植物类病变突变体的诱发与突变机制

植物类病变突变体（lesion mimic mutant,LMM）是在无明显逆境或病原物侵染时,植物自发地形成类似病斑的一类突变体。它涉及到细胞程序性死亡（programmed cell death,PCD）,往往能提高植物的抗病能力。因此,它对于揭示植物抗病反应机制,增加植物的广谱抗性具有重要意义。现就植物类病变突变体的诱发与表型特点、突变基因的分子定位与克隆及类病变表型的形成机制研究进展作一简要综述,以期为植物细胞程序性死亡机制和抗病分子作用机制研究提供有益的信息。     

水稻类病斑突变体研究进展

植物类病变突变体是一类在没有病原物侵染情况下就能自发产生坏死斑的突变体。这类突变往往导致植株的抗病增强和防御相关基因的组成性表达。水稻中已报道了将近200个来源不同的类病变突变体,截至2014年12月73个水稻类病变突变体已被鉴定和命名,其中11个控制类病变性状的基因被克隆,它们分别编码不同的蛋白,包括热激蛋白转录因子、E3泛素连接酶、质膜蛋白激酶、锌指蛋白、酰基转移酶。尽管这些蛋白不是直接与植物抗病途径相关,但是在已鉴定的水稻类病变突变体中,绝大多数提高了对白叶枯病或稻瘟病的抗性,表明这些类病变基因的突变激活了植株的防御系统,并且不同的类病变基因可能参与了不同的抗病信号传导途径。深入研究水稻类病变突变体对作物抗病的分子机理研究和栽培品种的遗传改良都具有重要的意义。     

灰葡萄孢侵染垫缺失突变体的筛选及其相关生物学特性的研究

【目的】从农杆菌介导获得的灰葡萄孢RoseBC-3的突变体库中筛选侵染垫缺失突变体菌株,并明确其相关生物学特性。【方法】将菌株接种于洋葱表皮,利用棉兰染色观察侵染垫形成情况,筛选得到一个侵染垫缺失突变体(AT19)。采用形态学方法、离体叶片接种法、钌红染色法、小麦种子幼芽生长抑制法分别对该菌株的菌落培养性状、侵染垫产生情况、致病力、产果胶酶能力以及产植物毒性代谢产物能力进行测定。【结果】筛选灰葡萄孢突变体168株,根据侵染垫形成可分为三类：快速形成侵染垫型(158株)、缓慢形成侵染垫型(9株)和侵染垫形成缺陷型(1株,AT19)。AT19在接种洋葱120 h后依然无法形成成熟侵染垫。该菌株生长较为缓慢,菌落扩展均匀,可以产生分生孢子,对烟草、草莓、蚕豆和豌豆叶片均不能致病,可以产生果胶酶和植物代谢毒性物质。【结论】突变体菌株AT19可以产生果胶酶和植物代谢毒性物质,其致病力缺失与侵染垫产生缺陷相关。研究结果为了解灰葡萄孢侵染垫形成分子机制提供基础材料。     

黄瓜花叶病毒2a基因上的两个氨基酸共同决定其在豆科植物上引发的过敏反应

过敏反应(hypersensitive response, HR)是植物对病原物侵染的一种抗性反应. 对黄瓜花叶病毒(Cucumber mosaic virus, CMV)在豆科植物上引发HR反应的相关因子进行了研究, 证明CMV 2a基因中的243个碱基决定了过敏反应. 进一步对该区域进行了分析和点突变研究, 用豆科系统侵染株系CMV-P1的631位氨基酸酪氨酸(Tyr)替换豆科局部坏死株系CMV-Fny的相应位点氨基酸苯丙氨酸(Phe)后得到的突变体(Fny-F/Y)在豆科植物上引发HR反应, 但过敏枯斑能够向外扩展; 用CMV-P1的641位氨基酸丝氨酸(Ser)替换CMV-Fny的相应位点氨基酸丙氨酸(Ala)得到的突变体(Fny-A/S)在豆科植物上引发的HR反应与CMV-Fny引发的症状没有差异; 当这两个位点同时被置换后得到的突变体(Fny-FA/YS)在豆科植物接种叶上就不再引发HR反应, 而是产生系统侵染, 说明CMV在豆科植物上引发的过敏反应是由CMV 2a基因上的631和641位氨基酸共同决定的.     

