Screening promoters for Anthurium transformation using transient expression.

KEY MESSAGE : There are multiple publications on Anthurium transformation, yet a commercial product has not been achieved. This may be due to use of non-optimum promoters here we address this problem. Different promoters and tissue types were evaluated for transient ß-glucuronidase (GUS) expression in Anthurium andraeanum Hort. 'Marian Seefurth' following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35S promoter from Cauliflower Mosaic Virus fused to a GUS reporter gene were bombarded into in vitro grown anthurium lamina, somatic embryos and roots. The number of GUS foci and the intensity of GUS expression were evaluated for each construct. Ubiquitin promoters from rice and maize resulted in the highest number of expressing cells in all tissues examined. Due to the slow growth of anthurium plants, development of transgenic anthurium plants takes years. This research has rapidly identified multiple promoters that express in various anthurium tissues facilitating the development of transformation vectors for the expression of desirable traits in anthurium plants.

Genetic diversity and evidence for recent modular recombination in Hawaiian Citrus tristeza virus.

The Hawaiian Islands are home to a widespread and diverse population of Citrus tristeza virus (CTV), an economically important pathogen of citrus. In this study, we quantified the genetic diversity of two CTV genes and determined the complete genomic sequence for two strains of Hawaiian CTV. The nucleotide diversity was estimated to be 0.0565 + or - 0.0022 for the coat protein (CP) gene (n = 137) and 0.0822 + or - 0.0033 for the p23 gene (n = 30). The genome size and organization of CTV strains HA18-9 and HA16-5 were similar to other fully sequenced strains of CTV. The 3'-terminal halves of their genomes were nearly identical (98.5% nucleotide identity), whereas the 5'-terminal halves were more distantly related (72.3% nucleotide identity), suggesting a possible recombination event. Closer examination of strain HA16-5 indicated that it arose through recent recombination between the movement module of an HA18-9 genotype, and the replication module of an undescribed CTV genotype.

Safety of virus-resistant transgenic plants two decades after their introduction: lessons from realistic field risk assessment studies.

Potential safety issues have been raised with the development and release of virus-resistant transgenic plants. This review focuses on safety assessment with a special emphasis on crops that have been commercialized or extensively tested in the field such as squash, papaya, plum, grape, and sugar beet. We discuss topics commonly perceived to be of concern to the environment and to human health--heteroencapsidation, recombination, synergism, gene flow, impact on nontarget organisms, and food safety in terms of allergenicity. The wealth of field observations and experimental data is critically evaluated to draw inferences on the most relevant issues. We also express inside views on the safety and benefits of virus-resistant transgenic plants, and recommend realistic risk assessment approaches to assist their timely deregulation and release.

Development of genetically engineered resistant papaya for papaya ringspot virus in a timely manner: a comprehensive and successful approach.

Papaya orchards throughout most of the world are severely damaged by the destructive disease caused by the papaya ringspot virus (PRSV). PRSV-resistant papaya expressing the coat protein gene (CP) of PRSV have been used in Hawaii to control PRSV since 1998. This chapter presents the experimental steps involved in the development of transgenic papaya, including transgene construction, transformation, and analysis for virus resistance of the transformed papaya. We also describe the important factors that enabled deregulation, commercialization, and adoption of transgenic papaya to occur in Hawaii in a timely manner. Transfer of this technology to other countries with the similar goal and the development of transgenic papaya in other regions of the world also are described.

Incidence, Spatial Patterns, and Associations Among Viruses in Snap Bean and Alfalfa in New York.

