| Induced resistance is an important mechanism of plant defense against disease and herbivorous insects. Because of its broad-spectrum and durability against pests, induced plant resistance was paid much attention by scientists. Exploiting synthetic chemical elicitors that trigger plant defense and new molecular markers that can be used to screen elictiors are of importance in elucidating mechanism of plant defense responses and in pest management. The work has been extensively carried out in the interaction between plants and pathogens. However, little to nothing was done in this regard in the field of plant induced defense against herbivores. In this study, we chosed a Linalool synthase gene (OsLIS), whose production linalool is a chemical that is attractive to the parasitoid, as molecular marker and established a high-throughput screening system, LISp::GUS homozygous rice plants with expression of the OsLIS promoter plus a reporter gene GUS, for synthetic chemical elicitors. Using this system, we identified one chemical,2,4-Dicholorophenoxyacetic acid (2,4-D) that can induce rice plant defense and thus we studied its elicitation mechanisms. We also cloned an herbivore specifically induced xylanase inhibitor gene OsHI-XIP. Overexpression of this gene increased rice resistance to the rice striped stem borer (SSB) Chilo suppressalis. This gene may be used as a new molecular marker for screening plant defense elicitors. The results are as follows:A 1.5kb promoter sequence upstream of OsLIS was amplified from rice genome based on the sequence of this gene and named it LISp. An expression vector LISp::GUS was conducted by fused LISp to GUS gene and transformed into rice by Agrobacterium-based transformation. Using transgenic lines, we established a specific high-throughput system for screening defense elicitors that trigger plant defense responses. One chemical,2,4-D, efficiently enhanced GUS activities in the transgenic lines. Chemical and transcriptional analysis show that exogenous application of 2,4-D up-regulated transcript levels of defense-related genes, such as three MAPK genes (OsMPK3,OsMPK6 and OsMEK3), OsWRKY53, OsHI-LOX, OsACS and OsNPR1, and enhanced levels of Jasmonic acid (JA) and ethylene (ET), and defense chemicals, Trypsin protease inhibitors (TrypPIs) and herbivore-induced volatiles but decreased Salicylic acid (SA) levels in herbivore-infested and non-infested plants. Moreover, the induction of 2,4-D on levels of OsLIS transcripts and TrypPIs decreased in mutants with impaired JA (as-lox and as-pld) and ethylene (as-acs) biosynthesis. Bioassay showed that exogenous application of 2,4-D increased rice plant direct resistance to the rice striped stem borer (SSB) Chilo suppressalis and indirect resistance to the rice brown planthopper (BPH) Niaparvata lugens, but decreased plant direct resistance to BPH. A field experiment found that there were higher population densities of BPH adults and higher parasitisms of BPH eggs by the parasitoid Anagrus nilaparvatae on rice plants sprayed with 2,4-D, which could be used to control BPH as one of "push-pull" means. These findings suggest that 2,4-D modulates rice defense responses by regulating the biosynthesis of JA, ET and SA.A 1315 bp cDNA of an herbivore specifically induced xylanase inhibitor gene OsHI-XIP, containing an open reading frame of 891bp which encodes a protein of 297 amino acids with a calculated molecular weight of 37.4KDa, was cloned. OsHI-XIP localized in endoplasmic reticulum (ER), and its expression levels in roots were higher than those in other organs. SSB feeding, BPH feeding, wounding and JA treatment up-regaulated expression levels of OsHI-XIP. Overexpression of OsHI-XIP enhanced rice resistance to SSB, but did not influence JA and TrypPI levels. These results suggested that OsHI-XIP was involved in rice plant defense against chewing herbivores and can be used as a new molecular marker for screening chemical elicitors.
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