Obtaining transgenic rice resistant to rice fungal blast disease by controlled cell death strategy

The strategy of the two-component system,composed of Barnase and Barstar which encode RNase and a specific inhibitor to the RNase respectively, is adopted to obtain transgenic rice resistant to rice fungal blast disease. In this study, two chimeric promoters, induced by rice blast fungus pathogen (Magnaporthe grisea), are fused with Barnase respectively to construct two plant expression vectors, pWBNBS and pPBNBS together with the Barstar driven by CaMV 35S promoter. The resistance of the transgenic rice lines to rice blast fungus disease and rice blight disease are evaluated. The results show that (1) the expression of Barnase is induced in rice leaves when inoculated with the spores of Magnaporthe grisea; (2) the induced expression level of Barnase surpasses the level of Barstar, which elicits a similar hypersensitive response (HR) in the leaves, and the transgenic plant shows high resistance to the rice fungal blast disease; and (3) transgenic rice plants also show obvious resistance to rice bacterial blight disease. Taken together, these results suggest that the transgenic rice plants harboring this two-component system acquire relatively broad spectrum resistance against pathogens, especially high resistance to rice fungal pathogen.     

A P-type ATPase required for rice blast disease and induction of host resistance

To cause diseases in plants, pathogenic microorganisms have evolved mechanisms to deliver proteins directly into plant cells, where they suppress plant defences and facilitate tissue invasion. How plant pathogenic fungi, which cause many of the world's most serious plant diseases, deliver proteins during plant infection is currently unknown. Here we report the characterization of a P-type ATPase-encoding gene, MgAPT2, in the economically important rice blast pathogen Magnaporthe grisea, which is required for exocytosis during plant infection. Targeted gene replacement showed that MgAPT2 is required for both foliar and root infection by the fungus, and for the rapid induction of host defence responses in an incompatible reaction. DeltaMgapt2 mutants are impaired in the secretion of a range of extracellular enzymes and accumulate abnormal Golgi-like cisternae. However, the loss of MgAPT2 does not significantly affect hyphal growth or sporulation, indicating that the establishment of rice blast disease involves the use of MgApt2-dependent exocytotic processes that operate during plant infection.     

两个稻瘟病菌假想蛋白的纯化和特性分析

稻瘟病菌(Magnaporthe Grisea)引起的水稻稻瘟病是世界水稻生产最具毁灭性的病害之一,也是研究植物与病原物相互作用分子机理的模式系统之一.真菌分泌蛋白由于其本身所具有的分泌特性极有可能成为被宿主植物识别的作用病菌分泌蛋白,该研究到目前为止还比较有限. 文章证明了稻瘟病菌分泌蛋白的存在并且检验了其中几个蛋白在其宿主植物水稻中的诱导因子作用. 通过同源基因的时空表达技术,表明分泌蛋白在稻瘟病菌中大量表达.其中两个在稻瘟病菌中的未知蛋白被测试出具有诱导因子作用.     

The genome sequence of the rice blast fungus Magnaporthe grisea

Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.     

Blast fungus-induction and developmental and tissue-specific expression of a rice P450<Emphasis Type="Italic">CYP72A</Emphasis> gene cluster

Cytochrome P450 gene superfamily is widely involved in diverse processes of plant development and environmental responses including defense response to pathogens.We previously isolated a rice cDNA fragment in a DD-PCR screening for blast fungus-induced genes. In the current study, we isolated a CYP72A gene cluster consisting of 7 P450 CYP72A genes (CYP72A17-23) with the conserved cDNA sequence through the public rice genome data. There are total 14 putative CYP72A members in the rice genome, with high diversity at N-terminal sequences while high homology at C-terminal sequences of those 14 putative proteins. We analyzed expression profiles of the cloned 7 CYP72A genes during pathogen infection and development. The results showed that expression of CYP72A18, 19, 22 and 23 was differentially regulated in the incompatible and compatible interactions between rice and blast fungus. Except CYP72A20, a pseudogene, other 6 CYP72A genes also exhibited temporal and spatial expression patterns, respectively.These findings provide fundamental data for rice P450 gene function analysis.     

