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Grapevine fanleaf virus elicits a hypersensitive response above and below ground in a model host

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Brandon G. Roy at Cornell University
Brandon G. Roy
Marc Fuchs at Cornell University
Marc Fuchs
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Abstract

Grapevine fanleaf virus (GFLV, genus Nepovirus, family Secoviridae) causes fanleaf degeneration in in most vineyards worldwide (Andret-Link et al. 2004). GFLV is transmitted by the ectoparasitic dagger nematode Xiphinema index (Andret-Link et al. 2004) and is mechanically transmissible to herbaceous hosts (Roy and Fuchs 2022). GFLV determinants of vein clearing symptoms in Nicotiana benthamiana map to the amino acid in position 802 of the RNA-dependent RNA polymerase (protein 1EPol) (Osterbaan et al. 2019). Profiling proteome and transcriptome changes during GFLV infection revealed the activation of immune responses, including a hypersensitive reaction, and the dysregulation of biochemical pathways during symptom expression (Roy et al. 2023). Although these findings contributed to our understanding of leaf symptom development, how GFLV impacts plant host root systems is unknown. With advances in root-phenotyping technologies and a library of GFLV strains we set out to explore the impact of the virus on N. benthamiana root system architecture (RSA) traits. We hypothesized phenotypic RSA differences upon infection with different GFLV strains.
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Proceedings of the 20th Congress of ICVG, Thessaloniki, Greece 25-29 September, 2023
129
O42. Grapevine fanleaf virus elicits a hypersensitive response above and below
ground in a model host
Brandon G. Roy1* and Marc Fuchs1
1Plant Pathology and Plant-Microbe Biology Section, Cornell University, Cornell Agritech at the New York
State Experiment Station, Geneva, NY 14456
*Corresponding author: bgr36@cornell.edu
INTRODUCTION
Grapevine fanleaf virus (GFLV, genus Nepovirus, family Secoviridae) causes fanleaf degeneration in
in most vineyards worldwide (Andret-Link et al. 2004). GFLV is transmitted by the ectoparasitic
dagger nematode Xiphinema index (Andret-Link et al. 2004) and is mechanically transmissible to
herbaceous hosts (Roy and Fuchs 2022). GFLV determinants of vein clearing symptoms in Nicotiana
benthamiana map to the amino acid in position 802 of the RNA-dependent RNA polymerase (protein
1EPol) (Osterbaan et al. 2019). Profiling proteome and transcriptome changes during GFLV infection
revealed the activation of immune responses, including a hypersensitive reaction, and the
dysregulation of biochemical pathways during symptom expression (Roy et al. 2023). Although these
findings contributed to our understanding of leaf symptom development, how GFLV impacts plant
host root systems is unknown. With advances in root-phenotyping technologies and a library of GFLV
strains we set out to explore the impact of the virus on N. benthamiana root system architecture (RSA)
traits. We hypothesized phenotypic RSA differences upon infection with different GFLV strains.
MATERIALS AND METHODS
N. benthamiana plants were maintained in controlled growth chambers at 25 °C and 70% humidity.
GFLV wildtype strains GHu and F13, as well as single amino acid mutants GFLV-GHu 1EPolK802G and
GFLV-F13 1EPolG802K (Osterbaan et al. 2019) were utilized to inoculate N. benthamiana plants.
Inoculated plants were tested for the presence of GFLV by DAS-ELISA, RT-PCR, and Sanger
sequencing of RT-PCR amplicons (Osterbaan et al. 2019). Infected plants were uprooted at 4-, 17-, and
26-days post inoculation (dpi). Crown root systems were washed off with distilled water, dried, and
imaged on an EPSON Expression 12000XL scanner. Images were analyzed in Rhizovision Explorer
(v2.0.3, Noble Research Institute, LLC). Statistical analysis of RSA traits was performed in RStudio
(v4.2.2) using packages ‘stats’ (v4.1.3) and ‘corplot’ (v0.92). RNA was extracted from roots and
relative GFLV titer was measured by RT-qPCR against the FBOX housekeeping gene (Osterbaan et al.
2019) using the Luna® Universal One Step kit. Quantification was done using the output Cq values of
SYBR green detection. Statistical analysis was performed using log(2^-∆∆Cq) values compared to
mock inoculated control plants through the Wilcoxon geometric comparison of means. 3’RNA-Seq
analysis of N. benthamiana root tissue at 4-, 17-, and 26 dpi was performed as previously described
(Roy et al. 2023). Gene set enrichment analysis contrasting 17 dpi root samples to 7 dpi apical leaf
transcriptomes was performed to identify overlap of these datasets.
RESULTS AND DISCUSSION
Plants infected with wildtype GFLV-GHu exhibited vein clearing symptoms in apical tissue at 8 dpi
and plants recovered by 14 dpi, as expected. No other GFLV strains elicited foliar symptoms (Fig 1A).
