Functional Characterization of Leucine-rich Repeat Receptor-
发布时间：2022-01-07 06:50
卵菌属于假藻界,进化上与光合藻类相近。卵菌中有800个种,其中包括诸多植物病原菌,如疫霉属与腐霉属。在卵菌排名第五,引起巨大的经济与危害性。辣椒疫霉（P.capsici）是一种丝状的植物病原卵菌,其寄主范围广泛,对茄属、豆科、瓜果类植物均有极大的危害。真核生物通过受体蛋白激酶（RPKs）来感应、感知与传导来自细胞表面接收到的信号。基于RPKs的蛋白结域的不同,它们被分为不同的类别。在多个基因组中均发现富含亮氨酸重复的类受体蛋白激酶（LRR-RLKs）,它们具有影响生长、发育与对生物与非生物胁迫的免疫调节等多种生物学功能。我们在卵菌的基因组也发现含有LRR结构域的受体蛋白编码基因,这些基因在侵染阶段表达模式不同。但是这些基因在卵菌中的功能尚无相关报道。为明确这些基因的功能,我们进行了以下研究:首先,我们从4个分类群体（植物、藻类、真菌、卵菌）中选择14个具有代表性的物种,通过比较这些物种的完整基因组序列并进行仔细分析,发现卵菌的LRR-RLKs家族由111个成员组成。所有物种中的LRR-RLK蛋白质序列理论上都形成一个典型的LRR-RLKs结构,包括LRRs、跨膜区、激酶域。在卵菌中,我...
【文章来源】：南京农业大学江苏省 211工程院校 教育部直属院校
【文章页数】：178 页
【学位级别】：博士
【文章目录】：
ACKNOWLEDGEMENTS
摘要
ABSTRACT
CHAPTER ONE: INTRODUCTION
1.1 Objectives
1.2 Research Background
1.2.1 Introduction to Phytophthora plant pathogen threatening agriculture and natural ecosystem
1.2.2 Evolution
1.2.3 Biology
1.2.4 Genes for host-Phytophthora interaction
1.2.5 Phytophthora capsici-a model oomycete species with broad host range
1.2.6 Leucine-rich repeat receptor-like kinases(LRR-RLKs)
CHAPTER TWO: GENOME WIDE IDENTIFICATION OF LEUCINE-RICH REPEAT RECEPTOR-LIKE KINASES (LRR-RLKs) IN OOMYCETES
2.1 INTRODUCTION
2.2 MATERIAL AND METHODS
2.2.1 Identification and distribution of LRR-RLKs in oomycetes genome
2.2.2 Preparation of P.capsici cultures and plants
2.2.3 Collection of zoospore suspension and inoculation assay
2.2.4 RNA extraction and real-time RT-PCR assay
2.3 RESULTS
2.3.1 Identification of LRR-RLKs in oomycetes
2.3.2 Evolutionary analysis of LRR-RLKs in oomycetes
2.3.3 Protein structure and conserved motif analysis of LRR-RLKs in oomycetes
2.3.4 Differential expression profiles of P. capsici LRR-RLKs
2.4 DISCUSSION
2.5 CONCLUSION
CHAPTER THREE: PcLRR-RK1,AN LRR RECEPTOR KINASE REGULATESGROWTH, DEVELOPMENT AND PATHOGENESIS IN PHYTOPHTHORA CAPSICI
3.1 INTRODUCTION
3.2 MATERIAL AND METHODS
3.2.1 Identification and distribution of LRR-RLKs in P. capsici genome
3.2.2 Preparation of P. capsici cultures and plants
3.2.3 Construction and transformation of recombinant plasmids
3.2.4 RNA extraction and real-time RT-PCR assay
3.2.5 Analysis of colony growth, zoosporangium development and production
3.2.6 Inoculation assays on N. benthamiana and A. thaliana
3.2.7 DAB staining and callose deposition assay
3.2.8 Microscopic observation of cyst germination and pathogen establishment
3.3 RESULTS
3.3.1 Identification and distribution of LRR-RLKs in P. capsici genome
3.3.2 PcLRR-RK1 is required for vegetative growth of P. capsici
3.3.3 PcLRR-RK1 is essential for zoosporangium development and production
3.3.4 Silencing of PcLRR-RK1 results in loss of zoospores infection abilities into hosttissues
3.3.5 PcLRR-RK1 results into reduced germination of zoospores into host tissues
3.3.6 Silencing of PcLRR-RK1 results in penetration reduction of P. capsici mycelium into the host tissues
