The Influence Of Tobacco Curly Shoot Virus And Its Betasatellite On Photosynthesis Of Nicotiana Benthamiana And Responses Of Resistance Genes Based On RNAseq

Tobacco curly shoot virus?TbCSV?is a monopartite virus belonging to the genus Begomovirus in the family Geminiviridae.TbCSV is an important pathogen causing leaf curl disease on tomato and tobacco,widely spreaded and have caused great losses to crops in Yunnan and Sichuan province.The evolutionary relationship and interaction mechanism between monopartite begomovirus and betasatellite is complicated in China.TbCSV is considered to be an evolutionary intermediate between the betasatellite requiring begomoviruses and the truly monopartite begomoviruses.Therefore,the study of TbCSV is of great significance for elucidating the origin and genetic evolution mechanism of geminivirus.At present,some studies has been carried out on TbCSV and TbCSB,mainly focusing on virus detection,pathogenicity,gene function,protein localization,population variation analysis,gene silencing vector construction.However,interaction mechanism of TbCSV with host,influence on host gene expression and biological processes and TbCSV-triggered disease resistance are still unclear.Making clear of above questions will help to resolve the pathogenesis of the TbCSV.In this study,the TbCSVon the endogenous gene the impacts of TbCSV infection on the endogenous genes expression of N.benthamiana,the functional roles and involved biological processes of these differential expression genes?DEGs?were analysed by transcriptome sequencing technology.Based on bioinformatics results,changes in photosynthesis pathway,responses of PR and LRR-RLK genes and BR and JA pathways involvement post TbCSV+TbCSB infection were explicated by gene silencing and overexpression,and research results of this study are summarized as follows:1.Effects of TbCSV and TbCSV+TbCSB infection on the endogenous genes expression of N.benthamianaThrough transcriptome sequencing,59814 genes were identified from both TbCSV-infected and TbCSV+TbCSB-infected plants.Among them,3196 differentially expressed genes?DEGs?were identified in group Y35A vs CK,5.3%of the total number of genes.Among them,1391 genes were up-regulated and 1,805 genes were down-regulated.4081 DEGs were identified in group Y35AB vs CK,6.8%of the total number of genes.Among them,1775 genes were up-regulated and 2306 genes were down-regulated.The DEGs in group Y35AB vs CK were enriched in total of 3074 GO terms with 2215 GO terms from up-regulated genes and 2658 GO terms from down-regulated genes,respectively.124 GO terms were significantly enriched,85 GO terms from up-regulated DEGs and 166 GO terms from down-regulated DEGs.The DEGs in group Y35A vs CK were enriched in total of 2,966 GO terms,among them the up-regulated and down-regulated DEGs enriched 2041 and 2033 GO terms,respectively.76 GO terms were significantly enriched,78 GO terms from up-regulated DEGs and 66GO terms from down-regulated DEGs.These DEGs mainly involved in biological process,metabolic process,cellular process,organic substance metabolic process,primary metabolic process,and play roles in biological pathways including ribosome,plant hormone signal transduction,metabolic pathways,DNA replication,carbon fixation in photosynthetic organisms.12 genes were randomly selected for RT-qPCR,and the expression trends of these genes were consistant with sequencing data.Upon TbCSV and TbCSV+TbCSB infection,the expression levels of defense-related genes and genes involved in photosynthesis were altered.How these genes function during virus infection needs further investigation.2 Effects of TbCSV infection on the photosynthesisAt 20 dpi,the contents of chlorophyll a in TbCSV-infected and TbCSV+TbCSB-infected plants was 458.7?g/g and 475.0?g/g,respectively,of which significantly lower than that in control plants?579.7?g/g?.Compared to control plants?141.6?g/g?,the content of chlorophyll b in TbCSV-and TbCSV+TbCSB-infected plants were 122.3?g/g and 126.7?g/g,respectively,of which were decreased without significant differences.The determination of photosynthetic physiological characteristic in N.benthamiana plants after TbCSV-and TbCSV+TbCSB-infected