Functional Characterization of AtLecRK-VI.2 in Nicotiana benthamiana and Tomato Plants and Screening of Cysteine-rich Receptor-like Kinases Involved in Arabidopsis Defense Responses

病原菌相關分子模式(pathogen-associated molecular patterns, PAMPs) 會被植物細胞表面的模式辨識受體(pattern-recognition receptors, PRR)所辨 識而引發植物的免疫反應。模式誘發免疫反應(pattern-triggered immunity, PTI)是植物免疫中最初步限制病原菌入侵增生的機制。先前研究顯示，利用基因 轉殖技術，將阿拉伯芥的EFR(一種PRR)轉殖到菸草(Nicotiana benthamiana) 及番茄(Solanum lycopersicum)中，會使得該基因轉殖植物能廣泛地抵禦不同種 的病原菌，這顯示著PTI 的訊息傳遞機制，即使在不同科的植物中仍是保守的。 除此之外，這項研究也為利用PTI 來進行基因改造，培育抗病作物設立了典範。 我們先前發現阿拉伯芥的凝集素受體激酶VI.2(AtLecRK-VI.2)是PTI 的正向調 控者，為了瞭解AtLecRK-VI.2 在其他植物物種中是否也是PTI 的正向調控者， 我培育了過度表現AtLecRK-VI.2 的菸草及蕃茄。初步的實驗結果顯示，菸草轉 殖株較野生型能抵抗病菌Pseudomonas syringae pv. syringae B728a (Pss)。 進一步分析顯示，轉殖菸草的抗性可能來自於較強的PTI反應，如較胝質的累積、 活性氧自由基的產生及PTI 標的基因的表現等。這些初步的結果，顯示了菸草可 能帶有AtLecRK-VI.2 訊息傳遞的相關元件，來正向調控PTI。 在阿拉伯芥的類受體蛋白激酶中，富含半胱胺酸類受體激酶(Cysteine-rich receptor-like kinases, CRKs)被認為參與植物抵抗病原菌的調控。藉由結合阿拉伯芥凝集素受體激酶VI.2的過度表現轉殖株的轉錄組學分析及公開的微陣列資料，我們發現有12個CRK可能參與植物的防禦反應。我們藉由基因功能喪失的篩選，這些CRK並沒有顯現與野生種相異的表現型，因此，我們將藉由讓基因過度表現的方式來對這些CRK進行分析。Recognition of pathogen-associated molecular patterns (PAMPs) by host cell surface-localized pattern-recognition receptors (PRRs) activates plant innate immunity. PAMP-triggered immunity (PTI) is the first layer of plant immunity. Crucial experiments demonstrated that the transfer of the Arabidopsis PRRs EFR to N. benthamiana and tomato (S. lycopersicum) gave the transgenic plants broad-spectrum resistance against pathogens, suggesting PTI signaling machinery is conserved across different plant families and setting an example utilizing PTI to confer disease resistance in crops. We have recently identified Arabidopsis LecRK-VI.2 as a positive regulator of PTI. To test whether AtLecRK-VI.2 can also be a positive regulator of PTI in other plant species, I generated transgenic N. benthamiana and tomato plants over-expressing AtLecRK-VI.2. Preliminary test on transgenic N. benthamiana T2 plants showed that they are resistant against bacteria P. syringae pv. syringae (Pss) B728a. Additional analyses suggest their resistance is due to enhanced PTI responses, such as callose deposition, ROS production, and expression of PTI marker genes. These preliminary findi ngs indicate N. benthamiana contains the components required for AtLecRK-VI.2 signaling to positively modulate the PTI response. Of the receptor-like kinases (RLKs) in Arabidopsis, cysteine-rich receptor-like kinases (CRKs) have been suggested to play important roles in the regulation of pathogen defense. Combining transcriptome analysis from a AtLecRK-VI.2 overexpression line and publicly available microarray data revealed 12 CRKs that could potentially be involved in plant defense. Our results show that loss-of-function analysis on these CRKs did not reveal an altered phenotype; thus further analysis is being conducted using gain-of-function analysis