Figures and data in Apoptotic neurodegeneration in whitefly promotes the spread of TYLCV

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5 figures, 1 table and 4 additional files

Figures

Figure 1 with 1 supplement

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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 *OBP*s, *CSP*s, 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

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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

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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, n_{Uninfected plant}=21, n_{Infected plant}=60, n_{Artificial diet}=21, n_Diet+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

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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

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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

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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.

Figure 3

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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, n_TYLCV+dsGFP=30, n_{TYLCV+dsCaspase1}=31, n_{TYLCV+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

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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, n_TYLCV+dsGFP=40, n_{TYLCV+dsNLRL4}=49, n_{TYLCV+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

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Virus transmission bioassay.

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

Author response image 1

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Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Tomato yellow leave curl virus)	Tomato yellow leave curl virus isolate SH2 infectious clone	Xueping Zhou, Institute of Plant Protection, CAAS
Strain, strain background (Tomato yellow leave curl virus)	Mutant Tomato yellow leave curl virus isolate SH2 infectious clone	Xiaowei Wang, Zhejiang University
Antibody	Mouse monoclonal anti-TYLCV CP	Jianxiang Wu, Zhejiang University		IF (1:500)
Antibody	Mouse monoclonal anti-GAPDH	Proteintech	Cat# 60004–1-Ig; RRID:AB_2107436	WB (1:5000)
Antibody	Rabbit polyclonal anti-Caspase3b p10	This paper		Immunogen: YFRPKRPAIDL*C WB (1:3000) IF (1:500)
Antibody	Rabbit polyclonal anti-Caspase3b p20	This paper		Immunogen: LSQEDHSDADC WB (1:2000)
Antibody	Alexa 488 goat anti-mouse IgG	Abcam	Cat#ab150113; RRID:AB_2576208	IF (1:500)
Antibody	Alexa 555 goat anti-rabbit IgG	Abcam	Cat#ab150078; RRID:AB_2722519	IF (1:500)
Commercial assay or kit	One Step TUNEL Apoptosis Assay Kit	Beyotime	Cat#C1088
Commercial assay or kit	Absolutely RNA Nanoprep Kit	Agilent	Cat#400753
Commercial assay or kit	RoomTemp Sample Lysis Kit	Vazyme	Cat#P073
Commercial assay or kit	TRIzol Reagent	Ambion	Cat#15596018
Commercial assay or kit	FastQuant RT Kit with gDNase	Tiangen	Cat#KR106
Commercial assay or kit	PowerUp SYBR Green Master Mix	Applied Biosystems	Cat#A25742
Commercial assay or kit	T7 RiboMAX Express RNAi System	Promega	Cat#P1700
Software, algorithm	SPSS	SPSS	RRID:SCR_002865
Software, algorithm	GraphPad Prism software	GraphPad Prism (https://graphpad.com)	RRID:SCR_015807