Specificity of a promoter from the rice tungro bacilliform virus for expression in phloem tissues.

The major promoter region for the transcription of the genome of rice tungro bacilliform virus (RTBV), a newly described badnavirus, has been identified. Fragments of the RTBV genome upstream of the site of transcription initiation were isolated and tested for promoter activity using a beta-glucuronidase receptor gene (gusA). Assays of transient gusA expression were performed following introduction of the chimeric gene into protoplasts via electroporation. The chimeric RTBV-promoter: gusA gene was more active in rice protoplasts than in maize or tobacco protoplasts, but was weaker than gusA controlled by an enhanced 35S promoter from cauliflower mosaic virus. Analysis of gusA gene expression following introduction of chimeric reporter genes into intact leaves via micro-projectile bombardment indicated that the GUS activity is present primarily in vascular tissues. Transgenic rice plants carrying the chimeric gusA gene had GUS activity only in the phloem of the vascular bundles in the leaf. Tissue printing studies demonstrated that RTBV accumulates in the vascular bundles of infected rice leaves. The results of our study indicate that phloem-specific expression from the RTBV promoter is an intrinsic property of the viral promoter.

Sequence-specific interaction with the viral AL1 protein identifies a geminivirus DNA replication origin.

The bipartite geminiviruses such as tomato golden mosaic virus (TGMV) and squash leaf curl virus (SqLCV) have two single-stranded circular genomic DNAs, the A and B components, thought to be replicated from double-stranded circular DNA intermediates. Although it has been presumed that the origin sequences for viral replication are located in the highly conserved 200-nucleotide common region (CR) present in both genomic components and that the viral-encoded AL1 protein interacts with these sequences to effect replication, there has been no evidence that this is in fact so. We have investigated these questions, demonstrating selectivity and sequence specificity in this protein-DNA interaction. Simple component switching between the DNAs of TGMV and SqLCV and analysis of replication in leaf discs showed that whereas the A components of both TGMV and SqLCV promote their own replication and that of their cognate B component, neither replicates the noncognate B component. Furthermore, using an in vivo functional replication assay, we found that cloned viral CR sequences function as a replication origin and direct the replication of nonviral sequences in the presence of AL1, with both circular single-stranded and double-stranded DNA being synthesized. Finally, by the creation of chimeric viral CRs and specific subfragments of the viral CR, we demonstrated sequence-specific recognition of the replication origin by the AL1 protein, thereby localizing the origin to an approximately 90-nucleotide segment in the AL1 proximal side of the CR that includes the conserved geminiviral stem-loop structure and approximately 60 nucleotides of 5' upstream sequence. By deletional analysis, we further demonstrated that the conserved stem-loop structure is essential for replication. These studies identify the functional viral origin of replication within the CR, demonstrating that sequence-specific recognition of this origin by the AL1 protein is required for replication.

Characterization of the genome of rice tungro bacilliform virus: comparison with Commelina yellow mottle virus and caulimoviruses.

Rice tungro disease is caused by an infection of two different viruses, rice tungro spherical virus (a (+) sense RNA virus) and rice tungro bacilliform virus (RTBV) with a genome of circular double-stranded DNA. The genome of an RTBV isolate from the Philippines was cloned, sequenced, and found to be 8000 bp in length. It contains four open reading frames (ORFs) on a single strand, with ORF 1 having an internal termination codon (TAA). The 5' and 3' ends of a polyadenylated viral RNA transcript, of genome length, were mapped by primer extension and cDNA sequence analysis, respectively. The transcript is terminally redundant by 265-268 nucleotides. Purified virus particles contain two major proteins with molecular masses of 37 and 33 kDa, although only the 37-kDa protein was detected in the infected rice tissues. The N-terminal amino acid sequence of the 33-kDa protein was determined and its coding region was identified on the RTBV genome. The identity of the coat protein gene was further confirmed by expressing a region of the genome in Escherichia coli, the products of which reacted with anti-RTBV antibody. The unusually long ORF 3 of RTBV is predicted to encode a polyprotein of 194.1 kDa that includes: the coat protein(s), viral proteinase, reverse transcriptase, and ribonuclease H. The sections of the polyprotein show varying degrees of similarity to the counterparts of Commelina yellow mottle virus (a member of the proposed badnavirus group) and caulimoviruses. The functions of the other three ORFs are unknown.

Molecular cloning and characterization of genes expressed in shoot apical meristems.

