Exploiting the Combination of Natural and Genetically Engineered Resistance to Cassava Mosaic and Cassava Brown Streak Viruses Impacting Cassava Production in Africa

Cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) are currently two major viral diseases that severely reduce cassava production in large areas of Sub-Saharan Africa. Natural resistance has so far only been reported for CMD in cassava. CBSD is caused by two virus species, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). A sequence of the CBSV coat protein (CP) highly conserved between the two virus species was used to demonstrate that a CBSV-CP hairpin construct sufficed to generate immunity against both viral species in the cassava model cultivar (cv. 60444). Most of the transgenic lines showed high levels of resistance under increasing viral loads using a stringent top-grafting method of inoculation. No viral replication was observed in the resistant transgenic lines and they remained free of typical CBSD root symptoms 7 month post-infection. To generate transgenic cassava lines combining resistance to both CBSD and CMD the hairpin construct was transferred to a CMD-resistant farmer-preferred Nigerian landrace TME 7 (Oko-Iyawo). An adapted protocol allowed the efficient Agrobacterium-based transformation of TME 7 and the regeneration of transgenic lines with high levels of CBSV-CP hairpin-derived small RNAs. All transgenic TME 7 lines were immune to both CBSV and UCBSV infections. Further evaluation of the transgenic TME 7 lines revealed that CBSD resistance was maintained when plants were co-inoculated with East African cassava mosaic virus (EACMV), a geminivirus causing CMD. The innovative combination of natural and engineered virus resistance in farmer-preferred landraces will be particularly important to reducing the increasing impact of cassava viral diseases in Africa.     

Silencing Homologous RNA Recombination Hot Spots with GC-Rich Sequences in Brome Mosaic Virus

It has been observed that AU-rich sequences form homologous recombination hot spots in brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants (P. D. Nagy and J. J. Bujarski, J. Virol. 71:3799–3810, 1997). To study the effect of GC-rich sequences on the recombination hot spots, we inserted 30-nucleotide-long GC-rich sequences downstream of AU-rich homologous recombination hot spot regions in parental BMV RNAs (RNA2 and RNA3). Although these insertions doubled the length of sequence identity in RNA2 and RNA3, the incidence of homologous RNA2 and RNA3 recombination was reduced markedly. Four different, both highly structured and nonstructured downstream GC-rich sequences had a similar “homologous recombination silencing” effect on the nearby hot spots. The GC-rich sequence-mediated recombination silencing mapped to RNA2, as it was observed when the GC-rich sequence was inserted at downstream locations in both RNA2 and RNA3 or only in the RNA2 component. On the contrary, when the downstream GC-rich sequence was present only in the RNA3 component, it increased the incidence of homologous recombination. In addition, upstream insertions of similar GC-rich sequences increased the incidence of homologous recombination within downstream hot spot regions. Overall, this study reveals the complex nature of homologous recombination in BMV, where sequences flanking the common hot spot regions affect recombination frequency. A replicase-driven template-switching model is presented to explain recombination silencing by GC-rich sequences.     

CMV抑制PVYN CP基因沉默及其介导的抗病性

以前曾报道用RNA介导的抗病毒策略,获得了高度抗病的表达马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)的转基因烟草,并对T1、T2代转基因植株进行了遗传和抗病性分析。此次以T,代转基因植株为试验材料,在筛选高度抗病植株并证明其抗病性是基于转基因沉默的基础上,采用Northern杂交的方法,证明CMV侵染抑制了转基因植株中PVY^N CP基因的沉默,而且CMV对PVY^N CP基因沉默的抑制部位是发生在接种后的新生叶上,接种叶及其下部叶片中PVY^N CP基因沉默则未受到影响。采用ELISA方法对CMV PVY^N复合接种的转基因植株进行PVY^N检测,结果表明,接种叶及下部叶没有检测到PVY^N,植株叶片对PVY^N表现为抗病。而在CMV接种后植株新生叶中则检测出了高滴度的PVY^N,植株叶片对PVY^N表现为感病。该文报道了在表达PVY^N CP基因的RNA介导抗性转基因植株中,异源病毒侵染抑制了转基因的沉默,并导致转基因植株的抗病性丧失。     

African Cassava Mosaic Virus (ACMV): Stability of Purified Virus and Improved Conditions for its Detection in Cassava Leaves by ELISA

African Cassava Mosaic Virus (ACMV) was purified by a method which allowed the separation of monomer from dimcr virus particles. Optimal conditions for storing purified virus to be used for immunization were determined by ELISA and inoculation on Nicotiana benthamiana. Purified virus could be stored without loss of infectivity and serological activity for more than 145 days at 4 °C or frozen at –20 °C, but not longer than 40 days in the presence of 50 % redistilled glycerol. Rabbit and chicken immunoglobulins were used to detect ACMV in cassava leaves by direct and indirect ELISA. To obtain the same absorbance values, it was necessary to use longer incubation times with the indirect method, but the virus detection end-point m sap from infected plants was the same for the two methods (1/512). Conditions for improving virus detection tn cassava samples were determined. The virus was better detected when leaves from diseased plants were ground in 100 mM Tris-HCl containing 1 % polyvinylpyrrolidone at pH 8.5 than in phosphate buffer. Plant inhibitors were the restricting factor in the detection of virus by ELISA, but this difficulty was avoided when leaves to be tested were harvested from the top of the cassava plants.     

