拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象, 该文通过建立拟南芥-大豆疫霉菌(Arabidopsis thaliana-Phytophthora sojae)非寄主互作系统, 筛选对大豆疫霉菌感病的拟南芥突变体, 为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T-DNA插入突变体离体叶片, 从代表12 000个独立转化株系的40 000株T₃代T-DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定, 离体叶片接菌后3天内出现明显的水渍状病斑, 4–5天后产生大量卵孢子和/或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明, 581-51突变体含有4个T-DNA插入事件, 其感病性状可能由隐性单基因控制。     

银杏叶片三种酶活性变化与抗疫霉菌关系的研究

将疫霉菌离体接种于银杏叶片,发现不同龄期银杏叶片对疫霉菌的抗病性有差异,叶龄为100d的抗病性较强,叶龄为20d的抗病性较弱。不同银杏品种叶片对疫霉菌的抗性也有差异,潮田1号和大佛手2号较抗病,大佛手1号和桂G86-1较感病。不同龄期叶片接种后,其过氧化物酶(POD)和苯丙氨酸解氨酶(PAL)的酶活性变化与抗病性成正相关,而多酚氧化酶(PPOD)的酶活性变化与抗病性没有相关性;不同银杏品种叶片接种后,品种间的抗病性与PPOD酶活性变化成正相关,而POD和PAL的酶活性变化与品种间的抗病性没有相关性。可见,POD、PPOD和PAL的酶活性变化没有明显规律性,不能作为衡量银杏叶片抗病性的生化指标。     

大豆疫霉菌的EMS化学诱变

以甲基磺酸乙酯(ethylmethane sulfonate,EMS)为诱变剂,通过其对大豆疫霉菌Phytophthora sojae休止孢萌发的影响,确定化学诱变条件。通过收集单卵孢子,建立了包含640个单卵孢子系的突变体库,其中约有50%的诱变菌系在培养性状和菌落形态方面发生了明显变化,菌落形态多样,表现出较紧密或松散,近圆形或不规则;气生菌丝减少,生长速度较慢或快;在卵孢子产量方面,8.13%的菌系有增加,20.41%的菌系减少,27.82%的菌系极少或者没有卵孢子产生,43.64%的菌系卵孢子产量类似野生型。以质膜氢离子泵蛋白基因PsPMA1(plasma membrane H+-ATPase1)为对象,通过TILLING技术,从320个大豆疫霉菌突变体中获得9个突变体,进一步确认了EMS对大豆疫霉菌的诱变效果,并且估算EMS对大豆疫霉菌的诱变频率至多每115kb发生一个核苷酸变异。新构建的突变体库为开展大豆疫霉病菌的功能基因组研究奠定了遗传材料基础。     

大豆疫霉对寄主大豆和非寄主菜豆根及根分泌物的响应与其对寄主选择性的关系

【背景】大豆疫霉根腐病作为大豆生产上的一种毁灭性病害已被美国、加拿大等多国报道,其病原菌大豆疫霉(Phtophthora sojae Kaufmann and Gerdemann)为典型的土传病原菌。近年来,土传病原菌与植物根系的互作成为研究土传病原菌寄主选择机制的主要方向。【目的】探究寄主大豆和非寄主菜豆根及根分泌物对大豆疫霉的不同影响,阐明这种影响与大豆疫霉对寄主选择的关系。【方法】应用原位土培法种植大豆疫霉感病品种Sloan、抗病品种Williams82和非寄主菜豆一点红,测定了单个大豆疫霉游动孢子对寄主大豆和非寄主菜豆幼根的侵染行为,收集了寄主及非寄主根分泌物,测定了根分泌物对大豆疫霉游动孢子的趋化作用,包括吸引游动孢子的能力以及对游动孢子成囊、孢囊萌发和芽管生长的影响。【结果】大豆疫霉单个游动孢子对寄主大豆幼根表现强烈趋向性,沿着根面进行多次试探性接触后在根尖伸长区快速成囊并萌发,产生的芽管顶端贴附在幼根表面,在感病大豆品种根面上的芽管比抗病大豆品种上的短且粗,而对非寄主菜豆幼根则不具有趋向性,接触一次后即远离,最终在距离幼根75μm的位置成囊萌发,且芽管生长不具有方向性。此外,大豆疫霉游动孢子对抗病、感病大豆和非寄主菜豆幼根的侵染行为差异完全在根分泌物试验中重现,即寄主大豆根分泌物对大豆疫霉游动孢子具有较强的趋向作用,能够有效吸引游动孢子,促进游动孢子快速成囊及萌发,抑制芽管的伸长,而非寄主菜豆根分泌物不具有上述作用。【结论】大豆疫霉对寄主的选择性与根分泌物有关,为进一步了解大豆疫霉的寄主选择机制提供理论依据。     

