Comparative approach to decipher the molecular basis of pattern-triggered immunity in Brassica rapa pekinensis

다국어 초록 (Multilingual Abstract)

Chinese cabbage (Brassica rapa ssp. pekinensis) is an important horticultural crop which is particularly popular in Korea. Several bacterial and fungal diseases can significantly impair Chinese cabbage production. Plants rely on their innate immune system to defend themselves against pathogens. The surface-localized pattern-recognition receptors (PRRs) perceive pathogen-associated molecular patterns (PAMPs), such as flagellin, elongation factor Tu or fungal cell wall component chitin, and induce a series of defense responses called pattern-triggered immunity (PTI). PTI efficiently restricts pathogen growth and provide durable broad-spectrum disease resistance. PRRs and PTI signaling have been intensively investigated in the model species Arabidopsis thaliana, however, the comprehensive understanding is scarce in Chinese cabbage.
In order to decipher the molecular basis of PTI in Chinese cabbage, PAMP responsiveness was investigated by assessing typical defense responses. BraNHL10 was identified as a reliable marker gene whose expression was significantly and rapidly induced by several PAMPs. ROS production was rapidly induced upon flg22, elf18 and chitin treatments in Chinese cabbage.
Comparative analysis revealed the presence of putative homologs of Arabidopsis FLS2, EFR and CERK1 in B. rapa genome, and revealed the high similarity between protein sequences of BraPRR and AtPRR. The putative BraFLS2, BraEFR1, BraEFR2 and BraCERK1 were cloned using Golden Gate cloning strategy.
Transient expression of BraFLS2, BraEFRs and BraCERK1 in Nicotiana benthamiana showed all BraPRRs were plasma membrane-localized and BraFLS2 mediated flg22-triggered endocytosis, BraEFR2 mediated elf18-triggered ROS burst and BraCERK1 led to chitin-independent cell death. BraEFR1 expression could not induce ROS burst in N. benthamiana, suggesting that BraEFR1 might not function.
Subsequently, BraFLS2, BraEFR1, BraEFR2 and BraCERK1 were further characterized by functional complementation in Arabidopsis fls2 single mutant or fls2 efr cerk1 triple mutant. The expression of BraFLS2, BraEFR2 and BraCERK1 conferred gain of flg22, elf18 and chitin-responsiveness, respectively, in transgenic lines. Expression of BraFLS2 and BraEFR2 led to flg22 and elf18-induced seedling growth inhibition, respectively, in transgenic BraFLS2 or BraEFR2 expressing lines. Expression of BraFLS2, BraEFR2 and BraCERK1 induced ROS production upon flg22, elf18 and chitin elicitation in transgenic BraPRR expressing lines, respectively. Kinase activity of BraPRRs was also assessed in this study via construction of BraPRR dead kinase versions.
All results indicated that BraFLS2, BraEFR2 and BraCERK1 most likely function as PRRs in Chinese cabbage in a partial kinase-dependent manner.

국문 초록 (Abstract)

배추 (Brassica rapa spp. pekinensis)는 중요한 원예 작물의 하나로 국내에서 인기가 많다. 배추의 생산에 있어 가장 큰 피해는 박테리아와 곰팡이로부터 발생하며, 이 때문에 면역체계에 대한 관련 연구가 활발히 일어나고 있다. 식물은 병원체로부터 자신을 보호하기 위해 선천적인 면역계에 의존한다. 세포 표면에 있는 패턴 인식 수용체 (PRR; Pattern-Recognition Receptor)는 flagellin, elongation factor Tu 혹은 곰팡이 세포벽 성분 chitin 같은 병원체 관련 분자 패턴 (PAMP; Pathogen-Associated Molecular Pattern)을 인지하고 패턴 유발 면역 (PTI; Pattern Triggered Immunity)이라고 불리는 일련의 방어 반응을 유도한다. PTI는 병원체의 성장을 효율적으로 억제시키고 내구성이 있는 광범위한 스펙트럼의 질병에 대한 저항성을 제공한다. 모델 식물 애기장대에서 PRR과 PTI에 대해 연구가 집중적으로 되었지만, 배추에서는 포괄적인 연구가 많이 부족하다.
이에 본 연구는 배추의 PTI 분자적 기초를 탐구하기 위해 대표적인 방어반응을 평가함으로 PAMP 대응성을 조사하였다. 우선 신뢰성 있는 마커 유전자 BraNHL10을 발견 및 확인하였다. BraNHL10 은 여러 PAMP에 의해 그 발현이 현저하고 신속하게 유도되었다. ROS 생산은 배추에서 flg22, elf18 및 chitin 처리시 신속하게 유도됨을 확인할 수 있었다.
비교분석 결과 애기장대의 FLS2, EFR, CERK1의 첨가 호몰로그가 B. rapa 게놈에 존재하며, BraPRR과 AtPRR의 단백질 배열이 높은 유사성을 보이는 것으로 나타났다. 그 다음으로 BraFLS2, BraEFR1, BraEFR2 및 BraCERK1를 골든 게이트 클로닝 전략으로 복제한 후Nicotiana benthamiana에서BraFLS2, BraEFR1, BraEFR2 및 BraCERK1의 일시적인 발현을 통해 모든 BraPRRs이 세포막에 정착함을 확인할 수 있었으며, BraFLS2 발현에 따라flg22 처리시 endocytosis가, BraEFR2발현에 따라elf18 처리시 ROS burst가, BraCERK1발현에 따라 chitin처리와 무관한 세포예정사가 유발되었다. BraEFR1의 발현은N. benthamiana에서 ROS burst을 유도하지 않았으므로 BraEFR1은 기능하지 않는 것으로 추정되었다.
이어서 BraFLS2, BraEFR1, BraEFR2 및 BraCERK1이 애기장대 단일 돌연변이 fls2 혹은 트리플 돌연변이 fls2 efr cerk1에서 진행한 기능보완시험을 통해 기능을 재확인하였다. BraFLS2, BraEFR2 및 BraCERK1의 발현은 flg22, elf18 및 chitin에 대한 인식과 관련된 면역 반응을 각각 보완하였다. BraFLS2와BraEFR2의 발현으로 형질전환 식물에서 각각 flg22 및 elf18으로 유도된 묘목 성장 억제를 보였다. BraFLS2, BraEFR2 및 BraCERK1의 발현은 형질전환체에서flg22, elf18 및 chitin 처리시 또 각각 ROS 생산을 유도하였다. 그리고 BraPRR의 kinase 활성이 부여한 기능을 연구하기 위해 BraPRR의 dead kinase 버전을 구축해서 면역 반응을 평가하였다.
이 모든 결과로부터 BraFLS2, BraEFR2 및 BraCERK1이 배추에서 PRR 역할을 담당하고 그들의 기능은 kinase의 활성에 부분적 의지하고 있을 것으로 추정되었다.

