Search

Pigeonpea (Cajanus cajan), an important food legume crop in the semi-arid regions of the world and the second most important pulse crop in India, has an average crop productivity of 780 kg/ha. The relatively low crop yields may be attributed to non-availability of improved cultivars, poor crop husbandry and exposure to a number of biotic and abiotic stresses in pigeonpea growing regions. Narrow genetic diversity in cultivated germplasm has further hampered the effective utilization of conventional breeding as well as development and utilization of genomic tools, resulting in pigeonpea being often referred to as an ‘orphan crop legume’. To enable genomics-assisted breeding in this crop, the pigeonpea genomics initiative (PGI) was initiated in late 2006 with funding from Indian Council of Agricultural Research under the umbrella of Indo-US agricultural knowledge initiative, which was further expanded with financial support from the US National Science Foundation’s Plant Genome Research Program and the Generation Challenge Program. As a result of the PGI, the last 3 years have witnessed significant progress in development of both genetic as well as genomic resources in this crop through effective collaborations and coordination of genomics activities across several institutes and countries. For instance, 25 mapping populations segregating for a number of biotic and abiotic stresses have been developed or are under development. An 11X-genome coverage bacterial artificial chromosome (BAC) library comprising of 69,120 clones have been developed of which 50,000 clones were end sequenced to generate 87,590 BAC-end sequences (BESs). About 10,000 expressed sequence tags (ESTs) from Sanger sequencing and ca. 2 million short ESTs by 454/FLX sequencing have been generated. A variety of molecular markers have been developed from BESs, microsatellite or simple sequence repeat (SSR)-enriched libraries and mining of ESTs and genomic amplicon sequencing. Of about 21,000 SSRs identified, 6,698 SSRs are under analysis along with 670 orthologous genes using a GoldenGate SNP (single nucleotide polymorphism) genotyping platform, with large scale SNP discovery using Solexa, a next generation sequencing technology, is in progress. Similarly a diversity array technology array comprising of ca. 15,000 features has been developed. In addition, >600 unique nucleotide binding site (NBS) domain containing members of the NBS-leucine rich repeat disease resistance homologs were cloned in pigeonpea; 960 BACs containing these sequences were identified by filter hybridization, BES physical maps developed using high information content fingerprinting. To enrich the genomic resources further, sequenced soybean genome is being analyzed to establish the anchor points between pigeonpea and soybean genomes. In addition, Solexa sequencing is being used to explore the feasibility of generating whole genome sequence. In summary, the collaborative efforts of several research groups under the umbrella of PGI are making significant progress in improving molecular tools in pigeonpea and should significantly benefit pigeonpea genetics and breeding. As these efforts come to fruition, and expanded (depending on funding), pigeonpea would move from an ‘orphan legume crop’ to one where genomics-assisted breeding approaches for a sustainable crop improvement are routine.

Abstract Background In today's age of genomic discovery, no attempt has been made to comprehensively sequence a gymnosperm genome. The largest genus in the coniferous family Pinaceae is Pinus, whose 110-120 species have extremely large genomes (c. 20-40 Gb, 2N = 24). The size and complexity of these genomes have prompted much speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed to be highly repetitive, but there is little information available on the nature and identity of repetitive units in gymnosperms. The pines have extensive genetic resources, with approximately 329000 ESTs from eleven species and genetic maps in eight species, including a dense genetic map of the twelve linkage groups in Pinus taeda. Results We present here the Sanger sequence and annotation of ten P. taeda BAC clones and Genome Analyzer II whole genome shotgun (WGS) sequences representing 7.5% of the genome. Computational annotation of ten BACs predicts three putative protein-coding genes and at least fifteen likely pseudogenes in nearly one megabase of sequence. We found three conifer-specific LTR retroelements in the BACs, and tentatively identified at least 15 others based on evidence from the distantly related angiosperms. Alignment of WGS sequences to the BACs indicates that 80% of BAC sequences have similar copies (≥ 75% nucleotide identity) elsewhere in the genome, but only 23% have identical copies (99% identity). The three most common repetitive elements in the genome were identified and, when combined, represent less than 5% of the genome. Conclusions This study indicates that the majority of repeats in the P. taeda genome are 'novel' and will therefore require additional BAC or genomic sequencing for accurate characterization. The pine genome contains a very large number of diverged and probably defunct repetitive elements. This study also provides new evidence that sequencing a pine genome using a WGS approach is a feasible goal.

