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Imaging viral infection: studies on Nicotiana benthamiana plants infected with the pepper mild mottle tobamovirus

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Abstract

We have studied by kinetic Chl-fluorescence imaging (Chl-FI) Nicotiana benthamiana plants infected with the Italian strain of the pepper mild mottle tobamovirus (PMMoV-I). We have mapped leaf photosynthesis at different points of the fluorescence induction curve as well as at different post-infection times. Images of different fluorescence parameters were obtained to investigate which one could discriminate control from infected leaves in the absence of symptoms. The non-photochemical quenching (NPQ) of excess energy in photosystem II (PSII) seems to be the most adequate chlorophyll fluorescence parameter to assess the effect of tobamoviral infection on the chloroplast. Non-symptomatic mature leaves from inoculated plants displayed a very characteristic time-varying NPQ pattern. In addition, a correlation between NPQ amplification and virus localization by tissue-print was found, suggesting that an increase in the local NPQ values is associated with the areas invaded by the pathogen. Changes in chloroplast ultrastructure in non-symptomatic leaf areas showing different NPQ levels were also investigated. A gradient of ultrastructural modifications was observed among the different areas.

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Abbreviations

AS:

asymptomatic leaf

C:

chloroplast

CCD:

charge coupled device

Chl a :

chlorophyll a

Chl-FI:

chlorophyll fluorescence imaging

CP:

viral coat protein

CW:

cell wall

dpi:

days post-infection

F :

actual fluorescence

F′ M :

maximum fluorescence in the light-adapted state

F M :

maximal fluorescence in the dark-adapted state

G:

grana

L:

Sharp and narrow band marking the border between leaf areas with different NPQ values in NPQ30 images from AS leaves.

La:

stroma lamellae

LHCII:

light harvesting complex II

Mt:

mitochondrion

M:

outer region of the mesophyll in AS leaves

NPQ:

non-photochemical quenching

NPQ t :

non-photochemical quenching at referred kinetics time, t

OEC:

oxygen-evolving complex

PAR:

photosynthetically active radiation

Pg:

plastoglobuli

Pm:

plasmatic membrane

PMMoV-I:

pepper mild mottle virus Italian strain

PSI:

photosystem I

PSII:

photosystem II

PVX:

potato virus X

ROI:

region of interest

S:

starch grains

TMV:

tobacco mosaic virus

V:

areas surrounding the main veins in AS leaves

Va:

vacuole

VP:

virus particles

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Acknowledgments

We are grateful to María Teresa Serra (CIB, CSIC, Madrid) for the generous gift of PMMoV-I coat protein antiserum; to Greet Clerx, Jan Daenen and Natascha Steffanie for skilful technical assistance (Limburgs Universitair Centrum), as well as to Carlota Sajnani and Mónica Pineda (EEZ, CSIC, Granada) for their encouraging discussions providing their most recent data in the N. benthamiana-PMMoV system. This work was supported by the Spanish Ministry of Science and Technology, Ministry of Science and Education (Grants BIO2001-1937-C02 and BIO2004-04968-C02), Comunidad Autónoma de Madrid (CAM) and the BOF Funds of the Slimme Region of the Province of Limburg. MLPB was the recipient from a MCyT fellowship.

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Authors and Affiliations

  1. Plant Biology Department, Centro Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, Madrid, 28040, Spain

    María Luisa Pérez-Bueno & Isabel García-Luque

  2. Dept. SBG, Centre for Environmental Sciences, Laboratory of Molecular and Physical Plant Physiology, Hasselt University, Agoralaan, Bldg. D, Diepenbeek, B-3590, Belgium

    Massimo Ciscato, Martin vandeVen & Roland Valcke

  3. Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, Granada, 18008, Spain

    María Luisa Pérez-Bueno & Matilde Barón

Authors
  1. María Luisa Pérez-Bueno
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  2. Massimo Ciscato
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  3. Martin vandeVen
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  4. Isabel García-Luque
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  5. Roland Valcke
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  6. Matilde Barón
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Correspondence to Matilde Barón.

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The paper is dedicated to the memory of Prof. Dr López Gorgé (1935–2004)

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Pérez-Bueno, M.L., Ciscato, M., vandeVen, M. et al. Imaging viral infection: studies on Nicotiana benthamiana plants infected with the pepper mild mottle tobamovirus. Photosynth Res 90, 111–123 (2006). https://doi.org/10.1007/s11120-006-9098-0

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