Assembly Of Paulownia Fortunei Genome And Functional Analysis Of Paulownia Witches’ Broom Related Gene PfCYP714A

Paulownia is a deciduous tree of Paulowniaceae,which is native to China.Paulownias have fast growing,good quality and strong adaptability,which made them often highly prized worldwide in gardening and landscaping for their opulent and fragrant flowers,elegant heart-shaped leaves,and large canopies.However,this tree species is easy to be infected with Paulownia witches’ broom disease(Pa WB),an infectious disease caused by phytoplasma.Paulownias with phytoplasma infection often have some symptoms such as witches’ broom,yellowing leaves and internode shortening,etc.,resulting in slow growth and even death of trees,seriously hindering the industrial development of paulownia.Therefore,a systematic and comprehensive study on the genes related to the occurrence of Pa WB will help to better clarify the molecular mechanism of witches’ broom disease and develop a scientific and effective methods to prevention and control it.However,although a series of researches had been conducted,the mechanism of Pa WB has not been fully clarified for the limitation of the genetic information of the host and the difficulty to cultivate phytoplasmas in vitro.Thus,in this study,a combination of short-read sequencing,long-read sequencing,and chromosome conformation capture sequencing(Hi-C)were performed to sequence the genome of Paulownia fortunei.At the same time,we also conducted a proteogenomics analysis to re-annotation the P.fortunei genome.Based on these results,the proteins related to Pa WB were screened,and the function of Pf CYP714 A was verified.Furthermore,the potential binding partners was identified,which preliminary clarified the molecular mechanism of dwarf symptom,and a model diagram was drawn.The main results are summarized as follows:1.The high-quality chromosome-scale nuclear genome and proteome map of P.fortunei was drawn for the first time.In this study,a combination of short sequencing,long-read sequencing,and chromosome conformation capture sequencing was performed to sequence the genome of P.fortunei.The final genome size of P.fortunei is 511.6 M,Contig N50 is 852.4 K,with 92.88%genome reads can anchor into the pseudomolecules.A total of 31,985 protein coding genes were predicted.Repetitive sequence analysis showed that the P.fortunei genome containing approximately 50% repetitive elements,of which Ty3/gypsy and Ty1/copia accounted for a high proportion,and no obvious LTR-RTs burst had occurred in P.fortunei within the past eight million years.At the same time,combined label free proteomics technology and protein genomics research technology,666 novel protein coding genes were identified,208 genes were revised,and three single amino acid variant and 390 genes with alternative splicing were identified,which improved the genome annotation of P.fortunei,and laid a solid foundation for the biological analysis of paulownia.2.The protein PfCYP714 A specifically related to the occurrence of Paulownia witches’ broom disease was screened and its function was verified.Based on the the Paulownia witches’ broom disease simulation system,a lable-free quantitative proteomics technology was applied,and a total of 71 proteins were considered as the closely related to Pa WB.Co-expression network analysis of these proteins revealed that Pf CYP714 A may be associated with the occurrence of witches’ broom disease.Pf CYP714 A encodes a cytochrome P450 protein,which is located on the endoplasmic reticulum with a constitutive expression.Overexpression of Pf CYP714 A can interfere the normal growth of plants,which make them show dwarfing symptoms,and reduce the content of GA in plants;In addition,endogenous hormone measurements showed that the levels of gibberellin(GA1 and GA4)in the internodes of transgenic plants were significantly lower than those of wild type plants.These results provided a theoretical basis for the study of the pathogenesis of Pa WB.3.Screened the interaction proteins Pf ACO1 and Pf SAM2 of Pf CYP714 A and verified a relationship between endogenous hormones GA and ET that mediated by Pf CYP714 A.Using yeast two hybrid assay(Y2H),18 proteins interacting with Pf CYP714 A were screened.Y2 H,Bi FC,and GST pull-down showed that the 1-aminocyclopropane-1-carboxylic acid oxidase gene(Pf ACO1)and S-adenosylmethionine synthetase 2(Pf SAM2)that related to hormone metabolism are the interactors of Pf CYP714 A.Further analysis of endogenous hormone content showed that overexpression of Pf CYP714 A significantly changed the content of endogenous hormones ethylene in plants compared to wild type plants,and Pf CYP714 A affected the expression of ethylene synthesis genes Pf ACO1 and Pf SAM2.Subcellular localization analysis of Pf ACO1 and Pf SAM2 showed that they were co-localized with Pf CYP714 A.These results suggest that there is a synergistic effect between endogenous hormones GA and ET in the occurring of dwarf symptom,which laid the foundation for elucidating the pathogenesis of Pa WB.4.Discovery of transcription factors that regulate the expression of Pf CYP714 A and preliminary elucidation of the molecular mechanism of stunting symptoms in witches’ broom disease.The Pf CYP714 A promoter activation region that located upstream 316-896 of the ATG was screened by using the transient expression analysis.Through the yeast single hybrid assay,ten proteins that may interact with the Pf CYP714 A promoter were screened,including chromatin remodeling factor SWP73(Pf SWP73)and transcription factor PIF8(Pf PIF8).Y1 H and Dual-Luc experiments have demonstrated that Pf PIF8 can bind the promoter and activate its expression of Pf CYP714 A.Meanwhile,through yeast two hybrid and Bi FC,we found that Pf PIF8,Pf RGA1,Pf RGA2,and Pf SWP73 may form a transcriptional regulatory complex that co-regulates the expression of Pf CYP714 A.The results elucidate the molecular mechanism of dwarfing symptoms of witches’ broom disease and providing data support for the cultivation of new varieties.In conclusion,we presented a chromosome-level P.fortunei genome assembly,and re-annotated the genome using proteogenomics technology.Finally,the key proteins Pf CYP714 A that related to the occurrence of Pa WB dwarf symptoms were obtained.At the same time,the transcription factors that regulate the expression of Pf CYP714 A and the proteins that interact with them were screened.Based on the comprehensive research results,a model map for the molecular mechanism of dwarfing symptoms occurence of Pa WB was drawn,which provides an important theoretical basis for clarify the occurrence of Pa WB and also a solid foundation for exploration of excellent genes and the cultivation of new varieties of paulownia with Pa WB resistance.