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.
Highlights
Rice blast is a devastating disease threatening rice production worldwide, causing about 30%estimated yield losses annually [1,2,3]
The sequencing results confirmed the successful construction of the CRISPR/Cas9 binary vector having both the sgRNAs sequences (Supplementary file 1, Figure S5E)
Hygromycin phosphotransferase primers (HPT-F/R) were used to select mutant plants (Supplementary file 1, Table S1), and the amplified product was confirmed in 25 regenerated mutant lines by gel electrophoresis (Supplementary file 1, Figure S5F)
Summary
Rice blast is a devastating disease threatening rice production worldwide, causing about 30%estimated yield losses annually [1,2,3]. The most practical approach for managing the blast disease is the development of cultivars with durable resistance by introducing resistance (R)-genes and it is the most economical, sustainable, and environment-friendly [1,4]. Genes 2020, 11, 735 has evolved sophisticated defense mechanisms against the various invading pathogens. The plant immune system consists of PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI response activated by pattern recognition receptors (PRRs), which detects pathogen-associated molecular patterns (PAMPs), is relatively weak and restricts colonization of invading pathogens. The ETI-mediated resistance is highly specific and expected with low durability [5]. The R-gene mediated defense mechanism still needs to be elucidated because it is likely to be broken down by highly variable pathogenicity of M. oryzae, which makes the rice breeding difficult for durable blast resistance [2]
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