Characterization and Proteomic Analysis of Novel Rice Lesion Mimic Mutant with Enhanced Disease Resistance

doi:10.1016/j.rsci.2021.07.007

Abstract

Abstract:

Lesion mimic mutants (LMMs) are plants that spontaneously form lesions without pathogen infection or external stimulus and exhibit resistance to pathogens. Here, a rice LMM was created by ethyl methane sulfonate mutagenesis, named as hpil (hydrogen peroxide induced lesion). Diaminobenzidine and trypan blue staining showed that large amounts of H₂O₂ were produced and cell death was occurred at and around the parts of lesion mimic in the rice leaves. The phenotype of hpil is controlled by a single recessive gene, localized at a 2 Mb interval on chromosome 2. The data suggested that hpil is a novel LMM with enhanced bacterial and fungal disease resistance, and multiple pathogenesis-related proteins (PRs) were up-regulated. The proteomes of leaves at three positions (different degrees of lesion mimic severity) were characterized in hpil compared with its wild type plant. Differentially expressed proteins were detected by two dimensional difference gel electrophoresis and 274 proteins were identified by MALDI TOF/TOF^TM. These proteins were related to metabolic process, cellular process and response to stimulus, with mostly down-regulated in hpil leaves. Many of these proteins were related to the Calvin cycle, photosynthetic electron transport chain, glycolysis/gluconeogenesis and phosphonates pathways. Some resistance-related proteins including 14-3-3 proteins, OsPR10 and antioxidases such as peroxidase, superoxide dismutase and ascorbate peroxidase were up-regulated in leaves with lesion mimic. These results provide the foundation for cloning of the target gene and shed light on the mechanism involved in autoimmunity of rice.

Key words: lesion mimic mutant, H₂O₂, disease resistance, pathogenesis-related protein, resistance- related pathway

Yong Yang, Qiujun Lin, Xinyu Chen, Weifang Liang, Yuwen Fu, Zhengjin Xu, Yuanhua Wu, Xuming Wang, Jie Zhou, Chulang Yu, Chengqi Yan, Qiong Mei, Jianping Chen. Characterization and Proteomic Analysis of Novel Rice Lesion Mimic Mutant with Enhanced Disease Resistance[J]. Rice Science, 2021, 28(5): 466-478.

Figures/Tables 12

Fig. 1. Characterization of hpil and relative expression of pathogenesis-related proteins (PRs). A, Phenotypes of Zhejing 22 (ZJ22) and hpil. B, Leaves of ZJ22 and hpil. Shaded region is indicated by red box. C, Trypan blue staining of leaves of ZJ22 and hpil. Shaded region is indicated by red box. D, Diaminobenzidine staining of leaves of ZJ22 and hpil. Shaded region is indicated by red box.E, Relative expression of PRs in ZJ22 and hpil. Data are Mean ± SE (n = 3). **, P < 0.01.

Fig. 2. High resistance of hpil to different races of Xoo and M. oryzae.A, Leaves of Zhejing 22 (WT) and hpil inoculated with 10 different races of Xoo. B, Lesion lengths caused by Xoo infection. Data are Mean ± SD (n = 6); **, P < 0.01.C, Leaves of WT and hpil inoculated with two different races of M. oryzae. D, Disease severity of leaves inoculated with M. oryzae. Data are Mean ± SD (n = 10); **, P < 0.01.

Fig. S1. Genotype frequency profile of progeny on chromosome 2.The x-coordinate represents the coordinates of the chromosomes. The ordinate represents the frequency of the mutant genotype. The red line represents the corresponding frequency values of the sliding window in different regions of the chromosome.

Table S1 Unique protein spots identified by MS/MS. Ratios are the fold changes of DEPs in leaf A, leaf B and leaf C.

Fig. S2. Numbers of differentially expressed protein spots on hpil in comparison with wild-type plants.Protein spots (≥ 1.5-fold, P < 0.05) detected in leaves from three positions (1, 2 and 3).

Fig. S3. Gene ontology classification of differently expressed proteins.Red bar represent up-regulated spots, green bar represent down-regulated spots.

Fig. S4. Kyoto Encyclopedia of Genes and Genomes enrichment of differently expressed proteins.

Fig. 3. Quantitative real-time PCR analysis of relative mRNA levels of differentially expressed proteins (DEPs) in leaves of WT and hpil.AAA type ATPase, ATPase associated with various cellular activities; SOD, Superoxide dismutase.WT, Zhejing 22; Leaf A, The first leaf from the top with no lesion mimic; Leaf B, The second leaf from the top with mild lesion mimic; Leaf C, The third leaf from the top with severe lesion mimic.Data are Mean ± SD (n = 3).

Fig. S5. Differently expressed proteins in the major metabolic pathways of rice.Three squares from left to right represent differential changes in leaf A, leaf B, leaf C in wild plant and hpil. Up-regulated (red), down-regulated (green), no change (black). The ratios of DEPs are listed in Table S2. SBPase (Sedoheptulose-1,7- bisphosphatase), TK (Transketolase), RPI 3 (Ribose 5-phosphate isomerase 3), PRK (Phosphoribulose kinase), RPE (Ribulosephosphate 3-epimerase), RBCL (Ribulose bisphosphate carboxylase large chain), FBA (Fructose bisphosphate aldolase), TPI (Triosephosphate isomerase), GAPDH (3-phosphate dehydrogenase), PGK (Phosphoglycerate kinase), FBPase (Fructose 1,6-bisphosphatase), MDH (Malate dehydrogenase), SuCLβ（Succinyl-CoA ligase beta-chain）, S7P (Sedoheptulose 7-phosphate), R5P (Ribose 5 phosphate), Ru5P (Ribulose 5 phosphate), RUBP (Ribulose-1,5- bisphosphate), 3GP (glycerol-3-phoshpate), BPG (1,3 diphosphoglyceric acid), G3P (glyceraldehyde 3-phosphate), FBP (β-D Fructose 1,6-biphosphate), F6P (β-D Fructose 6-phosphate), G6P (α-D-Glucose 6-phosphate), G1P (α-D-Glucose 1 phosphate), E4P (Erythrose 4-phosphate), X5P (Xylulose 5-phosphate), OAA (Oxalaetic acid), Suc (Succinate), Mal (Malic acid).

Fig. S6. Detection on chorophyll content, antioxidases activity and photosynthetic parameters.A, Chorophyll content, SOD and APX activity of leaf A, B and C in wild-type and hpil.B, Net photosynthetic rate (Pn), stomatal conductance (Gs) and intercellular CO2 (Ci) of leaf A, B and C in wild-type and hpil.Asterisks indicate significant differences (*, **, P < 0.05 and P < 0.01 by the Student’s t-test).

Table S3 Grade description for screening against M. Oryzae.

Table S4 List of primers used for quantitative real-time PCR.

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