1. Microbiology and Infectious Disease
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Apoptotic neurodegeneration in whitefly promotes the spread of TYLCV

  1. Shifan Wang
  2. Huijuan Guo
  3. Feng Ge  Is a corresponding author
  4. Yucheng Sun  Is a corresponding author
  1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, China
  2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, China
  3. Maoming Branch, Guangdong Laboratory of Lingnan Modern Agriculture, China
Research Article
Cite this article as: eLife 2020;9:e56168 doi: 10.7554/eLife.56168
5 figures, 1 table, 1 data set and 4 additional files

Figures

Figure 1 with 1 supplement
TYLCV impairs the host selectivity of whitefly between infected and uninfected host plants.

(A) Whitefly preferences change after TYLCV acquisition. (B–D) Whitefly attraction index (feed on infected or uninfected plants) of (B) free-choice assay with plants, n = 8, (C) dual-choice assay with plant odors, n = 12, and (D) dual-choice assay with colors (green and yellow), n = 6. (E–G) Whitefly attraction index (feed on artificial diet with/without purified virions) of (E) free-choice assay with plants, n = 8, (F) dual-choice assay with plant odors, n = 12, (G) dual-choice assay with plant colors, n = 12, (H) Responding time to the same plant odor in 12 min-monitoring was recorded. The difference of means ± SEM is labeled. Whiteflies on infected or uninfected plants were collected separately after 2 days, 4 days, or 6 days of feeding. (I) Relative gene expression of whitefly OBPs, CSPs, and ORco, n = 3–5. (J) Relative gene expression of whitefly neuron membrane receptors, n = 3–5. Box plots represent the median (bold black line), quartiles (boxes), as well as the minimum and maximum (whiskers). Values in bar plots represent mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Figure 1—figure supplement 1
Whitefly immune system and nervous system response to TYLCV in head transcriptome analysis.

Blue frame represents immune-related changes and red frame represents nerve-related changes in (A) KEGG enrichment pathway and (B) differentially expressed gene enrichment analysis.

Figure 2 with 3 supplements
TYLCV induces apoptotic neurodegeneration in the brain of whitefly.

(A–B) Neurodegeneration of whitefly feeding on plant diet (infected or uninfected) and artificial diet (with or without TYLCV virions) were observed (A) and quantified (B) in head sections, nUninfected plant=21, nInfected plant=60, nArtificial diet=21, nDiet+TYLCV=60. (C–D) Both whitefly bodies with head and dissected heads were collected, and the relative gene expression of whitefly Caspase1 and Caspase3b were analyzed using qRT-PCR, n = 5. (E–F) A 2 hr time-course UV treatment was considered as a positive control to monitor Caspase3b cleavage and activation by non-reduced denaturing polyacrylamide gel electrophoresis (E). Both purified virions and infected plants would induce Caspase3b cleavage and activation (F). All bands of each sample were imaged from the same blot, n = 3. (G) Whitefly head sections were fixed and labeled with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Green indicates TUNEL staining of the apoptotic cells. (H) Head sections were labeled with anti-TYLCV CP and anti-Caspase3b antibodies. All confocal images of the head section were dissected from whiteflies feeding on artificial diet. Scale bar = 100 μm, n > 15. Values in bar plots represent mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Figure 2—figure supplement 1
Relative gene expression in qRT-PCR analysis.

(A–B) RNA interference efficiency in (A) whole bodies and (B) heads. (C) TYLCV from infected benthamiana can also induce whitefly Caspase1, Caspase3b, NLRL4, and Spaetzle1 and 2 expression. (D) TYLCV can induce whitefly Caspase1, Caspase3b, NLRL4, and Spaetzle1 and 2 expression with only 12 hr virus acquisition. n = 5. Values represent mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Figure 2—figure supplement 2
Dynamics of Caspase3b cleavage.

An anti-Caspase3b p20 antibody labeled p20 domain was also used in the time-course UV treatment experiment to monitor the dynamics of Caspase3b cleavage and the result was confirmed in Figure 2E using anti-Caspase3b p10 antibody.

Figure 2—figure supplement 3
Localization of genome DNA and CP of TYLCV.

Localization of genome DNA and CP of TYLCV was observed in the whitefly brain using both (A) fluorescence in situ hybridization and (B) immunofluorescence. All samples were collected from artificial diet-containing TYLCV virions. Scale bar = 100 μm, n > 15.

Silencing caspases alleviates virus-induced neurodegeneration.

(A–B) Whitefly attraction index (fed on artificial diet with dsRNA) of (A) free-choice assay with plants, n = 8, (B) dual-choice assay with plant odors, n = 8. (C) Caspase3b of whitefly treated with virions and dsRNA was detected using western blot. (D–E) Neurodegeneration of whitefly fed with virions and dsRNA was observed (D) and quantified (E) in head sections, nTYLCV+dsGFP=30, nTYLCV+dsCaspase1=31, nTYLCV+dsCaspase3b=41. (F–G) Head section images of whitefly feed with TYLCV and dsRNA. Interference with Caspase1 and Caspase3b alleviates brain apoptosis in TUNEL assay (F) and Caspase3b in immunofluorescence. Scale bar = 100 μm, n > 12. Box plots represent the median (bold black line), quartiles (boxes), as well as the minimum and maximum (whiskers). Values in bar plots represent mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Figure 4 with 1 supplement
NLRL4 responses to TYLCV and induces neurodegeneration in whitefly.

