Proteomic analysis of a disease-resistance-enhanced lesion mimic mutant spotted leaf 5 in rice
Xifeng Chen
0
Shufang Fu
0
Pinghua Zhang
0
Zhimin Gu
0
Jianzhong Liu
0
Qian Qian
1
Bojun Ma
0
0
College of Chemistry & Life Sciences, Zhejiang Normal University
, Jinhua 321004,
China
1
China National Rice Research Institute, Chinese Academy of Agricultural Sciences
, Hangzhou 310006,
China
Background: A lesion-mimic mutant in rice (Oryza sativa L.), spotted leaf 5 (spl5), displays a disease-resistance-enhanced phenotype, indicating that SPL5 negatively regulates cell death and resistance responses. To understand the molecular mechanisms of SPL5 mutation-induced cell death and resistance responses, a proteomics-based approach was used to identify differentially accumulated proteins between the spl5 mutant and wild type (WT). Results: Proteomic data from two-dimensional gel electrophoresis showed that 14 candidate proteins were significantly up- or down-regulated in the spl5 mutant compared with WT. These proteins are involved in diverse biological processes including pre-mRNA splicing, amino acid metabolism, photosynthesis, glycolysis, reactive oxygen species (ROS) metabolism, and defense responses. Two candidate proteins with a significant up-regulation in spl5 - APX7, a key ROS metabolism enzyme and Chia2a, a pathogenesis-related protein - were further analyzed by qPCR and enzyme activity assays. Consistent with the proteomic results, both transcript levels and enzyme activities of APX7 and Chia2a were significantly induced during the course of lesion formation in spl5 leaves. Conclusions: Many functional proteins involving various metabolisms were likely to be responsible for the lesion formation of spl5 mutant. Generally, in spl5, the up-regulated proteins involve in defense response or PCD, and the down-regulated ones involve in amino acid metabolism and photosynthesis. These results may help to gain new insight into the molecular mechanism underlying spl5-induced cell death and disease resistance in plants.
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Background
In plants, one of the most common and effective defense
responses to pathogen attack is the hypersensitive
response (HR), which prevents further spread of pathogens
to adjacent cells (Morel and Dangl 1997). Lesion mimic
mutants (lmms), displaying HR-like lesions in the
absence of pathogen attacks, have been identified from
maize (Johal et al. 1995), Arabidopsis (Dietrich et al.
1994), barley (Wolter et al. 1993), and rice (Takahashi
et al. 1999). Most lmms constitutively activate immune
responses, including callose deposition, induction of
Pathogenesis-related (PR) genes, production of reactive
oxygen species (ROS), and accumulation of phytoalexins
(Staskawicz et al. 1995). Therefore, lmms are very useful
genetic tools to dissect molecular mechanisms of
programmed cell death (PCD) and defense responses in
plants.
In rice, more than 43 lmms have been isolated, most
of which display enhanced resistance to rice blast and/or
bacterial blight pathogens (Takahashi et al. 1999; Yin
et al. 2000; Mizobuchi et al. 2002; Jung et al. 2005; Mori
et al. 2007; Wu et al. 2008; Qiao et al. 2010). So far, at
least 11 lmms have been functionally characterized,
including spl7 (Yamanouchi et al. 2002), spl11 (Zeng et al.
2004), Spl18 (Mori et al. 2007), spl28 (Qiao et al. 2010),
sl (Fujiwara et al. 2010), ttm1 (Takahashi et al. 2007),
rlin1 (Sun et al. 2011), NPR1 (Chern et al. 2005), lsd1
(Wang et al. 2005), acdr1 (Kim et al. 2009), and edr1
(Shen et al. 2011). Interestingly, these LMM genes
encode different proteins with distinct functions. For
example, SPL7 is a heat stress transcription factor
(Yamanouchi et al. 2002); SPL11 is a E3 ubiquitin ligase
(Zeng et al. 2004); SPL18, a acyltransferase (Mori et al.
2007); SPL28, a clathrin-associated adaptor protein
complex 1 medium subunit 1 (Qiao et al. 2010). These
findings indicate that numerous proteins with distinct
functions in multiple signaling pathways and/or
processes are involved to prevent inappropriate activation of
PCD. Thus, lmms have helped to gain an in-depth
insight into regulatory mechanisms of PCD and defense
responses in plants.
Rice spotted leaf 5 (spl5) is a lmm with spontaneous
HR-like lesions on its leaves, and broadly enhanced
resistance to rice blast and bacterial blight pathogens (Yin
et al. 2000; Mizobuchi et al. 2002). The spl5 gene was
previously mapped into a 36.4-cM region on rice
chromosome 7 (Iwata et al. 1978). Recently, we finely
mapped and isolated spl5 by a map-based cloning, and
surprisingly, it was found that the protein encoding by
SPL5 gene (GeneBank accessioin: KC128660) shares a
certain degree of homology with a human splicing factor
3b subunit 3 (SF3b3), one subunit of the SF3 protein
complex involved in binding of U2 snRNP to the branch
site in the splicing reaction of pre-mature RNAs (Chen
et al. 2009, Chen et al. 2012). Therefore, it is likely that
the SPL5 regulated cell death and resistance responses
post-transcriptionally.
Two-dimensional gel electrophoresis (2-DE) is a most
commonly used proteomics tec (...truncated)