showed that the Net photosynthetic rate?Pn?,Stomatal conductance?Gs?,Transpiration rate?Tr?,Value of stomatal limits?LS?,Water use efficiency?WUE?,Light use efficiency?LUE?and CO₂use efficiency?CUE?of TbCSV-or TbCSV+TbCSB-infected plants were significantly reduced when compared to control plants,while Intercellular carbon dioxide concentration?Ci?was significantly increased.The results of chlorophyll fluorescence characteristics measurement showed that the Non-photochemical quenching?qN?and Excitation capture efficiency of PS??F_v'/F_m'?in TbCSV-or TbCSV+TbCSB-infected plants were increased,Electron transport rate?ETR?,Quantum efficiency of PS???PSII?and Photochemical quenching?qP?were reduced when compared to control plants.Ultrastructural observation of chloroplast revealed decreased number of chloroplast in both TbCSV-infected and TbCSV+TbCSB-infected plants.Medium-sized starch granules were accumulated in the chloroplast in TbCSV-infected plants.Swelling chloroplast,bending deformation,and squeezed lamellar structure inside the chloroplast were observed in TbCSV+TbCSB-infected plants.Among tweenty-three DGEs that induced upon both TbCSV and TbCSV+TbCSB infection,15 genes?65%?were related to chloroplast.After TRV-or PVX-mediated expression of candidate genes NbGDCSa,NbGDCSb,NbrbcS,NbChlHa,NbChlHb,NbDXS,NbHemA,NbBchP,NbGRP and NbPU in N.benthamiana,plants exhibiting yellowing,albino,chlorisis,mottle,vein clearing,wilting and necrosis,leaf curling,bulging and dwarfing.These results verified that infection with TbCSV or TbCSV+TbCSB could reduce the photosynthesis of plants.3 Responses of PR and LRR-RLK genes upon TbCSV infectionTo clarify the involvement of PR and LRR-RLK genes during virus infection,11diffrentially expressed PR genes and 12 diffrentially expressed LRR-RLK genes were randomly selected for quantification at 4,8 and 12 dpi in TbCSV-infected and TbCSV+TbCSB-infected plants.RT-qPCR results showed that the expression levels of genes NbPR2,NbPR3,NbPR11?PR genes?and Nb1g56140,Nb1g67720,Nb2g16250,NbEI-LRR?LRR-RLK genes?all were up-regulated at 4 dpi,8 dpi and 12 dpi in both TbCSV-infected and TbCSV+TbCSB-infected plants.The expression levels of NbPR1,NbPR4,NbPR5,NbPR9,NbPR10a,NbPR17?PR genes?and Nb1g06840,Nb3g47570a,Nb4g26540,Nb4g30520,NbGSO1?LRR-RLK genes?were up-regulated at some time points and not significantly expressed at other time points in TbCSV-infected or TbCSV+TbCSB-infected plants.The expression levels of NbPR6,NbPR10b?PR genes?and Nb3g47570b,Nb4g36180,NbFbLRR-MAX2?LRR-RLK genes?were up-regulated at some time points after TbCSV-or TbCSV+TbCSB-infection and down-regulated at other time points.Post TRV-mediated expression of NbPR2,NbPR6,NbPR9,NbPR10a and NbPR11,plants were agro-infiltrated with TbCSV or TbCSV+TbCSB and the accumulation of TbCSV and TbCSB were detected at 4 dpi,8 dpi and 12 dpi.The results showed that after silencing of NbPR2,NbPR6,NbPR9 and NbPR10a,the accumulation of TbCSV was significantly increased at 4 dpi in TbCSV-infeted and TbCSV+TbCSB-infected plants,respectively.The accumulation of TbCSB was significantly increased in both NbPR9-silenced and NbPR10a-silenced plants.While in NbPR11-silenced plants,the accumulation of TbCSV was significantly decreased at 4dpi and 8 dpi in both TbCSV-infected and TbCSV+TbCSB-infected plants,and the accumulation of TbCSB also was significantly decreased at 4 dpi.These results uncovered that PR and LRR-RLK genes response to TbCSV or TbCSV+TbCSB infection and partial PR genes regulate the accumulation of TbCSV and TbCSB during infection.4 Responses of BR and JA pathway-related genes to TbCSV infectionTo identify the potential roles of BR and JA pathway in virus infection,6 BR pathway-related genes and 10 JA pathway-related genes were screened and quantified by RT-qPCR in TbCSV-or TbCSV+TbCSB-infected plants at 4,8 and 12 dpi.The expression levels of NbBZR1,NbCYCD3-X2?BR pathway?and NbDOX1,NbHPL1,NbKAT2,NbJAR1,NbJAZ1?NbJAZ2?JA pathway?were up-regulated upon TbCSV-or TbCSV+TbCSB infection at some time points and not significantly expressed at other time points compared to the control.The expression levels of NbCYP90C1/D1,NbBRI1,NbBZR2,NbCYCD3-X1?BR pathway?and NbSAMT1,NbSAMT2,NbCOI1,NbMYC2?JA