RESISTANCE TO TOMATO YELLOW LEAF CURL GEMINIVIRUS IN NICOTIANA-BENTHAMIANA PLANTS TRANSFORMED WITH A TRUNCATED VIRAL C1 GENE

The C1 gene of tomato yellow leaf curl geminivirus (TYLCV) encodes a m ultifunctional protein (Rep) involved in replication, A truncated form of this gene, capable of expressing the N-terminal 210 amino acids (a a) of the Rep protein, was cloned under the control of the CaMV 35S pr omoter and introduced into Nicotiana benthamiana using Agrobacterium t umefaciens. The same sequence was also cloned in antisense orientation . When self-pollinated progeny of 19 primary transformants were tested for resistance to TYLCV by agroinoculation, some plants proved to be resistant, particularly in the sense lines. Two such lines were furthe r studied. The presence of the transgene was verified and its expressi on was followed at intervals. AII plants that were resistant to TYLCV at 4 weeks postinoculation (wpi) contained detectable amounts of trans genic mRNA and protein at the time of infection. Resistance was overco me in a few plants at 9 wpi, and in most at 15 wpi. Infection of leaf discs derived from transgenic plants showed that expression of the tra nsgene correlated with a substantial reduction of viral DNA replicatio n. Cotransfections of tobacco protoplasts demonstrated that inhibition of viral DNA replication requires expression of the truncated Rep pro tein and suggested that the small ORF C4, also present in our construc t, plays no role in the resistance observed. The results obtained usin g both transient and stable gene expression systems show that the expr ession of the N-terminal 210 aa of the TYLCV Rep protein efficiently i nterferes with virus infection. (C) 1996 Academic Press, Inc.