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.     

Comprehensive analysis of the expression of twenty-seven beta-1, 3-glucanase genes in rice (Oryza sativa L.)

Plant beta-1, 3-glucanases are involved in plant defense and in development. Very little data are available on the expression of rice glucanases both in developmental tissues and under various stresses. In this study, we cloned and characterized twenty-seven rice beta-1, 3-glucanases (OsGlu) from at total of 71 putative glucanases. The OsGlu genes were obtained by PCR from a cDNA library and were classified into seven groups (Group I to VII) according to their DNA or amino acid sequence homology. Analysis of the expression of the twenty-seven OsGlu genes by Northern blotting revealed that they were differentially expressed in different developmental tissues as well as in response to plant hormones, biotic stress, high salt etc. OsGlu11 and 27 in Group IV were clearly expressed only in stem and leaf and were also induced strongly by SA (5 mM), ABA (200 microM), and M. grisea. OsGlu1, 10, 11, and 14 were induced earlier and to higher levels in incompatible M. grisea interaction than in compatible one. Taken together, our findings suggest that the twenty-seven rice OsGlu gene products play diverse roles not only in plant defense but also in hormonal responses and in development.     

Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata

Inhibition of jasmonic acid (JA) signaling has been shown to decrease herbivore resistance, but the responsible mechanisms are largely unknown because insect resistance is poorly understood in most model plant systems. We characterize three members of the lipoxygenase (LOX) gene family in the native tobacco plant Nicotiana attenuata and manipulate, by antisense expression, a specific, wound- and herbivory-induced isoform (LOX3) involved in JA biosynthesis. In three independent lines, antisense expression reduced wound-induced JA accumulation but not the release of green leaf volatiles (GLVs). The impaired JA signaling reduced two herbivore-induced direct defenses, nicotine and trypsin protease inhibitors (TPI), as well as the potent indirect defense, the release of volatile terpenes that attract generalist predators to feeding herbivores. All these defenses could be fully restored by methyl-JA (MeJA) treatment, with the exception of the increase in TPI activity, which was partially restored, suggesting the involvement of additional signals. The impaired ability to produce chemical defenses resulted in lower resistance to Manduca sexta attack, which could also be restored by MeJA treatment. Expression analysis using a cDNA microarray, specifically designed to analyze M. sexta-induced gene expression in N. attenuata, revealed a pivotal role for LOX3-produced oxylipins in upregulating defense genes (protease inhibitor, PI; xyloglucan endotransglucosylase/hydrolase, XTH; threonine deaminase, TD; hydroperoxide lyase, HPL), suppressing both downregulated growth genes (RUBISCO and photosystem II, PSII) and upregulated oxylipin genes (alpha-dioxygenase, alpha-DOX). By genetically manipulating signaling in a plant with a well-characterized ecology, we demonstrate that the complex phenotypic changes that mediate herbivore resistance are controlled by a specific part of the oxylipin cascade.     

Root-derived oxylipins promote green peach aphid performance on Arabidopsis foliage

Oxylipins function as signaling molecules in plant growth and development and contribute to defense against stress. Here, we show that oxylipins also facilitate infestation of Arabidopsis thaliana shoots by the phloem sap-consuming green peach aphid (GPA; Myzus persicae), an agronomically important insect pest. GPAs had difficulty feeding from sieve elements and tapping into the xylem of lipoxygenase5 (lox5) mutant plants defective in LOX activity. These defects in GPA performance in the lox5 mutant were accompanied by reduced water content of GPAs and a smaller population size of GPAs in the mutant compared with the wild-type plant. LOX5 expression was rapidly induced in roots in response to infestation of shoots by GPAs. In parallel, levels of LOX5-derived oxylipins increased in roots and in petiole exudates of GPA-colonized plants. Application of 9-hydroxyoctadecadienoic acid (an oxylipin produced by the LOX5 enzyme) to roots restored water content and GPA population size in lox5 plants, thus confirming that a LOX5-derived oxylipin promotes infestation of the foliage by GPAs. Micrografting experiments demonstrated that GPA performance on foliage is influenced by the LOX5 genotype in roots, thus demonstrating the importance of root-derived oxylipins in colonization of aboveground organs by an insect.     