Readily transformable Nicotiana tabacum cv. SR1 (Petite Havana) was evaluated as a host for the bioassay of Xylella fastidiosa strains. Plant growing conditions and inoculation methods were optimized to enhance symptom expression 4 to 6 weeks post inoculation. Tobacco plants were inoculated with X. fastidiosa strains associated with almond leaf scorch disease (ALSD) and Pierce's disease (PD) of grapevine in California. All PD strains and the ALSD strain Dixon caused characteristic leaf scorch symptoms, whereas two other ALSD-associated strains (M12 and M23) caused severe leaf chlorosis followed by necrosis, leaf death, and drooping of older leaves. Symptoms began to develop 10 to 14 days post inoculation and proceeded to resemble those of X. fastidiosa-infected grape and almond. The presence of X. fastidiosa in affected plants was confirmed by reisolation of the pathogen, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction (QPCR), and observation of X. fastidiosa cells by transmission and scanning electron microscopy, as well as by confocal laser scanning microscopy, in the xylem cells of inoculated plants. The pathogenicity of selected reisolated strains was confirmed by inoculation of grape plants in the greenhouse. The average levels of X. fastidiosa cells/g of tissue, estimated by QPCR, were higher for PD strains than for ALSD strains and reflected the relative titers of these strains in economic hosts. No symptoms were observed and bacteria were not detected in untreated tobacco or in tobacco inoculated with Xanthomonas campestris pv. campestris or water. Symptoms induced by Xylella fastidiosa in this bioassay were fully expressed within 2 months following inoculation. The described bioassay, under optimized environmental conditions, provides a useful system for studying X. fastidiosa strains (e.g., confirmation of pathogenicity and differentiation of PD and ALSD pathotypes) and for investigating X. fastidiosa-host interactions. N. tabacum cv. SR1 tobacco was a better bioassay host for X. fastidiosa than N. tabacum cvs. Havana, RP1, and TNN described previously. [ABSTRACT FROM AUTHOR]

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1 -transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity., Competing Interests: Competing interests statement:The authors declare no competing interest.

Root-knot nematodes (RKN; Meloidogyne spp.) can parasitize over 2,000 plant species and are generally considered to be the most agriculturally damaging group of plant-parasitic nematodes worldwide. Infective juveniles (J2) are non-feeding and must locate and invade a host before their reserves are depleted. However, what attracts J2 to appropriate root entry sites is not known. An aim of this research is to identify semiochemicals that attract RKN to roots. J2 of the three RKN species tested are highly attracted to root tips of both tomato and Medicago truncatula. For both hosts, mutants defective in ethylene signaling were found to be more attractive than those of wild type. We determined that cell-free exudates collected from tomato and M. truncatula seedling root tips were highly attractive to M. javanica J2. Using a pluronic gel-based microassay to monitor chemical fractionation, we determined that for both plant species the active component fractionated similarly and had a mass of ~400 based on size-exclusion chromatography. This characterization is a first step toward identification of a potent and specific attractant from host roots that attracts RKN. Such a compound is potentially a valuable tool for developing novel and safe control strategies.

Pierce's disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms.

We postulated that a synergistic combination of two innate immune functions, pathogen surface recognition and lysis, in a protein chimera would lead to a robust class of engineered antimicrobial therapeutics for protection against pathogens. In support of our hypothesis, we have engineered such a chimera to protect against the gram-negative Xylella fastidiosa (Xf), which causes diseases in multiple plants of economic importance. Here we report the design and delivery of this chimera to target the Xf subspecies fastidiosa (Xff), which causes Pierce disease in grapevines and poses a great threat to the wine-growing regions of California. One domain of this chimera is an elastase that recognizes and cleaves MopB, a conserved outer membrane protein of Xff. The second domain is a lytic peptide, cecropin B, which targets conserved lipid moieties and creates pores in the Xff outer membrane. A flexible linker joins the recognition and lysis domains, thereby ensuring correct folding of the individual domains and synergistic combination of their functions. The chimera transgene is fused with an amino-terminal signal sequence to facilitate delivery of the chimera to the plant xylem, the site of Xff colonization. We demonstrate that the protein chimera expressed in the xylem is able to directly target Xff, suppress its growth, and significantly decrease the leaf scorching and xylem clogging commonly associated with Pierce disease in grapevines. We believe that similar strategies involving protein chimeras can be developed to protect against many diseases caused by human and plant pathogens.

Extreme resistance of Arlington line cowpea (Vigna unguiculata) to Cowpea mosaic virus (CPMV) is under control of a dominant locus designated Cpa. We transiently expressed, using Tomato bushy stunt virus (TBSV) vectors and Agrobacterium tumefaciens, in nearly isogenic Cpa/Cpa and cpa/cpa cowpea lines, sequences from RNA1, the larger of two CPMV genomic RNAs. Activation of a Cpa-specific response mapped to the CPMV 24K protease (24KPro). Mutational analysis of the 24KPro gene implicated protease activity, rather than 24KPro structure, in Cpa-mediated recognition of CPMV invasion. A 24KPro with alanine replacing the active site cysteine [24KPro(C-A)], but not wildtype 24KPro, accumulated after agroinfiltration of the corresponding binary vector constructions into Cpa/Cpa cowpea. In cpa/cpa cowpea, both protease versions accumulated, with 24KPro(C-A) in greater abundance. Thus, enzymically active 24KPro was recognized by both cowpea genotypes, but in Cpa/Cpa cowpea the suppression of 24KPro accumulation was very strong, consistent with extreme resistance to CPMV., (Copyright © 2011 Elsevier Inc. All rights reserved.)