梨黑星菌粗毒素对抗病和感病梨离体叶片生理特性的影响

以不同抗病性梨品种为材料,研究了梨黑星菌粗毒素对梨离体叶片的过氧化物酶(POD)活性、苯丙氨酸解氨酶(PAL)活性、丙二醛(MDA)含量、细胞膜相对透性以及叶绿素含量变化的影响.研究表明,毒素接种后抗病和感病品种叶片的POD和PAL活性、MDA含量和相对电导率均呈上升趋势,其间会有1个或2个峰值出现,且抗病品种叶片的POD和PAL活性高于感病品种,而细胞内MDA含量和相对电导率增加比率低于感病品种;同时,毒素使梨离体叶片的叶绿素、类胡萝卜素含量下降,且感病品种的下降幅度大于抗病品种.总之,梨离体叶片POD和PAL的活性变化与梨品种抗病性呈正相关,叶片MDA含量和相对电导率变化与梨品种抗病性呈负相关,抗病品种离体叶片对毒素的毒害有更强的抵抗力.     

聚乙二醇模拟水分胁迫筛选拟南芥突变体的新方法

在以聚乙二醇（PEG）模拟水分胁迫的条件下,分别在拟南芥种子萌发期和幼苗期以25％和30％为选择浓度,根据一种子是否萌发和幼苗能否存活为指标,筛选拟南芥抗水分胁迫突变体。此法操作简单,方便快捷,特别适合实验室进行大规模筛选。我们用这种方法筛选了T—DNA插入的拟南芥突变体库,得到78株可能是拟南芥抗水分胁迫的突变体。     

化学诱导激活型拟南芥突变体库的构建及分析

利用化学诱导激活XVE（LexA-VP16-ER）系统构建了一个包含40000余个独立转化株系的拟南芥突变体库,并对其中的18000余个株系进行了初步的遗传学和表型分析鉴定。卡那霉素抗性分离比表明,51．6％的株系为单位点插入株系,T-DNA插入的平均拷贝数为每株系1．38个。部分T1代和T2代植株表现出了可见的形态变异,包括下胚轴长度、根长度、植株大小和颜色、叶子颜色和形态、开花时间、种皮颜色及结实情况等对数个代表性突变株系表型及T—DNA插入位点侧翼序列进行了分析,结果表明突变体的表型是由于T—DNA的插入造成的,而且这些突变体中包括前人发现的AP2和AGAMOUS的等位基因。由于T-DNA标记或相邻的基因可被XVE系统诱导性的激活,或被T-DNA破坏导致功能缺失,该突变体库可以用于大规模筛选鉴定功能缺失性和功能获得性突变体。     

转PcRS基因拟南芥的抗炭疽病研究

为确定虎杖(Polygonum cuspidatum Sieb.et Zucc.)白藜芦醇合酶基因PcRS(Polygonum cuspidatum resveratrol synthase)提高植物抗病性的有效性,以转PcRS基因拟南芥为材料,对其进行了分子鉴定和抗炭疽病(Colletotrichum higginsianum)研究。利用Southern blot和Northern blot验证了外源PcRS基因在转基因拟南芥基因组中的整合和表达。对经过选育得到的T3代纯合株系进行离体的抗病性试验。转基因拟南芥甲醇提取物对炭疽病菌具有明显抑制作用。进一步采用点滴法接种生长4周的转基因拟南芥离体叶片,与野生型植株相比,转基因植株叶片坏死斑直径和坏死斑上的孢子数目都显著减少,表明转基因拟南芥通过抑制孢子的繁殖来限制炭疽病菌的定植,从而提高转基因植株对炭疽病的抗性。     