The green peach aphid,Myzus persicae,acquires a LIPOXYGENASE5-derived oxylipin from Arabidopsis thaliana,which promotes colonization of the host plant

Oxylipins derived from lipoxygenase (LOX) activity play important roles in plant growth, development and stress response. In a recent study, we provided evidence that infestation of Arabidopsis thaliana foliage by the green peach aphid (GPA; Myzus persicae), a phloem sap-consuming insect, was promoted by plant LOX5-derived oxylipins. In comparison to the wild-type (WT) plant, GPA population was smaller on the Arabidopsis lox5 mutant. The insect spent less time feeding from the sieve element and xylem of the lox5 mutant compared with the WT plant. In addition, compared with insects feeding on the WT plant, when on the lox5 mutant, the GPA was unable to suppress an antibiotic activity that is present in Arabidopsis vascular sap. Roots are the critical source of a LOX5-derived oxylipin(s) that promotes colonization of the foliage by GPA. Here we show that the 9-hydoxy-10E, 12Z-octadecadienoic acid (9-HOD), a LOX5-derived oxylipin, accumulated in GPA that were reared on the WT, but not the lox5 mutant plant. However, 9-HOD accumulated in insects reared on lox5 mutant plants that were irrigated with 9-HOD, thus indicating that the insect ingests oxylipins from the host plant. We further demonstrate that the host plant requires LOX5 function to promote expression of the defense regulatory gene PHYTOALEXIN-DEFICIENT4 in the foliage. Taken together, our previous observations and results presented here indicate that while the host plant utilizes LOX5-dependent factors for promoting defense mechanisms, GPA has evolved to utilize plant 9-LOX-derived oxylipins as cues to facilitate infestation, thus suggesting a complex involvement of oxylipins in Arabidopsis interaction with GPA.     

Constitutive expression of an inducible lipoxygenase in transgenic tobacco decreases susceptibility to Phytophthora parasitica var. nicotianae

Plant lipoxygenases (LOXs) are key enzymes involved in the generation of fatty acid derivatives, called oxylipins. In tobacco, LOX gene expression and activity are very low in healthy tissues and are highly enhanced in response to infection by Phytophthora parasitica nicotianae and to elicitor treatment. We previously showed, using antisense-LOX1 plants, that expression of the tobacco LOX1 gene is required for the race-cultivar specific resistance of tobacco to Phytophthora parasitica nicotianae. In order to investigate the effect of over-expressing a LOX gene on plant resistance, we transformed tobacco plants with the LOX1 coding sequence fused to the CaMV 35S promoter. Four transgenic lines with enhanced levels of LOX protein and specific activity over control plants were selected for further analysis. These plants were macroscopically indistinguishable from WT plants. Upon stem inoculation, the sense-LOX1 plants displayed a significantly decreased susceptibility to virulent races of Phytophthora parasitica nicotianae, stem lesions being 2- to 3-fold shorter in the transgenic lines than in WT plants. Using a root inoculation assay, the survival rate of sense-LOX1 seedlings was increased about 4-fold compared to their WT counterparts, with 60 to 80% of transgenic plants vs 15 to 20% of WT controls remaining healthy following inoculation with Phytophthora parasitica nicotianae. This is the first demonstration that the over-expression of a LOX gene is sufficient to reduce the susceptibility of a host plant to an oomycete pathogen.     

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     

Disruption of a maize 9-lipoxygenase results in increased resistance to fungal pathogens and reduced levels of contamination with mycotoxin fumonisin

Plant oxylipins, produced via the lipoxygenase (LOX) pathway, function as signals in defense and development. In fungi, oxylipins are potent regulators of mycotoxin biosynthesis and sporogenesis. Previous studies showed that plant 9-LOX-derived fatty acid hydroperoxides induce conidiation and mycotoxin production. Here, we tested the hypothesis that oxylipins produced by the maize 9-LOX pathway are required by pathogens to produce spores and mycotoxins and to successfully colonize the host. Maize mutants were generated in which the function of a 9-LOX gene, ZmLOX3, was abolished by an insertion of a Mutator transposon in its coding sequence, which resulted in reduced levels of several 9-LOX-derived hydroperoxides. Supporting our hypothesis, conidiation and production of the mycotoxin fumonisin B1 by Fusarium verticillioides were drastically reduced in kernels of the lox3 mutants compared with near-isogenic wild types. Similarly, conidia production and disease severity of anthracnose leaf blight caused by Colletotrichum graminicola were significantly reduced in the lox3 mutants. Moreover, lox3 mutants displayed increased resistance to southern leaf blight caused by Cochliobolus heterostrophus and stalk rots caused by both F. verticillioides and C. graminicola. These data strongly suggest that oxylipin metabolism mediated by a specific plant 9-LOX isoform is required for fungal pathogenesis, including disease development and production of spores and mycotoxins.     

