Fig 1.
(A) S. rostratum plants infiltrated with Agrobacteria carrying the empty TRV vector were imaged two weeks after inoculation. (B) RT-PCR using TRV CP-specific primers and S. rostratum; ACT1 expression from cDNA samples of TRV-infiltrated and control S. rostratum plants. (C) A schematic depiction of VIGS treatment in S. rostratum plants using a needleless syringe for infiltration method.
Fig 2.
Cloning of SrPDS and SrChlH genes from S. rostratum and the construction of TRV-SrPDS and TRV-SrChlH.
(A) Amplification of SrPDS and SrChlH from S. rostratum plants. Primers for PDS and ChlH were designed from conserved regions of PDS or ChlH based on the alignment of tomato and potato CDS sequences (red arrows in panel). S. rostratum cDNA was used as a template to amplify SrPDS (479-bp) and SrChlH (346-bp) PCR products. An RT reaction without reverse transcriptase served as a control. (B, D) Amino acid sequences of PDS and ChlH from tomato and potato were used in an alignment analysis of S. rostratum. (C) Diagrams of TRV-SrPDS and TRV-SrChlH. The amplified S. rostratum gene fragments were inserted into pTRV2 to obtain the pTRV-SrPDS and pTRV-SrChlH silencing vectors.
Fig 3.
Silencing of PDS and ChlH genes in S. rostratum plant seedlings.
(A) The SrPDS and SrChlH silencing phenotypes in S. rostratum plants. Agrobacterium containing TRV alone, pTRV2-SrPDS or pTRV2-SrChlH was infiltrated into the first true leaves of S. rostratum seedlings together with pTRV1. Two weeks after inoculation, photo-bleaching was observed in the PDS-silenced plants and yellowing was observed in the ChlH-silenced plants. Control plants inoculated with TRV alone were green in color. (B) RT-PCR analysis of mRNA levels of SrPDS and SrChlH in TRV control and PDS or ChlH gene-silencing plant. Error bars show +/-SD.
Fig 4.
Silencing of PDS genes in the flowers and fruit of S. rostratum.
(A) The phenotype of SrPDS silencing in S. rostratum flowers and fruit. Agrobacterium containing TRV alone or TRV-SrPDS was co-infiltrated with pTRV1 into the first true leaves of S. rostratum seedlings together. Photo-bleaching was observed in the flowers and fruit that developed 44–45 days after infection on the PDS-silenced S. rostratum plants. Control plants inoculated with TRV alone produced yellow flowers and green fruit. (B) Real-time RT-PCR analysis of SrPDS transcript levels in the flowers and fruit of the TRV control and TRV-SrPDS plants. Error bars show +/- SD.
Fig 5.
Comparison of agroinoculation methods in S. rostratum.
(A) Effects of three agroinoculation methods. Agrobacterium cultures containing pTRV1 and pTRV2 alone (TRV) or TRV2-SrPDS were mixed in a 1:1 ratio and infiltrated into the true leaves and cotyledons of S. rostratum with a needleless syringe, or into sprouts using a vacuum. (B) Efficiency of SrPDS gene silencing of S. rostratum plants and the first appearance of SrPDS silencing in leaves for each treatment. Silencing efficiency was assessed 14 days after TRV inoculation. The data are representative of three independent experiments, with N = 100 for each experiment.