RT-PCR based detection of resistance conferred by an insensitive GS in glufosinate-resistant maize cell lines

Selection of maize (Zea mays L.) cell lines resistant to glufosinate was carried out using cell suspension cultures induced from an embryo. The cell suspension was cultured on MS liquid medium supplemented with 150 mg/l asparagine, 1 mg/l thiamine, 3 mg/l 2,4-D and 2% sucrose (pH 5.8) and treated with different concentrations of glufosinate herbicide. In vitro selection from these cell suspension cultures resulted in the identification of a maize cell line resistant to glufosinate. The resistance index of the resistant cell line was 120-fold more than the normal cells. Moreover, the resistance of the cell line remained stable for at least 6 months when kept in herbicide-free medium. Glutamine synthetase (GS) activity in the resistant cells was higher than the normal cells by about 2.30-fold at 5 days after treatment of 10⁻⁶ M glufosinate. The low sensitivity of GS in resistant cells resulted in the non-inhibition of the enzyme activity, hence, the high resistance of the cell line to the herbicide. Using RT-PCR to amplify the GS mRNA, a substitution of 12 nucleotides was observed in resistant maize cells compared to that of normal cells. Conceptual translation of this sequence shows a substitution of 10 amino acids in the GS protein sequence from Genbank database of NCBI. The GS gene sequence of the newly identified glufosinate-resistant maize cell was submitted to Genbank and was given the Accession No. AY339214. The results of this study indicate a functional role of mutation in the evolution of glufosinate-resistant plants. It also supports the view that glufosinate resistance in plants was primarily due to the alteration of GS making it less sensitive to inhibitory effect of the herbicide.     

Fully "Recombinant Enzyme-Linked Immunosorbent Assays" Using Genetically Engineered Single-Chain Antibody Fusion Proteins for Detection of Citrus tristeza virus

ABSTRACT Recombinant single-chain variable fragment antibodies (scFv) that bind specifically to Citrus tristeza virus (CTV), which cause the most detrimental viral disease in the citrus industry worldwide, were obtained from the hybridoma cell lines 3DF1 and 3CA5. These scFv were genetically fused with dimerization domains as well as with alkaline phosphatase, respectively, and diagnostic reagents were produced by expressing these fusion proteins in bacterial cultures. The engineered antibodies were successfully used for CTV diagnosis in plants by tissue print enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich-ELISA. The fully recombinant ELISAs were as specific and sensitive as conventional ELISAs performed with the parental monoclonal antibodies, showing the usefulness of recombinant antibodies for routine detection of a virus in woody plants for the first time.     

Resistance to Plum pox virus in plants expressing cytosolic and nuclear single‐chain antibodies against the viral RNA NIb replicase

The expression of engineered single‐chain variable fragments specific to the NIb RNA replicase of Plum pox virus (PPV) (scFv2A) in transgenic plants was successfully used as a strategy to interfere with viral infection. Different scFv2A fusion proteins were constructed to target those subcellular compartments, such as the cytosol, endoplasmic reticulum (ER) membrane structures and the nucleus, where NIb protein presumably accumulates. Several transgenic lines of Nicotiana benthamiana plants expressing the scFv2A targeted to the cytosol (2A lines), ER (6K2 lines) and nucleus (NLS lines) were obtained. The protective effect of scFv expression was determined by mechanical virus inoculation in five 2A, three 6K2 and four NLS transgenic lines. The strongest resistance was afforded with the 2A‐3 (six non‐infected plants out of 10), 6K2‐1 (17 out of 33) and NLS‐11 (16 out of 19) transgenic lines. The success of this interference with PPV infection opens new possibilities for the control of this RNA virus and could be exploited not only to confer resistance in transgenic plants, but also to elucidate the role of the non‐structural NIb protein in different cell compartments during viral infection.     

Generation and evaluation of movement protein‐specific single‐chain antibodies for delaying symptoms of Tomato spotted wilt virus infection in tobacco

This study investigated whether single‐chain antibodies (scFvs) specific for a viral movement protein could accumulate in the plant cell cytosol and restrict viral systemic infection in plants. Nine chicken scFv fragments against the Tomato spotted wilt virus (TSWV) movement protein (NS_M) were isolated by phage display. Soluble scFvs were produced in bacteria and the NS_M binding activity of purified scFvs was confirmed. The nine scFv genes were cloned into a plant expression vector enabling recombinant protein accumulation in the plant cell cytosol. Immunoblot analysis demonstrated that two of the nine chicken scFvs accumulated to high levels (5·9 and 8·0% of total soluble protein). Bioassays of viral infection using transgenic tobacco plants producing NS_M‐specific chicken scFvs showed delayed symptom development when compared to non‐transgenic control plants, indicating that expression of antibodies recognizing the TSWV movement protein is a potential strategy for generating resistant plants.     

