Cellular reactions in Arabidopsis following challenge by strains of Pseudomonas syringae: From basal resistance to compatibility

Reactions to strains of the bean halo-blight pathogen Pseudomonas syringae pv. phaseolicola (Pph) strain 1448A and the Arabidopsis and tomato pathogen P.s. pv. tomato (Pst) strain DC3000 were examined by transmission electron microscopy. Wild-type and hrpL mutant strains of Pph failed to multiply in the accession Columbia (Col)-5, but did not cause a hypersensitive reaction (HR). Symptomless non-host resistance to 1448A and the hrpL mutant was associated with the progressive alteration of the plant cell wall adjacent to bacteria, following the accumulation of membrane bound vesicles within the cytoplasm at reaction sites. Large papillae containing callose accumulated within challenged plant cells. Papillae also formed in the pmr4–1 mutant of Col-0 which lacks an inducible callose synthase but immunocytochemical labelling demonstrated that they contained very little β-1, 3 glucan. Some papillae formed in Col-5 in response to the virulent pathogen DC3000, but they dispersed during cell collapse and lesion formation. Transconjugants of Pph expressing the avirulence genes avrPpiA and avrPphB matching the RPM1 and RPS5 resistance genes, caused rapid and slow HR development, respectively. Although corpse morphology was observed our observations suggest that in Arabidopsis, plant cell death during the HR is programmed but represents a variant of necrosis rather than apoptosis. Cerium chloride staining revealed the accumulation of H₂O₂ at reaction sites. The strongest H₂O₂ response was found during the HR activated by avrPpiA but localised generation of peroxide was also found at sites of papilla deposition next to 1448A or the hrpL mutant. Accumulation of H₂O₂ during the HR, but not during wall alterations, was strongly suppressed by inhibition of NADPH oxidase. The differential effect of the inhibitor suggests an alternative source of H₂O₂ to modify the plant wall. Extension of peroxide-driven cross-linking reactions to bacterial cell walls may contribute to the restriction of bacterial multiplication. The lowest level of H₂O₂ occurred during the compatible reaction to DC3000. Characterisation of the cellular co-ordination of basal (non-host) resistance has revealed several potential targets for bacterial effector proteins.     

Histochemical studies on the accumulation of reactive oxygen species (O2 and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.     

小麦抗白粉病基因Pm2的SSR标记筛选

为筛选与小麦抗白粉病基因Pm2紧密连锁的分子标记,将感病品种Chancellor与Pm2的近等基因系杂交,获得F1、F2分离群体,采用分离群体分组法对Pm2进行了微卫星(microsatellite,又称simple sequence repeats,SSR)标记分析.结果表明,定位于小麦5D染色体上的71对SSR引物中有12对引物能在Pm2的近等基因系、Chancellor间稳定地揭示出多态性差异,7对引物Xcfd189、Xcfd29、Xcfd8、Xcfd102、Xcfd7、Xcfd57和Xgwm190分别能在抗病、感病池间和F2分离群体的抗病、感病单株间稳定地扩增出特异性产物.7对引物所扩增的特异谱带分别为:Xcfd189360、Xcfd29190、Xcfd8160、Xcfd102250、Xcfd7200、Xcfd57245和Xgwm190210,它们与Pm2基因间的遗传距离分别为0、1.5、2.3、5.4、10.2、31.5和54.3 cM,其中标记Xcfd189360与Pm2共分离,标记Xcfd29190、Xcfd8160和Xcfd102250与Pm2紧密连锁,可用于Pm2的标记辅助选择.     

Oxygen metabolism in plant cell culture/bacteria interactions: role of bacterial concentration and H2O2-scavenging in survival under biological and artificial oxidative stress

