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

Abstract

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

A single region of the Phytophthora infestans avirulence effector Avr3b functions in both cell death induction and plant immunity suppression

“…To perform biomass measurement, total gDNA was extracted using a hexadecyltrimethylammonium bromide method (May & Ristaino, 2004) and quantified by 1% agarose gel electrophoresis. Quantitative PCR of gDNA was performed using CFX Connect (BioRad) as described by Gu et al (2021) to obtain biomass measurements. Genomic DNA abundance or relative gene expression was measured by a 2 −ΔΔCt method using the Actin genes from both P. infestans and N. benthamiana as endogenous controls.…”

Divergent sequences of tetraspanins enable plants to specifically recognize microbe-derived extracellular vesicles

“…For double knockout mutants, we began with the single knock out mutants. After culturing the single knock out mutants for at least three generations in antibiotic-free plates for more than 2 weeks, the mutants could no longer grow on G418 antibiotic plates 45 , 46 . Using a single knock out mutant of PsTET1 that had lost the resistance to G418 (KOT1-9), we conducted another CRISPR-mediated gene replacement to knock out the other gene PsTET3 as described 45 , 46 .…”

Molecular mechanisms of Phytophthora sojae avirulence effectors escaping host recognition

“…This indicates the presence of a genetic mechanism, gene silencing due to the destruction of transcription elements, by which avirulence is lost. The naturally occurring Avr1b -silenced also suggests that Avr1b is not an essential effectors, and the gene knockout assay also demonstrated that PsAvr1b is not required for full virulence of P. sojae ( Gu et al, 2021 ).…”