Improved method for assaying maize plant resistance to maize rough dwarf disease by artificial inoculation and real-time RT-PCR

The study of maize rough dwarf disease (MRDD) and breeding for resistance requires inoculation of maize plants by means of planthoppers. The plant age, insect density and inoculation duration are main factors in the success of maize rough dwarf disease inoculation. These parameters were tested using a susceptible maize inbred line Ye478. Using one or two-leaf plants, 15 planthoppers per plant and a five day inoculation duration, the line Ye478 was the most susceptible with 100% diseased plants; F112132 was moderately susceptible with 60% diseased plants and 90110 and F022411 were resistant without any disease. The results were consistent with those from six years of field studies. Using enzyme-linked immunosorbent assay (ELISA) and real-time quantitative RT-PCR, rice black-streaked dwarf virus was detected in severely diseased plants. The plants were rated from 0 to 3 according to their symptoms at the time of flowering. Plants scoring 0, 1 and 2 could not be distinguished by ELISA, only by real-time quantitative RT-PCR. All of the plants with a score of 3 were positive by ELISA and real-time quantitative RT-PCR. The significant differences in the average viral contents in plants with different symptom ratings could be distinguished by using real-time RT-PCR.     

抗对硫磷单链抗体在昆虫细胞中的表达及活性鉴定

利用Bac-to-Bac杆状病毒表达系统,在Sf9昆虫细胞中表达抗对硫磷单链抗体,并评价该重组抗体scFv-4C6的分子识别活性。以分泌能特异性识别对硫磷的单克隆抗体的杂交瘤细胞株4C6为RNA来源,采用RT-PCR方法扩增抗体的重链和轻链可变区基因,经重叠延伸PCR方法串联拼接获得单链抗体基因片段(scFv-4C6)。构建包含目的片段的重组杆粒Bacmid-scFv-4C6,转染Sf9细胞表达目的蛋白,采用免疫印迹法(Western blotting)检测表达产物,间接竞争酶联免疫吸附(ic-ELISA)法评价产物的生物活性。结果表明:scFv-4C6基因片段拼装正确,成功转染Sf9细胞,并在转染后72 h表达量最高,表达的单链抗体大小为28.3 kD;表达产物能特异性识别对硫磷,IC50值为7.9 ng/mL,对甲基对硫磷和杀螟硫磷分别有12%和1.8%的交叉反应率,与亲本单克隆抗体的识别性能相似。该研究表明,具有生物活性的抗对硫磷单链抗体scFv-4C6可在昆虫细胞中成功得到表达。     

Induction of phenylalanine ammonia-lyase (PAL) in insect damaged and neighboring undamaged cotton and maize seedlings

There has been scepticism about plants responding to information from their damaged neighbor plants since it was first reported, although a growing body of evidence indicates that interplant communication may play a key role in the resistance of plants. Cotton aphids and corn borer are important insects in cotton and maize, respectively. The resistance of healthy cotton and maize plants induced by their neighbor damaged plants was investigated in this study. Phenylalanine ammonia-lyase (PAL) is a chemical marker of induced resistance in many plants. The enzymatic activity and gene expression of PAL were determined in cotton and maize seedlings damaged by wounding, aphid and corn borer attack and in intact undamaged maize and cotton seedlings growing nearby. The enzymatic activity and gene expression of PAL were significantly increased in the cotton and corn seedlings damaged by wounding or by aphid and corn borer. Furthermore, undamaged neighboring cotton and maize seedlings also showed elevated enzymatic activity and gene expression of PAL. These inductions of PAL enzymatic activity and gene expression in the damaged and undamaged seedlings suggest inter-species communication between cotton and maize.     

