Broad-Spectrum Resistance to Different Geographic Strains of Papaya ringspot virus in Coat Protein Gene Transgenic Papaya

Papaya ringspot virus (PRSV) is a major limiting factor for cultivation of papaya (Carica papaya) in tropical and subtropical areas throughout the world. Although the coat protein (CP) gene of PRSV has been transferred into papaya by particle bombardment and transgenic lines with high resistance to Hawaii strains have been obtained, they are susceptible to PRSV isolates outside of Hawaii. This strain-specific resistance limits the application of the transgenic lines in other areas of the world. In this investigation, the CP gene of a local strain isolated from Taiwan, designated PRSV YK, was transferred into papaya via Agrobacterium-mediated transformation. A total of 45 putative transgenic lines were obtained and the presence of the transgene in papaya was confirmed by polymerase chain reaction amplification. When the plants of transgenic lines were challenged with PRSV YK by mechanical inoculation, they showed different levels of resistance ranging from delay of symptom development to complete immunity. Molecular analysis of nine selected lines that exhibited different levels of resistance revealed that the expression level of the transgene is negatively correlated with the degree of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism. The segregation analysis showed that the transgene in the immune line 18-0-9 has an inheritance of two dominant loci and the other four highly resistant lines have a single dominant locus. Seven selected lines were tested further for resistance to three PRSV heterologous strains that originated in Hawaii, Thailand, and Mexico. Six of the seven lines showed varying degrees of resistance to the heterologous strains, and one line, 19-0-1, was immune not only to the homologous YK strain but also to the three heterologous strains. Thus, these CP-transgenic papaya lines with broad-spectrum resistance have great potential for use in Taiwan and other geographic areas to control PRSV.

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Engineered Resistance Against Papaya ringspot virus in Venezuelan Transgenic Papayas

Plant Disease ◽

10.1094/pdis.2004.88.5.516 ◽

2004 ◽

Vol 88 (5) ◽

pp. 516-522 ◽

Cited By ~ 28

Author(s):

Gustavo Fermin ◽

Valentina Inglessis ◽

Cesar Garboza ◽

Sairo Rangel ◽

Manuel Dagert ◽

...

Keyword(s):

Amino Acid ◽

Agrobacterium Tumefaciens ◽

Coat Protein ◽

Amino Acid Sequence ◽

Sequence Similarity ◽

Ringspot Virus ◽

Amino Acid Sequence Similarity ◽

Papaya Ringspot Virus ◽

Local Varieties ◽

Cp Gene

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.

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Field Evaluation of Transgenic Papaya Lines Carrying the Coat Protein Gene of Papaya ringspot virus in Taiwan

Plant Disease ◽

10.1094/pdis.2004.88.6.594 ◽

2004 ◽

Vol 88 (6) ◽

pp. 594-599 ◽

Cited By ~ 27

Author(s):

Huey-Jiunn Bau ◽

Ying-Huey Cheng ◽

Tsong-Ann Yu ◽

Jiu-Sherng Yang ◽

Pan-Chi Liou ◽

...

Keyword(s):

Coat Protein ◽

Transgenic Plants ◽

Rainy Season ◽

Field Evaluation ◽

Weather Conditions ◽

Fruit Production ◽

Ringspot Virus ◽

Papaya Ringspot Virus ◽

Transgenic Papaya ◽

Transgenic Lines

Four transgenic papaya lines expressing the coat protein (CP) gene of Papaya ringspot virus (PRSV) were evaluated under field conditions for their reaction to PRSV infection and fruit production in 1996 to 1999. Plants were exposed to natural virus inoculation by aphids in two adjacent fields in four different plantings at the same sites. None of the transgenic lines showed severe symptoms of PRSV whereas control nontransgenic plants were 100% severely infected 3 to 5 months after planting. In the first and second trials, 20 to 30% of the transgenic plants showed mild symptoms consisting of confined mottling or chlorotic spots on leaves. The number of transgenic plants with mild symptoms fluctuated according to the season and weather conditions, with a tendency to increase in the winter or rainy season and decrease in the summer. Also, the incidence of the mild symptoms in the third trial increased significantly due to infection by root rot fungi during the rainy season. Interestingly, there was no apparent adverse effect on fruit yield and quality in transgenic plants with mild symptoms. In the first and second experiments, transgenic lines yielded 10.8 to 11.6 and 54.3 to 56.7 times more marketable fruit, respectively, than controls. All transgenic plants produced fruit of marketable quality with no ringspots or distortion.

