Cloning of the Arabidopsis RTM1 gene, which controls restriction of long-distance movement of tobacco etch virus

Abstract

The locus RTM1 is necessary for restriction of long-distance movement of tobacco etch virus in Arabidopsis thaliana without causing a hypersensitive response or inducing systemic acquired resistance. The RTM1 gene was isolated by map-based cloning. The deduced gene product is similar to the alpha-chain of the Artocarpus integrifolia lectin, jacalin, and to several proteins that contain multiple repeats of a jacalin-like sequence. These proteins comprise a family with members containing modular organizations of one or more jacalin repeat units and are implicated in defense against viruses, fungi, and insects.

Figure 1

Cloning of RTM1. (A) PCR-based markers that flank or cosegregate with RTM1 are indicated at the top. The number of recombination events per total number of meiotic events scored is given below each marker. A BAC contig spanning RTM1 is shown (open boxes). Cosmids (solid lines) derived from BACs F2M5 (cosmids with prefix 1), F25G24 (prefix 2), and F26O9 (prefix 3) were introduced into Arabidopsis ecotype C24 (rtm1/rtrm1). The complementing interval is shaded. Putative ORFs are indicated by boxes in the bottom expanded region. The HindIII (H) and BsaAI (B) restriction sites used to generate 2–63H and 2–8XB, respectively, are indicated. The number of putative C24 transformants that restricted long-distance movement of TEV-GUS per total plants tested is shown adjacent to each clone. (B) GUS activity assays of selected TEV-GUS-infected T2 C24 lines containing complementing cosmids (2–8 #3, 2–9 #11, 2–41 #1, 2–63 #1, 2–72 #12, 2–79 #8), a noncomplementing cosmid (1–31 #1), or empty vector (pSLJ755I5), or wild-type susceptible (C24) and nonsusceptible (Col-0) lines. Inflorescence tissue from 10 T2 individuals was tested at 20 days postinoculation. The mean GUS activity value (+ SD) is shown. (C) Representation of the RTM1 cDNA, with the nucleotide positions of the start codon (nucleotide 55), 5′ intron splice site (nucleotide 262), stop codon (nucleotide 577), and 3′ terminal nucleotide (nucleotide 729) indicated. Arrows indicate positions of amino acid substitutions in rtm1 mutant alleles.

Figure 2

Predicted RTM1 amino acid sequence for Col-0, C24, and La-er alleles, and mutant alleles rtm1–1, rtm1–2, and rtm1–5. Differences relative to the Col-0 sequence are shaded. Dashes indicate amino acid residues absent from the deduced La-er product.

Figure 3

Alignment of the deduced Col-0 RTM1 sequence, amino acids 61–217 of the jacalin prepropeptide, and JRs from B. napus MBPs 1 and 6. Gray and black shading indicate conserved and identical residues, respectively. The arrow indicates the first residue in the jacalin α-chain. Amino acids corresponding to the mature jacalin β-chain are underlined. * indicate residues involved in carbohydrate binding in the jacalin α polypeptide (22). Dashes indicate gaps in the alignment.

Figure 4

Organization of proteins containing JRs. Boxes indicate JRs. Color of boxes corresponds to the position of the repeat in the cladogram (Fig. 5). Open boxes indicate repeats that are not grouped within a clade with other JRs in Fig. 5. Gray arrow indicates the Kelch repeats of A. th. F25I18–19. A. in., A. integrifolia; A. th., A. thaliana; B. n., B. napus; f-, floral; M. p., M. pomifera.

Figure 5

Cladistic analysis of JRs from various proteins. Repeats were aligned and assembled into a single tree by a heuristic search using bootstrap analysis (100 replicates) with simple sequence addition, tree-bisection-reconnection branch swapping, steepest descent off, and mulpars on using paupsearch and paupdisplay. Jacalin and M. pomifera agglutinin were assigned to the outgroup. Numbers above branches are bootstrap percentage values. The relative positions of JRs in proteins containing multiple repeats are indicated (e.g., JR1, JR2). Colored lines highlight each clade. For abbreviations, see Fig. 4 legend.