OsBIRH1, a DEAD-box RNA helicase with functions in modulating defence responses against pathogen infection and oxidative stress

DEAD-box proteins comprise a large protein family with members from all kingdoms and play important roles in all types of processes in RNA metabolism. In this study, a rice gene OsBIRH1, which encodes a DEAD-box RNA helicase protein, was cloned and characterized. The predicted OsBIRH1 protein contains a DEAD domain and all conserved motifs that are common characteristics of DEAD-box RNA helicases. Recombinant OsBIRH1 protein purified from Escherichia coli was shown to have both RNA-dependent ATPase and ATP-dependent RNA helicase activities in vitro. Expression of OsBIRH1 was activated in rice seedling leaves after treatment with defence-related signal chemicals, for example benzothiadiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid, and jasmonic acid, and was also up-regulated in an incompatible interaction between a resistant rice genotype and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants that overexpress the OsBIRH1 gene were generated. Disease resistance phenotype assays revealed that the OsBIRH1-overexpressing transgenic plants showed an enhanced disease resistance against Alternaria brassicicola and Pseudomonas syringae pv. tomato DC3000. Meanwhile, defence-related genes, for example PR-1, PR-2, PR-5, and PDF1.2, showed an up-regulated expression in the transgenic plants. Moreover, the OsBIRH1 transgenic Arabidopsis plants also showed increased tolerance to oxidative stress and elevated expression levels of oxidative defence genes, AtApx1, AtApx2, and AtFSD1. The results suggest that OsBIRH1 encodes a functional DEAD-box RNA helicase and plays important roles in defence responses against biotic and abiotic stresses.     

Functional analysis reveals pleiotropic effects of rice RING-H2 finger protein gene OsBIRF1 on regulation of growth and defense responses against abiotic and biotic stresses

RING finger proteins comprise a large family and play key roles in regulating growth/developmental processes, hormone signaling and responses to biotic and abiotic stresses in plants. A rice gene, OsBIRF1, encoding a putative RING-H2 finger protein, was cloned and identified. OsBIRF1 encodes a 396 amino acid protein belonging to the ATL family characterized by a conserved RING-H2 finger domain (C-X2-C-X15-C-X1-H-X2-H-X2-C-X10-C-X2-C), a transmembrane domain at the N-terminal, a basic amino acid rich region and a characteristic GLD region. Expression of OsBIRF1 was up-regulated in rice seedlings after treatment with benzothaidiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid and jasmonic acid, and was induced differentially in incompatible but not compatible interactions between rice and Magnaporthe grisea, the causal agent of blast disease. Transgenic tobacco plants that constitutively express OsBIRF1 exhibit enhanced disease resistance against tobacco mosaic virus and Pseudomonas syringae pv. tabaci and elevated expression levels of defense-related genes, e.g. PR-1, PR-2, PR-3 and PR-5. The OsBIRF1-overexpressing transgenic tobacco plants show increased oxidative stress tolerance to exogenous treatment with methyl viologen and H2O2, and up-regulate expression of oxidative stress-related genes. Reduced ABA sensitivity in root elongation and increased drought tolerance in seed germination were also observed in OsBIRF1 transgenic tobacco plants. Furthermore, the transgenic tobacco plants show longer roots and higher plant heights as compared with the wild-type plants, suggesting that overexpression of OsBIRF1 promote plant growth. These results demonstrate that OsBIRF1 has pleiotropic effects on growth and defense response against multiple abiotic and biotic stresses.     

Sucrose-mediated priming of plant defense responses and broad-spectrum disease resistance by overexpression of the maize pathogenesis-related PRms protein in rice plants

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. The PRms gene encodes a fungal-inducible PR protein from maize. Here, we demonstrate that expression of PRms in transgenic rice confers broad-spectrum protection against pathogens, including fungal (Magnaporthe oryzae, Fusarium verticillioides, and Helminthosporium oryzae) and bacterial (Erwinia chrysanthemi) pathogens. The PRms-mediated disease resistance in rice plants is associated with an enhanced capacity to express and activate the natural plant defense mechanisms. Thus, PRms rice plants display a basal level of expression of endogenous defense genes in the absence of the pathogen. PRms plants also exhibit stronger and quicker defense responses during pathogen infection. We also have found that sucrose accumulates at higher levels in leaves of PRms plants. Sucrose responsiveness of rice defense genes correlates with the pathogen-responsive priming of their expression in PRms rice plants. Moreover, pretreatment of rice plants with sucrose enhances resistance to M. oryzae infection. Together, these results support a sucrose-mediated priming of defense responses in PRms rice plants which results in broad-spectrum disease resistance.     

