Precise CRISPR-Cas9 Mediated Genome Editing in Super Basmati Rice for Resistance Against Bacterial Blight by Targeting the Major Susceptibility Gene

Abstract

Basmati rice is famous around the world for its flavor, aroma, and long grain. Its demand is increasing worldwide, especially in Asia. However, its production is threatened by various problems faced in the fields, resulting in major crop losses. One of the major problems is bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xoo hijacks the host machinery by activating the susceptibility genes (OsSWEET family genes), using its endogenous transcription activator like effectors (TALEs). TALEs have effector binding elements (EBEs) in the promoter region of the OsSWEET genes. Out of six well-known TALEs found to have EBEs in Clade III SWEET genes, four are present in OsSWEET14 gene's promoter region. Thus, targeting the promoter of OsSWEET14 is very important for creating broad-spectrum resistance. To engineer resistance against bacterial blight, we established CRISPR-Cas9 mediated genome editing in Super Basmati rice by targeting 4 EBEs present in the promoter of OsSWEET14. We were able to obtain four different Super Basmati lines (SB-E1, SB-E2, SB-E3, and SB-E4) having edited EBEs of three TALEs (AvrXa7, PthXo3, and TalF). The edited lines were then evaluated in triplicate for resistance against bacterial blight by choosing one of the locally isolated virulent Xoo strains with AvrXa7 and infecting Super Basmati. The lines with deletions in EBE of AvrXa7 showed resistance against the Xoo strain. Thus, it was confirmed that edited EBEs provide resistance against their respective TALEs present in Xoo strains. In this study up to 9% editing efficiency was obtained. Our findings showed that CRISPR-Cas9 can be harnessed to generate resistance against bacterial blight in indigenous varieties, against locally prevalent Xoo strains.

Keywords: CRISPR-Cas9; Xanthomonas oryzae; bacterial blight; genome editing; rice improvement.

FIGURE 1

(A) Schematic figure of the promoter showing EBEs location on the promoter against which three gRNAs were designed. gRNA1 was designed to target PthXo3 and AvrXa7, gRNA2 for TalC and gRNA3 targeted TalF. (B) Schematic figure of developing resistance by editing the OsSWEET14 gene. The intact promoter was susceptible to Xoo whereas the EBE edited promoter can be resistant to Xoo.

FIGURE 2

Schematic diagram of the construct used for genome editing. All three gRNAs were cloned in the same way. The construct was expressing gRNA under the OsU3 promoter. The rice codon-optimized Cas9 was expressing under the Ubi promoter.

FIGURE 3

(A) Sanger sequencing of AvrXa7 and PthXo3 EBE edited plants. The plants were named as SB-WT (Super Basmati wild type), SB-E1 (Super Basmati edited 1) and SB-E2 (Super Basmati edited 2). Green colored bases AGG represents the PAM sequence and red dashes symbolize the deletion at the target site. SB-E1 and SB-E2 have 24 and 4 bp deletion at the target site, respectively. (B) Sanger sequencing of TalF EBE edited plants. The plants were named SB-WT (Super Basmati wild type), SB-E3 (Super Basmati edited 3), SB-E4 (Super Basmati edited 4), and SB-E5 (Super Basmati edited 5). SB-E3 and SB-E4 have 18 and 4 bp deletion at the target site, respectively, disrupting TalF EBE, whereas SB-E5 had 5 bp deletion at the target site but TalF EBE remained intact. (C) T7 Endonuclease assay of T₀ edited and wild type plants. Red arrows showed the bands from edited plants that are absent in wild type plants (E1 = SB-E1, E2 = SB-E2, E3 = SB-E3, E4 = SB-E4, E5 = SB-E5, W1 and W2 = Wild Type Super Basmati). (D) Lesions induced by Xoo after 14 days post-inoculation on edited and control plants. SB-E1 and SB-E2 has reduced lesion length as compared to control plants. Negative = Plant was inoculated with scissors dipped in water. All the other lines were inoculated with scissors dipped in Xoo suspension. (E) Average of Percentage disease leaf area (% DLA) of the edited and control plants. The lowest DLA 10.12% was observed in the case of SB-E1 plants where 24 bp deletion was detected. The highest DLA 88.47% was present in IR-24 which was used as a susceptible check.

FIGURE 4

OsSWEET14 Induction by Xoo strain: Relative mRNA levels (2^–ΔΔCt) of OsSWEET14 in leaves of wild type and edited rice lines were compared. qRT-PCR was conducted in wild type Super Basmati and edited lines (SB-E2, SB-E3, SB-E4). Samples were harvested after 24 h of Inoculation with a locally virulent Xoo strain (mean ± s.e.m., n = 3 leaf samples from biological replicates) with expression normalized to rice SPS levels; repeated independently three times with comparable results.

FIGURE 5

Root length and shoot length of wild type and edited plants. Edited plants show normal root and shoot length just like wild type plants (two representative plants are shown in figure out of three).