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Dennis Gonsalves

Publications and source records attributed to Dennis Gonsalves.

12 recordsLinked to original sources
PubMed2007

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.

Base Sequence↗
PubMed2006

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.

Animals↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2001

Comparative reactions of recombinant papaya ringspot viruses with chimeric coat protein (CP) genes and wild-type viruses on CP-transgenic papaya.

Transgenic papaya cultivars SunUp and Rainbow express the coat protein (CP) gene of the mild mutant of papaya ringspot virus (PRSV) HA. Both cultivars are resistant to PRSV HA and other Hawaii isolates through homology-dependent resistance via post-transcriptional gene silencing. However, Rainbow, which is hemizygous for the CP gene, is susceptible to PRSV isolates from outside Hawaii, while the CP-homozygous SunUp is resistant to most isolates but susceptible to the YK isolate from Taiwan. To investigate the role of CP sequence similarity in overcoming the resistance of Rainbow, PRSV HA recombinants with various CP segments of the YK isolate were constructed and evaluated on Rainbow, SunUp and non-transgenic papaya. Non-transgenic papaya were severely infected by all recombinants, but Rainbow plants developed a variety of symptoms. On Rainbow, a recombinant with the entire CP gene of YK caused severe symptoms, while recombinants with only partial YK CP sequences produced a range of milder symptoms. Interestingly, a recombinant with a YK segment from the 5' region of the CP gene caused very mild, transient symptoms, whereas recombinants with YK segments from the middle and 3' parts of the CP gene caused prominent and lasting symptoms. SunUp was resistant to all but two recombinants, which contained the entire CP gene or the central and 3'-end regions of the CP gene and the 3' non-coding region of YK, and the resulting symptoms were mild. It is concluded that the position of the heterologous sequences in the recombinants influences their pathogenicity on Rainbow.

Capsid↗
PubMed2000

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.

Artificial Gene Fusion↗
PubMed2000

Evidence that resistance in squash mosaic comovirus coat protein-transgenic plants is affected by plant developmental stage and enhanced by combination of transgenes from different lines.

Three transgenic lines of squash hemizygous for the coat protein genes of squash mosaic virus (SqMV) were shown previously to have resistant (SqMV-127), susceptible (SqMV-22) or recovery (SqMV-3) phenotypes. Post-transcriptional gene silencing (PTGS) was the underlying mechanism for resistance of SqMV-127. Here, experiments conducted to determine the mechanism of the recovery phenotype and whether enhanced resistance could be obtained by combining transgenes from susceptible and recovery plants are reported. Upper leaves of SqMV-3 plants were sampled for Northern analysis at 17, 31 and 45 days after germination (DAG) and a proportion of plants were inoculated with SqMV. SqMV-3 plants inoculated at a young stage (17 DAG) showed susceptible or recovery phenotypes. However, a number of plants inoculated at later developmental stages (31 or 45 DAG) were resistant to infection. Resistance of recovery plants was due to PTGS that was activated at a later developmental stage, independent of virus infection. Similar results were observed with plants grown under field conditions. To investigate the interactions of transgenes, progeny of crosses between SqMV-127, -3 and -22 were inoculated with SqMV. Progeny with the transgene of line 127 were resistant. However, a number of plants with transgenes from the recovery and susceptible lines or the self-pollinated recovery line were resistant even when inoculated at a young stage. Northern analysis suggested that resistance was due to PTGS. The results reveal that the timing of PTGS and consequent resistance of the transgenic plants were affected by their developmental stage and the interaction of transgene inserts.

Blotting, Northern↗
PubMedDate not supplied

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.

Capsid Proteins↗
PubMedDate not supplied

Comparative fitness of a wild squash species and three generations of hybrids between wild x virus-resistant transgenic squash.

We compared some fitness components of the wild squash species Cucurbita pepo spp. ovifera var. texana (C. texana) and three generations of hybrids (F1, BC1, and BC2) between C. texana and commercial transgenic squash CZW-3 over three consecutive years under field conditions of low (LDP) and high disease pressure (HDP) by Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV). Transgenic squash CZW-3 expresses the coat protein (CP) genes of CMV, ZYMV, and WMV, and is resistant to these three aphid-borne viruses. Across all HDP trials, transgenic BC1 and BC2 hybrids expressing the three CP genes grew more vigorously, displayed resistance to CMV, ZYMV, and WMV, and produced a greater number of mature fruits and viable seeds than nontransgenic hybrid segregants and C. texana. Transgenic F1 hybrids behaved similarly to BC1 and BC2 hybrids but grew less vigorously than C. texana. In contrast, across all LDP trials, C. texana outperformed the transgenic and nontransgenic hybrid segregants. Further, only one back cross was necessary to recover individuals with most of the C. texana characteristics and yet maintain virus resistance. Our data suggest that C. texana acquiring CP transgenes upon hybridization and introgression could have a selective advantage if CMV, ZYMV, and WMV are severely limiting the growth and reproductibility of wild squash populations.

Animals↗