Isolation and characterisation of a new Venezuelan strain of cassava common mosaic virus

A new strain of cassava common mosaic virus, designated C_sCMV-ve, was isolated from cassava (Manihot esculenta Crantz) plants growing in Venezuela. The 520 nm long semi-flexuous virus particles sedimented in sucrose gradients as one component and contained a single coat protein molecule of c. 28 kDa, and a ssRNA of c. 2.1 times 10⁶. A single dsRNA species of c. 4.2 times 10⁶kDA was isolated from virus-infected cassava leaves. In double antibody sandwich enzyme-linked immunosorbent assay, C_sCMV-ve reacted strongly with antisera to C_sCMV and potato virus X, but not with those to other typical members of the potexvirus group. Ultrastructural studies of C_sCMV-ve infected cells showed the presence of large bundles of inclusion bodies in the cytoplasm, a proliferation of the rough endoplasmic reticulum, and large crystals in the peroxisomes. C_sCMV-ve in crude buffered sap or in purified preparations was not mechanically transmitted to known hosts of the Chaya and Brazilian strains of C_sCMV. Isolated virus RNA also failed to infect a range of test-plant species. This is the first report of the presence of C_sCMV in Venezuela.     

The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters

Here we report the effect of the 35S promoter sequence on activities of the tissue- and organ-specific gene promoters in tobacco plants. In the absence of the 35S promoter sequence the AAP2 promoter is active only in vascular tissues as indicated by expression of the AAP2:GUS gene. With the 35S promoter sequence in the same T-plasmid, transgenic plants exhibit twofold to fivefold increase in AAP2 promoter activity and the promoter becomes active in all tissue types. Transgenic plants hosting the ovary-specific AGL5:iaaM gene (iaaM coding an auxin biosynthetic gene) showed a wild-type phenotype except production of seedless fruits, whereas plants hosting the AGL5:iaaM gene along with the 35S promoter sequence showed drastic morphological alterations. RT-PCR analysis confirms that the phenotype was caused by activation of the AGL5:iaaM gene in non-ovary organs including roots, stems and flowers. When the pollen-, ovule- and early embryo-specific PAB5:barnase gene (barnase coding a RNase gene) was transformed, the presence of 35S promoter sequence drastically reduced transformation efficiencies. However, the transformation efficiencies were restored in the absence of 35S promoter, indicating that the 35S promoter might activate the expression of PAB5:barnase in non-reproductive organs such as calli and shoot primordia. Furthermore, if the 35S promoter sequence was replaced with the NOS promoter sequence, no alteration in AAP2, AGL5 or PAB5 promoter activities was observed. Our results demonstrate that the 35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter. Xuelian Zheng and Wei Deng contributed equally to this work and are considered co-first authors.     

The effects of cassava mosaic virus disease on yield and compensation in mixed stands of healthy and infected cassava

The effects of cassava mosaic virus disease (CMD) on yield in fully and partly infected stands of cassava were investigated in field trials in Uganda in 1990-91 and 1991-92. Three cultivars (Ebwanateraka, Bao and Bukalasa 1 l), each at three levels of cutting infection (O%, 50% and 100%) and harvested 510 and 15 months after planting (MAP) were used in a randomised block design with split-split plots and four replicates. Moreover, yield and growth data for individual infected and uninfected plants were considered in relation to the health status of their nearest neighbours. In each experiment, fresh tuberous root yields of plants from 100% infected plots gave sigdicantly lower yields than those from 0% or 50% infected plots at each harvest date and the losses were greatest in cv. Bao. Yields of plants from 0% and 50% plots for each of the three cultivars were not significantly different, 10 and 15 MAP. The loss in yield differed between cultivars and harvest dates. Fresh stem, leaf and root yields and the number of tuberous roots were influenced by the health status of the plants harvested and that of their nearest neighbours. Uninfected plants surrounded by infected ones had more roots and heavier total fresh root, stem and leaf weights than those surrounded by uninfected ones. Overall, 26% and 42% compensation was recorded in 1990-91 and 1991-92, respectively. The effects of CMD on cassava production and of compensation in mixed stands of infected and uninfected plants are discussed, especially in relation to control strategies such as roguing.     

