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Research Overview
Research Group: Molecular Genetics of Plant-Pathogen Interactions
I. Introduction
The objective of the group is to understand the mechanisms of plant-pathogen
interactions, and the signal transduction pathways leading to the induction of disease
resistance responses. Employing genetic and genomic approaches, we focus in signal
transduction of rice (Oryza sativa) disease resistance, and pathogenicity of
Xanthomonas campestris pv. Campestris (Xcc) and X. oryzae pv. Oryzae (Xoo).
Group Members
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Principle investigator: Chao-Zu He, Professor, Ph.D. (1996, University of
Queensland, Australia)
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Other members:
Dr. Wei Qian (Research Associate)
Mr. Gui-Fu Liu (Research Associate)
And 10 graduate students
II. Background and Significance
Plants have developed sophisticated mechanism to protect themselves from
microbial attack in their natural environment. One of the highly evolved strategies is
the gene-for-gene interactions, in which a specific resistance gene in plants recognizes
a corresponding avirulence (avr) gene of the pathogen. This recognition process
activates a cascade of defense genes that inhibit the pathogen's ingress. During the last
15 years, over 50 disease resistance genes have been cloned from different plants and
many pathogenicity-related genes have been cloned from plant pathogens. This has
dramatically advanced our understanding of the molecular basis of plant-pathogen
interactions. Thus, in dissecting the disease resistance pathway, one might ask how
many genes are required in the signal transduction pathway before defense genes are
activated, and secondly, what the function of each gene is in switching on the
signaling pathway. On the other side, how many genes are required in pathogenicity
of a pathogen and how the pathogenesis processes are regulated.
III. Major Achievements
In last 4 years, the group conducted several research projects on signal
transduction of rice disease resistance and functional genomics of Xcc/Xoo, resulting
in publication of research papers in international journals. Professor Chaozu He gave
oral presentations in two international conferences. Three graduate students of the
group obtained their Ph.D degree successfully from the Institute. Major achievements
are summarized below.
1. Map-based cloning of the rice lesion mimic gene Spl1
The rice lesion mimic mutant, spl1 (spotted leaf 1), was first identified in rice
cultivar Asahi in 1965. This mutant displayed spontaneous disease-like lesions in the
absence of any pathogen and was found to confer resistance to multi-isolates of rice
blast. We employed a map-based cloning strategy to isolate the Spl1 gene. A total of
ten cleaved amplified polymorphic sequence (CAPS) markers linked to the Spl1
gene were identified and mapped into an 8.5 cM region on chromosome 12. A
high-resolution genetic map was developed using these ten CAPS markers and a
segregating population consisting of 3202 individuals. A BAC contig containing four
BAC clones was constructed and Spl1 was located within a 423-kb region. Seven
spl1 allelic mutants were obtained from the IR64 deletion mutant collection.
Molecular analysis using these mutants delimited the Spl1 gene in a 70-kb interval,
covered by two BAC clones. Four candidate genes were identified from this region.
Genetic complementation and functional analysis of these genes are in progress.
2. Functional analysis of a rice zinc finger protein OsLSD1
The Arabidopsis LSD1 and LOL1 proteins both contain three conserved zinc
finger domains and have antagonistic effects on plant programmed cell death (PCD).
We have identified a rice functional homolog of LSD1, designated as OsLSD1 (for
Oryza sativa LSD1). The expression of OsLSD1 was light-induced or
dark-suppressed. Overexpression of OsLSD1 driven by the cauliflower mosaic virus
35S promoter accelerated callus differentiation in transformed rice tissues and
increased chlorophyll b content in transgenic rice plants. Antisense transgenic rice
plants exhibited lesion mimic phenotype, increased expression of PR-1 mRNA, and
an accelerated hypersensitive response (HR) when inoculated with avirulent isolates
of blast fungus. Both sense and antisense transgenic rice plants conferred
significantly enhanced resistance against a virulent isolate of blast fungus. Moreover,
ectopic overexpression of OsLSD1 in transgenic tobacco (Nicotiana tabacum)
enhanced the tolerance to fumonisins B1 (FB1), a PCD-eliciting toxin.
OsLSD1-green fluorescent protein fusion protein was located in the nucleus of
tobacco cells. Our results suggest that OsLSD1 plays a negative role in regulating
plant PCD whereas a positive role in callus differentiation. Further experiments are
carrying out to dissect mechanisms how OsLSD1 regulates these pathways in plant
cell.
3. Comparative and functional genomic analysis for the pathogenicity of
Xanthomonas campestris pv. campestris
Gram-negative bacterium Xanthomonas campestris pathovar campestris (Xcc) is
one of the model organisms for studies on plant-microbe interactions. It is the
causative agent of black rot disease of cruciferous plants, including the model plant
Arabidopsis thaliana. Xcc could cause symptoms of blackened veins and foliar
marginal V-shaped necrotic lesions, resulting in severe agricultural production loss
every year.
Employing a whole genome shotgun strategy, we sequenced the genome of Xcc
8004. It consists of a single circular chromosome (5,148,708 bp) with a G + C content
of 64.9%, larger than the published genome of the Xcc ATCC 33913 (5,076,187 bp).
