| Harpin proteins are elicitors secreted through type Ⅲ secretion system in gram-negative plant pathogenic bacteria, they are acidic, glycine-rich, heat-stable and protease-sensitive, generally affect virulence in host plants, induce hypersensitive cell death in non-host plants and elicit diverse beneficial responses in plants. Exogenous applications of harpins can induce systemic acquired resistance, enhance defense response against diverse pathogens and insects, enhancement of drought tolerance, as well as stimulate plants growth.In a previous study, we cloned popW from Ralstonia solanacearum strain ZJ3721, coding PopW, a harpin protein, like other known harpins, it is acidic, rich in glycine and serine, heat-stable and protease-sensitive. The procaryotically expressed PopW can induce resistance to tobacco mosaic virus (TMV), enhance growth and improve quality of tobacco, when sprayed onto tobacco leaves. To further investigate the function of popW, we transferred popW into tobacco in previous study. Here, kanamycin resistance tests, PCR and RT-PCR analysis, PCR-Southern blot and GUS staining analysis were used to screen and identify T1 to T3 progeny of popW-transgenic tobacco lines, and 21 T3 lines were identified as positive transgenic plants. Genomic intergration and expression of the transferred gene were determined by PCR, PCR-Southern blot and RT-PCR. And GUS staining analysis indicated that the protein expressed in transgenic tobacco was bioactive and exhibited different expression levels among lines. Growth assays showed that expression of popW in tobacco exhibited a plant growth enhancement.15 d after sown on MS medium, the root length of transgenic tobacco seedlings was 1.67 times longer than that of non-transgenic tobacco plants, and 60 d after transplanting to pots, the height, fresh weight and dry weight of transgenic tobacco were 1.57,1.83 and 1.81 times larger than that of non-transgenic tobacco plants, respectively.Previous study showed that PopW induced systemic acquired resistance by priming SA-dependent pathway. In order to investigate the function of PopW, we studied the popW-transgenic tobacco plants. We detected the resistance of transgenic tobacco lines to TMV and Ralstonia solanacearum YN10 under greenhouse conditions and analyzed the mechanisms underlying the enhanced resistance to TMV and Ralstonia solanacearum of transgenic tobacco lines. The result of resistance detection to TMV showed that transgenic tobacco lines had enhanced resistance to TMV with biocontrol efficiency up to 54.25%. The change of H2O2 content in transgenic tobacco lines showed a typical bimodal pattern, and the content of H2O2 in non-transgenic tobacco plants was lower than that of transgenic tobacco lines after inoculation with TMV. The activities of defense-related enzymes POD, PAL and PPO in transgenic tobacco lines were also higher than that in non-transgenic tobaccos after inoculation with TMV. Erexpression of popW in tobacco also significantly enhanced the relative transcript levels of SA-related and HR-related genes, and after inoculation with TMV, the transcript levels of these genes were significantly induced. The grafting experiment further demonstrated that the resistant signals in transgenic tobacco plants were systemic signal which could be transmitted. The result of resistance detection to Ralstonia solanacearum showed that transgenic tobacco lines had also enhanced resistance to Ralstonia solanacearum and the biocontrol efficiency of transgenic tobacco line 1-1-2 was up to 81.32%. There were much more H2O2 and HR in transgenic tobacco leaves after inoculation with Ralstonia solanacearum, which limited the growth of the pathogen. We also analyzed the relative transcript levels of defense-related genes before and after Ralstonia solanacearum inoculation, it showed that expression of popW in tobacco enhanced the expression of SA, ET, JA and HR-related genes.During our study to the transgenic tobacco, we found that the seeds of transgenic tobacco lines germinated normally on MS plates with low concentration of H2O2, but the seed germination of non-transgenic tobacco was suppressed. So we infer that expression of popW in tobacco may enhanced tolerance to oxidative stress.We studied their tolerance to oxidative stress under greenhouse conditions and analyzed the molecular mechanisms underlying the enhanced oxidative stress tolerance of transgenic tobacco lines. The results showed that the transgenic tobacco lines reduced the inhibitory effect of hydrogen peroxide (H2O2) on seedlings growth and exhibited a reduction in membrane damage. The transgenic tobacco lines have displayed lower malondialdehyde (MDA), relative electrical conductivity (REC) and higher relative chlorophyll content than non-transgenic tobacco plant under oxidative stress conditions. Furthermore, transgenic tobacco lines displayed a significant increase in peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) activities and ascorbic acid (AsA) content compared to non-transgenic tobacco plant under methyl viologen-mediated oxidative stress conditions, and these levels were up to 1.95, 1.68,1.33 and 1.34 times higher than those of non-transgenic tobacco plants, respectively. Overexpression of pop Win tobacco also significantly enhanced the relative transcript levels of oxidative stress-responsive genes NtAPX, NtCATl, NtGST and NtCu/Zn-SOD under oxidative stress conditions. The relative transcript levels of these genes were up to 1.94, 2.36,5.24 and 3.62 times higher than those of non-transgenic plants, respectively.These results indicate that ectopic expression of popW resulted in dual resistance to disease and oxidative stress. On one hand, it activated the disease resistance signaling pathways and induced the expression of defense-related genes, thus enhanced resistance to TMV and Ralstonia solanacearum in tobacco; On the other hand, popW transgenic tobacco lines increase tolerance to oxidative stress due to enhanced ROS-scavenging ability. |
PDF Full Text Request