Geminivirus coat protein gene replacement alters insect specificity

Chimeric clones have been constructed in which the coat protein encoded by DNA A of the bipartite genome of the geminivirus African cassava mosaic virus (ACMV) has been replaced by that of beet curly top virus (BCTV). Constructs containing the coding region inserted in either orientation were infectious when co-inoculated with ACMV DNA B onto Nicotiana benthamiana, producing symptoms typical of ACMV infection. The onset of symptom production was delayed relative to plants inoculated with parental ACMV clones and remission of symptoms was observed. When inserted in the correct orientation for expression from the ACMV coat protein promoter, the BCTV gene was expressed in plants and the coat protein synthesized encapsidated ssDNA of both ACMV genomic components. The BCTV leafhopper vector, Circulifer tenellus (Baker), transmitted both BCTV and the chimeric virus but not ACMV when injected with virus preparations and transferred to N. benthamiana seedlings. The results show that the specificity of leafhopper transmission from insect to plant resides with the coat protein.     

Expression of full-length and truncated Rep genes from Mungbean yellow mosaic virus-Vigna inhibits viral replication in transgenic tobacco

Mungbean yellow mosaic virus-Vigna (MYMV-Vig) is a bipartite geminivirus that causes a severe yellow mosaic disease in blackgram. An assay was developed to study MYMV-Vig replication by agroinoculation of tobacco leaf discs with partial dimers of the virus. This assay, in a non-host model plant, was used to evaluate pathogen-derived resistance contributed by MYMV-Vig genes in transgenic plants. Viral DNA accumulation was optimum in tobacco leaf discs cultured for 10 days after infection with Agrobacterium tumefaciens strain Ach5 containing partial dimers of both DNA A and DNA B of MYMV-Vig. Transgenic tobacco plants with MYMV-Vig genes for coat protein (CP), replication-associated protein (Rep)-sense, Rep-antisense, truncated Rep (T-Rep), nuclear shuttle protein (NSP) and movement protein (MP) were generated. Leaf discs from transgenic tobacco plants, harbouring MYMV-Vig genes, were agroinoculated with partial dimers of MYMV-Vig and analyzed for viral DNA accumulation. The leaf discs from transgenic tobacco plants harbouring CP and MP genes supported the accumulation of higher levels of MYMV-Vig DNA. However, MYMV-Vig accumulation was inhibited in one transgenic plant harbouring the Rep-sense gene and in two plants harbouring the T-Rep gene. Northern analysis of these plants revealed a good correlation between expression of Rep or T-Rep genes and inhibition of MYMV-Vig accumulation.     

Identification and molecular characterization of a single-stranded circular DNA virus with similarities to Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1

A putative circular single-stranded DNA (ssDNA) virus was recovered from Hypericum japonicum collected in Vietnam. The viral isolate was tentatively named Hypericum japonicum-associated circular DNA virus (HJasCV). HJasCV shares 58.7-65.4% nucleotide sequence identity with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) and SsHADV-1-like viruses. Like this group of viruses, the genome of HJasCV (2 200 nt) has two large ORFs, one in the virion-sense and the other in the complementary-sense DNA. The proteins encoded in the virion-sense and complementary-sense ORFs share 39-46 % and 45-67 % amino acid sequence identity with the putative capsid and replication-associated proteins (Reps), respectively, of SsHADV-1 and SsHADV-1-like viruses. The putative Rep of HJasCV contains all of the motifs related to rolling-circle replication. Its 111-bp intergenic region (IR) contains a hairpin structure with a geminivirus-like nonanucleotide sequence, TAATGTTAT, at the apex of the loop. Phylogenetic analysis revealed that HJasCV forms a monophyletic clade with SsHADV-1 and SsHADV-1-like viruses.     

