CRISPR/Cas9‐mediated editing of GmTAP1 confers enhanced resistance to Phytophthora sojae in soybean

Soybean root rot disease caused by Phytophthora sojae seriously constrains soybean yield. Knocking out the susceptibility gene GmTAP1 in soybean created new soybean lines resistant to several P. sojae strains and these lines showed no agronomic penalties in the field. Soybean (Glycine max) is an economically important oil-bearing crop that is widely planted worldwide (Babu et al., 2017). Many diseases seriously limit soybean yield. Soybean root rot disease, one of the most destructive soybean diseases, occurs throughout the growth period, resulting in considerable yield losses (Kamoun et al., 2015). Phytophthora sojae (P. sojae), a major pathogen causing soybean root rot, is difficult to control by chemical treatment (Tyler, 2007). Cultivating disease-resistant varieties is an effective strategy for preventing and controlling soybean root rot. P. sojae secretes numerous effectors that target host factors to enhance infection. Both effectors and their host targets are essential for pathogenicity (Wang and Wang, 2018). Genetic engineering of these host targets is a promising strategy for boosting soybean resistance. CRISPR/Cas9 is a rapidly developing gene editing method that can be used to alter the traits of various species quickly and efficiently. CRISPR/Cas9-mediated editing of susceptibility genes was recently used in disease-resistant crop breeding efforts (Zaidi et al., 2018). However, to date, no reports describe the editing of soybean susceptibility genes to enhance resistance to soybean root rot disease. We previously demonstrated that the key P. sojae effector PsAvh52 recruits an acetyltransferase GmTAP1 (Glyma.18G216900.1) into the nucleus, where it acetylates histones H2A and H3, thereby increasing the transcription of susceptibility genes to promote P. sojae infection. Resistance evaluation in soybean hairy roots indicated that GmTAP1 is an important susceptibility factor that positively regulates P. sojae infection of soybean (Li et al., 2018). In the current study, we used CRISPR/Cas9 gene editing to knock out GmTAP1 and created novel germplasm with increased resistance to soybean root rot. We constructed a pFGC5941-Cas9-GmTAP1 vector targeting exon 4 of GmTAP1 (Figure 1A). We transferred this vector into Agrobacterium tumefaciens strain AGL1 to produce soybean transgenic plants. After screening the progeny by PCR and Sanger sequencing, we obtained two Cas9-null homozygous mutants with frameshifts in the coding region of GmTAP1: tap1-1 (2-bp deletion) and tap1-2 (1-bp deletion; Figure 1B). We also blasted the SgRNA of GmTAP1 and found four possible off-target sites (Table S1). Sequencing analysis of these potential off-target sites showed that none of them were edited in the two mutants (Figure S1). We evaluated the resistance of these two mutants to P. sojae. PsAvh52 promotes P. sojae infection by targeting GmTAP1 to enhance the transcription of susceptibility genes (Li et al., 2018). We therefore measured the expression levels of these susceptibility genes, which play negative roles in soybean immunity, in wild-type (WT, Williams 82), tap1-1, and tap1-2 seedlings after inoculation with P. sojae. Specifically, we measured the expression levels of MtN3 (Glyma.08G010000, the soybean ortholog of SWEET) (Oliva and Quibod, 2017), PG (Glyma.08G287500, encoding a cell wall-degrading polygalacturonase) (Cantu et al., 2008), and the lipoxygenase gene LOX-2 (Glyma.08G189200) (Van Schie and Takken, 2014) by RT-qPCR. These susceptibility genes were significantly downregulated in tap1-1 and tap1-2 (Figure 1C), indicating that knockout of GmTAP1 repressed their expression. Genome editing of GmTAP1 confers enhanced resistance to Phytophthora sojae (A) The newly constructed vector pFGC5941-Cas9-GmTAP1 and the target site. The protospacer adjacent