The Annexin Gene ANN8 Negatively Regulates RPW8.1-mediated Cell Death And Disease Resistance In Arabidopsis

Arabidopsis RPW8 has a broad spectrum resistance against powdery mildew. This gene locus contains two genes, named RPW8.1 and RPW8.2. RPW8.2 protein is specifically targeted to then extrahaustorial membrane (EHM) for its function against powdery mildew; while RPW8.1 locates around chloroplasts in the mesophyll cells. To better understand the molecular mechanisms of RPW8.1-mediated broad spectrum resistance, the transgenic line R1Y4 that expresses RPW8.1-eYFP from the native promoter was used for mutagenesis by EMS. Quite a few mutants wiht enhanced cell death were obtained and the analyses on the mutant b7-6 were described.The mutant b7-6 exhibited more severe cell death compared with that of R1Y4. By crossing to the Arabidopsis ectoype Ler, we obtained an F2 population. Positional cloning identified a A-G mutation at the nucleotide position 280 resulting in amino acid substitution of Alanine(A) to Threonine(T) in At5gl2380 (encoding ANNEXIN, ANN8). The cell death phenotype of b7-6 was recovered to that of R1Y4 by introducing genomic DNA of At5g12380 fused with eYFP into b7-6. Thus, At5g12380 is the gene responsible for the mutant phenotypes observed in b7-6, and we renamed b7-6 as ann8-1/R1Y4.ANN8 is a member of Annexin gene family that contains 8 members in Arabidopsis. To characterize the authentic role ofANN8 in disease resistance or cell death, ann8-1/R1Y4 was crossed to Col-gl and the sigle mutant ann8-lwas obtained (within the Col-gl background). Meanwhile, we made RNAi construct to knock-down the expression of ANN8 and obtained ANN8 knock-down lines named m\ANN8 and m\ANN8/R1Y4 (miANN8 within R1Y4 background obtained by crossing miANN8 to R1Y4).Subsequently, we evaulatied the disease resistance to powdery mildew pathogen strain UCSC1 and Pseudomonas syringae strains DC3000 and DC3000-hrcC- between ann8-1 and ann8-1/R1Y4, miANN8 and miAnn8/R1Y4 together with Col-gl (wild type) as a reference. Defense responses were monitored and compared by methodologies including using confocal scanning laser microscopy, trypan blue staining, DAB staining, aniline blue staining and ROS burst detection. Furthermore, quantitative real-time PCR was applied to examine the expression patterns of defense-related genes. Finally, we also examined the expression patterns of the 8 annexin genes in different tissues and upon infection by different strains of Pseudomonas syringae.Our data demonstrate that ANN8 nagtively regulates RPW8.1-mediated cell death and disease resistance.First, ann8-1/R1Y4 exhibited a stronger resistance than R1Y4 against both powdery mildew UCSC1, DC3000 and DC3000-hrcC. Similarly, defense responses such as programmed cell death, peroxide accumulation, callose deposition and ROS burst were stronger in ann8-1/R1Y4 than in R1Y4. However, ann8-1 mutants showed no significant differences on resistance and defense responses compared with Col-gl. The data of ANN8 knock-down mutant miANN8 and miANN8/R1Y4 were similar to that annn8-1 and ann8-1/R1Y4, respectively. These data also suggest that mutation in ANN8 leads to RPW8.1-dependent cell death, disease resistance and defense responses.Second, ANN8 negativeiy regulates RPW8.1’s mRNA expression level and protein accumulation. We found that the fluorescence intensity of RPW8.1-eYFP and RPW8.1 transcript level were significantly higher in ann8-1/R1Y4 than that in R1Y4. Upon inoculation of powdery mildew, more induction of PR1 expression was observed in ann8-1/R1Y4. FRK1, a marker gene in PTI defense signaling, was also induced to a higher level by powdery mildew in ann8-1/R1Y4 than in R1Y4.Third, ANN8 probably be the resistance associated member of the eight Annexin genes in Arabidopsis. The expression pattern of ANN8 was distinct with those of other members in Annexin gene family. Its expression pattern showed as follows:expressing in the cytoplasm of particular plant cells, displayed high expression level in leaves, and induced by powdery mildew and P. syringae. These results suggest a specific role ofANN8 in disease resistance.Taken together, we concluded that ANN8 loss-of-function mutant ann8-1 probably elevates disease resistance through enhancing the expression of RPW8.1, in turn activating the defense-related genes and responses, then enhanced plant disease resistance.