Comparative Proteomics Study Of The Response Of Rice To Brown Planthopper

The brown planthopper (Nilaparvata lugens Stal, BPH) is a typical vascular feeder and considered one of the most serious pests of rice(Oryza sativa L.). BPH causes reductions in leaf area, photosynthetic rate, leaf and stem nitrogen concentrations, chlorophyll contents and organic dry weight; the infestation ratios, egg numbers, and survival ratios of eggs and nymphs are high on susceptible rice plants. In the field, feeding by large numbers of BPHs usually causes "hopperburn", i.e. drying of the leaves and wilting of the tillers, resulting dramatic reductions in the yield of susceptible rice varieties. The BPH also transmits "grassy stunt" and "ragged" diseases as a virus vector. Nymph development is generally disrupted, oviposition is severely inhibited and the survival rate of nymphs is significantly lower on resistant rice plants than on susceptible plants. Thus population growth is suppressed, and a little or no physiological stress and yield loss are caused to resistant rice variety. For rice crops, growing resistant varieties is the most effective and environment-friendly way to control the BPH.Proteomics has emerged as a powerful tool for gaining insight into physiological changes at the protein level, but few attempts have been made to apply this technique to study the response of rice to BPH attack. Relative quantification proteomic analysis, using highly sensitivity mass spectrometry techniques, is becoming increasingly popular, due to the high-throughputs, reproducibility and sensitivity it offers. To investigate the role of defense-responsive proteins in susceptible and resistant rice lines against BPH, we used an iTRAQ (isobaric tag for relative and absolute quantitation) proteomic analysis to screen for the pathological levels of proteins in rice. Proteins were extracted from leaf sheath 96 hours after BPH feeding. Several proteins involved in multiple pathways showed significant changes in expression in response to BPH feeding. The iTRAQ results identified them as three JA synthesis proteins (alpha-DOX, AOS, AOC), seven oxidative stress response proteins (CATA, APX2 and five POXs), three P-glucanases (Gns1, Gns4 and Gns5), three protein kinases (CRK5, CRK6 and atypical RLK), clathrin heavy chain protein, glycine cleavage system H protein, five photosynthesis proteins and four aquaporins. Of these proteins, AOC, CATA, three POXs, Gns1, Gns5, three protein kinases and clathrin heavy chain protein were induced more in compatible than in incompatible line. The corresponding genes of eight important proteins were further analyzed by quantitative RT-PCR. Proteomic and transcript responses that were related to wounding, oxidative and pathogen stress overlapped considerably between BPH-resistant (carrying the resistance gene BPH15) and susceptible rice lines. In contrast, proteins and genes related to callose metabolism remained unchanged and glycine cleavage system protein was up-regulated in the BPH resistant lines, indicating that they have an efficient and specific defense mechanism. We proposed that compatible and incompatible rice lines use different defense stratigies to defense BPH and the BPH resistance gene BPH15 is a decisive factor.The phloem is the route for the translocation and distribution of organic metabolites assimilated during photosynthesis of plant. It is also the target of sucking and piercing insects. In addition to small molecules like sugars and amino acids, phloem sap of higher land plants contains proteins. Many phloem sap proteins have potential roles in wound and defense responses and may influence plant-insect interactions. The mass tagging strategy iTRAQ was applied to compare the phloem sap proteins of leaf sheath of the BPH compatible rice 9311 and the BPH incompatible rice B5. We found that the phloem sap proteins were significantly affected by feeding of BPH. In total,238 phloem sap proteins are identified; 47 showed significant differences in quantity between 9311 and B5 belong to function groups in sugar metabolism, reactive oxygen/redox, protein modification and signal transduct. The amount of 54 proteins changed significantly in 9311 after 96 hrs feeding by BPH and 40 in B5. Eleven of them changed in an opposite direction in the compatible and incompatible varieties. Our results show that 9311 and B5 respond to BPH differently at the protein levels. We further carried out in situ hybridization analysis for five important proteins to verify the expression of their corresponding proteins in rice phloem at the mRNA level. Three of them showed positive signals in the rice sieve elements.To further screening phloem specific expressed genes, we applied laser capture microdissection to collect the rice phloem cells and parenchyma cells. Their RNA were extracted and though one round of in vitro RNA linear amplification we got sufficient pure phloem/parenchyma RNA for RT-PCR analysis. Four phloem specific expressed genes were identified:RGAP, Serpin, WDS and TCTP. The result showed that they were specific expressed in the phloem. The promoters of RGAP and Serpin were analyzed by constructing different length of 5'end deletion vectors, aiming at identifying domains and motifs that regulate genes of interest specifically expressed in rice phloem.