Yunfang Zhang, Laureen Blahut-Beatty, Suqin Zheng, Steven J. Clough, Daina H. Simmonds

ABSTRACT

The role of soybean 14-3-3 gene (Glyma05g29080) in defense against white mold and in nodulation was investigated by loss-of-gene-function with CRISPR-Cas9 editing and RNAi silencing. Particle bombardment was used to introduce the CRISPR expression cassette to target the soybean 14-3-3 gene and an RNAi construct to silence gene transcription. Transmission of the edited14-3-3 gene and the RNAi construct was confirmed in their respective progeny. The recovered transgenic plants and their progeny were significantly more susceptible to Sclerotinia sclerotiorum infection and showed a significant reduction in nodulation, thus confirming the role of the 14-3-3 (Glyma05g29080) gene in both nodulation and defense.

See https://apsjournals.apsnet.org/doi/epdf/10.1094/MPMI-07-22-0157-R

Figure 1. Editing of Glyma05g29080.  A. Diagram of pGRE31 with 14-3-3 gRNA. The promoters driving gRNA and Cas9 were snoRNA U3 and CaMV 35s, respectively. B. Illustration of the sequence of an edited T0 event. The double peaking in the chromatogram indicates presence of both the hybridized edited and unedited sister DNA. C. Chromatograms and DNA sequences of X5 WT and mutations in 5 of 49 transgenic events. Deletions are shown as green dashes and base substitutions are shown as red letters.