Recent epidemics in snap bean (Phaseolus vulgaris) characterized by virus-like symptoms prompted a survey of commercial fields for Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), and the Bean yellow mosaic virus (BYMV)/Clover yellow vein virus (ClYVV) complex in 2002 and 2003. Snap bean fields were either remote from or adjacent to alfalfa (Medicago sativa), a putative source of these viruses. Bean fields were sampled at the bloom stage in both years. Model-adjusted mean incidences of infection by AMV, BYMV/ClYVV, and CMV were 41.96, 6.56, and 6.69%, respectively, in alfalfa, and 6.66, 6.38, and 17.20% in snap bean. In 2002, 25.9% of snap bean plants were infected by more than one virus; <1% had more than one virus in 2003. Virus incidences did not differ between snap bean adjacent to or remote from alfalfa, but incidence of infection by AMV and BYMV/ClYVV was significantly higher in snap bean planted later in the season rather than earlier. In 2002, there was a positive association between AMV and CMV in the tendency to find both viruses in the same snap bean plant. In some years, infection by aphid-transmitted viruses can become widespread in snap bean in New York.

Comparative development and impact of transgenic papayas in Hawaii, Jamaica, and Venezuela.

We present data concerning the creation of transgenic papayas resistant to Papaya ringspot virus (PRSV) and their adoption by three different countries: the United States (e.g., Hawaii), Jamaica, and Venezuela. Although the three sets of transgenic papayas showed effective resistance to PRSV, the adoption rate in each country has varied from full utilization in Hawaii to aggressive testing but delay in deregulating of the product in Jamaica to rejection at an early stage in Venezuela. Factors that contributed to the rapid adoption in Hawaii include a timely development of the transgenic product, PRSV causing severe damage to the papaya industry, close collaboration between researchers and the industry, and the existence of procedures for deregulating a transgenic product. In Jamaica, the technology for developing the initial field-testing of the product progressed rather rapidly, but the process of deregulation has been slowed down owing to the lack of sustained governmental efforts to complete the regulatory procedures for transgenic crops. In Venezuela, the technology to develop and greenhouse test the transgenic papaya has moved abreast with the Jamaica project, but the field testing of the transgenic papaya within the country was stopped very early on by actions by people opposed to transgenic products. The three cases are discussed in an effort to provide information on factors, other than technology, that can influence the adoption of a transgenic product.

Genome sequences and structures of two biologically distinct strains of Grapevine leafroll-associated virus 2 and sequence analysis.

Grapevine leafroll-associated virus 2 (GLRaV-2), a member of the genus Closterovirus within Closteroviridae, is implicated in several important diseases of grapevines including "leafroll", "graft-incompatibility", and "quick decline" worldwide. Several GLRaV-2 isolates have been detected from different grapevine genotypes. However, the genomes of these isolates were not sequenced or only partially sequenced. Consequently, the relationship of these viral isolates at the molecular level has not been determined. Here, we group the various GLRaV-2 isolates into four strains based on their coat protein gene sequences. We show that isolates "PN" (originated from Vitis vinifera cv. "Pinot noir"), "Sem" (from V. vinifera cv. "Semillon") and "94/970" (from V. vinifera cv. "Muscat of Alexandria") belong to the same strain, "93/955" (from hybrid "LN-33") and "H4" (from V. rupestris "St. George") each represents a distinct strain, while Grapevine rootstock stem lesion-associated virus.

The wayward Hawaiian boy returns home.

This chapter represents a travelog of my life and career and the philosophical points I acquired along the way. I was born on a sugar plantation on the island of Hawaii and early on had a stuttering problem. I attended the Kamehameha Schools and received my BS and MS degrees from the University of Hawaii and my Ph.D. from the University of California at Davis. I link my life and career to various principles and events, some of which are: the importance of positioning oneself; going for the big enchilada; music, the international language; the red zone of biotechnology; the human side of biotechnology; the transgenic papaya story; and my leadership time at USDA in Hawaii. The guiding light throughout my career were the words from Drs. Eduardo Trujillo and Robert Shepherd, respectively, "Dennis, don't just be a test tube scientist, do something to help people" and "Now tell me, what have you really accomplished?"

First Detection of Rupestris stem pitting associated virus Particles by Antibody to a Recombinant Coat Protein.

