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Specific cells in the primary salivary glands of the whitefly Bemisia tabaci control retention and transmission of begomoviruses - PubMed

Specific cells in the primary salivary glands of the whitefly Bemisia tabaci control retention and transmission of begomoviruses

Jing Wei et al. J Virol. 2014 Nov.

Abstract

The majority of plant viruses are vectored by arthropods via persistent-circulative or noncirculative transmission. Previous studies have shown that specific binding sites for noncirculative viruses reside within the stylet or foregut of insect vectors, whereas the transmission mechanisms of circulative viruses remain ambiguous. Here we report the critical roles of whitefly primary salivary glands (PSGs) in the circulative transmission of two begomoviruses. The Middle East Asia Minor 1 (MEAM1) species of the whitefly Bemisia tabaci complex efficiently transmits both Tomato yellow leaf curl China virus (TYLCCNV) and Tomato yellow leaf curl virus (TYLCV), whereas the Mediterranean (MED) species transmits TYLCV but not TYLCCNV. PCR and fluorescence in situ hybridization experiments showed that TYLCCNV efficiently penetrates the PSGs of MEAM1 but not MED whiteflies. When a fragment of the coat protein of TYLCCNV was exchanged with that of TYLCV, mutated TYLCCNV accumulated in the PSGs of MED whiteflies, while mutant TYLCV was nearly undetectable. Confocal microscopy revealed that virion transport in PSGs follows specific paths to reach secretory cells in the central region, and the accumulation of virions in the secretory region of PSGs was correlated with successful virus transmission. Our findings demonstrate that whitefly PSGs, in particular the cells around the secretory region, control the specificity of begomovirus transmission.

Importance: Over 75% of plant viruses are transmitted by insects. However, the mechanisms of virus transmission by insect vectors remain largely unknown. Begomoviruses and whiteflies are a complex of viruses and vectors which threaten many crops worldwide. We investigated the transmission of two begomoviruses by two whitefly species. We show that specific cells of the whitefly primary salivary glands control viral transmission specificity and that virion transport in the glands follows specific paths to reach secretory cells in the central region and then to reach the salivary duct. Our results indicate that the secretory cells in the central region of primary salivary glands determine the recognition and transmission of begomoviruses. These findings set a foundation for future research not only on circulative plant virus transmission but also on other human and animal viruses transmitted by arthropod vectors.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Figures

FIG 1
FIG 1

TYLCCNV and TYLCV transmissibility and virus dynamics in MEAM1 and MED whiteflies. (A) TYLCCNV and TYLCV transmissibility in MEAM1 and MED whiteflies. Whiteflies were fed on virus-infected plants for a 48-h AAP and then were used for inoculation tests in groups of 10 insects per healthy tomato plant for a 48-h IAP. Plant infection was determined by monitoring disease symptoms and by PCR amplification of the viral DNA 30 days after inoculation. Data on virus transmission were obtained from both 40 tomato plants and 40 tobacco plants for each combination of a whitefly species and a virus. (B) Quantitative PCR analysis of TYLCCNV DNA in groups of 50 MEAM1 or MED whiteflies that were maintained on TYLCCNV-infected tobacco plants (continuous feeding) for the time intervals indicated. (C) Quantitative PCR analysis of TYLCCNV DNA in groups of 50 MEAM1 or MED whiteflies that were transferred to nonhost cotton plants after a 48-h AAP on TYLCCNV-infected tobacco plants (retention feeding). Whiteflies were sampled at the time intervals indicated after transfer onto cotton plants.

FIG 2
FIG 2

Retention of TYLCCNV in the MGs and PSGs of MEAM1 and MED whiteflies. Adult whiteflies were allowed a 48-h AAP on TYLCCNV-infected plants and then maintained on a virus nonhost cotton plant. (A) Different abundances of TYLCCNV DNA within whitefly PSGs. Twenty PSGs dissected from viruliferous MEAM1 and MED whiteflies were collected separately for qPCR analysis. The numbers on the x axis represent three independent experiments. The numbers on the y axis represent abundances of TYLCCNV DNA relative to that of whitefly β-actin DNA. Vertical bars on columns indicate standard errors. (B and C) Percentages of MGs (B) and PSGs (C) with fluorescence signals. The data were surveyed separately from 20 dissected MGs and PSGs. (D to M) MGs and PSGs were hybridized with a Cy3-labeled virus-specific probe (red), and nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (blue). Specimens were visualized under a confocal microscope. Panels D and I are bright-field images of panels E and J for better visualization. Annotations under the corresponding pictures represent whitefly species-virus combinations. CA, caeca; FC, filter chamber; DM, descending midgut; AM, ascending midgut; DS, ductal section of the central region; EC, endcap; SS, secretory section of the central region.

FIG 3
FIG 3

Comparison of amino acid sequences of TYLCV and TYLCCNV coat proteins. Alignments were done by GeneDoc. The exchanged region is indicated by underlining.

FIG 4
FIG 4

Transmission and localization of mutant viruses in MEAM1 and MED whiteflies. (A) Acquisition of wild-type and mutant viruses by MEAM1 and MED whiteflies. Adult whiteflies were allowed a 48-h AAP on virus-infected plants and then were collected for analysis of viral DNA abundance. Quantitative PCR analysis was used to evaluate viral DNA abundances in groups of 50 MEAM1 or MED whiteflies. Vertical bars on columns indicate standard errors. (B) mTYLCCNV and mTYLCV transmissibility by MEAM1 and MED whiteflies. For each combination of a whitefly species and a virus, data on transmission were obtained from 40 tomato and 40 tobacco plants. (C and D) Percentages of MGs (C) and PSGs (D) with viruses. The data were obtained separately from 20 dissected MGs and PSGs. (E to N) MGs and PSGs were hybridized with a virus-specific Cy3-labeled probe (red), stained with DAPI to detect nuclei (blue), and then visualized under a confocal microscope. Panels E and J are bright-field images of panels F and K for better visualization. Annotations under the corresponding pictures represent whitefly species-virus combinations. CA, caeca; FC, filter chamber; DM, descending midgut; AM, ascending midgut; DS, ductal section of the central region; EC, endcap; SS, secretory section of the central region.

FIG 5
FIG 5

TYLCV accumulation and entry into PSGs. MED whiteflies were continuously fed on TYLCV-infected plants for 72 h. At each selected time point, 10 PSGs were dissected. TYLCV virions were detected by use of a mouse anti-TYLCV monoclonal antibody and a goat anti-mouse secondary antibody conjugated to FITC (green). Nuclei were stained with DAPI (blue) and then examined with a confocal microscope.

FIG 6
FIG 6

TYLCCNV accumulation and entry into PSGs. MED whiteflies were continuously fed on TYLCCNV-infected plants for 72 h. At each selected time point, 10 PSGs were dissected. TYLCCNV virions were detected by use of a mouse anti-TYLCCNV monoclonal antibody and a goat anti-mouse secondary antibody conjugated to FITC (green). Nuclei were stained with DAPI (blue) and then examined with a confocal microscope.

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