Ectopic expression of MgSM1, a Cerato-platanin family protein from Magnaporthe grisea, confers broad-spectrum disease resistance in Arabidopsis

Proteins belonging to the newly identified Cerato-platanin (CP) family have been shown to have elicitor activity in inducing disease resistance responses in various plants. In this study, we characterized a gene, MgSM1 , from Magnaporthe grisea , encoding a putative small protein belonging to the CP family. MgSM1 was constitutively expressed not only in different fungal growth stages but also during its infection process in rice plants. Agrobacterium-mediated transient expression of MgSM1 in Arabidopsis resulted in hypersensitive response in the infiltrated local leaves and enhanced disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato ( Pst ) DC3000 in upper leaves of plants, accompanyed by up-regulated expression of defense genes ( PR-1 , PR-5 and PDF1.2 ). Transgenic Arabidopsis plants expressing MgSM1 under control of a dexamethasone (DEX)-inducible promoter were generated. Expression of MgSM1 in transgenic plants was induced by exogenous application of DEX. MgSM1- expressing plants showed normal growth with application of <10 μ m DEX. After DEX induction, the MgSM1 -expressing plants showed enhanced disease resistance against B. cinerea , Alternaria brassicicola and Psto DC3000 as well as up-regulated expression of some of defense genes. Moreover, accumulation of reactive oxygen species was observed in MgSM1 -expressing plants. These results collectively suggest that ectopic expression of MgSM1 in transgenic plants confers broad-spectrum resistance against different types of pathogens. Our study also provides a novel strategy to generate environment-friendly crops with enhanced broad-spectrum resistance through ectopic expression of microbe-derived disease resistance-inducing proteins.     

番茄多蛋白桥梁因子基因LeMBF1的克隆及抗病性分析

采用同源序列克隆法,从番茄中克隆了多蛋白桥梁因子基因LeMBF1,该基因包含一个完整的420 bp的开放阅读框,编码139个氨基酸,具有MBF1保守结构域.LeMBF1氨基酸序列与马铃薯StMBF1、烟草NtMBF1和葡萄VvMBF1的氨基酸序列相似度分别是99.3%、91.4%和84.2%.为了研究番茄多蛋白桥梁因子LeMBF1在植物抗病性中的作用,以LeMBF1超表达转基因番茄和野生型番茄为材料,对其进行接种病原细菌Pst.DC3000和尖孢镰刀菌Fusarium.oxysporum的生物胁迫实验.抗菌表型分析发现,LeMBF1超表达转基因番茄叶片上的菌斑数明显少于对照植株;实时定量PCR分析表明,LeMBF1超表达番茄植株中防卫基因PR1、PR6的表达水平明显增强.由此可见,LeMBF1可能通过激活部分PRs基因的表达提高了植物的抗病性.     

Overexpression of the Qc-SNARE gene OsSYP71 enhances tolerance to oxidative stress and resistance to rice blast in rice (Oryza sativa L.)

OsSYP71 is an oxidative stress and rice blast response gene that encodes a Qc-SNARE protein in rice. Qc-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), which function as important components of the vesicle trafficking machinery in eukaryotic cells. In this paper, 12 Qc-SNARE genes were isolated from rice, and expression patterns of 9 genes were detected in various tissues and in seedlings challenged with oxidative stresses and inoculated with rice blast. The expression of OsSYP71 was clearly up-regulated under these stresses. Overexpression of OsSYP71 in rice showed more tolerance to oxidative stress and resistance to rice blast than wild-type plants. These results indicate that Qc-SNAREs play an important role in rice response to environmental stresses, and OsSYP71 is useful in engineering crop plants with enhanced tolerance to oxidative stress and resistance to rice blast.     

Overexpression of Rice Sphingosine-1-Phoshpate Lyase Gene OsSPL1 in Transgenic Tobacco Reduces Salt and Oxidative Stress Tolerance

Sphingolipids, including sphingosine-1-phosphate (S1P), have been shown to function as signaling mediators to regulate diverse aspects of plant growth, development, and stress response. In this study, we performed functional analysis of a rice (Oryza sativa) S1P lyase gene OsSPL1 in transgenic tobacco plants and explored its possible involvement in abiotic stress response. Overexpression of OsSPL1 in transgenic tobacco resulted in enhanced sensitivity to exogenous abscisic acid (ABA), and decreased tolerance to salt and oxidative stress, when compared with the wild type. Furthermore, the expression levels of some selected stress-related genes in OsSPL1-overexpressing plants were reduced after application of salt or oxidative stress, indicating that the altered responsiveness of stress-related genes may be responsible for the reduced tolerance in OsSPL1-overexpressing tobacco plants under salt and oxidative stress. Our results suggest that rice OsSPL1 plays an important role in abiotic stress responses.     

