Wheat Ym2 originated from Aegilops sharonensis and confers resistance to soil-borne Wheat yellow mosaic virus infection to the roots

Abstract

Wheat yellow mosaic virus (WYMV) is a pathogen transmitted into its host's roots by the soil-borne vector Polymyxa graminis. Ym1 and Ym2 genes protect the host from the significant yield losses caused by the virus, but the mechanistic basis of these resistance genes remains poorly understood. Here, it has been shown that Ym1 and Ym2 act within the root either by hindering the initial movement of WYMV from the vector into the root and/or by suppressing viral multiplication. A mechanical inoculation experiment on the leaf revealed that the presence of Ym1 reduced viral infection incidence, rather than viral titer, while that of Ym2 was ineffective in the leaf. To understand the basis of the root specificity of the Ym2 product, the gene was isolated from bread wheat using a positional cloning approach. The candidate gene encodes a CC-NBS-LRR protein and it correlated allelic variation with respect to its sequence with the host's disease response. Ym2 (B37500) and its paralog (B35800) are found in the near-relatives, respectively, Aegilops sharonensis and Aegilops speltoides (a close relative of the donor of bread wheat's B genome), while both sequences, in a concatenated state, are present in several accessions of the latter species. Structural diversity in Ym2 has been generated via translocation and recombination between the two genes and enhanced by the formation of a chimeric gene resulting from an intralocus recombination event. The analysis has revealed how the Ym2 region has evolved during the polyploidization events leading to the creation of cultivated wheat.

Keywords: Bymovirus; molecular evolution; plant breeding; recombination; wild relatives.

Fig. 1.

The effect of allelic variation at Ym1 and Ym2 on P. graminis colonization and WYMV infection. The genotype of the WYMV resistant cv. Madsen and the WYMV susceptible cv. Hokushin is, respectively, Ym1/Ym2 and ym1/ym2. (A) Plant response to P. graminis-mediated virus infection. P. graminis colonization of the root epidermal and cortex layers is evidenced by cotton blue staining. WYMV RNA was visualized in transverse root sections using RNA in situ hybridization based on an antisense probe (AS); the same sections were also probed with a sense probe (S). The presence of WYMV coat protein in the root and leaf blade was visualized using an immunohistological stain: The coat protein fluoresces green, while membranes and chlorophyll fluoresce red. The adaxial surface of a leaf blade showing symptoms of WYMV infection. The arrow heads indicate the endodermis. st: stele. Scale bars: cotton blue stained root and immuno-stained leaf–50 μm; root transverse sections–100 μm; leaf blade surface–1 mm. (B) Plant response to the mechanical inoculation of leaves. WYMV RNA1 titer following inoculation was determined using a qRT-PCR assay. Dots represent individual plants and bars genotypic means. The WYMV copy number was log₁₀ transformed. **: means differ significantly (P < 0.01). “Virus inoculation”: leaves inoculated with virus suspended in buffer; “Buffer inoculation”: leaves inoculated with buffer only.

Fig. 2.

Map-based cloning of Ym2 and its pattern of transcription. (A) The Xwmc754-Xcfd79 interval flanking Ym2 (5), narrowed to an interval flanked by XHG610 and XHG621. Mbp numbers in parentheses refer to physical positions in the cv. Chinese Spring whole-genome sequence. The target gene region was further narrowed to between XCDS614.1 and XCDS619, a region which in cv. Madsen harbors six putative candidates (black arrows). CDS618 encodes a 940-residue CC-NBS-LRR protein. Exons are represented by open boxes and the coding sequence of conserved domains are shaded in gray. Loss-of-function polymorphisms between cv. Madsen and cv. Hokushin are shown in blue, and those between cv. Madsen and cv. Chinese Spring in orange. c: coding sequence, p: protein, del: deletion, fs: frameshift. (B) Complementation test using a CDS618 transgene. Quantification of the pUbi-CDS618 transcript and WYMV RNA1 in roots and leaf blades acquired using qRT-PCR. #02 and #06 refer to two independent T₂ families. Plants harboring (+) and lacking (–) the transgene were grown in virus-infested soil. Copy numbers of the CDS618 transcript and of WYMV RNA1 have been log₁₀ (transcript copy number) transformed. (C) An abundance of CDS618 transcript protects plants from WYMV infection. The quantification of CDS618 transcript and WYMV RNA1 in plants grown in virus-infested soil acquired using qRT-PCR. Copy numbers of the CDS618 transcript and of WYMV RNA1 have been log₁₀ transformed. (D) CDS618 transcript abundance in the root, leaf sheath, and leaf blade of plants carrying Ym2 and either Ym1 or ym1. Copy numbers of the CDS618 transcript have been log₁₀ transformed. (E) Transcription of two candidate genes for Ym2 in root tips determined by RNA in situ hybridization. The assay was based on the use of an antisense probe (AS) and a sense probe (S). st: stele, cp: cortex parenchyma, rc: root cap meristem. The arrow heads indicate the endodermis, in which a thick cell wall is laid down. (Scale bar: 100 μm.) **, ***: means differ significantly at, respectively, P < 0.01 and <0.001.

Fig. 3.

Sequence variation at Ym2 (CDS618) and its effect on WYMV resistance. (A) Variable nucleotide sites in the cv. Madsen sequence are represented in red are shown on the Left, mis-sense variants in magenta (shown above the coding sequence) and premature stop codons in blue (shown below the sequence). Accessions lacking any CDS618 product (null mutations) are shown in black to the Right, and those harboring a chimeric version of the gene are boxed. g: genome, c: coding sequence, p: amino acid position, fs: frameshift, *: stop codon, a: p.F291C, p.I306V, p.G345D, p.N360K, p.K361E, p.E381V,p.K445R, p.S464A and p.T508M specific for cultivar M45/66. Parentheses: duplicated gene products from multiple plant accessions. The cultivars Chogokuwase, Minaminokomugi, and Norin61 (two accessions) harbor two copies of CDS618, one encoding a truncated 213-residue protein and the other a chimera between CDS618 and TraesCS3B02G035800, predicted to encode a 940-residue protein. (B) WYMV copy number (log₁₀ transformed) in the roots of plants exposed to WYMV.

Fig. 4.

The origin of Ym2. (A) Phylogeny of the polypeptide products of NBS-LRR genes in Triticum spp. and related genera. B37500 is an A. sharonensis ortholog of cv. Chinese Spring TraesCS3B02G037500 and B35800 an A. speltoides ortholog of cv. Chinese Spring TraesCS3B02G035800. (B) The proposed mode of origin of Ym2. B37500 and B35800 (respectively the Ym2 ortholog and paralog) arose in the ancestors of, respectively, A. sharonensis and A. speltoides. Recombination between the two genes generated a concatenated sequence carried in A. speltoides and passed on to polyploid Triticum spp. A 1 bp deletion in B-37500 generated the truncated gene B-37500’, an inverted duplication of B35800 generated B35800’, and an intralocus recombination event between B35800’ and B37500 the chimeric gene harbored by cv. Norin61.