Fast-Suppressor Screening for New Components in Protein Trafficking,Organelle Biogenesis and Silencing Pathway in Arabidopsis thaliana Using DEX-Inducible FREE1-RNAi Plants  

作　　者：Qiong Zhao Caiji Gao PoShing Lee Lin Liu Shaofang Li Tangjin Hu Jinbo Shen Shuying Pan Hao Ye Yunru Chen Wenhan Cao Yong Cui Peng Zeng Sheng Yu Yangbin Gao Liang Chen Beixin Mo Xin Liu Shi Xiao Yunde Zhao Silin Zhong Xuemei Chen Liwen Jiang 

机构地区：[1]Centre for Cell & Developmental Biology and State Key Laboratory of Agrobiotechnology,School of Life Sciences,The Chinese University of Hong Kong [2]Department of Botany and Plant Sciences,Institute of Integrative Genome Biology,University of California [3]CUHK Shenzhen Research Institute,The Chinese University of Hong Kong [4]School of Life Sciences,The Chinese University of Hong Kong [5]Beijing Genomics Institute at Shenzhen [6]Section of Cell and Developmental Biology,University of California San Diego [7]Shenzhen Key Laboratory of Microbial Genetic Engineering,College of Life Sciences,Shenzhen University [8]State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources,School of Life Sciences,Sun Yat-sen University

出　　处：《Journal of Genetics and Genomics》2015年第6期319-330,共12页遗传学报（英文版）

基　　金：supported by grants from the NIH GM114660 to Y.Zhao;the Research Grants Council of Hong Kong (CUHK466011,465112,466613,CUHK2/CRF/11G,C401114R and Ao E/M-05/12);NSFC/RGC (N_CUHK406/12);NSFC (31270226 and 31470294);Shenzhen Peacock Project (KQTD201101) to L.Jiang

摘　　要：Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for transport）.FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body（MVB）,vacuolar protein transport and vacuole biogenesis,and autophagic degradation.FREE1 knockout plants show defective MVB formation,abnormal vacuolar transport,fragmented vacuoles,accumulated autophagosomes,and seedling lethality.To further uncover the underlying mechanisms of FREE1 function in plants,we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants.The obtained mutants are termed as suppressors of free1（sof）.To date,229 putative sof mutants have been identified.Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors.Also145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants.Through next-generation sequencing（NGS）of bulked DNA from F2 mapping population of two RNAi-related sof mutants,FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly.These FREE1-and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis,as well as for uncovering the new components involved in the regulation of silencing pathways in plants.Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for transport）.FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body（MVB）,vacuolar protein transport and vacuole biogenesis,and autophagic degradation.FREE1 knockout plants show defective MVB formation,abnormal vacuolar transport,fragmented vacuoles,accumulated autophagosomes,and seedling lethality.To further uncover the underlying mechanisms of FREE1 function in plants,we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants.The obtained mutants are termed as suppressors of free1（sof）.To date,229 putative sof mutants have been identified.Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors.Also145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants.Through next-generation sequencing（NGS）of bulked DNA from F2 mapping population of two RNAi-related sof mutants,FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly.These FREE1-and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis,as well as for uncovering the new components involved in the regulation of silencing pathways in plants.

关 键 词：SUPPRESSORS FREE1 Endomembrane trafficking Arabidopsis NGS 

分 类 号：Q945[生物学—植物学]