植物抗真菌和细菌病害基因工程的策略及其进展

本文从（1）在植物与病原物相互识别水平上调控而激活其保卫反应机制;（2）导入植物保卫反应相关基因;（）导人降解或抑制病原菌致病因子基因等方面讨论了植物抗真菌和细菌病害基因工程的策略,介绍了目前的主要进展,并对有关策略作了简要的评价。     

转基因改良水稻抗稻瘟病的策略及其进展

稻瘟病是由子囊菌引起的广泛发生在世界各水稻产区的主要真菌病害。由于病原菌致病性的高度分化,使得对稻瘟病很难控制和防治。长期实践证明,培育抗病品种是稻瘟病抗病育种的主要目标。随着基因工程的发展,利用转基因技术导入外源基因改良稻瘟病抗性已成为一条新途径。现有研究表明,通过某些抗病基因、抗真菌蛋白基因、杀菌肽基因的克隆和转育,可以培育出获得对稻瘟病广谱抗性的水稻品种(系)。     

植物抗真菌病害基因工程的研究进展

简述了转基因植物提高真菌性病害抗性方面的研究进展。     

植物抗真菌病害基因工程研究概述

真菌病害是作物损失的主要原因之一。作物病害的80％由病原真菌所引起。真菌病的危害性极大,如19世纪中期,爱尔兰曾因晚疫病严重爆发而导致马铃薯绝产,致使100万人挨饿、200万人被迫背井离乡远迁北美。过去,人们对作物真菌病害的控制有三条途径:(1)选育并采用抗病品种,育种方法为通过有性杂交利用作物本身或近缘种中的抗性基因;(2)施用化学杀菌剂;(3)采取预防措施,如轮作、     

抗真菌植物基因工程的策略和进展

所有高等植物都受多种真菌的侵害,水稻的240多种病害中真菌性痫害占90％。,可见真菌病害是世界范围内危害作物产蘑的主要因素之一,是长期以来作物育种学家一直在努力攻克的难题。目前国     

植物几丁质酶及其在抗真菌病害中的应用

植物几丁质酶的研究是抗真菌基因工程的热点之一。几丁质酶能够水解真菌细胞壁的主要成分几丁质,在植物抗真菌病害反应中发挥重要的作用。介绍了几丁质酶的基本生物学特性、基因的诱导表达,并对植物几丁质酶基因在抗真菌病害基因工程中的应用进行了阐述。     

植物抗真菌基因工程研究进展

自从人类开始种植农作物以来,真菌病害就是造成作物损失的主要原因之一。目前控制真菌病害的主要方式不外乎轮作、培育抗真菌品种、施用化学农药等。虽然这些方法在不同时期都发挥了各自的作用,但由此而产生的各种弊病日益显著。随着人们对环境的关注及降低生产成本的愿望的不断增强,激励着育种家们寻求新的育种途径。建立在分子生物学技术基础上的基因工程方法,是培育抗病植物品种的一条全新而有效的途径。近年来由于对植物抗病反应机制及植物病原真菌致病机理的深入研究,该领域的研究者们提出了较多利用基因工程技术控制植物真菌病害的设想。目前。要从以下几种途径获取和利用抗真菌基因：从常规育种已确知但其产物未知的小种一品种特异的抗性基因;参与对真菌有毒性的化合物的合成酶基因;对真菌生长具有直接抑制作用的蛋白质基因、真菌酶抑制物基因、植保素基因等。该文就此方面的策略及进展做一综述。     

核糖体灭活蛋白及其在植物抗真菌病基因工程中的应用

真菌病往往使作物产量造成严重损失,也使农产品品质下降。植物核糖体灭活蛋白(Ribosome-in-activating proteins,RIPs)是一种作用于真核或原核细胞的核糖体,抑制蛋白质生物合成的毒素。随着对其作用机理、生化特性、表达调控的深入研究,核糖体灭活蛋白基因转化植株显示出很高的抗真菌能力,正日益发展成为植物真菌病害防治的新途径。围绕RIPs在抗真菌病基因工程中的应用,本文对RIPs的功能、分类、分布及性质等进行了阐述。     