Unusually severe symptoms are a characteristic of the current epidemic of mosaic virus disease of cassava in Uganda

Mosaic disease (MD) is more severe in cassava plants infected within the area of the current epidemic in northern and central Uganda than to the south of the affected area. This difference in severity was recorded within a single cultivar as well as amongst the mixtures of cultivars found commonly in farmers' fields. An increase in severity also occurred as the epidemic passed through localities. Varietal or agroecological factors coincident with the area of the epidemic are therefore unlikely to cause the increased severity. The severe disease could also be graft and cutting transmitted and could super-infect mildly diseased plants. Both mildly and severely diseased plants gave positive reactions in ELISA tests to antisera prepared against African cassava mosaic virus (ACMV) and an unusually severe form of ACMV or a closely related geminivirus is likely to be the cause of the severe mosaic disease. The epidemic also involves increased populations of the whitefly vector of ACMV, Bemisia tabaci , and possible hypotheses are presented as to how these phenomena may be related.     

Particle purification and properties of cassava Ivorian bacilliform virus

A virus found in cassava from the north-west of the Ivory Coast was transmitted by inoculation with sap extracts to herbaceous species in six plant families. Chenopodium quinoa was used as a propagation host and C. murale was used for local lesion assays. The virus particles are bacilliform, c. 18 nm in diameter, with predominant lengths of 42,49 and 76 nm and a structure apparently similar to that found in alfalfa mosaic virus. Purified preparations of virus particles had A₂₆₀/A₂₈₀ of 1.7 ±0.05, contained one protein of M_rc. 22 000, and yielded three species of RNA with M_r (× 10^-6) of c. 0.7, 0.8 and 1.2. Although the virus particles were poorly immunogenic, an antiserum was produced and the virus was detected by enzyme-linked immunosorbent assay (DAS-ELISA) in leaf extracts at concentrations down to c. 6 ng/ml. Four other field isolates were also detected, including a strain which caused only mild systemic symptoms in C. quinoa instead of necrosis. The naturally infected cassava source plants were also infected with African cassava mosaic virus (ACMV) but when the new virus was cultured in Nicotiana benthamiana, either separately or together with ACMV, its concentration was the same. The new virus did not react with antisera to several plant viruses with small bacilliform or quasi-bacilliform particles, and alfalfa mosaic virus reacted only weakly and inconsistently with antiserum to the cassava virus. The new virus, for which the name cassava Ivorian bacilliform virus is proposed, is tentatively classified as the second member of the alfalfa mosaic virus group.     

Functional definition of ABA-response complexes: the promoter units necessary and sufficient for ABA induction of gene expression in barley (Hordeum vulgare L.)

Abscisic acid (ABA)-response promoter complexes (ABRCs), consisting of an ACGT core-containing element (ACGT box) and a coupling element (CE), have been shown to be necessary and sufficient for ABA induction of gene expression in cereal plants. In this work, the component elements of two ABRCs are defined in terms of base sequence, orientation, and distance from each other. The ACGT element requires the sequence 5-ACGTGGC-3 and the elements CE1 and CE3 require the sequences CCACC and GCGTGTC, respectively. The ACGT element and CE3 are next to each other in the barley ABA-inducible gene HVA1, and lengthening the distance between them gradually decreases their activity in conferring ABA response. On the other hand, the ACGT element and CE1 are separated by about 20 bp in the promoter of another ABA-inducible gene, HVA22, and need to be separated by multiples of 10 bp in order to confer high ABA induction, suggesting that these two elements have to be located in the same side of the DNA double helix. Although the coupling between an ACGT box and a CE is sufficient for ABA induction, two copies of the ACGT element are equally active. However, two copies of CE3 appear to be less active. Specific interactions between ABRC and nuclear proteins have been detected. In vitro binding activities of nuclear proteins to an ABRC and to its mutant forms appear to be proportional to the biological activities of these sequences in vivo. Our data suggest that the specific response to ABA is determined by the presence of two ACGT boxes or an ACGT box plus a CE as well as by the flanking sequences of the ACGT boxes and the CEs.     