Identification of the minimal sequence required for vascular-specific activity of Tomato mottle Taino virus Replication-associated protein promoter in transgenic plants

A 597 nt fragment from Tomato mottle Taino virus (ToMoTV) DNA-A, with 459 nt located upstream of the Replication-associated protein translation start codon, was tested for promoter activity in solanaceous plants. The promoter activity of this fragment (pRep(459::Rep)) was demonstrated when it was introduced upstream the uidA reporter gene into tobacco, potato and tomato plants by genetic transformation. It became active in 7-day-old transgenic tobacco seedlings as revealed by a vascular-specific pattern of gene expression which was maintained during the continued growth of the plant. Transformed potato and tomato plants also showed a vascular-specific pattern of expression. In comparative assays, pRep(459::Rep) showed an expression activity 10-40-fold less than the 35S promoter from Cauliflower mosaic virus. To delimit the minimal cis-acting elements necessary for vascular specificity of this promoter, a set of PCR deletion mutants of pRep(459::Rep) (pRep(459), pRep(324), pRep(203), pRep(145), pRep(132) and pRep(115)), were generated and used to transform tobacco plants. Transgenic tobacco plants belonging to all the pRep versions were blue stained in the vascular system except those from the pRep(115) version. The results described in this report demonstrate that the minimal sequences necessary for the pRep promoter activity are confined in a segment of 132 nts (located between the nts 2454 and 2585 of the ToMoTV DNA A) and that this promoter harbors those elements sufficient for vascular-specific expression.     

Adeno-associated virus type 2 nonstructural protein Rep78 suppresses translation in vitro

Adeno-associated virus type 2 nonstructural protein Rep78 [621 amino acids (aa) long] affects the expression of various cellular and viral genes. In this study we examined the effects of Rep78 on expression of the luciferase gene from the human cytomegalovirus immediate-early promoter in HeLa cells and on translation of RNA encoding luciferase in rabbit reticulocyte lysate. When Rep78 and luciferase were coexpressed, the luciferase activity decreased despite increased levels of luciferase mRNA in the cells. Purified Rep78 or Rep68 fused with Escherichia coli maltose binding protein suppressed translation of luciferase RNA in vitro, but Rep52/40 fusion proteins did not. A mutated Rep78, which is 520 aa long and truncated at its C-terminus, did suppress the in vitro translation, whereas a similarly truncated Rep78 of 420 aa did not. The results indicate that Rep78/68 function to suppress gene expression through translation inhibition, which requires the N-terminal region contained within aa 1-520.     

Inhibition of African cassava mosaic virus systemic infection by a movement protein from the related geminivirus tomato golden mosaic virus.

Plant viruses encode proteins that mediate their movement through the host plant leading to the establishment of a systemic infection. We have analyzed the effect of tomato golden mosaic virus (TGMV) genes BL1 and BR1, which are thought to be involved in the process of virus movement, on the infectivity of African cassava mosaic virus (ACMV) in Nicotiana benthamiana. Recombinant genomes were constructed by replacing the ACMV coat protein coding sequence with those of either BL1 or BR1. Replication of recombinants containing BL1 and BR1 coding sequences in the sense orientation with respect to the coat protein promoter was detected in the inoculated leaves only when the constructs were co-inoculated, suggesting that both genes are being expressed and act in a cooperative manner. Co-inoculated recombinants induced localized symptoms on inoculated leaves but did not spread systematically, either because of a defect in BL1 and/or BR1 expression or due to the inability of the TGMV gene products to functionally complement their ACMV counterparts. Systemic spread of ACMV was inhibited when the recombinant containing the BL1 coding sequence in the sense, but not in the antisense, orientation was co-inoculated with ACMV DNA B. Disruption of the BL1 coding sequence by a frameshift mutation restored the ability of the recombinant to spread systemically, suggesting that the gene product is responsible for the inhibitory effect. The inhibitory phenotype was mimicked by a chimera containing amino-terminal sequences of TGMV BL1 and carboxy-terminal sequences of its ACMV homologue, BC1. The chimera has characteristics of a dominant negative mutant. We suggest that dominant negative mutants of virus movement genes may provide a novel source for virus resistance genes.     