第20位脯氨酸对人肿瘤坏死因子的活性和结构的作用

采用寡聚核苷酸指导的体外基因定点突变方法,将重组人肿瘤坏死因子第２０位脯氨酸变为精氨酸。突变体在大肠杆菌中的表达量是ＴＮＦＰｒｏ２０的６０％。表达产物经一系列离子交换层析分离纯化以后,突变体的比活是ＴＮＦＰｒｏ２０的千分之。一突变体和ＴＮＦＰｒｏ２０在非变性非还原的条件下进行聚丙烯酰胺凝胶电泳,两者显示相同的区带,这表明突变体也可以形成三聚体活性形式,ＴＮＦＰｒｏ２０和突变体的荧光光谱分析揭示,Ａ     

十字花科黑腐病菌的XC1193基因与致病相关

十字花科黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)是一种重要植物病原细菌,在全球范围内侵染十字花科植物引起黑腐病,其σ因子在基因表达中起到重要调节作用。XC1193基因在Xcc 8004菌株编码一个σ~70因子,为进一步研究该σ因子在Xcc中的调控作用,利用自杀质粒p K18mobsac B构建了XC1193基因的缺失突变体DM1193。与野生型菌株的比较发现,XC1193基因突变不影响菌体在丰富培养基和基本培养基上的生长;突变体的胞外蛋白酶、纤维素酶、淀粉酶等生化表型也与野生型一致;植株实验表明XC1193突变不影响过敏反应。采用剪叶接种法进行致病性检测,突变体致病力与野生型一致。而采用喷雾接种法,突变体致病力显著降低,互补菌株致病力恢复至野生型水平。结果表明,该σ因子在十字花科黑腐病菌致病过程中发挥作用,并与Xcc侵染寄主的早期事件有关。     

植物细胞程序性死亡研究进展

植物细胞死亡分为坏死和程序性死亡。细胞程序性死亡是具有信号或一系列分子参与,并且由细胞内在的死亡程序介导的有序过程。它在植物生长发育和抵御外界胁迫中具有重要作用。简要介绍了植物PCD的特征,对植物PCD中的信号分子和类caspase的作用等进行了综述,并对植物PCD存在的问题进行分析和展望,为深入研究植物PCD提供参考。     

植物Tnt1反转录转座子突变体库的特征及应用

植物反转录转座子是转座子中的一大类。目前, 在构建突变体库中应用较多的反转录转座子包括Tnt1、Tos17、Tto1和LORE1。该文比较了利用不同反转录转座子构建的植物突变体库的特征, 并对目前应用较为广泛的Tnt1突变体库进行了详细的分析和总结, 重点对豆科模式植物蒺藜苜蓿(Medicago truncatula) Tnt1突变体库的构建及其在固氮、代谢和发育等方面的应用进行了系统概括。该综述将有助于全面了解蒺藜苜蓿Tnt1突变体库的特征, 并为豆科植物功能基因组学研究提供有益的参考。     

TMV 54K基因的3个突变体介导抗病性的研究

利用PCR方法分别构建了烟草花叶病毒（TMV）中一个推测为复制酶的54－kD蛋白基因（54K）缺失N端、C端和仅余基因中部261bp的3个缺失突变体,与野生型54K一起克隆入植物中间载体p208,并通过根癌土壤杆菌（Agrobacterium tumefaciens (SDmith et Townsend)Conn）介导的方法转化烟草（Nicotiana tabacum L.）。用TMV侵染转基因植物的R0代和R1代,结果显示这3个缺失突变体均能介导对TMV的抗病性。     

水稻斑点叶突变体spl101和spl102的筛选及候选基因鉴定

斑点叶突变体是指植物在正常生长条件下叶片或叶鞘上自发形成、且与病原菌侵染产生的病斑类似的一类突变体,筛选并研究斑点叶突变体对揭示植物抗病反应机理具有重要意义。为了进一步研究斑点叶的形成机制,本文通过EMS诱变品种宽叶粳(KYJ),筛选得到两个斑点叶突变体spl101和spl102。这两个突变体在生长发育晚期(抽穗期以后)形成严重的类病斑。遗传分析表明,spl101和spl102均受隐性单基因控制。利用Mutmap方法对候选基因进行克隆,结果显示,spl101和spl102的候选基因均为OsEDR1,该基因与类病斑发生有关。在spl101中,OsEDR1基因突变发生在第6外显子和第6内含子的交接处,该突变导致第6内含子的错误识别,最终造成移码突变。在spl102中,OsEDR1基因突变发生在第10外显子上,导致一个苯丙氨酸(F)变成半胖氨酸(C)。因此,本研究鉴定了两个新的OsEDR1等位突变,对OsEDR1抗病反应机理的进一步研究及丰富水稻种质资源具有积极意义。同时验证了利用Mutmap方法克隆水稻突变基因的有效性。     

Characterizing rice lesion mimic mutants and identifying a mutant with broad-spectrum resistance to rice blast and bacterial blight