Rupestris stem pitting associated virus (RSPaV), a member of the genus Foveavirus, is associated with the Rupestris stem pitting component of the Rugose wood (RW) disease complex of grapevines. Heretofore, particles of RSPaV have not been visualized. In this work, flexuous rod particles approximately 723 nm in length were detected in the sap of infected grapevines by immunosorbent electron microscopy (ISEM), using a polyclonal antiserum produced to a recombinant coat protein of RSPaV. Particles of RSPaV were detected in tissue culture-, greenhouse-, and field-grown grapevines infected with RSPaV, but not in healthy control plants. Detection of virus particles by ISEM corresponded with detection of RSPaV by Western blot, enzyme-linked immunosorbent assay, and reverse transcription-polymerase chain reaction. Virus particles were decorated with the antibodies specific to RSPaV but not with antibodies to Grapevine virus A or Grapevine virus B, two other viruses believed to be associated with RW. This definitive identification of RSPaV particles will help define the etiology of RW.

Antiserum to Recombinant Virus Coat Protein Detects Rupestris stem pitting associated virus in Grapevines.

Rupestris stem pitting (RSP) is the most widespread virus disease of grapevines. The genome of Rupestris stem pitting associated virus (RSPaV), the putative causal agent of RSP, was recently sequenced. Until recently, the only method to diagnose RSP was biological indexing on woody indicator plants, a process that takes 2 to 3 years to complete. This study reports on the production of a polyclonal antiserum to a recombinant coat protein of RSPaV. The antiserum was used effectively to detect RSPaV from various genotypes and tissues of grapevines by Western blot and indirect enzyme-linked immunosorbent assay. Virus antigens were consistently detected in the cambium of dormant canes and in actively growing leaves of grapevines. Moreover, plants of Vitis rupestris 'St. George', the standard biological indicator for RSP, tested positive for RSPaV. The serological methods developed in this study are advantageous as compared with biological indexing because they are more rapid, less expensive, as reliable, and more suitable for assays of a large number of samples.

Engineered Resistance Against Papaya ringspot virus in Venezuelan Transgenic Papayas.

Local varieties of papaya grown in the Andean foothills of Mérida, Venezuela, were transformed independently with the coat protein (CP) gene from two different geographical Papaya ringspot virus (PRSV) isolates, designated VE and LA, via Agrobacterium tumefaciens. The CP genes of both PRSV isolates show 92 and 96% nucleotide and amino acid sequence similarity, respectively. Four PRSV-resistant R0 plants were intercrossed or selfed, and the progenies were tested for resistance against the homologous isolates VE and LA, and the heterologous isolates HA (Hawaii) and TH (Thailand) in greenhouse conditions. Resistance was affected by sequence similarity between the transgenes and the challenge viruses: resistance values were higher for plants challenged with the homologous isolates (92 to 100% similarity) than with the Hawaiian (94% similarity) and, lastly, Thailand isolates (88 to 89% similarity). Our results show that PRSV CP gene effectively protects local varieties of papaya against homologous and heterologous isolates of PRSV.

Comparative Virus Resistance and Fruit Yield of Transgenic Squash with Single and Multiple Coat Protein Genes.

Five transgenic squash lines expressing coat protein (CP) genes from cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV 2) were analyzed in the field for their reaction to mixed infections by these three viruses and for fruit production. Test plants were exposed to natural inoculations via aphids in trials simulating the introduction of viruses by secondary spread from mechanically infected susceptible border row plants. Plants of transgenic line CZW-3 expressing the CP genes from CMV, ZYMV, and WMV 2 displayed the highest level of resistance with no systemic infection, although 64% exhibited localized chlorotic dots which were mainly confined to older leaves. CZW-3 plants had a 50-fold increase in marketable yield compared to controls and the highest predicted cash returns. Plants of transgenic line ZW-20 expressing the CP genes from ZYMV and WMV 2 displayed high levels of resistance to these two potyviruses, but 22% became infected by CMV. However, ZW-20 plants provided a 40-fold increase in marketable yield relative to controls and good estimated cash returns. Three transgenic lines expressing single CP genes from either ZYMV (line Z-33), WMV 2 (line W-164) or CMV (line C-14) developed systemic symptoms similar to those of controls but showed a delay of 2 to 4 weeks before the onset of disease. Plants of transgenic line Z-33 were highly resistant to ZYMV but not to WMV 2 and CMV. Interestingly, Z-33 plants had a 20-fold increase in marketable yield compared to controls and some predicted cash returns if market sale prices were high. This study indicates that virus-resistant transgenic lines are economically viable even if they are affected by viruses other than those to which they are resistant.

