Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line

Abstract

Rice blast (Magnaporthe oryzae) is a devastating disease affecting rice production globally. The development of cultivars with host resistance has been proved to be the best strategy for disease management. Several rice-resistance genes (R) have been recognized which induce resistance to blast in rice but R gene-mediated mechanisms resulting in defense response still need to be elucidated. Here, mutant lines generated through CRISPR/Cas9 based targeted mutagenesis to investigate the role of Pi21 against blast resistance and 17 mutant plants were obtained in T₀ generation with the mutation rate of 66% including 26% bi-allelic, 22% homozygous, 12% heterozygous, and 3% chimeric and 17 T-DNA-free lines in T₁ generation. The homozygous mutant lines revealed enhanced resistance to blast without affecting the major agronomic traits. Furthermore, comparative proteome profiling was adopted to study the succeeding proteomic regulations, using iTRAQ-based proteomic analysis. We identified 372 DEPs, among them 149 up and 223 were down-regulated, respectively. GO analysis revealed that the proteins related to response to stimulus, photosynthesis, carbohydrate metabolic process, and small molecule metabolic process were up-regulated. The most of DEPs were involved in metabolic, ribosomal, secondary metabolites biosynthesis, and carbon metabolism pathways. 40S ribosomal protein S15 (P31674), 50S ribosomal protein L4, L5, L6 (Q10NM5, Q9ZST0, Q10L93), 30S ribosomal protein S5, S9 (Q6YU81, Q850W6, Q9XJ28), and succinate dehydrogenase (Q9S827) were hub-proteins. The expression level of genes related to defense mechanism, involved in signaling pathways of jasmonic acid (JA), salicylic acid (SA), and ethylene metabolisms were up-regulated in mutant line after the inoculation of the physiological races of M. oryzae as compared to WT. Our results revealed the fundamental value of genome editing and expand knowledge about fungal infection avoidance in rice.

Keywords: CRISPR/Cas9; M. oryzae; Pi21; homozygous; iTRAQ; proteomics; resistance; rice.

Figure 1

Detection of mutations induced by CRISPR/Cas9; (A) chromatograms showing the sequencing results for both the target sites in mutant lines; (B) inheritance of mutations in T₁ and T₂ and T₃ generations. Red hyphens and letters represent the deletions and blue letters represent insertions, respectively while the PAM sequence is highlighted in green.

Figure 1

Detection of mutations induced by CRISPR/Cas9; (A) chromatograms showing the sequencing results for both the target sites in mutant lines; (B) inheritance of mutations in T₁ and T₂ and T₃ generations. Red hyphens and letters represent the deletions and blue letters represent insertions, respectively while the PAM sequence is highlighted in green.

Figure 2

Phenotypic appearance of wild-type and mutant lines inoculated with M. oryzae (A) after 5 dpi and (B) Blast disease scoring in WT and mutant plants “**” denotes the significant difference, student’s t-test, p ≤ 0.01, n = 10.

Figure 3

Identified proteins and heatmap of wild type and CRISPR mutant line (GN-5). (A) The number of the total, and differentially expressed proteins (up and down-regulated). (B) Proteins with higher expression difference among WT and mutant. Red color denotes higher while the green color is representing the lower level of expression.

Figure 4

STRING predicted protein to protein interaction (PPI) of differentially expressed proteins (DEPs) between wild type and mutant lines (GN-5). Nodes with redder color show a higher co-expression level.

Figure 5

Gene ontology (GO) pathways of up-regulated proteins of WT and mutant line GN-5.

Figure 6

KEGG pathway enrichment histogram for up-regulated proteins (p-value ≤ 0.05).

Figure 7

The hub-proteins selected from the total DEPs of WT and GN-5. Every node represents proteins and the edges indicate connections between the nodes, the red color denotes the higher co-expression.

Figure 8

RT-qPCR results for target genes and proteomic data. (A) Pi21 and (B) eight selected DEPs responsive genes. (C) Relative expression of defense-response genes in WT and the homozygous mutant line inoculated with M. coryza. hpi; hours post-inoculation. “*” denotes the significant difference, p ≤ 0.05 and “**” denotes the significant difference, student’s t-test, p ≤ 0.01, n = 3.

Figure 8

RT-qPCR results for target genes and proteomic data. (A) Pi21 and (B) eight selected DEPs responsive genes. (C) Relative expression of defense-response genes in WT and the homozygous mutant line inoculated with M. coryza. hpi; hours post-inoculation. “*” denotes the significant difference, p ≤ 0.05 and “**” denotes the significant difference, student’s t-test, p ≤ 0.01, n = 3.