植物抗病毒基因工程育种策略及其进展

简要讨论了近年来植物抗病毒基因工程育种策略。这些策略包括利用植物自身的抗病毒基因;利用病毒宙蛋白基因、动物蛋白基因、复制酶基因、卫星ＲＮＡ、反义链ＲＮＡ和缺陷干扰型ＲＮＡ;利用抗体基因、核酶和干扰素。并以各种策略的抗病毒机理及其在农业生产上的应用前景进行了讨论。     

核糖体失活蛋白及其在植物抗真菌病基因工程中的应用

真菌病是农作物减产的主要原因之一。而植物界大量存在着具有离体抑制真菌生长增殖能力的蛋白质,核糖体失活蛋白（ＲＩＰ,ｒｉｂｏｓｏｍｅ ｉｎａｃｔｉｖａｔｉｎｇ ｐｒｏｔｅｉｎ）就是其一。它能特异地水解核糖体ＲＮＡ ３′－端茎环结构的腺嘌呤残基而导致核糖体失活,进而抑制蛋白合成。但它却不使自身的核糖体失活,只对其它物种核糖体显示高度特异性,这显然具有防止外来病原体侵染的功能。利用基因工程技术,使其在一些经济作物中高效表达,筛选具有抗性的转基因植株,这正日益成为植物真菌病防治的新途径。它克服了常规育种周期长,抗性种质缺乏的弊端,更避免了施用农药带来的环境污染等问题,其应用前景甚为广阔。围绕其在真菌病基因工程中的应用,本文对核糖体失活蛋白在植物体中的分布、分类、生化、结构、功能特性、作用机制以及应用前景等作简要、全面的     

Resistance and Susceptibility of Plants to Fungal Pathogens

Plants are under continuous threat of infection by pathogens endowed with diverse strategies to colonize their host. Comprehensive biochemical and genetic approaches are now starting to reveal the complex signaling pathways that mediate plant disease resistance. Initiation of defense signaling often involves specific recognition of invading pathogens by the products of specialized host resistance (R) genes. Potential resistance signaling components have been identified by mutational analyses to be required for specific resistance in the model Arabidopsis and some crop species. Strikingly, many of the components share similarity to that of innate immune systems in animals. Evidence is also accumulating that plant pathogens have a number of ways to evade host defenses during the early stages of infection, similar to animal pathogens. These strategies are becoming much better understood in a number of plant–pathogen interactions. In this review, we focus on the current knowledge of host factors that control plant resistance and susceptibility to fungal pathogens. The knowledge accumulated in these studies will serve a fundamental basis for combating diseases in strategic molecular agriculture.     

In Vitro Selection for Improved Plant Resistance to Toxin-Producing Pathogens

Simultaneously with the progress in plant biotechnology since the 1980s, new methods in plant pathology have been developed. This review summarizes papers that cover basic research on the effects of selective agents on in vitro cultures of host plants, as well as applications of agents in regeneration systems that result in lines with increased variability in resistance or susceptibility. The first part of the study deals with theoretical aspects of the interactions between plants and toxin‐producing pathogens, mode of phytotoxic action, and host‐ and non‐host‐selective toxins. The second part lists and describes various agents used for selections in vitro. In the last two decades more than 100 publications focused on these selections for the improvement of resistance to plant pathogens. Over 30 plant species were examined to utilise various selection agents extracted from about 40 plant pathogens. The review covers basic research studies and methods that elucidate the relationships between in vitro and in vivo mechanisms of resistance, but also try to develop practical applications to obtain resistant breeding lines. Such methods often utilise some type of explant cultures of the host plants that are treated with various selective agents (culture filtrates, toxins, elicitors), which then elicit typical reactions that parallel those by the pathogens. Their application successfully resulted in resistant lines in banana, carnation, grapevine, strawberry and wheat. Nowadays, these techniques are an important complement to classical breeding methods.     