Heterologous expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">NPR1</Emphasis> (<Emphasis Type="Italic">AtNPR1</Emphasis>) enhances oxidative stress tolerance in transgenic tobacco plants

In Arabidopsis, NPR1 (non-expressor of pathogenesis related genes 1, AtNPR1) functions downstream of salicylic acid (SA) and modulates the SA mediated systemic acquired resistance. It is also involved in a cross talk with the jasmonate pathway that is essential for resistance against herbivores and necrotrophic pathogens. Overexpression of AtNPR1 in transgenic plants resulted in enhanced disease resistance. Recently, tobacco transgenic plants expressing AtNPR1 were shown to be tolerant to the early instars of Spodoptera litura (Meur et al., Physiol Plant 133:765–775, 2008). In this communication, we show that the heterologous expression of AtNPR1 in tobacco has also enhanced the oxidative stress tolerance. The transgenic plants exhibited enhanced tolerance to the treatment with methyl viologen. This tolerance was associated with the constitutive upregulation of PR1, PR2 (glucanase), PR5 (thaumatin like protein), ascorbate peroxidase (APX) and Cu²⁺/Zn²⁺ superoxide dismutase (SOD). This is the first demonstration of the novel function of heterologous expression of AtNPR1 in oxidative stress tolerance in transgenic tobacco.     

The Arabidopsis AtNPR1 inversely modulates defense responses against fungal, bacterial, or viral pathogens while conferring hypersensitivity to abiotic stresses in transgenic rice

The nonexpressor of pathogenesis-related (PR) genes (NPR1) protein plays an important role in mediating defense responses activated by pathogens in Arabidopsis. In rice, a disease-resistance pathway similar to the Arabidopsis NPR1-mediated signaling pathway one has been described. Here, we show that constitutive expression of the Arabidopsis NPR1 (AtNPR1) gene in rice confers resistance against fungal and bacterial pathogens. AtNPR1 exerts its protective effects against fungal pathogens by priming the expression of salicylic acid (SA)-responsive endogenous genes, such as the PR1b, TLP (PR5), PR10, and PBZ1. However, expression of AtNPR1 in rice has negative effects on viral infections. The AtNPR1-expressing rice plants showed a higher susceptibility to infection by the Rice yellow mottle virus (RYMV) which correlated well with a misregulation of RYMV-responsive genes, including expression of the SA-regulated RNA-dependent RNA polymerase 1 gene (OsRDR1). Moreover, AtNPR1 negatively regulates the expression of genes playing a role in the plant response to salt and drought stress (rab21, salT, and dip1), which results in a higher sensitivity of AtNPR1 rice to the two types of abiotic stress. These observations suggest that AtNPR1 has both positive and negative regulatory roles in mediating defense responses against biotic and abiotic stresses.     

Molecular characterization of rice sphingosine-1-phosphate lyase gene OsSPL1 and functional analysis of its role in disease resistance response

Key message

Our results indicate that overexpression of OsSPL1 in transgenic tobacco plants attenuated disease resistance and facilitated programmed cell death.

Abstract

Long-chain base phosphates including sphingosine-1-phosphate have been shown to act as signaling mediators in regulating programmed cell death (PCD) and stress responses in mammals. In the present study, we characterized a rice gene OsSPL1, encoding a putative sphingosine-1-phosphate lyase that is involved in metabolism of sphingosine-1-phosphate. Expression of OsSPL1 was down-regulated in rice plants after treatments with salicylic acid, benzothiadiazole and 1-amino cyclopropane-1-carboxylic acid, but was induced by infection with a virulent strain of Magnaporthe oryzae, the causal agent of rice blast disease. Transgenic tobacco lines with overexpression of OsSPL1 were generated and analyzed for the possible role of OsSPL1 in disease resistance response and PCD. The OsSPL1-overexpressing tobacco plants displayed increased susceptibility to infection of Pseudomonas syringae pv. tabaci (Pst), the causal agent of wildfire disease, showing severity of disease symptom and bacterial titers in inoculated leaves, and attenuated pathogen-induced expression of PR genes after infection of Pst as compared to the wild-type and vector-transformed plants. Higher level of cell death, as revealed by dead cell staining, leakage of electrolyte and expression of hypersensitive response indicator genes, was observed in the OsSPL1-overexpressing plants after treatment with fumonisin B1, a fungal toxin that induces PCD in plants. Our results suggest that OsSPL1 has different functions in regulating disease resistance response and PCD in plants.     