pathway?were up-regulated at some time points after TbCSV or TbCSV+TbCSB infection,and down-regulated at some time points.The JA content detection results showed that upon TbCSV or TbCSV+TbCSB infection at 12 dpi,the JA contents in plants infected by TbCSV?14.04 pg/g?or TbCSV+TbCSB?12.33 pg/g?were increased when compared with control?8.89 pg/g?.Because BZR1 and CYCD3 are involved in the regulation of plant growth and development,and to clarify the potential roles of NbBZR1 and NbCYCD3-X1 in development and defense,they were overexpressed by PVX and results exhibited that 20 days after NbBZR1 overexpression,shorter plant height and pitch,downward crinkle,deformation and uniform distribution of necrotic spots were observed in overexpressed plants.NbCYCD3-X1 overexpressed plants showed slightly crinkle in the newly emerged leaves compared to control plants.After silencing or overexpression of the candidate genes,the accumulation of TbCSV and TbCSB were detected.No matter NbBZR1 was silenced or overexpressed,the accumulation of TbCSV and TbCSB were significantly decreased at 4 dpi in both treatments compared to control.After overexpression of NbCYCD3-X1,the accumulation of TbCSV was significantly decreased at 4 dpi in both treatments compared to control,but the accumulation of TbCSB was significantly increased at 4dpi.After silencing of NbCOI1,the accumulation of TbCSV was significantly decreased at 8 and 12 dpi in TbCSV-infected plants,but significantly increased at 4,8 and 12 dpi in TbCSV+TbCSB-infected plants,while the accumulation of TbCSB was significantly decreased at 4 dpi.After treatment with exogenous MeJA and BL,the accumulation trend of TbCSV and TbCSB was consistent in the two treatments.The accumulation of TbCSV was significantly increased at 4 dpi in TbCSV-infected plants,and was significantly decreased at 8 and 12 dpi.While the accumulation of TbCSV and TbCSB were significantly decreased at 4 dpi and 8 dpi,and was significantly increased at 12 dpi in TbCSV+TbCSB-infected plants.5 Interaction between BR and JA pathways in response to virus infectionTwelve hours post BL treatment,the expression level of NbCOI1 was significantly increased.The expression levels of NbCOI1 was increased at all three time points post TbCSV infection,and was increased at 4 dpi and 12 dpi in TbCSV+TbCSB-infected plants.Six and twelve hours after treatment with MeJA,the expression level of NbBRI1was significantly increased.The expression level of NbBRI1 was increased at 4 dpi and8 dpi in TbCSV-infected plants,and was increased at 4 dpi and 12 dpi in TbCSV+TbCSB-infected plants.After co-treatment with MeJA+BL 12 h,the expression level of NbCOI1 and NbBRI1 were significantly increased.The expression levels of NbCOI1 and NbBRI1 were both increased in TbCSV-infected plants at three time points,and were increased in TbCSV+TbCSB-infected plants at 4 dpi.The above results indicated that after treatment with MeJA,the BR pathway receptor gene NbBRI1was induced before or after infection with TbCSV or TbCSV+TbCSB.Similarly,after treatment with BL,the JA pathway receptor gene NbCOI1 was also induced before or after infection with TbCSV or TbCSV+TbCSB.Moreover,after co-treatment with MeJA+BL,the NbBRI1 and NbCOI1 were induced.It was speculated that the MeJA and BL treatments promoted the expression of these two the receptor genes in both signaling pathways.This indicated that mutual promotion between BR pathway and JA pathway plays a role in the regulation process of plant resistance to TbCSV or TbCSV+TbCSB infection.In this study,transcriptome analysis indicated that TbCSV or TbCSV+TbCSB infection affect the expression of host genes that involved in various physiological functions and biological processes.Upon TbCSV or TbCSV+TbCSB infection,the photosynthesis ability of host plants was reduced.At the same time,PR and LRR-RLK genes,as well as the BR and JA pathways,participate in defense to the infection of TbCSV and its betasatellite,and are involved in the regulation of viral and satellite accumulation.These findings provide a scientific basis for the molecular mechanisms between virus/betasatellite-host interactions,as well as host disease resistance mechanisms.