I recount the early influences that directed me toward a career in research and then describe some efforts investigating Cowpea mosaic virus and the satellite RNA of Tobacco ringspot virus. These descriptions have a common theme of surprise, how things often can be not as they are expected to be. Finally, I examine the widely held belief that a plant transgene derived from a distant taxonomic source presents a greater risk than a transgene derived from a closely related plant and contend that this also is a situation in which things may not be as they initially seem., (Copyright © 2011 by Annual Reviews. All rights reserved.)

Root-knot nematodes (Meloidogyne spp.) are obligate endoparasites of a wide range of plant species. The infective stage is attracted strongly to and enters host roots at the zone of elongation, but the compounds responsible for this attraction have not been identified. We developed a simple assay to investigate nematode response to chemical gradients that uses Pluronic F-127, a synthetic block copolymer that, as a 23% aqueous solution, forms a liquid at low temperature and a gel at room temperature. Test chemicals are put into a modified pipette tip, or 'chemical dispenser,' and dispensers are inserted into the gel in which nematodes have been dispersed. Meloidogyne hapla is attracted to pH gradients formed by acetic acid and several other Brønsted acids and aggregates between pH 4.5 and 5.4. While this pH range was attractive to all tested root-knot nematode strains and species, the level of aggregation depended on the species/strain assessed. For actively growing roots, the pH at the root surface is most acidic at the zone of elongation. This observation is consistent with the idea that low pH is an attractant for nematodes. Root-knot nematodes have been reported to be attracted to carbon dioxide, but our experiments suggest that the observed attraction may be due to acidification of solutions by dissolved CO(2) rather than to CO(2) itself. These results suggest that Pluronic F-127 gel will be broadly applicable for examining responses of a range of organisms to chemical gradients or to each other.

The cowpea (Vigna unguiculata) line Arlington, inoculated with Cowpea mosaic virus (CPMV), showed no symptoms, and no infectivity or accumulation of capsid antigen was detected at several days after inoculation. Coinoculation, but not sequential inoculation, of CPMV with similar concentrations of another Comovirus; Cowpea severe mosaic virus (CPSMV), resulted in reduced numbers of CPSMV-induced lesions. This apparent, CPMV-mediated reduction in number of CPSMV-induced infection centers was termed concurrent protection. We report results obtained by inoculating two nearly isogenic cowpea lines derived from a CPMV-susceptible cowpea crossed to Arlington, one line CPMV-susceptible and the other resistant. The CPMV virions B and M, encapsidating genomic RNAs 1 and 2, respectively, were extensively purified by gradient centrifugation. In the CPMV-resistant cowpea, either CPMV or CPMV B affected concurrent protection against CPSMV and against two distinct non-Comoviruses: Cherry leafroll virus and Southern bean mosaic virus. Adding CPMV M to the inoculum did not enhance CPMV-B-mediated protection. CPMV B was ineffective in protecting CPMV-susceptible cowpea. We postulate that CPMV-mediated concurrent protection is elicited in CPMV-resistant cowpea by a CPMV RNA-1-encoded factor and acts to reduce accumulation or spread of CPMV and certain coinoculated challenging viruses in or from the inoculated cell. Coinoculated CPMV did not protect CPMV-resistant cowpea against Tomato bushy stunt virus or Cucumber mosaic virus., (Copyright 2000 Academic Press.)

The most abundant form of the satellite RNA of tobacco ringspot virus (sTRSV RNA) is a linear, unit length molecule of 359 nucleotide residues, designated L-(+)M. A postulated replication scheme for the satellite RNA has as its first, and apparently virus-independent, step the ligation of L-(+)M into the corresponding circular form C-(+)M. We transiently expressed L-(+)M wild type and L-(+)M mutants in tobacco protoplasts using an African cassava mosaic geminivirus vector. Measured extents of C-(+)M accumulation were correlated with computer-predicted folding to suggest wild-type secondary structure elements that might be deleted without reducing ligation. A 127-nucleotide residue mutant L-(+)M was created by replacing, with 7 and 3 residues, respectively, nucleotide residues 53-211 and 268-350, each of which was predicted to form a set of three adjacent imperfect stem-loops in wild-type L-(+)M. The mutant L-(+)M was found to be extensively ligated to C-(+)M in protoplasts and to retain a calculated helix of the wild-type molecule that incorporates the 3' terminal sequence. A trinucleotide in the 3' region was mutated so as to disrupt and restore, respectively, the calculated helix, reducing and restoring, respectively, C-(+)M formation. These results suggest that the 3' stem contributes to the suitability of the small L-(+)M molecules as a substrate for a protoplast RNA ligase and that computed folding of sTRSV RNA may be predictive of sTRSV RNA structure in vivo.

