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Mechanisms of virus-vector interactions mediating disease transmission
Christiane Then, Fanny Bellegarde, Geoffrey Schivre, Tou Cheu Xiong, Martin Drucker, Stoyan Yordanov, Kaloian Koynov, Hans-Jürgen Butt,
Jaclyn S Zhou, James C. K. Ng
To cite this version:
Christiane Then, Fanny Bellegarde, Geoffrey Schivre, Tou Cheu Xiong, Martin Drucker, et al.. Mech-anisms of virus-vector interactions mediating disease transmission. 19. HFSP Awardees Meeting, Jul 2019, Tsukuba, Japan. 2019. �hal-02947634�
INRA Colmar (France): Christiane Then, Fanny Bellegarde, Géoffrey Schivre, Tou-Cheu Xiong, Martin Drucker
MPI Mainz (Germany): Stoyan Yordanov, Kaloian Koynov, Hans-Jürgen Butt
UC Riverside (USA): Jaclyn S. Zhou, James C.K. Ng
A
phid punctures trigger instant calcium elevations at the puncture site. They might be the first step in establishment of plant defense responses against these predators (see model above).M
any plant viruses are transmitted by aphids. There is evidence that viruses modify plant defenses, for example to modify interactions of plants with virus-transmitting aphids.S
uch modifications could effect the very first steps in virus-aphid interactions (transmission).T
herefore, we tested whether virus infection interferes with local calcium elevations.Experimental system
Method:
Recording of aphid activity by bright field microscopy
Recording of calcium concentration by fluorescence ratiometry Tempo-spatial analysis M. persicae Epidermis (1) (2) M es ophy ll (3) (4) (5) (6) (7) (8) (9) Apoplast Cytosol Vacuole Extracellular Ca2+entry Intracellular Ca2+release Plant defense GLR3.3 GLR3.6 BAK1 TPC1 Unknown receptor ? (1) Puncture (2) local [Ca2+] (3) HAMP (4) Activation (5) Ca2+influx (6) Activation (7) Ca2+flux (8) Activation (9) Aphid effector?
Adapted from Vincent, T. R. et al. Plant Cell 29, 1460–1479 (2017)
5s 10s 15s 25s 35s Healthy / M. p. 5s 10s 15s 25s 55s TuMV / M. p. 5s 10s 15s 20s 25s TuYV / M. p.
Analysis of calcium signal
Curtain by Freepik
Acknowledgments:
CaMV / M. p. 5s 10s 15s 20s 25s
0.2mm
Signal duration (frames)
Num ber of s ignal s 3 4 5 6 7 8 9 10 11 12 13 14 0 2 4 6 8 10 12 14 16
CaMV Healthy TuMV TuYV
Bimodal distribution for CaMV and TuMV, unimodal for TuYV and healthy
CaMV Healthy TuMV TuYV 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5
*
**
Sign al v elo c it yDecreased velocity for CaMV
Decreased surface for CaMV
CaMV Healthy TuMV TuYV 0 10000 20000 30000 40000 50000 60000
*
Sign al s ur fa c e( mm 2 )Mean velocity of signal
Viruses alter calcium signaling
differently
Signal duration (frames)
Num ber of s ignal s 3 4 5 6 7 8 9 10 11 12 13 14 0 2 4 6 8 10 12 14 16
CaMV Healthy TuMV TuYV
Bimodal distribution for CaMV and TuMV, unimodal for TuYV and healthy
CaMV Healthy TuMV TuYV 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5
*
**
Sign al v elo c it yDecreased velocity for CaMV
Decreased surface for CaMV
CaMV Healthy TuMV TuYV 0 10000 20000 30000 40000 50000 60000
*
Sign al s ur fa c e( mm 2 )Mean velocity of signal
Mechanisms of virus-vector interactions mediating disease transmission
Excitation 440 nm Visible Fluorescence 480/535 nm Bright field camera CCD Camera Plant leaf expressing the
calcium sensor YC3.6
Probing aphid Plant viruses
CaMV TuMV TuYV
Model
Results
Time lapse video
**
*
Plant hosts
of whiteflies
Virus Inoculation by
whitefly vector
Brassica napus
Brassica rapa
Experimental system
Membrane feeding: LIYV virion acquisitionB. napus
B. rapa
400 whiteflies 400 whiteflies V iru s Re ten tion Assa y s Collect 100 whiteflies Collect 100 whitefliesTransmitted light Widefield fluorescence
Results
Establish virus host (B. rapa) and non-host (B. napus)
Establish whitefly colonies
Can a plant virus perceive the presence of a
plant while being retained in its insect vector?
Do plant viruses perceive the presence of aphid
vectors?
Plant-to-plant transmission of lettuce infectious yellows virus (LIYV) to B. rapa or B. napus
1 kb ladd er H2O con trol Spray con trol 1 Spray con trol 2 B. rapa 1 B. rapa 2 B. rapa 3 B. rapa 4 B. rapa 5 B. rapa 6 B. rapa 7 B. rapa 8 B. rapa 9 B. rapa 10 No RT ( -) con trol LIYV (+ ) con trol
LIYV virion purified from B. rapa
LIYV (+ ) v irion -50ng LIYV (+ ) v irion -10ng LIYV (+ ) v irion -1ng B. Rapa v irion -1ul B. Rapa v irion -1ul 1/10 dilu ted 28 kDa CP 1 kb ladd er H2O con trol Spray con trol 1 Spray con trol 2 B. nap us 1 B. nap us 2 B. nap us 3 B. nap us 4 B. nap us 5 No RT ( -) con trol LIYV (+ ) con trol LIYV (+ ) con trol B. nap us 6 B. nap us 7 B. nap us 8 B. nap us 9 B. nap us 10 B. nap us 11 B. nap us 12 B. nap us 13 B. nap us 14 B. nap us 15 Spray con trol 3 Spray con trol 4 Reverse transcription (RT) – PCR Western Blot TEM Analysis
Virus Retention Assays
B. napus B. rapa Experiment Total number of whitefly heads viewed Number of whitefly heads with fluorescent signal % of whitefly heads with fluorescent signal Total number of whitefly heads viewed Number of whitefly heads with fluorescent signal % of whitefly heads with fluorescent signal % Relative Change 1 124 19 15.32% 149 11 7.38% -52% 2 81 19 23.46% 79 7 8.86% -62% 3 86 29 33.72% 94 6 6.38% -81% 4 84 15 17.86% 96 7 7.29% -59% 5 100 20 20.00% 97 11 11.3% -43% B. napus B. rapa 22.07 ±3.20 8.25 ±0.87 Ave. % relative -59.4%
% foreguts with fluorescent signals & relative
B. napus B. rapa
Transmitted light Widefield fluorescence
B. Rapa (>18 generations) B. Napus (>36 generations) Whole-mount, widefield Fluorescence microscopy of whiteflies (heads)