Allergenicity assessment of the papaya ringspot virus coat protein expressed in transgenic rainbow papaya.

The virus-resistant, transgenic commercial papaya Rainbow and SunUp (Carica papaya L.) have been consumed locally in Hawaii and elsewhere in the mainland United States and Canada since their release to planters in Hawaii in 1998. These papaya are derived from transgenic papaya line 55-1 and carry the coat protein (CP) gene of papaya ringspot virus (PRSV). The PRSV CP was evaluated for potential allergenicity, an important component in assessing the safety of food derived from transgenic plants. The transgene PRSV CP sequence of Rainbow papaya did not exhibit greater than 35% amino acid sequence homology to known allergens, nor did it have a stretch of eight amino acids found in known allergens which are known common bioinformatic methods used for assessing similarity to allergen proteins. PRSV CP was also tested for stability in simulated gastric fluid and simulated intestinal fluid and under various heat treatments. The results showed that PRSV CP was degraded under conditions for which allergenic proteins relative to nonallergens are purported to be stable. The potential human intake of transgene-derived PRSV CP was assessed by measuring CP levels in Rainbow and SunUp along with estimating the fruit consumption rates and was compared to potential intake estimates of PRSV CP from naturally infected nontransgenic papaya. Following accepted allergenicity assessment criteria, our results show that the transgene-derived PRSV CP does not pose a risk of food allergy.

Resistance to Grapevine leafroll associated virus-2 is conferred by post-transcriptional gene silencing in transgenic Nicotiana benthamiana.

Grapevine leafroll-associated virus-2 (GLRaV-2) is an important component of the leafroll disease complex in grapevine. We have previously sequenced the GLRaV-2 genome and identified the coat protein (CP) gene. The objective of this study is to test the concept of pathogen-derived resistance against a closterovirus associated with grapevine leafroll disease. Because GLRaV-2 is capable of infecting Nicotiana benthamiana, we decided to test the concept on this herbaceous host. Thirty-seven T(0) transgenic N. benthamiana plants expressing the GLRaV-2 CP gene were regenerated following Agrobacterium-mediated transformation. Disease resistance was evaluated in greenhouse-grown T(1) and T(2) plants by mechanical inoculation with GLRaV-2. Although all the inoculated non-transgenic plants showed symptoms 2-4 weeks post inoculation, various numbers of transgenic plants (16-100%) in 14 of 20 T(1) lines tested were not infected. In these resistant plants, GLRaV-2 was not detectable by enzyme linked immunosorbent assay. Although virus resistance was confirmed in T(2) progenies, the percentage of resistant plants was generally lower (0-63%) than that of the corresponding T(1) lines (0-100%). Northern blot and nuclear run-off results showed that virus resistance in the transgenic plants was consistently associated with the low level of transgene RNA transcript suggesting a post-transcriptional gene silencing. The success of pathogen-derived resistance to GLRaV-2 in transgenic N. benthamiana plants represents the first step towards eventual control of the leafroll disease in grapevines using this strategy.

Papaya ringspot virus-P: characteristics, pathogenicity, sequence variability and control.

