Defining Transgene Insertion Sites and Off-Target Effects of Homology-Based Gene Silencing Informs the Application of Functional Genomics Tools in Phytophthora infestans

Abstract

DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in Phytophthora spp. Since unplanned side-effects of these tools are not well-characterized, we used P. infestans to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spread to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5′ ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for 12 different gene targets indicated that neighbors within 500 nt were often cosilenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, this cis spreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not cosilenced with the target in all transformants, which informs how screens for silencing should be performed. We conclude that transformation and gene silencing can be reliable tools for functional genomics in Phytophthora spp. but must be used carefully, especially by testing for the spread of silencing to genes flanking the target.

The Basic Leucine Zipper Transcription Factor PlBZP32 Associated with the Oxidative Stress Response Is Critical for Pathogenicity of the Lychee Downy Blight Oomycete Peronophythora litchii

“…3A and B). Considering that homology-based transcriptional silencing might cause off-target effects, especially those affecting the expression of neighboring gene(s) (32,33), we searched the neighboring locus of PlBZP32 and found one gene (Pl105397; GenBank accession number MT396990) 875 nucleotides (nt) away from PlBZP32. Pl105397 encodes a structural maintenance of chromosomes (SMC) protein FIG 1 Phylogenetic analysis of PlBZP32 protein and its orthologs.…”

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

“…All incoming transgenes were embedded in the same upstream and downstream 1-kb flanking ends from the Avr1b-1 locus. Numerous studies have shown that sense as well as antisense constructs can readily induce silencing of both ectopic transgenes and endogenous genes in Phytophthora species (van West et al, 1999(van West et al, , 2008Vu et al, 2019). However, our results are unusual in that only a single copy of the transgene is inserted into the genome, at the Avr1b-1 locus.…”

Chemical genetic approach using β-rubromycin reveals that a RIO kinase-like protein is involved in morphological development in Phytophthora infestans

“…In humans, a previous study proved that β-rubromycin competitively interacts with the telomerase substrate primer, TS-A (K i = 0.74 μM), enabling β-rubromycin to interact with the telomerase RNA 22 . Although β-rubromycin showed nonspecific cytotoxicities, it inhibited the proliferation of both K-562 and Hela cells with IC 50 values of 19.5 μM and 22.7 μM, respectively 21 . While oomycetes do contain orthologs of human telomerase, it is possible that β-rubromycin affects other targets since it had no effect on vegetative growth on rye medium.…”

“…6B). Since neighbors within 500 nt of the target gene are often cosilenced 21 , we investigated the expression of PITG_04583, which resides within 540 nt of PITG_04584; there was no predicted gene on the other side of PITG_04584 within 40 kb. Since the expression of PITG_04583 in all transformants was not significantly different from wild-type ( Supplementary Fig.…”