高温处理西花蓟马若虫对其雌成虫寿命、繁殖力及后代发育的影响

温度是影响昆虫生长发育的重要生态因素之一,高温逆境作为一种绿色防控手段可以有效控制温室害虫种群发展。为了探究高温处理西花蓟马若虫对其种群发展的影响,本试验用41℃和43℃两个温度分别处理西花蓟马初孵若虫2、6、12、24和36h后,观察并记录其雌成虫寿命、繁殖力及后代发育指标的变化情况。结果表明,随着处理时间的延长,两个高温处理后西花蓟马雌成虫寿命,子代若虫总数、成虫总数和总存活率(若虫发育到成虫的存活率)均显著下降,后代雌雄性比呈总体下降趋势。41℃处理后,性比从2.30∶1降低到2.13∶1;43℃处理后从2.25∶1降低到2.07∶1,均明显低于对照的2.69∶1。另外,两性生殖种群相比于孤雌生殖种群更易遭受高温处理的影响。     

Application of Phage Display in Selecting Tomato spotted wilt virus-Specific Single-Chain Antibodies (scFvs) for Sensitive Diagnosis in ELISA

ABSTRACT A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S, which allowed the scFvs to be expressed as alkaline phosphatase fusion proteins, 17 different scFv antibodies were obtained. Of these, 12 scFvs were directed against the nucleoprotein (N) and 5, putatively, against the glycoproteins (G1 and G2). Five of the N-specific antibodies cross-reacted with two other tospoviruses (Tomato chlorotic spot virus and Groundnut ringspot virus), but none recognized the more distantly related tospoviruses Impatiens necrotic spot virus, Watermelon silverleaf mottle virus, Iris yellow spot virus, or Physalis severe mottle virus. The successful use of one of the antibodies as coating and detection reagent in a double-antibody sandwich enzyme-linked immunosorbent assay showed the potential of the phage display system in obtaining antibodies for routine TSWV diagnosis.     

Fusion proteins of single-chain variable fragments derived from phage display libraries are effective reagents for routine diagnosis of potato leafroll virus infection in potato

ABSTRACT A panel of 11 different single-chain variable fragment antibodies (scFv) that bind to potato leafroll virus (PLRV) has been studied to assess each one's suitability as practical diagnostic tools. The scFv, previously obtained from naive phage display libraries, were expressed in Escherichia coli as fusion proteins. The fusion proteins comprised scFv joined to either the human light chain kappa constant domain (C(L)), an amphipathic helix (Zip), a combination of C(L) and Zip, or alkaline phosphatase (AP/S). The fusion proteins were tested for their ability to detect, or trap on enzymelinked immunosorbent assay (ELISA) plates, PLRV in extracts of infected potato leaves. The tests done with the different scFv fusion proteins were compared with a standard triple-antibody sandwich (TAS)-ELISA that employs a rabbit polyclonal antibody preparation to coat microtiter plates and a monoclonal antibody, SCR3, to detect PLRV. Of 11 scFvC(L) fusion proteins, 7 detected PLRV as readily as SCR3 when used as detecting antibodies in TAS-ELISA. The limit of detection of purified PLRV for the different scFvC(L) fusion proteins ranged from 250 to 5 ng/ml; that for SCR3 is 5 ng/ml. Of the 11 scFv, 4 cross-reacted with some other luteoviruses. Several scFvC(L) and scFvC(L)Zip fusion proteins trapped PLRV from extracts of infected potato leaves as effectively as the polyclonal antibody preparation. Four scFv fusion proteins were used in a stem print assay to detect PLRV, and the results were similar to those obtained in tests using SCR3. The scFvC(L) fusion proteins retained activity for at least 6 months at 4 degrees C, and all scFv fusion proteins were fully active on reconstitution after lyophilization. A fully recombinant ELISA was devised that detected PLRV in extracts of infected potato, with results comparable to those obtained using the standard TAS-ELISA. The advantages of using scFv fusion proteins for the routine detection of plant viruses include the ability to produce large quantities of reagents cheaply in bacterial fermenters and to incorporate them into standardized tests.     