TAXONOMY: Papaya ringspot virus (PRSV) is an aphid-transmitted plant virus belonging to the genus Potyvirus, family Potyviridae, with a positive sense RNA genome. PRSV isolates belong to either one of two major strains, P or W. The P strains infect both papaya and cucurbits whereas the W strains infect only cucurbits. GEOGRAPHICAL DISTRIBUTION: PRSV-P is found in all major papaya-growing areas. PHYSICAL PROPERTIES: Virions are filamentous, non-enveloped and flexuous measuring 760-800 x 12 nm. Virus particles contain 94.5% protein and 5.5% nucleic acid. The protein component consists of the virus coat protein (CP), which has a molecular weight of about 36 kDa as estimated by Western blot analysis. Density of the sedimenting component in purified PRSV preparations is 1.32 g/cm(3) in CsCl. GENOME: The PRSV genome consists of a unipartite linear single-stranded positive sense RNA of 10 326 nucleotides with a 5' terminus, genome-linked protein, VPg. TRANSMISSION: The virus is naturally transmitted via aphids in a non-persistent manner. Both the CP and helper component (HC-Pro) are required for vector transmission. This virus can also be transmitted mechanically, and is typically not seed-transmitted. HOSTS: PRSV has a limited number of hosts belonging to the families Caricaceae, Chenopodiaceae and Cucurbitaceae. Propagation hosts are: Carica papaya, Cucurbita pepo and Cucumis metuliferus cv. accession 2459. Local lesion assay hosts are: Chenopodium quinoa and Chenopodium amaranticolor. CONTROL: Two transgenic papaya varieties, Rainbow and SunUp, with engineered resistance to PRSV have been commercially grown in Hawaii since 1998. Besides transgenic resistance, tolerant varieties, cross-protection and other cultural practices such as isolation and rogueing of infected plants are used to manage the disease. VIRUS CODE: 00.057.0.01.045. VIRUS ACCESSION NUMBER: 57010045. USEFUL LINK: http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/57010045.htm.

Comparative spatial spread overtime of Zucchini Yellow Mosaic Virus (ZYMV) and Watermelon Mosaic Virus (WMV) in fields of transgenic squash expressing the coat protein genes of ZYMV and WMV, and in fields of nontransgenic squash.

The spatial and temporal patterns of aphid-vectored spread of Zucchini Yellow Mosaic Virus (ZYMV) and Watermelon Mosaic Virus (WMV) were monitored over two consecutive years in plantings of nontransgenic and transgenic squash ZW-20H (commercial cv. Freedom II) and ZW-20B, both expressing the coat protein genes of ZYMV and WMV. All test plants were surrounded by nontransgenic plants that were mechanically inoculated with ZYMV or WMV, and served as primary virus source. Across all trials, none of the transgenic plants exhibited systemic symptoms upon infection by ZYMV and WMV but a few of them developed localized chlorotic dots and/or blotches, and had low mixed infection rates [4% (6 of 139) of ZW-20H and 9% (13 of 139) of ZW-20B], as shown by ELISA. Geostatistical analysis of ELISA positive transgenic plants indicated, (i) a lack of spatial relationship on spread of ZYMV and WMV for ZW-20H with flat omnidirectional experimental semivariograms that fitted poorly theoretical models, and (ii) some extent of spatial dependence on ZYMV spread for ZW-20B with a well structured experimental semivariogram that fitted poorly theoretical models during the first but not the second growing season. In contrast, a strong spatial dependence on spread of ZYMV and WMV was found for nontransgenic plants, which developed severe systemic symptoms, had prevalent mixed infection rates (62%, 86 of 139), and well-defined omnidirectional experimental semivariograms that fitted a spherical model. Geostatistical data were sustained by virus transmission experiments with Myzus persicae in screenhouses, showing that commercial transgenic squash ZW-20H alter the dynamics of ZYMV and WMV epidemics by preventing secondary plant-to-plant spread.

Complete genome sequences of two new variants of Grapevine rupestris stem pitting-associated virus and comparative analyses.

