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

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Abstract

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.

Translated abstract

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.

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Most cited references 28

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Genetic and biochemical dissection of transgenic RNA-mediated virus resistance.

J. Goodwin, K. Chapman, S Swaney … (1996)

RNA-mediated virus resistance has been observed in transgenic plants at varying frequencies, suggesting that a nuclear requirement or other pre-condition must be met. This study was undertaken to characterize genetically transgenes that confer a highly resistant state to infection by tobacco etch virus (TEV). Transgenic tobacco line 2RC-6.13, expressing an untranslatable mRNA containing the TEV coat protein open reading frame, had three distinct transgene integration events that segregated as two linkage groups. A genetic series of plants that contained zero, one, two, or all three transgene inserts in both homozygous and heterozygous conditions was produced and examined. Genetic and biochemical data suggested that RNA-mediated virus resistance is a multigenic trait in line 2RC-6.13; three or more transgenes were necessary to establish the highly resistant state. One or two transgene copies resulted in an inducible form of resistance (i.e., recovery). Transcription rates and steady state RNA levels of the transgene-derived transcript present in different members of the genetic series supported a post-transcriptional RNA degradation process as the underlying mechanism for transgene transcript reduction and virus resistance. This degradation process appeared to initiate via cleavage of specific sites within the target RNA sequence, as determined by RNA get blot and primer extension analyses of transgene-derived mRNA from various transgenic plant lines.