Boosting silica levels in wheat leaves reduces grazing by rabbits

Systemic application of sodium silicate can significantly enhance the levels of leaf silica in winter wheat (Triticum aestivum L. cv. Mercia), suggesting that this material could reduce the palatability of plants to vertebrate herbivores (e.g. rabbits, Oryctolagus cuniculus L.). A bioassay was developed using hydroponically grown wheat plants. Plants treated with sodium silicate were significantly more resistant to grazing by wild rabbits than untreated plants, with severe, potentially lethal feeding damage being reduced by over 50%. Further studies were carried out to develop more practical techniques for boosting silica levels in plants using silicon-rich 'fertilisers' including calcium silicate and calcium silicate slag (CSS). Silica levels were elevated in the plant 1.9-2.8 times over the control through the application of various silicon materials, in line with those of the hydroponic treatment. Encouragingly, levels of silica were elevated even in young wheat plants, which are most vulnerable to rabbit damage, and in a range of wheat varieties. The use of CSS is particularly promising because of its lower cost in comparison with calcium silicate, and it has a proven track record in slag fertilisation of rice and sugar cane crops. At the optimum CSS application rate of 3 g silicon L(-1) soil, wheat silica levels were approximately doubled, with no detrimental impacts on long-term growth or yield.     

Identification of a Maize Kernel Pathogenesis-Related Protein and Evidence for Its Involvement in Resistance to Aspergillus flavus Infection and Aflatoxin Production

ABSTRACT Aflatoxins are carcinogens produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops such as maize. Several aflatoxin-resistant maize genotypes have been identified and kernel proteins have been suggested to play an important role in resistance. In the present study, one protein (#717), which was expressed fivefold higher in three resistant lines compared with three susceptible ones, was identified using proteomics. This protein was sequenced and identified as a pathogenesis-related protein (PR-10) based on its sequence homology. To assess the involvement of this PR-10 protein (ZmPR-10) in host resistance of maize against fungal infection and aflatoxin production, the corresponding cDNA (pr-10) was cloned. It encodes a protein of 160 amino acids with a predicted molecular mass of 16.9 kDa and an iso-electric point of 5.38. The expression of pr-10 during kernel development increased fivefold between 7 and 22 days after pollination, and was induced upon A. flavus infection in the resistant but not in the susceptible genotype. The ZmPR-10 overexpressed in Escherichia coli exhibited a ribonucleolytic and antifungal activities. Leaf extracts of transgenic tobacco plants expressing maize pr-10 also demonstrated RNase activity and inhibited the growth of A. flavus. This evidence suggests that ZmPR-10 plays a role in kernel resistance by inhibiting fungal growth of A. flavus.     

Expression of Oryzacystatin I and II in Alfalfa Increases Resistance to the Root-Lesion Nematode

ABSTRACT Digestive cysteine proteinases have been isolated from plant-parasitic nematodes as well as coleopteran and hemipteran insects. Phytocystatins, inhibitors of cysteine proteinases, are found in a number of plants where they may play a role in defense against pathogens and pests. The cDNAs of the phytocystatins from rice, oryzacystatin I (OC-I) and oryzacystatin II (OC-II), were expressed in alfalfa (Medicago sativa) plants under the control of the potato protease inhibitor II (PinII) promoter and the plants were evaluated for resistance to the root-lesion nematode (Pratylenchus penetrans). A PinII-beta-glucuronidase (GUS) gene was introduced into alfalfa to determine the pattern of gene expression from this promoter. Constitutive GUS expression was observed in leaf and root vascular tissue, and in some plants, expression was observed in leaf mesophyll cells. Mechanical wounding of leaves increased GUS expression approximately twofold over 24 h. Inoculation with root-lesion nematodes resulted in localized GUS expression. Populations of root-lesion nematodes in alfalfa roots from one line containing the PinII::OC-I transgene and one line containing the PinII::OC-II transgene were reduced 29 and 32%, respectively, compared with a transgenic control line. These results suggest that oryzacystatins have the potential to confer increased resistance to the root-lesion nematode in alfalfa.     

Evaluation of Maize Inbreds for Maize stripe virus and Maize mosaic virus Resistance: Disease Progress in Relation to Time and the Cumulative Number of Planthoppers