Preliminary studies with plant cell suspensions indicated that high concentrations of bacterial inoculum can significantly reduce the H₂O₂levels detected immediately following inoculation. Further investigation suggested that bacterial concentration is an important factor in H₂O₂-scavenging, and is often overlooked in both plant/pathogen interactions as well asin vitrosurvival studies.In vitrostudies withPseudomonasspp. characterized the relationship between H₂O₂-scavenging and culturability of bacteria. Because H₂O₂permeates membranes rapidly, the intracellular scavenging activity of the bacteria could be detected as a decrease in the external H₂O₂concentration. Therefore, the effective scavenging activity of a given suspension of bacteria was proportional to the bacterial concentration. The culturability of populations of bacteria exposed to H₂O₂concentrations which are intolerable to individual cells (≥100 μm) was also dependent on bacterial concentration. Studies with a catalase-deficient mutant ofP. putidaindicated that catalase, due to its high K_m, had little effect on scavenging H₂O₂at the lower concentrations of H₂O₂(<100 μm) that are likely to be encountered in suspension cells and other plant tissues. Using high concentrations of bacteria (1×10⁹cfu ml^-1), there was little difference between the catalase-deficient mutant and various isolates ofPseudomonasspp. in the reduction of H₂O₂levels in suspension cells. The studies indicated that an additional scavenging system other than catalase exists in bacteria and this may play an important role in scavenging H₂O₂during plant/bacteria interactions.     

甘肃小麦白粉病抗源材料的筛选及抗病基因库的组建

2002-2005年,对收集到的30份已知抗白粉病基因载体品种在甘肃省的不同生态区进行了抗病性监测,结果表明:Pm1、Pm3 a、Pm3 b、Pm3 c、Pm3 f、Pm4 a、Pm5、Pm6、Pm7、Pm8、Pm17在田间抗病性丧失,失去利用价值;Pm2、Pm19、Pm4+8、Pm4、Pm5+6、Pm13在田间抗病性较低,不宜单独作为亲本利用;Pm1+2+9、Pm2+6、Pm2+mld、Pm2+talent、Pm4+2 x、Pm4 b、Pm4 b+5、Pm20、Pm21、Pmxbd在田间抗性表现良好,在今后的育种工作中应充分加以利用。同时经过4年抗病性监测,从省内外2 638份小麦品种(系)材料中筛选出了抗病性强、综合农艺性状优良的92R137、贵农21等优异材料10余份,初步组建了抗白粉病基因库。文中还对抗病基因现状和利用及今后如何避免由于抗源单一化带来的白粉病流行进行了初步探讨。     

Gene expression profile and physiological and biochemical characterization of hexaploid wheat inoculated with Blumeria graminis f. sp. tritici

Wheat (Triticum aestivum L.) powdery mildew has direct effects on photosynthesis- and energy-related pathways. Because it is prevalent in Sichuan Province, China, knowledge concerning Pm40-mediated wheat powdery mildew resistance is important for understanding host–pathogen interactions. The aim of this study was to understand physiological changes during the host–pathogen interaction, including gene expression, photosynthetic and chlorophyll fluorescence parameters, chlorophyll content, and antioxidant activity, specific to the resistance response to powdery mildew. The Pm40-expressing L693 and Pm40-deficient L1095 wheat lines were employed for comparison analyses. Fifty-eight and 26 expressed sequence tags (ESTs) were obtained from forward and reverse suppression subtractive hybridization cDNA libraries constructed from Bgt-infected L693 and L1095 leaves, respectively, and 69% of the ESTs in the forward library were related to photosynthesis- and energy metabolism-related pathways. Our data indicate that photosynthesis and carbon metabolism as well as nitrogen metabolism related to photosynthesis are the main pathways involved in Pm40-mediated wheat powdery mildew resistance and that an acetyl CoA-like gene might be a key regulatory factor in these pathways. This study provides new insight into the physiological and genetic mechanisms of resistance to wheat powdery mildew.     

<Emphasis Type="Italic">Sunflower Chlorotic Mottle Virus</Emphasis> in Compatible Interactions with Sunflower: ROS Generation and Antioxidant Response

Sunflower chlorotic mottle virus (SuCMoV) is a recently described potyvirus that causes systemic infections in sunflower plants leading to chlorotic mottling and important growth reductions and yield losses. Oxidative damage is expressed after symptom development in this host-pathogen combination. The involvement of antioxidant enzyme activities in disease susceptibility was studied in two sunflower lines differing in the intensity and rate of development of SuCMoV infections: L2 is more susceptible than L33. A transient superoxide production peak was detected in leaves of both lines before symptom development. H₂O₂ accumulation increased before symptom expression in infected plants of L33 but in L2 such increase was registered only after symptoms became evident. In healthy plants of both lines, superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) showed similar activity levels. In inoculated plants of line L2, but not in L33, SOD and CAT activities increased significantly before the appearance of symptoms, and APX increases were detected later. A 1 mM SA treatment effectively decreased SuCMoV accumulation in plants of L2 but it did not affect it in L33. This treatment increased H₂O₂ accumulation and prevented the increase in antioxidant enzyme activities in infected plants of L2. It is suggested that increases in antioxidant enzyme activities interrupted the signals generated by the increase in ROS, which may have otherwise triggered defence reactions in the host and thus, resulted in a compatible interaction.     