Many plant mutants develop spontaneous lesions that resemble disease symptoms in the absence of pathogen attack. In several pathosystems, lesion mimic mutations have been shown to be involved in programmed cell death, which in some instances leads to enhanced disease resistance to multiple pathogens. We investigated the relationship between spontaneous cell death and disease resistance in rice with nine mutants with a range of lesion mimic phenotypes. All nine mutations are controlled by recessive genes and some of these mutants have stunted growth and other abnormal characteristics. The lesion mimics that appeared on the leaves of these mutants were caused by cell death as measured by trypan blue staining. Activation of six defense-related genes was observed in most of the mutants when the mimic lesions developed. Four mutants exhibited significant enhanced resistance to rice blast. One of the mutants, spl11, confers non-race-specific resistance not only to blast but also to bacterial blight. The level of resistance in the spl11 mutant to the two pathogens correlates with the defense-related gene expression and lesion development on the leaves. The results suggest that some lesion mimic mutations in rice may be involved in disease resistance, and cloning of these genes may provide a clue to developing broad-spectrum resistance to diverse pathogens.     

Rice lesion mimic mutants with enhanced resistance to diseases

Lesion mimic mutants are characterized by the formation of necrotic lesions in the absence of pathogens. Such genetic defects often result in enhanced resistance to pathogen infection and constitutive expression of defense response genes. To understand the genetic mechanisms leading to these mutations, we characterized 21 lesion mimic mutants isolated from IR64 rice mutant populations produced by mutagenesis with diepoxybutane (D), gamma rays (G), and fast neutrons (F). Four mutations are controlled by single dominant genes, one of which is inherited maternally. Five lesion mimics are allelic to known spotted leaf (spl) mutants spl1, spl2, spl3, or spl6. In total, 11 new lesion mimic mutations, named spl16, spl17, and spl19 through Spl27, were established based on allelism tests. Two lesion mimics, spl17 and Spl26 showed enhanced resistance to multiple strains of Magnaporthe oryzae, the rice blast pathogen, and Xanthomonas oryzae pv. oryzae, the bacterial blight (BB) pathogen. Co-segregation analyses of blast and BB resistance and lesion mimic phenotypes in segregating populations of spl17 and Spl26 indicate that enhanced resistance to the two diseases is conferred by mutations in the lesion mimic genes. A double mutant produced from two independent lesion mimics showed more severe lesions and higher level of resistance to X. o. pv. oryzae than their single mutant parents indicating a synergistic effect of the two mutations. In mutants that exhibit enhanced disease resistance to both pathogens, increases in expression of defense response genes PR-10a, POX22.3, and PO-C1 were correlated with lesion mimic development and enhancement of resistance. These lesion mimic mutants may provide essential materials for a comprehensive dissection of the disease resistance pathways in rice.     

SDR7-6, a short-chain alcohol dehydrogenase/reductase family protein,regulates light-dependent cell death and defence responses in rice

Lesion mimic mutants resembling the hypersensitive response without pathogen attack are an ideal material to understand programmed cell death, the defence response, and the cross-talk between defence response and development in plants. In this study, mic, a lesion mimic mutant from cultivar Yunyin treated with ethyl methanesulphonate (EMS), was screened. By map-based cloning, a short-chain alcohol dehydrogenase/reductase with an atypical active site HxxxK was isolated and designated as SDR7-6. It functions as a homomultimer in rice and is localized at the endoplasmic reticulum. The lesion mimic phenotype of the mutant is light-dependent. The mutant displayed an increased resistance response to bacterial blight, but reduced resistance to rice blast disease. The mutant and knockout lines showed increased reactive oxygen species, jasmonic acid content, antioxidant enzyme activity, and expression of pathogenicity-related genes, while chlorophyll content was significantly reduced. The knockout lines showed significant reduction in grain size, seed setting rate, 1000-grain weight, grain weight per plant, panicle length, and plant height. SDR7-6 is a new lesion mimic gene that encodes a short-chain alcohol dehydrogenase with atypical catalytic site. Disruption of SDR7-6 led to cell death and had adverse effects on multiple agricultural characters. SDR7-6 may act at the interface of the two defence pathways of bacterial blight and rice blast disease in rice.     

Deterioration of Antioxidant Competence in Barley Lesion Mimic Mutant 194

A barley mutant, 194, was observed to exhibit a leaf spot phenotype over the whole course of its growing period. In this study, the phenotype and antioxidant competence were studied in the lesion mimic mutant 194. Plant height was slightly higher in mutant 194 than in the wild type (WT). In addition, leaf spot per plant in mutant 194 was significantly higher than in WT. Antioxidant competence, as indicated by reactive oxygen species (ROS) accumulation, antioxidant enzyme activity, and the expression of antioxidant enzyme-encoding genes was also assessed in mutant 194. Compared to the WT, mutant 194 displayed a relatively higher accumulation of ROS, accompanied by lower activities of some antioxidant enzymes and downregulation of antioxidant enzyme-encoding genes. This demonstrated reduced antioxidant competence in mutant 194. The results suggested that this lower antioxidant competence of mutant 194 could lead to the accumulation of excessive ROS. This excess of ROS could induce programmed cell death and has the potential to promote disease resistance in mutant 194.     

