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A single amino acid at position 31 in the N-terminus of the coat protein of cucumber mosaic virus determines its avirulence function for RCY1-conferred virus resistance

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Abstract

The coat protein (CP) of the cucumber mosaic virus (CMV) yellow strain [CMV(Y)], but not the CMV B2 strain [CMV(B2)], serves as an avirulence determinant against the NB-LRR class RCY1 of Arabidopsis thaliana. To investigate the avirulence function, a series of binary vectors were constructed by partially exchanging the CP coding sequence between CMV(Y) and CMV(B2) or introducing nucleotide substitutions. These vectors were transiently expressed in Nicotiana benthamiana leaves transformed with modified RCY1 cDNA. Analysis of hypersensitive resistance-cell death (HCD), CP accumulation, and defense gene expression at leaf sites infiltrated with Agrobacterium indicated that a single amino acid at position 31 of the CP seems to determine the avirulence function.

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Data availability

The data that support the findings of this study are available from the corresponding authors, HT and SM, upon reasonable request.

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Acknowledgements

This study was financially supported by the Japan Society for the Promotion of Science KAKENHI (grant numbers 21H04721, 19H02953, 22KK0081, 21H04721, 19K22300, and 23K18017).

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Authors and Affiliations

  1. Graduate School of Agricultural Science, Tohoku University, Sendai, 980-0845, Japan

    Hideki Takahashi, Yuki Ando, Yoshinori Kanayama & Shuhei Miyashita

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  1. Hideki Takahashi
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  2. Yuki Ando
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  3. Yoshinori Kanayama
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  4. Shuhei Miyashita
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Correspondence to Hideki Takahashi or Shuhei Miyashita.

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Supplementary Information

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705_2024_5961_MOESM1_ESM.pdf

Supplementary Fig. S1 Detection of transient expression of cDNA of each of the CMV(Y) RNAs that encode viral protein in a wild-type N. benthamiana #4-3 leaf. (A) Schematic diagrams of the binary vector constructs 35S::CY1, 35S::CY2, 35S::CY3, 35S::YCP, 35S::Y2b-HA, and 35S::GFP. The cDNAs encoding CMV RNA1, RNA2, and RNA3 were each cloned into the pRI201-AN binary vector under the control of the 35S promoter. The cDNA of the RNA encoding the CMV(Y) CP, which is the second ORF in RNA3, was independently cloned for expression under the control of 35S promoter into the pRI201-AN binary vector and designated 35S::YCP. To express the cDNA encoding the low-molecular-weight 2b protein, which is encoded by the small second ORF of RNA2, the HA-epitope-tagged 2b cDNA was cloned under the control of 35S promoter into the pRI201-AN binary vector and designated 35S::Y2b-HA. As controls for agroinfiltration, 35S::GFP, in which the GFP gene was cloned under the control of the 35S promoter in the pRI201-AN binary vector (RI201), and the empty RI201 vector were used. The positions of RT-PCR-amplified fragments corresponding to binary vector constructs are indicated by arrows indicating nucleotide positions. (B) Electrophoresis of cDNA fragments amplified by RT-PCR from total RNA isolated from each site infiltrated with Agrobacterium carrying 35S::CY1, 35S::CY2, 35S::CY3, 35S::YCP, or 35S::GFP in wild-type N. benthamiana. A 100-kb ladder marker was loaded at the right side of the gel. (C) Detection of an HA-epitope-tagged 2b protein of CMV(Y). Transient accumulation of the CMV(Y) 2b protein at the 35S::Y2b-HA-agroinfiltrated site of wild-type N. benthamiana was immunologically detected using an anti-HA monoclonal antibody (α-HA). The RuBisCO protein band is indicated as an internal control for overall protein expression; Supplementary Fig. S2 Pathogenesis-related protein 1 (PR-1) gene expression in P/P/C-HA-transformed N. benthamiana #4-3 leaves agroinfiltrated with a series of chimeric CP-coding sequences under the control of the CaMV 35S promoter. A P/P/C-HA-transformed N. benthamiana #4-3 leaf was agroinfiltrated with YCP, B2CP, CP#1, CP#2, CP#3, CP#4, CP#5, CP#6, CP#7, CP#8, CP#9, or CP#10. The transcript abundance of PR-1 in each agroinfiltration site of the P/P/C-HA-transformed N. benthamiana #4-3 leaf was analyzed by quantitative PCR (qPCR). PR-1 transcript abundances were normalized relative to the values of constitutively expressed UBQ5 mRNAs. Three independent P/P/C-HA-transformed N. benthamiana #4-3 plants were used for quantitative measurement of the abundance of each transcript, and gene expression is expressed as the average and SD of the value of the PR-1 transcript relative to that of UBQ5 mRNA. Data were subjected to analysis of variance, and treatment means were compared using Tukey’s test (p < 0.05). (PDF 83 KB)

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Takahashi, H., Ando, Y., Kanayama, Y. et al. A single amino acid at position 31 in the N-terminus of the coat protein of cucumber mosaic virus determines its avirulence function for RCY1-conferred virus resistance. Arch Virol 169, 61 (2024). https://doi.org/10.1007/s00705-024-05961-4

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