Family 19 chitinase of Streptomyces griseus HUT6037 increases plant resistance to the fungal disease

Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. In vitro, ChiC clearly inhibited hyphal extension of Trichoderma reesei but a rice family 19 chitinase did not. In order to investigate the effects of ChiC as an increaser of plant resistance to fungal diseases, the chiC gene was introduced into rice plants under the control of the increased CaMV 35S promoter and a signal sequence from the rice chitinase gene. Transgenic plants were morphologically normal. Resistance to leaf blast disease caused by Magnaporthe grisea was evaluated in R1 and R2 generations using a spray method. Ninety percent of transgenic rice plants expressing ChiC had higher resistance than non-transgenic plants. Disease resistance of sibling plants within the same line was correlated with the ChiC expression levels. ChiC produced in rice plants accumulated intercellularly and had the hydrolyzing activity against glycol chitin.     

Reciprocal oxylipin-mediated cross-talk in the Aspergillus–seed pathosystem

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans , oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene ( ZmLOX3 ) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans , and into a Δ ppoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2–3 expression was decreased when infected by A. nidulans Δ ppo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus –seed pathosystem.     

Reciprocal oxylipin-mediated cross-talk in the Aspergillus-seed pathosystem.

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans, oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene (ZmLOX3) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans, and into a DeltappoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2-3 expression was decreased when infected by A. nidulansDeltappo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus-seed pathosystem.     

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.     

Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus Giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.     

Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens

Plant oxylipins are a large family of metabolites derived from polyunsaturated fatty acids. The characterization of mutants or transgenic plants affected in the biosynthesis or perception of oxylipins has recently emphasized the role of the so-called oxylipin pathway in plant defense against pests and pathogens. In this context, presumed functions of oxylipins include direct antimicrobial effect, stimulation of plant defense gene expression, and regulation of plant cell death. However, the precise contribution of individual oxylipins to plant defense remains essentially unknown. To get a better insight into the biological activities of oxylipins, in vitro growth inhibition assays were used to investigate the direct antimicrobial activities of 43 natural oxylipins against a set of 13 plant pathogenic microorganisms including bacteria, oomycetes, and fungi. This study showed unequivocally that most oxylipins are able to impair growth of some plant microbial pathogens, with only two out of 43 oxylipins being completely inactive against all the tested organisms, and 26 oxylipins showing inhibitory activity toward at least three different microbes. Six oxylipins strongly inhibited mycelial growth and spore germination of eukaryotic microbes, including compounds that had not previously been ascribed an antimicrobial activity, such as 13-keto-9(Z),11(E),15(Z)-octadecatrienoic acid and 12-oxo-10,15(Z)-phytodienoic acid. Interestingly, this first large-scale comparative assessment of the antimicrobial effects of oxylipins reveals that regulators of plant defense responses are also the most active oxylipins against eukaryotic microorganisms, suggesting that such oxylipins might contribute to plant defense through their effects both on the plant and on pathogens, possibly through related mechanisms.     

差异显示法分离水稻抗稻瘟病相关基因

采用mRNA差异显示技术,分析水稻稻瘟病抗源材料“地谷”叶片受稻瘟病菌侵染前后的基因的表达差异,获得87个差异片段。对这87个差异片段进行了回收、重扩增与克隆,并对其中的81个片段进行了杂交鉴定。斑点杂交结果证实其中6个片段受稻瘟病菌诱导表达。进一步克隆测序并进行数据库比对分析表明其中一个与水稻4号染色体中一推测的苹果酸合成酶高度同源,一个与水稻11号染色体上的RPR1基因高度同源,RPR1基因具有保守的NBS-LRR结构,并与水稻防卫反应的信号传导有关;另一个与水稻第6号染色体上一推测的硫氧还蛋白高度同源,其余3个为新的cDNA片段。