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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (4): 857-870.doi: 10.3724/SP.J.1006.2024.32010

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Phenotypic identification and disease resistance mechanism analysis of rice lesion mutant lms1

YU Yao1(), WANG Zi-Yao1, ZHOU Si-Rui1, LIU Peng-Cheng1, YE Ya-Feng2, MA Bo-Jun1, LIU Bin-Mei2,*(), CHEN Xi-Feng1,*()   

  1. 1College of Life Science, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
    2Hefei Institutes of Physical Science, Chinese Academy of Science, Hefei 230031, Anhui, China
  • Received:2023-03-27 Accepted:2023-10-23 Online:2024-04-12 Published:2023-11-23
  • Contact: * E-mail: liubm@ipp.ac.cnl; E-mail: xfchen@zjnu.cn E-mail:2315318441@qq.com;liubm@ipp.ac.cnl;xfchen@zjnu.cn
  • Supported by:
    National Natural Science Foundation of China(32071987);National Natural Science Foundation of China(32101697);Natural Science Foundation of Zhejiang Province(LQ22C130005);Natural Science Foundation of Zhejiang Province(LZ23C130004)

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Abstract:

Lesion mimic mutants are important genetic materials for studying molecular mechanisms of plant cell death and disease resistance. Through radiation mutagenesis of a japonica rice cultivar ‘Wuyunjing 7’, a rare lesion mimic and disease-susceptible mutant lms1 was obtained. Compared to the wild type, the leaves of this mutant spontaneously appeared reddish-brown spots, and its plant height, panicle length, number of grains per panicle, and yield per plant decreased, but the weight of 1000-grain increased. In addition, the resistance of lms1 to rice bacterial blight decreased significantly, and tissue staining showed significant cell death and excessive accumulation of reactive oxygen species in the mutant leaves. Genetic analysis showed that the phenotype of the lms1 mutant was controlled by a single recessive nuclear gene, and the lms1 gene was finely located between two molecular markers, Indel7 and Indel8, on chromosome 9 of rice with a physical distance of 62 kb. PCR amplification and sequencing of candidate genes in the localization interval showed that a 654 bp sequence was inserted into the first exon of the OsLMP1 (Lesion Mimic Phenotype 1) gene, which encodes a ubiquitin carboxyl-terminal hydrolase, resulting in premature termination of protein translation. The protein compositions of lms1 mutants and WT controls were analyzed by a proteomics technology, which identified a total of 19 differentially accumulated proteins (7 upregulated and 12 down-regulated), mainly involved in redox, chlorophyll synthesis, photosynthesis, and other metabolic pathways. The above results provide a reference for further research on the function of the OsLMP1 gene and its molecular mechanism of regulating programmed cell death and disease resistance.

Key words: rice, lesion mimic, lms1, gene mapping, proteomic analysis

Table 1

Molecular markers and their sequences used for fine mapping"

名称
Name		 正向引物序列
Former primer (5′-3′)		 反向引物序列
Reverse primer (5′-3′)
Indel1 ATGCAAATGACCTCGCTTCC GCCTGCCACTGACTCTGTTC
Indel2 TACAGGGAGATAAGAGGAAG CAAGTCGGAGGAACGAACAA
Indel3 CTTACCTCCGTCTCCAAA GTCATATTAACAGTCCCATT
Indel4 GCTTCACCCGCTTCTTCAC CAGGTTCTCGGCCCAATC
Indel5 AGAGGGTTGAGGGAGGCTGA TTCCATGCGGTCCATAAGTG
Indel6 ATGGCTGGAGGAGTGAATGA CTCGTGAGCGGATTATAGTTG
Indel7 GTATTTCCCGATGACTTCC ACATTTATTGATTGCTCCC
Indel8 AGGCTTAGTGATGGAGTAGAA CATGATGCCATTTGATTTGA

Table 2

Primer pairs used for PCR amplification and identification of target gene"

名称
Name		 正向引物序列
Former primer (5′-3′)		 反向引物序列
Reverse primer (5′-3′)
OsLMP1-1 GGGGATTTCTGTTGGTGTAGG GTTGCATCATCAATAGGGTTTT
OsLMP1-2 GTCATGTGCTTCTGTGCCTAA CATGGACAGTGGGACCTCTTA
OsLMP1-3 AGTATGCCTTCCCATCTGTA AGGCCAACTGGACTCAATCAAA
lms1-Chr.9 AGCAGCAGCAGAAGAAGAAA AGCCCTCTGATTACACTACCAT
lms1-Chr.3 CAGTGATGAACACCTGAGAAC GCCGCAGTAGTTAGATGTTGT

Fig. 1

Lesion mimic and disease resistance phenotype of lms1 mutant A: plants at the seedling stage; B: plants at the mature stage; C: flag leaves. D: chlorophyll contents. E: leaves before staining; F-H: leaves stained by TB, NBT, and DAB, respectively; I: photographs of the leaves infected by Xoo strains; J: statistical results of the lesion length in the leaves infected by Xoo strains (Measure the length of the diseased spot with a graduated ruler, measure at least 15 sword leaves for each type of rice, and finally take the average value as its diseased spot length). A two-tailed unpaired Student’s t-test determined the statistical significance (hereafter). *: P < 0.05; **P < 0.01. Bar: 2 cm in (A, C-G, I-J), 10 cm in (B)."

