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, 12 (1), 68

Identification and Characterization of a Spotted-Leaf Mutant spl40 With Enhanced Bacterial Blight Resistance in Rice

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Identification and Characterization of a Spotted-Leaf Mutant spl40 With Enhanced Bacterial Blight Resistance in Rice

Atul Prakash Sathe et al. Rice (N Y).

Abstract

Background: Spotted leaf mutants show typical necrotic lesions that appear spontaneously in the absence of any pathogen attack. These mutants are often characterized to exhibit programmed cell death (PCD) and activation of plant defense responses resulting in enhanced disease resistance to multiple pathogens. Here, we reported a novel spotted-leaf mutant, spl40 that showed enhanced disease resistance response.

Results: Initially lesions appeared at leaf tips during seedling stage and gradually covered the whole leaf at the tillering stage. The lesion development was light-dependent. spl40 showed obvious cell death at and around the lesion, and burst of reactive oxygen species (ROS) was accompanied by disturbed ROS scavenging system. Photosynthetic capacity was compromised as evidenced by significant reductions in chlorophyll content, important photosynthesis parameters and downregulated expression of photosynthesis-related genes which ultimately led to poor performance of major agronomic traits. spl40 exhibited enhanced resistance to 14 out of 16 races of bacterial blight pathogen of rice, caused by Xanthomonas oryzae pv. oryzae, most probably though activation of SA and JA signaling pathways, owing to upregulated expression of SA and JA signaling genes, though the exact mechanism remain to be elucidated. The spotted-leaf phenotype was controlled by a novel single recessive nuclear gene. Genetic mapping combined with high throughput sequencing analysis identified Os05G0312000 as the most probable candidate gene. Sequencing of ORF revealed a single SNP change from C to T that resulted in non-synonymous change in amino acid residue from leucine to phenylalanine. Interestingly, the complementation plants did not display lesions before heading but showed lesions at the heading stage and the transgenic T1 progenies could be classified into 3 categories based on their lesion intensity, indicating the complex genetic nature of the spl40 mutation.

Conclusion: The results obtained here clearly show that genes related to defense and PCD were upregulated in accordance with enhanced disease resistance and occurrence of PCD, whereas the photosynthetic capacity and overall ROS homeostasis was compromised in spl40. Our data suggest that a novel spotted-leaf mutant, spl40, would help to elucidate the mechanism behind lesion development involving programmed cell death and associated defense responses.

Keywords: Bacterial blight; Defense response; Programmed cell death; Rice; Spotted-leaf.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Spotted leaf phenotype of spl40. a Lesions appear at 4-leaf stage (shown detached 3rd leaf with lesions) (bar = 5 cm). b WT and spl40 at maturity stage (Scale bar = 10 cm). c Effect of light on lesion formation under the natural condition (1–2) Zhongjian100 and spl40 before shading. (2–3) Zhongjian100 and spl40 shaded for 7 days. (5) spl40 re-instated for 5 d. (6) spl40 re-instated for 15 d. Shaded areas are boxed. d Lesion phenotype on matured seeds (bar = 2 cm). e Photosynthetic pigment contents at tillering stage. f Soluble protein content at seedling and tillering stage. Values are means ± SD (n = 3); ** indicates significance at P ≤ 0.01 and * indicates significance at P ≤ 0.05 by Student’s t test
Fig. 2
Fig. 2
Photosynthesis parameters of leaves at the tillering and heading stages. a Net photosynthetic rate (Pn). b Stomatal conductance (Gs). c Transpiration rate (Tr). d Intercellular CO2 concentration (Ci). e Expression profile of photosynthesis related genes in spl40 and WT at tillering stage. Values are means ± SD (n = 3); ** indicates significance at P ≤ 0.01 and * indicates significance at P ≤ 0.05 by Student’s t test
Fig. 3
Fig. 3
Histochemical staining of spl40 and WT. a and d Evans blue staining: a before lesion development. d after lesion development. b and e DAB staining: b before lesion development. e after lesion development. c and f NBT staining: c before lesion development. f after lesion development. g Malonaldehyde (MDA) content at seedling and tillering stage. h Membrane ion leakage rate at seedling and tillering stage. i Expression analysis of PCD-related genes at tillering stage. Values are means ± SD (n = 3); ** indicates significance at P ≤ 0.01 and * indicates significance at P ≤ 0.05 by Student’s t test
Fig. 4
Fig. 4
ROS scavenging enzymes and associated genes. ac Enzymatic activities of CAT, SOD and POD at seedling and tillering stage. SOD, superoxide dismutase; CAT, catalase; POD, peroxidase. d Expression levels of genes encoding ROS-generating enzymes. e Expression levels of ROS detoxification-related genes. f Expression levels of senescence-related genes. Values are means ± SD (n = 3); ** indicates significance at P ≤ 0.01 and * indicates significance at P ≤ 0.05 by Student’s t test
Fig. 5
Fig. 5
Evaluation of disease resistance to Xanthomonas oryzae pv. oryzae and expression of defense genes involved in JA and SA signaling pathway. a Lesion lengths after Xoo inoculation at 20 DPI. b Expression analysis of JA signaling pathway genes. c Expression analysis of SA signaling pathway genes. Values are means ± SD (A n = 9; B and C n = 3); ** indicates significance at P ≤ 0.01 and * indicates significance at P ≤ 0.05 by Student’s t test
Fig. 6
Fig. 6
MutMap assisted mapping of spl40 gene on the long arm of chromosome 5. a Fine mapping of the candidate region of spl40. b Manhattan plot depicting the distribution of SNPs along 12 rice chromosomes. c Confirmation of the spl40 mutation by sequencing
Fig. 7
Fig. 7
T1 transgenic complementation plants. a Phenotype of WT, spl40 and T1 complementation plants (13–4 and 1–6). b T1 leaves representing three phenotypic categories viz., lesion mimic type, intermediate type and wild type. c Distribution of T1 plants in three phentoypic categories

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