ABSTRACT Five tropical maize lines were tested and compared with the susceptible control line B73 for resistance to Maize stripe virus (MStV) and Maize mosaic virus (MMV), both propagatively transmitted by the planthopper Peregrinus maidis (Homoptera: Delphacidae). Resistance to each virus was evaluated separately by artificial inoculations with planthoppers viruliferous for either one virus or the other. Disease incidence and symptom severity progression were quantified in relation to time and the cumulative number of planthoppers. Line Hi40 was found to be susceptible to MStV and highly resistant to MMV. Generally, no MMV symptoms developed on Hi40, even under intense inoculation pressure by a large number of viruliferous planthoppers. Line Rev81 showed a partial but strong resistance to MStV, which mainly reduced disease incidence. Nevertheless, this resistance to MStV was the highest ever reported and held up, even when challenged by large numbers of planthoppers. The percentage of infected plants in line Rev81 never exceeded 30 to 40% in our experiments. Moderate levels of resistance to MStV, and to a lesser extent MMV, were found in lines 37-2, A211, and Mp705. However, resistance in these lines was completely overcome using a large number of insects transmitting either of the two viruses. These results suggest that different types of resistance to MMV and MStV are available in maize lines from Caribbean and Mascarene germ plasm. The expression of virus-specific resistance identified in Hi40 and Rev81 lines was not affected by intense inoculation pressure. In contrast, the moderate resistance in 37-2, A211, and Mp705 was partially effective against both viruses but not at high inoculation pressure. These different types of resistance, when present in the same genotype, could provide protection against both viruses.     

甘蔗花叶病毒VPg蛋白的原核表达及其在玉米中的免疫定位

 利用RT-PCR方法从感染甘蔗花叶病毒北京分离株(SCMV-BJ)的玉米叶片中扩增得到VPg基因,将VPg基因连接到原核表达载体pET-28a上。获得的重组子转化大肠杆菌BL21(DE3)后,用IPTG进行诱导表达。SDS-PAGE分析表明,VPg在大肠杆菌中获得了高效表达,产生的融合蛋白分子量为30 kD。将融合蛋白纯化后免疫兔子获得了特异性较高的抗血清。ACP-ELISA测定抗血清的效价为1/4096。利用该抗血清对健康玉米和病毒侵染的玉米茎、叶脉和叶片进行免疫金标记,结果表明在茎和叶脉的韧皮部筛管的细胞壁处有金颗粒。     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Chemical control of maize late wilt in the field

Late wilt, a severe vascular disease of maize caused by the fungus Harpophora maydis, is characterized by relatively rapid wilting of maize plants before tasseling and until shortly before maturity. In Israel, the disease has become a major problem in recent years. The pathogen is currently controlled using cultivars of maize having reduced sensitivity. In an earlier work, we modified a molecular method for use as a diagnostic tool to evaluate disease progression in field-infested plants and showed that several fungicides suppressed H. maydis in vitro. Here, we examine the effect of different fungicides on disease progression in a contaminated maize field in the spring and summer of 2009 and 2010. The field was watered using a drip irrigation line for each row and the fungicides were injected directly into the drip line. One of the four fungicides tested, azoxystrobin, was highly effective compared with the control, inhibited the development of wilt symptoms and recovered cob yield by 100%. Although this is the first success in preventing disease symptoms in infested fields in Israel, the azoxystrobin treatment did not reduce the amounts of pathogen DNA in host tissues or delay its spread. Attempts to reduce concentrations of this fungicide or to apply it by spraying were less effective than the triple full dosage treatment. The presence of the pathogen in the host tissues of the successfully treated plants and its ability to undergo pathogenic variations are increasing the risk of pathogen resistance and the urgent need to develop new ways of controlling late wilt.     

玉米茉莉酸信号途径参与抵抗玉米褪绿斑驳病毒的侵染

为研究茉莉酸（jasmonic acid, JA）信号途径在玉米响应玉米褪绿斑驳病毒（maize chloroticmottle virus, MCMV）侵染中的作用,利用外源喷施茉莉酸甲酯 （methyl jasmonate, MeJA）方法,采用病毒诱导的基因沉默技术以及玉米原生质体过表达探究JA信号途径是否参与玉米抗MCMV侵染。结果表明,相比于对照, MCMV在外源喷施MeJA的玉米植株上引起的褪绿和花叶症状明显减轻, MCMV基因组RNA积累水平下降了69%,外壳蛋白 （coat protein, CP）积累水平下降了43%,表明MeJA处理提升了玉米植株对MCMV的抗性。在沉默JA信号途径抑制基因 ZmJAZ5的玉米植株上,相比于对照植株, MCMV引起的褪绿及花叶症状也明显减轻, MCMV的基因组RNA积累水平下降了71%, CP积累水平下降了56%。在玉米原生质体中过表达ZmJAZ5后,与对照相比, MCMV基因组RNA积累水平上升了1.58倍, CP积累水平上升了1.34倍。表明JA信号途径在玉米抵抗MCMV侵染过程中发挥着关键作用。