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.     

A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens.

A purified preparation of antifungal protein (AFP) from Aspergillus giganteus exhibited potent antifungal activity against the phytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme, as well as the oomycete pathogen Phytophthora infestans. Under conditions of total inhibition of fungal growth, no toxicity of AFP toward rice protoplasts was observed. Additionally, application of AFP on rice plants completely inhibited M. grisea growth. These results are discussed in relation to the potential of the afp gene to enhance crop protection against fungal pathogens in transgenic plants.     

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.     

Cerebroside elicitors found in diverse phytopathogens activate defense responses in rice plants

Cerebrosides A and C, compounds categorized as glycosphingolipids, were isolated in our previous study from the rice blast fungus (Magnaporthe grisea) as novel elicitors which induce the synthesis of rice phytoalexins. In this paper, these cerebroside elicitors showed phytoalexin-inducing activity when applied to plants by spray treatment and also induced the expression of pathogenesis-related (PR) proteins in rice leaves. This elicitor activity of the cerebrosides showed the structural specificity as that for the induction of phytoalexins. Ceramides prepared from the cerebrosides by removal of glucose also showed the elicitor activity even in lower level compared to the cerebrosides. In field experiments, the cerebroside elicitors effectively protected rice plants against the rice blast fungus, an economically devastating agent of disease of rice in Japan. The cerebrosides elicitors protected rice plants from other disease as well and were found to occur in a wide range of different phytopathogens, indicating that cerebrosides function as general elicitors in a wide variety of rice-pathogen interactions.     

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants.     

转拟南芥AtNPR1基因增强水稻对水稻白叶枯病和稻瘟病的抗性

AtNPR1基因是拟南芥系统获得抗性的一个重要调节基因,在拟南芥中过量表达AtNPR1基因能使拟南芥对细菌和真菌的抗性同时增强.为了研究在水稻中过量表达AtNPR1基因对水稻抗病性的影响,将该基因转入到广西主栽籼稻恢复系品种桂99中.经PCR验证得到了79株转基因植株,DNA斑点杂交表明ATNPR1基因已经整合到桂99染色体DNA中.Northern杂交和RT-PCR分析表明,AtNPR1基因在桂99中已经表达;同时还检测了转基因植株对水稻白叶枯病和稻瘟病的抗性,结果表明转基因植株对该两种病害的抗性均显著增强.     

Cyanide, a coproduct of plant hormone ethylene biosynthesis, contributes to the resistance of rice to blast fungus

Rice (Oryza sativa) plants carrying the Pi-i resistance gene to blast fungus Magnaporthe oryzae restrict invaded fungus in infected tissue via hypersensitive reaction or response (HR), which is accompanied by rapid ethylene production and formation of small HR lesions. Ethylene biosynthesis has been implicated to be important for blast resistance; however, the individual roles of ethylene and cyanide, which are produced from the precursor 1-aminocyclopropane-1-carboxylic acid, remain unevaluated. In this study, we found that Pi-i-mediated resistance was compromised in transgenic rice lines, in which ethylene biosynthetic enzyme genes were silenced and then ethylene production was inhibited. The compromised resistance in transgenic lines was recovered by exogenously applying cyanide but not ethephon, an ethylene-releasing chemical in plant tissue. In a susceptible rice cultivar, treatment with cyanide or 1-aminocyclopropane-1-carboxylic acid induced the resistance to blast fungus in a dose-dependent manner, while ethephon did not have the effect. Cyanide inhibited the growth of blast fungus in vitro and in planta, and application of flavonoids, secondary metabolites that exist ubiquitously in the plant kingdom, enhanced the cyanide-induced inhibition of fungal growth. These results suggested that cyanide, whose production is triggered by HR in infected tissue, contributes to the resistance in rice plants via restriction of fungal growth.     

