AUTHOR=Gu Biao , Shao Guangda , Gao Wenxin , Miao Jianqiang , Wang Qinhu , Liu Xili , Tyler Brett M. 

TITLE=Transcriptional Variability Associated With CRISPR-Mediated Gene Replacements at the Phytophthora sojae Avr1b-1 Locus

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.645331

DOI=10.3389/fmicb.2021.645331

ISSN=1664-302X

ABSTRACT=<p>Transcriptional plasticity enables oomycetes to rapidly adapt to environmental challenges including emerging host resistance. For example, the soybean pathogen <italic>Phytophthora sojae</italic> can overcome resistance conferred by the host resistance gene <italic>Rps1b</italic> through natural silencing of its corresponding effector gene, <italic>Avr1b-1</italic>. With the <italic>Phytophthora</italic> 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 <italic>Avr1b-1</italic> gene was deleted from the <italic>P. sojae</italic> genome using a homology-directed recombination strategy that replaced <italic>Avr1b-1</italic> with a gene encoding the fluorescent protein mCherry. As expected, all selected KO transformants gained virulence on <italic>Rps1b</italic> plants, while infection of plants lacking <italic>Rps1b</italic> was not compromised. When a sgRNA-resistant version of <italic>Avr1b-1</italic> was reintroduced into the <italic>Avr1b</italic>-1 locus of an Avr1b KO transformant, KI transformants with a well-transcribed <italic>Avr1b-1</italic> gene were unable to infect <italic>Rps1b</italic>-containing soybeans. However, loss of expression of the incoming <italic>Avr1b-1</italic> gene was frequently observed in KI transformants, which resulted in these transformants readily infecting <italic>Rps1b</italic> 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 <italic>Avr1b-1</italic> may be unusually susceptible to transcriptional variation.</p>