custom-synthesized, mutation-containing dna fragments  (Twist Bioscience)

Journal: bioRxiv

Article Title: Rescue of tomato yellow leaf curl virus mutants with heterologous iterons through in planta evolution

doi: 10.1101/2025.05.27.656274

Figure Lengend Snippet: A . The m1, m2, and m3 mutants compromises SH2 single-cell replication to varying extents. The m1-G, m1-R, m2-G, m2-R, m3-G, m3-R constructs are derivatives of LM5-G and LM5-R, respectively, with corresponding m1, m2, and m3 mutations. The pairs of constructs were delivered into N. benthamiana epidermal cells via agro-infiltration. The cells were observed with a confocal microscope and photographed at 4 days post agro-infiltration. B. Semi-quantitative PCR assessing relative accumulation levels of viral genomic DNA in cells receiving constructs shown above the lanes. Note the PCR primers do not distinguish between the GFP- and mCherry-coding constructs. Also note that the non-replicating C1fs1 mutant (containing a frameshift mutation in Rep coding sequence) was included as a negative control. C . N. benthamiana plants showing symptoms of different SH2 mutants 6 and 9 weeks post inoculation (agro-infiltration) (6 and 9 wpi). D . Assessing the relative levels of viral genomic DNA using semi-quantitative PCR in plants treated with m1, m2, and m3 mutants. At 1 wpi, the agro-infiltrated leaves (IL) of two representative plants were analyzed, whereas at 6 and 9 wpi, the systemic leaves (SL) were exmined. The numbers of PCR cycles were also shown. The rDNA control were always amplified for 15 cycles.

Article Snippet: A few mutants were generated with custom-synthesized, mutation-containing DNA fragments (TWIST Biosciences).

Techniques: Construct, Microscopy, Real-time Polymerase Chain Reaction, Mutagenesis, Sequencing, Negative Control, Control, Amplification

Journal: bioRxiv

Article Title: Rescue of tomato yellow leaf curl virus mutants with heterologous iterons through in planta evolution

doi: 10.1101/2025.05.27.656274

Figure Lengend Snippet: De novo mutations identified in m1 and m2 descendants enhance m2 symptoms. A . Iteron portion sequences of new m2-based mutants incorporating the de novo mutations; and sequences of their descendants at 6 wpi. B . Relative accumulation levels of SH2, m1, m2, m2a, and m2b in IL at 1 wpi. C . Symptoms of infected plants at 6 wpi. D . Accumulation levels of viral DNA in SL at 6 wpi as measured with 35 cycles of PCR. Note the absence of viral DNA in three of the m2-infected plants, and the extremely low accumulation in the fourth.

Article Snippet: A few mutants were generated with custom-synthesized, mutation-containing DNA fragments (TWIST Biosciences).

Techniques: Infection

Journal: bioRxiv

Article Title: Rescue of tomato yellow leaf curl virus mutants with heterologous iterons through in planta evolution

doi: 10.1101/2025.05.27.656274

Figure Lengend Snippet: The TC-to-GT mutations of m2b are sufficient to rescue viral replication even if one or both of the flanking iteron motifs (of Y35 origin) are eliminated. A . Iteron portion sequences of the mutants, their absence/presence in 6 wpi SL, stability of the mutations, and SL symptoms. B . Semi-quantitative PCR assessing the viral DNA levels in IL for a selected set of mutants (m2g, m2b-g). C . Symptoms of representative mutants at 6 wpi. D . Detection of viral DNA in SL with PCR (35 cycles) at 6 wpi.

Article Snippet: A few mutants were generated with custom-synthesized, mutation-containing DNA fragments (TWIST Biosciences).

Techniques: Real-time Polymerase Chain Reaction