Delivery of Rice Gall Dwarf Virus Into Plant Phloem by Its Leafhopper Vectors Activates Callose Deposition to Enhance Viral Transmission

Abstract

Rice gall dwarf virus (RGDV) and its leafhopper vector Recilia dorsalis are plant phloem-inhabiting pests. Currently, how the delivery of plant viruses into plant phloem via piercing-sucking insects modulates callose deposition to promote viral transmission remains poorly understood. Here, we initially demonstrated that nonviruliferous R. dorsalis preferred feeding on RGDV-infected rice plants than viruliferous counterpart. Electrical penetration graph assay showed that viruliferous R. dorsalis encountered stronger physical barriers than nonviruliferous insects during feeding, finally prolonging salivary secretion and ingestion probing. Viruliferous R. dorsalis feeding induced more defense-associated callose deposition on sieve plates of rice phloem. Furthermore, RGDV infection significantly increased the cytosolic Ca²⁺ level in rice plants, triggering substantial callose deposition. Such a virus-mediated insect feeding behavior change potentially impedes insects from continuously ingesting phloem sap and promotes the secretion of more infectious virions from the salivary glands into rice phloem. This is the first study demonstrating that the delivery of a phloem-limited virus by piercing-sucking insects into the plant phloem activates the defense-associated callose deposition to enhance viral transmission.

Keywords: callose deposition; callose synthase genes; insect feeding behavior; phloem-limited virus; rice leafhopper.

Figure 1

Nonviruliferous Recilia dorsalis preferred to feed on infected rice plants. Selective preference of nonviruliferous (A) or viruliferous (B) R. dorsalis adults to rice gall dwarf virus (RGDV) infected or healthy rice plants. The histogram bars show the number of insects feeding on healthy or RGDV-infected rice plants. HP, healthy plant; IP, infected plant. Significant differences in gene expression are denoted as ^*(p, 0.05) or ^**(p, 0.01); multiple t-tests.

Figure 2

The effect of odors emitted by virus-infected rice plants on the feeding tendency of R. dorsalis. (A) Y-tube olfactometer diagram. (B) Selective odor preference of viruliferous or nonviruliferous R. dorsalis adults to RGDV infected or healthy rice plants. Each experimental group comprises 30 adults and is repeated five times in total. Viruliferous, viruliferous R. dorsalis; nonviruliferous, nonviruliferous R. dorsalis; HP, healthy plant; IP, infected plant; NR, no responding. Bars represent means ± SE.

Figure 3

Feeding behavior of viruliferous or nonviruliferous R. dorsalis on rice plants, as detected by electrical penetration graph (EPG) assay. (A) Characterization of the EPG waveforms produced by feeding R. dorsalis on rice plants, including waveforms NP, A, S, C, E, F, and R. NP, not probing. A, stylet movement in the tissue. S, intracellular salivation in mesophyll cells. C, active ingestion from the phloem. E, passive phloem sap ingestions. F, obstacle waveform. R, rest waveform. (B–I) Viral infection of insect vectors affected the feeding behavior of R. dorsalis. The data were electrically recorded during 5 h feeding period for viruliferous or nonviruliferous insects on healthy rice plants. V⁻, nonviruliferous adults feeding on healthy rice; V⁺, viruliferous adults feeding on healthy rice. Bars represent means ± SE. Significant differences in feeding behavior are denoted as *(p, 0.05), **(p, 0.01), or ***(p, 0.001); student’s t-test.

Figure 4

Rice gall dwarf virus distribution in rice phloem after infested by viruliferous R. dorsalis. (A–E) Immunofluorescence detection of RGDV in the cross-sections prepared from leaf phloem infested with nonviruliferous or viruliferous R. dorsalis. Nonviruliferous (B) or viruliferous (C–E) R. dorsalis adults were fed on healthy rice plants for 3 days, which were then processed for immunofluorescence with RGDV P8 antibody conjugated to fluorescein isothiocyanate (FITC). Non-infested healthy rice leaves served as a control. Xy, xylem; Ph, phloem; Mes, mesophyll. (F) The distribution of RGDV particles in rice phloem parenchyma was observed by electron microscopy. V, RGDV virions; PP, phloem parenchyma; SE, sieve elements; Va, vacuole; Mi, mitochondrion. Scale bars: 25 μm (A–E) and 500 nm (F).

Figure 5

Infection of RGDV by R. dorsalis activated the callose deposition in rice phloem. (A–F) Induced callose deposition in the phloem depicted by bright blue fluorescence in the cross-sections (A–C) and longitudinal-sections (D–F) prepared from leaf phloem infested with nonviruliferous or viruliferous R. dorsalis. The sample without insect infestation served as the control. The thin sections were stained with 0.1% aniline blue at 3 days after R. dorsalis feeding and examined under a fluorescence microscope. Xy, xylem; Ph, phloem; Mes, mesophyll; Epi, epidermis. Arrows indicate the bright blue fluorescence. Scale bars: 50 μm. (G) The average areas of sieve plates with callose deposition in nonviruliferous or viruliferous R. dorsalis-infested leaf sheaths were counted using Image J. Error bars denote ± SE of sieve plates with callose deposition observed in 46 cross-sections. Significant differences in callose deposition are denoted as ^***(p, 0.001); student’s t-test.

Figure 6

Real-time RT-PCR assay of the callose synthase and callose hydrolyzing genes in response to R. dorsalis feeding. The callose synthase-encoding gene GSL1 (A) and callose hydrolase-encoding gene Gns5 (B) were analyzed. Total RNA was extracted from rice leaf sheaths after different R. dorsalis feeding times. Expression of genes was quantified relative to the value obtained from 0 h samples (R. dorsalis-free plants). Each bar represents the mean ± SE of three replicates. Each RNA sample was extracted from approximately 100 mg of fresh leaf sheaths of five rice plants. Rice actin gene was used as a reference control. Significant differences in gene expression are denoted as ^*(p, 0.05), ^**(p, 0.01), or ^***(p, 0.001); student’s t-test.

Figure 7

Viruliferous R. dorsalis induced the elevated cytosolic Ca²⁺ level in the rice plant. (A) Fluochemical intracellular Ca²⁺ determination in leaves infested by viruliferous or nonviruliferous adults of R. dorsalis. The green fluorescence refers to the binding of Fluo-3 AM with Ca²⁺. A portion of rice leaf incubated with 5 μM Fluo-3 AM solution was infested by viruliferous or nonviruliferous adults for 1, 3, and 6 h. The sample without insect infestation served as the control. Untreated and wound-treated rice plants were used as controls. Scale bars: 100 μm. Arrows indicated the feeding sites. (B) Statistical analysis of feeding sites in infested-leaves by viruliferous and nonviruliferous adults. Significant differences of feeding sites were indicated with ^*(p, 0.05); student’s t-test.