3.3.7 DAB staining and Callose deposition assay
3.4 DISCUSSION
3.5 CONCLUSION
CHAPTER FOUR: AN LRR RECEPTOR KINASE,PCLRR-RK2,IS CRUCIALLYREQUIRED FOR VEGETATIVE GROWTH, DEVELOPMENT AND PATHOGENESIS IN PHYTOPHTHORA CAPSICI
4.1 INTRODUCTION
4.2 MATERIAL AND METHODS
4.2.1 Protein sequence selection and identification
4.2.2 Maintenance of microbial strains, plants and culture conditions
4.2.3 Plasmid construction and P. capsici transformation
4.2.4 RNA isolation and quantitative real-time PCR
4.2.5 Phenotypic characterization
4.2.6 Pathogenecity assays
4.2.7 DAB staining and Callose deposition assay
4.2.8 Assays for the germination and penetration of zoospores into host tissues
4.3 RESULTS
4.3.1 Protein sequence selection and identification
4.3.2 PcLRR-RK2 is required for vegetative development of P.capsici
4.3.3 Silencing of PcLRR-RK2 interferes with zoosporangium development of P.capsici
4.3.4 PcLRR-RK2 is important for zoospores infection abilities into host tissues
4.3.5 PcLRR-RK2 results into reduced germination of zoospores into host tissues
4.3.6 PcLRR-RK2 is required for penetration of P. capsici mycelium into host tissues
4.3.7 PcLRR RK2 is important for host penetration,H_2O_2 accumulation and callose deposition
4.4 DISCUSSION
4.5 CONCLUSION
CHAPTER FIVE: PcLRR-RK3, AN LRR RECEPTOR KINASE IS REQUIRED FORVEGETATIVE DEVELOPMENT AND IN-PLANTA INFECTION PROCESSES IN PHYTOPHTHORA CAPSICI
5.1 INTRODUCTION
5.2 MATERIAL AND METHODS
5.2.1 Protein sequence selection and identification
5.2.2 Maintenance of plant and P.capsici cultures
5.2.3 Plasmid Construction and transformation into P.capsici culture
5.2.4 RNA extraction and qRT-PCR assay
5.2.5 Phenotypic analysis of PcLRRK3-silenced transformants
5.2.6 Pathogenecity assay on N. benthamiana
5.2.7 Trypan blue staning,DAB staining and Callose deposition assay
5.2.8 Observation of zoospores germination and infection into host tissues
5.2.9 Agrobacterium-mediated transient gene expression in N. benthamiana
5.2.10 Confocal Microscopy
5.3 RESULTS
5.3.1 PcLRR-RK3 is required for vegetative growth of P. capsici
5.3.2 Impact of PcLRR-RK3 on the zoosporangium development and production
5.3.3 PcLRR-RK3 is required for full virulence and in planta growth of P. capsici
5.3.4 PcLRR-RK3 is essential for zoospores penetration and establishment into hostleaf tissues
5.3.5 Sub-cellular localization of PcLRR-RK3
5.4 DISCUSSION
5.5 CONCLUSION
CHAPTER SIX: RNA SEQUENCING FOR COMPARATIVE TRANSCRIPTOMICANALYSIS OF PHYTOPHTHORA CAPSICI AFTER PcLRR-RLKs-SILENCING
5.1 INTRODUCTION
5.2 MATERIALS AND METHODS
5.2.1 RNA extraction and qRT-PCR assay
5.2.2 Library Preparation and Transcriptome analysis
5.3 RESULTS
5.3.1 Overview of the transcriptome
5.3.2 Identification of differentially expressed genes(DEGs) in response to PcLRR-RLKs silencing
5.3.3 GO annotation of DEGs