杀菌剂三唑酮在我国农业中的应用研究进展

对三唑酮在我国小麦、水稻、玉米、水果、蔬菜、草坪、花卉、树木等方面的应用进行了综述。三唑酮可用于防治小麦的纹枯病、全蚀病、白粉病、根腐病和水稻的稻瘟病、稻叶尖枯病、稻曲病、黑粉病等多种病害。另外,三唑酮还是一种甾醇抑制剂和植物生长调节剂,在与一些农药混配后具有协同增效作用。     

The Recent Advances in Plant Protection Researches of China

There are eight examples briefly given in this paper, namely, (1) Polymyxa graminis and the cereal viruses it transmits; (2) the geographical types and facultative migration of cotton bollworm as well as the safety of Bt transgenic cotton; (3) development of crop near-isogenic lines with resistance to diseases; (4) molecular-biological researches induced resistance of rice by infection of blast fungus;(5) to use cytological and molecular-biological techniques for breeding wheat varieties resistant to barley yellow dwarf virus; (6) mass rearing and field releasing of Microplitis mediator for cotton bollworm control; (7) identification and recombination of insecticidal crystal genes of Bacillus thuringiensis; and (8) interplanting of diverse resistance rice varieties for sustainable control of blast disease; which reflect the general situation of recent advances in plant protection researches of China.     

稻瘟病菌对水稻PS Ⅱ的影响研究

采用在分蘖期接种稻瘟病菌,研究稻瘟病菌不同生理小种对水稻叶片光合色素和叶绿素荧光参数的影响.结果表明水稻接种稻瘟病菌孢子后,叶片出现不同程度发病,光合色素含量下降,叶片的Fv/Fm,Fv/F0,F′v/F′m,ΦPS Ⅱ,qP,qN,ETR和α较对照均显著降低.不同水稻品种对稻瘟病菌的敏感程度不同,R725接种ZA5后,叶片细胞结构遭到破坏,光合色素含量大幅下降,叶瘟发生最重;R720接种ZC8后发病最重,叶绿素a(Chla)和类胡萝卜素(Car)含量大幅下降,Fv/F0和ΦPS Ⅱ等叶绿素荧光参数也显著低于对照;R727接种ZG1后发病最重,光合色素显著下降,叶绿素荧光参数Fv/Fm,qP,ΦPSⅡ,ETR,qN和α也显著低于对照.     

用抑制差减杂交技术分离烯丙异噻唑诱导水稻特异表达的基因

烯丙异噻唑(PBZ)处理水稻根能使其产生对稻瘟病的系统获得性抗性,因此在东南亚稻区被广泛用于防治稻瘟病,然而关于其作用的分子机理还知之甚少．运用抑制差减杂交技术,试图通过分离鉴定受PBZ诱导调控的关键基因,探索其作用的分子机理．以PBZ处理后的水稻叶片cDNA为目标群体(tester),以未处理水稻叶片cDNA为对照群体(driver),用经过对照cDNA差减的、烯丙异噻唑处理的cDNA群体构建了一个含260个重组子的差减文库．通过差示筛选鉴定出了26个。PBZ诱导水稻特异表达和增强表达的候选克隆．对26个cDNA克隆进行了双向测序和同源性比较,发现其中3个克隆：rJAB1,rTAB2和蛋白磷酸酯酶2Aδ调节亚基同型物基因,位于抗病相关信号转导途径上,它们与哺乳动物和人类免疫途径上的信号因子有明显相似之处,因此推断可能与诱导抗性有关．另外8个克隆与已知基因同源性为70％～99％．经Northern杂交分析,其中rJAB1(编码c-jun激活区结合蛋白1)受烯丙异噻唑和稻瘟菌诱导表达;膜糖蛋白同源基因及肌动蛋白(actin)α1受烯丙异噻唑诱导表达,部分克隆为低丰度转录本．     

Resistance to rice blast (Pyricularia oryzae) caused by the expression of trichosanthin gene in transgenic rice plants transferred through agrobacterium method

The gene of trichosanthin has been transferred into rice plants through agrobacterium method. The single copy insertion and the expression of foreign gene have been proved in regenerated plants. In antifungal assay the degrees of rice blast (Pyricularia oryzae) infection of the transgenic plants expressing trichosanthin and expressingGUS gene as control have been evaluated. The differences such as the time of disease symptom observed, the number of infected plants and damaged leaves, the growth of infected plants of the two transgenic plants after being inoculated by rice blast (Pyricularia oryzae) are significant. The transgenic plants with trichosanthin gene grew faster than the plants withGUS gene, even when humidity environment was removed. The results show that the transgenic plants that expressed trichosanthin are able to delay the infection of rice blast compared with the plants as control. In addition, no damage caused by the expression of trichosanthin gene in transgenic plants has been observed.     

