Identification Of NtVQ35 Gene In Nicotiana Tabacum And Preliminary Study On Its Function Against Tobacco Bacterial Wilt Disease

Tobacco bacterial wilt disease is of economic importance for tobacco industry,which is worldwide distributed especially in tropical and subtropical regions,and can seriously impact and decrease the quality of tobacco plants.It was reported that huge economic loss caused by this disease ranks the fourth among all the tobacco diseases in China.This disease is caused by the bacteria-Ralstonia solanacearum(Rsc).The VQ proteins,identified by the presence of a conserved FxxhVQxhTG amino acid motif,have been shown to be major transcriptional regulators in plants working independently or in combination with other transcription factors to regulate diverse growth and developmental processes and responses to biotic and abiotic stresses.To date,little information is currently available on the VQ protein family in cultivated tobacco(Nicotiana tabacum L.),and on the role(s)of these transcriptional regulators in controlling plant growth and development and response to various biotic and abiotic stressors,particularly on the disease resistance against Rsc.Therefore,the VQ protein family in tobacco was identified using bioinformatics methods in this study.Furthermore,their gene and protein structural characteristics were systematically analyzed,and the responses of the family gene members under different stress factors were explored as well.After systematically screening,NtVQ35 gene was selected for further research.To explore its function in resistance to tobacco bacterial wilt,NtVQ35 expressed and overexpressed NtVQ35 plants were obtained by gene-editing of clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated genes(Cas9)or Gateway technology together with tobacoo leaf-disk transgenic technology.Observation of symptoms,statistics of disease index and statistical comparison of bacterial numbers in leaves of gene-editing lines,overexpressed lines and wild type(WT)after Rsc inoculation were carried out in this research.Meanwhile,comparative transcriptome analysis was performed to analyze the mechanism of NtVQ35 against bacterial wilt in tobacco plants at the transcriptional level.In addition,free-state salicylic acid was also mearsured in gene-editing plants,overexpressed plants and WT plants.The main results are as follows:1.NtVQ protein family of tobacco were classified into groups I-VIII,and NtVQ genes’ response to phytohormonal,abiotic stimuli and Rsc treatmentFifty-nine tobacco vq genes were identified and analyzed by bioinformatics methods in this research.Comprehensive analyses of the gene expression patterns were conducted in tobacco treated by salicylic acid(SA),jasmonate acid(JA),ethephon(ET),abscisic acid(ABA),auxin(2,4 D),and abiotic stimuli [cold(4?C),heat(35?C),wounding] and Rsc treatment,repectively.Results showed that 59 NtVQ protein family can be divided into eight groups I-VIII according to their amino acid sequences and all conservative domains structures.Members of the II,IV,V,VI and VIII groups can respond to one or more hormones.Group VII did not respond to any of the hormones.NtVQ proteins with similar domains have similar response patterns to different hormones.Group II and V were the most responsive to ethephen treatment,while group V and VIII were the most responsive to ABA.Most NtVQ genes can respond to SA treatment.During the cold and heat treatments,NtVQ33 and NtVQ34 were significantly induced.The genes NtVQ32,NtVQ46 and NtVQ58 were significantly induced under wounding treatment.More than half of NtVQ genes were significantly up-regulated by Rsc.NtVQ35 gene was significantly modulated by both SA and Rsc.In addition,the promoter region of NtVQ35 contains three TC-rich repeats,which is the cis-elements related to stress and defense response.Therefore,it is suggested that this gene may play a certain role in plant resistance against Rsc in tobacco.2.NtVQ35 gene of tobacco plays a negative regulatory role against RscNtVQ35 gene-editing and overexpressing transgenic plants were obtained with the CRISPR/Cas9 or Gateway technology by leaf-disk transgenic way.There was no phenotypic difference observed in the obtained NtVQ35 gene-editing transgenic lines(H4-8 and H7-8)and overexpressed lines(OE5,OE6 and OE8).Therefore,it was speculated that this gene would not affect the growth and phenotypic changes of plants.The results of inoculation of Rsc on gene-editing transgenic line H7-8,overexpressed line OE6 and WT K326 tobacco showed that the H7-8 plants(disease index 22.22%)wilted significantly compared with WT plants at 4 d post inoculation(dpi),while the H7-8 gene-editing plants showed no significant difference compared to the WT plants with the disease index 0.55%,4.45%,respectively.At 9 dpi,wilting symptom was shown on all leaves of the H7-8 plants with black stem base,on most leaves of the WT plants and half leaves of the H7-8 plants.The disease indexes of OE6,WT and H7-8 were 90%,71% and 48.25%,respectively.In addition,the symptoms of gene-editing line H4-8,overexpressing line OE6 and WT plants were also observed at 3 dpi.Results showed that OE6 occurred significant necrosis and chlorosis.WT plants showed apparent chlorosis and yellow circle around the inoculation locus while the H4-8 also occurred yellow circle around the inoculation locus.However,the symptoms on H4-8 generally were not as serious as on WT planlts.At 3 dpi,the numbers of Rsc on all leaves of tobacco were counted,the H4-8 plants had the least numbers of bacteria,H7-8 was less,while the OE6 plants had the most,indicating that the gene-editing plants were more resistant to Rsc than the overexpressing plants did.In conclusion,NtVQ35 gene plays a negative regulatory role in plant resistance against Rsc.3)The negative regulation of Rsc by NtVQ35 may be somehow related to SA signal transduction pathwayTranscriptomic data were analyzed and compared among OE6,H4-8 and WT plants sampled at 1 dpi.Results showed that the significant enrichment metabolic pathways in KEGG related to plant disease resistance are mainly flavonoids biosynthesis pathways,plant hormone signal transduction pathways and plants-pathogen interaction pathways.Particularly,the expression level of genes related to flavonoids synthesis in gene-editing plants after bacterial wilt infection were higher than that in overexpressed plants.In PTI pathway,gene expression of FLS2 and CNGC was significantly higher than that of overexpressed plants,so it was speculated that gene-editing plant could produce stronger basic immunity than overexpressed plants.On the other hand,in the ETI pathway,the R gene RPS2 was highly induced in the gene-editing plants compared with that in the overexpressed plants,indicating that they were more stimulated by the Avr gene(effector protein)of Rsc than those in the overexpressed plants.The expression of related genes in SA,ET and BR pathways in gene-editing plants were also significantly higher than that in overexpressed plants,indicating that the gene-editing plants were more resistant to bacterial wilt than overexpressed plants.In addition,in order to explore the potential role of SA in related to resistance of NtVQ35 to Rsc,high performance liquid chromatography(HPLC)was used to determine the content of SA in gene-editing H4-8,overexpression OE6 and WT plants at 4 dpi.The results showed that the content of free-state SA in gene-editing leaves(656.9 ng/g)was significantly higher than that in WT(204.6 ng/g)and overexpressed plants(164.2 ng/g)at 4 dpi,suggesting that the negative regulation of Rsc by NtVQ35 may be somehow related to SA signal transduction pathway.Further research is worthwhile to be addressed in the future.