Terpene Down-Regulation Triggers Defense Responses in Transgenic Orange Leading to Resistance against Fungal Pathogens

Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with d-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a d-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of d-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, d-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in d-limonene content and the decrease in the defense response suggests that d-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores.Plants are sessile organisms that produce and emit a vast array of volatile organic compounds (VOCs) to communicate between parts of the same plant and with other plants. It is generally accepted that the original role of these compounds in nature is related to defense functions (Degenhardt et al., 2003). Most VOCs are terpenoids, fatty acid degradation compounds, phenylpropanoids, and amino acid-derived products. Among these, terpenoids are likely to be the most abundant and expensive to produce (Gershenzon, 1994). Terpenoids are isoprenoid-derived compounds synthesized through the condensation of C5 isoprene units, a process that is catalyzed by a wide diversity of terpene synthases using geranyl diphosphate (GDP), farnesyl diphosphate (FDP), and geranylgeranyl diphosphate (GGDP) as substrates. These reactions give rise to the C5 hemiterpenes, the C10 monoterpenes, the C15 sesquiterpenes, and the C20 diterpenes (Dudareva et al., 2006).In green tissues, volatile terpenoid synthesis is either induced upon wounding or occurs constitutively; terpenes can be then stored in specific organs or tissues where they would be most effective in defense responses, such as leaf trichomes, resin ducts and lacticifers, pockets near the epidermis, or secretory cavities in Citrus spp. (Langenheim, 1994; Turner et al., 2000; Trapp and Croteau, 2001; Voo et al., 2012). Genetic engineering experiments have demonstrated that specific terpenoid compounds emitted by leaves can intoxicate, repel, or deter herbivores (Aharoni et al., 2003; Wu et al., 2006), or they may attract the natural predators and parasitoids of damaging herbivores to protect plants from further damage (Kappers et al., 2005; Schnee et al., 2006). These terpenoids are naturally found in complex mixtures, and it has been proposed that they can act synergistically, as in conifer resin, for simultaneous protection against pests and pathogens (Phillips and Croteau, 1999). Although fatty acid degradation products (such as jasmonates) and phenylpropanoids (such as salicylates) as well as their volatile and nonvolatile precursors are clearly involved in many induced defense responses against pests and pathogens (Glazebrook, 2005), much less is known regarding the participation of terpenoid volatiles in the defense against microorganisms in plants and about the possible interactions of these terpenoids with phytohormones.In contrast to their function in leaves, when released from flowers and mature fruits, the main function of terpenoid volatiles is in the attraction of pollinators (Pichersky and Gershenzon, 2002; Kessler et al., 2008; Junker and Blüthgen, 2010; Schiestl, 2010) and seed-dispersing animals (Lomáscolo et al., 2010; Rodríguez et al., 2011b), respectively. Fruit maturation and ripening are usually associated with large increases in the synthesis and accumulation of specific flavored volatiles, which are proposed to function as signals for seed dispersal (Auldridge et al., 2006; Goff and Klee, 2006; Rodríguez et al., 2013).Upon wounding, plant responses to biotic stresses are orchestrated locally and systemically by signaling molecules. Among these molecules, the jasmonates regulate defenses against arthropod herbivores and necrotroph fungal pathogens as well as biotrophic pathogens, such as some mildews (Ellis and Turner, 2001; Stintzi et al., 2001; Kessler et al., 2004; Li et al., 2005; Wasternack, 2007; Browse and Howe, 2008). In addition to jasmonates, molecules such as salicylic acid (SA) and ethylene appear to regulate distinct defense pathways and are major synergistic (Mur et al., 2006) or antagonistic (De Vos et al., 2005) regulators of plant innate immunity. Plants produce a specific blend of these alarm signals after pathogen or pest attacks, and the production of these molecules varies greatly in quantity, composition, and timing. These signals activate differential sets of defense-related genes that eventually determine the nature of the defense response against the attacker (Reymond and Farmer, 1998; Rojo et al., 2003; De Vos et al., 2005). All genes that encode enzymes involved in the biosynthesis of jasmonates are jasmonic acid (JA) inducible (Wasternack, 2006), indicating that JA biosynthesis is regulated by positive feedback. The precursor for the biosynthesis of JA is α-linolenic acid. The activity of the 13-lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) enzymes converts α-linolenic acid to cis-(+)-12-oxophytodienoic acid (OPDA). OPDA REDUCTASE3 catalyzes the reduction of OPDA (and dinor-OPDA) to oxo-pentenyl-cycloheptane-octanoic acid, which, in turn, undergoes three rounds of β-oxidation