목차 (Table of Contents)

• ABSTRACT i
• CONTENTS v
• LIST OF TABLES viii
• LIST OF FIGURES ix
• LIST OF ABBREVIATIONS x
• INTRODUCTION 1
• MATERIALS AND METHODS 8
• Plant materials and growth conditions 8
• PAMP solutions 9
• RNA isolation and cDNA synthesis 9
• Reverse transcription polymerase chain reaction (RT-PCR) 10
• Quantitative Real-time PCR (qRT-PCR or qPCR) 10
• Oxidative burst measurement 11
• Cloning and Construction of PRR library 11
• Agrobacterium-mediated transient expression in N. benthamiana 13
• Subcellular localization analysis 13
• Generation of transgenic PRR expressing lines 14
• PAMP-triggered seedling growth inhibition 14
• RESULTS 15
• Pattern-triggered immune responses in Chinese cabbage 15
• Multiple homologs of AtPRRs exist in Chinese cabbage genome. 15
• PAMPs upregulated the expression of defense marker genes in Chinese cabbage. 17
• flg22, elf18 and chitin triggered ROS burst in Chinese cabbage. 22
• Identification and characterization of BraFLS2, BraEFR and BraCERK1 candidates 27
• At least one B. rapa ortholog of AtFLS2, AtEFR and AtCERK1 express in Chinese cabbage. 27
• BraPRRs share a high similarity with their orthologous AtPRRs in protein sequences. 29
• BraFLS2, BraEFR, and BraCERK1 were expressed in 1, 2, 4, 6-week-old Chinese cabbage leaves. 38
• Cloning and expression library of PRRs were created using Golden Gate Assembly. 43
• Functional analysis of BraFLS2, BraEFR1, BraEFR2 and BraCERK1 by transient expression in N. benthamiana 47
• BraPRR-YFPs were localized at the plasma membrane of plant cells. 47
• BraFLS2 endocytosis was induced upon flg22 perception. 52
• BraEFR2 expression induced elf18-triggered ROS production. 53
• BraCERK1 expression led to chitin-independent cell death. 56
• Functional complementation in Arabidopsis transgenic BraPRR-expressing lines 59
• Screening of Arabidopsis transgenic PRR expressing lines 59
• BraFLS2 and BraEFR2 expression complemented flg22 and elf18-triggered seedling growth inhibition in fls2 and fec mutants. 61
• Expression of BraPRRs led to PAMP responsive ROS production in fls2 or fec mutant. 65
• Functional complementation in SGI assay and oxidative burst assay correlated with transgene expression. 69
• DISCUSSION 76
• The repertoire of PAMP-perception in Chinese cabbage 76
• Divergence in the expression of two BraFLS2 candidates 77
• Functionality between two BraEFR candidates 79
• BraCERK1: the PTI dogma breaker 80
• Kinase activity of BraFLS2, BraEFR2 and BraCERK1 for immune responses 81
• Diverse degrees of immune responses from T2 transgenic plants 83
• The conserved and sophisticated immune system of plant 83
• REFERENCES 85
• APPENDICES 96
• ABSTRACT IN KOREAN 105