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

Arabidopsis DXO1 possesses deNADding and exonuclease activities and its mutation affects defense-related and photosynthetic gene expression^FA

RNA capping and decapping tightly coordinate with transcription,translation,and RNA decay to regulate gene expression.Proteins in the DXO/Rai1 family have been implicated in mRNA decapping and decay,and mammalian DXO was recently found to also function as a decapping enzyme for NAD+-capped RNAs(NAD-RNA).The Arabidopsis genome contains a single gene encoding a DXO/Rai1 protein,AtDXO1.Here we show that AtDXO1 possesses both NAD-RNA decapping activity and 5?-3?exonuclease activity but does not hydrolyze the m7G cap.The atdxo1 mutation increased the stability of NAD-RNAs and led to pleiotropic phenotypes,including severe growth retardation,pale color,and multiple devel-opmental defects.Transcriptome profiling analysis showed that the atdxo1 mutation resulted in upregulation@of defense-related genes but downregulation of photo-synthesis-related genes.The autoimmunity phenotype of the mutant could be suppressed by either eds1 or npr1 mutation.However,the various phenotypes associated with the atdxo1 mutant could be complemented by an enzymatically inactive AtDXO1.The atdxo1 mutation ap-parently enhances post-transcriptional gene silencing by elevating levels of siRNAs.Our study indicates that AtDXO1 regulates gene expression in various biological and physiological processes through its pleiotropic mo-lecular functions in mediating RNA processing and decay.