Each of the two genomic RNAs of tobacco ringspot nepovirus is known to have a 5'-linked protein, the VPg. We report a simplified analysis of the covalent VPg-RNA connection that allowed us to identify the 5' nucleotide residue of each genomic RNA and its phosphodiester link to a specific serine residue of the VPg, without resorting to in vivo labeling with 32P, in vitro radioiodination, or separation of the two genomic RNAs. Unfractionated genomic RNA was incubated with an oligodeoxyribonucleotide specific for the 5' region of either RNA 1 or RNA 2 and ribonuclease H. Reaction products were 3'-end-labeled and were fractionated by gel electrophoresis. The most highly labeled product derived from each genomic RNA was identified as a VPg-oligoribonucleotide (VPg-5'-oligo) by its sensitivity to proteinase. In a presumed beta-elimination reaction that apparently was more rapid than phosphodiester cleavage, incubation in alkaline sodium bicarbonate released a rapidly migrating product, 5'-oligo. Phosphatase-treated 5'-oligo accepted 5'-label in a polynucleotide kinase-catalyzed reaction, and uridylate was identified as the 5' terminal residue for both RNA 1 and RNA 2. Results from Edman degradation of the VPg suggest that the VPg is linked at serine 5 to the 5' uridylate of each genomic RNA.

Cherry leafroll nepovirus (CLRV) genomic RNA 1 (8 kb) and genomic RNA 2 (7 kb) have 3' polyadenylate tracts and, extending 5' from the polyadenylate, nearly identical sequences of 1.6 kb termed the 3' common region. We observed RNAs 1 and 2 and a third RNA of 1.5 kb in nucleic acid extracts of CLRV-infected Nicotiana tabacum suspension cell protoplasts and Chenopodium quinoa plants, using a hybridization probe complementary to 1 kb of the 3' common region. The third RNA was partially purified by preparative gel electrophoresis and chromatography on an oligodeoxythymidylate column. Analyses of transcripts primed by a complementary oligodeoxyribonucleotide and of cDNA clones revealed that the third RNA corresponds to the 3' 1500 nucleotide residues of RNA 1. Hence we designate the newly characterized RNA as RNA 1A. RNA 1A was not detected as encapsidated RNA in extracts of either protoplasts or C. quinoa plants. The amount of accumulated RNA 1A declined between 24 and 48 hr after inoculation of protoplasts with CLRV virions, although CLRV RNAs 1 and 2 continued to accumulate. Other results were not consistent with cleaved RNA 1 being the origin of RNA 1A. RNA 1A has the properties of a subgenomic RNA, presumably synthesized from negative-sense RNA 1 as template.

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.

The small satellite RNA of tobacco ringspot virus (sTRSV RNA) is dependent on tobacco ringspot virus (TRSV) for replication and encapsidation. sTRSV RNA has appeared during serial passage of certain TRSV strains in some hosts. Co-inoculation of bean with TRSV and either of two related, nonaccumulating mutants of sTRSV RNA induced the appearance of sTRSV RNA in a single passage (van Tol et al., 1991, Virology 180, 23-30). The sTRSV RNA obtained after serial passage and after co-inoculation have the same nucleotide sequence, designated the endogenous sequence. The endogenous sTRSV RNA nucleotide sequence differs from that of each of the nonaccumulating sTRSV RNA at three positions. In order to detect possible trace amounts of endogenous satellite RNA in virion RNA preparations, RNA from two TRSV isolates was subjected to reverse transcription and polymerase chain reaction of the transcript (RT-PCR), using primers with sTRSV RNA terminal sequences. The yield of RT-PCR product suggests that the virion RNA preparations contained approximately 0.1 fg of sTRSV RNA per microgram of virion RNA. The nucleotide sequence of the RT-PCR product corresponded to that of the endogenous sTRSV RNA. The endogenous sTRSV RNA of TRSV inocula appears to be latent, being maintained in very small amounts during serial passage of TRSV in some hosts but capable of dramatic increase during serial passage in other hosts or when TRSV was co-inoculated with either of two specific sTRSV RNA mutants. Ten other nonaccumulating sTRSV RNA mutants did not induce a detected increase in sTRSV RNA.

A small satellite RNA of tobacco ringspot virus (sTRSV RNA) generates circular and linear molecules of unit length and repetitive sequence, linear multimers during replication. The phosphodiester junction joining the unit satellite RNA sequences in multimeric and circular RNA resisted base-catalyzed cleavage in circles but not in linear dimers. We postulate that junctions of multimeric satellite RNA form during synthesis of the polyribonucleotide chain, whereas those of circular RNA result from a ligation reaction that introduces a group blocking the junction 2'-hydroxyl. To test the relative effectiveness of linear and circular satellite RNAs in initiating replication, we inoculated onto bean (Phaseolus vulgaris cv Black Valentine) the four possible pairs of satellite RNA molecules, one member of each pair having the wild-type sTRSV RNA sequence and the other that of the replicating mutant 51AG/212CU, with each sequence provided as the unit circular or linear form. The relative amounts of wild-type and mutant satellite RNA sequence recovered from progeny virions reflected their relative abundances in the inoculum without regard to whether the sequence was supplied as a linear or a circular molecule. These results are consistent with models for the replication of the satellite RNA in which a circular form of the satellite RNA is a template for rolling circle transcription or is otherwise a replication intermediate or is readily converted to an intermediate. We also show that a circular form of a nonaccumulating satellite RNA mutant induced an increase in a satellite RNA that is endogenous to some tobacco ringspot virus virion preparations, as demonstrated previously for the linear form.

