The Molecular Responses Of Rice To Planthoppers Feeding And Dehydration Stress

Plant responses to herbivores are complex. 108 cDNA clones representing genes relating to plant responses to chewing insect-feeding, pathogen infection, wounding and other stresses were collected. Northern blot and cDNA array analysis were employed to investigate gene expression regulated by piercing-sucking insect, brown planthopper (BPH), Nilaparvata lugens (Homoptera: Dephacidae) on both the resistant and susceptible rice genotypes (B5 and Minghui63). After BPH feeding in rice for 72 h, the expression of most tested genes was affected. A two-fold or even higher difference in signal intensity between the treatment and the control was treated as significant in terms of transcript concentrations, based on this criterion, 14 genes in resistant rice variety B5 and 44 genes in susceptible Minghui63 were significantly up- or down-regulated. Most of the well-regulated genes were grouped in the categories of signaling pathways, oxidative stress/apoptosis, wound-response, drought-inducible and pathogen-related proteins. Those related to the flavonoid pathway, aromatic metabolidsm and the octadecanoid pathway were mostly kept unchanged or down-regulated. Our results indicate that BPH feeding induces plant responses which would take part in a jasmonic acid independent pathway and crosstalk with those related to abiotic stress, pathogen invasion and phytohormone signaling pathways.Brown planthopper (Nilaparvata lugens Stal, BPH) and whitebacked planthopper (Sogatella furcifera Horvath, WBPH) are the phloem-sucking herbivores of rice. We employed cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique to characterize the molecular responses of Oryza officinalis Wall ex Watt (at the late vegetative stage of rice growth) to BPH and WBPH feeding. After investigating about 7% of the insects-responsive transcriptome, we isolated 258 differential transcript-derived fragments (TDFs) and sequenced 112, of which 44 presented similarities to known or putative functional proteins. Among these 44 TDFs, 35 TDFs were altered by WBPH-feeding while 24 were by BPH-feeding. There were more down-regulated TDFs than the up-regulated ones found after fed by BPH and WBPH.Only 2 TDFs were co-induced but 12 were co-suppressed by two planthoppers feeding. The insect feeding would enhance the oxidative stress in O. qfficinalis. An overlap lies between the responses of O. qfficinalis to planthopper feeding and plants to pathogen invasion. Both planthoppers' feeding would activate the phosphorylation and dephosphorylation protein modification system. And WBPH-feeding would also activate the ubiqitination system in O. officinalis. The results revealed that O. qfficinalis responded differentially to these two phloem herbivores feeding.We also employed cDNA-AFLP technique to elucidate the characterization of molecular responses of O. sativa Minghui63 (at the late vegetative stage of rice growth) to BPH and WBPH feeding. Investigating about 7% of the insects-responsive transcriptome, we isolated 295 differential TDFs and sequenced 139, of which 50 presented similarities to known or putative functional proteins. Both phloem feeders could modulate the gene expressions of oxidative stress. However, more up-regulated TDFs were revealed in BPH-damaged Minghui63 while more down-regulated TDFs were found in WBPH-feeding rice. 43 TDFs were altered in rice by WBPH-feeding but 25 were by BPH-feeding. Only five TDFs were co-induced by both hoppers' feeding. BPH-feeding nearly up-regulated all the kinases detected in Minghui63, while WBPH-feeding down-regulated most of these kinases. When Minghui63 responded to BPH-feeding, it would take hydrogen peroxide as the second messenger to activate the downstream signaling pathway. But when Minghui63 responded to WBPH-feeding, it would take both calcium ion and hydrogen peroxide as the second messengers. The results revealed that Minghui63 responded differentially to these two phloem herbivores feeding.By cDNA-AFLP technique, we also analyze the molecular responses of O. officinalis and O. sativa Minghui63 to salinity stress and drought stress. We isolated and sequenced 148 TDFs, of which 89 TDFs are from O. officinalis, 59 are from Minghui63. 64 TDFs presented similarities to known or putative functional proteins. Among these 64 TDFs, 16 TDFs were up-regulated while 21 were down-regulated under salinity stress, and 23 were up-regulated while 25 were down-regulated under drought stress. 15 out of 64 TDFs were co-suppressed and only two (14M3 and 16H3, namely Cytochrome P450 and ABC transporter family protein) were co-induced by both stresses. Both stresses could induce the oxidative stress, which confirmed the results reported by others. Both stresses could suppress the responses of rice to extreme temperature stresses such as heat or chilling. The protein phosphorylation and dephosphorylation system would play an important role in the response of rice to salinity and drought stresses. One of the significant differences between the responses of O. officinalis and Minghui63 to stresses is that ABC transporter protein family is only activated in O. officinalis. However, its function in rice needs further work.We used the differential TDF 16M2 (Showing homology to rice MAR-binding protein) isolated by cDNA-AFLP procedure as probe to screen the Minghui63 full-length cDNA library. After screening for three times, we obtained two cDNA clones, which were designated as OsMARBPl and OsMARBP2. OsMARBPl is 2393bp in length, with an open reading frame (ORF) of 592aa. 0sMARBP2 is 1674bp in length, with an ORF of 360aa. After searching the homology in protein databases with BLAST tool, they show high similarities with rice MAR-binding protein. Southern blot result indicates that OsMARBP is a single-copy gene. By inquiring at the website http://www.gramene.org. we find that OsMARBPl and OsMARBP2 are different transcripts of the same gene. OsMARBPl comes from two exons, splicing out an intron of about 300bp. 0sMARBP2 comes from a single exon, and is a part of OsMARBPl sequence. Northern blot result indicate that this gene is induced by salinity stress (1% NaCl solution), 100 umol/L ABA treatment, needle piercing damage and feeding by Borbo cinnara Wallace. But it is not activated by BPH-feeding, WBPH-feeding, drought stress, 100 umol/L SA treatment, 0.4% (W/V) ethephon treatment or 100 umol/L MJ treatment. Meanwhile we analyze its gene expression pattern following the time course of salinity stress, and find that its strongest signal appears at the 3h time course, and the rest all present a little stronger expression than the control. All this work proves that OsMARBP from Minghui63 is a salinity-stress induced gene.