稻瘟病菌致病性的分子遗传学研究进展

由稻瘟病菌引起的稻瘟病是水稻生产上危害最为严重的真菌病害,对世界粮食生产造成巨大损失。稻瘟病菌成功侵染寄主包括分生孢子萌发、附着胞形成、侵染钉分化和侵染性菌丝扩展等一系列错综复杂的过程,其中每一环节都是由特定基因控制的。稻瘟病菌与水稻的互作符合经典的基因对基因学说,二者的不亲和互作是无毒基因与抗病基因相互作用的结果。近几十年来,世界各国的科学家对稻瘟病菌致病性的生物学及其遗传的分子机制进行了深入的研究。文章就稻瘟病菌致病性的分子遗传学及其遗传变异机制的研究进行了综述,同时对功能基因的研究方法进行了总结。     

Expression of the maize proteinase inhibitor (mpi) gene in rice plants enhances resistance against the striped stem borer (Chilo suppressalis): effects on larval growth and insect gut proteinases

The maize proteinase inhibitor (mpi) gene was introduced into two elite japonica rice varieties. Both constitutive expression of the mpi gene driven by the maize ubiquitin 1 promoter and wound-inducible expression of the mpi gene driven by its own promoter resulted in the accumulation of MPI protein in the transgenic plants. No effect on plant phenotype was observed in mpi-expressing lines. The stability of transgene expression through successive generations of mpi rice lines (up to the T(4) generation) and the production of functional MPI protein were confirmed. Expression of the mpi gene in rice enhanced resistance to the striped stem borer (Chilo suppressalis), one of the most important pests of rice. In addition, transgenic mpi plants were evaluated in terms of their effects on the growth of C. suppressalis larvae and the insect digestive proteolytic system. An important dose-dependent reduction of larval weight of C. suppressalis larvae fed on mpi rice, compared with larvae fed on untransformed rice plants, was observed. Analysis of the digestive proteolytic activity from the gut of C. suppressalis demonstrated that larvae adapted to mpi transgene expression by increasing the complement of digestive proteolytic activity: the serine and cysteine endoproteinases as well as the exopeptidases leucine aminopeptidase and carboxypeptidases A and B. However, the induction of such proteolytic activity did not prevent the deleterious effects of MPI on larval growth. The introduction of the mpi gene into rice plants can thus be considered as a promising strategy to protect rice plants against striped stem borer.     

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.     

Wheat puroindolines enhance fungal disease resistance in transgenic rice

Antimicrobial peptides play a role in the immune systems of animals and plants by limiting pathogen infection and growth. The puroindolines, endosperm-specific proteins involved in wheat seed hardness, are small proteins reported to have in vitro antimicrobial properties. Rice, the most widely used cereal crop worldwide, normally does not contain puroindolines. Transgenic rice plants that constitutively express the puroindoline genes pinA and/or pinB throughout the plants were produced. PIN extracts of leaves from the transgenic plants reduced in vitro growth of Magnaporthe grisea and Rhizoctonia solani, two major fungal pathogens of rice, by 35 to 50%. Transgenic rice expressing pinA and/or pinB showed significantly increased tolerance to M. grisea (rice blast), with a 29 to 54% reduction in symptoms, and R. solani (sheath blight), with an 11 to 22% reduction in symptoms. Puroindolines are effective in vivo in antifungal proteins and could be valuable new tools in the control of a wide range of fungal pathogens of crop plants.     

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.     

稻瘟病菌AVR-pita等位基因的遗传多样性研究(简报)

由真菌Magnaporthe grisea引起的稻瘟病是我国水稻三大病害之一．也是遍及世界各水稻产区的重要病害．每年均有不同程度的发生．流行年份一般减产10％-20％．严重的达40％-50％．局部田块甚至颗粒无收。稻瘟病菌在进化过程中形成了遗传多样性和毒性易变的特性．是水稻品种抗病性容易丧失的主要原因之一。对稻瘟病系统研究的证据表明．水稻与稻瘟病菌之间的互作．符合“基因对基因”假说。也就是说．水稻有一抗病基因,稻瘟病菌中就会有相对应的无毒基因．     

A fungal metallothionein is required for pathogenicity of Magnaporthe grisea

The causal agent of rice blast disease, the ascomycete fungus Magnaporthe grisea, infects rice (Oryza sativa) plants by means of specialized infection structures called appressoria, which are formed on the leaf surface and mechanically rupture the cuticle. We have identified a gene, Magnaporthe metallothionein 1 (MMT1), which is highly expressed throughout growth and development by M. grisea and encodes an unusual 22-amino acid metallothionein-like protein containing only six Cys residues. The MMT1-encoded protein shows a very high affinity for zinc and can act as a powerful antioxidant. Targeted gene disruption of MMT1 produced mutants that show accelerated hyphal growth rates and poor sporulation but had no effect on metal tolerance. Mmt1 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. Mmt1 appears to be distributed in the inner side of the cell wall of the fungus. These findings indicate that Mmt1-like metallothioneins may play a novel role in fungal cell wall biochemistry that is required for fungal virulence.