Cherry leafroll nepovirus (CLRV) is the causative agent of blackline disease, which results in a fatal necrosis of the graft union of English walnut scion on certain rootstocks. Tobacco suspension cell protoplasts were electroporated with plasmid constructions, bearing or not bearing RNA-derived sequences, and, subsequently, were electroporated with CLRV virions or virion RNA Replication of CLRV in protoplasts was demonstrated by accumulation of both positive- and negative-sense CLRV genomic RNAs 1 and 2, capsid antigen, and virions. Three plasmids were tested for antiviral action. These have inserts that were derived from the coat protein gene, inserted in both orientations, and from the 3' terminal sequence that is nearly identical in RNA 1 and RNA 2, oriented for expression of antisense RNA. Plasmids were introduced into protoplasts 12 hr prior to introducing the virions or virion RNA. CLRV accumulation was reduced significantly by prior electroporation of plasmids intended to express coat protein or 3' antisense RNA, but not by electroporation of plasmid without insert or plasmid with coat protein encoding sequences in the antisense orientation. These results demonstrate the utility of transient expression in a protoplast system for comparing the efficacy of a variety of virus-derived and other sequences for their potential application in virus control strategies.

Hybridization analyses of cDNA clones derived from the two genomic RNAs, RNA1 and RNA2, of the walnut strain of the nepovirus cherry leafroll nepovirus (wCLRV) demonstrated a long region of high homology between the two viral RNAs. Subsequent mapping and nucleotide sequencing revealed a long, noncoding, presumably untranslated, region (3' UTR) immediately 5' of the terminal polyadenylate, a region that is almost identical in the two RNAs. This 3' UTR is 1567 nucleotide residues long in RNA1. Homologies of about 80% were found with corresponding regions of genomic RNAs from other strains of CLRV, but not with the corresponding regions of other nepovirus genomic RNAs.

The ST9 strain of beet western yellows virus (BWYV ST9) is unique among BWYV strains because it encapsidates not only its 5.6-kb genomic RNA but also a 2.8-kb RNA of distinct nucleotide sequence, designated as the ST9-associated RNA. We obtained isolates of BWYV ST9 that are free of the associated RNA by transfecting Nicotiana tabacum protoplasts with transcripts of an ST9 genomic cDNA clone. Aphids were fed on extracts of infected protoplasts and were transferred to young Shepherd's Purse (Capsella bursa-pastoris) plants. When the protoplast inoculum was ST9 genomic transcript or virion RNA of the L-1 strain of BWYV (free of the associated RNA), symptoms were mild and characteristic of BWYV L-1. When ST9-associated RNA was included in the inoculum with genomic RNA of either source, subsequently infected Shepherd's Purse plants showed the severe symptoms that are characteristic of BWYV ST9. Inclusion of ST9-associated RNA in the inoculum with ST9 genomic RNA increased the accumulation of capsid antigen and ST9 genomic RNA, relative to infections initiated with ST9 genomic RNA alone. Using gold-labeled antibody and electron microscopy, we assessed the distribution of virions in Shepherd's Purse plants. Regardless of whether the associated RNA was present, sites showing immunoreactivity above background levels were restricted to the phloem, suggesting that the increased BWYV ST9 titer and symptom severity that are correlated with the presence of the ST9-associated RNA are not due to escape of the infection from phloem limitation.

The 359 nucleotide residue (nt) satellite RNA of tobacco ringspot virus increases detectably only in association with replicating tobacco ringspot virus and becomes encapsidated in the virus coat protein. Results from previous reports are consistent with participation of rolling circle transcription in replication of the satellite RNA: (i) both the more abundant plus polarity satellite RNA, s(+)RNA and the complementary s(-)RNA occur in multimeric forms that self-cleave to release the unit length, 359 nt satellite RNA, (ii) circles of both s(+)RNA and s(-)RNA are present in extracts of infected tissue, and (iii) s(-)RNA, but not s(+)RNA, spontaneously and efficiently circularizes in vitro. Our analyses of RNA in tissue extracts suggest that s(+)RNA of all forms is about 100-fold more abundant than s(-)RNA. Nucleic acids were purified rapidly to minimize interconversion of linear and circular forms. For s(+)RNA and for s(-)RNA, the circular and the linear forms were detected in about equal amounts in tissue extracts. The linear s(-)RNA from tissue extracts was found to have the same 5'-terminal sequence as previously was found for s(-)RNA self-cleaved from in vitro transcripts. Like s(-)RNA synthesized in vitro, the circular and linear s(-)RNA from tissue extracts spontaneously and readily interconverted during incubation in vitro. In contrast, the bulk of the circular and linear forms of s(+)RNA were stable. The very limited interconversion of s(+)RNA forms in vitro suggests that circulation in vivo is enzymically catalyzed. Encapsidated satellite RNA was found to be composed of linear, unit length and multimeric forms, including previously undocumented s(-)RNA present in approximately the same relative abundance compared to s(+)RNA as was observed for RNA from tissue extracts. Circles were not detected in encapsidated RNA. We interpret our results in the context of a rolling circle model for satellite RNA replication.