Hypersensitive reaction and P/HRGP accumulation is modulated by nitric oxide through hydrogen peroxide in pearl millet during Sclerospora graminicola infection

Priming of pearl millet seedlings with nitric oxide (NO) donors sodium nitroprusside (SNP) and S-nitrosoglutathione (SNOG) induced hypersensitive reactions (HR) and accumulation of Proline/Hydroxyproline-rich glycoprotein (P/HRGP) during infection by downy mildew pathogen Sclerospora graminicola. Such defense responses were specifically altered by concentration of NO donors resulting in the modulation of endogenous NO in seedling tissues. The stoichiometric interactions of NO and hydrogen peroxide (H₂O₂) when followed in relation to HR and P/HRGP accumulation, the degree of defense response varied with H₂O₂ level, the latter being largely influenced by NO concentration. Therefore, balancing NO and H₂O₂ is vital for optimum expression of defense responses for imparting disease resistance.     

葡萄应答霜霉病过程中多磷酸肌醇激酶基因与过氧化氢的作用机制

以对霜霉病不同抗性的葡萄品种左优红和霞多丽为材料,利用分子生物学和植物生理学试验手段,结合药理学试验,探讨葡萄在应答霜霉病过程中葡萄多磷酸肌醇激酶基因（VvIPK2）和H2O2的作用机制。接种霜霉病菌后15 h葡萄叶片VvIPK2表达量是正常水平的12倍,接种后3 hH2O2含量达最大值,同时苯丙氨酸解氨酶和几丁质酶活性升高;多磷酸肌醇激酶（IPK2）抑制剂、外源H2O2及H2O2清除剂均能改变霜霉病菌所引起的抗性葡萄品种左优红叶片PAL和几丁质酶活性的变化,同时可以影响不同抗性品种叶片的感病情况;IPK2抑制剂对葡萄霜霉病菌引起的H2O2水平变化没有影响;清除H2O2可减弱葡萄霜霉病菌对VvIPK2表达量的诱导效应。研究表明H2O2位于IPK2的上游,通过调控PAL和几丁质酶活性参与葡萄应答霜霉病过程。     

Histochemical studies on the accumulation of reactive oxygen species (O2− and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.     

陕西省小麦白粉菌群体毒性结构及遗传多样性

为明确近年来陕西省小麦白粉菌Blumeria graminis f. sp. tritici群体毒性及遗传变异情况,利用32份已知抗白粉病基因小麦品种(系)对2013年和2014年陕西省小麦白粉菌群体毒性结构进行测定,并应用扩增片段长度多态性(amplified fragment length polymorphism,AFLP)标记对2014年陕西省西安市、咸阳市、渭南市、宝鸡市及陕西省毗邻的甘肃省天水市5个小麦白粉菌地理群体共118株白粉菌菌株进行遗传多样性分析。毒性监测结果显示,供试小麦白粉菌群体对Pm1c、Pm2、Pm3d、Pm4a、Pm4b、Pm5b、Pm13、Pm21、Pm24、Pm30、Pm2+6、Pm2+Mld、Pm2+6+?、Pm4b+5b、Pm4+?、Pm5+6的平均毒性频率在0～17.23%之间,表明这些基因抗性保持较好;对Pm1a、Pm3b、Pm3c、Pm3e、Pm3f、Pm7、Pm8、Pm19、Pm1+2+9的平均毒性频率介于69.17%～99.60%之间,表明这些基因抗性已丧失。用筛选出的6对AFLP标记共扩增出831个多态性位点,多态性位点百分比为94.86%;小麦白粉菌群体遗传多样性指数和香农信息指数分别为0.1151和0.2036,遗传变异主要来源于群体内变异。群体间基因流为4.7939,表明5个小麦白粉菌群体间基因交流广泛。群体间遗传距离和地理距离两者显著正相关。     

