Map-based Cloning And Characterization Of Trichome Genes In Tomato

Trichomes are hair-like structures originating from epidermal cells that are widely present on the surface of the aerial organs of plants.They are classified as unicellular or multicellular,glandular or non-glandular,and branched or unbranched,depending on the species.As a first line of defense,trichomes function in protecting plants against various biotic and abiotic stress.Trichome formation has been well characterized as an excellent model system for studying cell differentiation and cell morphogenesis in Arabidopsis.However,the regulatory mechanisms of multicellular trichome in tomato are still largely unknown.To analyze the genetic and molecular mechanisms underlying trichome development in tomato(Solanum lycopercicum),linkage analysis and fine-mapping were conducted to map two key genes response for trichome development.The functions of Hl2 and Ln,through map-based cloning were analyzed and a regulatory network was predicted.The main results are as follows:1.Hairless-2(Hl-2)gene was cloned and its molecular mechanism was analyzed.1.1 Genetic analysis and identification of Hl-2The hairless phenotype was controlled by a single recessive gene in the F2 population constructed from the hairless mutant 3-417 and the hairy parent LA1589.The gene responsible for the hairless was named as Hairless-2(Hl-2)which was mapped to chromosome 2 using BSR-seq strategy.Then,Hl-2 was fine mapped to the region of 74.7 kb between the two markers NH8 and NH9.Sequence analysis revealed 11 predicted genes in this fragment.We amplified and sequenced the cDNA sequences of this candidate gene from 3-417 and Ailsa Craig.Nucleotide sequencing showed that there was a 100 bp fragment insertion of the cDNA from 658 to 659 bp in the mutant 3-417,resulting in a reading frame shift and premature stop codon.1.2 Functional verification and phenotype analysis of Hl-2The hairless phenotype was restored when OFR5 was overexpressed in hl-2 mutant.ORF5 was knocked out in Ailsa Craig by CRISPR/Cas9 mediated gene editing technology,resulting in the hairless phenotype similar to the mutant.Complementary test showed ORF5 was HI-2.SEM was used to compare the morphology of trichomes on stems and leaves of the hl-2 mutant with Alisa Craig.The most conspicuous phenotypes of the mutant were the absence of normal type I trichomes and the presence of highly twisted and swollen trichomes,indicating that Hl-2 plays an important role in the development of type I trichomes.Hl-2 was constitutively expressed in various tissues.The analysis of Hl-2 promoter fused to the GUS reporter gene demonstrated that GUS activity was detected in several tissues,including shoot apexes,stems,axillary meristems,and leaves,with stronger GUS-derived signal in shoot apexes,axillary meristems,young stems,and adaxial young leaves.1.3 SIHDZIV8 regulated trichome morphogenesis through binding the promoters of Hl-2 or Hl genesHl-2 directly interacted with Hl(SRA1)in vivo by yeast two hybrid and CoImmunoprecipitation to regulate type I trichome development.SlHDZIV8 encodes a HD ZIP IV protein.SlHDZIV8-RNAi plants exhibited reduced density of trichomes and the morphology of trichomes obviously changed.The expression of Hl-2 and Hl were downregulated in SIHDZIV8-RNAi plants,indicating that the two genes may be regulated by SlHDZIV8.SlHDZIV8 could bind to both the promoter of Hl-2 and Hl directly by yeast one hybrid and EMSA.1.4 Mutation in Hl-2 reduced mechanical strength and cellulose contentCompared to Alisa Craig,the stem of the hl-2 mutant showed obvious brittleness,reduced mechanical strength,thinner stem,smaller stem cells,hollow network structure of vascular tissue,and significantly reduced cellulose content.The relative expressions of related genes CESA4,CESA7 and CESA8 involved in the synthesis of secondary cell walls were significantly reduced in the mutant.2.Another novel hairy gene Ln was cloned and characterized in tomato.2.1 Map-based cloning and functional verification of LnSegregation populations were constructed through crossing of a trichome mutant Ln(LA3127)and a nontrichome accession LA1589(S.pimpinellifolium).Genetics analysis showed that the hairy trait in mutant LA3127 is controlled by one single dominant gene.The gene was delimited in the 136 kb region between markers LMP-12 and LM-5,which includes 14 predicted ORFs.After sequencing all the candidate genes,a mutation was found in an ORF7,indicating that ORF7 may be the target gene.We amplified and sequenced the cDNA sequences of this candidate gene from LA3127 and Ailsa Craig.There is a missense mutation(C-1676 to G)and results in an amino acid change of Ala to Glu in the mutant LA3127.Ln encodes a HD-zip IV,localizing in the nucleus.The expression pattern of Ln was analyzed by qRT-PCR using RNAs isolated from root,young stems,functional leaves,flowers,and green fruits from Alisa Craig.Ln is expressed in all tissues,with relatively high level in the functional leaves.Complementary test showed ORF7 was Ln.2.2 Ln directly interacts with Wo in vitro and in vivoGenetic analysis showed Wo genetically interacted Ln and there was an additive effect between them。Ln interacts with Wo to form complex to regulate Type Ⅰ trichome formation by yeast two hybrid、BIFC and Co-Immunoprecipitation.2.3 Ln regulated the expression of H and directly interacts with H to affected affect the formation of Type Ⅰ glandular trichomeGenetic analysis showed Ln genetically interacted H and h gene was recessive epistatic to the Ln.The expression level of H gene was up-regulated in Ln mutant,whereas down-regulated in the Ln-CR plants,indicating that H gene may be positively regulated by Ln.EMSA and yeast one hybrid revealed that Ln binds to H directly.Ln also interacts with H by yeast two hybrid and BIFC.In conclusion,Hl-2 and Ln genes were isolated and identified by map-based cloning.Hl-2 interacted with Hl and was regulated by SlHDZIV8,which jointly affected the trichome morphogenesis in tomato,and clarified the role of Hl-2 in the mechanical properties in tomato.The genetic and molecular interactions between the genes Ln and Wo and H,which regulate the formation of type I glandular trichome,were identified.