Development of an efficient expression system with large cargo capacity for interrogation of gene function in bamboo based on bamboo mosaic virus

Bamboo is one of the fastest growing plants among monocotyledonous species and is grown extensively in subtropical regions.Although bamboo has high economic value and produces much biomass quickly,gene functional research is hindered by the low efficiency of genetic transformation in this species.We therefore explored the potential of a bamboo mosaic virus(BaMV)-mediated expression system to investigate genotype-phenotype associations.We determined that the sites between the triple gene block proteins(TGBps)and the coat protein(CP)of BaMV are the most efficient insertion sites for the expression of exogenous genes in both monopodial and sympodial bamboo species.Moreover,we validated this system by individually overexpressing the two endogenous genes ACE1 and DEC1,which resulted in the promotion and suppression of intemode elongation,respectively.In particular,this system was able to drive the expression of three 2A-linked betalain biosynthesis genes(more than 4 kb in length)to produce betalain,indicating that it has high cargo capacity and may provide the prerequisite basis for the development of a DNA-free bamboo genome editing platform in the future.Since BaMV can infect multiple bamboo species,we anticipate that the system described in this study will greatly contribute to gene function research and further promote the molecular breeding of bamboo.

Multi-omics of Circular RNAs and Their Responses to Hormones in Moso Bamboo(Phyllostachys edulis)

Circular RNAs(circRNAs)are endogenous non-coding RNAs with covalently closed structures,which have important functions in plants.However,their biogenesis,degradation,and function upon treatment with gibberellins(GAs)and auxins(1-naphthaleneacetic acid,NAA)remain unknown.Here,we systematically identified and characterized the expression patterns,evolutionary conservation,genomic features,and internal structures of circRNAs using RNase R-treated libraries from moso bamboo(Phyllostachys edulis)seedlings.Moreover,we investigated the biogenesis of circRNAs dependent on both cis-and trans-regulation.We explored the function of circRNAs,including their roles in regulating microRNA(miRNA)-related genes and modulating the alternative splicing of their linear counterparts.Importantly,we developed a customized degradome sequencing approach to detect miRNA-mediated cleavage of circRNAs.Finally,we presented a comprehensive view of the participation of circRNAs in the regulation of hormone metabolism upon treatment of bamboo seedlings with GA and NAA.Collectively,our study provides insights into the biogenesis,function,and miRNA-mediated degradation of circRNAs in moso bamboo.

Allele-aware chromosome-scale assembly of the allopolyploid genome of hexaploid Ma bamboo(Dendrocalamus latiflorus Munro)

Dendrocalamus latiflorus Munro is a woody clumping bamboo with rapid shoot growth.Both genetic transformation and clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene editing techniques are available for D.latiflorus,enabling reverse genetic approaches.Thus,D.latiflorus has the potential to be a model bamboo species.However,the genome sequence of D.latiflorus has remained unreported due to its polyploidy and large genome size.Here,we sequenced the D.latiflorus genome and assembled it into three allele-aware subgenomes(AABBCC),representingthe largest genome of a major bamboo species.We assembled 70 allelic chromosomes(2,737 Mb)for hexaploid D.latiflorus using both singlemolecule sequencing from the Pacific Biosciences(Pac Bio)Sequel platform and chromosome conformation capture sequencing(Hi-C).Repetitive sequences comprised 52.65%of the D.latiflorus genome.We annotated 135231 protein-coding genes in the genome based on transcriptomes from eight different tissues.Transcriptome sequencing using RNA-Seq and Pac Bio singlemolecule real-time long-read isoform sequencing revealed highly differential alternative splicing(AS)between non-abortive and abortive shoots,suggesting that AS regulates the abortion rate of bamboo shoots.This high-quality hexaploid genome and comprehensive strand-specific transcriptome datasets for this Poaceae family member will pave the way for bamboo research using D.latiflorus as a model species.

Profiling of circular RNA N^(6)-methyladenosine in moso bamboo(Phyllostachys edulis)using nanopore-based direct RNA sequencing

N6-methyladenosine(m^(6)A)is a prevalent modification in messenger RNAs and circular RNAs that play important roles in regulating various aspects of RNA metabolism.However,the occurrence of the m^(6)A modification in plant circular RNAs has not been reported.A widely used method to identify m^(6)A modifications relies on m^(6)A-specific antibodies followed by next-generation sequencing of precipitated RNAs(MeRIP-Seq).However,one limitation of MeRIP-Seq is that it does not provide the precise location of m^(6)A at single-nucleotide resolution.Although more recent sequencing techniques such as Nanopore-based direct RNA sequencing(DRS)can overcome such limitations,the technology does not allow sequencing of circular RNAs,as these molecules lack a poly(A)tail.Here,we developed a novel method to detect the precise location of m^(6)A modifications in circular RNAs using Nanopore DRS.We first enriched our samples for circular RNAs,which we then fragmented and sequenced on the Nanopore platform with a customized protocol.Using this method,we identified 470 unique circular RNAs from DRS reads based on the back-spliced junction region.Among exonic circular RNAs,about 10%contained m^(6)A sites,which mainly occurred around acceptor and donor splice sites.This study demonstrates the utility of our antibody-independent method in identifying total and methylated circular RNAs using Nanopore DRS.This method has the additional advantage of providing the exact location of m^(6)A sites at single-base resolution in circular RNAs or linear transcripts from non-coding RNA without poly(A)tails.