Grapevine rupestris stem pitting-associated virus (GRSPaV), a member of the genus Foveavirus within the family Flexiviridae, is the putative causal agent of the disease Rupestris stem pitting (RSP) of grapevines. GRSPaV comprises a family of variants whose pathological characteristics have not been determined. Recently, many of the indicator "St George" plants (Vitis rupestris) used throughout the world to index RSP tested positive for GRSPaV. This finding questions the validity of past biological indexing results. In this work, a representative genomic region of GRSPaV was first sequenced from ten "St George" plants from two sources and it was demonstrated that nine of them carried a new variant, GRSPaV-SG1. The genomes of GRSPaV-SG1 and GRSPaV-BS from "Bertille Seyve 5563" plants were sequenced, revealing a genome structure identical to that of GRSPaV-1. It was demonstrated experimentally that infection of "St George" plants with GRSPaV-SG1 is asymptomatic and thus it is proposed that GRSPaV-SG1 infection should not have interfered with the outcome of past indicator indexing. This represents the first attempt to link a GRSPaV variant with pathological properties.

Movement of coat protein genes from a commercial virus-resistant transgenic squash into a wild relative.

We monitored pollen-mediated transgene dissemination from commercial transgenic squash CZW-3 into its wild relative Cucurbita pepo ssp. ovifera var. texana (C. texana). Transgenic squash CZW-3 expresses the neomycin phosphotransferase II (nptII) gene and the coat protein (CP) genes of Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV), and Watermelon mosaic virus (WMV); thereby, it is resistant to these three aphid-borne viruses. The rate of NPT II and CP transgene introgression increased with overlapping flowering patterns and a high ratio of transgenic F1 hybrids (C. texana x CZW-3) to C. texana. Transgene transfer also readily occurred from transgenic F1 hybrids into C. texana over three generations in field settings where test plants grew sympatrically and viruses were not severely limiting the growth, and fruit and seed production of C. texana. In contrast, introgression of the transgenes into C. texana was not sustained under conditions of high viral disease pressure. As expected, C. texana progeny that acquired the CP transgenes exhibited resistance to CMV, ZYMV, and WMV. This is the first report on transgene dissemination from a transgenic crop that exhibits disease resistance and hybridizes with a wild plant species without loss of fertility.

Complete nucleotide sequence and genome organization of Grapevine leafroll-associated virus 3, type member of the genus Ampelovirus.

This study reports on the complete genome sequence of Grapevine leafroll-associated virus 3, the type member of the genus Ampelovirus. The genome is 17 919 nt in size and contains 13 open reading frames (ORFs). Previously, the sequence of 13 154 nt of the 3'-terminal of the genome was reported. The newly sequenced portion contains a 158 nt 5' UTR, a single papain-like protease and a methyltransferase-like (MT) domain. ORF1a encodes a large polypeptide with a molecular mass of 245 kDa. With a predicted +1 frameshift, the large fusion protein generated from ORF1a/1b would produce a 306 kDa polypeptide. Phylogenetic analysis using MT domains further supports the creation of the genus Ampelovirus for mealy-bug-transmitted viruses in the family Closteroviridae.

A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing.

We showed previously that 218 and 110 bp N gene segments of tomato spotted wilt virus (TSWV) that were fused to the non-target green fluorescent protein (GFP) gene were able to confer resistance to TSWV via post-transcriptional gene silencing (PTGS). N gene segments expressed alone did not confer resistance. Apparently, the GFP DNA induced PTGS that targetted N gene segments and the incoming homologous TSWV for degradation, resulting in a resistant phenotype. These observations suggested that multiple resistance could be obtained by replacing the GFP DNA with a viral DNA that induces PTGS. The full-length coat protein (CP) gene of turnip mosaic virus (TuMV) was linked to 218 or 110 bp N gene segments and transformed into Nicotiana benthamiana. A high proportion (4 of 18) of transgenic lines with the 218 bp N gene segment linked to the TuMV CP gene were resistant to both viruses, and resistance was transferred to R(2) plants. Nuclear run-on and Northern experiments confirmed that resistance was via PTGS. In contrast, only one of 14 transgenic lines with the TuMV CP linked to a 110 bp N gene segment yielded progeny with multiple resistance. Only a few R(1) plants were resistant and resistance was not observed in R(2) plants. These results clearly show the applicability of multiple virus resistance through the fusion of viral segments to DNAs that induce PTGS.