A significantly increased root diameter and/or a significantly decreased number of root tips were
observed at 17 dpi on plants infected with wildtype GFLV-GHu and mutant GFLV-F13 1EPolG802K, both
containing a lysine at protein 1EPol residue 802 (Fig. 1B-C). Absent (Fig. 1C) or not significantly
reduced RSA traits were observed in plants infected with wildtype GFLV-F13 and mutant GFLV-GHu
1EPolK802, both containing a glycine at protein 1EPol residue 802. To our knowledge, this is a novel
GFLV phenotype induced by protein 1EPol802 in a plant host. Other RSA traits captured by Rhizovision
Proceedings of the 20th Congress of ICVG, Thessaloniki, Greece 25-29 September, 2023
130
Explorer were highly correlated with root tip number and average diameter, however, there was no
relationship between root symptomology and relative GFLV titer. Gene set enrichment after
differentially abundant transcript identification revealed overrepresentation of genes relating to
hypersensitive response, abiotic stress, and protein processing/gene regulation. Further work is needed
to assess the effect of modified plant host root systems on X. index-mediated transmission of GFLV,
changes in root phenotype by distinct GFLV strains in Vitis spp., as well as the molecular mechanism
of this root phenotype.
Figure 1. Grapevine fanleaf virus (GFLV) strains elicit differential symptomology in N. benthamiana depending on
the strain. (a) At 7 days post inoculation (dpi) vein clearing symptoms are observed for wildtype GFLV-GHu infected
plants while the other four treatments (mock control, wildtype GFLV-F13, mutant GFLV-F13 1EPolG802K, and mutant
GFLV-GHu 1EPolK802G) remain asymptomatic. (b) Root systems of N. benthamiana are altered by different GFLV
strains. (c) Number of root tips as calculated by Rhizovision Explorer show statistically supported phenotypic
differences among viral strains. (d) Comparative gene set enrichment analysis of wildtype GFLV-GHu vs mock
control in apical leaves and root tissue reveal major common gene ontologies relating to hypersensitive response and
inorganic molecules.
ACKNOWLEDGEMENTS
Funding for this project was supported by California Department of Food and Agriculture grants.
B.G.R. was supported by National Science Foundation Research Traineeship in Digital Plant Science
(Award #1922551). We are thankful to Dr. Matt Thomas of the Cornell Statistical Consulting Unit for
providing guidance in statistical analysis.
REFERENCES
Andret-Link, P., Laporte, C., Valat, L., Ritzenthaler, C., Demangeat, G., Vigne, E., Laval, V., Pfeiffer, P., Stussi-Garaud,
C., & Fuchs, M. (2004). Grapevine fanleaf virus: Still a major threat to the grapevine industry. Journal of Plant Pathology,
86(3), 183–195. https://www.jstor.org/stable/41992424
Osterbaan, L. J., Choi, J., Kenney, J., Flasco, M., Vigne, E., Schmitt-Keichinger, C., Rebelo, A. R., Heck, M., & Fuchs, M.
(2019). The identity of a single residue of the RNA-dependent RNA polymerase of grapevine fanleaf virus modulates vein
clearing in Nicotiana benthamiana. Molecular Plant-Microbe Interactions, 32(7), 790–801.
https://doi.org/10.1094/MPMI-12-18-0337-R
Roy, B.G. and Fuchs, M. 2022. Herbaceous plant hosts as supermodels for grapevine viruses: a historical perspective.
Journal of Plant Pathology, https://doi.org/10.1007/s42161-022-01267-z.
Roy, B. G., DeBlasio, S., Yang, Y., Thannhauser, T., Heck, M., & Fuchs, M. (2023). Profiling plant proteome and
transcriptome changes during grapevine fanleaf virus infection. Journal of Proteome Research, acs.jproteome.3c00069.
https://doi.org/10.1021/acs.jproteome.3c00069
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Profiling Plant Proteome and Transcriptome Changes during Grapevine Fanleaf Virus Infection
Article
Full-text available
  • Apr 2023
  • J PROTEOME RES
Viruses can elicit varying types and severities of symptoms during plant host infection. We investigated changes in the proteome and transcriptome of Nicotiana benthamiana plants infected by grapevine fanleaf virus (GFLV) with an emphasis on vein clearing symptom development. Comparative, time-course liquid chromatography tandem mass spectrometry and 3' ribonucleic acid sequencing analyses of plants infected by two wildtype GFLV strains, one symptomatic and one asymptomatic, and their asymptomatic mutant strains carrying a single amino acid change in the RNA-dependent RNA polymerase (RdRP) were conducted to identify host biochemical pathways involved in viral symptom development. During peak vein clearing symptom display at 7 days post-inoculation (dpi), protein and gene ontologies related to immune response, gene regulation, and secondary metabolite production were overrepresented when contrasting wildtype GFLV strain GHu and mutant GHu-1EK802GPol. Prior to the onset of symptom development at 4 dpi and when symptoms faded away at 12 dpi, protein and gene ontologies related to chitinase activity, hypersensitive response, and transcriptional regulation were identified. This systems biology approach highlighted how a single amino acid of a plant viral RdRP mediates changes to the host proteome (∼1%) and transcriptome (∼8.5%) related to transient vein clearing symptoms and the network of pathways involved in the virus-host arms race.