The replication origin site of Xcc 8004 genome was predicted at the intergenic region
of dnaA and dnaN. Approximately, 63% of the CDSs (2709 CDSs) has been assigned
biological functions. A large amount of phage-related genes, including two copies of
ΦLf filamentous phages that specifically infect Xcc, were identified in the genome. In
addition, there are 115 insertion sequence elements, ranging from 0.8 ~ 1.6 kb and
belonging to 15 groups, distributed throughout the genome.
Comparative analysis revealed high degree of conservation between Xcc 8004 and
the sequenced genome of Xcc ATCC 33913 in terms of gene content (97% of CDSs
were shared) However, strain-specific and dramatic genome-scale of rearrangements
were identified by comparison (Figure1). Interestingly, intra-species divergence
between Xcc ATCC 33913 and Xcc 8004 is more significant than that of inter-species
between Xcc ATCC 33913 and X. axonopodis pv. citri 306 (Xac), suggesting Xcc 8004
might be originated from recent recombination events.
We constructed a Xcc 8004 random insertional mutant library that covers
approximately 4 times of the genome. A total of 16,512 transformants were
inoculated on the susceptible host plant cabbage (Brassica oleracae) and 172
pathogenicity-deficient mutants were obtained, of which 75 non-redundant,
single-copy transposon insertional disrupted CDSs or intergenic regions were
identified by flanking sequence analysis. These mutant genes were found involved in
multiple physiological pathways, such as secretion systems, pili assembly,
lipopolysaccharide biosynthesis, fatty acid degradation and general metabolism.
Furthermore, function unknown genes and strain-specific genes were identified. Our
genome-scale analyses provided new insight into the relationship between host
specificity and genome evolution of Xcc, and generated a mutant profile as standing
point to investigate the molecular pathogenesis of black rot disease.
4. Other ongoing projects
Several ongoing projects are being conducted. These include functional
genomics of pathogenicity of Xoo and signal transduction and regulation of Xcc
Type II secretion.
Key Publications
1. Qihong Sun, Wei Wu, Wei Qian, Jun Hu, Rongxiang Fang and Chaozu He (2003).
High-quality mutant libraries of Xanthomonas oryzae pv. oryzae and X. campestris pv.
campestris generated by an efficient transposon mutagenesis system. FEMS Microbiology
Letters 226:145-150.
2. Youbao Sha, Shutian Li, Zhongyou Pei, Lijuan Luo, Yingchuan Tian and Chaozu He (2004).
Generation and flanking sequence analysis of a rice T-DNA tagged population. Theoretical
and Applied Genetics (TAG) 108: 306-314.
3. Lian-Hui Wang, Yawen He, Yunfeng Gao, Ji En Wu, Yi-Hu Dong, Chaozu He, Su Xing Wang,
Li-Xing Weng, Jin-Ling Xu, Leng Tay, Rong Xiang Fang, Lian-Hui Zhang (2004). A bacterial
cell-cell communication signal with cross-kingdom structural analogues. Molecular
Microbiology 51:903-912.
4. Qihong Sun, Jun Hu, Guixiu Huang, Chao Ge, Rongxiang Fang and Chaozu He (2005). The
type II secretion pathway structural gene xpsE is required for xylanase- and cellulasesecretion and virulence in Xanthomonas oryzae pv. oryzae. Plant Pathology 54:15-21.
5. Guifu Liu, Lijuan Wang, Zhuangzhi Zhou, Hei Leung, Guo-liang Wang and Chaozu He
(2004). Physical mapping of a rice lesion mimic gene, Spl1, to a 70-kb segment of rice
chromosome 12. Molecular Genetics and Genomics 272: 108-115.
6. Lijuan Wang, Zhongyou Pei, Yingchuan Tian and Chaozu He (2005). OsLSD1, a Rice Zinc
Finger Protein, Regulates Programmed Cell Death and Callus Differentiation. Molecular
Plant-Microbe Interactions 18: 375-384.
7. Wei Qian, Yantao Jia, Shuang-Xi Ren, Yong-Qiang He, Jia-Xun Feng, Ling-Feng Lu, Qihong
Sun, Ge Ying, Dong-Jie Tang, Hua Tang, Wei Wu, Pei Hao, Lifeng Wang, Bo-Le Jiang,
Shenyan Zeng, Wen-Yi Gu, Gang Lu, Li Rong, Yingchuan Tian, Zhijian Yao, Gang Fu,
Baoshan Chen, Rongxiang Fang, Boqin Qiang, Zhu Chen, Guo-Ping Zhao, Ji-Liang Tang, and
Chaozu He (2005). Comparative and functional genomic analyses of the pathogenicity of
phytopathogen Xanthomonas campestris pv. campestris. Genome Research 15: 757-767.
Invited presentations in International Conferences
1. Strategies to dissect the molecular basis for virulence in Xanthomonas campestris pv.
campestris and X. oryzae pv. oryzae. First International Rice Congress, 16-18 Sept. 2002; Beijing.
2. Functional analysis for pathogenicity-related genes of xanthomonads using mutagenesis
approach. 10th International Congress for Culture Collections, Tsukuba, Japan, 10-15 2004.
IV. Future Research Plan
Research is focuses on following projects next few years.
(1) The mechanisms of Spl1 gene and OsLSD1 gene in cell death of rice.
(2) Functional genomics of pathogenicity of Xcc and Xoo.
(3) Signal transduction and regulation of pathogenesis of Xcc and Xoo.