Infections of plants by subviral RNA agents, alone or in association with virus genomic RNA molecules, are well known. The ST9 strain of beet western yellows virus encapsidates not only the 5.6-kilobase genomic RNA that is typical of luteoviruses, but also a 2.8-kilobase-associated RNA that has a distinct nucleotide sequence. The ST9-associated RNA has been postulated to be a satellite RNA, which by definition would be capable of replicating only in coinfections with beet western yellows virus or closely related viruses. To characterize the associated RNA, we inoculated protoplasts and leaves with in vitro transcripts of the virus genomic RNA and the ST9-associated RNA separately and in combination. Surprisingly, the ST9-associated RNA alone replicated efficiently in both protoplasts and leaves, and it stimulated accumulation of the virus genomic RNA in protoplasts. Thus, the ST9-associated RNA is a newly discovered type of plant infectious agent, which depends on its associated virus, beet western yellows virus, for encapsidation but not for replication.

The replication properties of linker insertion-deletion mutants of tobacco ringspot virus satellite RNA have been studied by amplification in plants infected with the helper virus. Sequence analysis of the cDNAs corresponding to the replicated forms shows that only one of the original mutated molecules replicates unaltered, and in general new variants accumulate. Depending on the location of the original mutation three types of sequence modifications were observed: (i) deletion of the mutated region followed by sequence duplication, (ii) sequence duplication and deletion outside of the mutated region and (iii) limited rearrangements at the site of mutation. The mutant that replicates without sequence changes accumulates linear multimeric forms suggesting that self-cleavage is affected although the sequence alteration does not involve the hammerhead catalytic domain. Alternative RNA conformations are likely to play a role in the origin of this phenotype and in the formation of sequence duplications. These results demonstrate the great structural flexibility of this satellite RNA.

The less abundant polarity of the satellite RNA of tobacco ringspot virus, designated sTobRV(-)RNA, contains a ribozyme and its substrate. We demonstrate that the ribozyme can catalyze the ligation of substrate cleavage products and that oligoribonucleotides, termed 'mini-monomers' and containing little more than covalently attached ribozyme and substrate cleavage products, circularized spontaneously, efficiently and reversibly. The kinetics of ligation and cleavage of one such mini-monomer was consistent with a simple unimolecular reaction at some temperatures. Evidence suggests that the circular ligation product includes a 5 bp stem that is connected to a 4 bp stem by a bulge loop. Reduction of the bulge loop to one nt is expected to place the 4 and 5 bp helices in a nearly coaxial, rather than an angled or parallel, orientation. Such molecules did not circularize in a unimolecular reaction but did when incubated with second, trans-acting oligoribonucleotides that had either the original or a substituted 4 bp helix. These results suggest that a bulge loop that is too small prevents formation of geometry essential for unimolecular ligation. We suggest the term 'paperclip' to represent the arrangement of RNA strands in the region of sTobRV(-)RNA that participates in the cleavage and ligation reactions.

Cowpea severe mosaic virus (CPSMV) is a member of the comovirus group of messenger-sense RNA viruses with bipartite genomes, of which cowpea mosaic virus (CPMV) is the type member. Full-length copies of CPSMV RNA 1 were cloned in plasmids bearing a bacteriophage T7 promoter. Previously, similar clones of CPSMV RNA 2 had been obtained. A 5'-rUAUUAAAAUUUU sequence is common to RNA 1 and RNA 2. From two RNA 1 clones and four RNA 2 clones we excised non-CPSMV sequences so as to provide templates for in vitro transcripts that have only a single guanylate preceding CPSMV RNA sequences. Transcripts from the most active RNA 1 and RNA 2 clones, when mixed, showed about 5% of the infectivity of unfractionated CPSMV RNAs from virions. The longest, 1858 codon open reading frame of the 5957 nt CPSMV RNA 1 extends from an AUG at nt 257 to a UGA termination codon at nt 5831. The calculated molecular weight of the polyprotein is 208,000. Comparisons with the available amino acid residue (aa) sequence information from the complete CPMV RNA 1 sequence and the partial sequence of red clover mottle virus RNA 1 suggest that CPSMV RNA 1 specifies the expected set of five mature proteins: 32K proteinase cofactor, 58K presumed helicase, VPg 5'-linked protein of the genomic RNAs, 24K proteinase, and 87K presumed polymerase, separated by four cleavage sites. Of the determined and deduced cleavage sites of the three RNA 1 polyproteins, only that at the 24K/87K junction has a distinct aa pair in the CPSMV polyprotein. Of the five proteins, VPg and 87K show the greatest similarity between CPSMV and CPMV, with identities of 68 and 55%, respectively. Published mutational analysis of the CPMV 24K proteinase and alignment of aa sequences from three comoviruses suggest that cysteine-168, histidine-40 and glutamic acid-77 form the catalytic triad of the CPSMV 24K proteinase. Results are discussed in the context of the resistance that some cowpea (Vigna unguiculata) lines exhibit against CPMV but not against CPSMV.

