Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.     

Upregulation of jasmonate-inducible defense proteins and differential colonization of roots of Oryza sativa cultivars with the endophyte Azoarcus sp

The endophyte Azoarcus sp. strain BH72 expresses nitrogenase (nif) genes inside rice roots. We applied a proteomic approach to dissect responses of rice roots toward bacterial colonization and jasmonic acid (JA) treatment. Two sister lineages of Oryza sativa were analyzed with cv. IR42 showing a less compatible interaction with the Azoarcus sp. resulting in slight root browning whereas cv. IR36 was successfully colonized as determined by nifHi::gusA activity. External addition of JA inhibited colonization of roots and caused browning in contrast to the addition of ethylene, applied as ethephon (up to 5 mM). Only two of the proteins induced in cv. IR36 by JA were also induced by the endophyte (SalT, two isoforms). In contrast, seven JA-induced proteins were also induced by bacteria in cv. IR42, indicating that IR42 showed a stronger defense response. Mass spectrometry analysis identified these proteins as pathogenesis-related (PR) proteins (Prb1, RSOsPR10) or proteins sharing domains with receptorlike kinases induced by pathogens. Proteins strongly induced in roots in both varieties by JA were identified as Bowman-Birk trypsin inhibittors, germinlike protein, putative endo-1,3-beta-D-glucosidase, glutathion-S-transferase, and 1-propane-1-carboxylate oxidase synthase, peroxidase precursor, PR10-a, and a RAN protein previously not found to be JA-induced. Data suggest that plant defense responses involving JA may contribute to restricting endophytic colonization in grasses. Remarkably, in a compatible interaction with endophytes, JA-inducible stress or defense responses are apparently not important.     

Lesion mimic mutants of rice with alterations in early signaling events of defense

We screened 93 lesion mimic mutants of rice for resistance to the blast fungus, Magnaporthe grisea, and found eight mutants that exhibited significant resistance to the fungus. We called these mutants cdr (cell death and resistance) and further analyzed three of them. Two mutations, cdr1 and cdr2, were recessive and one, Cdr3, was dominant. Many small brownish lesions developed over the entire leaf of the mutants 20-50 days after sowing. TUNEL staining revealed that DNA fragmentation occurred in leaf blade cells of the homozygous Cdr3 mutants. Autofluorescence and callose deposition were visible in leaf cells of these three mutants. Activation of two defense-related genes, PBZ1 and PR1, was observed in the leaves of the mutants; high expression of PBZ1 was correlated with the lesion formation in the three mutants, whereas PR1 was constitutively expressed in the cdr2 and Cdr3 mutants irrespective of the lesion formation. Levels of momilactone A, a major phytoalexin of rice, in these mutants were increased approximately 100-400-fold relative to the wild-type levels. Suspension-cultured cells of the cdr1 and cdr2 but not Cdr3 produced higher levels of H2O2 than the wild type when treated with calyculin A, an inhibitor of protein phosphatase 1. These results suggest that biochemical lesions of cdr1 and cdr2 lie in the early signaling steps leading to activation of the NADPH oxidase and that type-1 protein phosphatase is operative in protein dephosphorylation involved in NADPH oxidase activation.     

Jasmonate-Inducible Genes Are Activated in Rice by Pathogen Attack without a Concomitant Increase in Endogenous Jasmonic Acid Levels

The possible role of the octadecanoid signaling pathway with jasmonic acid (JA) as the central component in defense-gene regulation of pathogen-attacked rice was studied. Rice (Oryza sativa L.) seedlings were treated with JA or inoculated with the rice blast fungus Magnaporthe grisea (Hebert) Barr., and gene-expression patterns were compared between the two treatments. JA application induced the accumulation of a number of pathogenesis-related (PR) gene products at the mRNA and protein levels, but pathogen attack did not enhance the levels of (-)-JA during the time required for PR gene expression. Pathogen-induced accumulation of PR1-like proteins was reduced in plants treated with tetcyclacis, a novel inhibitor of jasmonate biosynthesis. There was an additive and negative interaction between JA and an elicitor from M. grisea with respect to induction of PR1-like proteins and of an abundant JA-and wound-induced protein of 26 kD, respectively. Finally, activation of the octadecanoid signaling pathway and induction of a number of PR genes by exogenous application of JA did not confer local acquired resistance to rice. The data suggest that accumulation of nonconjugated (-)-JA is not necessary for induction of PR genes and that JA does not orchestrate localized defense responses in pathogen-attacked rice. Instead, JA appears to be embedded in a signaling network with another pathogen-induced pathway(s) and may be required at a certain minimal level for induction of some PR genes.     