Fig. 2

Comparison of main agronomic traits between the lms1 mutant and WT Statistical results of plant height (A), flag-leaf length (B) and width (C), effective tiller number (D), panicle length (E), grains number per panicle (F), setting ratio (G), grain length (H), grain width (I), grain thickness (J), 1000-grain weight (K), and yield per plant (L); photograph of 10 grains in length (M), width (N) and thickness (O) between WT and lms1 mutant, scale bar: 1 cm."

Fig. 3

Fine mapping and identification of the candidate gene of lms1 A and B: preliminary and fine mapping genetic map of the lms1 mutant; ‘n’ refers to the number of F2 mutants used; the number under each marker represents the recombinants detected by corresponding marker. C: annotation genes in the 62-kb region, and the mutation site of the candidate gene of lms1; a 654-bp fragment was inserted into target gene at the 625 bp from the first codon. D: sequencing results of lms1 gene in the mutation site comparing to WT, and the sequence boxed is the whole fragment inserted. E: model diagram of chromosome translocation in lms1 mutant. DSB, DNA double-strand break. NHEJ, non-homologous end joining; F: electrophoresis of PCR products amplified from WT and lms1 using primers pair flanking by the mutation sites. M: molecular marker DL2000."

Fig. 4

Conservation domain and phylogenetic tree analysis of OsLMP1 protein A: sequence alignment of OsLMP1 and its homologous proteins, and the conservation domain were underlined or boxed; B: phylogenetic tree of OsLMP1 proteins. The Maximum-Likelihood tree was generated by MEGA v7.0 with bootstrap-1000 repeats tests."

Table 3

Basic information of differentially accumulated proteins"

蛋白序号a
Protein IDa		 基因序号b
Gene IDb		 描述
Description		 表达量差异倍数
FC		 分数
Score		 趋势(下调/下调)
Trend (down/up)
Q8W250 LOC_Os01g01710 1-deoxy-D-xylulose 5-phosphate reductoisomerase 0 16.768 Down
Q9LGQ6 LOC_Os01g09010 Acyl transferase 9 0 11.008 Down
Q6Z2T6 LOC_Os02g51080 Geranylgeranyl diphosphate reductase 0 57.381 Down
Q75L11 LOC_Os05g02300 Histone H2A.6 0 139.17 Down
A2Y8E0 LOC_Os06g01850 Leaf-type ferredoxin-NADP+ oxidoreductase 0 5.7841 Down
Q69TY4 LOC_Os06g42000 Peroxiredoxin-2E-1 0 4.344 Down
Q53JF7 LOC_Os11g06720 Abscisic stress-ripening protein 5 0 323.31 Down
B6RGY0 LOC_Os01g37510 Peptide deformylase 0.2145 7.3262 Down
Q7XBW5 LOC_Os10g42500 Probable plastid-lipid-associated protein 3 0.2309 27.554 Down
Q71U98 LOC_Os03g27310 Histone H3.3 0.2328 203.14 Down
Q6ENC8 LOC_Os10g21358 50S ribosomal protein L23 0.2462 55.05 Down
Q53RM0 LOC_Os03g36540 Magnesium-chelatase subunit ChlI 0.3060 71.42 Down
P30298 LOC_Os06g09450 Sucrose synthase 2.0059 121.52 Up
Q9LSU0 LOC_Os01g59600 Proteasome subunit alpha type-3 2.5953 11.201 Up
Q7F1U0 LOC_Os07g48020 Peroxidase 3.3684 109.27 Up
Q84YK8 LOC_Os08g39850 Lipoxygenase 4.7947 20.507 Up
Q75IM9 LOC_Os05g03480 Isovaleryl-CoA dehydrogenase 8.1778 12.587 Up
Q6ZCR3 LOC_Os08g13440 Cupin domain containing protein 27468000 6.2905 Up
Q0JG75 LOC_Os01g71190 Photosystem II reaction center PSB28 protein 189290000 323.31 Up

Fig. 5

Proteomic comparison of lms1 mutant and WT A: GO analysis of the differentially-expressed proteins (DEPs) between lms1 mutant and WT; B: KEGG enrichment of the DEPs. The X-axis indicates the enrichment ratio, the Y-axis indicates the KEGG pathway, and the bubble size indicates the number of proteins. The color represents the enriched Q-value; C: content of H2O2. D: POD activity."

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