We report the nucleotide sequence of cowpea severe mosaic comovirus (CPSMV) genomic RNA 2. The molecule is composed of 3732 nucleotide (nt) residues, exclusive of the polyadenylate at the 3' end. Only one of the six reading frame registers has a long open reading frame, from nt 255 to nt 3260 in the polarity of encapsidated RNA and corresponding to a polyprotein of 1002 amino acid residues (aa). As has been reported for other comoviruses, a second in-frame AUG, at nt position 531, apparently also initiates translation, at least in vitro. Multiple alignments of the deduced CPSMV polyprotein aa sequence with those of bean pod mottle comovirus (BPMV), cowpea mosaic comovirus (CPMV), and red clover mottle comovirus (RCMV) were consistent with a similar size for each of the three genes: the putative movement protein, beginning at the second in-frame AUG, the large coat protein (L), and the small coat protein. Identical nucleotide sequences in the terminal noncoding regions of RNA 2 of the four viruses are limited to 9 nt at the 5' end and the 3' polyadenylate. However, extensive similarities in sequence and potential structure were found. For all three genes and the 5' untranslated region, CPSMV and BPMV are more similar to each other than either is to CPMV or RCMV, the last two being similar to each other. Observed similarities predict that both cleavage sites in the CPSMV RNA 2 polyprotein are at glutamine-serine dipeptides. A sequence of 16 aa at the amino terminus of L, determined by automated Edman degradation, matched a region of the deduced aa sequence in the polyprotein and is consistent with cleavage at the predicted glutamine-serine dipeptide.

Replication of the satellite RNA of tobacco ringspot virus (sTobRV RNA) has been postulated to require rolling circle transcription. The expected product of rolling circle transcription, multimeric sTobRV RNA, is known to undergo self-cleavage in vitro to release unit-length sTobRV RNA. A spontaneous, efficient, not enzymically-catalyzed in vitro circularization reaction is characteristic of unit-length sTobRV RNA of the less abundant, (-) polarity. We mutated sTobRV RNA at two sites that are distant from each other in the polyribonucleotide chain. A third form of the sTobRV RNA was mutated at both sites. Multimeric forms of the one-site mutants of sTobRV(+)RNA and sTobRV(-)RNA showed, respectively, undiminished and slightly diminished self-cleavage, whereas the spontaneous circularization of each one-site-mutated, unit-length sTobRV(-)RNA was greatly reduced, compared to the reactions of wild-type sTobRV RNA and the two-site mutant. The two-site mutant and the wild-type sTobRV RNAs replicated with similar efficiency. They reduced the titer of, and severity of, symptoms induced by coinoculated tobacco ringspot virus (TobRV). When coinoculated with TobRV, neither one-site mutant increased or provided protection against TobRV. Rather, each induced a substantial accumulation of what is apparently an endogenous form of sTobRV RNA. Our results are consistent with the formation of circular sTobRV(-)RNA as an essential step in sTobRV RNA replication.

Oligoribonucleotides with specific sequences derived from the satellite RNA of tobacco ringspot virus undergo autolytic cleavage at the CpA phosphodiester that is the junction between unit sequences of multimeric satellite RNA. Buzayan et al. (Nucleic Acids Res., 16, 4009-4023 (1988)) showed that an oligoribonucleotide with 97 satellite RNA-derived nucleotide residues self-cleaved with greatly reduced efficiency when it was synthesized in vitro from adenosine-5'-O-(1-thiotriphosphate) (abbreviated rATP alpha S) and three rNTPs. No other substitution of one rNTP by the corresponding rNTP alpha S had this effect, suggesting that a phosphorothioate CpA junction inhibits self-cleavage. Here, we replaced the usual CpA junction of a small self-cleaving oligoribonucleotide with a CpU junction. Self-cleavage of this molecule was reduced not only by rUTP alpha S-substitution, as expected, but also by partial and complete rATP alpha S-substitution. By analysis of the locations of rAMPS residues in cleavage products derived from partially rATP alpha S-substituted oligoribonucleotides, we identified A26 as the residue contributing the non-junction phosphorothioate diester that most strongly inhibited self-cleavage. Manganese ions strongly stimulated the self-cleavage of the rATP alpha S-substituted, CpU-junction oligoribonucleotide but was less effective when the junction was CpA.

We have developed a three-component system of host, tobacco ringspot virus (TobRV), and satellite tobacco ringspot virus RNA (sTobRV RNA) for investigating the specific contributions of host components or TobRV gene products to the propagative cycle of satellite RNA. Cowpea (Vigna unguiculata) protoplasts were inoculated with sTobRV and TobRV genomic RNAs by electroporation. An increase in sTobRV RNA was detected both by blot hybridization and by incorporation of [14C]uridine into material with the electrophoretic mobility of sTobRV RNA. DNA-dependent RNA synthesis in uninoculated protoplasts was effectively inhibited by 50 micrograms/ml actinomycin D (Act D) in the medium. Addition of Act D to protoplasts 24 or 48 hr after coinoculation with sTobRV RNA and TobRV genomic RNAs had little effect on accumulation of sTobRV RNA, whereas addition at 24 hr prior to coinoculation prevented any detected accumulation of sTobRV RNA of either polarity. Our results and previous findings of RNA complementary to encapsidated satellite RNA in extracts of infected tissue suggest that an RNA-dependent RNA polymerase is responsible for the synthesis of sTobRV RNA. The strongly inhibitory effect of Act D when added early implies a role for a host factor in the early phase of sTobRV RNA replication.

