Sheng-Zhi Pang

A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing

We showed previously that 218 and 110 bp N gene segments of tomato spotted wilt virus (TSWV) that were fused to the non-target green fluorescent protein (GFP) gene were able to confer resistance to TSWV via post-transcriptional gene silencing (PTGS). N gene segments expressed alone did not confer resistance. Apparently, the GFP DNA induced PTGS that targetted N gene segments and the incoming homologous TSWV for degradation, resulting in a resistant phenotype. These observations suggested that multiple resistance could be obtained by replacing the GFP DNA with a viral DNA that induces PTGS. The full-length coat protein (CP) gene of turnip mosaic virus (TuMV) was linked to 218 or 110 bp N gene segments and transformed into Nicotiana benthamiana. A high proportion (4 of 18) of transgenic lines with the 218 bp N gene segment linked to the TuMV CP gene were resistant to both viruses, and resistance was transferred to R(2) plants. Nuclear run-on and Northern experiments confirmed that resistance was via PTGS. In contrast, only one of 14 transgenic lines with the TuMV CP linked to a 110 bp N gene segment yielded progeny with multiple resistance. Only a few R(1) plants were resistant and resistance was not observed in R(2) plants. These results clearly show the applicability of multiple virus resistance through the fusion of viral segments to DNAs that induce PTGS.

A minimum length of N gene sequence in transgenic plants is required for RNA-mediated tospovirus resistance

We showed previously that transgenic plants with the green fluorescent protein (GFP) gene fused to segments of the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) displayed post-transcriptional gene silencing of the GFP and N gene segments and resistance to TSWV. These results suggested that a chimeric transgene composed of viral gene segments might confer multiple virus resistance in transgenic plants. To test this hypothesis and to determine the minimum length of the N gene that could trans-inactivate the challenging TSWV, transgenic plants were developed that contained GFP fused with N gene segments of 24-453 bp. Progeny from these plants were challenged with: (i) a chimeric tobacco mosaic virus containing the GFP gene, (ii) a chimeric tobacco mosaic virus with GFP plus the N gene of TSWV and (iii) TSWV. A number of transgenic plants expressing the transgene with GFP fused to N gene segments from 110 to 453 bp in size were resistant to these viruses. Resistant plants exhibited post-transcriptional gene silencing. In contrast, all transgenic lines with transgenes consisting of GFP fused to N gene segments of 24 or 59 bp were susceptible to TSWV, even though the transgene was post-transcriptionally silenced. Thus, virus resistance and post-transcriptional gene silencing were uncoupled when the N gene segment was 59 bp or less. These results provide evidence that multiple virus resistance is possible through the simple strategy of linking viral gene segments to a silencer DNA such as GFP.

Turnip mosaic potyvirus resistance in Nicotiana benthamiana derived by post-transcriptional gene silencing

The coat protein (CP) gene of turnip mosaic potyvirus isolate ESC8 (TuMV‐ESC8) was cloned and sequenced. Comparisons of the 867‐nucleotide (nt) CP region with those of 11 TuMV isolates showed 86.7–89.3% nucleotide identity and 92.4–95.5% amino acid identity. The CP gene was cloned into a plant expression vector and transformed into Nicotiana benthamiana plants via Agrobacterium tumefaciens‐mediated leaf disk transformation. Progeny from R0 lines was screened for resistance to TuMV‐ESC8. Five of 29 tested lines showed TuMV protection in more than 50% of their progeny. Interestingly, some of the resistant plants transformed with the CP gene of TuMV displayed mild mosaicism in the new growing leaves at the later stages of evaluation; but these mosaic symptoms disappeared when the leaves were fully expanded. Collective data from steady‐state RNA analysis and nuclear run‐on assay of a line showed that the resistance was RNA‐mediated through the post‐transcriptional gene silencing mechanism.

Evidence that resistance in squash mosaic comovirus coat protein-transgenic plants is affected by plant developmental stage and enhanced by combination of transgenes from different lines.

Three transgenic lines of squash hemizygous for the coat protein genes of squash mosaic virus (SqMV) were shown previously to have resistant (SqMV-127), susceptible (SqMV-22) or recovery (SqMV-3) phenotypes. Post-transcriptional gene silencing (PTGS) was the underlying mechanism for resistance of SqMV-127. Here, experiments conducted to determine the mechanism of the recovery phenotype and whether enhanced resistance could be obtained by combining transgenes from susceptible and recovery plants are reported. Upper leaves of SqMV-3 plants were sampled for Northern analysis at 17, 31 and 45 days after germination (DAG) and a proportion of plants were inoculated with SqMV. SqMV-3 plants inoculated at a young stage (17 DAG) showed susceptible or recovery phenotypes. However, a number of plants inoculated at later developmental stages (31 or 45 DAG) were resistant to infection. Resistance of recovery plants was due to PTGS that was activated at a later developmental stage, independent of virus infection. Similar results were observed with plants grown under field conditions. To investigate the interactions of transgenes, progeny of crosses between SqMV-127, -3 and -22 were inoculated with SqMV. Progeny with the transgene of line 127 were resistant. However, a number of plants with transgenes from the recovery and susceptible lines or the self-pollinated recovery line were resistant even when inoculated at a young stage. Northern analysis suggested that resistance was due to PTGS. The results reveal that the timing of PTGS and consequent resistance of the transgenic plants were affected by their developmental stage and the interaction of transgene inserts.

The coat protein genes of squash mosaic virus: cloning, sequence analysis, and expression in tobacco protoplasts

SummaryComplementary DNA of the middle-component RNA of the melon strain of squash mosaic comovirus (SqMV) was cloned. Clones containing the coat protein genes were identified by hybridization with a degenerate oligonucleotide synthesized according to the amino acid sequence of a purified peptide fragment of the SqMV large coat protein. A clone containing of 2.5 kbp cDNA insert of SqMV M-RNA was sequenced. The total insert sequence of 2510 bp included a 2373 bp open reading frame (ORF) (encoding 791 amino acids), a 123 bp 3′-untranslated region, and a poly(A) region. This ORF is capable of encoding both the 42 and 22 k SqMV coat proteins. Direct N-terminal sequence analysis of the 22 k coat protein revealed its presence at the 3′ end of this ORF and the position of the proteolytic cleavage site (Q/S) used to separate the large and small coat proteins from each other. A putative location of the N-terminal proteolytic cleavage site of the 42 k coat protein (Q/N) was predicted by comparisons with the corresponding coat proteins of cowpea mosaic virus, red clover mottle virus, and bean-pod mottle virus. Although the available nucleotide sequences of these viruses revealed little similarity, their encoded coat proteins shared about 47% identity. The identity of the encoded 42 k and 22 k peptides was confirmed by engineering the respective gene regions for expression followed by transfer into tobacco protoplasts using the polyethylene glycol method. Both SqMV coat proteins were expressed in vivo as determined by their reactivity to SqMV coat protein specific antibodies.