A role of hexokinases in plant resistance to oxidative stress and pathogen infection

Previously, we reported that mitochondria-associated hexokinases are active in controlling programmed cell death in plants (Plant Cell 18, 2341-2355). Here, we investigated their role under abiotic- and biotic-stress conditions. Expression ofNbHxk1, aNicotiana benthamiana hexokinase gene, was stimulated by treatment with salicylic acid or methyl viologen (MV), and was also up-regulated by pathogen infection. In response to MV-induced oxidative stress, NbHxk1-silenced plants exhibited increased susceptibility, while the HXK1— and HXK2-overexpressingArabidopsis plants had enhanced tolerance. Moreover, those overexpressing plants showed greater resistance to the necrotrophic fungal pathogenAlternaria brassicicola. HXK-over-expression also mildly protected plants against the bacterial pathogenPseudomonas syringae pv.tomato DC3000, a response that was accompanied by increased H₂O₂ production and elevatedPR1 gene expression. These results demonstrate that higher levels of hexokinase confer improved resistance to MV-induced oxidative stress and pathogen infection.     

Enhanced Botrytis cinerea Resistance of Arabidopsis Plants Grown in Compost May Be Explained by Increased Expression of Defense-Related Genes,as Revealed by Microarray Analysis

Composts are the products obtained after the aerobic degradation of different types of organic matter waste and can be used as substrates or substrate/soil amendments for plant cultivation. There is a small but increasing number of reports that suggest that foliar diseases may be reduced when using compost, rather than standard substrates, as growing medium. The purpose of this study was to examine the gene expression alteration produced by the compost to gain knowledge of the mechanisms involved in compost-induced systemic resistance. A compost from olive marc and olive tree leaves was able to induce resistance against Botrytis cinerea in Arabidopsis, unlike the standard substrate, perlite. Microarray analyses revealed that 178 genes were differently expressed, with a fold change cut-off of 1, of which 155 were up-regulated and 23 were down-regulated in compost-grown, as against perlite-grown plants. A functional enrichment study of up-regulated genes revealed that 38 Gene Ontology terms were significantly enriched. Response to stress, biotic stimulus, other organism, bacterium, fungus, chemical and abiotic stimulus, SA and ABA stimulus, oxidative stress, water, temperature and cold were significantly enriched, as were immune and defense responses, systemic acquired resistance, secondary metabolic process and oxireductase activity. Interestingly, PR1 expression, which was equally enhanced by growing the plants in compost and by B. cinerea inoculation, was further boosted in compost-grown pathogen-inoculated plants. Compost triggered a plant response that shares similarities with both systemic acquired resistance and ABA-dependent/independent abiotic stress responses.     

Rice RING protein OsBBI1 with E3 ligase activity confers broad-spectrum resistance against Magnaporthe oryzae by modifying the cell wall defence

Emerging evidence suggests that E3 ligases play critical roles in diverse biological processes, including innate immune responses in plants. However, the mechanism of the E3 ligase involvement in plant innate immunity is unclear. We report that a rice gene, OsBBI1, encoding a RING finger protein with E3 ligase activity, mediates broad-spectrum disease resistance. The expression of OsBBI1 was induced by rice blast fungus Magnaporthe oryzae, as well as chemical inducers, benzothiadiazole and salicylic acid. Biochemical analysis revealed that OsBBI1 protein possesses E3 ubiquitin ligase activity in vitro. Genetic analysis revealed that the loss of OsBBI1 function in a Tos17-insertion line increased susceptibility, while the overexpression of OsBBI1 in transgenic plants conferred enhanced resistance to multiple races of M. oryzae. This indicates that OsBBI1 modulates broad-spectrum resistance against the blast fungus. The OsBBI1-overexpressing plants showed higher levels of H(2)O(2) accumulation in cells and higher levels of phenolic compounds and cross-linking of proteins in cell walls at infection sites by M. oryzae compared with wild-type (WT) plants. The cell walls were thicker in the OsBBI1-overexpressing plants and thinner in the mutant plants than in the WT plants. Our results suggest that OsBBI1 modulates broad-spectrum resistance to blast fungus by modifying cell wall defence responses. The functional characterization of OsBBI1 provides insight into the E3 ligase-mediated innate immunity, and a practical tool for constructing broad-spectrum resistance against the most destructive disease in rice.