Inhibition of adenovirus cytotoxicity, replication, and E2a gene expression by adeno-associated virus

Adeno-associated virus (AAV) and the other parvoviruses have long been known to inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we have begun an investigation into AAV's relationship with adenovirus (Ad), AAV's most efficient helper virus. AAV, but not UV-inactivated AAV, delayed Ad-induced cytotoxicity and inhibited Ad E2a gene expression. AAV, but not UV-inactivated AAV or a recombinant AAV vector, inhibited Ad DNA replication. To determine whether AAV or its replication (Rep) proteins alter Ad early gene expression, we measured steady state E2a mRNA levels in AAV and Ad coinfected cultures and in a cell line (Neo6) that inducibly expresses the Rep proteins. AAV, but not UV-AAV, and Rep expression resulted in diminution of E2a protein and mRNA levels. To determine whether the AAV Rep proteins directly affect the individual Ad early promoters, we constructed luciferase reporter plasmids containing each of the five early promoters. Cotransfection of Ad-luciferase and an AAV rep gene-expressing plasmid in HeLa cells demonstrated that Rep78 repressed the E1a, E2a, and E4 promoters but trans-activated the E1b and E3 promoters. In the presence of a cotransfected E1a-expressing plasmid, Rep78 repressed expression from all five promoters. These results indicate that Rep may have different effects on the Ad early promoters dependent upon the presence of the E1a trans-activating protein.     

African cassava mosaic virus DI DNA interferes with the replication of both genomic components.

Natural infections of the geminivirus African cassava mosaic virus (ACMV) are known to be associated with low levels of defective interfering (DI) DNAs. Recently it has been demonstrated that extrachromosomal copies of the DI DNA, mobilized and amplified from an integrated DI DNA dimer, can ameliorate ACMV symptoms in transformed Nicotiana benthamiana, providing a possible means for the control of cassava mosaic disease. To further understand the molecular basis of the interference phenomenon, we have compared the ability of ACMV and tomato golden mosaic virus (TGMV) genomic components to replicate in leaf discs derived from DI DNA-transformed and control plants. Results indicate that the ACMV DI DNA interferes with the replication of both genomic components of ACMV to a similar extent. TGMV DNA A replicates to normal levels in transformed leaf discs and plants because it is unable to mobilize and amplify ACMV DI DNA. Differences in the relative levels of ACMV genomic components in transformed leaf discs and plants are discussed in terms of DNA replication and the availability of the genomic components for spread throughout the plant.     

Tomato yellow leaf curl China virus: monopartite genome organization and agroinfection of plants.

The complete DNA sequence (2734 nucleotides) of the monopartite genome of tomato yellow leaf curl China virus (TYLCCNV), a begomovirus transmitted by the whitefly Bemisia tabaci, was determined. The circular genomic DNA contains six open reading frames (ORFs) encoding proteins of molecular weights >10 kDa, of which two (V1 and V2) are located on the virion-sense strand and four (C1, C2, C3 and C4) on the complementary-sense strand. The ORFs are comparable to those of other whitefly-transmitted begomoviruses with a monopartite genome and to those encoded by DNA-A of bipartite begomoviruses. Sequence comparisons with other geminiviruses showed that TYLCCNV belongs to Begomovirus from the Old World. No putative DNA-B genome was found. Nicotiana species and tomato plants agroinoculated with the TYLCCNV monopartite genome developed typical yellowing and leaf-curling symptoms. The cloned molecule carried all the information needed for virus replication and systemic infection of plants.     

