Transgenically expressed T-Rep of tomato yellow leaf curl Sardinia virus acts as a trans-dominant-negative mutant, inhibiting viral transcription and replication

Abstract

We have previously shown that transgenic expression of a truncated C1 gene of Tomato yellow leaf curl Sardinia virus (TYLCSV), expressing the first 210 amino acids of the replication-associated protein (T-Rep) and potentially coexpressing the C4 protein, confers resistance to the homologous virus in Nicotiana benthamiana plants. In the present study we have investigated the role of T-Rep and C4 proteins in the resistance mechanism, analyzing changes in virus transcription and replication. Transgenic plants and protoplasts were challenged with TYLCSV and the related TYLCSV Murcia strain (TYLCSV-ES[1]). TYLCSV-resistant plants were susceptible to TYLCSV-ES[1]; moreover, TYLCSV but not TYLCSV-ES[1] replication was strongly inhibited in transgenic protoplasts as well as in wild-type (wt) protoplasts transiently expressing T-Rep but not the C4 protein. Viral circular single-stranded DNA (cssDNA) was usually undetectable in transgenically and transiently T-Rep-expressing protoplasts, while viral DNAs migrating more slowly than the cssDNA were observed. Biochemical studies showed that these DNAs were partial duplexes with the minus strand incomplete. Interestingly, similar viral DNA forms were also found at early stages of TYLCSV replication in wt N. benthamiana protoplasts. Transgenically expressed T-Rep repressed the transcription of the GUS reporter gene up to 300-fold when fused to the homologous (TYLCSV) but not to the heterologous (TYLCSV-ES[1]) C1 promoter. Similarly, transiently expressed T-Rep but not C4 protein strongly repressed GUS transcription when fused to the C1 promoter of TYLCSV. A model of T-Rep interference with TYLCSV transcription-replication is proposed.

FIG. 1

Replication of TYLCSV and TYLCSV-ES[1] in wt and transgenic (102.22) N. benthamiana protoplasts. Southern blots of total nucleic acids extracted 72 h after transfection with either pTOM6 (TYLCSV) or pSP97 (TYLCSV-ES[1]) were hybridized with a C1-sense RNA probe. DNAs from mock-inoculated protoplasts and infected plants were included as controls. Slowly migrating DNAs are indicated with an asterisk (∗). scDNA, supercoiled DNA. When pSP97 was used, slight inhibition of viral replication was observed in most experiments (panel 1), but in one case (panel 2), consistent inhibition coupled with the presence of the slow-migrating DNAs was detected.

FIG. 2

Consequence of expressing T-Rep and C4 proteins on viral DNA replication. Wt N. benthamiana protoplasts were cotransfected with either pTOM6 (TYLCSV) or pSP97 (TYLCSV-ES[1]), together with the constructs indicated above, which can transiently express the viral protein indicated below (see Table 1 for description of constructs). The C4 protein may start at the first ATG codon (1^st ATG) of the ORF or at another nearby in-frame ATG (2^nd ATG) C4?, C4 protein potentially expressed from internal ATG codon. Other details as in Fig. 1.

FIG. 3

Characterization of slowly migrating DNAs, indicated with an asterisk (∗). The positions of scDNA and cssDNA forms of viral DNA as well as input DNA are indicated. (A) The slowly migrating DNAs do not hybridize with a plus-sense probe. Wt protoplasts were cotransfected with pTOM6 together with pTOM100 or pTOM100NT, and TNAs were analyzed 72 h posttransfection. The blot was hybridized with a C1-sense RNA probe (probe minus) and reprobed with a V1-sense RNA probe (probe plus). DNAs from mock-inoculated protoplasts and from infected plants were included as controls. (B) The slowly migrating DNAs are not DNA-protein complexes. TNAs extracted from protoplasts cotransfected as in panel A were incubated at 56°C for 2 h with (+) or without (−) proteinase K prior to Southern blot analysis. The blot was hybridized with a C1-sense RNA probe. (C) Alkali treatment converts the slowly migrating DNAs to DNA migrating as cssDNA. TNAs extracted from transgenic 102.22 protoplasts at 72 h posttransfection with pTOM6 were analyzed directly (no treatment), after denaturation with NaOH (alkali), or after restriction with DpnI followed by NaOH (DpnI + alkali). The blot was hybridized with a C1-sense RNA probe (probe minus) and reprobed with a C1-antisense RNA probe (probe plus).

FIG. 4

Slowly migrating DNAs are converted into ocDNA by Taq (A) or T4 (B) DNA polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) T4 DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with BglII to show migration of linear DNA.

FIG. 5

Slowly migrating DNAs also accumulate at early stages after transfection of wt protoplasts with TYLCSV. TNAs were extracted from protoplasts transfected with pTOM6. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slow-migrating viral DNAs are indicated with an asterisk (∗). HPT, hours posttransfection. (A) Time course analysis of viral DNA forms. Sample at 72 h was diluted 20-fold to give signals of satisfactory intensity on the autoradiographic film. (B) Taq DNA polymerase treatment of TNAs at 24 and 72 h after transfection. Samples were incubated for 10 min at 37°C with (+) or without (−) the polymerase. Lane C, sample at 72 h digested with BglII to show the linear DNA.

FIG. 6

Transcriptional repression assays. Values are the averages of at least three independent experiments assayed in triplicate, and error bars indicate the standard error of the mean. Mean absolute GUS activity measured in transfected wt protoplasts, expressed in picomoles of 4-methylumbelliferone (MU) per minute per milligram of protein is indicated in italics. Background GUS activities associated with untransfected protoplasts were 18 and 29 pmol of MU/min/mg for transgenic and wt protoplasts, respectively. (A) 102.22 transgenic and wt N. benthamiana protoplasts were transfected with the GUS reporter constructs indicated on the left. GUS activity was measured in protein extracts at 24 h posttransfection. For each construct, GUS activity in 102.22 protoplasts (open columns) is shown as a percentage of the activity recorded from transfection of wt protoplasts (shaded columns) (in bold). (B) Wt N. benthamiana protoplasts were cotransfected with the GUS reporter construct pTOM202 together with the plant expression cassettes indicated on the left. GUS activity was measured in protein extracts at 24 h posttransfection. GUS activity obtained in cotransfection of pTOM202 with pGEM-P was set at 100%, and other values were standardized to this level (in bold).