烟草抗黑胫病突变体的细胞筛选

经实验我们成功地建立了在细胞水平上筛选烟草抗黑胫病突变体的筛选体系。该体系的主要内容为:γ-射线500—2000拉德诱变高度感病品种的花药后用50—80％的黑胫病菌粗毒素为选择压力,筛选出抗毒素花粉植株,用离体叶片法测定选出抗病植株,再从后代鉴定中选出抗病性能够稳定遗传的突变系。γ-射线及高浓度毒素处理均能得到抗病植株。选自感病品种的花粉植株中约有9—50％是真正抗病的。这些抗病植株中有一部分的抗病性能够稳定遗传。用该法已从感病优质品种小黄金1025及乔庄黑苗中选出6个突变系。并自N.C.628(抗)×小黄金1025(感)及N.C.628(抗)×庆胜2号(感)的F_1花粉植株中选出4个抗病系。所有的抗病系经3—4代后均表现出稳定抗性。其中一个突变体(R400)的抗性似由不完全显性多基因控制。     

大豆疫霉菌一个DNA指纹分析重复序列探针的鉴定

【目的】大豆疫霉菌指纹分析的建立和黑龙江与新疆大豆疫霉菌群体的群体遗传分析。【方法】利用生物信息学方法寻找大豆疫霉菌(Phytophthora sojae)的中度重复序列,并对黑龙江和新疆大豆疫霉菌进行DNA指纹分析。【结果】分析得到一个中度重复序列,定名为PS1227。Southern blot分析表明PS1227在大豆疫霉菌基因组中约有34条可辨的介于1.5-23kb之间的杂交条带,其中21个杂交条带在49个供试菌系中表现多态性。单游动孢子分析表明PS1227指纹特征在病菌无性生殖阶段表现稳定。利用PS1227标记,本实验发现采自黑龙江HP4002、SY6和GJ0105菌系分别与新疆的DW303、71228和71222菌系具有完全相同的指纹特征。【结论】获得一个可用于大豆疫霉菌流行学和群体生物学研究的指纹分析序列PS1227,在分子水平证实了新疆大豆疫霉菌可能由黑龙江传入。     

Arabidopsis nonhost resistance gene PSS1 confers immunity against an oomycete and a fungal pathogen but not a bacterial pathogen that cause diseases in soybean

ABSTRACT: BACKGROUND: Nonhost resistance (NHR) provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species. Three Arabidopsis thaliana PEN (penetration deficient) genes, PEN1, 2 and 3 have been shown to provide NHR against the barley pathogen Blumeria graminis f. sp. hordei at the prehaustorial level. Arabidopsis pen1-1 mutant lacking the PEN1 gene is penetrated by the hemibiotrophic oomycete pathogen Phytophthora sojae, the causal organism of the root and stem rot disease in soybean. We investigated if there is any novel nonhost resistance mechanism in Arabidopsis against the soybean pathogen, P. sojae. RESULTS: The P. sojae susceptible (pss) 1 mutant was identified by screening a mutant population created in the Arabidopsis pen1-1 mutant that lacks penetration resistance against the non adapted barley biotrophic fungal pathogen, Blumeria graminis f. sp. hordei. Segregation data suggested that PEN1 is not epistatic to PSS1. Responses of pss1 and pen1-1 to P. sojae invasion were distinct and suggest that PSS1 may act at both pre- and post-haustorial levels, while PEN1 acts at the pre-haustorial level against this soybean pathogen. Therefore, PSS1 encodes a new form of nonhost resistance. The pss1 mutant is also infected by the necrotrophic fungal pathogen, Fusarium virguliforme, which causes sudden death syndrome in soybean. Thus, a common NHR mechanism is operative in Arabidopsis against both hemibiotrophic oomycetes and necrotrophic fungal pathogens that are pathogenic to soybean. However, PSS1 does not play any role in immunity against the bacterial pathogen, Pseudomonas syringae pv. glycinea, that causes bacterial blight in soybean. We mapped PSS1 to a region very close to the southern telomere of chromosome 3 that carries no known disease resistance genes. CONCLUSIONS: The study revealed that Arabidopsis PSS1 is a novel nonhost resistance gene that confers a new form of nonhost resistance against both a hemibiotrophic oomycete pathogen, P. sojae and a necrotrophic fungal pathogen, F. virguliforme that cause diseases in soybean. However, this gene does not play any role in the immunity of Arabidopsis to the bacterial pathogen, P. syringae pv. glycinea, which causes bacterial blight in soybean. Identification and further characterization of the PSS1 gene would provide further insights into a new form of nonhost resistance in Arabidopsis, which could be utilized in improving resistance of soybean to two serious pathogens.     

Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis

In contrast to gene-for-gene disease resistance, nonhost resistance governs defense responses to a broad range of potential pathogen species. To identify specific genes involved in the signal transduction cascade associated with nonhost disease resistance, we used a virus-induced gene-silencing screen in Nicotiana benthamiana, and identified the peroxisomal enzyme glycolate oxidase (GOX) as an essential component of nonhost resistance. GOX-silenced N. benthamiana and Arabidopsis thaliana GOX T-DNA insertion mutants are compromised for nonhost resistance. Moreover, Arabidopsis gox mutants have lower H(2)O(2) accumulation, reduced callose deposition, and reduced electrolyte leakage upon inoculation with hypersensitive response-causing nonhost pathogens. Arabidopsis gox mutants were not affected in NADPH oxidase activity, and silencing of a gene encoding NADPH oxidase (Respiratory burst oxidase homolog) in the gox mutants did not further increase susceptibility to nonhost pathogens, suggesting that GOX functions independently from NADPH oxidase. In the two gox mutants examined (haox2 and gox3), the expression of several defense-related genes upon nonhost pathogen inoculation was decreased compared with wild-type plants. Here we show that GOX is an alternative source for the production of H(2)O(2) during both gene-for-gene and nonhost resistance responses.     

Creation of a collection of morphological insertional mutants of Arabidopsis thaliana

A collection of transgenic Arabidopsis thaliana plants has been obtained by Agrobacterium-mediated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12-18% of the total number of A. thaliana genes. Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects. Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant.     

Organ identity and environmental conditions determine the effectiveness of nonhost resistance in the interaction between Arabidopsis thaliana and Magnaporthe oryzae

Mechanisms leading to nonhost resistance of plants against nonadapted pathogens are thought to have great potential for the future management of agriculturally important diseases. In this article, we report an investigation of nonhost resistance motivated by the advantages of studying an interaction between two model organisms, namely Arabidopsis thaliana and Magnaporthe oryzae. During the course of our studies, however, we discovered an unexpected plasticity in the responses of Arabidopsis against this ostensibly nonhost pathogen. Thus, we elucidated that certain experimental conditions, such as the growth of plants under long days at constantly high humidity and the use of high inoculum concentrations of M. oryzae conidia, forced the interaction in leaves of some Arabidopsis ecotypes towards increased compatibility. However, sporulation was never observed. Furthermore, we observed that roots were generally susceptible to M. oryzae, whereas leaves, stems and hypocotyls were not infected. It must be concluded, therefore, that Arabidopsis roots lack an effective defence repertoire against M. oryzae, whereas its leaves possess such nonhost defence mechanisms. In summary, our findings point to organ-specific determinants and environmental conditions influencing the effectiveness of nonhost resistance in plants.     

Gene cluster of Pseudomonas syringae pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity of nonhost plants

Loss of the ability of Pseudomonas syringae pv. "phaseolicola" NPS3121 to elicit a hypersensitive response on tobacco and other nonhost plants was associated with loss of pathogenicity on the susceptible host bean. Eight independent, prototrophic transposon Tn5 insertion mutants which had lost the ability to elicit a hypersensitive response on tobacco plants were identified. Six of these mutants no longer produced disease lesions on primary leaves of the susceptible bean cultivar Red Kidney and failed to elicit a hypersensitive response on the resistant bean cultivar Red Mexican and on the nonhost plants tomato, cowpea, and soybean. The two remaining mutants had reduced pathogenicity on Red Kidney bean and elicited variable hypersensitive responses on the other plants tested. Southern blot analysis indicated that each mutant carried a single independent Tn5 insertion in one of three EcoRI fragments of about 17, 7, and 5 kilobases. Marker exchange mutagenesis further supported the conclusion that the pleiotropic mutant phenotype was not associated with multiple Tn5 insertions. A genomic library of the wild-type strain was constructed in the cosmid vector pLAFR3. A recombinant plasmid, designated pPL6, that carried P. syringae pv. "phaseolicola" genomic sequences was identified by colony hybridization. This plasmid restored the wild-type phenotype to all but one mutant, suggesting that genes affected by the insertions were clustered. Structural analysis of pPL6 and the wild-type genome indicated that the 17- and 5-kilobase EcoRI fragments were contiguous in the strain NPS3121 genome.     

