Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice

Rice blast, caused by Magnaporthe grisea, is the most important fungal disease of cultivated rice worldwide. We have developed a strategy for creating disease resistance to M. grisea whereby pathogen-induced expression of the afp (antifungal protein) gene from Aspergillus giganteus occurs in transgenic rice plants. Here, we evaluated the activity of the promoters from three maize pathogenesis-related (PR) genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric gene fusions were prepared between the maize promoters and the beta-glucuronidase reporter gene (gus A). Histochemical assays of GUS activity in transgenic rice revealed that the ZmPR4 promoter is strongly induced in response to fungal infection, treatment with fungal elicitors, and mechanical wounding. The ZmPR4 promoter is not active in the seed endosperm. The mpi promoter also proved responsiveness to fungal infection and wounding but not to treatment with elicitors. In contrast, no activity of the PRms promoter in leaves of transgenic rice was observed. Transgenic plants expressing the afp gene under the control of the ZmPR4 promoter were generated. Transformants showed resistance to M. grisea at various levels. Our results suggest that pathogen-inducible expression of the afp gene in rice plants may be a practical way for protection against the blast fungus. Most agricultural crop species suffer from a vast array of fungal diseases that cause severe yield losses all over the world. Rice blast, caused by the fungus Magnaporthe grisea (Herbert) Barr (anamorph Pyricularia grisea), is the most devastating disease of cultivated rice (Oryza sativa L.), due to its     

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.     

A rice serine carboxypeptidase-like gene OsBISCPL1 is involved in regulation of defense responses against biotic and oxidative stress

Serine carboxypeptidase-like proteins (SCPLs) comprise a large family of protein hydrolyzing enzymes that play roles in multiple cellular processes. During the course of study aimed at elucidating the molecular basis of induced immunity in rice, a gene, OsBISCPL1, encoding a putative SCPL, was isolated and identified. OsBISCPL1 contains a conserved peptidase S10 domain, serine active site and a signal peptide at N-terminus. OsBISCPL1 is expressed ubiquitously in rice, including roots, stems, leaves and spikes. Expression of OsBISCPL1 in leaves was significantly up-regulated after treatments with benzothiadiazole, salicylic acid, jasmonic acid and 1-amino cyclopropane-1-carboxylic acid, and also up-regulated in incompatible interactions between rice and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants with constitutive expression of OsBISCPL1 were generated and disease resistance assays indicated that the OsBISCPL1-overexpressing plants showed an enhanced disease resistance against Pseudomonas syringae pv. tomato and Alternaria brassicicola. Expression levels of defense-related genes, e.g. PR1, PR2, PR5 and PDF1.2, were constitutively up-regulated in transgenic plants as compared with those in wild-type plants. Furthermore, the OsBISCPL1-overexpressing plants also showed an increased tolerance to oxidative stress and up-regulated expression of oxidative stress-related genes. The results suggest that the OsBISCPL1 may be involved in regulation of defense responses against pathogen infection and oxidative stress.     

Disease resistance against Magnaporthe grisea is enhanced in transgenic rice with suppression of omega-3 fatty acid desaturases

Linolenic acid (18:3) is the most abundant fatty acid in plant membrane lipids and is a source for various oxidized metabolites, called oxylipins. 18:3 and oxylipins play important roles in the induction of defense responses to pathogen infection and wound stress in Arabidopsis. However, in rice, endogenous roles for 18:3 and oxylipins in disease resistance have not been confirmed. We generated 18:3-deficient transgenic rice plants (F78Ri) with co-suppression of two omega-3 fatty acid desaturases, OsFAD7 and OsFAD8. that synthesize 18:3. The F78Ri plants showed enhanced resistance to the phytopathogenic fungus Magnaporthe grisea. A typical 18:3-derived oxylipin, jasmonic acid (JA), acts as a signaling molecule in defense responses to fungal infection in Arabidopsis. However, in F78Ri plants, the expression of JA-responsive pathogenesis-related genes, PBZ1 and PR1b, was induced after inoculation with M. grisea, although the JA-mediated wound response was suppressed. Furthermore, the application of JA methyl ester had no significant effect on the enhanced resistance in F78Ri plants. Taken together, our results indicate that, although suppression of fatty acid desaturases involves the concerted action of varied oxylipins via diverse metabolic pathways, 18:3 or 18:3-derived oxylipins, except for JA, may contribute to signaling on defense responses of rice to M. grisea infection.     