(A–B) FKPMs of NLRLs (A) and Spaetzles (B) in whitefly head transcriptome data. (C–D) Relative gene expression of whitefly bodies (C) or heads (D) NLRL4 and Spaetzle1 and 2 were analyzed using qRT-PCR, n = 5. (E) Conserved domains of NLRL4 was predicted by InterPro and SMART. Green represents leucine-rich repeats, dashed boxes represent domain scores that are less significant than the required threshold. (F–G) Neurodegeneration of whitefly fed with virions and dsRNA was observed (F) and quantified (G) in head sections, nTYLCV+dsGFP=40, nTYLCV+dsNLRL4=49, nTYLCV+dsSpaetzle1&2 = 53. (H) Caspase3b of whitefly treated with virions and dsRNA was detected using western blot. (I–J) Head section images of whitefly fed with TYLCV and dsRNA. Interference with NLRL4 and Spaetzle1 and 2 alleviates brain apoptosis in TUNEL assay (F) and Caspase3b in immunofluorescence. Scale bar = 100 μm, n > 12. (K–L) Interference with NLRL4 or Spaetzle1 and 2 suppressed caspases expression in both bodies (K) and heads (L), n = 5. (M–N) TYLCV cannot alter whitefly preference in (M) free-choice assay, n = 8, and (N) odorant dual-choice assay, n = 8, after interference with NLRL4 or Spaetzle1 and 2. (O) Rescue whitefly preference impairs TYLCV transmission after interference with Caspase1, Caspase3b, NLRL4, or Spaetzle1 and 2. Box plots represent the median (bold black line), quartiles (boxes), as well as the minimum and maximum (whiskers). Values in bar plots represent mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

Figure 4—figure supplement 1
Virus transmission bioassay.

TYLCV infected and uninfected tomato plants were alternately placed in a circle for 4 hr transmission.

Author response image 1

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (Tomato yellow leave curl virus)Tomato yellow leave curl virus isolate SH2 infectious cloneXueping Zhou, Institute of Plant Protection, CAAS
Strain, strain background (Tomato yellow leave curl virus)Mutant Tomato yellow leave curl virus isolate SH2 infectious cloneXiaowei Wang, Zhejiang University
AntibodyMouse monoclonal anti-TYLCV CPJianxiang Wu, Zhejiang UniversityIF (1:500)
AntibodyMouse monoclonal anti-GAPDHProteintechCat# 60004–1-Ig; RRID:AB_2107436WB (1:5000)
AntibodyRabbit polyclonal anti-Caspase3b p10This paperImmunogen: YFRPKRPAIDL*C
WB (1:3000)
IF (1:500)
AntibodyRabbit polyclonal anti-Caspase3b p20This paperImmunogen: LSQEDHSDADC
WB (1:2000)
AntibodyAlexa 488 goat anti-mouse IgGAbcamCat#ab150113; RRID:AB_2576208IF (1:500)
AntibodyAlexa 555 goat anti-rabbit IgGAbcamCat#ab150078; RRID:AB_2722519IF (1:500)
Commercial assay or kitOne Step TUNEL Apoptosis Assay KitBeyotimeCat#C1088
Commercial assay or kitAbsolutely RNA Nanoprep KitAgilentCat#400753
Commercial assay or kitRoomTemp Sample Lysis KitVazymeCat#P073
Commercial assay or kitTRIzol ReagentAmbionCat#15596018
Commercial assay or kitFastQuant RT Kit with gDNaseTiangenCat#KR106
Commercial assay or kitPowerUp SYBR Green Master MixApplied BiosystemsCat#A25742
Commercial assay or kitT7 RiboMAX Express RNAi SystemPromegaCat#P1700
Software, algorithmSPSSSPSSRRID:SCR_002865
Software, algorithmGraphPad Prism softwareGraphPad Prism (https://graphpad.com)RRID:SCR_015807

Data availability

Sequencing data have been deposited in SRA under accession ID PRJNA606896.

The following data sets were generated
  1. 1
    NCBI Sequence Read Archive
    1. S Wang
    (2019)
    ID PRJNA606896. MED whitefly head transcriptome.

Additional files

Supplementary file 1

KEGG and GO enrichment of head transcriptome, western blot labeled by a Caspase3b p20 antibody, immunofluorescence and FISH of TYLCV, RNA interference efficiency.

https://cdn.elifesciences.org/articles/56168/elife-56168-supp1-v1.docx
Supplementary file 2

The list of differentially expressed genes (DEGs).

https://cdn.elifesciences.org/articles/56168/elife-56168-supp2-v1.xls
Supplementary file 3

The list of all gene expression (FPKM).

https://cdn.elifesciences.org/articles/56168/elife-56168-supp3-v1.xls
Transparent reporting form
https://cdn.elifesciences.org/articles/56168/elife-56168-transrepform-v1.docx

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