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. 2004 Dec;78(24):13966-74.
doi: 10.1128/JVI.78.24.13966-13974.2004.

A DNAbeta associated with Tomato yellow leaf curl China virus is required for symptom induction

Xiaofeng Cui  1 , Xiaorong Tao, Yan Xie, Claude M Fauquet, Xueping Zhou
Affiliations

A DNAbeta associated with Tomato yellow leaf curl China virus is required for symptom induction

Xiaofeng Cui et al. J Virol. 2004 Dec.

Abstract

We report here that all 25 isolates of Tomato yellow leaf curl China virus (TYLCCNV) collected from tobacco, tomato, or Siegesbeckia orientalis plants in different regions of Yunnan Province, China, were associated with DNAbeta molecules. To investigate the biological role of DNAbeta, full-length infectious clones of viral DNA and DNAbeta of TYLCCNV isolate Y10 (TYLCCNV-Y10) were agroinoculated into Nicotiana benthamiana, Nicotiana glutinosa, Nicotiana. tabacum Samsun (NN or nn), tomato, and petunia plants. We found that TYLCCNV-Y10 alone could systemically infect these plants, but no symptoms were induced. TYLCCNV-Y10 DNAbeta was required, in addition to TYLCCNV-Y10, for induction of leaf curl disease in these hosts. Similar to TYLCCNV-Y10, DNAbeta of TYLCCNV isolate Y64 was also found to be required for induction of typical leaf curl diseases in the hosts tested. When the betaC1 gene of TYLCCNV-Y10 DNAbeta was mutated, the mutants failed to induce leaf curl symptoms in N. benthamiana when coinoculated with TYLCCNV-Y10. However, Southern blot hybridization analyses showed that the mutated DNAbeta molecules were replicated. When N. benthamiana and N. tabacum plants were transformed with a construct containing the betaC1 gene under the control of the Cauliflower mosaic virus 35S promoter, many transgenic plants developed leaf curl symptoms similar to those caused by a virus, the severity of which paralleled the level of betaC1 transcripts, while transgenic plants transformed with the betaC1 gene containing a stop codon after the start codon remained symptomless. Thus, expression of a betaC1 gene is adequate for induction of symptoms of viral infection in the absence of virus.

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Figures

FIG. 1.
FIG. 1.
Mutational strategy for the βC1 gene of TYLCCNV-Y10 DNAβ. The three possible start codons are underlined. Mutation sites are in italic.
FIG. 2.
FIG. 2.
Detection of viral DNAs in agroinoculated N. benthamiana plants. Total nucleic acids (5 μg) were extracted 20 days postinoculation from individual plants agroinoculated with clones of TYLCCNV-Y10 (lanes 1 and 2) or TYLCCNV-Y10 plus DNAβ (lanes 3 and 4). Lane 5 represents nucleic acids (15 μg) extracted from leaf curl-affected leaves of an N. tabacum Samsun NN plant infected with Bemisia tabaci. Total nucleic acids were fractionated in agarose gels, blots were probed with the CP gene sequence of TYLCCNV-Y10 (top) or the full-length sequence of TYLCCNV-Y10 DNAβ (bottom). The positions of single-stranded (ssDNA) and subgenomic (sgDNA) forms of TYLCCNV-Y10 and DNAβ are indicated.
FIG. 3.
FIG. 3.
Time course of accumulation of TYLCCNV-Y10 in N. benthamiana coinfected with or lacking DNAβ. The youngest infected leaves (about 200 mg) were collected at intervals from 6 to 65 days postinoculation (p.i.). Triple antibody sandwich ELISA was used to estimate viral content (A represents A405). The error bars indicate the standard deviation of each sample. A, TYLCCNV-Y10, A+β, TYLCCNV-Y10 and DNAβ.
FIG. 4.
FIG. 4.
(A) Symptoms in N. benthamiana plants induced by pBinPLUS-A-1.7 coinoculated with different βC1 mutants. From left to right, plants were coinoculated with pBinPLUS-2β, pBinPLUS-C1M-Fβ, pBinPLUS-C1M-Sβ, pBinPLUS-C1M-Bβ, and pBinPLUS-C1M-Tβ, respectively. The wild-type DNAβ induced severe downward leaf curling, stunting, vein swelling and darkening, and enations. When the first ATG of the βC1 gene was mutated, plants developed leaf curl symptoms but did not become stunted. Plants inoculated with any of the other βC1 mutants remained symptomless. (B) Southern blot analysis of plants inoculated with βC1 mutants of TYLCCNV-Y10 DNAβ. Samples (10 μg) of total nucleic acids were extracted from individual N. benthamiana plants infected by agroinoculation with TYLCCNV-Y10 (lanes 1 and 2), TYLCCNV-Y10 and DNAβ (lanes 3 and 4), TYLCCNV-Y10 and DNAβ-C1MF (lanes 5 and 6), TYLCCNV-Y10 and DNAβ-C1MS (lanes 7 and 8), TYLCCNV-Y10 and DNAβ-C1MB (lanes 9 and 10), and TYLCCNV-Y10 and DNAβ-C1MT (lanes 11 and 12). The blots were probed with the full-length sequence of TYLCCNV-Y10 DNAβ. ssDNA, single-stranded DNA; scDNA, supercoiled DNA.
FIG. 5.
FIG. 5.
Phenotypes of transgenic N. benthamiana and N. tabacum expressing the βC1 gene of TYLCCNV-Y10 DNAβ. (A) Example of a severely abnormal plant. (B) Example of a moderately abnormal plant. (C) Example of a plant showing the recovered phenotype. The white arrow marks a curled leaf. (D) Distorted leaf from a severely abnormal plant. (E) Upward curling and blistering of leaves from a moderately abnormal plant. Left, top side; right, underside. (F) Examples of severely abnormal plants. (G) Distorted leaf from a severely abnormal plant. Left, top side; right, underside. (H) Curled leaf from a moderately abnormal plant. Left, top side; right, underside. (I) Interveinal protuberances and small interveinal tissue outgrowths on the underside of a leaf. (J) Enlarged view of a protuberance on the leaf underside.
FIG. 6.
FIG. 6.
Northern blot analysis of RNA from transgenic plants containing the βC1 gene of TYLCCNV-Y10 DNAβ. Aliquots (40 μg) of plant total RNA extracted from transgenic leaf tissue of individual plants were loaded onto a 1.2% agarose gel containing formaldehyde. The blot was probed with a radiolabeled βC1 gene fragment. The ethidium bromide-stained gel shown below the blot indicates equal loading of RNA. The source plants were severely abnormal (SA), moderately abnormal (A), or symptomless (N). The arrow marks the location of βC1 mRNA.
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