Centro Nacional de Biotecnologia (C.S.I.C.), Campus de la Universidad Autónoma, 28049 Madrid, Spain
Nicotiana benthamiana plants were transformed with intact and mutated nuclear inclusion b (NIb) gene sequences of plum pox potyvirus, Rankovic isolate (PPV-R). The constructs included additional in-frame initiation and termination codons. All of the eight independently transformed lines showed some kind of protection against PPV; however, this protection was largely overcome at high inoculum doses. Interestingly, a phenotype of total recovery from the initial infection was observed in a high percentage of plants of two lines transformed with an NIb coding sequence that carried a Gly to Val mutation at the GDD motif typical of viral RNA replicases. Recovery frequently started with the emergence of dark green patches in the infected leaves that accumulated much less virus than the surrounding tissue. Newly developing leaves were symptomless and virus free, and showed highly effective, and very specific, resistance to virus reinoculation. Both in the dark green patches and in the symptomless leaves, virus decline was accompanied by a drastic reduction in the accumulation of transgene transcripts. Normal transgene mRNA levels and initial susceptibility to infection were recovered in the progeny plants coming from the autofecundation of a recovered plant, indicating that the recovery phenotype is not meiotically stable. The results are discussed in terms of a previously proposed model that links gene silencing and RNA-mediated virus resistance (J. J. English, E. Mueller, and D. C. Baulcombe, Plant Cell 8:179--188, 1996), adapted to explain the recovery phenomenon.