Over-expression Of ANN8 Compromises RPW8.1-mediated Cell Death And Disease Resistance In Arabidopsis

The Arabidopsis thaliana resistance gene RESISTANCE TO POWDERY MILDEW 8.1(RPW8.1) triggers the hypersensitive response(HR) at the infection site to restrict pathogenic infections. To further understand how plants’ resistance response is regulated by RPW8.1,the transgenic line R1Y4 that expresses RPW8.1-eYFP from the native promoter was used for mutagenesis by EMS. One mutant that enhances RPW8.1-mediated HR-like cell death was selected,which is named B7-6 through phenotypic analysis. The mutant exhibited stronger resistance to powdery mildew compared with that of R1Y4.The basal defense responses are enhanced in B7-6 when induced by PAMPs flg22 or chitin, including programmed progress cell death(PCD), callose deposition and ROS burst. Furthermore, the transcript level of RPW8.1 and the fluorescence intensity of RPW8.1-eYFP in mutant are significantly higher than those in R1Y4.Based on map-based cloning,we identified an A to G point mutation in At5g12380 that led to the amino acid residue Ala(A) at position 94 substituted with Thr(T) in the previous experiment. The mutant gene encode a membrane protein(ANNEXIN8,ANN8);The complementary test supported that mutation in At5g12380 is responsible for the enhanced cell death phenotype. Therefore, we hypothesis that ANN8 negatively regulates RPW8.1-mediated resistance responses.In this study, we made construct to expression ANN8 from the constitutive promoter 35S and introduced into Col-gl and R1Y4 background to make transgenic plants over-expression ANN8 (35s-ANN8/Col-gl and 35s-ANN8/R1Y4).We observed the phenotype of the over-expression transgenic plants and identified the disease resistance to powdery mildew pathogen of Arabidopsis and Pseudomonas syringae strains DC3000 via artificial infection. Then the quantitative real-time PCR was applied to examine the mRNA level of RPW8.1 and the expression patterns of defense-related genes induced by powdery mildew in over-expression transgenic lines(R1Y4 background).In the end,we explored the mutant ann8-Ⅰ’ (loss function of ANN8) function in abiotic-stress. The main data are described as follow:1、The over-expression of ANN8 transgenic lines suppress the phenotype of R1Y4The pit-phenotype of R1Y4 is suppressed in transgenic R1Y4 lines over-expressing ANN8,the plants exhibited phenotypes similar to WT.2、The transcription level of RPW8.1 was reduced after over-expressing ANN8We speculated that the expression of RPW8.1 can be suppressed by over-expressing ANN8 since the transgenic lines exhibited phenotypes similar to WT. Then we examined the expression level of RPW8.1 by qRT-PCR and found that the mRNA level of RPW8.1 was significant lower in 35s-ANN8/R1Y4 than that in R1Y4.The data indicate that over-expressing ANN8 can suppress the expression of RPW8.1.3、Over-expression of ANN 8 compromises RPW8.1-mediated disease resistanceWe vaccinated powdery mildew pathogen of Arabidopsis and Pseudomonas syringae strains DC3000 in over-expressing transgenic lines. The data demonstrate that transgenic R1 Y4 lines over-expressing ANN8 are susceptible to powdery mildew and the resistance-responses in R1Y4 are significant suppressed.The bacterial colonies are more in 35s-ANN8/R1Y4 than that in R1Y4.The 35s-ANN8/R1Y4 exhibited less cell death compared with that of R1Y4 after induced by powdery mildew.4、The transcription level of FRK1 and PR1 were reduced in over-expressing of ANN8 transgenic linesPlants defense responses are associated with the expression level of defense genes, then we examined the expression level of FRK1 and PR1 by qRT-PCR. The data indicate, the expression level of PR1 and FRK1(a marker gene in PTI defense signaling) are down regulated in 35s-ANN8/R1Y4 compared with that in R1Y4 after induced by powdery mildew.5、ANN8 loss of function enhanced the plant roots on salt sensitivityWe measured the length of roots of Col-gl,ann8-1and amiANN8 after we processing with different phytohormones and salt stress at low concentration. The results suggest, both ann8-1 and amiANN8 roots are suppressed at low concentration salt stress, nevertheless, they are non-sensitive to exogenous SA.Taken together, these data indicate that ANN8 not only negatively regulates RPW8.1-mediated cell death and disease resistance, but also be involved in abiotic-responses.