辣椒抗烟草花叶病毒相关基因研究进展

病毒病是危害辣椒生产的主要病害之一。烟草花叶病毒(TMV)是最早被发现的病毒,它引起的烟草花叶病毒病是多种作物的重要病害,给辣椒等茄科作物的生产带来重大损失。文中综述了辣椒抗TMV防御反应中的相关基因及其研究进展,为明确辣椒抗TMV机理,挖掘抗病基因,选育抗病材料提供参考。     

Evaluation of Cassava Cultivars for Resistance to Cassava Mosaic Disease and Yield Potential in Central African Republic

Eleven cassava genotypes were tested against cassava mosaic disease (CMD) and compared to a local susceptible cultivar in field conditions from June 2011 to July 2012 in Central African Republic (CAR) at two sites representative of the savanna (Damara) and forest (Pissa) zones of the country. The mean number of whiteflies observed on plants varied among genotypes within each site, but was found nearly three times higher at Damara than at Pissa, resulting in a CMD incidence nearly five times higher at Damara than at Pissa. However, no relation was observed between the number of insect on the plants and the level of susceptibility/resistance of the genotypes. The difference of disease pressure between the two sites revealed high level of resistance in several genotypes, while some other ones indicated rather only a partial resistance. Nevertheless, none of the genotypes tested was found immune, in the end, the virus being detected at least in one site in every genotype, including those ones presenting no symptoms in both sites. The impact of CMD on yield components was assessed on the local susceptible check and three partially resistant genotypes, showing that the disease has no significant effect on the tuberous roots number as well as their weight in both sites. The yield potential varied among different genotypes and between the two sites, the mean number of tuberous roots as well as their mean weight being higher in Damara than in Pissa. This study identified highly resistant genotypes such as ‘Gabon’ that performed well in both sites, and ‘91/02322’ that was symptomless and presented a yield potential equivalent to the local check. These genotypes could be distributed to growers with the main advantage to be resistant to CMD and, therefore, reducing the risk to spread sources of inoculum all over the cassava cropping areas in the country.     

Coat Protein-mediated Resistance to Pea Enation Mosaic Virus in Transgenic Pisum Sativum L.

Pea (Pisum sativum L.) plants were transformed in planta by injection/electroporation of axillary meristems with a chimeric pea enation mosaic virus (PEMV) coat protein gene contruct. R1 progenies of these plants were shown to harbor the transgene by polymerase chain reaction (PCR) and genomic Southern analysis, while transgene expression was demonstrated by western blot analysis. Transgenic R2, R3 and R4 plants displayed delayed or transient PEMV multiplication and attenuated symptoms as compated to control inoculated individuals.     

Induction of Resistance in Tobacco Leaves to Tobacco Mosaic Virus by Dodecylbenzenesulfonate

Application of dodecylbenzenesulfonate (DBS) to half leaves of Nicotiana tabacum cv. Xanthi nc. before TMV inoculation resulted in a marked decrease in lesion number and size as well as in virus content of the lesions in the untreated half leaves. Systemic induction of resistance in untreated leaves of the plants was not detected.     

利用美国SMV株系初步鉴定中国大豆鉴别寄主的抗性基因

参照美国的大豆花叶病毒(SMV)株系鉴别系统,利用G1、G2、G3、G5和G7 5个株系对24个中国鉴别寄主进行接种鉴定,初步对中国SMV大豆鉴别寄主的抗性基因进行了推断.结果表明:'Davis'、'文丰5号'、'齐黄1号'、'齐黄10号'、'徐豆1号'、'徐豆2号'、'吉林26'(PI 612720A,B)、'铁丰25'和'诱变30'的表现型与'York'相同,可能携带相同的抗性基因Rsv1-y;'合丰25'可能携带一个新的Rsv1-n基因;'吉林26'(PI 597411A)和'1138-2'(PI 592914)可能携带Rsv3基因;'吉林26'(PI 597411B)、'早18'、'早熟18'、'吉林21'、'鲁豆4号'抗所有鉴定的SMV株系,可能携带Rsv1-h、Rsv4、Rsv1+3、Rsv1+4或Rsv3+4基因.利用SMV接种鉴定的反应型是对大豆抗性基因进行初步筛选的有效方法,本研究结果对构建中国SMV株系通用鉴别系统有一定借鉴意义.     