Regulators of cell death in disease resistance

Cell death and disease resistance are intimately connected in plants. Plant disease resistance genes (R genes) are key components in pathogen perception and have a potential to activate cell death pathways. Analysis of R proteins suggests common molecular mechanisms for pathogen recognition and signal emission whereas the subsequent signalling unexpectedly involves a network of pathways of parallel, branching and converging action. Disease resistance signalling mutants have revealed novel types of regulatory proteins whose biochemical functions are still unknown. Accumulation of small molecules such as salicylic acid, reactive oxygen intermediates, and nitric oxide amplifies resistance responses and directs cells to initiate cell death programs. Genetic analyses of lesion mimic mutants provide a glimpse of how cell death thresholds are set via an interplay of positive and negative regulatory components.     

Systemic acquired resistance signal transduction

Systemic acquired resistance (SAR) is an inducible plant defense response in which a prior foliar pathogen infection activates resistance in noninfected foliar tissues. Salicylic acid (SA) accumulation is essential for the establishment of SAR. While SA is probably not the long‐distance systemic signal instrumental for SAR activation, it is required for transduction of the signal in noninfected tissues. Although SAR was first described as a response to necrogenic pathogen infection, synthetic chemicals have been identified that effectively activate SAR. Elucidation of SAR signal transduction has been facilitated by the identification and characterization of Arabidopsis mutants. Disease lesion mimic mutants exhibit constitutive SAR as well as spontaneous lesion formation similar to pathogen‐associated hypersensitive cell death. Some disease lesion mimic mutants do not exhibit a lesioned phenotype when SA accumulation is prevented, thereby providing evidence for a feedback loop in SAR signal transduction. Moreover, characterization of mutants compromised for SAR activation has provided additional evidence for common signaling components between SAR and gene‐for‐gene resistance.     

RLIN1, encoding a putative coproporphyrinogen Ⅲ oxidase, is involved in lesion initiation in rice

Lesion mimic is necrotic lesions on plant leaf or stem in the absence of pathogenic infection, and its exact biological mechanism is varied. By a large-scale screening of our T-DNA mutant population, we identified a mutant rice lesion initiation 1 (rlin1), which was controlled by a single nuclear recessive gene. Map-based cloning revealed that RLIN1 encoded a putative coproporphyrinogen Ⅲ oxidase in tetrapyrrole biosynthesis pathway. Sequencing results showed that a G to T substitution occurred in the second exon of RLIN1 and led to a missense mutation from Asp to Tyr. Ectopic expression of RLIN1 could rescue rlin1 lesion mimic phenotype. Histochemical analysis demonstrated that lesion formation in rlin1 was light-dependent accompanied by reactive oxygen species accumulated. These results suggest that tetrapyrrole participates in lesion formation in rice.     

Inducible expression of bacterio-opsin in transgenic tobacco and tomato plants

The development of new strategies to enhance resistance of plants to pathogens is instrumental in preventing agricultural losses. Lesion mimic, the spontaneous formation of lesions resembling hypersensitive response lesions in the absence of a pathogen, is a dramatic phenotype occasionally induced upon expression of certain transgenes in plants. These transgenes simulate the presence of a pathogen and, therefore, activate the plant anti-pathogen defense mechanisms and induce a state of systemic resistance. Lesion mimic genes have been successfully used to enhance the resistance of a number of different plants to pathogen attack. However, constitutive expression of these genes in plants is associated with the spontaneous formation of lesions on leaves and stems, reduced growth, and lower yield. We tested the possibility of using a wound-inducible promoter to control the expression of bacterio-opsin (bO), a transgene that confers a lesion mimic phenotype in tobacco and tomato plants when constitutively expressed. We found that plants with inducible expression of bO did not develop spontaneous lesions. Nevertheless, under controlled laboratory conditions, they were found to be resistant to infection by pathogens. The activation of defense mechanisms by the bO gene was not constitutive, and occurred in response to wounding or pathogen infection. Furthermore, wounding of transgenic tobacco plants resulted in the induction of systemic resistance to pathogen attack within 48 h. Our findings provide a promising initial assessment for the use of wound-inducible promoters as a new strategy to enhance pathogen resistance in transgenic crops by means of lesion mimic genes.