Probenazole-induced accumulation of salicylic acid confers resistance to Magnaporthe grisea in adult rice plants

Probenazole (PBZ) is the active ingredient of Oryzemate, an agrochemical which is used for the protection of rice plants from Magnaporthe grisea (blast fungus). While PBZ was reported to function upstream of salicylic acid (SA) in Arabidopsis, little is known about the mechanism of PBZ-induced resistance in rice. The role of SA in blast fungus resistance is also unclear. The recommended application period for Oryzemate is just before the Japanese rainy season, at which time rice plants in the field have reached the 8-leaf stage with adult traits. Thus, the involvement of SA in PBZ-induced resistance was studied in compatible and incompatible blast fungus-rice interactions at two developmentally different leaf morphology stages. Pre-treatment of inoculated fourth leaves of young wild-type rice plants at the 4-leaf stage with PBZ did not influence the development of whitish expanding lesions (ELs) in the susceptible interaction without the accumulation of SA and pathogenesis-related (PR) proteins. However, PBZ pre-treatment increased accumulation of SA and PR proteins in the eighth leaves of adult plants at the 8-leaf stage, resulting in the formation of hypersensitive reaction (HR) lesions (HRLs). Exogenous SA induced resistance in adult but not young plants. SA concentrations in blast fungus-inoculated young leaves were essentially the same in compatible and incompatible interactions, suggesting that PBZ-induced resistance in rice is age-dependently regulated via SA accumulation.     

Paclobutrazol mitigates salt stress in <Emphasis Type="Italic">indica</Emphasis> rice seedlings by enhancing glutathione metabolism and glyoxalase system

Stress-induced methylglyoxal (MG) functions as a toxic molecule, inhibiting plant physiological processes such as photosynthesis and antioxidant defense systems. In the present study, an attempt was made to investigate the MG detoxification through glutathione metabolism in indica rice [Oryza sativa L. ssp. indica cv. Pathumthani 1] under salt stress by exogenous foliar application of paclobutrazol (PBZ). Fourteen-day-old rice seedlings were pretreated with 15 mg L^?1 PBZ foliar spray. After 7 days, rice seedlings were subsequently exposed to 0 (control) or 150 mM NaCl (salt stress) for 12 days. Prolonged salt stress enhanced the production of MG molecules and the oxidation of proteins, leading to decreased activity of glyoxalase enzymes, glyoxalase I (Gly I) and glyoxalase II (Gly II). Consequently, the decreased glyoxalase activities were also associated with a decline in reduced glutathione (GSH) content and glutathione reductase (GR) activity. PBZ pretreatment of rice seedlings under salt stress significantly lowered MG production and protein oxidation, and increased the activities of both Gly I and Gly II. PBZ also increased GSH content and GR activity along with the up-regulation of glyoxalase enzymes, under salt stress. In summary, salinity induced a high level of MG and the associated oxidative damage, while PBZ application reduced the MG toxicity by up-regulating glyoxalase and glutathione defense system in rice seedlings.     

Comparison of two proteomics techniques used to identify proteins regulated by gibberellin in rice

