| RESISTANCE TO POWDERY MILDEW8 (RPW8) is a broad-spectrum resistance gene locus containing two closely linked genes, RPW8.1 and RPW8.2. RPW8.2 protein expressed under its own promoter is specifically targeted to the interfacial membrane encasing the fungal haustorium of powdery mildew. RPW8.1 shares a high homology with RPW8.2, is located around chloroplasts in mesophyll cells, and also possesses a broad-spectrum disease resistance. However, little is understood on the resistance mechanism of RPW8.1.Previously, we established an EMS (ethyl methane sulphonate) mutant library using seeds of Arabidopsis thaliana expressing RPW8.1-YFP (R1Y4) as the starting material. In this research, we selected four variegation mutants B20-1,B25m1-12,B26m1-3 and B26ml-4 for further analysis. By map-based cloning, we found that mutations in the gene At4g22260 (encoding IMMUTANS, M) was responsible for the variegation phenotype of the four mutants. B20-1 had a G to A mutation at 1718-nt, resulting in alternateive splicing of mRNA. B25m1-12 had a C to T mutation at 103-nt, resulting in the change of Arginine (R) to stop codon. B26m1-3 and B26m1-4 had a same mutation, i.e. G to A mutation at 1891-nt, resulting in alternative mRNA splicing. B26m1-4 was recovered to the phenotype of R1Y4 by introducing the genomic DNA of At4g22260 fused with eYFP. Thus, At4g22260 is the gene responsible for the mutant phenotypes observed in B26m1-4. There are three immutans have reported before,so we renamed B20-1 to im1-4/R1Y4,B25m1-12 to iml-5/R1Y4,B26m1-3 and B26m1-4 to iml-6/R1Y4.IM is localized on the chloroplast and functions in chlorophyll synthesis. As RPW8.1 is also targeted to surrounding chloroplast, we assume that IM may be functionally associated with RPW8.1. B26ml-4 was selected as a representative of im/R1Y4 mutant for subsequent experiments. Mutant im/R1Y4 was crossed with Col-gl and NahG, respectively. Phenotyping and genotyping results revealed that im mutant phenotype was controlled by a recessive single gene, and independent of RPW8.1 and SA signaling pathway. Disease assay was performed with inoculation of tobacco powdery mildew, Pseudomonas syringae pv. tomato DC3000, or P. syringae pv. tomato DC3000 (hrcC-) on im mutants. Anline Blue, DAB, Trypan Blue and other staining methodologies combined with confocal microscopy, and detection of ROS were carried out. The main results are as follows:1. Loss-of-function of IM showed enhanced disease susceptibility. In contrast to the powdery mildew resistance of R1Y4, imlR1Y4 mutants were more sensitive to tobacco powdery mildew than Col-gl, i.e. no Rl Y4-type cell death and more fungal growth. In addition, imlR1Y4 mutants showed higher sensitivity to P. syringae pv. tomato DC3000 and P. syringae pv. tomato DC3000 (hrcC-) than R1Y4. These data suggest that IM is required for full function of RPW8.1 in broad-spectrum resistance in Arabidopsis thaliana.2. TMgene mutation reduced the accumulation of hydrogen peroxide. Accumulation of hydrogen peroxide (H2O2) is one of RPW8.1-mediated defense responses. After inoculation of powdery mildew, H2O2 accumulation was less in iml-4/R1Y4 and iml-6/R1Y4 than in R1Y4.3、IM gene mutations reduced callose deposition. After inoculation of DC3000 and flg22 treatment, im/R1Y4 mutants produced less callose than R1Y4. |
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