Antioxidant,ethylene and membrane leakage responses to powdery mildew infection of near-isogenic barley lines with various types of resistance

Leaves of powdery mildew-susceptible barley (Hordeum vulgare cv. Ingrid) and related near-isogenic lines bearing various resistance genes (Mla12, Mlg or mlo5) were inoculated with Blumeria graminis f. sp. hordei race A6. Fungal attack induced several-fold increases in ethylene emission and electrolyte leakage in leaves of susceptible Ingrid beginning 3 days after inoculation. Activities of peroxidase, superoxide dismutase, glutathione S-transferase, ascorbate peroxidase and glutathione reductase enzymes were induced markedly in susceptible leaves 5–7 days after inoculation. Similar, but less pronounced pathogen-induced changes were detected in inoculated leaves of Mla-type resistant plants that show hypersensitive cell death upon inoculation, and, to an even lesser extent, in the Mlg and mlo lines, where no visible symptoms accompanied the incompatible interaction. Glutathione content increased only in susceptible barley 7 days after inoculation. Catalase activity, total ascorbate content and redox state were not influenced by inoculation in any of the genotypes. The activity of dehydroascorbate reductase was significantly reduced 3–5 days after inoculation in the susceptible parental plants and after 5 days in Mla and Mlg lines, while it was stable in the mlo barley. Slightly elevated levels of H₂O₂ were observed in the inoculated resistant plants. In contrast, H₂O₂ content decreased in the susceptible line 7 days after pathogen attack. These data indicate that high levels of antioxidants are involved in the compatible interaction of susceptible barley and powdery mildew by protecting the pathogen from oxidative damage.     

陕西省小麦白粉菌群体毒性结构及遗传多样性

 为明确陕西省小麦白粉菌群体的毒性频率和遗传多样性,利用34个含有已知抗白粉病基因的小麦品种(系)和5对多态性ISSR分子标记,分别对2016年渭南、西安、咸阳、宝鸡、汉中和安康等6市的15个乡镇160个小麦白粉菌单孢子堆菌株进行毒性频率分析。结果显示:供试小麦白粉菌群体对Pm1、Pm2、Pm3b、Pm3c、Pm3e、Pm3f、Pm6、Pm7、Pm8、Pm19和Pm1+2+19的毒性频率在60%~100%之间,表明这些抗性基因已丧失抗性,在生产上已经丧失利用价值,对Pm4b、Pm24、Pm2+6、Pm2+Mld、Pm2+6+?、Pm4b+Mli、Pm"Era"、Pm"XBD"和Pm21的毒性频率低于20%,表明这些抗性基因抗性良好,可在生产中利用。选取其中93个单孢子堆菌株进行遗传多样性分析,结果表明白粉菌地理群体间遗传距离在0.020 4~0.103 7之间,其中宝鸡和渭南群体的遗传距离最近,汉中和咸阳群体的遗传距离最远。群体间遗传变异占总体变异的12.82%,群体内遗传变异占87.12%,表明遗传变异主要来自于群体内。Mantel Test分析表明,小麦白粉菌群体间遗传距离与地理距离相关性不大。     

Differential necrotic lesion formation in soybean cultivars in response to soybean mosaic virus

In this study, we examined the necrosis phenotype on leaves of two cultivars of soybean (ZheA8901 and Nannong1138-2) that show varying level of resistance to soybean mosaic virus (SMV). The necrotic symptoms seen on inoculated and systemic leaves of soybean cultivar ZheA8901 were reminiscent of programmed cell death (PCD). The cell death phenotypes were evaluated using the TUNEL method, quantification of hydrogen peroxide (H₂O₂) and salicylic acid, callose production, as well as by monitoring expression of defence genes GmPR-1 and GmNPR1. Our results show that SMV inoculation induced PCD on ZheA8901 is associated with rapid increase in H₂O₂, increased SA and callose accumulation and higher defence gene expression.     