leading to jasmonyl-CoA formation. Jasmonyl-CoA is then cleaved by a putative thioesterase yielding (+)-7-iso-JA, which equilibrates to the more stable (−)-JA (Wasternack and Kombrink, 2010).The exogenous application of jasmonates on plants and the existence of mutant and/or transgenic plants altered in JA biosynthesis or signaling have led to altered susceptibility or resistance to pathogens. Impaired JA biosynthesis or signaling is generally associated with decreased levels of defensive compounds, including VOCs, and reduced plant biomass and/or fitness under insect attack (Howe et al., 1996; Halitschke and Baldwin, 2004). For example, Arabidopsis (Arabidopsis thaliana) mutants defective in JA perception (e.g. coronatine-insensitive1 [coi1]) or biosynthesis (e.g. aos and defective in anther dehiscence1) are susceptible to pathogen infections (Feys et al., 1994; Xie et al., 1998; Park et al., 2002; Turner et al., 2002). In contrast, mutants (e.g. constitutive expression of vegetative storage protein1 and Arabidopsis Ser/Thr phosphatase of type 2C1) with constitutive or wound-induced activation of the JA pathway exhibit enhanced resistance to fungal pathogens and pests and phenotypes characteristic of JA-treated plants (Ellis and Turner, 2001; Ellis et al., 2002; Schweighofer et al., 2007).Sweet orange (Citrus sinensis) is a perennial tree species that is exposed to recurrent biotic and abiotic challenges during its decades of growth in orchards. Orange fruits undergo a nonclimacteric maturation process in which the biochemistry, physiology, and structure of the organ are altered to complete the release of mature seeds. These changes typically include fruit growth and texture modification; color change through the degradation of chlorophylls and a parallel induction of carotenogenesis in the peel (flavedo) and pulp; flavonoid accumulation in the pulp; increases and decreases in the sugar and acid contents, respectively; and global accumulation and selective emission of volatile terpenoids (Spiegel-Roy and Goldschmidt, 1996). In nature, d-limonene accumulates gradually in the oil glands of the peel during fruit development and reaches its maximum level shortly before the breaker stage, followed by a steady decline during maturation (Attaway et al., 1967; Kekelidze et al., 1989; Rodríguez et al., 2011b). The high amount of d-limonene that accumulates in orange peels has a tremendous metabolic cost, suggesting an important biological role for this terpene and other related compounds in the interactions between fruits and the biotic environment.Previously, we examined the biological role of d-limonene by manipulating oil gland chemistry via the antisense (AS) overexpression of a d-limonene synthase gene from Satsuma mandarin (Citrus unshiu) in orange fruits. Compared with empty vector (EV) controls, fruit peels from AS transformants showed a dramatic reduction in d-limonene accumulation; decreased levels of other monoterpenes, sesquiterpenes, and monoterpene aldehydes; and increased levels of monoterpene alcohols. When challenged with the necrotroph fungus Penicillium digitatum, the causal agent of green mold rot, AS-transformed fruits were highly resistant to fungal infection. Full susceptibility to P. digitatum infection was restored when AS fruits were supplemented with d-limonene but not other monoterpene alcohols, indicating that d-limonene accumulation in the orange peel was required for the successful progress of this plant-pathogen interaction (Rodríguez et al., 2011a, 2011b). Green mold rot is the most important postharvest disease of citrus fruit worldwide, accounting for up to 60% to 80% of total losses during postharvest life of the fruit. P. digitatum is considered to be a specialist pathogen of citrus fruits that efficiently infects the peel through injuries in which ubiquitous fungal spores germinate and rapidly colonize the surrounding areas (Droby et al., 2008). The control of this pathogen relies heavily on the use of synthetic chemicals, but concerns regarding their potential negative effects on human health and also the generation of fungicide-resistant strains have encouraged finding alternatives, such as the generation of citrus trees with fruits that are genetically resistant to the pathogen.In this work, to better understand the mechanism underlying the constitutive resistance to P. digitatum conferred by the reduction of limonene in AS orange fruits, we analyzed the pattern of fruit growth and the morphological and biochemical developmental characteristics and performed a global analysis of gene expression using a 20K citrus microarray. The study is supplemented by examining the possible involvement of key hormone signals and isoprenoid precursors in the fruit peel. We report here that the reduced level of d-limonene in AS fruits is tightly associated with the constitutive activation of defense response signaling cascades. Our results establish, to our knowledge for the first time, a correlation between increased volatile terpene content and the decline of defense responses in a fleshy fruit during maturation, which would facilitate necrotroph fungal infections in citrus fruits.     