Disease symptoms in transgenic tobacco induced by integrated gene VI of cauliflower mosaic virus

A chimeric vector (pKR 612B1) containing the neomycin phosphotransferase (APH) gene from the Tn5 transposon under the control of the gene VI promoter of cauliflower mosaic virus (CaMV) and the cloned gene VI region (SalI-BstEII) of the same virus were used to cotransform tobacco protoplasts. Using the polyethylene glycol transformation procedure, a large number of protoplasts were transformed and proved to be resistant to kanamycin (Km). Whole Km-resistant plants were regenerated and shown to contain the integrated foreign genes. DNA from transformed clones was analyzed by Southern blot hybridization, showing the presence of the Tn5-derived gene and the viral gene. Transgenic plants containing the viral gene show mild mosaic patterns and fasciation. The expression of the gene VI product was detected by immunoblots.     

Dual infection by cassava begomoviruses in two leguminous species (Fabaceae) in Yangambi, Northeastern Democratic Republic of Congo

A study on cassava mosaic begomoviruses was conducted around Yangambi (DR Congo) by sampling 10 different leguminous species with or without symptoms similar to cassava mosaic disease. DNA was isolated to amplify CMBs using primers targeting AC2 and AC4 genes for virus detection by PCR. The results showed a dual infection by ACMV and EACMV in two weed species, Centrosema pubescens and Pueraria javanica, associated with mosaic symptoms. The DNA-A genome component of ACMV and EACMV from the infested weeds was sequenced. Seven ACMV and four EACMV isolates are reported. The major ACMV strains were closely related to ACMV-NGogo, ACMV-IC and ACMV-UGMld, whereas all EACMV strains were closely related to a Uganda variant, the most prevalent virus. This study shows that whiteflies may transmit CMBs to non-cassava plants under high epidemic pressure.     

HRE/CMV启动子的克隆和缺氧调控活性的测定

目的： 将具有缺氧调控作用的多种缺氧反应元件 (HRE)与CMV启动子组合，以获得缺氧应答启动子。将荧光素酶报告基因置于缺氧应答启动子的下游，通过检测荧光素酶活性的方法，方便地研究缺氧应答启动子缺氧诱导的作用。方法: 合成多种HRE核苷酸序列，并设立HRE突变对照，克隆入pCI-neo中，构建HRE/CMV启动子。扩增HRE/CMV序列，定向亚克隆入pGL3-Basic中，获得HRE/CMV荧光素酶报告载体。瞬时转染HeLa细胞，在0.1% O₂ 环境下培养细胞，并设立正常氧分压环境对照。检测荧光素酶的相对活性。结果: 成功构建了HRE/CMV荧光素酶报告载体，其中mPGK-HRE/CMV载体表现出很好的缺氧诱导作用和高水平的表达。结论: mPGK-HRE/CMV启动子能够有效地进行缺氧诱导和调控工作。     

Multiplex PCR for the detection of African cassava mosaic virus and East African cassava mosaic Cameroon virus in cassava

A multiplex PCR was developed for simultaneous detection of African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in cassava affected with cassava mosaic disease (CMD). One set of three primers consisting of an upstream primer common for both viruses and two down stream virus-specific primers were designed for simultaneous amplification of 368 base pair (bp) and 650 bp DNA fragments specific to the replicase gene of ACMV and EACMCV, respectively. Similarly, a second set of three primers were designed for simultaneous amplification of 540 bp and 655 bp fragments specific to the coat protein gene of EACMCV and ACMV, respectively. Primers that can amplify a 171 bp fragment of the large subunit of ribulose bisphosphate carboxylase oxygenase L were included as an internal control in these assays to determine the reliability of multiplex PCR. A simplified, cost-effective and rapid sample preparation method was adapted in place of the conventional plant DNA extraction procedure for multiplex PCR detection of ACMV and EACMCV. The method was validated using CMD-infected cassava samples obtained from farmers' fields in Nigeria. The multiplex PCR is useful for reliable assessment of the prevalence of CMBs in epidemiological studies and for crop improvement and phytosanitary programs in African countries.