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. 2020 Mar;18(3):655-667.
doi: 10.1111/pbi.13230. Epub 2019 Sep 4.

Extreme Resistance to Potato Virus Y in Potato Carrying the Ry sto Gene Is Mediated by a TIR-NLR Immune Receptor

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Free PMC article

Extreme Resistance to Potato Virus Y in Potato Carrying the Ry sto Gene Is Mediated by a TIR-NLR Immune Receptor

Marta Grech-Baran et al. Plant Biotechnol J. .
Free PMC article

Abstract

Potato virus Y (PVY) is a major potato (Solanum tuberosum L.) pathogen that causes severe annual crop losses worth billions of dollars worldwide. PVY is transmitted by aphids, and successful control of virus transmission requires the extensive use of environmentally damaging insecticides to reduce vector populations. Rysto , from the wild relative S. stoloniferum, confers extreme resistance (ER) to PVY and related viruses and is a valuable trait that is widely employed in potato resistance breeding programmes. Rysto was previously mapped to a region of potato chromosome XII, but the specific gene has not been identified to date. In this study, we isolated Rysto using resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences single-molecule real-time sequencing). Rysto was found to encode a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal TIR domain and was sufficient for PVY perception and ER in transgenic potato plants. Rysto -dependent extreme resistance was temperature-independent and requires EDS1 and NRG1 proteins. Rysto may prove valuable for creating PVY-resistant cultivars of potato and other Solanaceae crops.

Keywords: PVY; Ry sto; RenSeq; TIR-NLR immune receptor; extreme resistance; potato.