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Grapevine fanleaf virus: Still a major threat to the grapevine industry
Article
Full-text available
  • Nov 2004
SUMMARY Grapevine fanleaf virus (GFLV) is responsible for fan- leaf degeneration, which is one of the most severe virus diseases of grapevines worldwide. GFLV causes substan- tial crop losses, reduces fruit quality and shortens the longevity of grapevines in the vineyard. GFLV is trans- mitted specifically from grapevine to grapevine by the ectoparasitic nematode Xiphinema index, and belongs to the genus Nepovirus in the family Comoviridae. Since the discovery of the nematode vector in the late 1950's and the identification of GFLV as the agent responsible for fanleaf degeneration in the early 1960's, a wealth of in- formation has been accumulated on its transmission, bi- ological properties and serological characteristics, as well as on the structure and expression of the GFLV genome. Although dissemination of the virus through propaga- tion material has been drastically reduced over the past two decades by implementing rigorous certification schemes and establishing quarantine facilities, effective strategies are still needed to control GFLV in naturally infected vineyards. Recently, significant progress has been made on the elucidation of the function(s) of most GFLV proteins, in particular those involved in critical steps of the virus multiplication cycle, including RNA replication, cell-to-cell movement, and transmission by X. index. New insights have also been gained into the population structure and genomic variability among iso- lates from naturally infected vineyards, which have opened new avenues for designing alternative strategies to control this destructive virus. This review article offers a comprehensive overview of the most significant ad- vances made over the past 15 years on GFLV and dis- cusses novel control strategies for one of the major threats to the grapevine industry worldwide.
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Herbaceous plant hosts as supermodels for grapevine viruses: a historical perspective
Article
  • Dec 2022
  • J PLANT PATHOL
Herbaceous plant hosts are essential to grapevine virus research. Such plants offer numerous advantages as experimental hosts of viruses over Vitis species, the natural hosts. They grow faster, have a shorter generation time, and require little maintenance in the greenhouse or growth chamber in comparison with the natural hosts for which experiments are more technically challenging and last substantially longer. Local, pseudo-local, systemic, and pseudo-systemic herbaceous plants have been identified among infected experimental hosts and used for advances in grapevine virology. The discovery pipeline made possible by alternative model hosts has had a tremendous impact on the field. Species of Chenopodium, Nicotiana, Phaseolus, Cucumis, Petunia, and Cuscuta have served to document mechanical or graft transmission of grapevine viruses, fulfill Koch’s postulates, study pathogenesis, examine movement, determine replication, purify and observe virus particles, image virions at the atomic level, develop diagnostic assays, characterize vector-mediated transmission, assess defense and counter defense mechanisms, and engineer resistance, among many other critical milestones. Alternative hosts continue to have intrinsic value for discoveries, although many technologies, including high-throughput sequencing, have largely outperformed their use for diagnostic applications. This review focuses on the critical role of herbaceous plant hosts in important discoveries on the biology of grapevine viruses.
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The Identity of a Single Residue of the RNA-dependent RNA Polymerase of Grapevine Fanleaf Virus Modulates Vein Clearing Symptoms in Nicotiana benthamiana
Article
  • Jan 2019
  • MOL PLANT MICROBE IN
The mechanisms underlying host plant symptom development upon infection by viruses of the genus Nepovirus in the family Secoviridae, including grapevine fanleaf virus (GFLV), are poorly understood. In the systemic host Nicotiana benthamiana, GFLV strain GHu produces characteristic symptoms of vein clearing in apical leaves, unlike other GFLV strains such as F13, which cause an asymptomatic infection. In this study, we expanded on earlier findings and used reverse genetics to identify residue 802 (lysine, K) of the GFLV-GHu RNA1-encoded RNA-dependent RNA polymerase (1EPol) as a modulator of vein-clearing symptom development in N. benthamiana. Mutations to this site abolished (K to G, A, or Q) or attenuated (K to N or P) symptom expression. Noteworthy, residue 802 is necessary but not sufficient for vein clearing, as GFLV-F13 RNA1 carrying K802 remained asymptomatic in N. benthamiana. No correlation was found between symptom expression and RNA1 accumulation, as shown by reverse transcription-quantitative polymerase chain reaction. Additionally, the involvement of RNA silencing of vein clearing was ruled out by virus-induced gene silencing experiments and structure predictions for protein 1EPol suggested that residue 802 is flanked by strongly predicted stable secondary structures, including a conserved motif of unknown function (805LLKT/AHLK/RT/ALR814). Together, these results reveal the protein nature of the GFLV-GHu symptom determinant in N. benthamiana and provide a solid basis for probing and determining the virus-host proteome network for symptoms of vein clearing.
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