The above-ground portion of a plant develops from the shoot apical meristem. An abundant source of apical meristems was obtained from cauliflower heads. Meristematic cDNAs were identified by differential screening and used to isolate corresponding Arabidopsis thaliana genes. Transcriptional promoters from Arabidopsis clones were fused to the beta-glucuronidase (GUS) reporter gene and introduced into plants, and GUS expression was used to analyze temporal and spatial regulation of the promoters. One promoter (meri-5) directed GUS expression in the meristematic dome and not the surrounding leaf primordia. The meri-5 promoter also directed GUS expression at branching points in the shoot and root. A second meristematic gene was found to be a histone (H3) gene. The H3 promoter was isolated and fused to GUS. Expression of the H3-GUS fusion in transgenic tobacco showed preferential expression in the peripheral zone and a lack of noticeable staining in the central zone.

Expression of functional replication protein from tomato golden mosaic virus in transgenic tobacco plants.

The A component of the bipartite genome of the geminivirus tomato golden mosaic virus (TGMV) encodes the viral protein (AL1) that is required for viral DNA replication. We have constructed transgenic Nicotiana benthamiana plants in which the AL1 open reading frame is transcribed under the control of the cauliflower mosaic virus 35S promoter. The transgenic plants, which were phenotypically normal, produced a single transcript from the 35S-AL1 construct and a 40-kDa protein that cross-reacted with a polyclonal antiserum raised against AL1 protein overproduced in Escherichia coli. Six of nine transgenic lines complemented a TGMV A variant with a mutation in AL1 when coinoculated with the B component of the TGMV genome. Single- and double-stranded forms of the B component were synthesized in leaf discs from a complementing, transgenic line in the absence of TGMV A. These results establish that the transgenic plants express functional AL1 protein and show that this viral protein is not only required, but sufficient, for single- and double-stranded replication of TGMV DNA in the presence of host proteins. These results also show that the AL1 protein is not by itself a determinant of disease or pathogenesis.

Functional expression of the leftward open reading frames of the A component of tomato golden mosaic virus in transgenic tobacco plants.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. We have constructed Nicotiana benthamiana plants that are transgenic for the three overlapping open reading frames, AL1, AL2, and AL3, from the left side of TGMV A. In the transgenic plants, the AL open reading frames are under the control of the cauliflower mosaic virus (CaMV) 35S promoter. In TGMV infectivity assays, seven of 10 transgenic lines complemented TGMV A variants with mutations in AL1, AL2, or AL3 when co-inoculated with the B component. The 35S-AL construct was transcribed as a single RNA species in the transgenic plants, indicating that AL1, AL2, and AL3 were expressed from a polycistronic mRNA. This differs from the complex transcription pattern in TGMV-infected plants, which contains five AL transcripts. There was no quantitative correlation between the efficiency of complementation in the infectivity assay and the level of expression of transgenic AL RNA in the leaves of a transgenic line. One line that failed to complement defects in AL1, AL2, and AL3 in infectivity assays contained high levels of transgenic AL RNA and functional AL1 protein. These results provide evidence that chromosomal position can affect the cell- and tissue-specific transcription of the 35S promoter in transgenic plants. Comparison of the complementing plants and wild-type infected plants may provide insight into the TGMV infection process and the use of the CaMV 35S promoter for gene expression in transgenic plants.

Transient expression of heterologous RNAs using tomato golden mosaic virus.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. The A component contains the only virally-encoded function required for autonomous replication in infected plant cells. We used agroinoculation of petunia leaf discs with the A component to develop a transient expression system which permits direct examination of viral transcripts by S1 nuclease protection. The AR1 gene, which encodes the TGMV coat protein, was transcribed transiently in leaf discs after agroinoculation of TGMV a DNA. Synthesis of AR1 RNA was dependent on T-DNA transfer and TGMV DNA replication, demonstrating that it is a plant transcription product. The AL open reading frames of TGMV A were also expressed transiently in leaf discs. The ratio between AR1 RNA and the major leftward RNA was constant and was used to normalize AR1 transcription for viral DNA copy number. The bacterial genes encoding chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) were transiently expressed in leaf discs from the AR1 promoter in TGMV A. The levels of AR1 and GUS RNAs were similar in leaf discs after adjusting for viral DNA copy number, while CAT RNA was less abundant. The geminivirus transient expression system allows rapid analysis of RNAs transcribed from foreign genes and can serve as a preliminary screen in the construction of transgenic plants.

Genetic analysis of the tomato golden mosaic virus. II. The product of the AL1 coding sequence is required for replication.