Figures

Figure 1
Figure 1
Functional analysis of candidate genes using transient expression assays in Nicotiana benthamiana. (a) Schematic representation of candidate genes and a fragment of chromosome XII linked to the Ry sto gene. Potato superscaffolds (DMB034, DMB750 and DMB114; top panel) were aligned to the distal end of the longer arm of potato chromosome XII (57–60 Mb fragment shown; middle panel), which is linked to the Ry sto gene. Annotated NLRs/NLR clusters are depicted in orange (CNL) and magenta (TNL). Red lines indicate known markers linked to Ry sto. Distances are given in Mb according to the DM potato reference genome (Potato Genome Sequencing Consortium (95 authors), 2011), with numbers indicating proximal positions of marked sequences. Candidate contigs for Ry sto derived from SMRT RenSeq were aligned to the DM potato genome, and best hit positions are shown on the map. Map drawn to scale. Schematic structure of c630 transcripts (lower panel). Approximately 80% of cDNA‐RenSeq reads supported a version of c630 with four exons. An additional intron (depicted with inverted lines) positioned 24 nt upstream of the initial stop codon results in a fifth exon at the 3′ end. Exons are drawn in grey; colours indicate canonical NLR domains: TIR (magenta), NBARC (blue) and LRR (green). Schematic drawn to scale. (b) Relative levels of PVY RNA after infection of Nicotiana benthamiana leaves transiently expressing candidate contigs. Three N. benthamiana leaves were infiltrated with vector pICSLUS0003:35S overexpressing c124_1, c124_2, c359, c516, c630, c660, c692, c908 c1459 and pICSLUS0001 vector overexpressing c630 under the control of native regulatory elements or an empty vector. After 48 h, leaves were inoculated with PVYNTN. Seven days after PVY inoculation, mRNA was isolated from upper, non‐inoculated leaves and PVY RNA levels were quantified with qPCR. EF1 and L23 were used as standardization references. Error bars represent the standard deviation of the means, and medians are also presented. One‐way ANOVA with Dunnett's test was used for statistical analysis. Experiments were performed on 3–10 plants for each construct and were repeated three times. (c) HR response of N. benthamiana plants expressing c630 or Rx genes. Two fully developed leaves of N. benthamiana plants were infiltrated with a suspension of A. tumefaciens carrying pICSLUS0003:c630 (area marked with red) or pGBT‐Rx‐GFP (area marked with yellow) 14 days after PVYNTN (left) or PVX (right) infection. Seventy‐two hours after infiltration, expression of c630 in PVY‐infected plants resulted in HR symptoms similar to those observed when Rx was delivered into plants infected with PVX virus. Experiments were repeated three times.
Figure 2
Figure 2
Expression of Rysto leads to immunity of transgenic plants to PVY infection. (a) Illustration of ER ‐type response of PVY ‐inoculated leaves of Solanum tuberosum cv Russet Burbank plants expressing Rysto. Four‐week‐old transgenic potato plants cv. Russet Burbank carrying construct 35S:Rysto, and non‐transformed plants, were inoculated with PVYNTN . Two weeks after infection, chlorosis was observed on inoculated leaves of non‐transformed plants (right), whereas Rysto transgenic plants (left) remained symptomless. (b) Rysto expression completely blocks PVY spreading in transgenic potato plants. Four‐week‐old transgenic potato plants cv. Russet Burbank (upper graph) or Maris Piper (middle graph) carrying construct 35S:Rysto, Maris Piper (lower graph) plants carrying Rysto construct under the control of native 5′ and 3′ regulatory elements and non‐transformed plants were inoculated with PVYNTN . Three weeks after viral inoculation, mRNA was isolated from upper, non‐inoculated leaves. PVYRNA levels and expression of Rysto were quantified using qPCR , relative to EF 1 and Sec3 reference genes, and expressed as means ±  SD calculated from three biological replicates per plant line. One‐way ANOVA with Dunnetts test was used for statistical analysis.
Figure 3
Figure 3
Coat proteins of two closely related potyviruses elicit Ry sto‐mediated immunity. (a) Rysto recognizes PVY (upper panel) and PVA (lower panel) coat proteins as avirulence factors in transient expression assays. To identify the elicitor of the Ry sto‐mediated resistance against PVY or PVA viruses, open reading frames encoding putative viral proteins (Hc‐Pro, Nia, Nib and CP) were cloned into the pBINmCherry vector and transiently expressed in N. tabacum Ry sto transgenic and non‐transformed plants. As a control (C1, C2), Agrobacterium with empty vector or buffer infiltration was used. Three days after treatment, HR was observed only for PVY or PVA coat proteins (CPs). (b) Comparison of PVY and PVA coat protein amino acid sequences. PVY and PVA CP amino acid sequences were compared with PVYNTN CP. Identical residues are shaded in black. PVA CP shares >59% identity with the PVY sequence. The alignment was generated using MAFFT‐L‐INS‐I (Katoh and Toh, 2008) and visualized in Jalview 2,10,4b1 (Waterhouse et al., 2009). (c) Western blot analysis of viral protein expression. The indicated combinations of PVYCPmCherry, PVACPmCherry, PVY‐Hc‐Pro‐mCherry, PVYNIb‐mCherry and PVYNIa‐mCherry were transiently expressed in leaves of N. tabacum. Total proteins were extracted and analysed by protein gel blotting with polyclonal anti‐mCherry antibody (Abcam). Staining of RuBisCO with Ponceau S was used as a loading control. (d) Cellular localization of viral proteins. Confocal images show representative N. benthamiana leaf epidermal cells transiently expressing indicated proteins. Images were taken 72 h after Agro‐infiltration. For each variant, approximately 50 transformed cells were examined. Bars = 10 μm.
Figure 4
Figure 4
Ry sto‐dependent ER is epistatic to HR and is temperature‐independent. (a) Determination of epistatic effect of ER to HR. Potato Ry sto/Ny‐1 and Ny‐1 plants were inoculated with PVYNTN. Five days after infection, symptoms of local cell death occurred only in Ny‐1 plants. The experiment was repeated three times. (b) Ry sto‐dependent PVY resistance is not inhibited at elevated temperature. Potato Ry sto/Ny‐1 and Ny‐1 plants were inoculated with PVYNTN and divided into two groups, at 20 °C and 28 °C. Water‐treated plants were used as negative controls. Seven days after inoculation, samples of inoculated (lower graphs) and upper non‐inoculated leaves (upper graphs) were collected and PVY RNA levels were measured using qPCR. Values are expressed relative to EF1 and Sec3 reference genes and are expressed as means ± SD calculated from three biological replicates per plant line. A and C describe the names of transgenic Ry sto lines used. One‐way ANOVA with Tukey's test for statistical analysis was performed. (c) Stable transgenic N. tabacum plants carrying Ry sto under the control of a 35S promoter are resistant to PVY infection at elevated (32 °C) temperature. Seven‐week‐old N. tabacum 35S:Ry sto transgenic and non‐transformed plants were inoculated with a PVYN 205:GFP clone. Typical symptoms of PVY infection were observed 7 dpi in non‐transformed plants, whereas Ry sto lines remained symptomless. Image was taken at 14 dpi.
Figure 5
Figure 5
Downstream signalling components are crucial for Rystomediated immunity. (a) Fully developed leaves of Nicotiana benthamiana eds1‐1 or nrg1‐1 knockout plants, NahG plants or non‐transformed plants were infected with PVYNTN or were mock treated with water. Two weeks later, leaves with symptoms of PVY infection were infiltrated with A. tumefaciens suspensions carrying pICSLUS 0001:Rysto (highlighted in green) or pGBT : GFP (highlighted in red) as a control. Three days after infiltration, symptoms of cell death were observed in non‐transformed plants and NahG plants ( SA ‐free). Experiments were repeated three times with similar results. (b) Complementation tests for Rysto‐triggered HR in eds1‐1 or nrg1‐1 N. benthamiana mutants. PVY ‐infected leaves of N. benthamiana eds1‐1, nrg1‐1 knockouts or non‐transformed plants were infiltrated with Agrobacterium carrying Rysto alone or Rysto coexpressed with EDS 1 or NRG 1. Three days after infiltration, HR was observed only when Rysto was coexpressed with indicated proteins. Experiments were repeated three times with similar results. Transcripts expression was confirmed via semiquantitative RTPCR (c) Elongation factor1α ( EF 1α) was used as an internal control. Raw data are presented in the Figure S9.

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