Functional Analysis Of T1N6₂2 From Arabidopsis Thaliana Against Botrytis Cinerea

Gray mould caused by Botrytis cinerea is one of the important world diseases, and always results in significant economic losses. Identifying of genes related to resistance, studying the molecular mechanism and disease-resistant breeding are very important and meaningful contribution to the gray mould control. In previous research, a Botrytis susceptible mutant was achieved by screening from the Arabidopsis T-DNA insertion mutant library. A resistant-related gene of Arabidopsis against B. cinerea T1N6₂2 was isolated by TAIL-PCR and bioinformatics methods. To investigate the function and role in Arabidopsis in resistance to B. cinerea, t1n6₂2 was identified by PCR and RT-PCR technology. Homozygous mutant t1n6₂2 was obtained and it was inoculated with B. cinerea and Pst DC3000 respectively. The results showed t1n6₂2 was highly susceptible to B. cinerea but highly resistance to Pst DC3000. Complementation and overexpression confirmed that T1N6₂2 was the resistance to B. cinerea gene. Semi-quality RT-PCR and Real-Time PCR were used to confirm the expression level and tissue localization. RT-PCR and Real-Time PCR were used to confirm whether T1N6₂2 gene affected resistance of Arabidopsis through the salicylic acid （SA） and jasmonic acid （JA） signaling pathways. T1N6₂2 protein was tested to be located in the chloroplasts by YFP fusion protein technolog. Interactive proteins of T1N6₂2 were screened from Arabidopsis cDNA library through yeast two-hybrid system. These works would help us to better understand T1N6₂2 regulatory mechanism and effective control of B. cinerea.1. The t1n6₂2 mutant was identified by PCR and RT-PCR technology. Homozygous mutant t1n6₂2 was obtained and it was determined that the T1N6₂2 gene was knocked down. Homozygous t1n6₂2 mutants and wild-type Col-0 plants were inoculated with B. cinerea. The results showed Col-0 was immune and T-DNA mutants were highly susceptible to B. cinerea. The expression level of BcACTIN in t1n6₂2 was significantly higher than the wild-type. By Trypan blue and DAB dyeing staining, we found that the cell death and H2O2 accumulation in the t1n6₂2 leaves were more than wild-type. These results showed the t1n6₂2 was susceptible to B. cinerea. Disease phenotypes of the t1n6₂2 and wild-type were compared after inoculation with Pst DC3000. The t1n6₂2 mutant enhanced resistance to Pst DC3000.2. T1N6₂2 was constructed into the pCAMBIA1300 vector and complementation lines were obtained via Agrobacterium-mediated transformed to t1n6₂2 mutants. The T1N6₂2 was certainly integrated to the t1n6₂2 mutant as multiple copies based on the PCR and Southern blotting. RT-PCR analysis showed that the expression level of T1N6₂2, PR4, SOD, SOD1, CAT, PAL and PPO in T1N6₂2-complementing plants were similar to wild-type. T1N6₂2-complementing plants were inoculated with B. cinerea and Pst DC3000. The results showed that T1N6₂2-complementing plants appeared significantly smaller size of lesions during B. cinerea induction and obvious disease symptoms during Pst DC3000 induction. This result showed that the phenotypic reversion was caused by the expression of exogenous T1N6₂2.3. It was found that the gene expression of SA and its analogues BTH treatment for 12h significantly enhanced, while the ACC, and JA processing of the gene expression did not change significantly by RT-PCR technique to analyze the gene expression of T1N6₂2 on Col-0 treatment with BTH, ACC, SA and JA. Jar1, JA biosynthesis deletion mutant, treatment with JA, detect T1N6₂2 gene expression of Col-0 T1N6₂2 after JA treatment down regulated mutation, the T1N6₂2 expression did not change significantly in the jar1, these results indicated that expression of the T1N6₂2 gene in response to molecules regulated by JA signaling pathways. The mutants such as NahG, eds5, sid2 and npr1 beloning to SA signaling pathway were treatment with SA, the results found the expression of T1N6₂2 gene in NahG, eds5 and npr significantly decreased, suggesting expression of T1N6₂2 regulated also by SA signaling pathway.4. Using RT-PCR and Real-Time PCR technology to detect the wild-type, mutant and complemental strains gene expression of related-disease resistance after inoculation Botrytis cinerea, the genes PR1, PR3, PR5, and PDF1.2 gene expression of t1n6₂2 was different from the wild-type. It was found that the wild-type, mutant and complemental strains gene expression of related-disease resistance after inoculation Pst DC3000, the genes PR1, PR3, PR5, and NPR1 gene expression of t1n6₂2 was different from the wild-type, indicating the function of T1N6₂2 was loss to destruction of the salicylic acid （SA）- and jasmonic acid/ethylene （JA/ET）-dependent signaling pathways.5. T1N6₂2 were subcloned in the the binary expression vector pCAMBIA1300, and transformed into Arabidopsis through protoplasts transformation. Using fluorescence microscopy to identified the T1N6₂2 protein was localized in the chloroplast.6. We constructed a yeast expression vector used the T1N6₂2 gene as bait. T1N6₂2 had transcription activation in vitro by 3-AT method. Four proteins were obtained using two-hybrid assays and PCR verification. The four proteins were all nucleosome assembly protein by bioinformatic analysis.