Hypersensitive-like response to Grapevine fanleaf virus in Nicotiana occidentalis.

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Hypersensitive-like response to Grapevine fanleaf virus in Nicotiana occidentalis.

Emmanuelle Vigne, Isabelle Martin, Veronique Komar, Marc Fuchs, Olivier Lemaire, Corinne Schmitt-Keichinger

To cite this version:

Emmanuelle Vigne, Isabelle Martin, Veronique Komar, Marc Fuchs, Olivier Lemaire, et al..

Hypersensitive-like response to Grapevine fanleaf virus in Nicotiana occidentalis.. 18th Congress of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine (ICVG), Sep 2015, Ankara, Turkey. �hal-02739284�

Hypersensitive-like response to Grapevine fanleaf virus in Nicotiana occidentalis.

Emmanuelle Vigne^1* , Isabelle Martin¹, Véronique Komar¹, Marc Fuchs², Olivier Lemaire¹ and Corinne Schmitt-Keichinger³.

1UMR ‘Santé de la Vigne et Qualité du Vin’, INRA / Université de Strasbourg, 28 rue de Herrlisheim, 68000 Colmar, France.

* Corresponding author: emmanuelle.vigne@colmar.inra.fr

2Department of Plant Pathology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA.

3Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France.

Introduction

Grapevine fanleaf virus (GFLV) from the genus Nepovirus, family Secoviridae is the major agent of fanleaf degeneration, one of the most damaging viral diseases of grapevine worldwide (Andret-Link et al., 2004). It is specifically transmitted from grapevine to grapevine by the ectoparasitic nematode Xiphinema index. GFLV induces systemic infection in grapevine as well as in some Solanaceae species. The genome of GFLV is composed of two (+)ssRNAs, RNA1 and RNA2. Symptoms differing in type and severity consist of distorted leaves, yellow mosaic patterns, short internodes, and stunted plants, among others. Little is known about the mechanisms of GFLV symptom expression. Recently the viral determinant of mosaic on systemic-infected leaves of Nicotiana benthamiana and N. clevelandii plants was identified in the 3' coding region of the RNA-dependent RNA polymerase encoded by RNA1 (Vigne et al., 2013).

In contrast to this compatible interaction on N. benthamiana and N. clevelandii, a necrotic phenotype with some characteristics of an incompatible hypersensitive reaction (HR)-like response was observed on N. occidentalis inoculated with GFLV-F13 but not with GFLV-GHu strains. These characteristics include necrotic spots and a partial restriction of the virus to inoculated leaves. To get insights into GFLV sequences acting as effectors involved in virulence on N. occidentalis, we used a reverse genetics approach with infectious cDNA clones of strains F13 and GHu, and chimeric clones derived thereof, in combination with a biochemical characterization of major hallmarks of HR interactions.

Material and methods

Biological material, plant inoculation, and characterization of viral progeny

All infectious clones and recombinant procedures were performed as previously described (Vigne et al., 2013). Full-length cDNA clones of GFLV-F13 and -GHu RNA1 and RNA2 were used for in vitro synthesis of transcripts. Four leaves-stage Chenopodium quinoa plants were mechanically inoculated with purified transcripts. Crude sap of infected C. quinoa was then used for passages in N. occidentalis and symptoms were monitored. Viral accumulation on inoculated and apical leaves was measured by semi-quantitative DAS-ELISA. The progeny viral RNAs were checked by RT-PCR and direct sequencing.

Characterization of the HR-like response in N. occidentalis

The accumulation of phytoalexins was evaluated by observation of a typical blue fluorescence surrounding necrotic spots under ultraviolet light (UV) at 3 days post-inoculation (DPI) (Chong et al., 2002). Hydrogen peroxide (one of several Reactive Oxygen Species-ROS) was detected in leaves of N. occidentalis by 3,3'-diaminobenzidine (DAB) staining (Daudi et al., 2012). Pathogenesis-related (PR) proteins were detected by western blot analyses using specific antibodies against PR1 and total proteins (Heitz et al., 1994). Expression of the hsr203j gene (Pontier et al., 1994) was analyzed by semi quantitative RT-PCR on total RNA extracts.

cDNAs of GFLV sequences of interest were introduced in a binary vector for agroinfiltration assays in N. occidentalis in order to assess plant responses upon their ectopic expression.

Results and discussion

Typical blue fluorescence rings surrounding necrotic spots were observed under UV light on leaves inoculated with GFLV-F13, suggesting the accumulation of phytoalexins (Fig 1A). In contrast, no fluorescence was observed on leaves of N. occidentalis inoculated with GFLV-GHu. In vitro transcripts of homologous combinations of RNA1 and RNA2 reproduced the symptoms observed with the wild- type parental viruses. The use of assorted transcripts showed that the formation of necrotic lesions on N. occidentalis mapped to RNA2 (Fig 1B). By generating recombinant RNA2, for which individual

genes were swapped between F13 and GHu cDNAs, we could further map the viral determinant to one of the three coding sequence.

GFLV accumulation was low in inoculated leaves of plants exhibiting necrotic symptoms (1 µg/g) at DPI12. In contrast, GFLV accumulation was high in inoculated leaves of plants without symptoms (40 µg/g). These results suggest that the necrosis most probably restricts the virus spread and corresponds to an HR-like response of the plant. These necrotic spots also correlated with the over- accumulation of hydrogen peroxide, PR1 proteins and hsr203j transcripts.

A similar HR-like reaction was recapitulated when the F13 coding region was transiently expressed by agroinfiltration, a property that further identifies this sequence as a putative avirulence factor. The application of this transient bioassay enabled a more precise mapping to a 150 nucleotides stretch.

A comparable HR reaction was described for Tomato ringspot virus, another nepovirus, but the viral effector of this response remained elusive (Jovel et al., 2011). Our findings are the first evidences for identifying a nepoviral coding sequence inducing an HR-like reaction in a plant host.

References

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