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.
Highlights
Phytophthora sojae causes destructive root and stem rot diseases of soybeans and has been a model for molecular genetics research into oomycete plant pathogens (Tyler, 2007; Jiang and Tyler, 2012; Wang and Wang, 2018)
To confirm that Avr1b-1 is essential for conferring hypersensitive response (HR) on soybean plants carrying Rps1b, we employed clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9dependent mutagenesis to delete Avr1b-1 by gene replacement
The P. sojae isolate P7063 carries a single copy of Avr1b-1 and lacks the close paralog Avh1; this strain was used as the recipient strain for Avr1b-1 replacement
Summary
Phytophthora sojae causes destructive root and stem rot diseases of soybeans and has been a model for molecular genetics research into oomycete plant pathogens (Tyler, 2007; Jiang and Tyler, 2012; Wang and Wang, 2018). CRISPR-Mediated P. sojae Avr1b Mutagenesis (Tyler and Gijzen, 2014; Wang Y. et al, 2019) Among those diverse infection-associated factors, a large superfamily of small secreted hydrophilic proteins with a conserved RXLR motif (RXLR effectors) has been characterized as key contributors to virulence by P. sojae and other oomycete plant pathogens (Dou et al, 2008a,b; Jiang et al, 2008; Jiang and Tyler, 2012; Wang and Wang, 2018; Wang Y. et al, 2019). The same superfamily includes most oomycete avirulence (Avr) determinants, which are proteins that enable recognition by host intracellular receptors encoded by major disease resistance (R) genes (Tyler and Gijzen, 2014). Most RXLR effector genes display transcriptional dynamics during host infection and other developmental stages (Wang et al, 2011; Ah-Fong et al, 2017)
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