Tomato golden mosaic virus (TGMV) belongs to the geminivirus subgroup that is characterized by a split genome consisting of two single-stranded circular DNAs. The TGMV A genome component encodes the virus coat protein as well as all of the functions necessary for viral DNA replication. Analysis of the nucleotide sequence indicates that the TGMV A component has, in addition to the coat protein encoding ORF, four overlapping open reading frames (ORFs) with the potential to encode proteins of greater than 10 kD. We have investigated the functions of these putative proteins in both symptom formation and DNA replication by creating mutations in each of the ORFs. Our results show that the AL4 ORF, which is encoded within the N-terminal region of ORF AL1, is not essential for normal virus infection. In contrast, we find that disruption of the AL3 ORF results in delay and attenuation of symptom formation. We also report that the products of the AL1 and AL2 ORFs are absolutely required for symptom formation. Studies of DNA replication show that only the AL1 open reading frame is essential for viral DNA synthesis. The significance of these results for the development of vectors from the geminiviruses is discussed.

Genetic analysis of tomato golden mosaic virus: the coat protein is not required for systemic spread or symptom development.

The geminiviruses are a unique group of higher plant viruses that are composed of twin isometric particles which contain circular, single-stranded DNA. Tomato golden mosaic virus (TGMV), a whitefly-transmitted agent, belongs to the subgroup of geminiviruses whose members possess a bipartite genome. The TGMV A genome component has the capacity to encode at least four proteins. One of these is the viral coat protein, as inferred by homology with coat-protein, genes of other geminiviruses and by the observation of typical geminate particles in transgenic plants that contain inserts of TGMV A DNA. We have investigated the role of the coat protein in TGMV replication and report here that its coding sequence may be interrupted or substantially deleted without loss of infectivity. However, certain coat-protein mutants showed reproducible delays in time of symptom appearance as well as reduced symptom development, when inoculated onto transgenic Nicotiana benthamiana plants containing the TGMV B component. The most attenuated symptoms were seen with a mutant in which the coat-protein coding sequence was almost entirely deleted. The significance of these findings for the development of plant vectors from TGMV DNA is discussed.

Agrobacterium-mediated inoculation of plants with tomato golden mosaic virus DNAs.

We have adapted the "agroinfection" procedure of Grimsley and co-workers [4,5] to develop a simple, efficient, reproducible infectivity assay for the insect-transmitted, split-genome geminivirus, tomato golden mosaic virus (TGMV). Agrobacterium T-DNA vectors provide efficient delivery of both components of TGMV when used in mixed inoculation of wild-type host plants. A greater increase in infection efficiency can be obtained by Agrobacterium delivery of the TGMV A component to "permissive" transgenic plants. These "permissive" plants contain multiple tandem copies of the B component integrated into the host genome. An inoculum containing as few as 2000 Agrobacterium cells can produce 100% infection under these conditions. Further, our results show that there is a marked effect of the configuration of the TGMV A components within the T-DNA vector on time of symptom development. We have also found that transgenic plants carrying tandem copies of the A component do not complement the B component. Possible mechanisms to explain these results and the potential use of this system to further study the functions of the geminivirus components in infection are discussed.

Cholinergic receptor mutants of the nematode Caenorhabditis elegans.

Potential acetylcholine receptor (AChR) mutants of the nematode are selectable by resistance to the neurotoxic drug levamisole, a probable cholinergic agonist. To determine which mutants may have achieved resistance through loss of levamisole receptor function, we have assayed mutant extracts for specific 3H-meta-aminolevamisole binding activity in the presence and absence of mecamylamine. We find that mutants in 3 of the 7 genes associated with extreme levamisole resistance are obviously deficient in saturable specific 3H-meta-aminolevamisole binding activity. Mutants of the 4 other genes have abnormal binding activities that fail to undergo the apparent allosteric activation of saturable specific 3H-meta-aminolevamisole binding activity caused by mecamylamine. Thus, all 7 genes appear to be required to produce a fully functional levamisole receptor. Mutants of several other genes associated only with partial resistance to levamisole have at least grossly normal receptor binding activities.

Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants.

Phenotypically normal petunia plants carrying chromosomal inserts of either the tomato golden mosaic virus (TGMV) A or the B component DNA, as single or tandem inserts, were obtained using an Agrobacterium tumefaciens Ti plasmid-based transformation system. Southern hybridization analysis revealed that the tandem, direct-repeat A plants contained free single and double stranded A component DNAs. No free B component DNA was detected in plants carrying tandem repeats of the B component. Progeny of self-fertilized plants appeared normal. In contrast, one-quarter of the progeny from tandem A by tandem B plant crosses showed chlorotic lesions on their leaves similar to virus symptoms. The significance of these results and the use of this method for the study of virus functions involved in TGMV replication and symptom production are discussed.