抗稻瘟病几丁质酶产生菌的筛选及产酶条件的研究

稻瘟病具有传染快、危害大等特点,其微生物防治具有良好的发展前景.本实验采用以稻瘟病细胞壁为筛选培养基来筛选抗稻瘟病的几丁质酶产生菌,并对筛选到的优良茵株进行产酶条件优化.实验结果表明,从不同来源的土壤和植物器官中分离得到16株能检测到几丁质酶活性的几丁质酶产生茵,其中以H3和H9的产酶能力最强,具有良好的开发价值.     

Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride

Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.     

芍药红斑病的研究

<正>芍药红斑病主要为害叶片和绿色茎。叶片受害后出现针头状的小点,最后扩展成不规则形大斑,正面为暗紫红色,背面为栗褐色;茎上为暗紫红色长圆形小斑。经鉴定病原为 Cladosporium Paeoniae Pass.。     

Pathogenic fungus harbours endosymbiotic bacteria for toxin production

A number of plant pathogenic fungi belonging to the genus Rhizopus are infamous for causing rice seedling blight. This plant disease is typically initiated by an abnormal swelling of the seedling roots without any sign of infection by the pathogen. This characteristic symptom is in fact caused by the macrocyclic polyketide metabolite rhizoxin that has been isolated from cultures of Rhizopus sp.. The phytotoxin exerts its destructive effect by binding to rice beta-tubulin, which results in inhibition of mitosis and cell cycle arrest. Owing to its remarkably strong antimitotic activity in most eukaryotic cells, including various human cancer cell lines, rhizoxin has attracted considerable interest as a potential antitumour drug. Here we show that rhizoxin is not biosynthesized by the fungus itself, but by endosymbiotic, that is, intracellular living, bacteria of the genus Burkholderia. Our unexpected findings unveil a remarkably complex symbiotic-pathogenic relationship that extends the fungus-plant interaction to a third, bacterial, key-player, and opens new perspectives for pest control.     

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

光照条件对紫叶观赏稻生长的影响

为研究不同光照条件下紫叶观赏稻茎、叶、分蘖等性状的生长差异,制定紫叶观赏稻种植技术,拓宽其应用途径。选取叶色为紫色的观赏稻品种B151和GY7作为试验材料,模拟家居庭院光照条件,设置正常光照、遮阳网下和墙边3个光照处理,以及分蘖前期和分蘖中后期2个移动时间处理。通过对叶色、叶片数、叶面积、剑叶性状、分蘖、有效穗、株高、抗逆性等性状数据的分析,结果表明:正常光照时间的缩短和光照强度的减弱,导致紫叶观赏稻分蘖数、叶片数、有效穗减少,叶片紫色变浅,剑叶长宽增加,剑叶夹角增大,叶片披垂,稻株仍具有较强的观赏性,适宜在家居庭院中种植。     

一株拮抗稻瘟病内生霉菌OsiSh-10的筛选与鉴定

从湖南浏阳大围山种植的湘矮早7号水稻中,筛选到一株对水稻稻瘟病菌有显著抑制效果的水稻内生放线菌OsiSh-10菌株.由拮抗菌包覆种子和喷晒水稻叶面的实验表明将该菌喷晒于水稻叶面可以对叶瘟起到明显的控制效果,且喷一次的效果比喷两次的效果好,包覆种子反而使病情指数升高.根据形态特征、培养特征、生理生化、16s rRNA序列比对及进化树构建鉴定OsiSh-10菌株属于白色链霉菌(Streptomyces albus).OsiSh-10是一株具有稻瘟病生防潜力的菌株.     

水杉赤枯病病原初探

<正>水杉赤枯病在江苏各地普遍发生,危害水杉苗和大树。春季,