Identification of wheat blue dwarf phytoplasma effectors targeting plant proliferation and defence responses

The identification of effectors from pathogenic microbes is one of the most important subjects for elucidating infection mechanisms. Wheat blue dwarf (WBD) phytoplasma causes dwarfism, witches' broom, and yellow leaf tips in wheat plants, resulting in severe yield loss in northwestern China. In this study, 37 candidate effector proteins were transiently expressed in Nicotiana benthamiana. Plants expressing the SAP11‐like protein SWP1 exhibited typical witches' broom. Interestingly, another protein, SWP11, induced both cell death and defence responses, including H₂O₂ accumulation and callose deposition. Analysis by qRT‐PCR was used to show that a marker gene of the hypersensitive response, HIN1, and three pathogenesis‐related genes, PR1, PR2 and PR3, were significantly up‐regulated in leaves of N. benthamiana expressing SWP11. In addition, SWP12 and SWP21 (TENGU‐like) were shown to suppress SWP11‐, BAX‐, and/or INF1‐induced cell death. These results indicated that SWP21 has a distinct role in virulence compared with TENGU and that WBD phytoplasma possesses effectors that target plant proliferation and defence responses. The ability of these effectors to trigger or suppress plant immunity provides new insights into the phytoplasma–plant interaction.     

Silicon induced resistance against powdery mildew of roses caused by Podosphaera pannosa

Powdery mildew, caused by Podosphaera pannosa, is a very common disease in greenhouse potted roses, resulting in poor marketing value and hence economic losses. Alternatives to chemical control are necessary, and therefore the ability of silicon (Si) applied to roots to control the disease was investigated, as well as the mechanisms behind the observed disease reductions. Four genotypes of miniature potted roses representing different genetic backgrounds and susceptibility to disease were studied. Plants were watered with a nutrient solution containing either 3·6 mm Si (100 ppm) supplied as K₂SiO₃ (Si+) or no Si (Si?) before inoculation with P. pannosa. Si application increased leaf Si content two‐ to four‐fold compared to control plants. Confocal microscopy showed that Si deposition was larger in Si+ than in Si? plants and that deposition mainly occurred in the apoplast, particularly in epidermal cell walls. Si application delayed the onset of disease symptoms by 1–2 days and disease severity was reduced by up to 48·9%. The largest reduction was found in the two most resistant genotypes, which also had the highest increase in Si uptake. The Si‐induced disease protection was accompanied by increased formation of papillae and fluorescent epidermal cells (FEC) as well as deposition of callose and H₂O₂, especially at the sites of penetration and in FEC, which are believed to represent the hypersensitive response. Si treatment reduced powdery mildew development by inducing host defence responses and can therefore be used as an effective eco‐friendly disease control measure.     

Bentazone and bromoxynil induce H and H2O2 accumulation, and inhibit photosynthetic O2 evolution in Synechococcouselongatus PCC7942

Using a unicellular cyanobacterium, Synechococcouselongatus PCC7942, we have shown that cytosolic acidification, O₂⁻; H₂O₂ production and photosystem II-inactivation are the causes of cell death from bentazone/bromoxynil incubations. Butyric acid evoked solely pH lowering response and yet inhibited PS II activity indicating that herbicide-caused acidification is sufficient to kill the cyanobacterial cells, but other factors like excess H₂O₂ production due to an imbalance in the peroxide sequestration machinery might be contributory. While the activities of superoxide dismutase and pyrogallol peroxidase increased consequent to herbicide incubations and displayed oligomeric states with mobility shift, catalase and glutathione peroxidase though present remained insensitive.     

BTH处理对甜瓜叶片活性氧代谢的诱导作用

为了明确活性氧（reactive oxygen species,ROS）代谢在甜瓜抗病性诱导中的作用,以抗白粉病甜瓜品种Tam Dew和感病品种卡拉克赛幼苗为材料,通过盆栽试验研究了苯丙噻二唑（BTH）喷雾或白粉菌接种后甜瓜叶片超氧阴离子（O2.-）产生速率、过氧化氢（H2O2）含量及超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、苯丙氨酸解氨酶（PAL）活性的变化。BTH处理或白粉菌接种均可诱导甜瓜叶片SOD、PAL活性升高,抑制CAT活性,导致叶组织O2.-产生速率和H2O2含量增加,BTH喷雾＋白粉菌接种比二者单独处理效果更好。结果表明,BTH处理后叶片O2.-产生速率提高和H2O2积累是甜瓜抗白粉病能力提高的重要机制,BTH通过诱导ROS代谢酶活性调节H2O2含量,且BTH诱导的甜瓜抗病性与品种的基础抗性有关。