5.3.4 PcLRR-RLKs regulate the growth of P. capsici
5.3.5 PcLRR-RLKs regulate the infection process in P. capsici
5.3.6 Validation of the transcriptome data by qRT-PCR
5.4 DISCUSSION
5.5 CONCLUSION
SUMMARY AND CONCLUSION
REFERENCES
PUBLICATIONS
本文编号：3574044
【文章来源】：南京农业大学江苏省 211工程院校 教育部直属院校
【文章页数】：178 页
【学位级别】：博士
【文章目录】：
ACKNOWLEDGEMENTS
摘要
ABSTRACT
CHAPTER ONE: INTRODUCTION
1.1 Objectives
1.2 Research Background
1.2.1 Introduction to Phytophthora plant pathogen threatening agriculture and natural ecosystem
1.2.2 Evolution
1.2.3 Biology
1.2.4 Genes for host-Phytophthora interaction
1.2.5 Phytophthora capsici-a model oomycete species with broad host range
1.2.6 Leucine-rich repeat receptor-like kinases(LRR-RLKs)
CHAPTER TWO: GENOME WIDE IDENTIFICATION OF LEUCINE-RICH REPEAT RECEPTOR-LIKE KINASES (LRR-RLKs) IN OOMYCETES
2.1 INTRODUCTION
2.2 MATERIAL AND METHODS
2.2.1 Identification and distribution of LRR-RLKs in oomycetes genome
2.2.2 Preparation of P.capsici cultures and plants
2.2.3 Collection of zoospore suspension and inoculation assay
2.2.4 RNA extraction and real-time RT-PCR assay
2.3 RESULTS
2.3.1 Identification of LRR-RLKs in oomycetes
2.3.2 Evolutionary analysis of LRR-RLKs in oomycetes
2.3.3 Protein structure and conserved motif analysis of LRR-RLKs in oomycetes
2.3.4 Differential expression profiles of P. capsici LRR-RLKs
2.4 DISCUSSION
2.5 CONCLUSION
CHAPTER THREE: PcLRR-RK1,AN LRR RECEPTOR KINASE REGULATESGROWTH, DEVELOPMENT AND PATHOGENESIS IN PHYTOPHTHORA CAPSICI
3.1 INTRODUCTION
3.2 MATERIAL AND METHODS
3.2.1 Identification and distribution of LRR-RLKs in P. capsici genome
3.2.2 Preparation of P. capsici cultures and plants
3.2.3 Construction and transformation of recombinant plasmids
3.2.4 RNA extraction and real-time RT-PCR assay
3.2.5 Analysis of colony growth, zoosporangium development and production
3.2.6 Inoculation assays on N. benthamiana and A. thaliana
3.2.7 DAB staining and callose deposition assay
3.2.8 Microscopic observation of cyst germination and pathogen establishment
3.3 RESULTS
3.3.1 Identification and distribution of LRR-RLKs in P. capsici genome
3.3.2 PcLRR-RK1 is required for vegetative growth of P. capsici
3.3.3 PcLRR-RK1 is essential for zoosporangium development and production
3.3.4 Silencing of PcLRR-RK1 results in loss of zoospores infection abilities into hosttissues
3.3.5 PcLRR-RK1 results into reduced germination of zoospores into host tissues
3.3.6 Silencing of PcLRR-RK1 results in penetration reduction of P. capsici mycelium into the host tissues
3.3.7 DAB staining and Callose deposition assay
3.4 DISCUSSION
3.5 CONCLUSION
CHAPTER FOUR: AN LRR RECEPTOR KINASE,PCLRR-RK2,IS CRUCIALLYREQUIRED FOR VEGETATIVE GROWTH, DEVELOPMENT AND PATHOGENESIS IN PHYTOPHTHORA CAPSICI
4.1 INTRODUCTION
4.2 MATERIAL AND METHODS
4.2.1 Protein sequence selection and identification