motif (PAM) is indicated in bold. The sgRNA sequence is shown in the box. (B) Sequencing of the GmTAP1 sites in transgenic progeny. Black hyphens in the target sequence indicate base deletions. (C) Relative transcript levels of putative susceptibility genes in tap1-1 and tap1-2 and WT plants 24 h after P. sojae inoculation. Student's t-test was used for statistical analysis (*P ≤ 0.05, **P ≤ 0.01). (D) Inoculation of the hypocotyls of etiolated WT, tap1-1, and tap1-2 seedlings to evaluate resistance against P. sojae. Photographs were taken at 48 h post-inoculation. Bar, 0.5 cm. (E) Lesion size and relative biomass of WT, tap1-1, and tap1-2 seedlings after P. sojae inoculation. Three biological replicates, each containing three technical replicates, were averaged, and statistically analyzed. (F) Evaluation of the resistance of potted WT, tap1-1, and tap1-2 seedlings against P. sojae in the greenhouse. Photographs were taken 14 days post-inoculation. Bar, 4 cm. (G) Survival rates of WT, tap1-1, and tap1-2 seedlings after P. sojae infection. Student's t-test was used for statistical analysis (*P ≤ 0.05, **P ≤ 0.01). (H–I) XEG1- and flg22-induced ROS bursts in WT, tap1-1, and tap1-2 plants. The indicated leaves of 2-week-old seedlings were subjected to ROS examination, and the peak relative luminescence unit (RLU) value was recorded. (J) Quantitative reverse-transcription PCR (RT-qPCR) of PTI-related genes expression in WT, tap1-1, and tap1-2 seedlings following flg22, XEG1, or mock treatment. Student's t-test was used for statistical analysis (*P ≤ 0.05, **P ≤ 0.01). (K) flg22- or XEG1-induced MAPK activation. Equal loading was confirmed by Ponceau S staining. (L–N) Diagram of the growth and development of WT, tap1-1, and tap1-2 plants in the field. L: Bar, 20 cm; M: Bar, 15 cm; N: Bar, 1.5 cm. (O–S) Statistical analysis of pod length, hundred-grain weight, plant height, yield per plant, and total pods per plant in WT, tap1-1, and tap1-2 plants in the field. Student's t-test was used for statistical analysis. P-values are indicated. Genome editing of GmTAP1 confers enhanced resistance to Phytophthora sojae (A) The newly constructed vector pFGC5941-Cas9-GmTAP1 and the target site. The protospacer adjacent motif (PAM) is indicated in bold. The sgRNA sequence is shown in the box. (B) Sequencing of the GmTAP1 sites in transgenic progeny. Black hyphens in the target sequence indicate base deletions. (C) Relative transcript levels of putative susceptibility genes in tap1-1 and tap1-2 and WT plants 24 h after P. sojae inoculation. Student's t-test was used for statistical analysis (*P ≤ 0.05, **P ≤ 0.01). (D) Inoculation of the hypocotyls of etiolated WT, tap1-1, and tap1-2 seedlings to evaluate resistance against P. sojae. Photographs were taken at 48 h post-inoculation. Bar, 0.5 cm. (E) Lesion size and relative biomass of WT, tap1-1, and tap1-2 seedlings after P. sojae inoculation. Three biological replicates, each containing three technical replicates, were averaged, and statistically analyzed. (F) Evaluation of the resistance of potted WT, tap1-1, and tap1-2 seedlings against P. sojae in the greenhouse. Photographs were taken 14 days post-inoculation. Bar, 4 cm. (G) Survival rates of WT, tap1-1, and tap1-2 seedlings after P. sojae infection. Student's t-test was used for statistical analysis (*P ≤ 0.05, **P ≤ 0.01). (H–I) XEG1- and flg22-induced ROS bursts in WT, tap1-1, and tap1-2 plants. The indicated leaves of 2-week-old seedlings were subjected to ROS examination, and the peak relative luminescence unit (RLU) value was recorded. (J) Quantitative reverse-transcription PCR (RT-qPCR) of PTI-related genes expression in WT, tap1-1, and tap1-2 seedlings following flg22, XEG1, or mock treatment. Student's t-test was used for statistical analysis (*P ≤ 0.05, **P ≤ 0.01). (K) flg22- or XEG1-induced MAPK activation. Equal loading was confirmed by Ponceau S