The Relative Resistance of Cassava Cultivars to African Cassava Mosaic Disease (ACMD) as Determined by Two Methods: Rank-Sum and the Area Under the Disease Progress Curve

Twenty-five newly bred improved cassava cultivars, twenty-three improved from the International Institute of Tropical Agriculture and two local cultivars were evaluated for their relative resistance to African cassava mosaic begomovirus disease (ACMD) at Ibadan, in an area of high disease pressure representative of the forest/savanna transition zone of Nigeria. These cultivars were exposed to natural infection by the viruliferous whiteflies ( Bemisia tabaci Gennadius) and the disease incidence (DI) and index of symptom severity (ISS) were assessed for all clones. Results for the Rank-sum (i.e., sum of ranks for DI and ISS for each cultivar) and the area under the disease progress curve (AUDPC) were used to assess the relative resistance of the cassava clones. Those that showed low Rank-sum and AUDPC values were rated 'moderately resistant (MR)', 'resistant ( R )', and 'highly resistant (HR)' to ACMD depending on their respective values and deviation from the mean distribution curve. Clones M94/0121 and 94/0239 were rated HR under the two methods. Clone M94/0583 was rated HR under the AUDPC with a deviation from the mean distribution curve of m 2.00 while it was rated R under the Rank-sum method with a deviation from the mean distribution of m 1.99. Also plants of clones ISU and TMS 30572 were rated highly susceptible (HS) under both methods. Clone TME-1 was intermediate between Moderately resistant (MR) and Moderately susceptible (MS) under the AUDPC method with a deviation from the mean distribution of 0.00 but observed to be MS under the Rank-sum method with a deviation of + 0.2. The two methods of evaluation gave similar results as revealed by Spearman rank correlation ( r equals; 0.99, P <0.01). However, the AUDPC method is less cumbersome compared to the Rank-sum method. None of the clones was observed to be immune to the disease.     

Problems Encountered with the Selection of Cucumber Mosaic Virus (CMV) Isolates for Resistance Breeding Programs

Among the Chili breeding lines from the Asian Vegetable Research Center, two were chosen for the screening of a larger selection of Cucumber mosaic virus (CMV) isolates, mainly from Asian countries. The chili line (VC246) showed a resistance against several CMV‐isolates and was compared with chili line VC27a that was susceptible to CMV infection. Among the 28 CMV isolates, five were identified as resistance breaking (AN‐like) and non‐resistance breaking (P3613‐like) for the line VC246, whereas all isolates could establish a systemic infection on VC27a. However, further testing revealed that resistance in VC246 was also dependent on the way of inoculation and the inoculums itself. Graft inoculation could overcome the resistance, and the inoculation with isolated viral RNA resulted in no infection at all on the resistant chili line, independent of the virus isolate. Using a pseudo‐recombinant approach, we identified RNA2 of resistance breaking isolates as responsible for systemic infection and confined the area within RNA2 to the 3′ terminal part including the ORF 2b. Sequence alignments of that area revealed eight distinct mutations on amino acid level, which was present either in resistance or non‐resistance breaking isolates. A reversion from the P3613‐like to the AN‐like sequence of two of these mutations induced no effect on Capsicum sp., but induced symptoms on several tobacco species distinct from those induced by the wild‐type virus. However, pseudorecombinants, each generated from sets of two different AN‐like isolates, which were expected to infect VC246 systemically, did not indicating that probably RNA2 must be in a specific context to have the effect. In this case, a generalized attribution of functions to single amino acid exchanges might be impossible or at least extremely difficult.     

RNA-dependent RNA Polymerase Activities in Tomato Plants Susceptible or Resistant to Tobacco Mosaic Virus

RNA-dependent RNA polymerase activities were measured in healthyand tobacco mosaic virus (TMV)-infected tomato plants, to investigatethe possibility that altered activity might be involved in theoperation of the Tm-I gene for resistance to TMV. Healthy, susceptibleand resistant plants had similar levels of enzyme activity.Infection with TMV strain 0, which is inhibited by Tm-I, causeda 2-fold increase in activity in susceptible plants but no increasein Tm-I plants. Infection with a number of strain 1 isolates,which overcome Tm-I resistance, led to a 2 to 4-fold increasein enzyme activity in resistant plants. RNA-dependent RNA polymerase, Tm-I resistance gene, tobacco mosaic virus, tomato, Lycopersicon esculentum     

Types of the cerebral arterial circle (circle of Willis) in a Sri Lankan Population

Background  

The variations of the circle of Willis (CW) are clinically important as patients with effective collateral circulations have a lower risk of transient ischemic attack and stroke than those with ineffective collaterals. The aim of the present cadaveric study was to investigate the anatomical variations of the CW and to compare the frequency of prevalence of the different variations with previous autopsy studies as variations in the anatomy of the CW as a whole have not been studied in the Indian subcontinent.     

