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, 154 (1), 222-32

RTM3, Which Controls Long-Distance Movement of Potyviruses, Is a Member of a New Plant Gene Family Encoding a Meprin and TRAF Homology Domain-Containing Protein

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RTM3, Which Controls Long-Distance Movement of Potyviruses, Is a Member of a New Plant Gene Family Encoding a Meprin and TRAF Homology Domain-Containing Protein

Patrick Cosson et al. Plant Physiol.

Abstract

Restriction of long-distance movement of several potyviruses in Arabidopsis (Arabidopsis thaliana) is controlled by at least three dominant restricted TEV movement (RTM) genes, named RTM1, RTM2, and RTM3. RTM1 encodes a protein belonging to the jacalin family, and RTM2 encodes a protein that has similarities to small heat shock proteins. In this article, we describe the positional cloning of RTM3, which encodes a protein belonging to an undescribed protein family of 29 members that has a meprin and TRAF homology (MATH) domain in its amino-terminal region and a coiled-coil domain at its carboxy-terminal end. Involvement in the RTM resistance system is the first biological function experimentally identified for a member of this new gene family in plants. Our analyses showed that the coiled-coil domain is not only highly conserved between RTM3-homologous MATH-containing proteins but also in proteins lacking a MATH domain. The cluster organization of the RTM3 homologs in the Arabidopsis genome suggests the role of duplication events in shaping the evolutionary history of this gene family, including the possibility of deletion or duplication of one or the other domain. Protein-protein interaction experiments revealed RTM3 self-interaction as well as an RTM1-RTM3 interaction. However, no interaction has been detected involving RTM2 or the potyviral coat protein previously shown to be the determinant necessary to overcome the RTM resistance. Taken together, these observations strongly suggest the RTM proteins might form a multiprotein complex in the resistance mechanism to block the long-distance movement of potyviruses.

Figures

Figure 1.
Figure 1.
Genotyping and LMV infection phenotyping of the RTM3 KO lines and of the F1 populations produced by crossing each KO line with the mutant rtm3-1 line. KO-g, KO Gabi 801D05; KO-s, KO Salk N517845; H2O, negative control without genomic DNA; Wt, wild type. Numbers 1, 2, 3, and 4 indicate replicates. A, Genotyping of both KO lines and of the F1 populations revealed by PCR on genomic DNA. PCR 1, PCR using the Gabi-Kat-specific primer and the two At3g58350-specific primers (see “Materials and Methods”); PCR 2, PCR using Lba1 and the two Salk-designed-specific primers for N517845 (see “Materials and Methods”); L, 100-pb ladder. B, RT-PCR amplification of RTM1 and RTM3 transcripts from total RNA from wild-type and RTM3 KO lines. gDNA, Genomic DNA; L, 1-kb ladder. C, RT-PCR of a 277-bp fragment of LMV RNA from LMV-inoculated RTM3 KO and F1 line systemic leaves. L, A 100-pb ladder.
Figure 2.
Figure 2.
Secondary structure for the RTM3 protein. Prediction obtained using NPS@. The four lines under the RTM3 sequence correspond to the results of four different prediction methods (DSC, MLRC, PHD, and Predator), and the last line is the consensus secondary structure prediction. h indicates helix, e indicates β-strand, and c indicates random coil. Numbers above the sequences indicate amino acid positions.
Figure 3.
Figure 3.
Genomic distribution of the RTM3 homologous genes. This map was made using the chromosome map tool from The Arabidopsis Information Resource (http://www.arabidopsis.org/index.jsp). Black arrows indicate genes encoding protein with MATH and CC domains; gray arrows indicate genes encoding protein with only a CC domain. Numbers at the top of the chromosomes indicate the number of each Arabidopsis chromosome.
Figure 4.
Figure 4.
List and schematic organization of Arabidopsis RTM3 homologous proteins containing MATH and CC domains. White boxes represent the MATH domain, and gray boxes represent the CC domain.
Figure 5.
Figure 5.
Protein-protein interactions between the RTM proteins and the potyviral CP with the yeast two-hybrid system. A, Interactions among the wild-type RTM proteins. The growth of cotransformed yeast was assessed in SD/Leu-Trp (−LT) medium, and then after 3 d, six colonies for each cotransformation experiment were plated on SD/Leu-Trp (as a control), SD/Leu-Trp-His (−LTH), and SD/Leu-Trp-His-Ade (−LTHA) media and were left 4 d at 30°C. C−, Negative control; C+, positive control. The clones in pGADT7 are those indicated in the horizontal rows, and those in pGBKT7 are indicated in the vertical rows. B, Interactions between the RTM proteins and the PPV CP. For each pair of tested clones, the first is the clone in pGAD-T7 and the second is the clone in pGBK-T7. pGAD-T and pGBK-53 are the clones supplied in the Clontech kit to be used as positive controls. pGAD is the empty vector pGAD-T7 and pGBK is the empty vector pGBK-T7 used as negative controls. Only the yeast colonies (three colonies for each cotransformation experiment) plated in the –LTHA medium are shown. C, Interactions with the RTM3 mutant protein from the rtm3-1 mutant line. Details are as in B.
Figure 6.
Figure 6.
In planta interaction between RTM1 and RTM3 by BiFC. Confocal microscopy images of onion epidermal cells cobombarded with constructs expressing the different RTM proteins fused to the N-terminal fragment of YFP (YN) and the C-terminal fragment of YFP (YC) are shown.

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