4.2.2 Maintenance of microbial strains, plants and culture conditions
4.2.3 Plasmid construction and P. capsici transformation
4.2.4 RNA isolation and quantitative real-time PCR
4.2.5 Phenotypic characterization
4.2.6 Pathogenecity assays
4.2.7 DAB staining and Callose deposition assay
4.2.8 Assays for the germination and penetration of zoospores into host tissues
4.3 RESULTS
4.3.1 Protein sequence selection and identification
4.3.2 PcLRR-RK2 is required for vegetative development of P.capsici
4.3.3 Silencing of PcLRR-RK2 interferes with zoosporangium development of P.capsici
4.3.4 PcLRR-RK2 is important for zoospores infection abilities into host tissues
4.3.5 PcLRR-RK2 results into reduced germination of zoospores into host tissues
4.3.6 PcLRR-RK2 is required for penetration of P. capsici mycelium into host tissues
4.3.7 PcLRR RK2 is important for host penetration,H_2O_2 accumulation and callose deposition
4.4 DISCUSSION
4.5 CONCLUSION
CHAPTER FIVE: PcLRR-RK3, AN LRR RECEPTOR KINASE IS REQUIRED FORVEGETATIVE DEVELOPMENT AND IN-PLANTA INFECTION PROCESSES IN PHYTOPHTHORA CAPSICI
5.1 INTRODUCTION
5.2 MATERIAL AND METHODS
5.2.1 Protein sequence selection and identification
5.2.2 Maintenance of plant and P.capsici cultures
5.2.3 Plasmid Construction and transformation into P.capsici culture
5.2.4 RNA extraction and qRT-PCR assay
5.2.5 Phenotypic analysis of PcLRRK3-silenced transformants
5.2.6 Pathogenecity assay on N. benthamiana
5.2.7 Trypan blue staning,DAB staining and Callose deposition assay
5.2.8 Observation of zoospores germination and infection into host tissues
5.2.9 Agrobacterium-mediated transient gene expression in N. benthamiana
5.2.10 Confocal Microscopy
5.3 RESULTS
5.3.1 PcLRR-RK3 is required for vegetative growth of P. capsici
5.3.2 Impact of PcLRR-RK3 on the zoosporangium development and production
5.3.3 PcLRR-RK3 is required for full virulence and in planta growth of P. capsici
5.3.4 PcLRR-RK3 is essential for zoospores penetration and establishment into hostleaf tissues
5.3.5 Sub-cellular localization of PcLRR-RK3
5.4 DISCUSSION
5.5 CONCLUSION
CHAPTER SIX: RNA SEQUENCING FOR COMPARATIVE TRANSCRIPTOMICANALYSIS OF PHYTOPHTHORA CAPSICI AFTER PcLRR-RLKs-SILENCING
5.1 INTRODUCTION
5.2 MATERIALS AND METHODS
5.2.1 RNA extraction and qRT-PCR assay
5.2.2 Library Preparation and Transcriptome analysis
5.3 RESULTS
5.3.1 Overview of the transcriptome
5.3.2 Identification of differentially expressed genes(DEGs) in response to PcLRR-RLKs silencing
5.3.3 GO annotation of DEGs
5.3.4 PcLRR-RLKs regulate the growth of P. capsici
5.3.5 PcLRR-RLKs regulate the infection process in P. capsici
5.3.6 Validation of the transcriptome data by qRT-PCR
5.4 DISCUSSION
5.5 CONCLUSION
SUMMARY AND CONCLUSION
REFERENCES
PUBLICATIONS
本文编号：3574044
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