Functional analysis of the promoter region of a maize (Zea mays L.) H3 histone gene in transgenic Arabidopsis thaliana

A 1023 bp fragment and truncated derivatives of the maize (Zea mays L.) histone H3C4 gene promoter were fused to the ß-glucuronidase (GUS) gene and introduced via Agrobacterium tumefaciens into the genome of Arabidopsis thaliana. GUS activity was found in various meristems of transgenic plants as for other plant histone promoters, but unexplained activity also occurred at branching points of both stems and roots. Deletion of the upstream 558 bp of the promoter reduced its activity to an almost basal expression. Internal deletion of a downstream fragment containing plant histone-specific sequence motifs reduced the promoter activity in all tissues and abolished the expression in meristems. Thus, both the proximal and distal regions of the promoter appear necessary to achieve the final expression pattern in dicotyledonous plant tissues. In mesophyll protoplasts isolated from the transformed Arabidopsis plants, the full-length promoter showed both S phase-dependent and -independent activity, like other plant histone gene promoters. Neither of the 5-truncated nor the internal-deleted promoters were able to direct S phase-dependent activity, thus revealing necessary cooperation between the proximal and distal parts of the promoter to achieve cell cycle-regulated expression. The involvement of the different regions of the promoter in the different types of expression is discussed.     

Restriction of Virus Movement into Axillary Buds is an Important Aspect of Resistance in Cassava to African cassava mosaic virus

Axillary buds and bark samples of resistant, moderately resistant and susceptible (control) cassava genotypes either naturally infected under field conditions or experimentally inoculated by grafting were indexed for African cassava mosaic virus (ACMV). Virus detection was carried out using enzyme‐linked immunosorbent assay and polymerase chain reactions to determine the distribution of the virus within the plant and elucidate the genotypes response to virus movement. Significantly more bud and bark samples were positive for virus on the susceptible genotype TME 117 than resistant genotypes TMS 30001 and TMS 91/02319, or the moderately resistant genotype TMS 30572. Detectable virus concentration was significantly lower in the buds of moderately resistant and resistant genotypes than the susceptible control. Under field conditions, it was significant that more primary stem buds were infected than the buds of secondary and tertiary stems but such a gradient was not obvious with bark samples. Shoots that had asymptomic new leaves after the initial symptomatic leaves had no virus in their buds, but some of the bark samples from the same plants tested positive. A significant interaction was observed between year and stem type, and among year, genotype and stem type with respect to virus detection in bud and bark samples. Restriction of virus movement into axillary buds occurred in all the resistant and moderately resistant genotypes. This may explain ACMV‐infected stem cuttings of resistant genotypes producing healthy plants in subsequent generation.     

Harpin_(Xoo)及其功能域片段启动病原物诱导性启动子在转基因拟南芥中的活性(英文)

从烟草(Mcotiana tabacum L.)中克隆了3个病原物诱导性启动子PPP1、PPP2和PPP3,它们都含受细菌诱导的反应元件PPP1和PPP2,另外含有受生物激发子及水杨酸(SA)诱导的元件;PPP1内部还含串联重复的两段111bp的序列,而在PPP2中,这个重复序列中的一个111 bp的片段被定点删除。分别构建了含这三个启动子及花椰菜花叶病毒35S启动子的转化单元,用它们分别转化拟南芥(Arabidopsis thaliana L.),获得了转基因植株。PCR证明,几个启动子和所带的gus基因(uidA)已经整合到拟南芥基因组中。用青枯病菌接种转基因第二代植株,组织染色表明三个PPP启动子在拟南芥中都可以受青枯病菌诱导,说明克隆的PPP启动子是有活性的。随后分别用SA、来自水稻白叶枯病菌的蛋白质激发子halpinXoo以及hatpinXoo的3个具有不同功能的片段DEG(促进生长)、DIR(诱导抗虫)、DPR(诱导抗病)喷雾处理转基因植株,通过GUS的荧光定量分析,检测了启动子的活性。结果显示,青枯病菌诱导的PPP1、PPP2和PPP3活性分别为35S启动子活性的53、39和25倍。PPP1和PPP2可以受SA、harpinXoo、DEG、DIR和DPR的诱导,而PPP3则不能。这些结果说明了有关元件的可能作用。另外,PPP1的活性比PPP2高3倍,表明111bp的重复序列可以影响启动子活性水平。     

