Resistance to Tobacco mosaic virus and Botrytis cinerea in tobacco transformed with complementary DNA encoding an inhibitor of viral replication‐like protein

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AbstractWe have previously cloned a 1016‐bp‐long complementary DNA (cDNA) clone (named NC330) encoding an inhibitor of virus replication (IVR)‐like protein from induced‐resistant leaves of Nicotiana tabacum cv. Samsun NN. In the present work, NC330 cDNA was cloned in an Agrobacterium binary vector and introduced into the genome of N. tabacum cv. Samsun nn that is susceptible to Tobacco mosaic virus (TMV). Eight R0 were highly resistant, with TMV titres less than 2% of the controls. The progeny of these primary transformants resulted in four TMV‐resistant and four susceptible lines. The progeny of the plant with highest resistance for TMV was selfed during four generations. In the R2 generation, out of 112 plants tested, 3 plants showed excellent resistance and 5 a lesser degree of resistance. NC330 transcripts were detected in these R2 plants as well as in their R1 and R0 parents. Two R2 plants were self‐pollinated to obtain the R3 generation. All the 25 R3 progeny plants of one line contained PCR‐amplifiable NC330, compared with only 6 of the 23 progeny plants from the other line. Following TMV inoculation, virus was undetectable in seven of the eight tested R3 progeny of the first line and five of the six R3 progeny plants of the second line. NC330 transcripts were detected in the TMV‐resistant transgenic plants but also in some susceptible plants. TMV resistance was suppressed when the plants were kept at higher temperatures (32–34°C), whereas returning the plants to lower temperatures (24–26°C) restored resistance. Transgenic plants resistant to TMV were also resistant to B. cinerea following inoculation of seedlings or of whole plants. The presence of the NC330 DNA was correlated with changes in the physiology of the transgenic plant. Seed germination was inhibited at high temperatures and in the dark. Rootlets of germinating seeds of the transgenic plants grew significantly faster than those of control seeds. These observations were correlated with a significantly higher concentration of abscisic acid in the seeds and seedlings of the transgenic plants. In silico analysis suggests that the NC330 protein translated from the NC330 cDNA is a tetratricopeptide repeats protein and that it may be involved in protein–protein interaction as part of the pathogen resistance mechanism.