The Arabidopsis flavin-dependent monooxygenase FMO1 is an essential component of biologically induced systemic acquired resistance

Upon localized attack by necrotizing pathogens, plants gradually develop increased resistance against subsequent infections at the whole-plant level, a phenomenon known as systemic acquired resistance (SAR). To identify genes involved in the establishment of SAR, we pursued a strategy that combined gene expression information from microarray data with pathological characterization of selected Arabidopsis (Arabidopsis thaliana) T-DNA insertion lines. A gene that is up-regulated in Arabidopsis leaves inoculated with avirulent or virulent strains of the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) showed homology to flavin-dependent monooxygenases (FMO) and was designated as FMO1. An Arabidopsis knockout line of FMO1 proved to be fully impaired in the establishment of SAR triggered by avirulent (Psm avrRpm1) or virulent (Psm) bacteria. Loss of SAR in the fmo1 mutants was accompanied by the inability to initiate systemic accumulation of salicylic acid (SA) and systemic expression of diverse defense-related genes. In contrast, responses at the site of pathogen attack, including increases in the levels of the defense signals SA and jasmonic acid, camalexin accumulation, and expression of various defense genes, were induced in a similar manner in both fmo1 mutant and wild-type plants. Consistently, the fmo1 mutation did not significantly affect local disease resistance toward virulent or avirulent bacteria in naive plants. Induction of FMO1 expression at the site of pathogen inoculation is independent of SA signaling, but attenuated in the Arabidopsis eds1 and pad4 defense mutants. Importantly, FMO1 expression is also systemically induced upon localized P. syringae infection. This systemic up-regulation is missing in the SAR-defective SA pathway mutants sid2 and npr1, as well as in the defense mutant ndr1, indicating a close correlation between systemic FMO1 expression and SAR establishment. Our findings suggest that the presence of the FMO1 gene product in systemic tissue is critical for the development of SAR, possibly by synthesis of a metabolite required for the transduction or amplification of a signal during the early phases of SAR establishment in systemic leaves.     

Pseudomonas syringae elicits emission of the terpenoid (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene in Arabidopsis leaves via jasmonate signaling and expression of the terpene synthase TPS4

Volatile, low-molecular weight terpenoids have been implicated in plant defenses, but their direct role in resistance against microbial pathogens is not clearly defined. We have examined a possible role of terpenoid metabolism in the induced defense of Arabidopsis thaliana plants against leaf infection with the bacterial pathogen Pseudomonas syringae. Inoculation of plants with virulent or avirulent P. syringae strains induces the emission of the terpenoids (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT), beta-ionone and alpha-farnesene. While the most abundant volatile, the C16-homoterpene TMTT, is produced relatively early in compatible and incompatible interactions, emission of both beta-ionone and alpha-farnesene only increases in later stages of the compatible interaction. Pathogen-induced synthesis of TMTT is controlled through jasmonic acid (JA)-dependent signaling but is independent of a functional salicylic acid (SA) pathway. We have identified Arabidopsis T-DNA insertion lines with defects in the terpene synthase gene TPS4, which is expressed in response to P. syringae inoculation. The tps4 knockout mutant completely lacks induced emission of TMTT but is capable of beta-ionone and alpha-farnesene production, demonstrating that TPS4 is specifically involved in TMTT formation. The tps4 plants display at least wild type-like resistance against P. syringae, indicating that TMTT per se does not protect against the bacterial pathogen in Arabidopsis leaves. Similarly, the ability to mount SA-dependent defenses and systemic acquired resistance (SAR) is barely affected in tps4, which excludes a signaling function of TMTT during SAR. Besides P. syringae challenge, intoxication of Arabidopsis leaves with copper sulfate, a treatment that strongly activates JA biosynthesis, triggers production of TMTT, beta-ionone, and alpha-farnesene. Taken together, our data suggest that induced TMTT production in Arabidopsis is a by-product of activated JA signaling, rather than an effective defense response that contributes to resistance against P. syringae.