Observations on the nesting habits of Oreochromis mossambicus (Peters) (Pisces: Cichlidae) in Sri Lankan reservoirs

The nest-building characteristics of Oreochromis mossambicus (Peters) were studied in five manmade lakes (reservoirs) Sri Lanka. The nests were always found in clusters (arenas) and were generally located in or near coves and/or bays in shallow water. The density of nests in an arena varied between arenas in a reservoir and between reservoirs. The overall mean density of nests ranged between 0.47 and 6.31 m ² in the five reservoirs. The nests ranged from 11 to 110 cm in diameter, two size groups of nests being recognizable: small, with diameter 10–50 cm, and large with diameter >50 cm. At any nesting site only one size group of nests was found.
Nest diameter ( ND ) was curvilinearly related to mean nest density ( D ) and linearly to mean distance between nests ( Dis ), the respective statistical relationships being
The diameter of nests was also related to nest depth ( Dep ) as
There was no apparent relationship between nesting characteristics and morphometric characters of the reservoirs.     

Silencing of the Host Factor eIF(iso)4E Gene Confers Plum Pox Virus Resistance in Plum

Plum pox virus (PPV) causes the most economically-devastating viral disease in Prunus species. Unfortunately, few natural resistance genes are available for the control of PPV. Recessive resistance to some potyviruses is associated with mutations of eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. In this study, we used an RNA silencing approach to manipulate the expression of eIF4E and eIF(iso)4E towards the development of PPV resistance in Prunus species. The eIF4E and eIF(iso)4E genes were cloned from plum (Prunus domestica L.). The sequence identity between plum eIF4E and eIF(iso)4E coding sequences is 60.4% at the nucleotide level and 52.1% at the amino acid level. Quantitative real-time RT-PCR analysis showed that these two genes have a similar expression pattern in different tissues. Transgenes allowing the production of hairpin RNAs of plum eIF4E or eIF(iso)4E were introduced into plum via Agrobacterium-mediated transformation. Gene expression analysis confirmed specific reduced expression of eIF4E or eIF(iso)4E in the transgenic lines and this was associated with the accumulation of siRNAs. Transgenic plants were challenged with PPV-D strain and resistance was evaluated by measuring the concentration of viral RNA. Eighty-two percent of the eIF(iso)4E silenced transgenic plants were resistant to PPV, while eIF4E silenced transgenic plants did not show PPV resistance. Physical interaction between PPV-VPg and plum eIF(iso)4E was confirmed. In contrast, no PPV-VPg/eIF4E interaction was observed. These results indicate that eIF(iso)4E is involved in PPV infection in plum, and that silencing of eIF(iso)4E expression can lead to PPV resistance in Prunus species.     

Identification of Genes for Resistance to Bean Common Mosaic Virus and Bean Common Mosaic Necrosis Virus in Snap Bean (Phaseolus vulgaris L.) Breeding Lines Using Conventional and Molecular Methods

Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) are among the biggest threats for snap bean production in Bulgaria due to their seed, aphid and mechanical transmission. Old valuable Bulgarian snap bean varieties are being neglected, because of the high percentage of virus‐infected seeds. Breeding resistant cultivars is the best way to solve the problem. The genetic control towards both viruses is assured by one dominant I gene and a number of recessive (bc‐u, bc‐1, bc‐1², bc‐2, bc‐2² and bc‐3) genes. Our aim was to identify resistance gene combinations in advanced F8 breeding lines, derived from two crosses (A‐8‐40‐7‐2‐1 × IVT 7214) and (Zaria × RH 26D), by the application of conventional and molecular approaches. Four methods were applied for the characterization of their resistance gene makeup: (i) leaf‐abscission infection test designed to identify I gene by direct inoculation with NL3 strain of BCMNV; (ii) intact‐plant infection test with strain NY15 of BCMV to separate immune genotypes, possessing bc‐ubc‐1², bc‐ubc‐2^2,bc‐ubc‐2bc‐3, I, Ibc‐1², Ibc‐2² or Ibc‐3; (iii) PCR analysis with the following markers: SCAR – SW13 (for I gene), SBD5 (for bc‐1²), ROC11 (for bc‐3) and CAPS – eIFE4 (for bc‐3); and 4) high‐temperature (more than 30°C) infection test with NL3 of BCMNV to provoke systemic necrosis in I, Ibc‐1, Ibc‐1², Ibc‐1²bc‐2² or Ibc‐3. The four methods applied worked properly and complemented each other. Valuable gene combination (Ibc‐3) was established in seven breeding lines with immune reaction to BCMNV. They will be included in the snap bean breeding programme for virus resistance.