staining. (L–N) Diagram of the growth and development of WT, tap1-1, and tap1-2 plants in the field. L: Bar, 20 cm; M: Bar, 15 cm; N: Bar, 1.5 cm. (O–S) Statistical analysis of pod length, hundred-grain weight, plant height, yield per plant, and total pods per plant in WT, tap1-1, and tap1-2 plants in the field. Student's t-test was used for statistical analysis. P-values are indicated. We then inoculated hypocotyls and roots of WT, tap1-1, and tap1-2 etiolated seedlings with three different P. sojae strains: P231, P233, and P234. The WT seedlings showed serious rot and brown phenotype on their inoculated regions, whereas tap1-1 and tap1-2 only showed slight browning (Figures 1D, S2A). The lesion area and biomass of P. sojae were significantly lower in tap1-1 and tap1-2 seedlings than in the WT (Figures 1E, S2B). We also evaluated the resistance of the mutants against P. sojae by examining potted seedlings grown in the greenhouse (Figure 1F). The survival rates of tap1-1 and tap1-2 were significantly higher than that of the WT after P. sojae infection (Figure 1G). Thus, the loss of function of GmTAP1 led to enhanced resistance to P. sojae. To explore the potential effects of the loss of function of GmTAP1 on plant basal immunity, we examined reactive oxygen species (ROS) production induced by XEG1 and flg22 in WT, tap1-1, and tap1-2 seedlings. ROS levels in both mutants were not significantly different from those of the WT (Figure 1H, I). Moreover, the expression levels of pattern-triggered immunity (PTI)-responsive genes, such as GmPR1, GmACS2, and GmERF, were not significantly different among WT, tap1-1, and tap1-2 seedlings following treatment with XEG1 and flg22 (Figure 1J). Finally, there were no marked differences in MAPK activity induced by XEG1 and flg22 in WT, tap1-1, and tap1-2 seedlings (Figure 1K). These results indicate that the loss of function of GmTAP1 had little effect on plant basal immunity. To evaluate whether knockout of GmTAP1 resulted in agronomic penalties, we investigated the agronomic traits of the tap1 mutants in the field. There were no significant differences in plant height, pod number per plant, hundred-grain weight, or yield per plant between WT and the tap1 mutants, indicating that knockout of GmTAP1 had no obvious agronomic penalty (Figure 1L–S). In summary, we succeeded in knocking out the susceptibility gene GmTAP1 in soybean via CRISPR/Cas9 and obtained a homozygous, non-transgenic mutant that exhibited enhanced resistance to different strains of P. sojae. Creating disease-resistant germplasm resources by editing host susceptibility factors proved to be more efficient compared to traditional breeding. As the number of characterized pathogen effectors and host targets increases, gene editing of host targets may offer a new avenue to improve plant resistance in the future. We thank Dr. Yan Wang for her constructive suggestions on manuscript writing. This work was supported by grants from the National Science Foundation of China (NSFC; 32172499, 31901957), China National Funds for Innovative Research Groups (31721004), the Chinese Modern Agricultural Industry Technology System (CARS-004-PS14), and Fundamental Research Funds for the Central Universities (JCQY201903). The authors declare no conflict of interest. Y.W. and K.D. designed the experiments; T.L., J.J., Y.C., S.Z., and Z.W. performed the experiments; T.L. wrote the manuscript; Y.W. and K.D. revised the manuscript. All authors read and approved of the final manuscript. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13476/suppinfo Material and methods. Supplementary references. Figure S1. Sequencing analysis of the potential off-target sites of GmTAP1 Figure S2. Inoculation of the roots of WT, tap1-1, and tap1-2 seedlings to evaluate resistance against P. sojae Table S1. Potential off-target sites of GmTAP1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.