Elicitation of hypersensitive responses in Nicotiana glutinosa by the suppressor of RNA silencing protein P0 from poleroviruses
Plant disease resistance (R) proteins that confer resistance to viruses recognize viral gene products with diverse functions, including viral suppressors of RNA silencing (VSRs). The P0 protein from poleroviruses is a VSR that targets the ARGONAUTE1 (AGO1) protein for degradation, thereby disrupting...
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| Published in: | Molecular plant pathology Vol. 16; no. 5; pp. 435 - 448 |
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| Main Authors: | , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
England
Blackwell Science in collaboration with the British Society of Plant Pathology
01-06-2015
Blackwell Publishing Ltd John Wiley & Sons, Inc John Wiley and Sons Inc |
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| Online Access: | Get full text |
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| Summary: | Plant disease resistance (R) proteins that confer resistance to viruses recognize viral gene products with diverse functions, including viral suppressors of RNA silencing (VSRs). The P0 protein from poleroviruses is a VSR that targets the ARGONAUTE1 (AGO1) protein for degradation, thereby disrupting RNA silencing and antiviral defences. Here, we report resistance against poleroviruses in Nicotiana glutinosa directed against Turnip yellows virus (TuYV) and Potato leafroll virus (PLRV). The P0 proteins from TuYV (P0ᵀᵘ), PLRV (P0ᴾᴸ) and Cucurbit aphid‐borne yellows virus (P0Cᴬ) were found to elicit a hypersensitive response (HR) in N. glutinosa accession TW59, whereas other accessions recognized P0ᴾᴸonly. Genetic analysis showed that recognition of P0ᵀᵘby a resistance gene designated RPO1 (Resistance to POleroviruses 1) is inherited as a dominant allele. Expression of P0 from a Potato virus X (PVX) expression vector transferred recognition to the recombinant virus on plants expressing RPO1, supporting P0 as the unique Polerovirus factor eliciting resistance. The induction of HR required a functional P0 protein, as P0ᵀᵘmutants with substitutions in the F‐box motif that abolished VSR activity were unable to elicit HR. We surmised that the broad P0 recognition seen in TW59 and the requirement for the F‐box protein motif could indicate detection of P0‐induced AGO1 degradation and disruption of RNA silencing; however, other viral silencing suppressors, including the PVX P25 that also causes AGO1 degradation, failed to elicit HR in N. glutinosa. Investigation of P0 elicitation of RPO1 could provide insight into P0 activities within the cell that trigger resistance. |
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| Bibliography: | http://dx.doi.org/10.1111/mpp.12201 istex:EA03037B2D365B4A9AEC7539DB90CCBF983916E6 California State University Program for Education and Research in Biotechnology ark:/67375/WNG-88Q8XPBR-2 Fig. S1 Turnip yellows virus (TuYV)-induced hypersensitive response (HR)-like cell death in Nicotiana glutinosa accession TW59. Nicotiana glutinosa leaves of 4-week-old plants were infiltrated with Agrobacterium carrying the infectious clone of TuYV-FL1 or with empty pBIN61 vector. TW59 underwent HR-like cell death beginning at 2 days post-infiltration, whereas other N. glutinosa accessions demonstrated mild yellowing or some chlorosis with small patches of necrosis that appeared by 6 dpi, as shown by leaves representative of these two phenotypes from accessions TW61 and TW66. Fig. S2 Polerovirus detection in Nicotiana glutinosa-agroinfected leaves. Nicotiana glutinosa accessions TW59 and TW61 were infiltrated with Agrobacterium carrying the infectious clone of Turnip yellows virus (TuYV)-FL1 (A, B) or Potato leafroll virus (PLRV) (C, D) or with empty pBIN61 vector. RNA was extracted from leaves at 48 h post-infiltration for real-time reverse transcription-polymerase chain reaction (RT-PCR) amplification to confirm that the virus could be amplified from RNA extracted from N. glutinosa. No inherent inhibition of the RT-PCR using RNA from this plant as template was observed. Plants were assayed in duplicate with the standard deviation determined for three technical replicates and PCRs were normalized to actin. Fig. S3 Detection of hydrogen peroxide accumulation elicited by P0 in Nicotiana glutinosa. Leaves from N. glutinosa accessions TW59 and TW61 were infiltrated with Agrobacterium carrying the infectious clone of Turnip yellows virus (TuYV)-FL1 or Potato leafroll virus (PLRV) (A, B) or the P0 genes (C, D) together with empty pBIN61 vector. The Tobacco mosaic virus (TMV) P50 gene was included as a positive control for TW61, which carries the N gene. Leaves were removed from plants 40 h (A, B) or 50 h (C, D) post-infiltration and immersed in 3,3′-diaminobenzidine (DAB) stain for 4 h in the dark prior to ethanol destaining and storage in 70% glycerol before being photographed. Fig. S4 Pathogenesis-related 1 (PR-1) gene induction in Nicotiana glutinosa induced by poleroviruses. Nicotiana glutinosa leaves were infiltrated with Agrobacterium carrying the infectious clone of Turnip yellows virus (TuYV)-FL1 (A) or Potato leafroll virus (PLRV) (B) or with empty pBIN61 vector. RNA was extracted from leaves at 48 h post-infiltration for real-time reverse transcription-polymerase chain reaction (RT-PCR) amplification to detect PR-1 gene induction. Higher levels of PR-1 transcripts were observed in TW59 agroinfected with TuYV and in TW61 agroinfected with PLRV, the two combinations that demonstrate the most rapid and robust hypersensitive responses (HRs). Plants were assayed in duplicate with three technical replicates, and transcripts were normalized to actin. Fig. S5 Pathogenesis-related 2 (PR-2) protein accumulation in Nicotiana glutinosa induced by poleroviruses. Nicotiana glutinosa leaves were infiltrated with Agrobacterium carrying the infectious clone of Turnip yellows virus (TuYV)-FL1 or Potato leafroll virus (PLRV), with P0 genes or with empty pBIN61 vector. The Tobacco mosaic virus (TMV) P50 gene was included as a positive control on TW61, which carries the N gene, and non-infiltrated leaves were used as negative controls (Cont). Proteins were extracted from leaves at 48 h post-infiltration, immunoblotting with PR-2 (Class I β-1,3-glucanase) rabbit polyclonal antibodies (Agrisera), followed by mouse anti-rabbit horseradish peroxidase (HRP) conjugate. The blot was stained with Coomassie brilliant blue following visualization using ECL chemiluminescence. The positions of the closest protein standards from the PageRuler Plus Prestained Protein Ladder (Thermo Scientific) are given on the immunoblots. Fig. S6 Detection of Potato virus X (PVX) carrying P0 in local and systemic leaves of resistant Nicotiana glutinosa. Virus stocks of PVX or PVX carrying P0Tu or P0PL were rub inoculated onto N. benthamiana (NB) or N. glutinosa accession TW59 or TW61. Local leaves (A, B) and non-inoculated upper leaves (C, D) were collected at 9 days post-inoculation for RNA extraction and real-time polymerase chain reaction (PCR) detection of PVX. Leaves from uninfected N. glutinosa plants were included as negative controls for non-specific amplification. PVX-specific PCRs were normalized with actin, and relative levels are shown for three biological replicates for PVX and two biological replicates for the controls, and error bars show standard deviations for three technical replicates. ArticleID:MPP12201 US National Science Foundation - No. 1122256 |
| ISSN: | 1464-6722 1364-3703 |
| DOI: | 10.1111/mpp.12201 |