Expression Of Tomato Resistance Gene Tm-2～2 In Tobacco And Determination Of Amino Acid Specificity For Resistance To ToMV

Tomato (Lycopersicon. esculentum Miller) Tm-22 gene is a member of NBS-LRR class of proteins with durable resistance to Tobamovirus, it located at the long arm of 9th chromosome closed to centromere and closely linked to nv gene. Both Tm-22 and Tm-2 derived from L.chilense. Tomato plants with Tm-22 gene are resistance to 5 strains in Tobamovirus and susceptible to ToMV-2a. Tm-22 and its alleles Tm-2, tm-2 present a stretch of 2586 bp of ORF in genomic DNA and give a protein of 861 amino acid with 98.8kDa in molecular weight and at PI 8.4. Tm-22 has 99.7% identical to Tm-2 and 97.7% identical to tm-2. No amino acid differences were indicated in the first 90 aa at N terminal of Tm-22 and its alleles. 2 and 6 aa differences of Tm-22 to Tm-2 and tm-2 respectively, in NBS domain from 91 to 483 aa. The characteristics of inhomogeneous distribution indicate the specificity of Tm-22 resistance. In order to insight to the function, property and mechanism of Tm-22, 3 steps works were considerable. First, to confirm the possibility of the functional expression, and transfer Tm-22 into tobacco SRI; second, to analyze the specificity of amino acid differences in recognition for both protein Tm-22 and ToMV-MP with comparison of sequences and structure. Finally, to exchange the DNA fragments of domain swapped in Tm-22 and tm-2, and built up chimeric constructs for chimera. Determinants of amino acid identified for recognition in resistance reaction. The results observed as follows: 1.13 homozygous transgenic lines were obtained with Tm-22 own promoter and 14 lines with 35S promoter. Both lines showed resistance to ToMV-0, ToMV-2, ToMV-1, TMV-U, TMV-Cg and susceptible to ToMV-2a. This result indicated that Tm-22 was functionally expression in receptor cell with distinct genetic background. Virus specificity and gene express can function in distinct genus within a family. The characteristics of Tm-22 expression presented in transgenic tobacco: 1). Virus specificity in either homozygote or heterozygote; 2) Tm-22 gene integrated in tobacco genomic DNA with single copy and in inheritance and segregation to progenies on the Mendel role; 3). Transgenic line with Tm-22 promoter (pTM47) showed infected symptoms with cell death distinguished to one with 35S promoter (pTM49) after inoculation with ToMV-2a.2. To further understand Tm-22 response reaction to ToMV, MP interacting Protein gene (MIP204), PR related gene (PR-la), RNA dependent RNA polymerase gene (RdRP), 3 genes mRNA test were performed after inoculation different strains of ToMV in both pTM47and pTM49 transgenic lines. 1). PR-la mRNA was tested in transcription in pTM47 transforraants after inoculation with ToMV-0,ToMV-1, ToMV-2, TMV-U, ToMV-Cg, except to ToMV-2a infection. 2). MJP204 mRNA presented in transcription in both pTM47 and pTM49 transformants after inoculation of ToMV-0, ToMV-2 and ToMV-2a. But without RdRP transcription were tested. So it is suggested that Tm-22 is a relative to multi-protein transcriptional coactivator. MIP204 and PR-la gene expression can be induced in resistant response and do not relate to RdRP gene transcription. Based on the Guard hypothesis, Tm-22 and MIP204 could be a guardee and a guardian cooperated in PR gene induction in resistance activity.3. Sequence analysis of amino acid and DNA for Tm-22 and it alleles, and comparison of the homologous of R genes indicate that: V). Tm-22 and it alleles encode a classical LZ-(NB-ARC)-LRR of membrane proteins. 4 of LZ (Leucine Zipper) motifs are located in first 90 aa of CC domain; 2). 9 ARC structures consist of NBS domain. In N terminal, 3 conserved sequences , WXIXGMXGXGKTTLA, L(I/V)(V/L)LDDV(W/D) and SRIIXTTRD XXV are in term of P-loop, kinase 2 and kinase 3a. Those are highly identical to the homologous of human(Apaf-1) and namenode(CED-4). It revealed that Tm-22 encodes a membrane protein with a receptor structure and kinase property and may function in ion flow, phosphorylation and proteins interaction. Amino acid differences are not found in conservative mot...