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Sequences of the coat protein gene from brazilian isolates of Papaya ringspot virus

Fitopatologia Brasileira ◽

10.1590/s0100-41582002000200009 ◽

2002 ◽

Vol 27 (2) ◽

pp. 174-180 ◽

Cited By ~ 7

Author(s):

ROBERTO C. A. LIMA ◽

MANOEL T. SOUZA JR. ◽

GILVAN PIO-RIBEIRO ◽

J. ALBERSIO A. LIMA

Keyword(s):

Coat Protein ◽

Carica Papaya ◽

Protein Gene ◽

Average Degree ◽

Ringspot Virus ◽

Plant Genome ◽

Papaya Ringspot Virus ◽

Transgenic Papaya ◽

Cp Gene ◽

Worldwide Production

Papaya ringspot virus (PRSV) is the causal agent of the main papaya (Carica papaya) disease in the world. Brazil is currently the world's main papaya grower, responsible for about 40% of the worldwide production. Resistance to PRSV on transgenic plants expressing the PRSV coat protein (cp) gene was shown to be dependent on the sequence homology between the cp transgene expressed in the plant genome and the cp gene from the incoming virus, in an isolate-specific fashion. Therefore, knowledge of the degree of homology among the cp genes from distinct PRSV isolates which are present in a given area is important to guide the development of transgenic papaya for the control of PRSV in that area. The objective of the present study was to assess the degree of homology among the PRSV cp genes of several Brazilian isolates of this virus. Papaya and PRSV are present in many different ecosystems within Brazil. Twelve PRSV isolates, collected in eight different states from four different geographic regions, were used in this study. The sequences of the cp gene from these isolates were compared among themselves and to the gene used to generate transgenic papaya for Brazil. An average degree of homology of 97.3% at the nucleotide sequence was found among the Brazilian isolates. When compared to 27 isolates from outside Brazil in a homology tree, the Brazilian isolates were clustered with Australian, Hawaiian, and Central and North American isolates, with an average degree of homology of 90.7% among them.

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Development of virus resistant transgenic papayas expressing the coat protein gene from a Brazilian isolate of Papaya ringspot virus

Fitopatologia Brasileira ◽

10.1590/s0100-41582005000400004 ◽

2005 ◽

Vol 30 (4) ◽

pp. 357-365 ◽

Cited By ~ 15

Author(s):

Manoel T. Souza Júnior ◽

Osmar Nickel ◽

Dennis Gonsalves

Keyword(s):

Coat Protein ◽

Carica Papaya ◽

Protein Gene ◽

Transformation Efficiency ◽

Ringspot Virus ◽

Zygotic Embryos ◽

Papaya Ringspot Virus ◽

Secondary Somatic Embryo ◽

Embryo Cultures ◽

Transgenic Lines

Translatable and nontranslatable versions of the coat protein (cp) gene of a Papaya ringspot virus (PRSV) isolate collected in the state of Bahia, Brazil, were engineered for expression in Sunrise and Sunset Solo varieties of papaya (Carica papaya). The biolistic system was used to transform secondary somatic embryo cultures derived from immature zygotic embryos. Fifty-four transgenic lines, 26 translatable and 28 nontranslatable gene versions, were regenerated, with a transformation efficiency of 2.7%. Inoculation of cloned R0 plants with PRSV BR, PRSV HA or PRSV TH, Brazilian, Hawaiian and Thai isolates, respectively, revealed lines with mono-, double-, and triple-resistance. After molecular analysis and a preliminary agronomic evaluation, 13 R1 and R2 populations were incorporated into the papaya-breeding program at Embrapa Cassava and Tropical Fruits, in Cruz das Almas, Bahia, Brazil.