Proteomics has become an essential methodology for large-scale analysis of proteins in various fields of plant biology. We compared two proteomics techniques, two-dimensional liquid chromatography (2D-LC) and fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), for their ability to identify proteins regulated by gibberellin (GA) in rice. Two-week-old rice seedlings were treated with or without 5 microM GA3 for 48 h and proteins extracted from the basal region of the leaf sheath. After separation of the proteins by the two techniques, the amino acid sequences of GA3-responsive proteins were analyzed using a protein sequencer and mass spectrometry. 2D-LC and 2D-DIGE were able to resolve 1248 protein fractions and 1500 proteins, respectively. Out of these, 2D-LC identified 9 proteins that were up-regulated and 9 that were down-regulated by GA treatment; 2D-DIGE identified 4 up-regulated and 4 down-regulated proteins. The two techniques detected overlapping sets of proteins. For example, cytosolic glyceraldehyde-3-phosphate dehydrogenase and photosystem II oxygen-evolving complex protein were identified as GA3-regulated proteins by both methods. In addition, these two methods uncovered GA3-regulated unknown proteins which had not been reported previously, and novel proteins which are not detected in 2D-PAGE followed by Coomassie brilliant blue staining. These results suggest that these two methods are among some of the very useful tools for detecting proteins that may function in various physiological and developmental processes in plants.     

Novel insight into kinetin-inducible stress responses in rice seedlings

Kinetin (KN) action in rice self-defense mechanism was studied using our established 2-week-old rice (Oryza sativa L. japonica-type cv. Nipponbare) seedling in vitro model system. It was strikingly observed that KN caused formation of brownish necrotic microlesions in leaves, suggesting it triggers a stress response in rice. Subsequent northern analyses revealed differential regulation (both up-and down-regulations) of 10 prominent defense/stress-related marker genes, including the critical pathogenesis-related (PR) protein genes of class 1, 5 and 10. A systemic effect of KN in leaves was shown using OsPR1b (basic) and OsPOX (peroxidase) genes as representatives. KN also exclusively triggered potent accumulation of PR proteins (OsPR5 and OsPR10), and a phytoalexin, sakuranetin. Interestingly, as KN failed to induce jasmonic acid (JA) inducible genes (OsPR1a and JIOsPR10), and had almost no effect on accumulated endogenous JA level due to wounding by cut, KN might act through a yet unknown (and JA-independent) pathway. These results provide a new aspect on the role of KN as a potent activator of the stress responses in the rice plant.     

表达广谱抗真菌蛋白转基因水稻秸秆的化学成分与纤维素结构

为了给表达广谱抗真菌蛋白转基因水稻的安全性评估提供基础数据,本文以表达广谱抗真菌蛋白转基因水稻转品1和转品8及非转基因七丝软粘(对照)为试材,对其秸秆化学成分进行分析,同时,采用傅立叶变换红外光谱(FTIR)和X-射线衍射(XRD)方法观察秸秆中不同部位纤维素结构的变化情况。研究结果显示:(1)转基因水稻转品1和转品8与对照在秸秆相同部位的化学成分含量不存在显著差异;(2)转基因水稻转品1和转品8及对照在秸秆相同部位纤维素的红外吸收峰形状基本一致,且品种间的吸收峰强度和结晶指数(O'KI和N·O'KI)也无显著差异;(3)转品1和转品8秸秆的X-射线衍射图与对照相似,且结晶度(CrI)与对照无显著差异。综上所述,广谱抗真菌基因的导入不会对水稻的化学成分、纤维素结构及晶体结构产生明显影响。     

Gene-Expression Patterns and Levels of Jasmonic Acid in Rice Treated with the Resistance Inducer 2,6-Dichloroisonicotinic Acid

Acquired disease resistance can be induced in rice (Oryza sativa) by a number of synthetic or natural compounds, but the molecular mechanisms behind the phenomenon are poorly understood. One of the synthetic inducers of resistance, 2,6-dichloroisonicotinic acid (INA), efficiently protected rice leaves from infection by the rice blast fungus Magnaporthe grisea (Hebert) Barr. A comparison of gene-expression patterns in plants treated with INA versus plants inoculated with the compatible pathogen M. grisea or the incompatible pathogen Pseudomonas syringae pv syringae revealed only a marginal overlap: 6 gene products, including pathogenesis-related proteins (PR1-PR9), accumulated in both INA-treated and pathogen-attacked leaves, whereas 26 other gene products accumulated only in INA-treated or only in pathogen-attacked leaves. Lipoxygenase enzyme activity and levels of nonconjugated jasmonic acid (JA) were enhanced in leaves of plants treated with a high dose of INA (100 ppm). Exogenously applied JA enhanced the gene induction and plant protection caused by lower doses of INA (0.1 to 10 ppm) that by themselves did not give rise to enhanced levels of endogenous (-)-JA. These data suggest that INA, aside from activating a pathogen-induced signaling pathway, also induces events that are not related to pathogenesis. JA acts as an enhancer of both types of INA-induced reactions in rice.     

