Molecular Reponse And CDNA Cloning In Brown Planthopper Exposure To Host Rice Resistance

The brown planthopper (BPH) (Nilaparvata lugens Stal, Homoptera: Delphacidae) is an important pest of rice. The BPH feeds mainly on the rice stems and sucks assimilates from the phloem. BPH feeding interferes with the translocation of photosynthates, hence, affects the rice plant growth and development. Feeding by a large number of BPH may result in drying of the leaves and wilting of the tiller, resulting in a loss of harvest called hopperburn. It is also a vector for virus diseases of rice. In terms of rice-BPH interaction, a broad range of changes in physiology, biochemistry and molecular genetics have been studied in rice infested by BPH. But the overall molecular response of BPH exposure to different rice varieties is not well understood, and looking at the "insect" side of an insect-plant interaction has been too frequently missing from the literature. This work will focus on understanding molecular response of BPH to rice resistance.Firstly, cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique was carried out to identify the differentially expressed genes in the fourth BPH instar nymphs feeding on three different rice varieties: a highly resistant rice line B5, a rice variety MH63 harboring a minor resistance gene, and a susceptible rice Taichung Native 1 (TN1). Of 2800 transcripts analyzed, 76 cDNA bands were up-regulated and eight bands down-regulated qualitatively, in BPH on B5 and MH63 plants, compared with that on TN1. The 84 transcript-derived fragments (TDFs) were isolated, cloned and sequenced. The identified genes were involved in signaling, stress defense, gene expression regulation and metabolism. Four of the TDFs homologous to genes encoding a putative B subunit of phosphatase PP2A (PP2Ab), nemo kinase (nemo), cytochrome P450 monooxygenase (CYP6AX1) and prolyl endopeptidase (PEP) were further characterized. Northern blot hybridization proved that expression pattern of nemo gene was slowly up-regulated in BPH feeding on B5 and MH63 plants, suggesting that some stimuli from B5 and MH63 plants might induce the gene, which might be involved in cell apoptosis in BPH. Theexpression of CYP6AX1 gene was activated rapidly in BPH by feeding on the two rice varieties, and maintained for a constant level during the time course, implying that CYP6AX1 gene is associated with detoxify allelochemicals in the sap of B5 and MH63. The elevated expression pattern of PEP gene indicating that more PEPs are produced to work in concert to convert larval peptides to free amino acids, to meet the need for synthesizing new proteins, in the case that BPH reduced ingesting of diet when meeting resistant rice. The gene coding for PP2Ab was up-regulated in BPH feeding on B5 solely suggests that some PP2A inhibitors probably exist in the highly resistant B5 plants.Secondly, a homology-based cloning strategy was employed to isolate cDNA fragments of detoxification and toxin-tolerance related genes encoding carboxylesterase (CAR), trypsin (TRY), cytochrome P450 monooxygenase (CYP6AY1), NADH-quinone oxidoreductase (NQO), acetylcholinesterase (ACE) and Glutathione S-transferase (GST) in BPH. The expression profiles of these genes were also monitored on the fourth BPH instar nymphs feeding on TNI, B5 and MH63 plants. Northern hybridization showed that P450 gene and CAR gene were up-regulated, TRY gene mRNA decreased in BPH feeding on both resistant rice varieties B5 and MH63, compared to BPH on TNI plants. GST gene had two transcripts, all of which having an up-regulated pattern in BPH feeding on B5, but in BPH fed MH63, this gene was enhanced and its expression reached a maximum level at 24h, then decreased slightly. The expression of NQO gene was strengthened in BPH on B5 plants while this gene remained a constant expression pattern in BPH on MH63. However, these five genes showed a constant expression level in BPH feeding on TNI. No difference in ACE gene expression between BPH on B5 and that on MH63 was detected by RT-PCR method. The data implies sophisticated gene profile alterations occurred in BPH response to the two different rice varieties, and BPH needed to change more in gene expression to deal with the more severe stress from the high resistant variety B5 than that from Minghui 63.Thirdly, full-length cDNAs of TRY, NQO, ACE, nemo, CYP6AX1 and CYP6AY1 genes were cloned by performing 5' and 3' RACE. The TRY cDNA is 1902 bp having an open reading frame (ORF) coding for a putative protein containing a functional peptide with 303 aa and a signal peptide with 18 aa at the N termius. The 5' untranslational region (UTR) of this cDNA is 283 bp and the 3' UTR is 491 bp. The NQO cDNA is 1930 bp having an ORF coding for a putative protein with 462 amino acid residues. The 5' UTR of this cDNA is 9 bp and the 3' UTR is 532 bp. The ACE cDNA is 2467 bp having an ORF coding for a putative protein containing a functional peptide with 576 aa and atransmembrane peptide with 30 aa at the N terminus. The 5' UTR and 3' UTR are 403 bp and 123 bp, separately. The nemo cDNA is 3455 bp having an ORF coding for a putative protein with 449 amino acid residues. The 5' UTR and 3' UTR are 770 bp and 1335 bp, respectively. The CYP6AX1 cDNA is 2395 bp with an ORF coding for a putative protein containing a functional peptide with 444 aa and a 35-aa membrane anchor at the N terminus. The 5' UTR and 3' UTR are 109 bp and 641 bp, separately. The CYP6AY1 cDNA is 2720 bp having an ORF coding for a putative protein containing a functional peptide with 431 aa and a 25-aa membrane anchor at the N terminus. The 5' UTR and 3' UTR are 85 bp and 1129 bp, respectively. The six full cDNAs are newly cloned ones from BPH.Finally, TUNEL methodology was used to detect the midgut apoptosis in BPH exposure to B5 and MH63 plants. No fluorescence signals representing apoptosis cells were observed in BPH on TNI. But in BPH feeding on B5 and MH63, there were obvious signals in the midgut detected under fluorescent microscope. Furthermore, more apoptosis cells were found in the midgut of BPH feeding on B5, which induced more cells undergoing apoptosis than that on MH63, suggesting more and severer stimuli were produced in B5 than in MH63. Meanwhile, DAPI staining was used to observe the midgut sections of BPH nymphs. Obviously athological changes in the midgut of BPH were observed following exposure to resistant rice. The cell nucleus swelled and chromatin became concentrated. Additionally, the midgut tissue was cankerously changed. But in the midgut of BPH feeding on TNI, the cell nucleus was round and full and the tissue was regularly arranged. These results are closely related to the gene expression regulation studied in the former work, especially the induction of nemo gene.The results obtained in this study are helpful in understanding the mechanism of interactions between N. lugens and its host, and provide clues for designing effective control programs as well.