Functional Analysis Of Herbivore Resistance-Related Genes OsICS And OsHPL3in Rice

Herbivore induced plant defense response, a complex physiological and biochemical processes, can produce when exposed to pathogen and phytophagous insects. Salicylic acid and green leaf volatiles, as signal molecules, play an important role in the process of herbivore induced plant defense responses in dicotyledonous plants such as Arabidopsis and tobacco. However, little to nothing is known in this aspect in rice, a model monocot species. So further research was made on whether isochorismate synthase was also involved in the synthesis of salicylic acid, and whether this pathway played an important role in rice defense responses. Also, little is known about functional analysis of OsHPL3, whose product competing the same substrate with jasmonic acid, against WBPH resistance. Therefore, we isolated two genes as the research object, one is isochorismate synthase gene OsICS, induced by brown planthopper feeding, and the other is hydroperoxide lyase gene OsHPL3, whose production green leaves volatile, combining with the methods of reverse genetics, molecular biology, chemical analysis and bioassay, in order to clarify OsICS function in rice herbivore induced resistance and OsHPL3against WBPH resistance defense responses. The main results are as follows:A2445bp open reading frame of an herbivore specifically induced isochorismate synthase gene OsICS, which encodes a protein of814amino acids with a calculated molecular weight of88.024KDa and pI5.92, was cloned. OsICS coding protein is located in the chloroplasts. Tissue specific expression analysis revealed OsICS transcript levels in rice leaves was higher than that of other parts. Induced expression analysis showed that the feeding of Nilaparvata lugens (brown planthopper, BPH), Sogatella furrifera (white back planthopper, WBPH), Chilo suppressalis (rice striped stem borer, SSB) and mechanical wounding treatment can induce OsICS gene expression, and exogenous jasmonic acid (JA) and salicylic acid (SA) can not induce the expression, but have an opposite effect. Through agrobacterium-mediated transgenic method, we obtained the OsICS mutant plants. The OsICS expression levels in transgenic lines were decreased by46.23%-50.86%than the wild type. Phenotypic results of different lines revealed OsICS transgenic mutants are significantly shorter than the wild type during germination and grown in the field. Besides, the level of SA in transgenic mutants are significantly lower than the wild type both grown in the field and green house induced by the feeding of brown planthopper and rice stem borer. The bioassay of different pests results show that, the OsICS transgenic lines could obviously increase the feeding choice of BPH and WBPH, and the treatment of exogenous SA can restore BPH and WBPH feeding choice. However, the OsICS transgenic lines can not affect herbivore induced rice JA, trypsin inhibitor (TrypPIs) and ethylene (ET). And the transgenic lines has no significant effect on the nymph developmental duration and population growth of BPH and WBPH.OsHPL3is a hydroperoxide lyase gene. Expression levels of OsHPL3could be significantly enhanced by mechanical wounding, SSB, BPH and WBPH infestation, whereas exogenous hormones JA and SA treatment had no significant effect. We use the same method to aquire the OsHPL3transgenic lines. The emission of GLV levels (Z)-3-hexenal,(Z)-3-Hexen-l-ol and (E)-2-hexenal were significantly lower in transgenic lines than in wild type rice plants after WBPH infestation and mechanical wounding treatment. At the same time, the transgenic lines can increase the resistance to WBPH. Bioassay results show that adult WBPH preferred to feed and oviposite on the transgenic lines. The nymph developmental duration in the transgenic lines were significantly longer, the survival rate was significantly decreased, and the number of eggs per day were significantly lower than in the wild type. While the treatment of exogenous (Z)-3-hexenal can restore the0sHPL3transgenic lines to the resistance of WBPH. The result of Chromatograms of volatiles obtained from various lines show that transgenic strains can increase the release of limonene after WBPH damage, while the decrease expression of OsHPL3in transgenic lines had no effect on other WBPH-induced plant volatiles. Compared with transgenic plants, Anagrus nilaparvatae, which is an egg parasitoid of WBPH, preferred the odors from wild type plants, releasing more GLV after WBPH infestation. However, exogenous different concentrations of GLV,(Z)-3-hexenal,(Z)-3-Hexen-l-ol and (E)-2-hexenal treatment can restore the Anagrus nilaparvatae selectively behavior between different lines.So GLV, the product of OsHPL3, play an important role in the rice resistance to WBPH.