Root Defense Responses to Fungal Pathogens: A Molecular Perspective

This review will focus on the molecular and genetic mechanisms underlying defense responses of roots to fungal pathogens. Soil-borne pathogens, including Phytophthora, Pythium, Fusarium, and Bipolaris, represent major sources of biotic stress in the rhizosphere and roots of plants. Molecular recognition and signaling leading to effective resistance has been demonstrated to occur between host and Phytophthora, or Pythium. The hypersensitive response and apoptotic cell death, two oxidative processes that limit biotrophic pathogens, generally act to exacerbate disease symptoms induced by necrotrophic organisms. Although pathogenesis-related proteins can be expressed in roots during pathogen challenge, salicylic acid has not been implicated in root-mediated interactions. Jasmonic acid and ethylene have been found to mediate parallel as well as synergistic pathways that confer partial tolerance to necrotrophic pathogens, as well as induced systemic resistance to root and foliar pathogens. Genomics approaches are revealing new networks of defense-signaling pathways, and have the potential of elucidating those pathways that are important in root-defense responses.     

内生真菌天然活性产物的研究进展

药用植物内生真菌能够产生许多结构新颖的活性次级代谢产物,已成为发现新天然活性物质的重要源泉。结合作者的工作,文中通过对内生真菌天然产物的分类,对这一领域的研究进展进行综述。     

Antimicrobial Phytoprotectants and Fungal Pathogens: A Commentary

Many plants produce antifungal secondary metabolites. These may be preformed compounds which are found in healthy plants and which may represent in-built chemical barriers to infection by potential pathogens (preformed antimicrobial compounds or phytoanticipins). Alternatively they may be synthesized in response to pathogen attack as part of the plant defence response (phytoalexins). If these molecules do play a role in protecting plants against pathogen attack, then successful pathogens are presumably able to circumvent or tolerate these defences. Strategies may include avoidance, enzymatic degradation, and/or nondegradative mechanisms. This review outlines the different ways in which fungal pathogens may counter the antifungal compounds produced by their host plants and summarizes the evidence for and against these compounds as antimicrobial phytoprotectants.     

Transgenic indica Rice Expressing ns-LTP-Like Protein Shows Enhanced Resistance to Both Fungal and Bacterial Pathogens

Antimicrobial peptides (AMPs) from plant seeds, known to inhibit pathogen growth have a great potential in developing transgenic plants resistant to disease. Some of the nonspecific-lipid transfer proteins (ns-LTP) that facilitate in vitro transport of lipids, show antimicrobial activity in vitro. Rice seeds also contain ns-LTPs; however, these genes are expressed weakly in seedlings. We have transformed Pusa Basmati 1, an elite indica rice cultivar, with the gene for Ace-AMP1 from Allium cepa, coding for an effective antimicrobial protein homologous to ns-LTPs. The gene for Ace-AMP1 was cloned under an inducible rice phenylalanine ammonia-lyase (PAL) or a constitutive maize ubiquitin (UbI) promoter. Ace-AMP1 was expressed in transgenic lines and secreted in the apoplastic space. Protein extracts from leaves of transgenic plants inhibited three major rice pathogens, Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae, in vitro. Enhanced resistance against these pathogens was observed in in planta assays, and the degree of resistance correlating with the levels of Ace-AMP1 with an average increase in resistance to blast, sheath blight, and bacterial leaf blight disease by 86%, 67%, and 82%, respectively. Importantly, transgenic rice plants, with stable integration and expression of Ace-AMP1, retained their agronomic characteristics while displaying enhanced resistance to both fungal and bacterial pathogens.