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 sprea

Transgenic papaya story: still a public-sector anomaly?

Papaya is the cheapest, most nutritious fruit in Hawaii. When papaya in Hawaii was threatened by the PRS virus. Efforts were made to save the crop, but all conventional methods failed, threatening a major basic food source for people not just in Hawaii, but all over the developing world. Eventually virus resistance was introduced through genetic engineering. This was achieved entirely through public sector research and the resistant plant material provided to farmers in Hawaii for free

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 viru

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 T0 transgenic N.benthamiana plants expressing the GLRaV-2 CP gene were regenerated following Agrobacterium-mediated transformation. Disease resistance was evaluated in greenhouse-grown T1 and T2 plants by mechanical inoculation with GLRaV-2. Although all the inoculated non-transgenic plants showed symptoms 2-4weeks post inoculation, various numbers of transgenic plants (16-100%) in 14 of 20 T1 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 T2 progenies, the percentage of resistant plants was generally lower (0-63%) than that of the corresponding T1 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 strateg

Transgenic Melon and Squash Expressing Coat Protein Genes of Aphid-borne Viruses do not Assist the Spread of an Aphid Non- transmissible Strain of Cucumber Mosaic Virus in the Field

Transgenic melon and squash containing the coat protein (CP) gene of the aphid transmissible strain WL of cucumber mosaic cucumovirus (CMV) were grown under field conditions to determine if they would assist the spread of the aphid non-transmissible strain C of CMV, possibly through heterologous encapsidation and recombination. Transgenic melon were susceptible to CMV strain C whereas transgenic squash were resistant although the latter occasionally developed chlorotic blotches on lower leaves. Transgenic squash line ZW-20, one of the parents of commercialized cultivar Freedom II, which expresses the CP genes of the aphid transmissible strains FL of zucchini yellow mosaic (ZYMV) and watermelon mosaic virus 2 (WMV 2) potyviruses was also tested. Line ZW-20 is resistant to ZYMV and WMV 2 but is susceptible to CMV. Field experiments conducted over two consecutive years showed that aphid-vectored spread of CMV strain C did not occur from any of the CMV strain C-challenge inoculated transgenic plants to any of the uninoculated CMV-susceptible non- transgenic plants. Although CMV was detected in 3% (22/764) of the uninoculated plants, several assays including ELISA, RT- PCR-RFLP, identification of CP amino acid at position 168, and aphid transmission tests demonstrated that these CMV isolates were distinct from strain C. Instead, they were non-targeted CMV isolates that came from outside the field plots. This is the first report on field experiments designed to determine the potential of transgenic plants expressing CP genes for triggering changes in virus-vector specificity. Our results indicate that transgenic plants expressing CP genes of aphid transmissible strains of CMV, ZYMV, and WMV 2 are unlikely to mediate the spread of aphid non-transmissible strains of CMV. This finding is of practical relevance because transgenic crops expressing the three CP genes are targeted for commercial release, and because CMV is economically important, has a wide host range, and is widespread worldwid

A protocol for efficient transformation and regeneration of Carica papaya L

Summary: A reproducible and effective biolistic method for transforming papaya (Carica papaya L.) was developed with a transformation-regeneration system that targeted a thin layer of embryogenic tissue. The key factors in this protocol included: 1) spreading of young somatic embryo tissue that arose directly from excised immature zygotic embryos, followed by another spreading of the actively growing embryogenic tissue 3 d before biolistic transformation; 2) removal of kanamycin selection from all subsequent steps after kanamycin-resistant clusters were first isolated from induction media containing kanamycin; 3) transfer of embryos with finger-like extensions to maturation medium; and 4) transferring explants from germination to the root development medium only after the explants had elongating root initials, had at least two green true leaves, and were about 0.5 to 1.0 cm tall. A total of 83 transgenic papaya lines expressing the nontranslatable coat protein gene of papaya ringspot virus (PRSV) were obtained from somatic embryo clusters that originated from 63 immature zygotic embryos. The transformation efficiency was very high: 100% of the bombarded plates produced transgenic plants. This also represents an average of 55 transgenic lines per gram fresh weight, or 1.3 transgenic lines per embryo cluster that was spread. We validated this procedure in our laboratory by visiting researchers who did four independent projects to transform seven papaya cultivars with coat protein gene constructs of PRSV strains from four different countries. The method is described in detail and should be useful for the routine transformation and regeneration of papay