Activity of promoters of carnation etched ring virus and dahlia mosaic virus in tobacco protoplasts and transgenic plants

Promoters of carnation etched ring virus (CERV) and dahlia mosaic virus (DMV) were cloned into binary vectors pCambia 1304, pCambia 1281Z, and pCambia 1291Z with reporter GFP and GUS genes. Activities of these promoters in tobacco protoplasts and transgenic plants were determined using these constructs. Histochemical GUS analysis demonstrated the absence of tissue-specificity in transgenic plants transformed with these promoters. The quantitative analysis of these promoter activities in transgenic tobacco plants, using 4-methylumbelliferone as a substrate, showed that 35S CaMV, CERV, and DMV promoters displayed approximately similar activities in transgenic tobacco plants.     

獐茅HAK基因表达调控区的分离及其在水稻中的功能分析

獐茅高亲和性K+转运蛋白基因(AlHAK1)是从单子叶禾本科盐生植物獐茅(Aeluropus littoralis(Gouan)Parl)中克隆,对于细胞营养和离子渗透调节起关键作用。为了进一步了解AlHAK1基因的表达调控机制,文章采用基因组步移法分离了AlHAK1基因转录起始位点上游长度约1.3 kb的启动子区域。启动子顺式元件分析显示该序列具有典型的TATA和CAAT盒,以及一些与植物生长发育和环境响应相关的顺式元件。为了明确AlHAK1启动子的功能,将其与GUS基因融合构建到植物表达载体pCAMBIA1301上,通过农杆菌介导转化法导入水稻中。对转基因植株进行GUS组织化学染色,结果显示在转化AlHAK1启动子水稻的根、茎、叶、花药和内外稃部位均检测到GUS活性。GUS荧光定量分析显示AlHAK1启动子调节GUS表达活性低于组成型启动子CaMV35S和Ubiquitin,但其根部和茎部的GUS活性相对较高。对转化植株进行不同胁迫处理后检测GUS活性,结果表明受到ABA、干旱、高温的诱导后其茎部和根部GUS活性有所提高,推测位于该启动子-682 bp的HSE元件和-1 268 bp的MybBS元件可能在高温、ABA和干旱诱导的表达调控中起作用。     

Functional properties of a chitinase promoter from cabbage （Brassica oleracea var.capitata）

The 5‘-region of the chitinase gene cabch29,derived from Brassica oleracea var.capitata,has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants.Different 5‘-deletion fragments were linked to reporter gene β-glucuronidase (GUS) as translational fusions,and the expression of these chimeric genes was analyzed in vegetative organs and tissues.Sequences up to-651 showed some basal GUS activity with nearly equal levels in wounded and intact tissues.The addition of further upstream sequences(-651 to-1284) enhanced expression level,and the expression driven by this fragment was inducible by a factor of two to three-fold by wounding.Histochemical analysis of different tissue from transgenic plants that contain cabch29 promoter-gus fusion gene demonstrated woundinducible and tissue-specific cabch29 promoter activity in plants containing the 1308 base pair fragment.The location of GUS activity appears to be cell-specific,being highest in vascular cells and epidermal cells of stem,leaf and roots.Meanwhile,the temporal and spatial expression of cabch29-GUS fusion gene has been investigated.Among the different vegetative organs,a high level of GUS activity was observed in stem and a moderate one in roots;whereas,wounding stress led to a high level of GUS in stem and moderate one in leaf.