Cotton leaf curl disease (CLCuD) is caused by Cotton leaf curl virus (CLCuV) DNA A, a geminivirus and a single-stranded satellite DNA β responsible for inducing symptoms. Antisense RNA technology was used to develop transgenic plants resistant to CLCuD. Three regions D1/4 (466 bp), Dl (543 bp) and D1/d2/d3 (526 bp) of AC1-4 genes of CLCuV DNA A were amplified by PCR using specific pairs of primers. Sequence and protein translation analysis showed that these three regions encompassed CLCuV DNA A (i) Dl/4-the 5 half of the replication gene ACl, (encodes the amino acids 1-213 of the 360 long Rep protein) and all of the AC4 ORF (ii) D1 -the3' half or ACI (excluding overlap with AC2 and AC 4), (encodes amino acids 212-330 of Rep, but with an internal methionine residue for translational initiation, only at position 274) and (iii) D1/d2/d3- AC2, a transcription activator, and AC3, a replication enhancer (plus the last 97 bases of the AC 1, and excluding the initiation codon of AC2 and 23 bases or the 3 end or 1\C3 (encodes amino acids 331-360 of Rep [ no methionine], amino acids 2-118 of the 134 long TrAP with methionine 112 [encoded by AC2] and amino acids 1-127 of the 134 long REn.
D1/4, D1 and D1/d2/d3 fragments or CLCuV DNA A were individually cloned in sense and antisense orientations in plasmid pJIT 60 under control of enhanced Cauliflower mosaic virus (CaMY) 35S promoter and poly A signal sequence for constructing six plant expression cassettes. The sense and antisense orientations of these clones were determined by restriction and sequence analysis. These six plant expression cassettes were subcloned in plant transformation binary vector pG1\482. Recombinant plasmids were individually transformed into Agrobacterium tumefaciens strain LBA4404 by electroporation.
Agrobacterium mediated transformation of tobacco leaf discs via direct shoot induction and cotton hypocotyl sections via somatic embryogenesis were carried out. Explants were placed on shoot (tobacco leaf discs) and callus (cotton hypocotyl) induction medium containing kanamycin for npt II gene selection. Transformations of tobacco and cotton were carried out in three different batches for each construct. In tobacco and cotton, about 20-25 putative independent transgenic (kanamycin resistant) lines per construct were produced. Twenty phenotypically normal selected T0 lines for each construct were selfed to produce T1, plants. The transformation efficiency of tobacco and cotton explants was about 65% and 20% respectively.
PCR results indicated that almost all-independent lines of tobacco and cotton contained both specific (sense and antisense gene of D1/4, D1 and D1/d2/d3) and npt 11 genes. Southern analysis of the selected transgenic lines showed that 1-3 copies of transgenes were present in tobacco and cotton. Northern analysis indicated that transgenes were expressed at transcriptional level and transgene mRNA accumulation in cytoplasm varied among transgenic lines.
Seventy two (thirty six each for sense and antisense) lines of tobacco and cotton for all the six constructs were tested for CLCuD resistance in containment. Eighteen lines of tobacco (twelve sense and six antisense) and eleven lines of cotton (seven sense and four antisense) showed stable heritable resistance in T1-T3 generations. The percentage resistance of these lines varied between 70-100%. No recovery phenomenon was observed in susceptible plants. Multiplex PCR revealed the presence of both CLCuV-26 and CLCuV-72b in the infected tobacco plants. Southern blots showed DNA A and DNA β replication in control and susceptible plant lines while resistant plant lines contained no detectable amounts of DNAs of either CLCuD components. No transcripts (geminiviral and transgenes) were detected by Northern analysis of selected resistant plants after exposure to viruliferous whiteflies as compared to susceptible lines and controls.
Resistance in D 1 antisense transgenic lines of cotton was further evaluated in field trials over two growing seasons. Plants of resistant lines showed 90-100% stable resistance to CLCuD under field conditions, which confirmed the containment data. The resistant plants neither developed symptoms nor showed replication of CLCuD components in randomly selected resistant plants. No geminiviral transcripts were detected in these resistant plants in contrast to those detected in susceptible control plants. The double-stranded RNAs (dsRNAs) were detected in the uninfected (unexposed to whitet1ies) resistant sense transgenic tobacco lines.
No viral and transgene RNA was detected in resistant lines following infection and selected uninfected resistant tobacco sense lines revealed dsRNAs, indicating that the most likely mechanism of resistance is via post-transcriptional gene silencing.