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.     

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.     

Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2-benzisothiazol-3 (2H)-one 1,1-dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis-related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1-1 and coi1-1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR-1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1-1 and coi 1-1 mutant plants. Thus SA and NPR1 appear to be required for PBZ- and BIT-mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1-mediated defense signaling pathway upstream of SA.     

Expression of a bacterial flagellin gene triggers plant immune responses and confers disease resistance in transgenic rice plants

Flagellin is a component of bacterial flagella and acts as a proteinaceous elicitor of defence responses in organisms. Flagellin from a phytopathogenic bacterium, Acidovorax avenae strain N1141, induces immune responses in suspension-cultured rice cells. To analyse the function of flagellin in rice, we fused the N1141 flagellin gene to the cauliflower mosaic virus 35S promoter and introduced it into rice. Many of the resulting transgenic rice plants accumulated flagellin at various levels. The transgenic rice developed pale spots in the leaves. The expression of a defence-related gene for phenylalanine ammonia-lyase was induced in the transgenic plants, and H(2)O(2) production and cell death were observed in some plants with high levels of gene expression, suggesting that the flagellin triggers immune responses in the transgenic rice. Transgenic plants inoculated with Magnaporthe grisea, the causal agent of rice blast, showed enhanced resistance to blast, suggesting that the flagellin production confers disease resistance in the transgenic rice.     

Brassinosteroid functions in a broad range of disease resistance in tobacco and rice

Brassinolide (BL), considered to be the most important brassinosteroid (BR) and playing pivotal roles in the hormonal regulation of plant growth and development, was found to induce disease resistance in plants. To study the potentialities of BL activity on stress responding systems, we analyzed its ability to induce disease resistance in tobacco and rice plants. Wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with BL and the pathogen infection assays using NahG transgenic plants indicate that BL-induced resistance does not require SA biosynthesis. BL treatment did not induce either acidic or basic pathogenesis-related (PR) gene expression, suggesting that BL-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible disease resistance. Analysis using brassinazole 2001, a specific inhibitor for BR biosynthesis, and the measurement of BRs in TMV-infected tobacco leaves indicate that steroid hormone-mediated disease resistance (BDR) plays part in defense response in tobacco. Simultaneous activation of SAR and BDR by SAR inducers and BL, respectively, exhibited additive protective effects against TMV and Pst, indicating that there is no cross-talk between SAR- and BDR-signaling pathway downstream of BL. In addition to the enhanced resistance to a broad range of diseases in tobacco, BL induced resistance in rice to rice blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. Our data suggest that BDR functions in the innate immunity system of higher plants including dicotyledonous and monocotyledonous species.     

Expression of a harpin-encoding gene in rice confers durable nonspecific resistance to Magnaporthe grisea

Engineering durable nonspecific resistance to phytopathogens is one of the ultimate goals of plant breeding. However, most attempts to reach this goal fail as a result of rapid changes in pathogen populations and the sheer diversity of pathogen infection mechanisms. In this study, we show that the expression of a harpin-encoding gene ( hrf1 ), derived from Xanthomonas oryzae pv. oryzae, confers nonspecific resistance in rice to the blast fungus Magnaporthe grisea . Transgenic plants and their T₁–T₇ progenies were highly resistant to all major M. grisea races in rice-growing areas along the Yangtze River, China. The expression of defence-related genes was activated in resistant transgenic plants, and the formation of melanized appressoria, which is essential for foliar infection, was inhibited on plant leaves. These results suggest that harpins may offer new opportunities for generating broad-spectrum disease resistance in other crops.     

Overexpression of a rice diacylglycerol kinase gene OsBIDK1 enhances disease resistance in transgenic tobacco

A rice diacylglycerol kinase (DGK) gene, OsBIDK1, which encodes a 499-amino acid protein, was cloned and characterized. OsBIDK1 contains a conserved DGK domain, consisting of a diacylglycerol kinase catalytic subdomain and a diacylglycerol kinase accessory subdomain. Expression of OsBIDK1 in rice seedlings was induced by treatment with benzothiadiazole (BTH), a chemical activator of the plant defense response, and by infection with Magnaporthe grisea, causal agent of blast disease. In BTH-treated rice seedlings, expression of OsBIDK1 was induced earlier and at a higher level than in water-treated control seedlings after inoculation with M. grisea. Transgenic tobacco plants that constitutively express the OsBIDK1 gene were generated and disease resistance assays showed that overexpression of OsBIDK1 in transgenic tobacco plants resulted in enhanced resistance against infection by tobacco mosaic virus and Phytophthora parasitica var. nicotianae. These results suggest that OsBIDK1 may play a role in disease resistance responses.     