Both polarities of the satellite RNA of tobacco ringspot virus occur in infected cells in multimeric forms which are capable of autolytic processing, using different sequences and structures [Feldstein, P.A., et al., Proc. Nat. Acad. Sci. USA (1990) 87 (in press)]. These transesterification reactions generate a 2',3'-cyclophosphate and a 5'-hydroxyl as the two new end groups. Cleavage is at a CpA for the (+) polarity RNA and at an ApG for the (-) polarity RNA. We enzymically synthesized oligoribonucleotides with processing capability and with specific 35S-labeled phosphorothioate diesters in the Rp configuration. After processing had occurred, the terminal nucleoside-2',3'-cyclophosphorothioate diester residues were recovered from the appropriate product by digestion with nuclease and phosphatase. Comparisons with specially prepared endo- and exoisomer reference compounds by thin layer chromatography and autoradiography revealed that the [35S]cytidine- and [35S]adenosine-2',3'-cyclophosphorothioate both were endo-isomers. The results are consistent with transesterification occurring by an inline SN2(P) attack of the 2'-hydroxyl group in the autolytic processing reactions of both polarities of the satellite RNA.

Both polarities of the satellite RNA of tobacco ringspot virus are sources of self-cleaving sequences. RNA of the less abundant, negative polarity, designated sTobRV-(-)RNA, has cleaving activity that was mapped previously to two noncontiguous regions of the polyribonucleotide chain. Endoribonucleolytic oligoribonucleotides (E) corresponding to the larger of the two regions cleaved smaller substrate oligoribonucleotides, at the ApG phosphodiester that is cleaved in sTobRV(-)RNA. An analogue of the substrate, which has a 2'-5' ApG phosphodiester, was not cleaved by E but acted as a competitive inhibitor of the cleavage of substrate. The analogue served as a primer, and E served as template, for reverse transcriptase-catalyzed copying of specific E sequences. The sequences transcribed suggest base pairing between the 5' region of E and a portion of the substrate that is located 3' to, but does not include, the ApG phosphodiester. Results from other experiments indicate this base pairing is a part of the functional cleavage complex. The association of the ends of E and substrate anticipates a second, 4-base-pair association between E and a portion of substrate that is 5' to, but does not include, the ApG phosphodiester. The effects of compensating mutations in E and substrate oligoribonucleotides support the existence of this second association in the active cleavage complex.

The satellite RNA of tobacco ringspot virus depends upon tobacco ringspot virus for its replication and source of coat protein. The satellite RNA reduces virus accumulation and the severity of virus-induced symptoms. Repetitive sequence, dimeric, and higher forms of the satellite RNA are known to autolytically process to form biologically active monomeric RNA of 359 nucleotide residues [Prody, G. A., Bakos, J. T., Buzayan, J. M., Schneider, I. R. & Bruening, G. (1986) Science 231, 1577-1580], with a 5'-hydroxyl and a 2',3'-cyclic phosphodiester as the new terminal groups. We show here that transcripts of full-length and truncated DNA clones of the satellite RNA sequence also process in a nonenzymic reaction. One such transcript was an RNA that has about one-fourth of the satellite RNA sequence, representing the 3'-terminal and 5'-terminal portions of monomeric RNA joined in the junction that is cleaved in dimeric RNA. This RNA autolytically processed more efficiently than molecules with a larger proportion of the satellite RNA nucleotide sequence.

Previously, a protein has been reported to be associated with the RNAs of cowpea mosaic virus. Here we present evidence for genome-associated proteins of other comoviruses: squash mosaic virus, Echtes Ackerbohnemosaik-Virus, and another strain of cowpea mosaic virus. Although the proteins and protein-oligonucleotide complexes derived from the RNAs of these viruses showed similar patterns of electrophoretic mobility, two-dimensional chromatograms of their tryptic peptides were distinct. Chromatograms of peptides from preparations of cowpea mosaic virus that were isolated from two hosts were very similar, indicating that the genome-associated protein may be virus specified. Upon digestion with ribonuclease T1, RNAs from the viruses gave rise to a distribution of large oligonucleotides that is consistent with the presence of polyriboadenylate.

The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates and becomes encapsidated in association with tobacco ringspot virus. Previous results show that the infected tissue produces multimeric STobRV RNAs of both polarities. RNA that is complementary to encapsidated STobRV RNA, designated as having the (-) polarity, cleaves autolytically at a specific ApG bond. Purified autolysis products spontaneously join in a non-enzymic reaction. We report characteristics of this RNA ligation reaction: the terminal groups that react, the type of bond in the newly formed junction and the nucleotide sequence of the joined RNA. The nucleotide sequence of the ligated RNA shows that joining of the reacting RNAs restored an ApG bond. The junction ApG has a 3'-to-5' phosphodiester bond. Thus the net ligation reaction of STobRV (-)RNA is the precise reversal of autolysis. We discuss this new type of RNA ligation reaction and its implications for the formation of multimeric STobRV RNAs during replication.