Proteomic analysis of pathogen-responsive proteins from rice leaves induced by rice blast fungus, Magnaporthe grisea

Proteomic approaches using two-dimensional gel electrophoresis (2-DE) were adopted to identify proteins from rice leaf that are differentially expressed in response to the rice blast fungus, Magnaporthe grisea. Microscopic observation of inoculated leaf with M. grisea revealed that callose deposition and hypersensitive response was clearly visible in incompatible interactions but excessive invading hypha with branches were evident in compatible interactions. Proteins were extracted from leaves 24, 48, and 72 hours after rice blast fungus inoculation. Eight proteins resolved on the 2-DE gels were induced or increased in the inoculated leaf. Matrix-assisted laser desorption/ionization-time of flight analysis of these differentially displayed proteins showed them to be two receptor-like protein kinases (RLK), two beta-1.3-glucanases (Glu1, Glu2), thaumatin-like protein (TLP), peroxidase (POX 22.3), probenazole-inducible protein (PBZ1), and rice pathogenesis-related 10 (OsPR-10). Of these proteins, RLK, TLP, PBZ, and OsPR-10 proteins were induced more in the incompatible interactions than in compatible ones. A phytohormone, jasmonic acid also induced all eight proteins in leaves. To confirm whether the expression profile is equal to the 2-DE data, seven cDNA clones were used as probes in Northern hybridization experiments using total RNA from leaf tissues inoculated with incompatible and compatible rice blast fungal races. The genes encoding POX22.3, Glu1, Glu2, TLP, OsRLK, PBZ1, and OsPR-10 were activated in inoculated leaves, with TLP, OsRLK, PBZ1, and OsPR-10 being expressed earlier and more in incompatible than in compatible interactions. These results suggest that early and high induction of these genes may provide host plants with leading edges to defend themselves. The localization of two rice PR-10 proteins, PBZ1 and OsPR-10, was further examined by immunohistochemical analysis. PBZ1 accumulated highly in mesophyll cells under the attachment site of the appressorium. In contrast, OsPR-10 expression was mainly localized to vascular tissue.     

Paclobutrazol pre-treatment enhanced flooding tolerance of sweet potato

The objective of this experiment was to study changes of antioxidants and antioxidative enzymes in the flooding-stressed sweet potato leaf, as affected by paclobutrazol (PBZ) treatment at 24 h prior to flooding. Sweet potato 'Taoyuan 2' were treated with 0 and 0.5 mg/plant of PBZ, afterwards subjected to non-flooding and flooding-stress conditions for 0, 1, 3, and 5 d, followed by a 2 d drainage period. The study was conducted as a factorial experiment in completely randomized blocks with three replications maintained within a screen house. Plants with various antioxidative systems responded differently to flooding stress according to the duration of the flooding period and subsequent drainage period. The increased levels of antioxidants and antioxidative enzymes observed on different days of flooding afforded the sweet potato leaf with improved flooding tolerance. Glutathione reductase activity in the leaf was significantly enhanced over 5 d continuous flooding followed by a drainage period, in comparison with non-flooding conditions. Under non-flooding conditions, antioxidative system of leaf was regulated and elevated by PBZ pre-treatment. PBZ treatment may enable sweet potato 'Taoyuan 2' to maintain the balance between the formation and the detoxification of activated oxygen species. Our results also show that under flooding-stress conditions, the level of 'Taoyuan 2' antioxidative system is linked to PBZ treatment. Pre-treating with PBZ may increase levels of various components of antioxidative systems after exposure to different durations of flooding and drainage, thus inducing flooding tolerance. PBZ exhibited the important function of enhancing the restoration of leaf oxidative damage under flooding stress after the pre-application of 0.5 mg/plant. These findings may have greater significance for farming in frequently flooded areas.     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.