| Harpin proteins can stimulate hypersensitive response (HR) in non-host plants and the HR is accompanied with programmed cell death (PCD) happen. However, how plants regulate the mechanism of hypersensitive response and PCD is unclear. Indole-3-aceticacid (IAA) is the first natural auxin discovered and it participates and regulates many physiological activities throughout the whole life of plants such as defense responses against plant pathogen infection. IAA can regulate the expression of some genes on the molecular level and it may play a role in the plant defense. Early research reveals that IAA can reverse harpin proteins infiltrated HR in plants, but, whether IAA can reverse harpin proteins induced PCD is unclear. In this article, we choosed the tobacco plants as object and explored harpinxoo and harpinXm proteins induced PCD, the influence of IAA acted on harpin proteins induced PCD in tobacco plants and the analysis of IAA act on harpin proteins induced TMV resistance in tobacco plants.Our laboratory early research proves that10mol/L IAA can reverse harpin proteins infiltrated HR in tobacco leaves and500?M TIB A (2,3,5-trllodobenzolc acid) can inhibit the effect of IAA. In this article, we also choose10-4mol/L IAA and500?M TIBA as materialsThe phenotype of harpin proteins on tobacco leaves showed that IAA can reverse the PCD stimulated by harpinxoo and harpinXm protein respectively on tobacco leaves and the effect can be inhibited by the applying of500?M TIBA at the same time. The observation of DAPI-strained DNA on tobacco leaves by fluorescence microscope showed that chromatin condensed and DNA fragments come into being following the treatment of harpinxoo and harpinxm protein respectively. The visible of DNA fragments was delayed following the treatment of harpin protein and IAA on tobacco leaves, and, the visible of DNA fragments on tobacco leaves treated with IAA, TIBA and harpin protein was earlier than the treatment of IAA and harpin protein, but later than the treatment of harpin protein. In order to further determine tha the result of DAPI-stained DNA is caused by the change of chromatin DNA, the author analyzed DNA isolated from tobacco leaves in agarose gel electrophoresis which so-called DNA Laddering. The analysis of DNA Laddering showed that a large degree of DNA degradation on tobacco leaves following the treatment of harpinxoo and harpinxm protein respectively. The extent of DNA degradation on tobacco leaves following the treatment of IAA and harpin protein relieved, and, the extent of DNA degradation on tobacco leaves following the treatment of IAA, TIBA and harpin protein recovered. The detection of oxidative burst was amid at insuring whether PCD process companys oxygen burst phenomenon and the result was in accordance with plant assays. It showed that IAA could also reduce the accumulation of ROS (reactive oxygen species, ROS) induced by harpin proteins. The analysis of semi-quantitative PCR of anti-apoptosis genes NtDADl, BI-1and PCD-related genes tpal, aoxl showed that the expressions of NtDADl, BI-1was down-regulated and the expressions of tpal, aoxl was up-regulated following the treatment of harpinXoo and harpinxm respectively. The expressions of NtDAD1, BI-1was up-regulated and the expressions of tpal, aoxl down-regulated following the treatment of the interaction of IAA and harpin protein. The expressions of NtDADl, BI-1following the treatment of the interaction of IAA, TIB A and harpin protein was higher than the treatment of harpin protein but lower than the treatment of IAA and harpin protein. The expressions of tpal, aoxl following the treatment of the interaction of IAA, TIB A and harpin protein was lower than the treatment of harpin protein but higher than the treatment of IAA and harpin protein. Result of RT-PCR revealed harpin protein could regulate the expression of anti-apoptosis genes NtDADl, BI-1and PCD-related genes tpal, aoxl and IAA had different effects on the expressions of these genes. The analysis of TMV resistance showed that harpinXoo and harpinXm proteins have resistance to TMV on tobacco. The vision of TMV spots on tobacco leaves induced by harpinXoo and harpinxm proteins respectively was earlier than tobacco leave induced by harpin proteins and the number of TMV spots was more than tobacco leave induced by harpin proteins. The vision of TMV spots on tobacco leaves induced by IAA? TIB A and harpin proteins was earlier than tobacco leaves induced by IAA and harpin proteins, but later than tobacco leaves induced by harpin proteins, and, the number of TMV spots was between them. TMV resistance analysis showed that harpinXoo and harpinxm proteins have resistance to TMV on tobacco and IAA can weaken the effect of harpin proteins.According to the result of plant assays, DAPI staining observation, DNA Laddering detection, oxidative burst detection, PCD related gene detection and analysis of TMV resistance, it draw a conclusion that harpin proteins can induce PCD and IAA can delay harpin proteins induced PCD. Otherwise, harpin proteins have resistance to TMV on tobacco and IAA can weaken the effect of harpin proteins. |
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