RPA-Assisted Cas12a System for Detecting Pathogenic Xanthomonas oryzae, a Causative Agent for Bacterial Leaf Blight Disease in Rice

doi:10.1016/j.rsci.2021.11.005

Abstract

Abstract:

Xanthomonas oryzae pv. oryzae (Xoo) is a widespread pathogen causing bacterial leaf blight (BLB) disease, devastating rice productivity in many cultivated areas of Thailand. A specific and simple method for Xoo detection is required to improve surveillance of disease transmission and outbreak. This study developed a recombinase polymerase amplification (RPA) assay assisted with CRISPR-cas12a assay (RAC) for Xoo detection from bacterial cell suspension of infected rice samples without DNA extraction. The efficiency of the RAC system for Xoo detection using either Xoo80 or Xoo4009 locus was optimized to amplify and determine the sensitivity and specificity using a Xoo DNA template from bacterial cell suspension of infected rice samples without DNA extraction. The RAC system using the Xoo4009 locus gave a higher specificity than Xoo80 locus, because only Xoo species was amplified positive RPA product with fluorescence signal by cas12a digestion, which indicated no cross reactivity. Optimal RAC using the Xoo4009 locus enabled diagnosis of Xoo presence from both plant extracted samples of Xoo artificially inoculated rice leaves within 3 d post-inoculation without symptomatic BLB appearance, and Xoo naturally infected rice. Findings exhibited that RAC using the Xoo4009 locus offered sensitivity, specificity and simplicity for Xoo detection, with low intensities of Xoo-DNA (1 × 10³ copies/µL) and Xoo-cell (2.5 × 10³cfu/mL). This developed RAC system showed significantly potential for Xoo detection at point-of-care application for early signs of BLB disease outbreak in rice fields.

Key words: bacterial leaf blight, CRISPR-cas12a, recombinase polymerase amplification, rice

Kittisak Buddhachat, Nattaporn Sripairoj, Onchira Ritbamrung, Phithak Inthima, Kumrop Ratanasut, Thanita Boonsrangsom, Tepsuda Rungrat, Pongsanat Pongcharoen, Kawee Sujipuli. RPA-Assisted Cas12a System for Detecting Pathogenic Xanthomonas oryzae, a Causative Agent for Bacterial Leaf Blight Disease in Rice[J]. Rice Science, 2022, 29(4): 340-352.

Figures/Tables 7

Fig. 1. Guide RNA (gRNA) synthesis and in vitro cas12a digestion. A and B, Tertiary complexes for cas12a: gRNA:DNA targets, DNA templates for gRNA synthesis using in vitro transcription with T7 RNA polymerase and DNA targets containing gRNA binding sites for Xoo80 (A) and Xoo4009 (B) were amplified by recombinase polymerase amplification (RPA). Protospacer adjacent motif (PAM) was used as a proto-adjacent motif. C, Synthesized gRNA-Xoo80 and gRNA- Xoo4009 were visualized under 3% agarose gel electrophoresis. D?F, Experimental procedures were conducted under in vitro digestion to assess the efficiency of concentration ratios of cas12a and gRNA varied from 33:33 to 330:330 nmol/L. E, Sensitivity of various amounts of RPA-Xoo4009 product ranging from 2 to 10 µL. N represents negative control (without RPA product). F, dsDNA digestibility with different chemical modifications (gRNA-Xoo4009, cas12a and RPA-Xoo4009 product).

Fig. 2. Optimization of recombinase polymerase amplification (RPA) reaction for RPA-DNA amplification based on Xoo80 or Xoo4009 primer pairs. A and B, RPA was performed under various temperatures ranging from 15 ºC to 50 ºC for RPA-Xoo80 (A) and RPA-Xoo4009 (B) primer pairs. C and D, Time duration for DNA amplification was conducted at different periods of 0?60 min for RPA-Xoo80 (C) and RPA-Xoo4009 (D) primer pairs after obtaining the optimal temperature of 39 ºC. N represents negative control which was the reaction without DNA templates.

Fig. 3. Specific and sensitive assessments of RAC system for Xoo detection compared to RPA and PCR systems. A and B, Specificity test of RAC system using Xoo80 (A) and Xoo4009 loci (B) was performed with different Xanthomonas species and other bacterial species. C?F, Sensitivity test of RAC system using Xoo80 (C and E) and Xoo4009 loci (D and F) was determined with DNA templates from plasmid DNA (pUC57-Xoo80 or pUC57-Xoo4009) ranging from 0 to 1 × 108 copies/µL (C and D), and bacterial number ranged in 0?2.5 × 108 cfu/mL (E and F). RPA, Recombinase polymerase amplification; RAC, RPA-assisted cas12a. N refers to an RPA reaction without DNA templates as a negative control.

Fig. 4. Compatibility test of optimal RAC system using Xoo4009 locus. Detection feasibility of RAC used 11 Xoo isolates (K1E, K2, M6, K4G, E, K4C, M2, F, D, K1G and SK2-3) distributed in the endemic area. The resulting products were visualized by agarose gel electrophoresis (A) and an LED transilluminator (B), and the fluorescence measurement was quantified using a real-time machine (C). N refers to an RPA reaction without DNA templates as a negative control. RPA, Recombinase polymerase amplification; RAC, RPA-assisted cas12a.

Fig. 5. Sensitivity of RAC system for Xoo detection in artificially infected rice leaf, RD47 variety, with different bacterial cell intensities. Sixty-day-old rice leaves were inoculated with XooD at different concentrations ranging from 25 to 2.5 × 108 cfu/mL for 7 d post- inoculation. External bacterial leaf blight symptoms were photographed (A). Leaf extracts were used as DNA templates in an in vitro RAC system and fluorescent signals from the RAC products were visualized by an LED transilluminator (B), and quantified by a real-time PCR machine (C). N refers to an RPA reaction without DNA templates as a negative control. RPA, Recombinase polymerase amplification; RAC, RPA-assisted cas12a.

Fig. 6. Sensitivity of RAC system for Xoo detection at different days post-inoculation (dpi) of rice leaves. Leaf samples of rice variety RD47 were artificially inoculated with XooD at 2.5 × 108 cfu/mL by the clipping method. Leaves were collected at different time courses of 1-28 dpi for Xoo detection. External bacterial leaf blight symptoms were photographed (A). Leaf extracts were used as DNA templates in an in vitro RAC system and fluorescent signals from the RAC products were visualized by an LED transilluminator (B), and quantified by a real-time PCR machine (C). N refers to an RPA reaction without DNA templates as a negative control. RPA, Recombinase polymerase amplification; RAC, RPA-assisted cas12a.

Fig. 7. Diagnosis of Xoo pathogen using RAC system in naturally infected rice samples. A total of 23 leaf samples were collected from rice fields in lower northern of Thailand. Plant extracts were used for DNA templates in the RAC and PCR reactions. The cas12a-mediated trans-cleavage fluorescence signals from the RAC assay were detected under an LED transilluminator (A), PCR products were visualized by agarose gel electrophoresis (B), and their intensities were quantified by a real-time PCR machine (C). N refers to an RPA reaction without DNA templates as a negative control. RPA, Recombinase polymerase amplification; RAC, RPA-assisted cas12a.

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