OsGLP3-7 positively regulates rice immune response by activating hydrogen peroxide,jasmonic acid,and phytoalexin metabolic pathways

Although germin-like proteins (GLPs) have been demonstrated to participate in plant biotic stress responses, their specific functions in rice disease resistance are still largely unknown. Here, we report the identification and characterization of OsGLP3-7, a member of the GLP family in rice. Expression of OsGLP3-7 was significantly induced by pathogen infection, jasmonic acid (JA) treatment, and hydrogen peroxide (H₂O₂) treatment. OsGLP3-7 was highly expressed in leaves and sublocalized in the cytoplasm. Overexpression of OsGLP3-7 increased plant resistance to leaf blast, panicle blast, and bacterial blight, whereas disease resistance in OsGLP3-7 RNAi silenced plants was remarkably compromised, suggesting this gene is a positive regulator of disease resistance in rice. Further analysis showed that OsGLP3-7 has superoxide dismutase (SOD) activity and can influence the accumulation of H₂O₂ in transgenic plants. Many genes involved in JA and phytoalexin biosynthesis were strongly induced, accompanied with elevated levels of JA and phytoalexins in OsGLP3-7-overexpressing plants, while expression of these genes was significantly suppressed and the levels of JA and phytoalexins were reduced in OsGLP3-7 RNAi plants compared with control plants, both before and after pathogen inoculation. Moreover, we showed that OsGLP3-7-dependent phytoalexin accumulation may, at least partially, be attributed to the elevated JA levels observed after pathogen infection. Taken together, our results indicate that OsGLP3-7 positively regulates rice disease resistance by activating JA and phytoalexin metabolic pathways, thus providing novel insights into the disease resistance mechanisms conferred by GLPs in rice.     

Characterization of a novel rice gene OsATX and modulation of its expression by components of the stress signalling pathways

In our search to identify gene(s) involved in the rice self-defense responses, we cloned a novel rice ( Oryza sativa L. cv. Nipponbare) gene, OsATX , a single copy gene, from the JA treated rice seedling leaves cDNA library. This gene encodes a 69 amino acid polypeptide with a predicted molecular mass of 7649.7 and a pI of 5.6. OsATX was responsive to cutting (wounding by cutting the excised leaf), over its weak constitutive expression in the healthy leaves. The critical signalling molecules, jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), and hydrogen peroxide, together with protein phosphatase inhibitors, effectively up-regulated the OsATX expression with time, over the excised leaf cut control, whereas ethylene had no affect. Furthermore, copper, a heavy metal, also up-regulated OsATX expression. Moreover, induced expression of OsATX mRNA was influenced by light signal(s), and showed a requirement for de novo synthesized protein factors. Additionally, co-application of either JA or ABA with SA drastically suppressed the induced OsATX mRNA level. Finally, the blast pathogen, Magnaporthe grisea , triggered OsATX mRNA accumulation. These results strongly suggest a function/role(s) for OsATX in defense/stress responses in rice.     

Identification of rice genes induced in a rice blast-resistant mutant

To clarify mechanisms of rice blast resistance in rice plants we used suppression subtractive hybridization (SSH) to isolate genes induced upon rice blast inoculation in a rice blast-resistant mutant. A total of 26 rice cDNAs were isolated and found to have elevated expression upon rice blast infection in a rice blast-resistant derivative, SHM-11, of the rice cultivar, Sanghaehyanghyella. Sequencing of the cDNAs revealed that many of the proteins they encoded had been previously described as involved in plant responses against pathogen attack. Two interesting groups of the defense-related proteins consisted of three different PR5 homologues and four different protease inhibitors, all highly expressed in the rice blast mutant. Genes encoding proteins involved in signal transduction and regulation were also identified, including translation initiation factor eIF5A, C2 domain DNA binding protein, putative rice EDS and putative receptor like kinase. Most of the identified cDNAs were highly expressed 24 h after blast inoculation. Our results suggest that a pathway regulating defense gene expression may be altered in the mutant, resulting in early induction of the defense genes upon fungal infection.