Study On The Mechanisms Of The Production Of Herbivore-induced Rice Trypsin Proteinase Inhibitors

Plant proteinase inhibitors (PIs) are a group of important defense compounds,which can be induced by herbivore infestation who activate multiple signalingpathways in plants. Previous studies had shown that PIs of rice can be induced by leaffolder infestation and herbivores with different feeding habits, such as Chilo.Suppressalia (Walker), Cnaphalocrocis medinalis (Guen(?)e) and Nilaparvata lugens(St(?)l) can activite a different set of signal molecules in rice plants. However, little isknown about the difference in the induction of the rice PIs by herbivores withdifferent feeding habits, and the roles of their elicited signal molecules in this process.Thus, using four rice pests with different feeding habits, including twophloem-feeding herbivores (N. lugens (St(?)l) & Sogatella furcifera (Horvath)), astembore (C. Suppressalia (Walker)) and a leaf folder (C medinalis (Guen(?)e)) as aresearch system, based on the previous results, the timing profile of trypsin proteinaseinhibitor (trypPI) levels in rice plants infested by the four pests were determined.Moreover, we also analyzed the levels of trypPIs and three defense-related signalmolecules (JA, SA, ET) in different organs of rice plants infested by leaf folder. Theroles of SA and ET signaling pathways in the production of trypPI induced by leaffolder infestation were tested by using exogenous application of signal molecules, aswell as reverse genetic technology. The main results are as follows:1) TrypPI levels in healthy rice stems were higher than leaves and were significantlyaffected by the photoperiod: TrypPI levels in stems significantly increased in darkperiod. After infestation by leaf folder, trypPI levels in leaves at tillering, bootingand flowering stages but not yellow ripening stage were significantly increasedcompared to the control plants, whereas trypPI levels in stems did not at all of thefour developmental stages. By contraries, at booting, flowering and yellowripening stages, trypPI levels were significantly lower in stems of infested plantsthan uninfested plants.2) Changes in trypPI levels of rice plants at tillering stage infested by the four herbivores were compared. The results showed that leaf folder infestation onlyenhanced trypPI levels in rice leaves, whereas the infestation of other threeherbivorous insects could significantly increase trypPI levels in both leaves andstems. This suggests that the production of trypPIs in plants infested by herbivoreinsects was systemic to some extent, and the transduction of the wound-relatedsignals may transmit from lower to upper. Furthermore, chewing herbivores like C.Suppressalia (Walker) can induce higher trypPI levels than sucking herbivores,such as N. lugens (St(?)l) and S. furcifera (Horvath).3) Levels of trypPIs and three defense-related signals (JA, SA, ET) in different riceorgans were determined after leaf folder infestation. Results showed that trypPIlevels were obviously increased not only in the damaged leaves but also in theyounger and older leaves after infestation by leaf folder. However, the increases intrypPI levels in the older leaves were later than the younger leaves. Moreover,trypPI levels in stems and roots of the leaf folder-infested plants did not changecompared to the control plants, confirming that the wound signals mainly conductfrom lower to upper. Leaf folder infestation could activate SA and ET signalingpathways but not JA pathway. Exogenous application of SA or ET on rice plantselicited the increases in TrypPI levels, suggesting a possible involvement of SAand ET signaling pathways in the production of the herbivore-induced riceTrypPIs.4) According to the published sequence, full sequence of gene ACS was PCRamplified. By using the ACS gene fragment, antisense expression vectorpCambia1301-antiACS was constructed. By using Agrogacterium-mediatedtransformation system, 15 T₁ lines with antisense inhibition of the gene ACS(as-ACS) were obtained. Chemical analysis showed that the amount of ethylenereleased from as-ACS lines were significantly lower than that from wide typeplants (Xiushui 11) when rice plants were infested by leaf folder. However, trypPIlevels didn't show obvious differences between as-ACS lines and wide type plants.These findings demonstrate that the gene ACS plays an important role in the production of the herbivore-elicited rice ethylene, whereas ethylene signalingpathway is not implicated in the biosynthesis of the herbivore-induced trypPIs.5) According to the published sequence, full sequence of the gene COI1 was PCRamplified. By using the COI1 gene fragment, antisense expression vectorpCambia1301-antiCOI1 was constructed. By using Agrobacterium-mediatedtransformation system, 8 homozygous lines with antisense inhibition of the geneCOI1 were obtained, which lays the foundation for elucidating the role of JAsignaling pathway in the production of the herbivore-elicited trypPIs.