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Field Resistance of Coat Protein Transgenic Papaya to Papaya ringspot virus in Jamaica

Plant Disease ◽

10.1094/pd-89-0841 ◽

2005 ◽

Vol 89 (8) ◽

pp. 841-847 ◽

Cited By ~ 20

Author(s):

Paula Tennant ◽

M. H. Ahmad ◽

D. Gonsalves

Keyword(s):

Disease Resistance ◽

Coat Protein ◽

Carica Papaya ◽

Virus Disease ◽

Field Resistance ◽

Ringspot Virus ◽

Papaya Ringspot Virus ◽

Two Generations ◽

Transgenic Lines ◽

Severe Leaf

Transgenic papayas (Carica papaya) containing translatable coat protein (CPT) or nontranslatable coat protein (CPNT) gene constructs were evaluated over two generations for field resistance to Papaya ringspot virus in a commercial papaya growing area in Jamaica. Reactions of R0 CPT transgenic lines included no symptoms and mild or severe leaf and fruit symptoms. All three reactions were observed in one line and among different lines. Trees of most CPNT lines exhibited severe symptoms of infection, and some also showed mild symptoms. R1 offspring showed reactions previously observed with parental R0 trees; however, reactions not previously observed or a lower incidence of the reaction were also obtained. The transgenic lines appear to possess virus disease resistance that can be manipulated in subsequent generations for the development of a product with acceptable commercial performance.

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RNA Silencing of the Introduced Coat Protein Gene of Turnip mosaic virus Confers Broad-Spectrum Resistance in Transgenic Arabidopsis

Phytopathology ◽

10.1094/phyto.2004.94.7.730 ◽

2004 ◽

Vol 94 (7) ◽

pp. 730-736 ◽

Cited By ~ 16

Author(s):

Ken Nomura ◽

Kazusato Ohshima ◽

Toyoaki Anai ◽

Hidetoshi Uekusa ◽

Nobuhiro Kita

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Broad Spectrum ◽

Rna Silencing ◽

Transgenic Arabidopsis ◽

Turnip Mosaic Virus ◽

Small Interfering Rnas ◽

Cp Gene ◽

Broad Spectrum Resistance ◽

Successive Generations

The coat protein (CP) gene derived from Turnip mosaic virus (TuMV) isolate JO was introduced into Arabidopsis thaliana and the resulting transgenic progenies were analyzed for resistance to TuMV. Transgenic Arabidopsis plants with no detectable transcripts of the introduced CP gene exhibited complete resistance to TuMV. There was no significant correlation between the resistance and the copy number of the transgene. Instead, small interfering RNAs (siRNAs) were detected in these resistant plants, indicating that the resistance is attributed to RNA silencing. The RNA-mediated resistance was not only inherited over successive generations but also effective against 17 worldwide TuMV isolates with different pathogenicity. Comparative analysis of the CP genes among the 17 TuMV isolates revealed that the 380-nt in the 3′ region is highly conserved, suggesting the importance of the 3′ conserved region for broad-spectrum resistance. These results indicate that introduction of the TuMV-CP gene into the target Brassicaceae plants followed by selecting transformants that show RNA silencing for the transgenes can be an effective and reliable strategy for developing crucifer crops with a broad spectrum of resistance to TuMV.

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

Pesquisa Agropecuária Brasileira ◽

10.1590/s0100-204x2005000500009 ◽

2005 ◽

Vol 40 (5) ◽

pp. 479-486 ◽

Cited By ~ 1

Author(s):

Manoel Teixeira Souza Júnior ◽

Dennis Gonsalves

Keyword(s):

Comparative Analysis ◽

Coat Protein ◽

Transgenic Plants ◽

Sequence Similarity ◽

Ringspot Virus ◽

Papaya Ringspot Virus ◽

High Sequence Similarity ◽

Mechanism Of Resistance ◽

Cp Gene ◽

Do So

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.

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

Journal of General Virology ◽

10.1099/0022-1317-82-11-2827 ◽

2001 ◽

Vol 82 (11) ◽

pp. 2827-2836 ◽

Cited By ~ 32

Author(s):

Chu-Hui Chiang ◽

Ju-Jung Wang ◽

Fuh-Jyh Jan ◽

Shyi-Dong Yeh ◽

Dennis Gonsalves

Keyword(s):

Coat Protein ◽

Sequence Similarity ◽

Transcriptional Gene Silencing ◽

Papaya Ringspot Virus ◽

Wild Type ◽

Coding Region ◽

Transgenic Papaya ◽

Cp Gene ◽

Post Transcriptional Gene Silencing

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.

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