Disease Resistance Improvement Of LK638S Via Gene Editing Of BSR-d1,PI21 And OSERF922

Longke 638S（LK638S）is an indica TGMS line with stable fertility and strong combining ability.LK638S has a weak resistance to rice blast which affect the promotion and application of hybrid rice.Previous studies showed that the loss of function of Bsr-d1,Pi21 and Os ERF922 can enhance resistance to rice blast.Therefore,we intends to use CRISPR/Cas9 technology to knock out Bsr-d1,Pi21 and Os ERF922 genes of LK638S to improve the resistance to rice blast of LK638S.The main research results are as follows:（1）In this study,a 20bp long guide RNA（g RNA）target sites driven by the rice U3promoter was designed to target and edit the sequence of the first of Bsr-d1,the second exon of Pi21 and the first exon of Os ERF922,respectively.Then connected to the expression vector to obtain rice transgenic lines.Among the positive transgenic plants in T₀transgenic generation,the mutation efficiency of the single gene editing sites of Bsr-d1,Pi21 and Os ERF922 were 82%,68%and 73%,respectively.Among them,there were 7,15 and 8 homozygous genotypes lines.The homozygous mutation efficiency were 25%,33%,and 24%,respectively.There were 3 lines of Bsr-d1,Pi21 and Os ERF922 genes all being homozygous mutations.The mutation types of T₁lines without transformed company of single-gene or triple-gene knockouts of Bsr-d1,Pi21 and Os ERF922 are consistent with the homozygous mutation types of T₀lines.（2）The identification results of the nature Nursery showed that the area of rice blast lesions of the single-gene homozygous mutant lines bsr-d1-9,bsr-d1-12,pi21-1,pi21-2,erf922-1,erf922-2 and triple-gene homozygous mutant lines bsr-d1pi21erf922-1 and bsr-d1pi21erf922-2 was significantly reduced compared with LK638S.The lesion area of the erf922 mutant and the three-gene bsr-d1/pi21/erf922 mutant was better than that of the bsr-d1 and pi21 mutants.In order to further evaluate the resistance of Bsr-d1,Pi21and Os ERF922 gene deletion to rice blast,we used different M.oryzae strains to inoculation test The results showed that compared with LK638S,the lesion area of the mutant lines was significantly reduced.The lesion area of lines of erf922-1 and bsr-d1pi21erf922-1 mutants was significantly lower than that of bsr-d1-9 and pi21-1.Consistent with the identification results of natural Nursery.（3）The expression levels of catalase genes LOC＿Os01g73170,LOC＿Os05g04470and LOC＿Os10g39170 in mutants bsr-d1-9 and bsr-d1-12 were significantly decreased,and the content of H₂O₂was significantly increased.The expression levels of defense-related genes in the SA and JA signaling pathways in the mutants after inoculation with Magnaporthe grisea were significantly higher than that of LK638S 24hours after inoculation with Magnaporthe grisea.（4）In this study,6 bacterial blight strains were collected for identification of bacterial blight inoculation of mutants.Compared with LK638S,pi21-1 has significantly enhanced resistance to the four physiological races Fu J,PXO99,PXO61 and PXO71;erf922-1 has significantly enhanced resistance to the four physiological races Fu J,PXO86,PXO61 and PXO71;bsr-d1-9 did not significantly increase the resistance to the six physiological races.Therefore,we speculate that knocking out Pi21 or Os ERF922genes in the background of LK638S can increase the resistance of LK638S to bacterial blight.The expression results of SA and JA signaling pathway defense-related genes showed that 72 hours after inoculation with Xanthomonas oryzae,Os PR1a,Os PR1b,Os WRKY45 and Os PR4 in pi21-1 and erf922-1 were significantly up-regulated,while in bsr-d1-9,Only Os WRKY45 was up-regulated in bsr-d1-9,indicating that knocking out Pi21 or Os ERF922 enhanced the resistance of LK638S to bacterial blight,which may be related to the activation of defense genes related to the SA and JA signaling pathways.（5）The inspection results of agronomic characteristics of LK638S and mutants showed that,compared with LK638S,the three-gene knockout mutant bsr-d1pi21erf922-1 had significantly lower plant height and tiller number than LK638S,and there was no significant difference in ear length and grain number.The single gene knockout mutants bsr-d1-9,pi21-1 and erf922-1 had no significant differences in plant height,tillers,ear length and number of grains per ear compared with LK638S.