Sequence similarity between the viral cp gene and the transgene in transgenic papayas

O gene da capa protéica (cp) do vírus da mancha anelar do mamoeiro (Papaya ringspot virus, PRSV), presente nos mamoeiros 'Rainbow' e 'SunUp', tem alta similaridade de seqüência (>89%) com o gene cp dos isolados PRSV BR e TH. Apesar deste alto grau de similaridade, ambos isolados são capazes de quebrar a resistência observada em 'Rainbow', ao passo que TH quebra a resistência em 'SunUp'. O objetivo deste trabalho foi avaliar o grau de similaridade de seqüência entre o gene cp do vírus desafiante e do transgene em mamoeiros transgênicos resistentes a PRSV. Produziu-se um vírus híbrido contendo o genoma do isolado PRSV HA até o sítio de restrição Apa I no gene NIb, e, a partir deste ponto, este vírus continha o genoma do isolado PRSV TH. PRSV HA/TH foi utilizado para desafiar plantas de 'Rainbow', 'SunUp' e de uma população R2 derivada da linha 63-1, todas resistentes a PRSV HA. PRSV HA/TH quebrou a resistência em todas essas plantas, demonstrando que a similaridade da seqüência é um fator preponderante no mecanismo de resistência utilizado pelos mamoeiros transgênicos expressando o gene cp de PRSV. Também foi realizada análise comparativa do transgene cp das plantas derivadas das linhas transgênicas 55-1 e 63-1, e do gene cp dos isolados PRSV HA, BR e TH.The Papaya ringspot virus (PRSV) coat protein transgene present in 'Rainbow' and 'SunUp' papayas disclose high sequence similarity (>89%) to the cp gene from PRSV BR and TH. Despite this, both isolates are able to break down the resistance in 'Rainbow', while only the latter is able to do so in 'SunUp'. The objective of this work was to evaluate the degree of sequence similarity between the cp gene in the challenge isolate and the cp transgene in transgenic papayas resistant to PRSV. The production of a hybrid virus containing the genome backbone of PRSV HA up to the Apa I site in the NIb gene, and downstream from there, the sequence of PRSV TH was undertaken. This hybrid virus, PRSV HA/TH, was obtained and used to challenge 'Rainbow', 'SunUp', and an R2 population derived from line 63-1, all resistant to PRSV HA. PRSV HA/TH broke down the resistance in both papaya varieties and in the 63-1 population, demonstrating that sequence similarity is a major factor in the mechanism of resistance used by transgenic papayas expressing the cp gene. A comparative analysis of the cp gene present in line 55-1 and 63-1-derived transgenic plants and in PRSV HA, BR, and TH was also performed

Cantaloupe line CZW-30 containing coat protein genes of cucumber mosaic virus, zucchini yellow mosaic virus, and watermelon mosaic virus-2 is resistant to these three viruses in the field

Cantaloupe line CZW-30 containing coat protein gene constructs of cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV-2) was investigated in the field over two consecutive years for resistance to infections by CMV, ZYMV, and/or WMV-2. Resistance was evaluated under high disease pressure achieved by mechanical inoculations and/or natural challenge inoculations by indigenous aphid vectors. Across five different trials, homozygous plants were highly resistant in that they never developed systemic symptoms as did the nontransformed plants but showed few symptomatic leaves confined close to the vine tips. Hemizygous plants exhibited a significant delay (2-3 weeks) in the onset of disease compared to control plants but had systemic symptoms 9-10 weeks after transplanting to the field. Importantly, ELISA data revealed that transgenic plants reduced the incidence of mixed infections. Only 8% of the homozygous and 33% of the hemizygous plants were infected by two or three viruses while 99% of the nontransformed plants were mixed infected. This performance is of epidemiological significance. In addition, control plants were severely stunted (44% reduction in shoot length) and had poor fruit yield (62% loss) compared to transgenic plants, and most of their fruits (60%) were unmarketable. Remarkably, hemizygous plants yielded 7.4 times more marketable fruits than control plants, thus suggesting a potential commercial performance. This is the first report on extensive field trials designed to assess the resistance to mixed infection by CMV, ZYMV, and WMV-2, and to evaluate the yield of commercial quality cantaloupes that are genetically engineere