then viewed with a Hitachi H-7650 or a JEM-1230 transmission electron microscope operated at 80 kV.

Immune Electron Microscopy (IEM) was conducted as previously report (Folimonova et al., 2008). The leaf samples were fixed using phosphate buffer containing 1.8% paraformaldehyde and 0.25% glutaraldehyde, and imbedded in LR White resin (London Resin Company, Hampshire, United Kingdom). Immunogold labeling of ultrathin sections (70- to 80-nm) was performed using a purified mouse monoclonal antibody against APV1-CP (concentration: 2 mg/ml, diluted 1:500) as the primary antibody, followed by incubation with goat anti-mouse 12-nm gold conjugate (diluted 1:25; Sigma). The specimens were meticulously observed using a HT7800/HT7700 transmission electron microscope (HITACHI, Japan).

To investigate the mechanism underlying the yellowing symptoms induced by APV1 in betel palm, Betel palm seedlings were inoculated with through APV1 through F. virgate mealybugs. At 60 days post inoculation (IS-1), leaf yellowing symptoms initially appeared at the leaflet tips. Over time, the yellowing gradually extended toward the petiole, resulting in most of the leaves turning yellow, while the midribs remained green, forming a distinct green-yellow border at 180 days post-inoculation (IS-2) (Figure 1A). This observation aligns with previous reports (Wang et al., 2020; Zhang et al., 2022). In addition to leaf yellowing, APV1-infected seedlings exhibited significant growth retardation compared to the control (Figure 1B; Supplementary Figure S1). Western blot analysis using a monoclonal antibody against APV1-CP revealed the accumulation of APV1 in the inoculated betel palm seedlings (Figure 1C). Measurement results indicated a significant decrease in photosynthesis-related pigments. The contents of chlorophyll a, chlorophyll b, and carotenoids, decreased from 0.693, 0.374, and 0.128 mg/g in control sample to 0.318, 0.209, and 0.067 mg/g in APV1-infected samples, respectively (Figure 1D).

Chlorophyll fluorescence can serve as an indicator of plant photosynthesis efficiency and can be used to detect early-stage biotic and abiotic stresses (Moustaka and Moustakas, 2023). Measurement results showed that four crucial indicators of chlorophyll fluorescence, e.g., photosynthetic electron transport rate (ETR), actual photochemical efficiency in photosystem II (Y_II), photochemical quenching (qP), and non-photochemical quenching (NPQ), were significantly lower in APV1-infected samples (IS-2) than that in the mock control (Figure 2). Furthermore, under conditions of saturated light intensity (1,000 μmol m⁻² s⁻¹), the net photosynthetic rate (Pn) and stomatal conductance (Gs) were significantly lower in APV1-infected samples (IS-2), while the intercellular CO₂ concentration (Ci) was higher than that of the control (Figure 3). A higher Ci value indicates lower photosynthesis efficiency (Tominaga et al., 2018). In summary, APV1 infection reduced chlorophyll pigmentation, resulted in fluctuation of chlorophyll fluorescence, and led to decreased photosystem efficiency.

To investigate the causes of yellowing symptoms, reduced chlorophyll, and decreased photosynthesis efficiency induced by APV1, leaves from APV1-infected and healthy control samples were collected for tissue sectioning and transmission electron microscopy (TEM) observation. In healthy control cells, chloroplasts were oval in shape and evenly distributed, with starch grains (SG) surrounded by grana lamellae (GL). In the early stages of virus infection (IS-1), the chloroplast membrane separated from the cell wall (CW), and the size and number of osmiophilic granules (OG) reduced. The GL became less organized, and SG were separated from each other, with many unknown particles and white holes appearing in the cytoplasm. In the late stage (IS-2), chloroplasts were nearly completely degraded, with a sharp reduction in size, and large starch grains (SG) accumulating in the cytoplasm (Figure 4). These findings confirm that APV1 infection severely damages the chloroplast structure and other cytoplasmic organelles in leaf cells. In this work, immune electron microscopy (IEM) was used to examine the distribution of APV1 in leaf. IEM revealed that APV1 mainly localized in chloroplast and occasionally in cytoplasm, confirming that APV1 has invaded chloroplast and caused degradation of chloroplast (Figure 5).

To identify genes regulated by APV1 infection, we conducted RNA-seq and analyzed differentially expressed genes (DEGs) between APV1-infected samples and healthy controls. A total of 166,590 unigenes were obtained through de novo assembly from the clean RNA-seq reads. DEG analysis revealed 12,553 up-regulated DEGs and 5,698 down-regulated DEGs in response to APV1 infection (Supplementary Figure S2). Gene ontology (GO) annotation enrichment showed that DEGs were distributed across 45 GO terms, with the most DEGs associated with terms such as Binding, Protein binding, and Intracellular organelle (Supplementary Figure S3). Notably, key genes involved in chlorophyll biosynthesis, such as chlorophyll synthase (CS) and chlorophyll b reductase (CbR), as well as genes related to carotene metabolism, such as zeta-carotene desaturase (zCD) and phytoene synthase (PS), were down-regulated in APV1-infected samples (Supplementary Figure S4). This was further confirmed by qRT-PCR, with the expression of zCD, PS, CS and CbR significantly down-regulated by APV1 infection (Figure 6). These findings suggest that APV1 infection has a global impact on gene expression patterns, suppressing the expression of key genes involved in chloroplast and photosynthesis, which were nuclear-encoded.

To investigate whether yellowing symptoms might attract transmission vectors for the plant virus, the APV1 vector F. virgate was used in host-choosing experiments among three types of betel palm seedlings. As expected, there was no significant difference in mealybug distribution between APV1-positive seedlings without yellowing symptoms and healthy seedlings (t = 0.458, P > 0.05), or between two groups of healthy seedlings (t = 0.266, P > 0.05). However, a significant difference was observed between APV1-positive seedlings with yellowing symptoms and healthy seedlings (t = 3.800, P < 0.05), with more mealybugs choosing the seedlings displaying yellowing symptoms as hosts (Figure 7). These results support the hypothesis that yellowing symptoms act as attractants for APV1 transmission vectors.