Transcriptional Variability Associated With CRISPR-Mediated Gene Replacements at the Locus.
Gu Biao,Shao Guangda,Gao Wenxin,Miao Jianqiang,Wang Qinhu,Liu Xili,Tyler Brett M
Frontiers in microbiology
Transcriptional plasticity enables oomycetes to rapidly adapt to environmental challenges including emerging host resistance. For example, the soybean pathogen can overcome resistance conferred by the host resistance gene through natural silencing of its corresponding effector gene, . With the CRISPR/Cas9 genome editing system, it is possible to generate site-specific knock-out (KO) and knock-in (KI) mutants and to investigate the biological functions of target genes. In this study, the gene was deleted from the genome using a homology-directed recombination strategy that replaced with a gene encoding the fluorescent protein mCherry. As expected, all selected KO transformants gained virulence on plants, while infection of plants lacking was not compromised. When a sgRNA-resistant version of was reintroduced into the -1 locus of an Avr1b KO transformant, KI transformants with a well-transcribed gene were unable to infect -containing soybeans. However, loss of expression of the incoming gene was frequently observed in KI transformants, which resulted in these transformants readily infecting soybeans. A similar variability in the expression levels of the incoming gene was observed with AVI- or mCherry-tagged Avr1b-1 constructs. Our results suggest that may be unusually susceptible to transcriptional variation.